Ps. putida

Farmaco, 2005 Jan, 60(1), 23 - 26 Epub 2004 Dec 19.
Synthesis and antibacterial activity of 4-benzoyl-1-methyl-5-phenyl-1H-pyrazole-3-carboxylic acid and derivatives; Akbas E et al.; Some new 1H-pyrazole-3-carboxylic acid and pyridazinone derivatives were synthesized and evaluated for their antibacterial activities against Bacillus cereus ATCC 7064, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 4230 and Pseudomonas putida using tube dilution method . The minimal inhibitory concentrations (MICs) experiments revealed that all chemical compounds showed inhibitor effects on the growth of the test microorganisms . Moreover, the results of this research showed that the compound named as 5c was the best compound in the series, exhibiting antibacterial activity against both Gram-positive and Gram-negative bacteria.

Can J Microbiol, 2004 Oct, 50(10), 861 - 7
Influence of substrate composition and flow rate on growth of Azospirillum brasilense Cd in a co-culture with 3 sorghum rhizobacteria; Lippi D et al.; The ability of Azospirillum brasilense Cd to colonize the niche occupied by 3 bacterial strains previously isolated from sorghum rhizosphere was studied by means of the Biolog system . The isolates were identified by different methods as strains belonging to Pseudomonas putida, Stenotrophomonas maltophilia, and Klebsiella terrigena species . Several C sources, also chosen among the constituents of sorghum root exudates, were used to evaluate the metabolic profiles of Azospirillum and the sorghum rhizobacteria . Azospirillum brasilense Cd exploited the same class of C compounds as the sorghum rhizobacteria and overlapped in their niche requirements . Since structure and functioning of a microbial community are largely affected by the flow rate of nutrient supply, the competitive behavior of A . brasilense Cd was studied in a chemostat mixed culture under C-limited conditions using disodium succinate as C source . Only at high growth rates, i.e., when the C source was highly supplied, A . brasilense Cd appeared to be a good competitor and it became the dominant species, whereas at low growth rates, it was outnumbered by the other species . However, the coexistence of all the strains was always maintained, thus suggesting that interactions other than competition or a potential cross-feeding might occur within the mixed culture.

Environ Microbiol, 2005 Jan, 7(1), 88 - 97
Actinide and metal toxicity to prospective bioremediation bacteria; Ruggiero CE et al.; Summary Bacteria may be beneficial for alleviating actinide contaminant migration through processes such as bioaccumulation or metal reduction . However, sites with radioactive contamination often contain multiple additional contaminants, including metals and organic chelators . Bacteria-based bioremediation requires that the microorganism functions in the presence of the target contaminant, as well as other contaminants . Here, we evaluate the toxicity of actinides, metals and chelators to two different bacteria proposed for use in radionuclide bioremediation, Deinococcus radiodurans and Pseudomonas putida, and the toxicity of Pu(VI) to Shewanella putrefaciens . Growth of D . radiodurans was inhibited at metal concentrations ranging from 1.8 microM Cd(II) to 32 mM Fe(III) . Growth of P . putida was inhibited at metal concentrations ranging from 50 microM Ni(II) to 240 mM Fe(III) . Actinides inhibited growth at mM concentrations: chelated Pu(IV), U(VI) and Np(V) inhibit D . radiodurans growth at 5.2, 2.5 and 2.1 mM respectively . Chelated U(VI) inhibits P . putida growth at 1.7 mM, while 3.6 mM chelated Pu(IV) inhibits growth only slightly . Pu(VI) inhibits S . putrefaciens growth at 6 mM . These results indicate that actinide toxicity is primarily chemical (not radiological), and that radiation resistance does not ensure radionuclide tolerance . This study also shows that Pu is less toxic than U and that actinides are less toxic than other types of metals, which suggests that actinide toxicity will not impede bioremediation using naturally occurring bacteria.

Appl Environ Microbiol, 2005 Jan, 71(1), 51 - 7
Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa: Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies; Arango Pinedo C et al.; The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp . was investigated by examinations of filter matings . A Pseudomonas putida KT2442-derived strain carrying a gfp-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients . The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer . We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators . Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer . At standard donor-to-recipient ratios (10(-3) for PAO1162N and 2 x 10(1) for PAO2002N) and total cell densities (10(5) cells/mm(2) for PAO1162N and 10(6) cells/mm(2) for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10(-7) (events/mm(2))(-1) for PAO1162N and 10(-11) (events/mm(2))(-1) for PAO2002N based on in situ observations of conjugation events . The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower . Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (P < 0.05) but not for the restriction-deficient strain PAO1162N . On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency . Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.

Appl Microbiol Biotechnol . 2005 Jan 6; {Epub ahead of print}
Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp . by an intrinsic phosphopantetheinyl transferase; Gross F et al.; We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv . tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase . The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue . The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin . The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts . The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography . Each purified CP was analysed using MALDI/TOF for the expected mass increase . Of the seven CPs tested, six could be purified from P . putida, which was chosen as the general host strain . Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form . Four domains were also expressed in the other hosts.

Biosens Bioelectron, 2005 Feb 15, 20(8), 1648 - 51
Respiratory response of cell-based sensors to toxicants measured by using pseudo-random signals; Hikuma M et al.; Cell-based sensors using such as Pseudomonas putida and Bacillus subtilis were applied to examine the toxicity of chemicals . Both toxicant and substrate solutions were introduced into the sensor system according to M-series and anti-symmetric M-series pseudo-random binary signals, X(n) (n = 15, minimal pulse width 4min, period 60min) and Y(n) (n = 30, minimal pulse width 4min, period 120min) . Toxicants such as Ag(+), formaldehyde, azide, and hypo-chlorite were used to demonstrate the proposed method . The individual responses of substrate and toxicant were obtained at a time by calculating the cross-correlation function between the input and the output signals . Effects of toxicant on the response of substrate and the response of toxicant itself can be observed at a time, so that the method appears to be useful in studying the behavior of microorganisms in the presence of toxicants.

Acta Crystallogr D Biol Crystallogr, 2005 Jan, 61(Pt 1), 75 - 9 Epub 2004 Dec 17.
The atomic resolution structure of methanol dehydrogenase from Methylobacterium extorquens; Williams PA et al.; The crystal structure of methanol dehydrogenase (MDH) from Methylobacterium extorquens has been refined without stereochemical restraints at a resolution of 1.2 A . The high-resolution data have defined the conformation of the tricyclic pyrroloquinoline quinone (PQQ) cofactor ring as entirely planar . The detailed definition of the active-site geometry has shown many features that are similar to the quinohaemoprotein alcohol dehydrogenases from Comamonas testosteroni and Pseudomonas putida, both of which possess MDH-like and cytochrome c-like domains . Conserved features between the two types of PQQ-containing enzyme suggest a common pathway for electron transfer between MDH and its physiological electron acceptor cytochrome c(L) . A pathway for proton transfer from the active site to the bulk solvent is also suggested.

Biotechnol Lett, 2004 Oct, 26(20), 1585 - 8
Cloning and expression of the polyhydroxyalkanote depolymerase gene from Pseudomonas putida, and characterization of the gene product; Jiang Y et al.; A polyhydroxyalkanote (PHA) depolymerase gene ( pha Z) was cloned by PCR from Pseudomonas putida and over-expressed in Escherichia coli as inclusion bodies . Nucleotide sequence analysis predicted an 852 bp open reading frame encoding a protein of 283 amino acids with a predicted molecular weight of 31283 Da . The deduced amino acid sequence had at least 80% homology to the PHA depolymerase from other Pseudomonas strains and consisted a conserved lipase box-like sequence (G-X-S(102)-X-G) . The inclusion bodies were refolded and biochemically characterized . The depolymerase activity was optimal at 40 degrees C and pH 8.

Org Biomol Chem, 2005 Jan 7, 3(1), 57 - 64 Epub 2004 Nov 18.
Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450(cam) and P450(BM-3); Sowden RJ et al.; The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties . Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450(cam) from Pseudomonas putida, and of P450(BM-3) from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance . Wild type P450(cam) did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products . Wild type P450(BM-3) and mutants had higher activities (up to 43 min(-1)) than P450(cam) but were much less selective . Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised . The selectivity patterns suggest that (+)-valencene has one binding orientation in P450(cam) but multiple orientations in P450(BM-3).

J Bacteriol, 2005 Jan, 187(1), 396 - 9
Assimilation of nitrogen from nitrite and trinitrotoluene in Pseudomonas putida JLR11; Caballero A et al.; Pseudomonas putida JLR11 releases nitrogen from the 2,4,6-trinitrotoluene (TNT) ring as nitrite or ammonium . These processes can occur simultaneously, as shown by the observation that a nasB mutant impaired in the reduction of nitrite to ammonium grew at a slower rate than the parental strain . Nitrogen from TNT is assimilated via the glutamine syntethase-glutamate synthase (GS-GOGAT) pathway, as evidenced by the inability of GOGAT mutants to use TNT . This pathway is also used to assimilate ammonium from reduced nitrate and nitrite . Three mutants that had insertions in ntrC, nasT, and cnmA, which encode regulatory proteins, failed to grow on nitrite but grew on TNT, although slower than the wild type.

J Bacteriol, 2005 Jan, 187(1), 155 - 67
Regulation of the Pseudomonas sp . strain ADP cyanuric acid degradation operon; Garcia-Gonzalez V et al.; Pseudomonas sp . strain ADP is the model strain for studying bacterial degradation of the s-triazine herbicide atrazine . In this work, we focused on the expression of the atzDEF operon, involved in mineralization of the central intermediate of the pathway, cyanuric acid . Expression analysis of atzD-lacZ fusions in Pseudomonas sp . strain ADP and Pseudomonas putida showed that atzDEF is subjected to dual regulation in response to nitrogen limitation and cyanuric acid . The gene adjacent to atzD, orf99 (renamed here atzR), encoding a LysR-like regulator, was found to be required for both responses . Expression of atzR-lacZ was induced by nitrogen limitation and repressed by AtzR . Nitrogen regulation of atzD-lacZ and atzR-lacZ expression was dependent on the alternative sigma factor sigmaN and NtrC, suggesting that the cyanuric acid degradation operon may be subject to general nitrogen control . However, while atzR is transcribed from a sigmaN-dependent promoter, atzDEF transcription appears to be driven from a sigma70-type promoter . Expression of atzR from a heterologous promoter revealed that although NtrC regulation of atzD-lacZ requires the AtzR protein, it is not the indirect result of NtrC-activated AtzR synthesis . We propose that expression of the cyanuric acid degradation operon atzDEF is controlled by means of a complex regulatory circuit in which AtzR is the main activator . AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system.

J Bacteriol, 2005 Jan, 187(1), 125 - 34
m-xylene-responsive Pu-PnifH hybrid sigma54 promoters that overcome physiological control in Pseudomonas putida KT2442; Carmona M et al.; The sequences surrounding the -12/-24 motif of the m-xylene-responsive sigma54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH sigma54 promoter variants known to have various degrees of efficacy and affinity for sigma54-RNA polymerase (RNAP) . In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P . putida KT2442 with a tailored vector system . The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the -12/-24 region of the wild-type PnifH and its higher sigma54-RNAP affinity variants PnifH049 and PnifH319 . As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well . When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant . Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.

J Bacteriol, 2005 Jan, 187(1), 85 - 91
Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases; van Beilen JB et al.; Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes . First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host . In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P . putida AlkB) or W58 (in the case of A . borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine . The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I) . Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes . Subsequent selection for growth on longer alkanes restored the leucine codon . A structure model of AHs based on these results is discussed.

J Microbiol Immunol Infect, 2004 Dec, 37(6), 343 - 9
Clinical experiences of bacteremia caused by metallo-beta-lactamase-producing gram-negative organisms; Lee NY et al.; The emergence of acquired metallo-beta-lactamase (MBL) in gram-negative bacilli is regarded as a therapeutic challenge since such enzymes are capable of hydrolyzing all beta-lactams in vitro except the monobactams . The clinical characteristics and outcome of 8 episodes of gram-negative bacteremia caused by MBL-producing isolates from January 1997 through December 2000 (Klebsiella pneumoniae, 6 isolates; Pseudomonas stutzeri, 4; Pseudomonas aeruginosa, 1; and Pseudomonas putida, 1) were analyzed . The median age of the patients was 61 years (range, 2-95 years) . Most patients (n = 6, 75%) had more than 1 comorbid illness or condition and 6 patients acquired bacteremia in the intensive care unit . The median time from admission to the first positive culture was 34.5 days (range, 1-99 days) . Pneumonia was the most common site of infection . Five patients (62.5%) received a carbapenem to treat bacteremia . The median time to defervescence was 6 days (range, 2-12 days) . No bacteriologic failure was noted during or after antimicrobial therapy . The overall mortality rate from bacteremia caused by gram-negative, MBL-producing organisms was nil at 14 or 28 days.

FEMS Microbiol Lett, 2004 Dec 15, 241(2), 163 - 9
Evidence for past integration of IncP-1 plasmids into bacterial chromosomes; Chiu CM et al.; Plasmids of the IncP-1 incompatibility group are self-transmissible between and stably maintained in a very broad range of Gram-negative bacteria . A characteristic feature of IncP-1 genomes is the existence of multiple binding sites (O(B)) for the KorB protein which plays a dual role in active partitioning of plasmid and coordinate regulation of expression of genes for replication, maintenance and transfer . A search of the available bacterial genome sequences revealed a significant number (70 out of 322) with one or more putative KorB binding sites . Binding of KorB to such a site was demonstrated by chromatin immunoprecipitation (ChIP) for Pseudomonas putida KT2440 . While such a site may arise by chance, this is unlikely for Pseudomonas aeruginosa UCBPP-PA14 whose genome sequence contains four clustered O(B) sites and several regions have more than 80% nucleotide identity to traJ, trbJ and trbL of IncP-1 plasmids . A number of other bacterial genomes also contain integrated partial IncP-1 genomes or their remnants . These data provide evidence for multiple past integration events of IncP-1 plasmids into bacterial chromosomes and provide new evidence for IncP-1 plasmids being important elements in gene mobility.

Biochim Biophys Acta, 2004 Dec 1, 1703(1), 11 - 9
Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion; Hontzeas N et al.; The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to alpha-ketobutyrate and ammonium . This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association . A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified . It showed selectivity and high activity towards the substrate ACC: K(M)=3.4+/-0.2 mM and k(cat)=146+/-5 min(-1) at pH 8.0 and 22 degrees C . The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe tapering in activity at higher pH resulting in loss of activity at pH>10 . The major component of the enzyme's secondary structure was determined to be alpha-helical by circular dichroism (CD) . P . putida UW4 ACCD unfolded at 60 degrees C as determined by its CD temperature profile as well as by differential scanning microcalorimetry (DSC) . Enzyme activity was knocked out in the point mutant Gly44Asp . Modeling this mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to the active site . This enzyme's biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC deaminases from other organisms can be compared.

J Bacteriol, 2004 Dec, 186(24), 8267 - 75
Genetic evidence that catabolites of the Entner-Doudoroff pathway signal C source repression of the sigma54 Pu promoter of Pseudomonas putida; Velazquez F et al.; Glucose and other C sources exert an atypical form of catabolic repression on the sigma54-dependent promoter Pu, which drives transcription of an operon for m-xylene degradation encoded by the TOL plasmid pWW0 in Pseudomonas putida . We have used a genetic approach to identify the catabolite(s) shared by all known repressive C sources that appears to act as the intracellular signal that triggers downregulation of Pu . To this end, we reconstructed from genomic data the pathways for metabolism of repressor (glucose, gluconate) and nonrepressor (fructose) C sources . Since P . putida lacks fructose-6-phosphate kinase, glucose and gluconate appear to be metabolized exclusively by the Entner-Doudoroff (ED) pathway, while fructose can be channeled through the Embden-Meyerhof (EM) route . An insertion in the gene fda (encoding fructose-1,6-bisphosphatase) that forces fructose metabolism to be routed exclusively to the ED pathway makes this sugar inhibitory for Pu . On the contrary, a crc mutation known to stimulate expression of the ED enzymes causes the promoter to be less sensitive to glucose . Interrupting the ED pathway by knocking out eda (encoding 2-dehydro-3-deoxyphosphogluconate aldolase) exacerbates the inhibitory effect of glucose in Pu . These observations pinpoint the key catabolites of the ED route, 6-phosphogluconate and/or 2-dehydro-3-deoxyphosphogluconate, as the intermediates that signal Pu repression . This notion is strengthened by the observation that 2-ketogluconate, which enters the ED pathway by conversion into these compounds, is a strong repressor of the Pu promoter.

Appl Environ Microbiol, 2004 Dec, 70(12), 6968 - 76
Use of Pseudomonas putida EstA as an anchoring motif for display of a periplasmic enzyme on the surface of Escherichia coli; Yang TH et al.; The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications . In this report, we investigated the N-terminal fusion display of the periplasmic enzyme beta-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes . To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain . The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting . The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability . Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active . These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E . coli . This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.

J Biol Chem . 2004 Nov 23; {Epub ahead of print}
The putative malate/lactate dehydrogenase from Pseudomonas putida is a NADPH-dependent delta1-piperideine-2-carboxylate/delta1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline; Muramatsu H et al.; A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence databases was disrupted by homologous recombination . The resultant dpkA(-) mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source . The dpkA gene was cloned and overexpressed in Escherichia coli and the gene product was characterized . The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as delta1-piperideine-2-carboxylate and delta1-pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively . NADH also served as a hydrogen donor for both substrates although the reaction rates were less than 1% of those with NADPH . The reverse reactions were also catalyzed by the enzyme, but at much lower rates . Thus, the enzyme has dual metabolic functions, and we named the enzyme delta1-piperideine-2-carboxylate/delta1-pyrroline-2-carboxylate reductase: the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.

J Hazard Mater, 2004 Dec 10, 116(1-2), 167 - 71
Use of poultry litter for biodegradation of soil contaminated with 2,4- and 2,6-dinitrotoluene; Gupta G et al.; Pseudomonas sp . and Pseudomonas putida can utilize dinitrotoluene (DNT) as N-source after the enzymatic removal of nitro groups from the aromatic ring . Addition of nutrients is known to stimulate the biodegradation process . Poultry litter has consortia of microorganisms (including Pseudomonas) along with many nutrients . The objective of this research was to study the biodegradation of 2,4- and 2,6-DNT contaminated soil (from Badger Army Ammunition Plant) using poultry litter . Complete biodegradation of both 2,4- and 2,6-DNT in the soil was observed after 1-day interaction with poultry litter . No degradation was observed using autoclaved litter.

Environ Microbiol, 2004 Dec, 6(12), 1264 - 86
Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440; Dos Santos VA et al.; A major challenge in microbiology is the elucidation of the genetic and ecophysiological basis of habitat specificity of microbes . Pseudomonas putida is a paradigm of a ubiquitous metabolically versatile soil bacterium . Strain KT2440, a safety strain that has become a laboratory workhorse worldwide, has been recently sequenced and its genome annotated . By drawing on both published information and on original in silico analysis of its genome, we address here the question of what genomic features of KT2440 could explain or are consistent with its ubiquity, metabolic versatility and adaptability . The genome of KT2440 exhibits combinations of features characteristic of terrestrial, rhizosphere and aquatic bacteria, which thrive in either copiotrophic or oligotrophic habitats, and suggests that P . putida has evolved and acquired functions that equip it to thrive in diverse, often inhospitable environments, either free-living, or in close association with plants . The high diversity of protein families encoded by its genome, the large number and variety of small aralogous families, insertion elements, repetitive extragenic palindromic sequences, as well as the mosaic structure of the genome (with many regions of 'atypical' composition) and the multiplicity of mobile elements, reflect a high functional diversity in P . putida and are indicative of its evolutionary trajectory and adaptation to the diverse habitats in which it thrives . The unusual wealth of determinants for high affinity nutrient acquisition systems, mono- and di-oxygenases, oxido-reductases, ferredoxins and cytochromes, dehydrogenases, sulfur metabolism proteins, for efflux pumps and glutathione-S-transfereases, and for the extensive array of extracytoplasmatic function sigma factors, regulators, and stress response systems, constitute the genomic basis for the exceptional nutritional versatility and opportunism of P . putida , its ubiquity in diverse soil, rhizosphere and aquatic systems, and its renowned tolerance of natural and anthropogenic stresses . This metabolic diversity is also the basis of the impressive evolutionary potential of KT2440, and its utility for the experimental design of novel pathways for the catabolism of organic, particularly aromatic, pollutants, and its potential for bioremediation of soils contaminated with such compounds as well as for its application in the production of high-added value compounds.

J Biol Chem . 2004 Nov 12; {Epub ahead of print}
Succinate complex crystal structures of the alpha -ketoglutarate-dependent dioxygenase AtsK: Steric aspects of ernzyme self-hydroxylation; Muller I et al.; The alkylsulfatase AtsK from Pseudomonas putida S-313 is a member of the non-heme iron(II)-alpha-ketoglutarate dependent dioxygenase superfamily . In the initial step of their catalytic cycle, enzymes belonging to this widespread and versatile family coordinate molecular oxygen to the iron center in the active site . The subsequent decarboxylation of the cosubstrate alpha-ketoglutarate yields carbon dioxide, succinate, and a highly reactive ferryl (IV) species, which is required for substrate oxidation via a complex mechanism involving the transfer of radical species . Non-productive activation of oxygen may lead to harmful side-reactions, and such enzymes therefore an effective built-in protection mechanism . One of the ways of controlling undesired side reactions is the self-hydroxylation of an aromatic side-chain, which leads to an irreversibly inactivated species . Here we describe the crystal structure of the alkylsulfatase AtsK in complexes with succinate and with Fe(II)/succinate . In the crystal structure of the AtsK-Fe(II)-succinate complex the side chain of Tyr168 is coordinated to the iron, suggesting that Tyr168 is the target of enzyme self-hydroxylation . This is the first structural study of an Fe(II)-alpha-ketoglutarate dependent dioxygenase that presents an aromatic side chain coordinated to the metal center, thus allowing structural insight into this protective mechanism of enzyme self-inactivation.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(11-12), 2983 - 91
Effect of combined nutrients on biosurfactant produced by Pseudomonas putida; Amezcua-Vega C et al.; This work investigated biosurfactant production by Pseudomonas putida in combined C/P, C/Ninorganic, C/Fe, C/Mg nutrient ratios and peptone concentration . Analysis of the 2(5-1) fractional factorial experimental design showed that only the C/Fe ratio had a significant (p<0.02) effect on biosurfactant production . The highest amount of biosurfactant was obtained at low C/Fe ratios, but net surface tension did not show significant differences . In addition, low amounts of peptone and the C/P-C/Mg nutrient ratios interaction significantly (p < 0.05) enhanced the biomass produced by P . putida . Analysis of biosurfactant by gas chromatography (GC) showed that the hydrophilic fraction was composed by rhamnose and the hydrophobic fraction, mainly by palmitic (C16), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) fatty acids.

Plasmid, 2004 Nov, 52(3), 169 - 81
Transcriptional regulation of pWW0 transfer genes in Pseudomonas putida KT2440; Lambertsen LM et al.; The conjugative IncP-9 plasmid pWW0 (TOL) carries transfer genes, many of whose functions can be predicted from sequence similarities to the well-studied IncW and IncP-1 plasmids, and that are clustered with the replication and maintenance genes of the plasmid core . In this study we show that the IncP-9 transfer genes are transcribed from at least three promoter regions . The promoters for traA and traD act divergently from the region found to encode the origin of transfer, oriT . These promoters regulate expression of traA, B, and perhaps traC in one direction and traD in the other, all of whose gene products are predicted to be involved in relaxasome formation and DNA processing during transfer, and they are repressed by TraA . The third promoter region, upstream of mpfR, is responsible for transcription of mpfR and mpfA to mpfJ, encoding proteins involved in mating pair formation . Transcription from this region is negatively autoregulated by MpfR . Thus the pWW0 transfer genes, like those of the IncP-1 plasmids, are expressed at all times, but kept in control by a negative feed back loop to limit the metabolic burden on the host . Although many of the related mating pair formation systems are, as in pWW0, transcribed divergently from an operon for replication and/or stable inheritance functions, MpfR is not related to the known regulatory proteins of these other transfer systems outside those of the IncP-9 family and indeed the regulators tend to be specific for each plasmid family . This suggests that the general pattern of genetic organisation exhibited by these systems has arisen a number of times independently and must therefore be highly favourable to plasmid survival and spread.

Appl Microbiol Biotechnol . 2004 Oct 23; {Epub ahead of print}
Production of 3-hydroxy- n-phenylalkanoic acids by a genetically engineered strain of Pseudomonas putida; Sandoval A et al.; Overexpression of the gene encoding the poly-3-hydroxy- n-phenylalkanoate (PHPhA) depolymerase ( phaZ) in Pseudomonas putida U avoids the accumulation of these polymers as storage granules . In this recombinant strain, the 3-OH-acyl-CoA derivatives released from the different aliphatic or aromatic poly-3-hydroxyalkanoates (PHAs) are catabolized through the beta-oxidation pathway and transformed into general metabolites (acetyl-CoA, succinyl-CoA, phenylacetyl-CoA) or into non-metabolizable end-products (cinnamoyl-CoA) . Taking into account the biochemical, pharmaceutical and industrial interest of some PHA catabolites (i.show $132#e., 3-OH-PhAs), we designed a genetically engineered strain of P . putida U ( P . putida U DeltafadBA-phaZ) that efficiently bioconverts (more than 80%) different n-phenylalkanoic acids into their 3-hydroxyderivatives and excretes these compounds into the culture broth.

Appl Biochem Biotechnol, 2004 Oct, 119(1), 51 - 70
High-cell-density cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates; Diniz SC et al.; We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHAMCL) using an equimolar mixture of glucose and fructose as carbon sources . Kinetics studies of P . putida growth resulted in a maximum specific growth rate of 0.65 h(-1) . Limitation and inhibition owing to NH4+ ions were observed, respectively, at 400 and 3500 mg of NH4+/L . The minimum concentration of dissolved oxygen in the broth must be 15% of saturation . Fed-batch strategies for high-cell-density cultivation were proposed . Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHAMCL content . Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHAMCL . Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHAMCL content . Using the last strategy, 21% PHAMCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH4+ limitation . Using phosphate limitation, 50 g/L cell concentration, 63% PHAMCL and a productivity of 0.8 g/(L x h) were obtained in 42 h of fed-batch operation . The PHAMCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.

Mol Microbiol, 2004 Nov, 54(3), 795 - 807
The ColR-ColS two-component signal transduction system is involved in regulation of Tn4652 transposition in Pseudomonas putida under starvation conditions; Horak R et al.; Bacteria use two-component signal transduction pathways to sense both extracellular and intracellular environment and to coordinate cellular events according to changing conditions . Adaptation can be either physiological or genetical . Here, we present evidence that a genome reorganization process such as transposition can be controlled by certain environmental cues sensed by a two-component signal transduction system . We demonstrate that transposition-dependent accumulation of phenol-utilizing mutants is severely decreased in Pseudomonas putida defective in a two-component system colRS . Translocation of Tn4652 is decreased both in colR- and colS-defective strains, indicating that signal transduction from a histidine kinase ColS to a response regulator ColR is necessary for the activation of Tn4652 in bacteria starving on phenol . However, overexpression of ColR in a colS-defective strain restores Tn4652 transposition, suggesting that absence of the signal from ColS can be compensated by an elevated amount of ColR . In vitro analysis of purified ColR and ColS proteins evidenced that they constitute a functional phosphorelay . Site-directed mutagenesis revealed that a conserved H221 can be the phosphoryl-accepting residue in ColS and that aspartate residues D8 and D51 of ColR are necessary for the phosphotransfer from ColS to ColR . To our knowledge, Tn4652 is the first bacterial transposon regulated by a two-component system . This finding indicates that transpositional activity can respond to signals sensed and processed by the host.

J Bacteriol, 2004 Nov, 186(21), 7353 - 63
TouR-mediated effector-independent growth phase-dependent activation of the sigma54 Ptou promoter of Pseudomonas stutzeri OX1; Solera D et al.; Transcription of the catabolic touABCDEF operon, encoding the toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1, is driven by the sigma(54)-dependent Ptou promoter, whose activity is controlled by the phenol-responsive NtrC-like activator TouR . In this paper we describe for the first time a peculiar characteristic of this system, namely, that Ptou transcription is activated in a growth phase-dependent manner in the absence of genuine effectors of the cognate TouR regulator . This phenomenon, which we named gratuitous activation, was observed in the native strain P . stutzeri OX1, as well as in a Pseudomonas putida PaW340 host harboring the reconstructed tou regulatory circuit . Regulator-promoter swapping experiments demonstrated that the presence of TouR is necessary and sufficient for imposing gratuitous activation on the Ptou promoter, as well as on other sigma(54)-dependent catabolic promoters, whereas the highly similar phenol-responsive activator DmpR is unable to activate the Ptou promoter in the absence of effectors . We show that this phenomenon is specifically triggered by carbon source exhaustion but not by nitrogen starvation . An updated model of the tou regulatory circuit is presented.

Appl Microbiol Biotechnol . 2004 Oct 9; {Epub ahead of print}
Carbon isotope fractionation during cis- trans isomerization of unsaturated fatty acids in Pseudomonas putida; Heipieper HJ et al.; The molecular mechanism of the unique cis to trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12 was studied . For this purpose, the carbon isotope fractionation of the cis- trans isomerase was estimated . In resting cell experiments, addition of 3-nitrotoluene for activation of the cis- trans isomerase resulted in the conversion of the cis-unsaturated fatty acids into the corresponding trans isomers . For the conversion of C16:1 cis to its corresponding trans isomer, a significant fractionation was measured . The intensity of this fractionation strongly depended on the rate of cis- trans isomerization and the added concentration of 3-nitrotoluene, respectively . The presence of a significant fractionation provides additional indication for a transition from the sp(2) carbon linkage of the cis-double bond to an intermediate sp(3) within an enzyme-substrate complex . The sp(2) linkage is reconstituted after rotation to the trans configuration has occurred . As cytochrome c plays a major role in the catabolism of Cti polypeptide, these findings favour a mechanism for the enzyme in which electrophilic iron (Fe(3+)), provided by a heme domain, removes an electron of the cis double bond thereby transferring the sp(2) linkage into sp(3).

Environ Microbiol, 2004 Nov, 6(11), 1186 - 96
Design of new promoters and of a dual-bioreporter based on cross-activation by the two regulatory proteins XylR and HbpR; Tropel D et al.; The HbpR protein is the sigma54-dependent transcription activator for 2-hydroxybiphenyl degradation in Pseudomonas azelaica . The ability of HbpR and XylR, which share 35% amino acid sequence identity, to cross-activate the PhbpC and Pu promoters was investigated by determining HbpR- or XylR-mediated luciferase expression and by DNA binding assays . XylR measurably activated the PhbpC promoter in the presence of the effector m-xylene, both in Escherichia coli and Pseudomonas putida . HbpR weakly stimulated the Pu promoter in E . coli but not in P . azelaica . Poor HbpR-dependent activation from Pu was caused by a weak binding to the operator region . To create promoters efficiently activated by both regulators, the HbpR binding sites on PhbpC were gradually changed into the XylR binding sites of Pu by site-directed mutagenesis . Inducible luciferase expression from mutated promoters was tested in E . coli on a two plasmid system, and from mono copy gene fusions in P . azelaica and P . putida . Some mutants were efficiently activated by both HbpR and XylR, showing that promoters can be created which are permissive for both regulators . Others achieved a higher XylR-dependent transcription than from Pu itself . Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild-type PhbpC, while keeping the same maximal induction level . On the basis of these results, a dual-responsive bioreporter strain of P . azelaica was created, containing both XylR and HbpR, and activating luciferase expression from the same single promoter independently with m-xylene and 2-hydroxybiphenyl.

Gene, 2004 Oct 27, 341, 167 - 79
Characterization of a second functional gene cluster for the catabolism of phenylacetic acid in Pseudomonas sp . strain Y2; Bartolome-Martin D et al.; Pseudomonas sp . strain Y2 is a styrene degrading bacterium that mineralises this compound through its oxidation to phenylacetic acid (PAA) . We previously identified a complete gene cluster (paa1 cluster) for the degradation of phenylacetate, but, surprisingly, some paa1 deletion mutants were still able to catabolize styrene (STY) suggesting that this strain contained a second catabolic pathway . We report here the characterization of a second and novel paa2 gene cluster comprising 17 genes related to the catabolism of phenylacetate . We have identified a new gene (paaP) that is most likely involved in a transport process . Remarkably, the organization of the paa2 gene cluster is more similar to that of Pseudomonas putida KT2440 than to the paa1 gene cluster . Two new genes of undefined function were located inside the paa2 cluster . Sequence comparison between the paa2 genes and the paa1 and paa clusters of Pseudomonas sp . strain Y2 and P . putida KT2440, respectively, revealed a similar degree of divergence among the three sets of genes . Differences in the gene organization between paa1 and paa2 clusters of Pseudomonas sp . strain Y2 can be explained by an independent evolutionary history, probably associated with the adjacent sty genes . Deletion of either the first (paa1) or the second (paa2) gene cluster did not affect the ability of strain Y2 to grow in phenylacetate, whereas the deletion of both clusters led to the loss of this ability . The co-existence of two functional gene clusters for the degradation of phenylacetic acid in a bacterium has not been reported so far.

Biodegradation, 2004 Aug, 15(4), 275 - 80
Bioremediation of textile azo dyes by aerobic bacterial consortium; Senan RC et al.; An aerobic bacterial consortium consisting of two isolated strains (BF1, BF2) and a strain of Pseudomonas putida (MTCC1194) was developed for the aerobic degradation of a mixture of textile azodyes and individual azodyes at alkaline pH (9-10.5) and salinity (0.9-3.68 g/l) at ambient temperature (28 +/- 2 degrees C) . The degradation efficiency of the strains in different media (mineral media and in the Simulated textile effluent (STE)) and at different dye concentrations were studied . The presence of a H2O2 independent oxidase-laccase (26.5 IU/ml) was found in the culture filtrate of the organism BF2 . The analysis of the degraded products by TLC and HPLC, after the microbial treatment of the dyes showed the absence of amines and the presence of low molecular weight oxidative degradation products . The enzymes present in the crude supernatant was found to be reusable for the dye degradation.

Can J Microbiol, 2004 Aug, 50(8), 595 - 604
Field and soil microcosm studies on the survival and conjugation of a Pseudomonas putida strain bearing a recombinant plasmid, pADPTel; Hirkala DL et al.; Pseudomonas putida CR30RNS (pADPTel) is an antibiotic-resistant strain with a recombinant plasmid that confers resistance to tellurite and the ability to catabolize atrazine . The survival of this strain as well as its ability to transfer genes for atrazine degradation and tellurite resistance to indigenous soil bacteria were tested in both fallow soil and canola (Brassica napus) rhizosphere by the use of parallel field and laboratory releases . Culturable CR30RNS (pADPTel) were enumerated in field and microcosm soils at 7- to 14-day intervals over 49 d . Strain CR30RNS (pADPTel) survived for up to 7 weeks in microcosm soils at a density of 10(4) CFU/g soil, whereas in field soils the population declined to 10(3) CFU/g soil by the fourth week . In contrast, when CR30RNS (pADPTel) was introduced into the soil as a seed coating of canola (B . napus 'Karoo'), the bacterium established at higher cell densities in the rhizosphere (10(6)-10(5) CFU/g fresh root mass), with no subsequent decrease in numbers . The presence of selective pressure (i.e., atrazine) had no significant effect on the survival of CR30RNS (pADPTel) in either field or microcosm soils . One year postinoculation field sites were examined for the presence of CR30RNS (pADPTel) and no evidence of culturable parental cells was observed when samples were plated onto selective media . However, the atzC and telAB gene segments were amplified from the field soils at that time . Under laboratory conditions, indigenous soil bacteria were capable of receiving and expressing the engineered plasmid construct at frequencies ranging from 1 to 10(-3) transconjugants per donor . However, no plasmid transfer to indigenous soil bacteria was detected in the field or microcosm soils regardless of the presence of canola rhizosphere and (or) the application of atrazine . Our results show that the survival and population size of P . putida CR30RNS (pADPTel) might be sufficient for degradation of environmental pollutants but that the transfer frequency was too low to be detected under the conditions of this study.

Appl Environ Microbiol, 2004 Oct, 70(10), 6092 - 7
Involvement of linear plasmids in aerobic biodegradation of vinyl chloride; Danko AS et al.; Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions . Strains AJ and TD also use ethene and ethylene oxide as growth substrates . Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids . While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained . The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers . Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene . As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost . Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes . Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase . Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).

J Bacteriol, 2004 Oct, 186(20), 6983 - 98
ParB of Pseudomonas aeruginosa: interactions with its partner ParA and its target parS and specific effects on bacterial growth; Bartosik AA et al.; The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell . Key properties of the ParB protein have been identified and are associated with different parts of the protein . The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role . The dimerization domain of P . aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group) . The C-terminal part of ParB is also involved in ParB-ParA interactions . Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor . The overproduction of ParB was shown to inhibit the function of genes placed near parS . This "silencing" was dependent on the parS sequence and its orientation . The overproduction of P . aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P . aeruginosa and P . putida but not Escherichia coli cells . Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P . aeruginosa ParB in interactions with host cell components.

J Bacteriol, 2004 Oct, 186(20), 6815 - 23
Transcriptional regulation of the ant operon, encoding two-component anthranilate 1,2-dioxygenase, on the carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10; Urata M et al.; The carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10 has two gene clusters, carAaAaBaBbCAcAdDFE and antABC, which are involved in the conversions of carbazole to anthranilate and anthranilate to catechol, respectively . We proved that the antABC gene cluster, encoding two-component anthranilate 1,2-dioxygenase, constitutes a single transcriptional unit through Northern hybridization and reverse transcription-PCR (RT-PCR) analyses . The transcription start point of antA was mapped at 53 bp upstream point of its translation start point, and the -10 and -35 boxes were homologous to conserved sigma70 recognition sequence . Hence the promoter of the ant operon was designated Pant . 5' Deletion analyses using luciferase as a reporter showed that the region up to at least 70 bp from the transcription start point of antA was necessary for the activation of Pant . Luciferase expression from Pant was induced by anthranilate itself, but not by catechol . Two probable AraC/XylS-type regulatory genes found on pCAR1, open reading frame 22 (ORF22) and ORF23, are tandemly located 3.2 kb upstream of the antA gene . We revealed that the product of ORF23, designated AntR, is indispensable for the stimulation of Pant in Pseudomonas putida cells . Northern hybridization and RT-PCR analyses revealed that another copy of Pant, which is thought to be translocated about 2.1 kb upstream of the carAa gene as a consequence of the transposition of ISPre1, actually drives transcription of the carAa gene in the presence of anthranilate, indicating that both ant and car operons are simultaneously regulated by AntR.

Mol Microbiol, 2004 Oct, 54(1), 212 - 22
Widespread distribution of a lexA-regulated DNA damage-inducible multiple gene cassette in the Proteobacteria phylum; Abella M et al.; The SOS response comprises a set of cellular functions aimed at preserving bacterial cell viability in front of DNA injuries . The SOS network, negatively regulated by the LexA protein, is found in many bacterial species that have not suffered major reductions in their gene contents, but presents distinctly divergent LexA-binding sites across the Bacteria domain . In this article, we report the identification and characterization of an imported multiple gene cassette in the Gamma Proteobacterium Pseudomonas putida that encodes a LexA protein, an inhibitor of cell division (SulA), an error-prone polymerase (DinP) and the alpha subunit of DNA polymerase III (DnaE) . We also demonstrate that these genes constitute a DNA damage-inducible operon that is regulated by its own encoded LexA protein, and we establish that the latter is a direct derivative of the Gram-positive LexA protein . In addition, in silico analyses reveal that this multiple gene cassette is also present in many Proteobacteria families, and that both its gene content and LexA-binding sequence have evolved over time, ultimately giving rise to the lexA lineage of extant Gamma Proteobacteria.

Mikrobiol Z, 2004 May-Jun, 66(3), 64 - 71
{Peculiarities of solid materials colonization by pure and mixed cultures of methanotrophs}; Mycobacterium sp . et al.; Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, 1049-001 Lisboa, PortugalThis work aimed at studying the behavior and tolerance of Mycobacterium sp . NRRL B-3805, Rhodococcus erythropolis DCL14 and Pseudomonas putida S12 cells in the presence of various concentrations of water miscible (ethanol, butanol, and dimethylformamide, up to 50% v/v) and water immiscible solvents (dodecane, bis(2-ethylhexyl) phthalate and toluene, up to 5% v/v) . When incubated in the presence of these solvents, the cells were found to have lower tolerance to butanol and toluene than to the remaining solvents . Nevertheless, the concentrations of solvents endured by the tested strains show that they are quite solvent-tolerant, confirming their potential as biocatalysts in nonconventional systems . Microscopic observation of samples showed that the hydrophobic Mycobacterium sp . and R . erythropolis cells were able to aggregate to protect the population under stress conditions . Comparison of the results obtained at the single cell level by fluorescence microscopy and colony development on agar plates indicated that the primary effects of most solvents tested were on the cell membrane and replicating capability of the cells .

Mol Plant Microbe Interact, 2004 Sep, 17(9), 1009 - 18
Stimulation of the lipoxygenase pathway is associated with systemic resistance induced in bean by a nonpathogenic Pseudomonas strain; Ongena M et al.; Systemic defense reactions induced in bean by the non-pathogenic Pseudomonas putida BTP1 strain reduced disease caused by Botrytis cinerea . Phenylalanine ammonialyase activity and the level of endogenous free salicylic acid were compared in plant growth-promoting rhizobacteria-treated versus control plants, but no significant differences were detected . Furthermore, no enhanced fungitoxicity was detected in methanolic leaf extracts, suggesting that accumulation of bean phytoalexins was not part of the stimulated defense mechanisms . However, BTP1-inoculated plants showed increased levels of both linoleic and linolenic acids . On this basis, we further investigated whether the lipoxygenase pathway, leading to antifungal phytooxylipins, could have been stimulated . Two key enzymatic activities of this metabolic route, namely lipoxygenase and hydroperoxide lyase, were significantly stimulated during the first four days after challenging BTP1-treated plants with the pathogen . This was observed in parallel with a more rapid consumption of the respective substrates of these enzymes, as revealed by measurements of endogenous concentrations of linolenic acid and their hydroperoxide derivatives . Moreover, headspace-gas chromatography analyses showed significantly higher concentrations of the fungitoxic final product Z-3-hexenal in leaves from BTP1-inoculated beans as compared with control plants . Taken together, these results strongly suggest that the oxylipin pathway can be associated with enhanced disease resistance induced in bean plants by nonpathogenic rhizobacteria.

Can J Microbiol, 2004 Jul, 50(7), 499 - 508
Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtration efficiency in soilless cultivation; Deniel F et al.; In tomato soilless culture, slow filtration allows one to control the development of diseases caused by pathogenic microorganisms . During the disinfecting process, microbial elimination is ensured by mechanical and biological factors . In this study, system efficacy was enhanced further to a biological activation of filter by inoculating the pozzolana grains contained in the filtering unit with 5 selected bacteria . Three strains identified as Pseudomonas putida and 2 as Bacillus cereus came from a filter whose high efficiency to eliminate pathogens has been proven over years . These 5 bacteria displayed either a plant growth promoting activity (P . putida strains) or antagonistic properties (B . cereus strains) . Over the first months following their introduction in the filter, the bacterial colonisation of pozzolana grains was particularly high as compared to the one observed in the control filter . Conversely to Bacillus spp . populations, Pseudomonas spp . ones remained abundant throughout the whole cultural season . The biological activation of filter unit very significantly enhanced fungal elimination with respect to the one displayed by the control filter . Indeed, the 6-month period needed by the control filter to reach its best efficacy against Fusarium oxysporum was shortened for the bacteria-amended filter; in addition, a high efficacy filtration was got as soon as the first month . Fast colonization of pozzolana grains by selected bacteria and their subsequent interaction with F . oxysporum are likely responsible for filter efficiency . Our results suggest that Pseudomonas spp . act by competition for nutrients, and Bacillus spp . by antibiosis and (or) direct parasitism . Elimination of other fungal pathogens, i.e., Pythium spp., seems to differ from that of Fusarium since both filters demonstrated a high efficacy at the experiment start . Pythium spp . elimination appears to mainly rely on physical factors . It is worth noting that a certain percentage of the 5 pozzolana-inoculated bacteria failed to colonise the filter unit and were, thus, driven to the plants by the nutrient solution . Their contribution to the establishment of a beneficial microbial community in the rhizosphere is discussed.

Proteomics, 2004 Sep, 4(9), 2640 - 52
Insights into Pseudomonas putida KT2440 response to phenol-induced stress by quantitative proteomics; Santos PM et al.; To gain insight into the global mechanism underlying phenol toxicity and tolerance in bacteria, we have generated a two-dimensional protein reference map and used it to identify variations in protein expression levels in Pseudomonas putida KT2440 following exposure to sub-lethal inhibitory concentrations of this solvent . Inspection of the two-dimensional gel electrophoresis gels revealed that 1 h following sudden cell exposure to two different concentrations of phenol, leading to the inhibition of exponential growth (600 mg/L) or to growth arrest for, at least, 4 h before inhibited growth resumption (800 mg/L), the amount of 68 proteins was increased while the amount of 13 proteins was reduced . The up-regulated proteins include proteins involved in the: (i) oxidative stress response (AhpC, SodB,Tpx and Dsb); (ii) general stress reponse (UspA, HtpG, GrpE and Tig); (iii) energetic metabolism (AcnB, AtpH, Fpr, AceA, NuoE, and MmsA-1); (iv) fatty acid biosynthesis (FabB, AccC-1 and FabBx1); (v) inhibition of cell division (MinD); (vi) cell envelope biosynthesis (LpxC, VacJ, and MurA); (vii) transcription regulation (OmpR and Fur); and (viii) transport of small molecules (TolC, BraC, AotJ, AapJ, FbpA and OprQ) . Among the down-regulated proteins are those involved in nucleotide biosynthesis (PurM, PurL, PyrH and Dcd) and cell motility (FliC) . The information emerging from this genome expression profiling and the detailed investigation of the biological role of candidate genes, as targets of phenol toxicity or as determinants of phenol resistance in P . putida KT2440, will allow more rationale strategies for developing bacteria with greater solvent tolerance with impact in bioremediation and whole-cell biotransformations in media with organic solvents.

Microbiology, 2004 Sep, 150(Pt 9), 2889 - 98
Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid; Zhang XX et al.; Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3' to 5' exoribonuclease specific for small oligoribonucleotides . The deduced amino acid sequence of the plasmid-derived orn (orn(pl)) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes . Deletion of orn(pl) generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440 . This defect was fully restored by complementation with orn from Escherichia coli (orn(E.coli)) . Plasmid-derived orn(pl) was capable of partially complementing the P . putida orn mutant, demonstrating functionality of orn(pl) . Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria . A survey of orn(pl) from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity . These data suggest that the orn(pl) was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.

Appl Environ Microbiol, 2004 Sep, 70(9), 5493 - 502
Regulation of the N-acyl homoserine lactone-dependent quorum-sensing system in rhizosphere Pseudomonas putida WCS358 and cross-talk with the stationary-phase RpoS sigma factor and the global regulator GacA; Bertani I et al.; Quorum sensing is a cell population-density dependent regulatory system which in gram-negative bacteria often involves the production and detection of N-acyl homoserine lactones (AHLs) . Some Pseudomonas putida strains have been reported to produce AHLs, and one quorum-sensing locus has been identified . However, it appears that the majority of strains do not produce AHLs . In this study we report the identification and regulation of the AHL-dependent system of rhizosphere P . putida WCS358 . This system is identical to the recently identified system of P . putida strain IsoF and very similar to the las system of Pseudomonas aeruginosa . It is composed of three genes, the luxI family member ppuI, the putative repressor rsaL, and the luxR family member ppuR . A genomic ppuR::Tn5 mutant of strain WCS358 was identified by its inability to produce AHLs when it was cross-streaked in close proximity to an AHL biosensor, whereas an rsaL::Tn5 genomic mutant was identified by its ability to overproduce AHL molecules . Using transcriptional promoter fusions, we studied expression profiles of the rsaL, ppuI, and ppuR promoters in various genetic backgrounds . At the onset of the stationary phase, the autoinducer synthase ppuI gene expression is under positive regulation by PpuR-AHL and under negative regulation by RsaL, indicating that the molecules could be in competition for binding at the ppuI promoter . In genomic rsaL::Tn5 mutants ppuI expression and production of AHL levels increased dramatically; however, both processes were still under growth phase regulation, indicating that RsaL is not involved in repressing AHL production at low cell densities . The roles of the global response regulator GacA and the stationary-phase sigma factor RpoS in the regulation of the AHL system at the onset of the stationary phase were also investigated . The P . putida WCS358 gacA gene was cloned and inactivated in the genome . It was determined that the three global regulatory systems are closely linked, with quorum sensing and RpoS regulating each other and GacA positively regulating ppuI expression . Studies of the regulation of AHL quorum-sensing systems have lagged behind other studies and are important for understanding how these systems are integrated into the overall growth phase and metabolic status of the cells.

Appl Environ Microbiol, 2004 Sep, 70(9), 5190 - 8
Cell density-dependent gene contributes to efficient seed colonization by Pseudomonas putida KT2440; Espinosa-Urgel M et al.; We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440 . The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function . Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene . Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant . Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase . It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth . Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates . This is the first evidence suggesting the existence of a quorum-sensing system in P . putida KT2440 . The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed.

Environ Microbiol, 2004 Oct, 6(10), 1021 - 31
Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida; Duque E et al.; The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase . A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo . The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain . This effect was even more pronounced when toluene was supplied in the gas phase . In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain . This effect was particularly exacerbated in cells that reached the stationary phase . The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(8), 2093 - 104
Biodegradation of phenol by acclimatized Pseudomonas putida cells using glucose as an added growth substrate; Mamma D et al.; Biodegradation of phenol, a pollutant derived from many industrial processes, was achieved through acclimatized Pseudomonas putida cells . The strategy to overcome the inhibitory effect of phenol on microbial growth involved the addition of glucose, a conventional carbon source . A factorial experimental design was employed in order to optimize the initial phenol and glucose concentrations . The optimum conditions found were applied in 2-lt bioreactors . The development of acclimatized cells and the use of glucose as an added growth substrate resulted in a significant phenol degradation rate of 60.7 mg L(-1) h(-1) with a complete removal of 1200 mg L(-1) phenol.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(8), 2071 - 80
Contribution of cell outer membrane and inner membrane to Cu2+ adsorption by cell envelope of Pseudomonas putida 5-x; Wang L et al.; The role of outer and inner membrane in the Cu2+ adsorption process by gram-negative bacterium Pseudomonas putida 5-x, which was isolated from local electroplating effluent with high Cu2+ accumulating capability, was studied . The results indicate that both the outer and inner membrane exhibited high Cu2+ adsorption capacity . Outer and inner membrane contributed about 30-35% and 20-25% parts of adsorption capacity in Cu2+ adsorption by cell envelope of Pseudomonas putida 5-x, respectively . The total contribution of outer and inner membrane to Cu2+ adsorption by cell envelope was much greater than that of peptidoglycan layer . The relatively high phospholipid content in the outer membrane might result in its greater heavy metal ions adsorption capacity . The Cu2+ binding process by the outer and inner membrane of Pseudomonas putida 5-x is the adsorption processes and can be described with Freundlich isotherms.

Org Biomol Chem, 2004 Sep 7, 2(17), 2530 - 7 Epub 2004 Aug 10.
Dioxygenase-catalysed oxidation of alkylaryl sulfides: sulfoxidation versuscis-dihydrodiol formation; Boyd DR et al.; Toluene- and naphthalene-dioxygenase-catalysed sulfoxidation of nine disubstituted methylphenyl sulfides, using whole cells of Pseudomonas putida, consistently gave the corresponding enantioenriched sulfoxides . Using the P . putida UV4 mutant strain, and these substrates, differing proportions of the corresponding cis-dihydrodiol sulfides were also isolated . Evidence was found for the concomitant dioxygenase-catalysed cis-dihydroxylation and sulfoxidation of methyl para-tolyl sulfide . A simultaneous stereoselective reductase-catalysed deoxygenation of (S)-methyl para-tolyl sulfoxide, led to an increase in the proportion of the corresponding cis-dihydrodiol sulfide . The enantiopurity values and absolute configurations of the corresponding cis-dihydrodiol metabolites from methyl ortho- and para-substituted phenyl sulfides were determined by different methods, including chemoenzymatic syntheses from the cis-dihydrodiol metabolites of para-substituted iodobenzenes . Further evidence was provided to support the validity of an empirical model to predict, (i) the stereochemistry of cis-dihydroxylation of para-substituted benzene substrates, and (ii) the regiochemistry of cis-dihydroxylation reactions of ortho-substituted benzenes, each using toluene dioxygenase as biocatalyst .

Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1798 - 800
Catechol 2,3-dioxygenase from Pseudomonas sp . strain ND6: gene sequence and enzyme characterization; Jiang Y et al.; The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp . ND6 was cloned and sequenced . The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues . The C23O of Pseudomonas sp . ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively . The Pseudomonas sp . ND6 C23O gene was overexpressed in Escherichia coli DH 5alpha using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography . The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp . ND6 were better than those of C23O from Pseudomonas putida G7.

Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1722 - 7
The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase; Fujii Y et al.; A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction . This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase . The activity at 29 degrees C of the variant was 1.7-fold higher than that of the wild type . The K(m) values of the mutant at 37 degrees C were 0.40 mM for NAD(+) and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD(+) and 2.1 mM for formaldehyde . The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme . The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 degrees C, and 47 degrees C, respectively . The thermal stability of the mutant was lower than that of the wild type.

J Bacteriol, 2004 Sep, 186(17), 5661 - 71
Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2; Muryoi N et al.; The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures . The AFP is unusual in that it also exhibits a low level of ice nucleation activity . A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP . The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function . Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria . Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein . The 164-kDa AFP previously purified from P . putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity . Therefore, the recombinant protein may not have been properly posttranslationally modified . The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs) . Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice . To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.

J Mol Biol, 2004 Sep 3, 342(1), 183 - 94
Structure and action of urocanase; Kessler D et al.; Urocanase (EC 4.2.1.49) from Pseudomonas putida was crystallized after removing one of the seven free thiol groups . The crystal structure was solved by multiwavelength anomalous diffraction (MAD) using a seleno-methionine derivative and then refined at 1.14 A resolution . The enzyme is a symmetric homodimer of 2 x 557 amino acid residues with tightly bound NAD+ cofactors . Each subunit consists of a typical NAD-binding domain inserted into a larger core domain that forms the dimer interface . The core domain has a novel chain fold and accommodates the substrate urocanate in a surface depression . The NAD domain sits like a lid on the core domain depression and points with the nicotinamide group to the substrate . Substrate, nicotinamide and five water molecules are completely sequestered in a cavity . Most likely, one of these water molecules hydrates the substrate during catalysis . This cavity has to open for substrate passage, which probably means lifting the NAD domain . The observed atomic arrangement at the active center gives rise to a detailed proposal for the catalytic mechanism that is consistent with published chemical data . As expected, the variability of the residues involved is low, as derived from a family of 58 proteins annotated as urocanases in the data banks . However, one well-embedded member of this family showed a significant deviation at the active center indicating an incorrect annotation.

Biochemistry, 2004 Aug 24, 43(33), 10692 - 700
Role of glycine 81 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate specificity and oxidase activity; Dewanti AR et al.; (S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids . Despite a high degree of sequence and structural similarity, this family can be divided into three subgroups based on the different oxidants utilized in the second oxidative half-reaction . Only the oxidases show high reactivity with molecular oxygen . Structural data indicate that the relative position of a peptide loop and the isoalloxazine ring of the FMN is slightly different in the oxidases compared to the dehydrogenases; the last residue on this loop is either an alanine or glycine . We examined the effect of the G81A, G81S, G81V, and G81D mutations in MDH on the overall reaction and especially on the suppression of activity with oxygen . G81A had a higher specificity for small substrates compared to that of wtMDH, though the affinity for (S)-mandelate was relatively unchanged . The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity . Redox-potential measurements indicate that the reduction in activity is due to the decrease in electrophilicity of the FMN . The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A . The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen . These results suggest that one of the ways the oxidase activity of MDH is controlled is through steric effects because of the relative positions of the FMN and the Gly81 loop.

Appl Microbiol Biotechnol, 2004 Aug, 65(3), 336 - 43 Epub 2004 Feb 04.
Combined effects of pH and biosurfactant addition on solubilization and biodegradation of phenanthrene; Shin KH et al.; Phenanthrene solubilization and biodegradation with a biosurfactant (rhamnolipid) solution were investigated as a function of pH . Batch phenanthrene solubilization experiments were performed in the pH range 4-8 and the highest solubilities with the biosurfactant were detected around a pH of 4.5-5.5 . The apparent solubility at pH 5.5 was 3.8 times greater than at pH 7 in the presence of 240 ppm rhamnolipid, probably due to the rhamnolipid-an anionic surfactant-forming different pH-dependent structures . Biodegradation experiments using Pseudomonas putida CRE 7 were performed in the absence and the presence of the rhamnolipid solution . Without the biosurfactant, the specific growth rate (mu) at pH 6 was higher than at other pH values, and analysis for the total phenanthrene loss confirmed the trends in mu, with the greatest phenanthrene removal at pH 6 . In presence of the rhamnolipid, the maximum mu value shifted to around pH 5, which showed maximum enhancement of solubility in the abiotic experiment . Although there was an increase in the observed specific growth rate with the biosurfactant, this increase was not as great as the increase in solubilization . For example, the 1.44 times increase in the mu value at pH 5 was lower than the 3.8 times enhancement in the solubility at the same pH . Thus, as observed by others, not all of the solubilized phenanthrene was bioavailable to the microorganisms . Interestingly, the results of a size distribution experiment showed that a large portion of the phenanthrene-rhamnolipid aggregates existed at a molecular weight of >300,000 . Furthermore, this fraction appeared to be the most available for biodegradation, although not all the phenanthrene was bioavailable.

Biochemistry, 2004 Aug 17, 43(32), 10424 - 34
Crystal structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida, a fumarase class II type cycloisomerase: enzyme evolution in parallel pathways; Yang J et al.; 3-Carboxy-cis,cis-muconate lactonizing enzymes (CMLEs), the key enzymes in the protocatechuate branch of the beta-ketoadipate pathway in microorganisms, catalyze the conversion of 3-carboxy-cis,cis-muconate to muconolactones . We have determined the crystal structure of the prokaryotic Pseudomonas putida CMLE (PpCMLE) at 2.6 A resolution . PpCMLE is a homotetramer and belongs to the fumarase class II superfamily . The active site of PpCMLE is formed largely by three regions, which are moderately conserved in the fumarase class II superfamily, from three respective monomers . It has been proposed that residue His141, which is highly conserved in all fumarase class II enzymes and forms a charge relay with residue Glu275 (both His141 and Glu275 are in adenylosuccinate lyase numbering), acts as the general base in most fumarase class II superfamily members . However, this charge relay pair is broken in PpCMLE . The residues corresponding to His141 and Glu275 are Trp153 and Ala289, respectively, in PpCMLE . The structures of prokaryotic MLEs and that of CMLE from the eukaryotic Neurospora crassa are completely different from that of PpCMLE, indicating MLEs and CMLEs, as well as the prokaryotic and eukaryotic CMLEs, evolved from distinct ancestors, although they catalyze similar reactions . The structural differences may be related to recognition by substrates and to differences in the mechanistic pathways by which these enzymes catalyze their respective reactions.

Acta Crystallogr D Biol Crystallogr, 1997 May, 53(Pt 3), 352 - 3
Characterization of two crystal forms of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida; Hasson MS; Two crystal forms of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida have been characterized . Form A is in space group P6, with unit-cell dimensions a = b = 232, c = 79 A, alpha = beta = 90, gamma = 120 degrees . Form B is orthorhombic, with cell dimensions a = 163, b = 139, c = 90 A alpha = beta = gamma = 90 degrees.

Acta Crystallogr D Biol Crystallogr, 1997 Sep, 53(Pt 5), 619 - 21
Crystallization and preliminary diffraction studies of morphinone reductase, a flavoprotein involved in the degradation of morphine alkaloids; Moody PC; Morphinone reductase from Pseudomonas putida M10, a flavoprotein involved in the degradation of morphine alkaloids, was purified from an overexpressing strain of Escherichia coli and crystallized using the hanging-drop vapour-diffusion method . Diffraction data were collected to 2.5 A . The I-centred orthorhombic cell has a monomer in the asymmetric unit . Preliminary molecular replacement calculations have been performed using Old Yellow Enzyme as the search model.

Environ Sci Technol, 2004 Jul 15, 38(14), 3864 - 70
Quantification of bacterial chemotaxis in porous media using magnetic resonance imaging; Olson MS et al.; Bacterial chemotaxis has the potential to enhance biodegradation of organic contaminants in polluted groundwater systems . However, studies of bacterial chemotaxis in porous media are scarce . In this study we use magnetic resonance imaging (MRI) for the noninvasive measurement of changes in bacterial-density distributions in a packed column at a spatial resolution of 330 microm as a function of time . We analyze both the diffusive and the chemotactic behavior of Pseudomonas putida F1 in the presence of the chemical stimulus trichloroethylene (TCE) . The migration of motile bacteria in experiments without TCE was described using an effective motility coefficient, whereas the presence of TCE required addition of a nonzero chemotactic sensitivity coefficient, indicating a significant response to TCE . The need for a chemotactic sensitivity term was justified by a test for statistical significance . This study represents the first quantification of bacterial chemotactic parameters within a packed column . For conditions under which chemotaxis occurs in porous media, it may potentially be exploited to significantly improve rates of in situ pollutant biodegradation in the subsurface environment, particularlyfor pollutants dissolved in water trapped in low-permeability formations or lenses.

Structure (Camb), 2004 Aug, 12(8), 1425 - 35
Active site geometry and substrate recognition of the molybdenum hydroxylase quinoline 2-oxidoreductase; Bonin I et al.; The soil bacterium Pseudomonas putida 86 uses quinoline as a sole source of carbon and energy . Quinoline 2-oxidoreductase (Qor) catalyzes the first metabolic step converting quinoline to 2-oxo-1,2-dihydroquinoline . Qor is a member of the molybdenum hydroxylases . The molybdenum ion is coordinated by two ene-dithiolate sulfur atoms, two oxo-ligands, and a catalytically crucial sulfido-ligand, whose position in the active site was controversial . The 1.8 A resolution crystal structure of Qor indicates that the sulfido-ligand occupies the equatorial position at the molybdenum ion . The structural comparison of Qor with the allopurinol-inhibited xanthine dehydrogenase from Rhodobacter capsulatus allows direct insight into the mechanism of substrate recognition and the identification of putative catalytic residues . The active site protein variants QorE743V and QorE743D were analyzed to assess the catalytic role of E743.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1062 - 8
Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures; Losen M et al.; Shake-flask cultures are widely used for screening of high producing strains . To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions . A novel method of measuring respiratory activity in shake-flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions . Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks . Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited . A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake-flask cultures at shaking frequencies below 400 rpm . These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E . coli SG13009, resulting in a 10-fold increase in volumetric enzyme production . This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity . This can help to efficiently optimize screening conditions for E . coli.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 41 - 6
Organomercurial removal from vaccine production wastewaters in a supported liquid membrane bioreactor; Fortunato R et al.; Vaccine production effluents are strongly polluted with thiomersal, a highly toxic organomercurial compound, for which there is presently no remediation technology available . This work describes a new remediation process based on the extraction of thiomersal from the wastewater to a biological compartment, where it is degraded by a microbial strain . The selective extraction of thiomersal is achieved by using an ionic liquid immobilized in a porous membrane . In the biological compartment, thiomersal is degraded to metallic mercury, under aerobic conditions, by a Pseudomonas putida strain . The utilization of ionic liquids in supported liquid membranes for thiomersal transport, and the kinetics of thiomersal biodegradation by a Pseudomonas putida strain are presented and discussed.

Appl Environ Microbiol, 2004 Aug, 70(8), 5019 - 25
Strategy for cloning large gene assemblages as illustrated using the phenylacetate and polyhydroxyalkanoate gene clusters; Garcia B et al.; We report an easy procedure for isolating chromosome-clustered genes . By following this methodology, the entire set of genes belonging to the phenylacetic acid (PhAc; 18-kb) pathway as well as those required for the synthesis and mobilization of different polyhydroxyalkanoates (PHAs; 6.4 kb) in Pseudomonas putida U were recovered directly from the bacterial chromosome and cloned into a plasmid for the first time . The transformation of different bacteria with these genetic constructions conferred on them the ability to either degrade PhAc or synthesize bioplastics (PHAs).

Appl Environ Microbiol, 2004 Aug, 70(8), 4544 - 50
Oxidation of methyl tert-butyl ether by alkane hydroxylase in dicyclopropylketone-induced and n-octane-grown Pseudomonas putida GPo1; Smith CA et al.; The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE) . We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity . Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA) . Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol . Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches . First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK . Second, no TBA production from MTBE was observed in DCPK-treated cells of P . putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid . Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells . Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process . Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE . Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells . Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K(s) value (20 to 40 mM) for MTBE.

Appl Environ Microbiol, 2004 Aug, 70(8), 4440 - 8
Polyhydroxyalkanoate (PHA) accumulation in sulfate-reducing bacteria and identification of a class III PHA synthase (PhaEC) in Desulfococcus multivorans; Hai T et al.; Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs) . During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) {poly(3HB)} . Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter . Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate . Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected . When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained . The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE(Dm) and phaC(Dm) genes . PhaC(Dm) and PhaE(Dm) were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases . Constructs of phaC(Dm) alone (pBBRMCS-2::phaC(Dm)) and of phaE(Dm)C(Dm) (pBBRMCS-2::phaE(Dm)C(Dm)) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB(-)4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively . In cells of the recombinant strains harboring phaE(Dm)C(Dm) small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected . This indicated that the cloned genes encode functionally active proteins . Hybrid synthases consisting of PhaC(Dm) and PhaE of Thiococcus pfennigii or Synechocystis sp . strain PCC 6308 were also constructed and were shown to be functionally active.

J Mol Biol, 2004 Aug 13, 341(3), 753 - 68
Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4; Dennis JJ et al.; The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature . Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence . The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments . Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species . Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis . Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.

Appl Microbiol Biotechnol, 2004 Dec, 66(2), 209 - 16 Epub 2004 Dec.
Characterization in Pseudomonas putida Cg1 of nahR and its role in bacterial survival in soil; Park W et al.; Sequencing, RFLP analyses and experiments utilizing a lacZ transcriptional reporter fused to the promoter regions of nahR and nahG in Pseudomonas putida Cg1 confirmed that regulation of naphthalene degradation in both P . putida Cg1 and the type strain, P . putida NCIB 9816-4, is consistent with that of NAH7 from P . putida G7 . Two nahR knockout strains (RK1 and Cg1-NAHR from P . putida NCIB 9816-4 and Cg1, respectively) showed a growth defect in the presence of naphthalene as sole carbon and energy source . We hypothesized that nahR influences ecological fitness of bacteria in naphthalene-contaminated soil and tested this hypothesis using both parent and nahR-knockout strains introduced to soil microcosms with and without added naphthalene . After 21 days, loss of cell viability was pronounced in the presence of added naphthalene crystals for nahR mutants of both test bacteria, relative to the wild types . Diminished viable counts were attributed to toxicity . Thus, our data indicated that NahR in P . putida Cg1 is virtually identical to its homologues in other pseudomonads and that nahR is required for resistance to naphthalene toxicity, hence the persistence of bacterial cells in soil with high concentrations of naphthalene.

FEMS Microbiol Lett, 2004 Aug 1, 237(1), 1 - 7
Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa; Hoffmann N et al.; Regulation of medium-chain-length polyhydroxyalkanoate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida was studied conducting PHA accumulation experiments and transcriptional analysis of PHA biosynthesis genes with wild type strains and rpoN-negative mutants . In P . putida PHA accumulation was RpoN-independent, whereas in P . aeruginosa PHA accumulation was RpoN-dependent . Transcriptional analysis applying reverse transcriptase-polymerase chain reaction showed strong induction of phaG, encoding the transacylase, under nitrogen starvation in P . putida KT2440 and the respective rpoN-negative mutant, indicating an RpoN-independent regulation of phaG . No transcription of phaG and no PHA accumulation was detected in the rpoN-negative mutant of P . aeruginosa neither from gluconate nor from octanoate as carbon source . Alginate-overproducing mutant P . aeruginosa FRD1 showed strongly decreased PHA accumulation from gluconate but no difference in phaC1 (encoding the PHA synthase) transcription, indicating that alginate biosynthesis competes with PHA biosynthesis regarding acetyl-CoA as precursor for both biopolymers . Transcription of phaF and phaI-F was nitrogen independent.

J Bacteriol, 2004 Aug, 186(15), 5062 - 77
The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida; Arias-Barrau E et al.; Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate . Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate . Whereas the phh, tyr, and hpd genes are not linked in the P . putida genome, the hmgABC genes appear to form a single transcriptional unit . Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule . Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site . The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway . We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source . Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P . putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.

Environ Microbiol, 2004 Aug, 6(8), 851 - 60
Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction; Ackerley DF et al.; Chromate {Cr(VI)} is a serious environmental pollutant, which is amenable to bacterial bioremediation . NfsA, the major oxygen-insensitive nitroreductase of Escherichia coli, is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III) . We show that this process involves single-electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF . However, NfsA generates less ROS than a known one-electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni- and di-valent electron transfer steps . The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH-catalysed chromate reduction reaction decreased ROS generation by c . 65, 40, or 20%, respectively, suggesting that these enzymes can pre-empt ROS generation by LpDH . We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E . coli to this compound.

Biochemistry, 2004 Jul 13, 43(27), 8744 - 53
Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101); Meilleur F et al.; Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida, has been characterized concerning thermal stability and structural dynamics . For the first time, Fourier transform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein . The secondary structure compositions were determined from the fitted amide I' spectral region, giving band populations at 10 degrees C for the perdeuterated protein of 22% between 1605 and 1624 cm(-1) (beta-sheets), 47% between 1633 and 1650 cm(-1) (alpha-helix (29%) plus unordered/3(10)-helix (18%)), and 28% between 1657 and 1677 cm(-1) (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm(-1) (beta-sheets), 52% between 1640 and 1658 cm(-1) (alpha-helix (41%) plus unordered/3(10)-helix (11%)), and 24% between 1665 and 1680 cm(-1) (turns).Thermal unfolding experiments revealed that perdeuterated P450cam was less stable than the hydrogenated protein . The midpoint transition temperatures were 60.8 and 64.4 degrees C for the perdeuterated and hydrogenated P450cam, respectively . Step-scan time-resolved FTIR was applied to the P450cam-CO complex to study the ligand-rebinding process after flash photolysis . Rebinding of the ligand occurred with the same kinetics and rate constants k(on), 8.9 x 10(4) and 8.3 x 10(4) M(-1) s(-1) for the perdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutron crystallographic study to determine the specific hydration states and hydrogen-bonding networks at the active site . The analyses presented here show that perdeuterated P450cam is structurally similar to its hydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained from the neutron structure will be representative of the normal hydrogenated P450cam.

Biotechnol Bioeng, 2004 Jul 20, 87(2), 219 - 27
Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors; Chung TP et al.; The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol . The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined . It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process . The cells P . putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers . Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation . At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system . At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate . The process development in an HFMBR system was also discussed .

Biochem J, 2004 Sep 15, 382(Pt 3), 967 - 73
Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B; Jang do S et al.; KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate . A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI . In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy . The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e . 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability . The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis . The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network . The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability . These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network . The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI . The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.

J Biochem (Tokyo), 2004 Jun, 135(6), 721 - 30
Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity; Ishida T et al.; Catechol 2,3-dioxygenase {EC 1.13.11.2} from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde . The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5 . The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols . Mpc preferred catechols with non-ionic substituents at the C3 or C4 position . 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not . The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols . The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol . These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents . However, k(cat) did not correlate significantly with pK(1) . The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.

J Bacteriol, 2004 Jul, 186(13), 4177 - 84
Altering the substrate specificity of polyhydroxyalkanoate synthase 1 derived from Pseudomonas putida GPo1 by localized semirandom mutagenesis; Sheu DS et al.; The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Pp), class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis . The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR . According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA- as the host . The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB(-)4 supplemented with octanoate . Notably, the amount of 3-hydroxybutyrate (short-chain-length {SCL} 3-hydroxyalkanoate {3-HA} unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%) . Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R . eutropha PHB(-)4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length {MCL} 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates . The 3-HA and MCL 3-HA units of the PHA produced by R . eutropha PHB(-)4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.

Anal Biochem, 2004 Jul 15, 330(2), 264 - 71
Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation; Li S et al.; Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life . The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the -amino group of lysines) . To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine alpha,gamma-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida . rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1 . The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation . This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide . The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction . The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1 . Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6-8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase . Approximately 15-20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule . Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase . Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.

Biochemistry, 2004 Jun 22, 43(24), 7725 - 35
Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family; Gopalakrishna KN et al.; A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia . Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad . Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH . The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity . The His-tagged variant showed no significant change in kinetic constants . Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m) . As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates . MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk . Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered . Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values . Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.

Environ Microbiol, 2004 Jul, 6(7), 726 - 32
Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins; Lambertsen L et al.; The mini-Tn7 transposon system is a convenient tool for site-specific tagging of bacteria in which the tagging DNA is inserted at a unique and neutral chromosomal site . We have expanded the panel of mini-Tn7 delivery plasmids expressing different fluorescent proteins (stable and unstable) from the Escherichia coli lac derived promoter, P(A1/04/03), or from the growth-rate-dependent Escherichia coli promoter PrrnB P1 . The mini-Tn7 transposons were inserted and tested in the soil bacterium, Pseudomonas putida KT2440 . Successful and site-specific tagging was verified by Southern blots as well as by PCR . Furthermore, the effect of fluorescent protein expression on the cellular growth rate was tested by growth competition assays.

Microbiology, 2004 Jun, 150(Pt 6), 1661 - 9
Characterization of Pseudomonas putida genes responsive to nutrient limitation; Syn CK et al.; The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation and biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high availability of nutrients and oxygen . Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 were examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate or oxygen, and in the rhizosphere . Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7 and NRM17, were selected for identification of the tagged genes . In strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased 4.9-26.4-fold under various low-nutrient conditions . In NRM7, expression of the novel NADPH : quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low-nutrient and anoxic conditions . The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low-nutrient conditions but its absolute expression level was still amongst the highest . Additionally, it was independent of oxygen availability, in contrast to that in Escherichia coli.

Appl Environ Microbiol, 2004 Jun, 70(6), 3637 - 43
Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols; Rojas A et al.; Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP(ow) (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of > or =2.5 . Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase . We tested P . putida DOT-T1E tolerance to different aliphatic alcohols with a logP(ow) value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP(ow) around 1.5 are produced . P . putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons . These defense mechanisms allow P . putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised . Our results support that the logP(ow) of aliphatic alcohols correlates with their toxic effects, as octanol (logP(ow) = 2.9) has more negative effects in P . putida cells than 1-nonanol (logP(ow) = 3.4) or 1-decanol (logP(ow) = 4) . A P . putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP(ow) = 3.2) into 3-methylcatechol (logP(ow) = 1.8) . The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.

Appl Environ Microbiol, 2004 Jun, 70(6), 3205 - 12
In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF; Moldes C et al.; A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria . The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs . This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports . The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications . Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule . The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase . This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

J Exp Bot, 2004 Aug, 55(404), 1939 - 45 Epub 2004 Jun 04.
The role of soil microbes in plant sulphur nutrition; Kertesz MA et al.; Chemical and spectroscopic studies have shown that in agricultural soils most of the soil sulphur (>95%) is present as sulphate esters or as carbon-bonded sulphur (sulphonates or amino acid sulphur), rather than inorganic sulphate . Plant sulphur nutrition depends primarily on the uptake of inorganic sulphate . However, recent research has demonstrated that the sulphate ester and sulphonate-pools of soil sulphur are also plant-bioavailable, probably due to interconversion of carbon-bonded sulphur and sulphate ester-sulphur to inorganic sulphate by soil microbes . In addition to this mineralization of bound forms of sulphur, soil microbes are also responsible for the rapid immobilization of sulphate, first to sulphate esters and subsequently to carbon-bound sulphur . The rate of sulphur cycling depends on the microbial community present, and on its metabolic activity, though it is not yet known if specific microbial species or genera control this process . The genes involved in the mobilization of sulphonate- and sulphate ester-sulphur by one common rhizosphere bacterium, Pseudomonas putida, have been investigated . Mutants of this species that are unable to transform sulphate esters show reduced survival in the soil, indicating that sulphate esters are important for bacterial S-nutrition in this environment . P . putida S-313 mutants that cannot metabolize sulphonate-sulphur do not promote the growth of tomato plants as the wild-type strain does, suggesting that the ability to mobilize bound sulphur for plant nutrition is an important role of this species.

Biotechnol Bioeng, 2004 Jun 30, 86(7), 801 - 8
Effect of ethanol, acetate, and phenol on toluene degradation activity and tod-lux expression in Pseudomonas putida TOD102: evaluation of the metabolic flux dilution model; Lovanh N et al.; The reporter strain Pseudomonas putida TOD102 (with a tod-lux fusion) was used in chemostat experiments with binary substrate mixtures to investigate the effect of potentially occurring cosubstrates on toluene degradation activity . Although toluene was simultaneously utilized with other cosubstrates, its metabolic flux (defined as the toluene utilization rate per cell) decreased with increasing influent concentrations of ethanol, acetate, or phenol . Three inhibitory mechanisms were considered to explain these trends: (1) repression of the tod gene (coding for toluene dioxygenase) by acetate and ethanol, which was quantified by a decrease in specific bioluminescence; (2) competitive inhibition of toluene dioxygenase by phenol; and (3) metabolic flux dilution (MFD) by all three cosubstrates . Based on experimental observations, MFD was modeled without any fitting parameters by assuming that the metabolic flux of a substrate in a mixture is proportional to its relative availability (expressed as a fraction of the influent total organic carbon) . Thus, increasing concentrations of alternative carbon sources "dilute" the metabolic flux of toluene without necessarily repressing tod, as observed with phenol (a known tod inducer) . For all cosubstrates, the MFD model slightly overpredicted the measured toluene metabolic flux . Incorporating catabolite repression (for experiments with acetate or ethanol) or competitive inhibition (for experiments with phenol) with independently obtained parameters resulted in more accurate fits of the observed decrease in toluene metabolic flux with increasing cosubstrate concentration . These results imply that alternative carbon sources (including inducers) are likely to hinder toluene utilization per unit cell, and that these effects can be accurately predicted with simple mathematical models .

J Bacteriol, 2004 Jun, 186(11), 3439 - 46
The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid; Revelles O et al.; Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere . Lysine is one of the major compounds in root exudates, and P . putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy . Lysine is channeled to delta-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products . We show that the davDT genes form an operon transcribed from a single sigma70-dependent promoter . The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium . However, the true inducer of this operon seems to be delta-aminovaleric acid because in a mutant unable to metabolize lysine to delta-aminovaleric acid, this compound, but not lysine, acted as an effector . Effective induction of the P . putida P(davD) promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters . Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.

J Hosp Infect, 2004 May, 57(1), 88 - 91
Outbreak of Pseudomonas putida bacteraemia in a neonatal intensive care unit; Bouallegue O et al.; During the period of 9-27 March 2001, Pseudomonas putida strains were recovered from 10 neonates hospitalized in the neonatal intensive care unit of Farhat Hached Hospital, Sousse (Tunisia) . Seven neonates developed bacteraemia, and three had an umbilical catheter-related infection (without bacteraemia) . A total of 18 isolates were cultured from blood {Formula: see text} and catheters {Formula: see text} These isolates were identified as P . putida by routine biochemical methods (API 20 NE, bioMerieux, Lyon, France) . Restriction endonuclease DNA profiles were determined by pulsed-field gel electrophoresis using two endonucleases XbaI and SpeI . They yielded the same patterns showing that the outbreak was caused by a single clone of P . putida . Although the antiseptic solutions used to clean the umbilicus were implicated circumstantially as probable sources, they were not sampled and so this could not be confirmed.

Environ Microbiol, 2004 Jun, 6(6), 605 - 10
Pseudomonas putida mutants in the exbBexbDtonB gene cluster are hypersensitive to environmental and chemical stressors; Godoy P et al.; The genes in the exbBexbDtonB cluster of Pseudomonas putida DOT-T1E are co-transcribed . We have generated non-polar mutants in each of the genes by inserting an aphA3 cassette encoding kanamycin resistance . All three mutants show similar phenotypes: the mutants are unable to grow on minimal medium under iron deficiency conditions . Furthermore, regardless of iron conditions, all mutants are hypersensitive to antibiotics, p-hydroxybenzoate and toluene, chemicals that are extruded from the cell by efflux pumps . These findings are discussed in terms of the involvement of the TonB system in the energization of outer membrane functions necessary for the import or export of different compounds in P . putida.

Environ Microbiol, 2004 Jun, 6(6), 574 - 83
Repression of Pseudomonas putida phenanthrene-degrading activity by plant root extracts and exudates; Rentz JA et al.; The phenanthrene-degrading activity (PDA) of Pseudomonas putida ATCC 17484 was repressed after incubation with plant root extracts of oat (Avena sativa), osage orange (Maclura pomifera), hybrid willow (Salix alba x matsudana), kou (Cordia subcordata) and milo (Thespesia populnea) and plant root exudates of oat (Avena sativa) and hybrid poplar (Populus deltoides x nigra DN34) . Total organic carbon content of root extracts ranged from 103 to 395 mg l(-1) . Characterization of root extracts identified acetate (not detectable to 8.0 mg l(-1)), amino acids (1.7-17.3 mg l(-1)) and glucose (1.6-14.0 mg l(-1)), indicating a complex mixture of substrates . Repression was also observed after exposure to potential root-derived substrates, including organic acids, glucose (carbohydrate) and glutamate (amino acid) . Carbon source regulation (e.g . catabolite repression) was apparently responsible for the observed repression of P . putida PDA by root extracts . However, we showed that P . putida grows on root extracts and exudates as sole carbon and energy sources . Enhanced growth on root products may compensate for partial repression, because larger microbial populations are conducive to faster degradation rates . This would explain the commonly reported increase in phenanthrene removal in the rhizosphere.

Appl Environ Microbiol, 2004 May, 70(5), 3143 - 5
Plastic encapsulation of stabilized Escherichia coli and Pseudomonas putida; Manzanera M et al.; Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials . Bacteria are recovered by rehydration after physical disruption of the plastic . P . putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination.

Appl Environ Microbiol, 2004 May, 70(5), 2830 - 5
Addition of aromatic substrates restores trichloroethylene degradation activity in Pseudomonas putida F1; Morono Y et al.; The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of Pseudomonas putida F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports . However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase . After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediated by TDO molecules that were present before the cessation of TCE degradation . The addition of benzene and cumene, which are also substrates of TDO, also caused restoration of TCE degradation activity: TCE was degraded simultaneously with cumene, and a larger amount of TCE was degraded after cumene was added than after toluene or benzene was added . But substrates that were expected to supply the cells with NADH or energy did not restore TCE degradation activity . This cycle of pseudoinactivation and restoration of TCE degradation was observed repeatedly without a significant decrease in the number of viable cells, even after six additions of toluene spread over 30 h . The results obtained in this study demonstrate a new type of restoration of TCE degradation that has not been previously reported.

J Bacteriol, 2004 May, 186(10), 2921 - 7
TtgV bound to a complex operator site represses transcription of the promoter for the multidrug and solvent extrusion TtgGHI pump; Guazzaroni ME et al.; The TtgGHI efflux pump of Pseudomonas putida extrudes a variety of antibiotics and solvents . We show that the ttgGHI operon is transcribed in vitro and in vivo from a single promoter and not from two overlapping promoters as previously proposed . The expression of this promoter is controlled by the TtgV repressor, whose operator expands through four helical turns that overlap the -10 region of the promoter . We also show that TtgV is released from its operator on binding of effectors such as aliphatic alcohols . Mutational analysis of the ttgGHI promoter revealed that substitutions at -13, -12, and -8 yielded promoters that were unable to drive transcription whereas certain mutations at -9, -11, and -6 to -3 increased expression in vivo . The cause of the increased expression was either a decrease in the affinity of the TtgV protein for its operator or an increase in the affinity of RNA polymerase for the mutant promoters.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 957 - 61 Epub 2004 Apr 21.
Crystallization and preliminary analysis of xenobiotic reductase A and ligand complexes from Pseudomonas putida II-B; Orville AM et al.; Diffraction-quality crystals have been obtained of the xenobiotic reductase A (XenA) from Pseudomonas II-B, which was originally cultured from the contaminated soil of a World War II era munitions-manufacturing plant . Several complete X-ray diffraction data sets have been collected and analyzed . The native XenA data set includes reflections between 35 and 1.65 A . Four-wavelength MAD data sets from selenomethionine-enriched XenA and from three different ligand complexes are also reported . The XenA crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 84, b = 158, c = 57 A . Experimental phasing from analysis of the MAD data from selenomethionine-enriched XenA reveals the presence of two molecules in the asymmetric unit . They are related by a non-crystallographic 2(1) screw axis nearly parallel to the c axis, but offset by a quarter unit-cell translation . Thus, the local symmetry produces approximate systematic absences along the (00l) principal axis and complicates the space-group determination.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 941 - 4 Epub 2004 Apr 21.
Crystallization and X-ray diffraction analysis of ornithine cyclodeaminase from Pseudomonas putida; Alam S et al.; Ornithine cyclodeaminase (OCD) is a member of the micro-crystallin protein family, the biological activity of which is the conversion of L-ornithine to L-proline and ammonia . In order to elucidate the functional groups of this enzyme that are involved in catalysis, the crystallization of OCD from Pseudomonas putida was undertaken . Using microbatch-under-oil screening at the high-throughput crystallization laboratory (HTC) at the Hauptman-Woodward Medical Research Institute Inc . (HWI Buffalo, NY, USA), numerous crystallization conditions were rapidly identified . Several conditions could be reproduced on a larger scale as vapor-diffusion experiments in-house . The best diffraction-quality crystals were obtained from solutions of 40%(v/v) 2-methyl-2,4-pentanediol buffered at pH 6.0 with 0.1 M MES and diffracted X-rays to 1.68 A resolution . Crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.0, b = 78.3, c = 119.4 A . The V(M) was 2.1 A(3) Da(-1), corresponding to 42% solvent, which is consistent with two 38.5 kDa molecules per asymmetric unit . The structure determination is under way using experimental phasing methods.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 816 - 22 Epub 2004 Apr 21.
Structure of C73G putidaredoxin from Pseudomonas putida; Smith N et al.; The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form . The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein . The final structure with R = 0.19 and R(free) = 0.21 has been deposited in the Protein Data Bank with accession code 1r7s . The C2 crystal contains three Pdx molecules in the asymmetric unit, giving three independent models of the protein that are very similar (r.m.s.d . < 0.3 A for the 106 C(alpha) atoms) . The unusually high solvent fraction of 80% results in comparatively few crystal-packing artifacts . The structure is briefly compared with the recently reported crystal structures of the C73S and C73S/C85S mutants . In general, the eight independent molecules in the three crystal structures (three in C73G, three in C73S and two in C73S/C85S) are much more similar to each other than to the previously reported NMR structure of wild-type Pdx in solution . The present findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106 . In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation, in all three molecules in the crystal.

Biochemistry, 2004 Apr 27, 43(16), 4646 - 54
Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida; Wong BJ et al.; Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole . In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned . Focusing on the orthologues from Pseudomonas aeruginosa and P . putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA . The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase . Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues . Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.

J Mol Biol, 2004 Feb 27, 336(4), 889 - 902
Crystal structure of putidaredoxin reductase from Pseudomonas putida, the final structural component of the cytochrome P450cam monooxygenase; Sevrioukova IF et al.; The crystal structure of recombinant putidaredoxin reductase (Pdr), an FAD-containing NADH-dependent flavoprotein component of the cytochrome P450cam monooxygenase from Pseudomonas putida, has been determined to 1.90 A resolution . The protein has a fold similar to that of disulfide reductases and consists of the FAD-binding, NAD-binding, and C-terminal domains . Compared to homologous flavoenzymes, the reductase component of biphenyl dioxygenase (BphA4) and apoptosis-inducing factor, Pdr lacks one of the arginine residues that compensates partially for the negative charge on the pyrophosphate of FAD . This uncompensated negative charge is likely to decrease the electron-accepting ability of the flavin . The aromatic side-chain of the "gatekeeper" Tyr159 is in the "out" conformation and leaves the nicotinamide-binding site of Pdr completely open . The presence of electron density in the NAD-binding channel indicates that NAD originating from Escherichia coli is partially bound to Pdr . A structural comparison of Pdr with homologous flavoproteins indicates that an open and accessible nicotinamide-binding site, the presence of an acidic residue in the middle part of the NAD-binding channel that binds the nicotinamide ribose, and multiple positively charged arginine residues surrounding the entrance of the NAD-binding channel are the special structural elements that assist tighter and more specific binding of the oxidized pyridine nucleotide by the BphA4-like flavoproteins . The crystallographic model of Pdr explains differences in the electron transfer mechanism in the Pdr-putidaredoxin redox couple and their mammalian counterparts, adrenodoxin reductase and adrenodoxin.

Appl Microbiol Biotechnol, 2004 Nov, 65(6), 727 - 33 Epub 2004 Apr 17.
Adhesion of a Pseudomonas putida strain isolated from a paper machine to cellulose fibres; Rochex A et al.; The adhesion to cellulose fibres of a strain of Pseudomonas putida isolated from a paper machine was studied under different environmental conditions . The physicochemical properties of both P . putida cells and cellulose fibres were also determined to better understand the adhesion phenomenon . Adhesion was rapid (1 min) and increased with time, cell concentration and temperature (from 25 to 40 degrees C), indicating that bacterial adhesion to cellulose fibres is essentially governed by a physicochemical process . The P . putida cell surface was negatively charged, as shown by electrophoretic mobility measurements, and was hydrophilic due to a strong electron-donor character, as shown by the microbial adhesion to solvents method . Cellulose fibres were shown to be hydrophilic by contact angle measurements using the capillary rise method . These results suggest the importance of Lewis acid-base interactions in the adhesion process . In various ionic solutions (NaCl, KCl, CaCl(2) and MgCl(2)), adhesion increased with increasing ionic strength up to 10-100 mM, indicating that, at low ionic strength, electrostatic interactions were involved in the adhesion process . An increase in the C/N ratio of the growth medium (from 5 to 90) decreased adhesion but this could not be related to changes in physicochemical properties, suggesting that other factors may be involved . In practice, temperature, ionic strength and nitrogen concentration must be taken into consideration to reduce bacterial contamination in the paper industry.

Gene, 2004 Apr 28, 331, 177 - 88
Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation; Carl B et al.; Although quinoline 2-oxidoreductase (Qor) and 1H-2-oxoquinoline 8-monooxygenase (OxoOR), which catalyse the first two steps of quinoline degradation by Pseudomonas putida 86, and their genes have been investigated in some detail, the genetic organization and regulation of the catabolic pathway are not known yet . A gene cluster involved in quinoline degradation was characterized . Upstream of oxoO encoding the oxygenase component of OxoOR, the gene oxoS coding for a XylS-type protein is located . The DNA region downstream of oxoO comprises potential open reading frames (ORFs) that may code for further catabolic enzymes (an alpha/beta-hydrolase fold protein, and an amidase), and for accessory proteins presumably required for the assembly of metal cofactor containing holoenzymes (XdhC-like protein, MoeC- and MobA-like protein(s), IscS and IscU) . The potential iscU gene is followed by the genes qorMSL that encode the structural subunits of Qor . Three potential ORFs (ORFs7-9) are located between qorMSL and oxoR, which codes for the reductase component of OxoOR . ORFs7-9 have counterparts in the cox (CO oxidizing system) and nic (nicotine degradation) gene clusters . Transcription of all these genes and ORFs located downstream of oxoS is induced by quinoline or 1H-2-oxoquinoline . Insertional inactivation of oxoS abolished quinoline-induced transcription . However, weak transcription of ORFs7-9 also occurred independent of quinoline and OxoS . The typical tandem recognition site for a XylS-type transcriptional activator was identified in the putative promoter region of qorM, and archetypal XylS indeed was found to activate synthesis of Qor . Motifs corresponding to single half-sites of a XylS-type binding site are located upstream of oxoO, the xdhC-like gene, and oxoR . Putative quinoline-specific transcriptional start sites were identified for these genes, and for qorM . The gene cluster probably is transcribed from several promoters, resulting in multiple overlapping polycistronic mRNAs.

Environ Pollut, 2000 Feb, 107(2), 239 - 44
Evaluation of the Galega-Rhizobium galegae system for the bioremediation of oil-contaminated soil; Suominen L et al.; The bioremediation potential of a nitrogen-fixing leguminous plant, Galega orientalis, and its microsymbiont Rhizobium galegae was evaluated in BTX (benzene, toluene, xylene)-contaminated soils in microcosm and mesocosm scale . To measure the intrinsic tolerance of the organisms to m-toluate, a model compound representing BTX, G . orientalis and R . galegae were cultivated under increasing concentrations of m-toluate alone and in association with Pseudomonas putida pWWO, a bacterial strain able to degrade toluene-derived compounds . The test plants and rhizobia remained viable in m-toluate concentrations as high as 3000 ppm . Plant growth was inhibited in concentrations higher than 500 ppm, but restituted when plants were transferred into m-toluate-free medium . Nodulation was blocked under the influence of m-toluate, but was restored after the plants were transferred into the non-contaminated media . In the mesocosm assay the Galega plants showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 ppm m-toluate . Thus, this legume system has good potential for use on oil-contaminated sites

Environ Pollut, 2000 May, 108(2), 219 - 23
Biodegradation and biosorption of acid anthraquinone dye; Walker GM et al.; The acid anthraquinone dye Tectilon Blue (TB4R) is a major coloured component from the aqueous effluent of a carpet printing plant in Northern Ireland . The aerobic biodegradation of TB4R has been investigated experimentally in batch systems, using three strains of bacteria, namely, Bacillus gordonae (NCIMB 12553), Bacillus benzeovorans (NCIMB 12555) and Pseudomonas putida (NCIMB 9776) . All three strains successfully decolourised the dye, and results were correlated using Michaelis-Menten kinetic theory . A recalculation of the reaction rate constants, to account for biosorption, gave an accurate simulation of the colour removal over a 24-h period . Up to 19% of the decolorisation was found to be caused by biosorption of the dye onto the biomass, with the majority of the decolorisation caused by utilisation of the dye by the bacteria . The reaction rate was found to be intermediate between zero and first order at dye concentrations of 200-1000 mg/l.

J Bacteriol, 2004 May, 186(9), 2766 - 73
Bdellovibrio bacteriovorus strains produce a novel major outer membrane protein during predacious growth in the periplasm of prey bacteria; Beck S et al.; Bdellovibrio bacteriovorus is a predatory bacterium that is capable of invading a number of gram-negative bacteria . The life cycle of this predator can be divided into a nonreproductive phase outside the prey bacteria and a multiplication phase in their periplasm . It was suggested that during the reproduction phase, B . bacteriovorus reutilizes unmodified components of the prey's cell wall . We therefore examined the outer membranes of B . bacteriovorus strains HD100 (DSM 50701) and HD114 (DSM 50705) by using Escherichia coli, Yersinia enterocolitica, and Pseudomonas putida as prey organisms . The combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses revealed novel and innate major outer membrane proteins (OMPs) of B . bacteriovorus strains . An incorporation of prey-derived proteins into the cell wall of B . bacteriovorus was not observed . The corresponding genes of the B . bacteriovorus strains were elucidated by a reverse-genetics approach, and a leader peptide was deduced from the gene sequence and confirmed by Edman degradation . The host-independent mutant strain B . bacteriovorus HI100 (DSM 12732) growing in the absence of prey organisms possesses an OMP similar to the major OMPs of the host-dependent strains . The similarity of the primary structure of the OMPs produced by the three Bdellovibrio strains is between 67 and 89% . The leader peptides of all OMPs have a length of 20 amino acids and are highly conserved . The molecular sizes of the mature proteins range from 34.9 to 37.6 kDa . Secondary-structure predictions indicate preferential alpha-helices and little beta-barrel structures.

J Bacteriol, 2004 May, 186(9), 2735 - 44
Involvement of error-prone DNA polymerase IV in stationary-phase mutagenesis in Pseudomonas putida; Tegova R et al.; In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the dinB gene) in stationary-phase mutagenesis in Pseudomonas putida . For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g., 1-bp deletions and different base substitutions) separately . A significant effect of Pol IV became apparent when the frequency of accumulation of 1-bp deletion mutations was compared in the P . putida wild-type strain and its Pol IV-defective dinB knockout derivative . Pol IV-dependent mutagenesis caused a remarkable increase (approximately 10-fold) in the frequency of accumulation of 1-bp deletion mutations on selective plates in wild-type P . putida populations starved for more than 1 week . No effect of Pol IV on the frequency of accumulation of base substitution mutations in starving P . putida cells was observed . The occurrence of 1-bp deletions in P . putida cells did not require a functional RecA protein . RecA independence of Pol IV-associated mutagenesis was also supported by data showing that transcription from the promoter of the P . putida dinB gene was not significantly influenced by the DNA damage-inducing agent mitomycin C . Therefore, we hypothesize that mechanisms different from the classical RecA-dependent SOS response could elevate Pol IV-dependent mutagenesis in starving P . putida cells.

Water Res, 2004 Apr, 38(8), 2027 - 34
Mass transfer and bioremediation of naphthalene particles in a roller bioreactor; Purwaningsih IS et al.; Naphthalene particles in a water slurry have been bioremediated in a sealed, roller bioreactor using a pure strain of Pseudomonas putida . High stripping losses of particles due to both splashing and aeration made the use of the traditional CSTR bioreactor unsuitable for bioremediation of naphthalene particles . The overall dissolution mass transfer coefficient of naphthalene particles in the roller bioreactor was low, 0.055 h(-1) at 50 RPM . The dissolution mass transfer rate was the limiting step for bioremediation . Although mass transfer was identified as the rate limiting step, the addition of hydroxypropyl-beta-cyclodextrin (a solubility enhancer) failed to improve naphthalene slurry bioremediation . In order to successfully bioremediate naphthalene particles at concentrations over 300 mg/L, intermittent aeration was applied in the sealed roller bioreactor on a daily basis . By operating in sequential batch mode with intermittent aeration, the roller bioreactor was successfully used to continuously bioremediate naphthalene particles at concentrations up to 1000 mg/L and at rates up to 10 mg/Lh.

Ultrason Sonochem, 2004 May, 11(3-4), 177 - 82
Application of power ultrasound for azo dye degradation; Rehorek A et al.; Power ultrasound of 850 kHz at 60, 90 and 120 W was used for the degradation of industrial azo dyes Acid Orange 5 and 52, Direct Blue 71, Reactive Black 5 and Reactive Orange 16 and 107 . The results show that power ultrasound is able to mineralize azo dyes to non-toxic end products, which was confirmed by respiratory inhibition test of Pseudomonas putida . All investigated dyes have been decolorized and degraded within 3-15 h at 90 W and within 1-4 h at 120 W, respectively . Mass spectrometric investigations show, that hydroxyl radicals attack azo dyes by simultaneous azo bond scission, oxidation of nitrogen atoms and hydroxylation of aromatic ring structures . A volumetric scale-up showed a correlation between the energy input and the absolute amount of degraded dye . Up to an energy input of about 90 W no enzymatic deactivation of laccase was observed which might be helpful for a simultaneous action of sonochemical and enzymatic treatments.

Environ Sci Technol, 2004 Mar 15, 38(6), 1858 - 65
Microbial removal of ionic mercury in a three-phase fluidized bed reactor; Deckwer WD et al.; The reductive biotransformation of mercuric ions to elemental mercury was studied by applying a model system with a genetically engineered Pseudomonas putida strain in a lab scale three-phase fluidized bed (TPFB) . The aim was to demonstrate the suitability of the TPFB to demercurize effluent streams containing up to 10 mg Hg2+ dm(-3) . The TPFB is used, first, to carry out the biotransformation on the alginate immobilized biocatalyst and, second, to remove the produced Hg0 by volatilization into the gas phase followed by its recovery through fast oxidative absorption . Targeted experiments with the immobilized biocatalyst were designed and carried out to determine mercury adsorption data on the biomass and all relevant mass transport rates at conditions prevailing in the TPFB . The evaluation of the performance data in the TPFB revealed almost complete reaction control and hence negligibility of mass transfer resistances . This simplifies the scale-up of larger TPFB reactors for mercury removal as it can be based on the known kinetics alone . The measured biotransformation capacities in the TPFB are similar to those reported for the fixed bed technology which has already proven its applicability at an industrial scale in long time runs . However, the TPFB offers some advantages over the fixed bed and could therefore possibly be a favorable, reliable, and less costly alternative to the existing technology.

Appl Environ Microbiol, 2004 Apr, 70(4), 2452 - 63
Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts; Martinez A et al.; The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts . Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector . In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E . coli . To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities . These tools include derivatives of S . lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P . putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E . coli to both S . lividans and P . putida by high-throughput conjugation . Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.

Eur J Biochem, 2004 Apr, 271(8), 1580 - 90
A novel R-stereoselective amidase from Pseudomonas sp . MCI3434 acting on piperazine-2-tert-butylcarboxamide; Komeda H et al.; A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp . MCI3434 and characterized . The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase . The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1 . The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp . MCI3434 and sequenced . Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase . This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P . aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on . The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp . MCI3434 . The intact cells of the E . coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide . The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E . coli transformant overexpressing the ramA gene . On gel-filtration chromatography, the enzyme appeared to be a monomer . It had maximal activity at 45 degrees C and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, or Pb2+ . RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide . The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.

Appl Microbiol Biotechnol, 2004 Sep, 65(4), 391 - 400 Epub 2004 Apr 03.
Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S; Matcher GF et al.; The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding D-amino acids or L-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase ( N-carbamoylase) . Pseudomonas putida strain RU-KM3S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and L-selective N-carbamoylase activity . This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source . Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and beta-ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain . dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup . Two mutant strains were isolated with increased levels of dihydropyrimidinase but not beta-ureidopropionase activity . Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source . However, these mutations had no effect on either the dihydropyrimidinase or beta-ureidopropionase activity . Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P . putida RU-KM3S cells.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 347 - 52
High survival and stability rates of Escherichia coli dried in hydroxyectoine; Manzanera M et al.; The tetrahydropyramidine hydroxyectoine acts as an osmolyte in a range of bacterial species, but its use as a desiccation protectant is less well explored . Recently, it was demonstrated that hydroxyectoine provides effective stabilisation of the Gram-negative species Pseudomonas putida, which is only relatively poorly preserved by the better-characterised protectant, trehalose . It is now shown that hydroxyectoine also protects the paradigmatic bacterium, Escherichia coli: osmotically-preconditioned E . coli dried in hydroxyectoine exhibited a high degree of desiccation tolerance, similar to that achieved using trehalose in this species . Hydroxyectoine is apparently accumulated from hypersaline medium in preference to trehalose biosynthesis, but E . coli loaded with hydroxyectoine in this way showed reduced stability in the dry state . This suggests that, although both hydroxyectoine and trehalose perform equally well as extracellular protectants, trehalose is preferred for intracellular protection.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 307 - 14
Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil; Wang G et al.; The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils . The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp) . P . putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp . Addition of P . putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days . The RTm-PCR estimations of P . putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation . However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts . These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted.

J Am Chem Soc, 2004 Apr 7, 126(13), 4181 - 91
Substrate binding favors enhanced NO binding to P450cam; Franke A et al.; Ferric cytochrome P450cam from Pseudomonas putida (P450cam) in buffer solution at physiological pH 7.4 reversibly binds NO to yield the nitrosyl complex P450cam(NO) . The presence of 1R-camphor affects the dynamics of NO binding to P450cam and enhances the association and dissociation rate constants significantly . In the case of the substrate-free form of P450cam, subconformers are evident and the NO binding kinetics are much slower than in the presence of the substrate . The association and dissociation processes were investigated by both laser flash photolysis and stopped-flow techniques at ambient and high pressure . Large and positive values of S and V observed for NO binding to and release from the substrate-free P450cam complex are consistent with the operation of a limiting dissociative ligand substitution mechanism, where the lability of coordinated water dominates the reactivity of the iron(III)-heme center with NO . In contrast, NO binding to P450cam in the presence of camphor displays negative activation entropy and activation volume values that support a mechanism dominated by a bond formation process . Volume profiles for the binding of NO appear to be a valuable approach to explain the differences observed for P450cam in the absence and presence of the substrate and enable the clarification of the underlying reaction mechanisms at a molecular level . Changes in spin state of the iron center during the binding/release of NO contribute significantly to the observed volume effects . The results are discussed in terms of relevance for the biological function of cytochrome P450 and in context to other investigations of the related reactions between NO and imidazole- and thiolate-ligated iron(III) hemoproteins.

Can J Microbiol, 2001 Jan, 47(1), 41 - 8
Competitiveness in root colonization by Pseudomonas putida requires the rpoS gene; Miller CD et al.; The rpoS gene in Pseudomonas putida was essential for plant root colonization under competitive conditions from other microbes . The RpoS- mutant survived less well than the wild-type strain in culture medium, and unlike the wild-type, failed to colonize the roots in a peat matrix containing an established diverse microflora . The RpoS-deficient P . putida isolate was generated by insertion of a glucuronidase-npt cassette into the rpoS gene . The RpoS mutant had dose-dependent increased sensitivity to oxidative stress and produced Mn-superoxide dismutase activity earlier than the parent . While extracts from wild-type P . putida stationary-phase cells contained three isozymes of catalase (CatA, CatB, and CatC), the sigma38-deficient P . putida lacked CatB . These results are consistent with previous findings that CatB is induced in stationary-phase.

Antonie Van Leeuwenhoek, 2004 Feb, 85(2), 93 - 101
Production of 3-hydroxydecanoic acid by recombinant Escherichia coli HB101 harboring phaG gene; Zheng Z et al.; Heterogenous expression of (R)-3-hydroxydecanoyl-ACP:CoA transacylase gene (phaG) isolated from Pseudomonas putida in Escherichia coli HB101 led to the extracellular production of 3-hydroxydecanoic acid (3HD) in a growth medium consisting of carbon source non-related to 3HD structure, while no 3HD was detected in the growth media inoculated with wild type E . coli HB101 and recombinant E . coli HB101 harboring vector pBluescript SK- only . 3HD production by E . coli HB101 (pLZZGPp) harboring phaG from fructose was 587 mg/l, approximately three times that from cultivation in glucose under same culture conditions . 3HD production was affected by timing of fructose addition and fructose concentration in the culture . As an inhibitor of fatty acid de novo synthesis, the presence of triclosan in the culture could increase 3HD production by E . coli HB101 (pLZZGPp) by about 20-40% . The results further confirmed that (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG) provides 3HD precursors for medium-chain-length polyhydroxyalkanoate synthesis . At the same time, this phenomenon showed that recombinant organisms can be used for production of certain fine chemicals such as hydroxyalkanoic acids.

Biochemistry, 2004 Mar 23, 43(11), 3075 - 88
Crystal structure of the alkylsulfatase AtsK: insights into the catalytic mechanism of the Fe(II) alpha-ketoglutarate-dependent dioxygenase superfamily; Muller I et al.; The alkylsulfatase AtsK from Pseudomonas putida S-313 belongs to the widespread and versatile non-heme iron(II) alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes the oxygenolytic cleavage of a variety of different alkyl sulfate esters to the corresponding aldehyde and sulfate . The enzyme is only expressed under sulfur starvation conditions, providing a selective advantage for bacterial growth in soils and rhizosphere . Here we describe the crystal structure of AtsK in the apo form and in three complexes: with the cosubstrate alpha-ketoglutarate, with alpha-ketoglutarate and iron, and finally with alpha-ketoglutarate, iron, and an alkyl sulfate ester used as substrate in catalytic studies . The overall fold of the enzyme is closely related to that of the taurine/alpha-ketoglutarate dioxygenase TauD and is similar to the fold observed for other members of the enzyme superfamily . From comparison of these structures with the crystal structure of AtsK and its complexes, we propose a general mechanism for the catalytic cycle of the alpha-ketoglutarate-dependent dioxygenase superfamily.

Mol Microbiol, 2004 Mar, 51(6), 1773 - 85
IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase; Ilves H et al.; Transpositional activity of mobile elements is not constant . Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements . We have demonstrated previously that transposition of Tn4652 in Pseudomonas putida is a stationary phase-specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445-5448) . We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P . putida, might contribute to the transposition of Tn4652 as well . Here, we demonstrate that transposition of Tn4652 in stationary phase P . putida is essentially limited by the amount of IHF . No transposition of Tn4652 occurs in a P . putida ihfA-defective strain . Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild-type strain . This indicates that the amount of IHF is a bottleneck in Tn4652 transposition . Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends . In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF . The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase.

Environ Microbiol, 2004 Apr, 6(4), 416 - 23
Fatty acid biosynthesis is involved in solvent tolerance in Pseudomonas putida DOT-T1E; Segura A et al.; The unusual tolerance of Pseudomonas putida DOT-T1E to toluene is based on the extrusion of this solvent by constitutive and inducible efflux pumps and rigidification of its membranes via phospholipid alterations . Pseudomonas putida DOT-T1E-109 is a solvent-sensitive mutant . Mutant cells were less efficient in solvent extrusion than the wild-type cells, as shown by the limited efflux of 14C-1,2,4-trichlorobenzene from the cell membranes, despite the fact that the efflux pumps are overexpressed as a result of increased expression of the ttgDEF and ttgGHI efflux pump operons . This limitation could be the result of alterations in the outer membrane because the mutant cells released more beta-lactamase to the external medium than the wild-type cells . The mutant P . putida DOT-T1E-109 showed negligible synthesis of fatty acids in the presence of sublethal concentrations of toluene as revealed by analysis of 13CH3-13COOH incorporation into fatty acids . In contrast, the mutant strain in the absence of solvents, and the wild-type strain, both in the presence and in the absence of toluene, incorporated 13CH3-13COOH at a high rate into de novo synthesized lipids . The mutation in P . putida DOT-T1E-109 increases sensitivity to the solvent because of a limited efflux of the solvent from the cell membranes with the concomitant inhibition of fatty acid biosynthesis.

Appl Environ Microbiol, 2004 Mar, 70(3), 1804 - 10
Construction of chimeric catechol 2,3-dioxygenase exhibiting improved activity against the suicide inhibitor 4-methylcatechol; Okuta A et al.; Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols . C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III) . In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated . To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes . These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI) . When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O . However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized . The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE . Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE . These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.

Appl Environ Microbiol, 2004 Mar, 70(3), 1593 - 9
Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates; Matz C et al.; We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans . A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates . Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested . High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates . The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h . In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains . Purified violacein from cell extracts of C . violaceum showed high toxicity to nanoflagellates . In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains . Pigment synthesis in C . violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system . An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C . violaceum AHL could restore toxicity . Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth . Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.

J Mol Biol, 2004 Mar 19, 337(2), 399 - 416
Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism; Yoshimoto T et al.; Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily . The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution . The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively . We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry . The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2) . Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1) . In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules . Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule . The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119 . The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme . The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes . We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.

J Bacteriol, 2004 Mar, 186(6), 1905 - 9
Transcriptional phase variation at the flhB gene of Pseudomonas putida DOT-T1E is involved in response to environmental changes and suggests the participation of the flagellar export system in solvent tolerance; Segura A et al.; Frameshift mutations in a poly(G) track at the flhB gene of Pseudomonas putida DOT-T1E are responsible for the diminished swimming of this strain on semisolid medium, which contrasts with the high swimming ability of P . putida KT2440, which does not exhibit a poly(G) track at the flhB gene . We previously showed that a mutant lacking FlhB was more sensitive to solvents than the wild-type strain (Segura et al., J . Bacteriol., 183:4127-4133, 2001) . In this study, we show that swimming ability correlates with solvent tolerance in P . putida DOT-T1E, so that growth conditions favoring a functional flhB gene (growth on semisolid medium) resulted in increased innate tolerance to a sudden toluene shock.

J Bacteriol, 2004 Mar, 186(6), 1898 - 901
Cellular XylS levels are a function of transcription of xylS from two independent promoters and the differential efficiency of translation of the two mRNAs; Gonzalez-Perez MM et al.; XylS controls the expression of the meta-cleavage pathway for the metabolism of benzoates in Pseudomonas putida KT2440 . The xylS gene is expressed from two promoters, Ps1 and Ps2 . Transcription from Ps2 is low and constitutive, whereas transcription from Ps1 is induced in the presence of toluene . In this study, we also show that translation of mRNA generated from Ps1 is 10 times more efficient than that generated from Ps2 . This pattern of transcription and translation of xylS gives rise to two modes of activation of the promoter of the meta pathway operon (Pm) according to the concentration of XylS in the cell . In cells growing with benzoate, with small amounts of XylS, the activated XylS regulator binds the effector and stimulates transcription from Pm, whereas in cells growing with toluene, the high levels of XylS suffice to stimulate transcription from Pm even in the absence of XylS effectors.

Biochemistry, 2004 Mar 9, 43(9), 2524 - 32
Hydrophobic nature of the active site of mandelate racemase; St Maurice M et al.; Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, stabilizing the altered substrate in the transition state by approximately 26 kcal/mol . We have used a series of substrate analogues (glycolates) and intermediate analogues (hydroxamates) to evaluate the contribution of the hydrophobic cavity within the enzyme's active site to ligand binding . Free energy changes accompanying binding of glycolate derivatives correlated well with the hydrophobic substituent constant pi and the van der Waals surface areas of the ligands . The observed dependence of the apparent binding free energy on surface area of the ligand was -30 +/- 5 cal mol(-1) A(-2) at 25 degrees C . Free energy changes accompanying binding of hydroxamate derivatives also correlated well with pi values and the van der Waals surface areas of the ligands, giving a slightly greater free energy dependence equal to -41 +/- 3 cal mol(-1) A(-2) at 25 degrees C . Surprisingly, mandelate racemase exhibited a binding affinity for the intermediate analogue benzohydroxamate that was 2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions . This suggests that there are additional specific interactions that stabilize the altered substrate in the transition state . Mandelate racemase was competitively inhibited by (R,S)-1-naphthylglycolate (apparent K(i) = 1.9 +/- 0.1 mM) and (R,S)-2-naphthylglycolate (apparent K(i) = 0.52 +/- 0.03 mM), demonstrating the plasticity of the hydrophobic pocket . Both (R)- (K(m) = 0.46 +/- 0.06 mM, k(cat) = 33 +/- 1 s(-1)) and (S)-2-naphthylglycolate (K(m) = 0.41 +/- 0.03 mM, k(cat) = 25 +/- 1 s(-1)) were shown to be alternative substrates for mandelate racemase . These kinetic results demonstrate that no major steric restrictions are imposed on the binding of this bulkier substrate in the ground state but that steric factors appear to impair transition state/intermediate stabilization . 2-Naphthohydroxamate was identified as a competitive inhibitor of mandelate racemase, binding with an affinity (K(i) = 57 +/- 18 microM) that was reduced relative to that observed for benzohydroxamate and that was in accord with the approximately 10-fold reduction in the value of k(cat)/K(m) for the racemization of 2-naphthylglycolate . These findings indicate that, for mandelate racemase, steric constraints within the hydrophobic cavity of the enzyme-intermediate complex are more stringent than those in the enzyme-substrate complex.

Biosci Biotechnol Biochem, 2004 Feb, 68(2), 317 - 23
Pseudomonas putida NCTC 10936 balances membrane fluidity in response to physical and chemical stress by changing the saturation degree and the trans/cis ratio of fatty acids; Loffhagen N et al.; This study explored the capability of Pseudomonas putida NCTC 10936 to maintain homeoviscosity after changing the growth temperature, incubating resting cells at different temperatures or at a constant temperature in the presence of 4-chlorophenol (4-CP) . After raising the growth temperature from 20 to either 30 or 35 degrees C, the degree of saturation of the organism's fatty acids increased and the ratio of trans to cis unsaturated fatty acids decreased somewhat . In contrast, after the incubation temperature of resting cells was raised (grown at 30 degrees C) from 20 to 30 or 35 degrees C the degree of saturation of the fatty acids remained nearly constant, while the ratio of trans to cis unsaturated fatty acids increased . Incubating resting cells (grown at 30 degrees C) at 20 degrees C in the presence of 4-CP again caused no major changes in the degree of saturation, but cis to trans conversion of unsaturated fatty acids was induced, with a corresponding increase in the trans/cis ratios . Increases in both the saturation degree of the fatty acids and the trans/cis ratio of the unsaturated fatty acids correlated with increases in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene intercalated in the bilayers of liposomes prepared from the cells of P . putida NCTC 10936 . Electron transport phosphorylation (ETP) could be stabilized by adaptive adjustments in the fluidity of the cytoplasmic membrane mediated by changes in fatty acid composition such as those observed . Whether changes in the degree of saturation or in the trans/cis ratio are more effective can be decided by studying P . putida NCTC 10936.

J Bacteriol, 2004 Mar, 186(5), 1337 - 44
The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds; Morales G et al.; The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression) . Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown . To better understand the role of Crc, the proteome profile of two otherwise isogenic P . putida strains containing either a wild-type or an inactivated crc allele was compared . The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons . This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P . putida . It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc . However, the pathway for phenylacetate appeared to be unaffected by Crc . These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P . putida.

Biochemistry, 2004 Feb 24, 43(7), 1883 - 90
Esters of mandelic acid as substrates for (S)-mandelate dehydrogenase from Pseudomonas putida: implications for the reaction mechanism; Dewanti AR et al.; (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes (S)-mandelate to benzoylformate . In this work, we show that the ethyl and methyl esters of (S)-mandelic acid are substrates for MDH . Although the binding affinity of the neutral esters is 25-50-fold lower relative to the negatively charged (S)-mandelate, they are oxidized with comparable k(cat)s . Substrate analogues in which the carbonyl group on the C-1 carbon is replaced by other electron-withdrawing groups were not substrates . The requirement of a carbonyl group on the C-1 carbon in a substrate suggests that the negative charge developed during the reaction is stabilized by delocalization to the carbonyl oxygen . Arg277, a residue that is important in both binding and transition state stabilization for the activity with (S)-mandelate, is also critical for transition state stabilization for the esters, but not for their binding affinity . We previously showed that the substrate oxidation half-reaction with (S)-mandelate has two rate-limiting steps of similar activation energies and proceeds through the formation of a charge-transfer complex of an electron-rich donor and oxidized FMN {Dewanti, A . R., and Mitra, B . (2003) Biochemistry 42, 12893-12901} . This charge-transfer intermediate was observed with the neutral esters as well . The observation of this electron-rich intermediate for the oxidation of an uncharged substrate to an uncharged product, as well as the critical role of Arg277 in the reaction with the esters, provides further evidence that the MDH reaction mechanism is not a concerted transfer of a hydride ion from the substrate to the FMN, but involves the transient formation of a carbanion/ene(di)olate intermediate.

Extremophiles, 2004 Jun, 8(3), 201 - 7 Epub 2004 Feb 11.
Degradation of phenol and toxicity of phenolic compounds: a comparison of cold-tolerant Arthrobacter sp . and mesophilic Pseudomonas putida; Margesin R et al.; Phenol degradation efficiency of cold-tolerant Arthrobacter sp . AG31 and mesophilic Pseudomonas putida DSM6414 was compared . The cold-tolerant strain was cultivated at 10 degrees C, while the mesophile was grown at 25 degrees C . Both strains degraded 200 mg and 400 mg phenol/l within 48-72 h of cultivation, but the cold-tolerant strain produced more biomass than the mesophile . Both strains oxidized catechol by the ortho type of ring fission . Catechol 1,2 dioxygenase (C1,2D) activity was found intra- and extracellularly in the absence and in the presence of phenol . In the presence of 200 mg phenol/l, C1,2D activity of the mesophile was about 1.5- to 2-fold higher than that of the cold-tolerant strain . However, an initial phenol concentration of 400 mg/l resulted in a comparable enzyme activity of the cold-tolerant and the mesophilic strain . The two strains differed significantly in their toxicity pattern towards 12 aromatic (mostly phenolic) compounds at different growth temperatures, which was determined via growth inhibition in the presence of nutrients and toxicants . For the cold-tolerant strain, toxicity was significantly lower at 10 degrees C than at 25 degrees C . The mesophile showed a significantly lower susceptibility to high hydrocarbon concentrations when grown at 25 degrees C compared to 10 degrees C .

Appl Environ Microbiol, 2004 Feb, 70(2), 873 - 82
Chromate-reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli; Ackerley DF et al.; Cr(VI) (chromate) is a toxic, soluble environmental contaminant . Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest . Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation . Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS) . Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III) . In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins--ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)-were examined . Both are dimers and reduce chromate efficiently to Cr(III) (kcat/Km = approximately 2 x 10(4) M(-1) x s(-1)) . The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS . However, the semiquinone was formed transiently and ROS diminished with time . Thus, ChrR probably generates Cr(V), but only transiently . Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation . ChrR is thus a suitable enzyme for further studies . During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS . The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen . As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored . The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.

Ecotoxicol Environ Saf, 2004 Feb, 57(2), 118 - 22
Recombinant Escherichia coli for the biomonitoring of benzene and its derivatives in the air; Berno E et al.; For protection against environmental deterioration, pollutants should be reduced as much as possible . Therefore, a sensitive detection method for air pollutants is required, particularly for benzene, a compound with mutagenic, teratogenic, and carcinogenic properties . Some microorganisms have a number of enzymes that degrade organic compounds . The genetic information for many of these enzymes is codified in plasmids that usually comprise some elements such as proteins and RNA for their replication, and proteins for cell division control . Some plasmids have been obtained from Pseudomonas putida, a ubiquitous microorganism that is able to degrade biologic and inorganic compounds . P . putida contains the TOL plasmid, responsible for the degradation of benzene and its derivatives . The TOL plasmid has been fused with the gene for firefly luciferase (pTNS316 plasmid) to produce a luminescent bacterium (Escherichia coli HB101), which can be applied to the environmental monitoring of these chemicals . The recombinant E . coli transformed with the plasmid (E . coli HB101-pTNS316) was applied to the environmental biosensing of benzene and its derivatives . Measurement of bacteria-generated photons was done using a color-coded device to estimate quantitatively the pollutant concentration . The expression of luciferase was induced in the presence of aromatic compounds but the E . coli sensitivity has a detection limit: this bacterium dies if the benzene concentration is higher than 0.5 ppm and at this concentration the luminescence decreases so it is impossible to detect . Another limit for this biodevice is that the microorganism, very likely, accumulates the benzene and its derivatives, and therefore it is very important to consider the time of exposure . This biomonitor potentially offers a rapid, inexpensive, and sensitive technique for environmental detection of aromatic pollutant compounds.

Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 610 - 4
Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450(cam); Prasad S et al.; The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450(cam) (P450(cam)) has eluded detection and kinetic analysis for several decades . Upon rapid mixing of peroxides-H(2)O(2) and m-CPBA with substrate-bound forms of P450(cam), we observed an intermediate with spectral features characteristic of compound I . Unlike in H(2)O(2), kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450(cam) and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site . The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450(cam).

Environ Toxicol Chem, 2003 Dec, 22(12), 2840 - 4
Combination of microautoradiography and fluorescence in situ hybridization for identification of microorganisms degrading xenobiotic contaminants; Yang Y et al.; One of the central topics in environmental bioremediation research is to identify microorganisms that are capable of degrading the contaminants of interest . Here we report application of combined microautoradiography (MAR) and fluorescence in situ hybridization (FISH) . The method has previously been used in a number of systems; however, here we demonstrate its feasibility in studying the degradation of xenobiotic compounds . With a model system (coculture of Pseudomonas putida B2 and Sphingomonas stygia incubated with {14C} o-nitrophenol), combination of MAR and FISH was shown to be able to successfully identify the microorganisms degrading o-nitrophenol . Compared with the conventional techniques, MAR-FISH allows fast and accurate identification of the microorganisms involved in environmental contaminant degradation.

Appl Environ Microbiol, 2004 Jan, 70(1), 285 - 92
Nitrobenzoates and aminobenzoates are chemoattractants for Pseudomonas strains; Parales RE; Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays . Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the beta-ketoadipate pathway, has a well-characterized beta-ketoadipate-inducible chemotactic response to aromatic acids . PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response . P . putida TW3 and Pseudomonas sp . strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (beta-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate . However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the beta-ketoadipate pathway for the degradation of benzoate . Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate . Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate . In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (beta-ketoadipate) of the chemotaxis system . The results suggest that strains TW3 and 4NT have a beta-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000 . The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.

Appl Environ Microbiol, 2004 Jan, 70(1), 43 - 51
Measurement of biologically available naphthalene in gas and aqueous phases by use of a Pseudomonas putida biosensor; Werlen C et al.; Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples . Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations . However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase . Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase . For this purpose, we reconstructed a bioluminescent Pseudomonas putida naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the sal promoter and the luxAB genes . Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase . Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively . The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase . In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase . This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 micro M) . Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices.

Luminescence, 2003 Nov-Dec, 18(6), 346 - 51
Spatial and temporal distribution of a bioluminescent-marked Pseudomonas putida on soybean root; Beauchamp CJ et al.; The ability of rhizobacteria to compete with other microorganisms for root colonization may be critical for its establishment on a root . Over a 6 day period, visualization of the spatial and temporal rhizosphere distribution of a bioluminescent-marked rhizobacterium, Pseudomonas putida, strain GR7.4lux, was examined on soybean grown in non-sterile soil conditions . Luminometry technologies showed a rapid root distribution of rhizobacteria where bioluminescence was particularly intense on the seed and upper root parts . The results provide new information on rhizobial root distribution, where, using enrichment broth, 50% of the root tips were still colonized by rhizobacteria up to 6 days after sowing . This suggests that rhizobial enrichment is required to detect low populations at the root tip . Bioluminescent technology represents a promising alternative to previous methods for studying rhizobial growth and distribution on roots .

Environ Microbiol, 2004 Jan, 6(1), 88 - 92
Plant-dependent active biological containment system for recombinant rhizobacteria; van Dillewijn P et al.; This work describes the construction of the rhizobacterium Pseudomonas putida strain CS-4, which contains an active biological containment (ABC) system that ensures bacterial suicide in the absence of proline . Maize plants exudate proline, which allows CS-4 to colonize the rhizosphere at a similar level to that of the wild-type strain . However, when the plants are removed, the CS-4 population decreased in bulk soil at a higher rate than the wild-type strain.

J Microsc, 2004 Jan, 213(Pt 1), 6 - 10
Oriented immobilization of Pseudomonas putida putidaredoxin at a gold (111)-buffer interface: a real time scanning tunnelling microscopy study; Mukhopadhyay R et al.; A scanning tunnelling microscopy study of adsorption of wild-type Pseudomonas putida putidaredoxin at a gold (111)-buffer interface has been made in real time . Reversible adsorption has been observed reflecting weak interaction of the wild-type protein with a gold (111) electrode . A genetically engineered mutant, C73S-D58C, which contains a surface thiol, has been used for 'immobilization' and 'orientated adsorption' on the gold surface . The implication of such orientated immobilization in development of a bio-electrode surface has been predicted.

J Biol Chem, 2004 Feb 27, 279(9), 7777 - 84 Epub 2003 Dec 12.
Novel physiological modulation of the Pu promoter of TOL plasmid: negative regulatory role of the TurA protein of Pseudomonas putida in the response to suboptimal growth temperatures; Rescalli E et al.; From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid . The knock-out inactivation of the turA gene resulted in enhanced transcription initiation from the Pu promoter, initially suggesting a negative regulatory role of TurA on Pu expression . Ectopic expression of TurA both in P . putida and in Escherichia coli reporter strains and transcription in vitro of the Pu promoter in the presence of purified TurA confirmed the TurA repressor role on Pu activity . turA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively . However, the growth at suboptimal temperatures resulted in a TurA-dependent increase of Pu repression . These results strongly suggest that a physiological significance of the negative role of TurA on Pu activity could be limitation of the expression of the toluene-degrading enzymes at suboptimal growth temperatures . Therefore, the identification of TurA as Pu-binding protein revealed a novel physiological modulation of Pu promoter that is different from those strictly nutritional described previously.

FEBS Lett, 2003 Dec 4, 555(2), 405 - 10
Prediction of the binding site on E1 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus; Jung HI et al.; The beta-subunit (E1beta) of the pyruvate decarboxylase (E1, alpha(2)beta(2)) component of the Bacillus stearothermophilus pyruvate dehydrogenase complex was comparatively modelled based on the crystal structures of the homologous 2-oxoisovalerate decarboxylase of Pseudomonas putida and Homo sapiens . Based on this homology modelling, alanine-scanning mutagenesis studies revealed that the negatively charged side chain of Glu285 and the hydrophobic side chain of Phe324 are of particular importance in the interaction with the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the complex . These results help to identify the site of interaction on the E1beta subunit and are consistent with thermodynamic evidence of a mixture of electrostatic and hydrophobic interactions being involved.

Environ Microbiol, 2003 Dec, 5(12), 1309 - 27
Characterization of a new solvent-responsive gene locus in Pseudomonas putida F1 and its functionalization as a versatile biosensor; Phoenix P et al.; A new gene cluster, designated sepABC and a divergently transcribed sepR, was found downstream of the two-component todST phosphorelay system that regulates toluene degradation (the tod pathway) in Pseudomonas putida F1 (PpF1) . The deduced amino acid sequences encoded by sepABC show a high homology to bacterial proteins known to be involved in solvent efflux or multidrug pumps . SepA, SepB and SepC are referred to be periplasmic, inner membrane and outer membrane efflux proteins respectively . Effects on growth of various PpF1 mutants compared to that of the wild type in the presence of toluene indicated a possible protective role of the solvent efflux system in a solvent-stressed environment . Growth tests with the complemented mutants confirmed the involvement of the Sep proteins in conferring solvent tolerance . The sepR gene encodes a 260-residue polypeptide that is a member of the E . coli IclR repressor protein family . The repressor role of SepR was established by conducting tests with a sep-lacZ transcriptional fusion in Escherichia coli and PpF1, expression of SepR as a maltose-binding fusion protein in a DNA binding assay, and mRNA analysis . Southern hybridization experiments and analysis of the P . putida KT2440 genome sequence indicated that sepR is a relatively rare commodity compared to homologues of the sepABC genes . We developed a whole-cell bioluminescent biosensor, PpF1G4, which contains a chromosomally based sep-lux transcriptional fusion . The biosensor showed significant induction of the sepABC genes by a wide variety of aromatic molecules, including benzene, toluene, ethylbenzene, and all three isomers of xylene (BTEX), naphthalene, and complex mixtures of aliphatic and aromatic hydrocarbons . PpF1G4 represents a second-generation biosensor that is not based on a catabolic promoter but is nonetheless inducible by aromatic pollutants and moreover functional under nutrient-rich conditions.

Environ Microbiol, 2003 Dec, 5(12), 1281 - 93
The sigma54 regulon (sigmulon) of Pseudomonas putida; Cases I et al.; sigma54 is unique among the bacterial sigma factors . Besides not being related in sequence with the rest of such factors, its mechanism of transcription initiation is completely different and requires the participation of a transcription activator . In addition, whereas the rest of the alternative sigma factors use to be involved in transcription of somehow related biological functions, this is not the case for sigma54 and many different and unrelated genes have been shown to be transcribed from sigma54-dependent promoters, ranging from flagellation, to utilization of several different carbon and nitrogen sources, or alginate biosynthesis . These genes have been characterized in many different bacterial species and, only until recently with the arrival of complete genome sequences, we have been able to look at the sigma54 functional role from a genomic perspective . Aided by computational methods, the sigma54 regulon has been studied both in Escherichia coli, Salmonella typhimurium and several species of the Rhizobiaceae . Here we present the analysis of the sigma54 regulon (sigmulon) in the complete genome of Pseudomonas putida KT2440 . We have developed an improved method for the prediction of sigma54-dependent promoters which combines the scores of sigma54-RNAP target sequences and those of activator binding sites . In combination with other evidence obtained from the chromosomal context and the similarity with closely related bacteria, we have been able to predict more than 80% of the sigma54-dependent promoters of P . putida with high confidence . Our analysis has revealed new functions for sigma54 and, by means of comparative analysis with the previous studies, we have drawn a potential mechanism for the evolution of this regulatory system.

Environ Microbiol, 2003 Dec, 5(12), 1270 - 80
Chaotropic solutes cause water stress in Pseudomonas putida; Hallsworth JE et al.; Low water availability is the most ubiquitous cause of stress for terrestrial plants, animals and microorganisms, and has a major impact on ecosystem function and agricultural productivity . Studies of water stress have largely focused on conditions that affect cell turgor, i.e . induce osmotic stress . We show that chaotropic solutes that do not affect turgor reduce water activity, perturb macromolecule-water interactions and thereby destabilize cellular macromolecules, inhibit growth, and are powerful mediators of water stress in a typical soil bacterium, Pseudomonas putida . Chaotropic solute-induced water stress resulted mostly in the upregulation of proteins involved in stabilization of biological macromolecules and membrane structure . Many environmental pollutants and agricultural products are chaotropic chemicals and thus constitute a previously unrecognised but common form of biological stress in water bodies and soils.

Environ Microbiol, 2003 Dec, 5(12), 1257 - 69
Proteome reference map of Pseudomonas putida strain KT2440 for genome expression profiling: distinct responses of KT2440 and Pseudomonas aeruginosa strain PAO1 to iron deprivation and a new form of superoxide dismutase; Heim S et al.; The genome sequence of Pseudomonas putida strain KT2440, a nutritionally versatile, saprophytic and plant root-colonizing Gram-negative soil bacterium, was recently determined by K . E . Nelson et al . (2002, Environ Microbiol 4: 799-808) . Here, we present a two-dimensional gel protein reference map of KT2440 cells grown in mineral salts medium with glucose as carbon source . Proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, in conjunction with an in-house database developed from the genome sequence of KT2440, and approximately 200 two-dimensional gel spots were assigned . The map was used to assess the genomic response of KT2440 to iron limitation stress and to compare this response with that of the closely related facultative human pathogen Pseudomonas aeruginosa strain PAO1 . The synthesis of about 25 proteins was affected in both strains, including four prominent upregulated ferric uptake regulator (Fur) protein-dependent proteins, but there were also striking differences in their proteome responses, for example in the expression of superoxide dismutases (Sod), which may indicate important roles of iron-responsive functions in the adaptation of these two bacteria to different lifestyles . The Sod enzyme of KT2440 was shown to be a novel heterodimer of the SodA and SodB polypeptides.

Environ Microbiol, 2003 Dec, 5(12), 1242 - 56
Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis; Canovas D et al.; The genome of Pseudomonas putida KT2440 encodes an unexpected capacity to tolerate heavy metals and metalloids . The availability of the complete chromosomal sequence allowed the categorization of 61 open reading frames likely to be involved in metal tolerance or homeostasis, plus seven more possibly involved in metal resistance mechanisms . Some systems appeared to be duplicated . These might perform redundant functions or be involved in tolerance to different metals . In total, P . putida was found to bear two systems for arsenic (arsRBCH), one for chromate (chrA), four to six systems for divalent cations (two cadA and two to four czc chemiosmotic antiporters), two systems for monovalent cations: pacS, cusCBA (plus one cryptic silP gene containing a frameshift mutation), two operons for Cu chelation (copAB), one metallothionein for metal(loid) binding, one system for Te/Se methylation (tpmT) and four ABC transporters for the uptake of essential Zn, Mn, Mo and Ni (one nikABCDE, two znuACB and one mobABC) . Some of the metal-related clusters are located in gene islands with atypical genome signatures . The predicted capacity of P . putida to endure exposure to heavy metals is discussed from an evolutionary perspective.

Appl Microbiol Biotechnol, 2004 May, 64(4), 486 - 92 Epub 2003 Nov 29.
Biotransformation of halophenols using crude cell extracts of Pseudomonas putida F6; Brooks SJ et al.; Crude cell extracts of Pseudomonas putida F6 transformed 4-substituted fluoro-, chloro-, bromo- and iodo-phenol without the exogenous addition of cofactors . The rate of substrate consumption decreased with increasing substituent size (F>Cl>Br>I) . Biotransformations resulted in greater than 95% utilisation of the halogenated substrate . Product accumulation was observed in incubations with 4-chloro, 4-bromo- and 4-iodo-phenol . These products were identified as the corresponding 4-substituted catechols . Transformation of 4-fluorophenol did not result in the accumulation of the corresponding catechol; however, manipulation of the reaction conditions by incorporation of ascorbic acid culminated in the formation of 4-fluorocatechol . Cell extracts of P . putida F6 also showed activity towards a 3-substituted phenol, namely 3-fluorophenol, resulting in the formation of a single product, 4-fluorocatechol .

Biochem Biophys Res Commun, 2003 Dec 5, 312(1), 235 - 40
Toluene-degrading Antarctic Pseudomonas strains from fuel-contaminated soil; Farrell RL et al.; Two psychrotolerant toluene-degrading Pseudomonas spp . were isolated from JP8 jet-fuel-contaminated soils, Scott Base, Antarctica . Isolates metabolized meta-toluate as sole carbon source at temperatures ranging from 6 to 30 degrees C . Large plasmids (>64kb) were isolated from both isolates . Sequence analysis of PCR products amplified using xylB (the gene encoding benzyl alcohol dehydrogenase) primers revealed that isolates 7/167 and 8/46 were 100% and 92% homologous, respectively, to the xylB gene of the meta-cleavage toluene degradative pathway encoded by the TOL plasmid (pWWO) of Pseudomonas putida mt-2 . Assays of cell-free extracts of 7/167 and 8/46 demonstrated activity of catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase, indicating that the isolates use the meta-cleavage pathway enzymes of toluene degradation typical of TOL type plasmids . As both isolates are able to grow at 6 degrees C ex situ it is feasible that they would be able to metabolize toluene in the Antarctic soils from where they were originally isolated.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6926 - 34
Transient XylR binding to the UAS of the Pseudomonas putida sigma54 promoter Pu revealed with high intensity UV footprinting in vivo; Valls M et al.; The binding of the transcriptional regulator XylR to its cognate upstream activating sequences (UAS) of the sigma54-dependent promoter Pu of Pseudomonas putida has been examined in vivo in single copy gene dose and stoichiometry . To this end, we have employed a novel in vivo genomic footprinting procedure that uses short exposures of bacterial cells to diffuse high intensity UV light that causes formation of TT or TC dimers . In contrast to simpler models for activation of sigma54-dependent promoters, our results clearly indicate that the XylR protein is not permanently bound in vivo to its target sites in Pu . On the contrary, the UAS appear to be mostly unoccupied at all growth stages . This is in contrast to the integration host factor (IHF), which binds Pu strongly in vivo at stationary phase, as also revealed by UV footprinting . Only overexpression of XylR altered the photoreactivity of the corresponding DNA region to report stable binding of the regulator to the UAS . However, the presence of aromatic XylR inducers reversed the forced occupation caused by increased levels of the activator . These results are compatible with the notion that XylR interacts very transiently with the UAS and detaches from the promoter during transcriptional activation of Pu.

Mol Genet Genomics, 2004 Feb, 271(1), 33 - 9 Epub 2003 Nov 18.
A functional gltB gene is essential for utilization of acidic amino acids and expression of periplasmic glutaminase/asparaginase (PGA) by Pseudomonas putida KT2440; Sonawane AM et al.; Pseudomonas putida KT2440, a root-colonizing fluorescent pseudomonad, is capable of utilizing acidic amino acids (Asp and Glu) and their amides (Asn and Gln) as its sole source of carbon and nitrogen . The uptake of Gln and Asn is facilitated by a periplasmic glutaminase/asparaginase (PGA), which hydrolyses Asn and Gln to the respective dicarboxylates . Here, we describe transposon mutagenesis of P . putida KT2440 with a self-cloning promoter probe vector, Tn 5-OT182 . Transconjugants defective in Glu-mediated PGA induction were selected for further studies . In most clones the transposon was found to have integrated into the gltB gene, which encodes the major subunit of the glutamate synthase (GOGAT) . The transconjugants were nonmotile, no longer showed a chemotactic response towards amino acids, and could not survive prolonged periods of starvation . The acidic amino acids and their amides supported growth of the transconjugants only when supplied together with glucose, suggesting that the gltB-mutants had lost the ability to utilize amino acids as a carbon source . To confirm that gltB inactivation was the cause of this phenotype, we constructed a mutant with a targeted disruption of gltB . This strain behaved like the clones obtained by random mutagenesis, and failed to express not only PGA but also a number of other Glu-induced proteins . In contrast to wild-type cells, the gltB(-) strain accumulated considerable amounts of both Glu and Gln during long-term incubation.

J Hazard Mater, 2003 Dec 12, 105(1-3), 157 - 67
Effect of additional carbon source on naphthalene biodegradation by Pseudomonas putida G7; Lee K et al.; Addition of a carbon source as a nutrient into soil is believed to enhance in situ bioremediation by stimulating the growth of microorganisms that are indigenous to the subsurface and are capable of degrading contaminants . However, it may inhibit the biodegradation of organic contaminants and result in diauxic growth . The objective of this work is to study the effect of pyruvate as another carbon source on the biodegradation of polynuclear aromatic hydrocarbons (PAHs) . In this study, naphthalene was used as a model PAH, ammonium sulfate as a nitrogen source, and oxygen as an electron acceptor . Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism . From a chemostat culture, the growth kinetics of P . putida G7 on pyruvate was determined . At concentrations of naphthalene and pyruvate giving similar growth rates of P . putida G7, diauxic growth of P . putida G7 was not observed . It is suggested that pyruvate does not inhibit naphthalene biodegradation and can be used as an additional carbon source to stimulate the growth of P . putida G7 that can degrade polynuclear aromatic hydrocarbons.

J Biol Chem, 2004 Jan 30, 279(5), 3749 - 57 Epub 2003 Nov 06.
High resolution structures of an oxidized and reduced flavoprotein . The water switch in a soluble form of (S)-mandelate dehydrogenase; Sukumar N et al.; The crystal structures of a soluble mutant of the flavoenzyme mandelate dehydrogenase (MDH) from Pseudomonas putida and of the substrate-reduced enzyme have been analyzed at 1.35-A resolution . The mutant (MDH-GOX2) is a fully active chimeric enzyme in which residues 177-215 of the membrane-bound MDH are replaced by residues 176-195 of glycolate oxidase from spinach . Both structures permit full tracing of the polypeptide backbone chain from residues 4-356, including a 4-residue segment that was disordered in an earlier study of the oxidized protein at 2.15 A resolution . The structures of MDH-GOX2 in the oxidized and reduced states are virtually identical with only a slight increase in the bending angle of the flavin ring upon reduction . The only other structural changes within the protein interior are a 10 degrees rotation of an active site tyrosine side chain, the loss of an active site water, and a significant movement of six other water molecules in the active site by 0.45 to 0.78 A . Consistent with solution studies, there is no apparent binding of either the substrate, mandelate, or the oxidation product, benzoylformate, to the reduced enzyme . The observed structural changes upon enzyme reduction have been interpreted as a rearrangement of the hydrogen bonding pattern within the active site that results from binding of a proton to the N-5 position of the anionic hydroquinone form of the reduced flavin prosthetic group . Implications for the low oxidase activity of the reduced enzyme are also discussed.

Int J Biol Macromol, 2003 Nov, 33(1-3), 101 - 6
Catechol 1,2-dioxygenase from Pseudomonas putida in organic media--an electron paramagnetic resonance study; Sanakis Y et al.; The ability of an isolated isozyme of catechol 1,2-dioxygenase from Pseudomonas putida DSM 437 to function in a non-aqueous environment was investigated . The lyophilized enzyme is able to keep its catalytic function catalyzing the oxidation of catechol in n-hexane . Electron paramagnetic resonance (EPR) spectroscopy at liquid helium temperatures was applied to compare the properties of the non-heme iron of the enzyme in the organic solvent and in the aqueous solution . The catalytic performance of the enzyme in the organic solvent is correlated with the spectroscopic properties of the non-heme iron.

Gene, 2003 Nov 13, 319, 71 - 83
Genetic characterization of the styrene lower catabolic pathway of Pseudomonas sp . strain Y2; Alonso S et al.; Pseudomonas sp . strain Y2 is a styrene-degrading bacterium, which initiates the catabolism of this compound via its transformation into phenylacetate by the sequential oxidation of the vinyl side chain . The styrene upper catabolic gene cluster (sty genes) had been localized in a 9.2-kb chromosomal region . This report describes the isolation, sequencing and analysis of an adjacent 20.5-kb chromosomal region that contains the genes of the styrene lower degradative pathway (paa genes), which are involved in the transformation of phenylacetate into aliphatic compounds that can enter the Krebs cycle . Hence, Pseudomonas sp . strain Y2 becomes the first microorganism whose entire styrene catabolic cluster has been completely characterized . Analysis of the paa gene cluster has revealed the presence of 17 open reading frames as well as gene duplications and gene reorganizations that are absent in other phenylacetate catabolic clusters described so far . The functionality of these genes has been proved by means of both complementation experiments on Pseudomonas putida mutants and in vitro enzymatic assays . Moreover, a DNA cassette encoding the whole styrene lower pathway has been constructed and has been used to expand the ability of Pseudomonas strains to degrade phenylacetic acid . For the first time, two functional phenylacetate-CoA ligases have been identified in an aerobic phenylacetic acid degradation pathway . Although the upper and lower styrene catabolic clusters are adjacent in the Pseudomonas sp . strain Y2 chromosome, their particular base composition and codon usage suggest a distinct evolutionary history.

Biochemistry, 2003 Nov 11, 42(44), 12893 - 901
A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida; Dewanti AR et al.; (S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids . The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction . In this study, we investigated the mechanism of this half-reaction in detail . At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction . This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin . The intermediate was not due to binding of anionic substrates or inhibitors . It was only observed with efficient substrates that have high k(cat) values . At higher temperatures, it was formed within the dead time of the stopped-flow instrument . The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect . This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton . This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I . E., and Mitra, B . (2000) Biochemistry 39, 10055-10065) . Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting . We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate . The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.

Environ Toxicol Chem, 2003 Nov, 22(11), 2599 - 604
Effect of nonaqueous-phase liquids on the availability of polycyclic aromatic hydrocarbons in soil for worm uptake and bacterial genotoxicity; Quinones-Rivera A et al.; A study was conducted to determine the effect of nonaqueous-phase liquids (NAPLs) on the bioavailability of benzo{a}pyrene (BaP) in soil . Sentry 19 oil and pristane reduced the availability of BaP for assimilation by the earthworm Eisenia fetida and for mutagenicity in a rifampicin-sensitive strain of Pseudomonas putida . As much as 80% of the compound could be rendered unavailable to the worms or for genotoxicity . Tests with five alkanes and an oil showed that the extent of reduction in genotoxicity of BaP varied with the identity, viscosity, and hydrophobicity of the NAPL . The magnitude of the decline in availability for genotoxicity differed in tests of three soils . Because little or no BaP was lost from the soil, the diminished bioavailability was not the result of a diminished total concentration of the compound . These findings show that exposure to hydrophobic toxicants can be appreciably altered in soils containing NAPLs.

J Ind Microbiol Biotechnol, 2003 Nov, 30(11), 636 - 42 Epub 2003 Oct 28.
Bioluminescent bioreporter integrated-circuit sensing of microbial volatile organic compounds; Ripp S et al.; A bioluminescent bioreporter for the detection of the microbial volatile organic compound p-cymene was constructed as a model sensor for the detection of metabolic by-products indicative of microbial growth . The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fused to a p-cymene/p-cumate-inducible promoter derived from the P . putida F1 cym operon . Exposure of strain UT93 to 0.02-850 ppm p-cymene produced self-generated bioluminescence in less than 1.5 h . Signals in response to specific volatile organic compounds (VOCs) such as m- and p-xylene and styrene, also occurred, but at two-fold lower bioluminescent levels . The bioreporter was interfaced with an integrated-circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures . This bioluminescent bioreporter integrated-circuit device was capable of detecting fungal presence within approximately 3.5 h of initial exposure to a culture of p-cymene-producing Penicillium roqueforti.

Genetika, 2003 Sep, 39(9), 1185 - 92
{Structural and functional variability of genetic systems for catabolizing polycyclic aromatic hydrocarbons in Pseudomonas putida strains}; Kosheleva IA et al.; The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils . These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1 operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon) . Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.

Biotechnol Bioeng, 2003 Nov 20, 84(4), 415 - 23
Enzymatic activity and stability of D-fructose dehydrogenase and sarcosine dehydrogenase immobilized onto giant vesicles; Kato K et al.; Stable vesicles with diameters between about 1 and 10 mum were prepared by a particular emulsification technology that involved the use of the surfactants Span 80 and Tween 80 and the phospholipid lecithin (phosphatidylcholine from soybeans) . Two membrane enzymes, d-fructose dehydrogenase from Gluconobacter sp . (FDH) and sarcosine dehydrogenase from Pseudomonas putida (SDH), were for the first time immobilized onto the bilayer membranes of these type of vesicles; and the catalytic activity and enzymatic stability were measured and compared with the enzymes in a vesicle-free solution . The enzyme activity as well as stability considerably increased upon immobilization . In particular, immobilized FDH at 25 degrees C was stable for at least 20 days, while the activity of the free enzyme dropped to about 20% of its initial value during the same period of time.In contrast to FDH and SDH, immobilization of sorbitol dehydrogenase from Gluconobacter suboxydans (SODH) was not successful, as no improved activity or stability could be obtained .

Biotechnol Bioeng, 2003 Nov 20, 84(4), 399 - 405
Degradation of xenobiotics in a partitioning bioreactor in which the partitioning phase is a polymer; Amsden BG et al.; Two-phase partitioning bioreactors (TPPBs) are characterized by a cell-containing aqueous phase and a second immiscible phase that contains toxic and/or hydrophobic substrates that partition to the cells at subinhibitory levels in response to the metabolic demand of the organisms . To date, the delivery phase in TPPBs has been a hydrophobic solvent that traditionally needed to possess a variety of important properties including biocompatibility, nonbioavailability, low volatility, and low cost, among others . In the present work we have shown that the organic solvent phase can be replaced by inexpensive polymer beads that function in a similar fashion as organic solvents, delivering a toxic substrate to cells based on equilibrium considerations . Specifically, 3.4 mm diameter beads of poly(ethylene-co-vinyl acetate) (EVA) were used to reduce the aqueous concentration of phenol in a bioreactor from toxic levels ( approximately 2,000 mg/L) to subinhibitory levels ( approximately 750 mg/L), after which Pseudomonas putida ATCC 11172 was added to the system and allowed to consume the total phenol loading . Thus, the beads absorbed the toxic substrate and released it to the cells on demand . The EVA beads, which could be reused, were able to absorb 14 mg phenol/g EVA . This work has opened the possibility of using widely mixed cultures in TPPB systems without concern for degradation of the delivery material and without concern of contamination .

Antonie Van Leeuwenhoek, 2003, 84(4), 261 - 8
Cis-2', 3'-dihydrodiol production on flavone B-ring by biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli; Kim SY et al.; Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones . Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments . The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC . Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively . The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy . Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2' and C3' on the B-ring of flavone, resulting in the formation of flavone cis-2', 3'-dihydrodiol (2-{3,4-dihydroxy-1.5-cyclohexadienyl}-4H-chromen-4-one) . Since this product is not found in Chemical Abstracts, this compound is considered a novel one . In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites.

J Contam Hydrol, 2003 Nov, 66(3-4), 219 - 37
The influence of substrate and electron acceptor availability on bioactive zone dynamics in porous media; Yolcubal I et al.; Two approaches were used to investigate the influence of dissolved oxygen (DO) and substrate availability on the formation and dynamics of "bioactive zones" in a water-saturated porous medium . A bioactive zone is defined as a region where a microbial community is sufficiently active to metabolize bioavailable substrates . In the first approach, microbial activity was characterized by monitoring the spatial and temporal variability of DO and aqueous substrate (salicylate and naphthalene) concentrations during miscible-displacement experiments . In the second approach, microbial activity was monitored using multiple fiber optics emplaced in the porous medium to detect luminescence produced by Pseudomonas putida RB1353, a bioluminescent reporter organism that produces light when salicylate (an intermediate of naphthalene degradation) is present . The results of both approaches show that the location and size of the bioactive zones were influenced by in situ DO and substrate availability . When DO was not a limiting factor (i.e., lower substrate input concentrations), the bioactive zone encompassed the entire column, with the majority of the microbial activity occurring between the inlet and midpoint . However, as the availability of DO became limiting for the higher substrate input experiments, the size of the bioactive zone shrank and was ultimately limited to the proximity of the column inlet.

Biotechnol Adv, 1990, 8(1), 59 - 96
Overproduction of glutathione and its derivatives by genetically engineered microbial cells; Murata K et al.; In order to improve the biotechnological potentials of Escherichia coli cells to produce glutathione, S-D-lactoylglutathione and other gamma-glutamyl compounds, the genes for enzymes {gamma-L-glutamyl-L-cysteine synthetase (GSH A) in E . coli B, glutathione synthetase (GSH B) in E . coli B, glyoxalase I (GLO I) in Pseudomonas putida} were cloned and amplified in E . coli . E . coli B cells transformed with both GSH A and GSH B genes exhibited a high activity in the synthesis of glutathione and other gamma-glutamyl compounds in bioreactor systems containing immobilized cells . E . coli C600 cells transformed with GLO I gene of P . putida showed a high GLO I activity and were used for the preparation of S-D-lactoylglutathione and other glutathione thiol esters.

Appl Environ Microbiol, 2003 Oct, 69(10), 5968 - 73
Bacterial chemotaxis to naphthalene desorbing from a nonaqueous liquid; Law AM et al.; Bacterial chemotaxis has the potential to increase the rate of degradation of chemoattractants, but its influence on degradation of hydrophobic attractants initially dissolved in a non-aqueous-phase liquid (NAPL) has not been examined . We studied the effect of chemotaxis by Pseudomonas putida G7 on naphthalene mass transfer and degradation in a system in which the naphthalene was dissolved in a model NAPL . Chemotaxis by wild-type P . putida G7 increased the rates of naphthalene desorption and degradation relative to rates observed with nonchemotactic and nonmotile mutant strains . While biodegradation alone influenced the rate of substrate desorption by increasing the concentration gradient against which desorption occurred, chemotaxis created an even steeper gradient as the cells accumulated near the NAPL source . The extent to which chemotaxis affected naphthalene desorption and degradation depended on the initial bacterial and naphthalene concentrations, reflecting the influences of these variables on concentration gradients and on the relative rates of mass transfer and biodegradation . The results of this study suggest that chemotaxis can substantially increase the rates of mass transfer and degradation of NAPL-associated hydrophobic pollutants.

J Mol Biol, 2003 Oct 17, 333(2), 377 - 92
Crystal structure of putidaredoxin, the {2Fe-2S} component of the P450cam monooxygenase system from Pseudomonas putida; Sevrioukova IF et al.; Stability of the {2Fe-2S}-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination . The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx . Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the {2Fe-2S} iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin . However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed . This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49 . In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx . The folding topology of the NMR model is similar to that of the X-ray structure of Pdx . The overall rmsd of Calpha positions between the two models is 1.59 A . The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus . The latter two peptides display conformational heterogeneity in the crystal structures . Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed . Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the {2Fe-2S} center of Pdx.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1612 - 4
Cell surface display of organophosphorus hydrolase in Pseudomonas putida using an ice-nucleation protein anchor; Shimazu M et al.; A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringe was used to localize organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida KT2440 . Cells harboring the shuttle vector pPNCO33 coding for the INP-OPH fusion were capable of targeting OPH onto the cell surface as demonstrated by whole cell ELISA . The whole cell activity of P . putida KT2440 was shown to be 10 times higher than those of previous efforts expressing the same fusion protein in Escherichia coli . The capability of expressing enzymes on the surface of a robust and environmentally benign P . putida KT2440 should open up new avenues for a wide range of applications such as in situ bioremediation.

Curr Microbiol, 2003 Aug, 47(2), 129 - 33
Direct detection and quantification of horizontal gene transfer by using flow cytometry and gfp as a reporter gene; Sorensen SJ et al.; A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated . The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry . Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein . A chromosomal encoded repressor (lacI(ql)) repressed expression of GFP in the donor bacteria . Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio-beta-D-galactoside (IPTG) . The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3067 - 72
Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR; Teran W et al.; Pseudomonas putida is well known for its metabolic capabilities, but recently, it has been shown to exhibit resistance to a wide range of antibiotics . In P . putida DOT-T1E, the TtgABC efflux pump, which has a broad substrate specificity, extrudes antibiotics such as ampicillin, carbenicillin, tetracycline, nalidixic acid, and chloramphenicol . We have analyzed the expression of the ttgABC efflux pump operon and its regulatory gene, ttgR, in response to several structurally unrelated antibiotics at the transcriptional level and investigated the role of the TtgR protein in this process . ttgABC and ttgR are expressed in vivo at a moderate basal level, which increases in the presence of hydrophobic antibiotics like chloramphenicol and tetracycline . In vitro experiments show that, in the absence of inducers, TtgR binds to a palindromic operator site which overlaps both ttgABC and ttgR promoters and dissociates from it in the presence of chloramphenicol and tetracycline . These results suggest that the TtgR repressor is able to bind to structurally different antibiotics, which allows induction of TtgABC multidrug efflux pump expression in response to these antimicrobial agents . This is the first case in which the expression of a drug transporter of the resistance-nodulation-division family has been shown to be regulated directly by antibiotics.

Bioprocess Biosyst Eng, 2003 Nov, 26(1), 11 - 7 Epub 2003 Jul 04.
Model description of bacterial 3-methylcatechol production in one- and two-phase systems; Husken LE et al.; Pseudomonas putida MC2 produces 3-methylcatechol from toluene in aqueous medium . A second phase of 1-octanol may improve total product accumulation . To optimise the design of such a biphasic process, a process model was developed, both for one- and two-phase applications . The insights obtained by the model predictions showed the importance of different process parameters (like growth substrate concentration and partition coefficient) on growth of biomass, accumulation of 3-methylcatechol and processing time . For future applications, the process model can be used to ensure enough extraction capacity from aqueous to octanol phase . It is a useful tool to define the optimum process conditions, depending on the desired optimisation parameter: product concentration or processing time.

Adv Colloid Interface Sci, 2003 Sep 18, 105, 201 - 328
Photon correlation spectroscopy investigations of proteins; Gun'ko VM et al.; Physical principles of photon correlation spectroscopy (PCS), mathematical treatment of the PCS data (converting autocorrelation functions to distribution functions or average characteristics), and PCS applications to study proteins and other biomacromolecules in aqueous media are described and analysed . The PCS investigations of conformational changes in protein molecules, their aggregation itself or in consequence of interaction with other molecules or organic (polymers) and inorganic (e.g . fumed silica) fine particles as well as the influence of low molecular compounds (surfactants, drugs, salts, metal ions, etc.) reveal unique capability of the PCS techniques for elucidation of important native functions of proteins and other biomacromolecules (DNA, RNA, etc.) or microorganisms (Escherichia coli, Pseudomonas putida, Dunaliella viridis, etc.) . Special attention is paid to the interaction of proteins with fumed oxides and the impact of polymers and fine oxide particles on the motion of living flagellar microorganisms analysed by means of PCS.

Biotechnol Lett, 2003 Jul, 25(14), 1147 - 50
Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane; Noda K et al.; Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane . By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells . The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.

J Clin Microbiol, 2003 Sep, 41(9), 4246 - 51
Presence of Pseudomonas putida strains harboring plasmids bearing the metallo-beta-lactamase gene bla(IMP) in a hospital in Japan; Yomoda S et al.; To determine the persistence and spread of antibiotic-resistant strains in Gunma University Hospital, 83 Pseudomonas putida strains (each from a different patient) were isolated from January 1997 through December 2001 . Of the 83 strains isolated, 27 were resistant to carbapenems . All 27 produced metallo-beta-lactamase and were found to be PCR positive for the bla(IMP) gene . Most (22 strains) were primarily isolated from the wards (W7 {9 strains} and W4 {8 strains}) . Another five bla(IMP)-positive P . putida strains from wards W7 and W4 were obtained by swabbing around the water pipes . A total of 32 bla(IMP)-positive P . putida strains were assessed by pulsed-field gel electrophoresis (PFGE) and testing of drug susceptibility to 10 chemotherapeutic agents . Both PFGE and MIC patterns revealed that there were long-term resident strains among inpatients and hospital environments . The bla(IMP) genes of 22 of 32 strains were all transferable to a recipient strain of Pseudomonas aeruginosa by conjugation or transformation and conferred resistance to carbapenems and cephems . The bla(IMP) plasmids were conjugally transmissible among P . aeruginosa strains and mediated resistance to amikacin as well as beta-lactams . Ten of the 22 plasmids mediated additional resistance to gentamicin and tobramycin . Plasmids with identical DNA and drug resistance patterns were found in P . putida strains with identical PFGE patterns and with different PFGE patterns . We presumed that P . putida was one of the resident species in inpatients and especially in hospital environments, spreading drug resistance genes via plasmids among P . putida strains and supplying them to more pathogenically important species, such as P . aeruginosa.

Appl Environ Microbiol, 2003 Sep, 69(9), 5120 - 7
Genetic engineering of a highly solvent-tolerant Pseudomonas putida strain for biotransformation of toluene to p-hydroxybenzoate; Ramos-Gonzalez MI et al.; The solvent-tolerant strain Pseudomonas putida DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA) . P . putida DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase . The todC1C2 genes encode the alpha and beta subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism . A DOT-T1EdeltatodC mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source . The P . putida pobA gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a Pseudomonas mendocina pobA1 pobA2 double mutant . This pobA gene was knocked out in vitro and used to generate a double mutant, DOT-T1EdeltatodCpobA, that was unable to use either toluene or 4-HBA as a carbon source . The tmo and pcu genes from P . mendocina KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of p-cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn5Tc and stably recruited in the chromosome of DOT-T1EdeltatodCpobA . Expression of the tmo and pcu genes took place in a DOT-T1E background due to cross-activation of the tmo promoter by the two-component signal transduction system TodST . Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed . Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant . The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24.

J Biol Chem, 2003 Nov 14, 278(46), 45305 - 10 Epub 2003 Sep 02.
A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin; Zhong Z et al.; Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart . The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts . Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein . With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P . aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity . Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44 . Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P . aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P . aeruginosa in vivo . A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P . putida helicase . Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P . aeruginosa helicase and significantly with the P . putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase . These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.

J Antimicrob Chemother, 2003 Oct, 52(4), 583 - 90 Epub 2003 Sep 01.
Genetic characterization of a novel metallo-beta-lactamase gene, blaIMP-13, harboured by a novel Tn5051-type transposon disseminating carbapenemase genes in Europe: report from the SENTRY worldwide antimicrobial surveillance programme; Toleman MA et al.; OBJECTIVE: In 2001, as part of the SENTRY worldwide antimicrobial surveillance programme, Pseudomonas aeruginosa 86-14571A was isolated at a hospital in Rome from a cancer patient with a bloodstream infection . The isolate was resistant to all antibiotics except amikacin, and displayed an imipenem MIC of 64 mg/L that decreased to 8 mg/L in the presence of EDTA . The resistance determinant was investigated . METHODS: The resistant determinant was cloned in Escherichia coli using a shotgun cloning approach . RESULTS: Sequence analysis revealed the presence of a novel IMP-type metallo-beta-lactamase (MBL) gene, blaIMP-13 . This encoded a protein displaying most identity to IMP variants: 93% and 92.3% identity, respectively, to IMP-8 and IMP-2 (previously identified in Italy) . The protein had 19 amino acid changes from IMP-2 and 17 amino acid changes from IMP-8 . The blaIMP-13 gene was found as a gene cassette in the first position of a class 1 integron . A 25 bp inverted repeat sequence IRi was identified 174 bp upstream of the class I integrase, which suggests that the integron is found on a Tn402-like transposon, or defective transposon derivative . This element, in turn, is located in the transposition locus (tnp region) of a Tn21 subfamily transposon that showed most identity to Tn5051, a transposon recently identified from a strain of Pseudomonas putida isolated in New York . Interestingly, the insertion point of the Tn402-like transposon and the sequence of the Tn5051-like genes were identical to those of the genetic element harbouring blaVIM-2 recently identified in Poland . CONCLUSIONS: The resistance determinant of P . aeruginosa 86-14571A is a novel IMP-type MBL carried on a composite transposon responsible for wide geographical dissemination of MBL genes in Europe.

J Mol Biol, 2003 Sep 5, 332(1), 287 - 301
Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution; Beuth B et al.; Creatinine amidohydrolase (creatininase; EC 3.5.2.10) from Pseudomonas putida, a homohexameric enzyme with a molecular mass of 28.4 kDa per subunit, is a cyclic amidohydrolase catalysing the reversible conversion of creatinine to creatine . The enzyme plays a key role in the bacterial degradation of creatinine . The three-dimensional structure of creatininase from P.putida was determined and refined to 2.1A . The structure shows the six subunits arranged as a trimer of dimers and definitely disproves previous reports that the enzyme has an octameric quaternary structure . Each monomer consists of a central, four-stranded, parallel beta-sheet flanked by two alpha-helices on both sides of the beta-sheet . This topology is unique within the superfamily of amidohydrolases . Moreover, creatininase possesses a novel fold with no close structural relatives within the Protein Data Bank . Each creatininase monomer contains a binuclear zinc centre near the C termini of the beta-strands and the N termini of the main alpha-helices . These zinc ions indicate the location of the active site unambiguously . The active site is entirely buried and is not accessible from the solution without movement of parts of the protein . The two zinc ions are bridged by a water molecule and by an aspartate residue, which acts as a bidentate ligand . They differ from each other in the number and the spatial arrangement of their ligands . One of them is tetrahedrally and the other trigonal-bipyramidally ligated . Using two water molecules of the first coordination sphere as anchor points, a creatinine-water adduct resembling the transition state of the hydrolysation reaction was modelled into the active site . The resulting complex in combination with structural comparisons with other amidohydrolases enabled us to identify the most probable candidate for the catalytic base and to suggest a putative reaction mechanism . Surprisingly these structural comparisons revealed a similarity in the active-site arrangement between creatininase and the hydantoinase-like cyclic amidohydrolases that was unexpected, given the completely unrelated primary and tertiary structures . In particular, the zinc-bridging aspartate residue of creatininase is a spatially and functionally analogue to a carboxylated lysine residue found in dihydroorotase and the hydantoinases . Hence, creatininase and the hydantoinase-like cyclic amidohydrolases represent a further example of convergent evolution within the enzyme class of hydrolases.

J Biochem (Tokyo), 2003 Jul, 134(1), 101 - 10
Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B; Nam GH et al.; Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit . The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme . The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A . Equilibrium unfolding analysis revealed that the DeltaG(U)(H(2)O) values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcal/mol, respectively, relative to that of the wild-type enzyme . The interface mutations not only decreased the k(cat)/K(M) value by about 8- to 96-fold, but also increased the K(D) value for d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold . The crystal structure of R72A determined at 2.5 A resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI . Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI.

Org Biomol Chem, 2003 Apr 21, 1(8), 1298 - 307
Stereochemical and mechanistic aspects of dioxygenase-catalysed benzylic hydroxylation of indene and chromane substrates; Boyd DR et al.; Toluene dioxygenase (TDO)-catalysed benzylic hydroxylation of indene substrates (8, 16 and 17), using whole cell cultures of Pseudomonas putida UV4, was found to yield inden-1-ol (14 and 22) and indan-1-one bioproducts (15 and 23) . The formation of these bioproducts is consistent with the involvement of carbon-centred radical intermediates . TDO-catalysed oxidation of indenes 8 and 16 also gave cis-diols 13 and 18 respectively . TDO and naphthalene dioxygenase (NDO), used as both whole-cell preparations and as purified enzymes, were found to catalyse the benzylic hydroxylation of chromane 30, deuteriated (+/-)-chromane 30D and enantiomers (4S)-30D and (4R)-30D to yield (4R)- and (4S)-chroman-4-ols 31/31D respectively . The mechanism of benzylic hydroxylation of chromane 30/30D involves the stereoselective abstraction of a pro-R (with TDO) or a pro-S (with NDO) hydrogen atom at C-4 and a marked preference for retention of configuration.

Org Biomol Chem, 2003 Mar 21, 1(6), 984 - 94
Dioxygenase-catalysed oxidation of monosubstituted thiophenes: sulfoxidation versus dihydrodiol formation; Boyd DR et al.; Toluene dioxygenase (TDO)-catalysed sulfoxidation, using Pseudomonas putida UV4, was observed for the thiophene substrates 1A-1N . The unstable thiophene oxide metabolites, 6A-6G, 6K-6N, spontaneously dimerised yielding the corresponding racemic disulfoxide cycloadducts 7A-7G, 7K-7N . Dimeric or crossed {4 + 2} cycloaddition products, derived from the thiophene oxide intermediates 6A and 6D or 6B and 6D, were found when mixtures of thiophene substrates 1A and 1D or 1B and 1D were biotransformed . The thiophene sulfoxide metabolite 6B was also trapped as cycloadducts 17 or 18 using stable dienophiles . Preferential dioxygenase-catalysed oxidation of the substituent on the thiophene ring, including exocyclic sulfoxidation (1H-1J) and cis-dihydroxylation of a phenyl substituent (1G and 1N), was also observed . An enzyme-catalysed deoxygenation of a sulfoxide in P . putida UV4 was noticed when racemic disulfoxide cyclo-adducts 7A, 7B and 7K were converted to the corresponding enantioenriched monosulfoxides 8A, 8B and 8K via a kinetic resolution process . The parent thiophene 1A and the 3-substituted thiophenes 1K-1N were also found to undergo ring dihydroxylation yielding the cis/trans-dihydrodiol metabolites 9A and 9K-9N . Evidence is provided for a dehydrogenase-catalysed desaturation of a heterocyclic dihydrodiol (9Kcis/9Ktrans) to yield the corresponding 2,3-dihydroxythiophene (24) as its preferred thiolactone tautomer (23) . A simple model to allow prediction of the structure of metabolites, formed from TDO-catalysed bacterial oxidation of thiophene substrates 1, is presented.

Appl Microbiol Biotechnol, 2004 Apr, 64(3), 429 - 35 Epub 2003 Aug 20.
Survival of naphthalene-degrading Pseudomonas putida NCIB 9816-4 in naphthalene-amended soils: toxicity of naphthalene and its metabolites; Park W et al.; Survival of naphthalene-degrading Pseudomonas putida NCIB 9816-4 was measured in nonsterile soil samples (coal tar-contaminated and pristine) with and without added crystalline naphthalene over a period of 21 days . A 2-3 log decrease in cfu occurred in the presence, but not absence, of added naphthalene . We used aqueous suspensions of crystalline naphthalene to explore potential mechanisms of its toxicity on the test bacterium under aerobic conditions . Measurements of dissolved naphthalene in medium indicated that uptake by P . putida NCIB 9816-4 maintained naphthalene at concentrations well below saturation . Accumulation of catechol was documented by high-performance liquid chromatography and gas chromatography/mass spectrometry in the presence of 0.5% (w/v) naphthalene crystals . Transient catechol accumulation was highest when cells entered stationary phase . A decrease in catechol concentration correlated with the development of brown color in the medium . Brown pigment accumulation correlated with a decrease in viable cell counts . These results suggested that catechol, related compounds, and their condensation products can accumulate to toxic levels in stationary phase P . putida NCIB 9816-4 cells . We hypothesize that the same mechanism of toxicity may occur under the nutrient-limited conditions expected in soil.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10181 - 6 Epub 2003 Aug 19.
The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv . tomato DC3000; Buell CR et al.; We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana . The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs . We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins . Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome . The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces . Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis . Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa, yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

Proteomics, 2003 Aug, 3(8), 1544 - 52
Use of proteomics and physiological characteristics to elucidate ecotoxic effects of methyl tert-butyl ether in Pseudomonas putida KT2440; Krayl M et al.; We monitored rates of growth, ATP-synthesis, respiration, and death to assess the sensitivity of the model organism Pseudomonas putida KT2440 to methyl tert-butyl ether (MTBE), and its degree of toxicity . The physiological data obtained suggested that the energy conservation system was the most sensitive site . However, with the help of proteomic analysis we obtained further information and deeper insight into the molecular mechanisms involved . This analysis indicated that sensitivity involves oxidative stress since alkylhydroperoxide reductase C (AhpC) and two superoxide dismutases (SodM, SodF) were amplified in the presence of MTBE . Thus, proteomics has major advantages in ecotoxicological investigations where the aims include elucidation of the molecular mechanisms as well as characterization of the ecostress and the potency of the stressor(s).

J Bacteriol, 2003 Sep, 185(17), 5279 - 86
Polynucleotide phosphorylase-deficient mutants of Pseudomonas putida; Favaro R et al.; In bacteria, polynucleotide phosphorylase (PNPase) is one of the main exonucleolytic activities involved in RNA turnover and is widely conserved . In spite of this, PNPase does not seem to be essential for growth if the organisms are not subjected to special conditions, such as low temperature . We identified the PNPase-encoding gene (pnp) of Pseudomonas putida and constructed deletion mutants that did not exhibit cold sensitivity . In addition, we found that the transcription pattern of pnp upon cold shock in P . putida was markedly different from that in Escherichia coli . It thus appears that pnp expression control and the physiological roles in the cold may be different in different bacterial species.

Appl Microbiol Biotechnol, 2004 Apr, 64(2), 284 - 8 Epub 2003 Aug 09.
Bioaugmentation of the phyllosphere for the removal of toluene from indoor air; De Kempeneer L et al.; The removal of airborne toluene by means of the phyllosphere of Azalea indica augmented with a toluene-degrading enrichment culture of Pseudomonas putida TVA8 was studied . The 95% disappearance time {DT95%; the time in which an initial toluene concentration of 90 ppmv (339 mg.m(3)) was removed in a batch experiment} was 75 h for Azalea plants . Under the same experimental conditions, DT95% of inoculated Azalea plants decreased remarkably to about 27 h . Subsequent additions of toluene further increased the removal efficiency of the bioaugmented system (DT95% decreased by a factor of four) . A decrease in DT95% was also recorded after repeated incubations of non-inoculated plants, but the toluene-removal rate was remarkably low, compared with the inoculated plants . Hence, inoculation of the leaf surface appeared essential for obtaining rapid removal rates . It was not possible to obtain comparable and sustained removal of airborne toluene by inoculating artificial plant surfaces . This is, to our knowledge, the first report on bioaugmentation of the leaf surface of plants to remove gaseous pollutants from air . The results presented are promising and could be of great practical importance in the field of indoor air pollution control.

Antonie Van Leeuwenhoek, 2003, 84(1), 17 - 24
Design of catabolic cassettes for styrene biodegradation; Lorenzo P et al.; A broad-host range metabolic cassette has been designed that, under the control of the Ptac promoter, expresses the sytABCD catabolic genes from Pseudomonas sp . Y2, which are responsible for the transformation of styrene into phenylacetic acid (styrene upper pathway) . This novel cassette confers to phenylacetic acid-degrading bacteria the ability to grow efficiently on styrene as the sole carbon and energy source . By combining both the sty cassette and the archetypal pWW0 TOL plasmid into the well-known Pseudomonas putida F1 aromatic biodegrader, we have constructed a novel derivative strain that shows one of the largest degradative potentials so far described for aromatic hydrocarbons, because it is able to use BTEX compounds (benzene, toluene, ethylbenzene and xylenes) and styrene as a source of carbon and energy . Furthermore, the sty cassette was engineered within a mini-transposon and endowed with a gene containment system, based on the toxic effect of the colicin E3 RNase, to reduce its lateral spread to other hosts . This contained cassette lacks defined transcriptional regulatory signals and, thus, it becomes an alternative strategy to select recombinant strains that efficiently express the desired phenotype from housekeeping regulatory elements.

Appl Environ Microbiol, 2003 Aug, 69(8), 4846 - 52
Dual labeling of Pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the TOL plasmid; Nancharaiah YV et al.; We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation . A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating . Green and red fluorescent proteins were coexpressed in donor P . putida cells . Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone . Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy . Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation . This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments . To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.

Appl Environ Microbiol, 2003 Aug, 69(8), 4727 - 31
A cytochrome c from a lupanine-transforming Pseudomonas putida strain is expressed in Escherichia coli during aerobic cultivation and efficiently exported and assembled in the periplasm; Kaderbhai MA et al.; We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain . Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm ( approximately 4 mg of hemoprotein/liter of culture) . The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine . Comparison of the N-terminal amino acid sequence of the isolated cytochrome c with that deduced from the DNA sequence indicated that the signal sequence was processed at the bond position predicted by the SigPep program . The molecular size of the cytochrome c determined by electrospray mass spectrometry (9,595) was in precise agreement with that predicted from the nucleotide sequence.

Chembiochem, 2003 Aug 4, 4(8), 721 - 6
Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution; Lingen B et al.; Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described . Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones . Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme . Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives . As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.

J Bacteriol, 2003 Aug, 185(16), 4772 - 8
Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures; Dinamarca MA et al.; Expression of the genes of the alkane degradation pathway encoded in the Pseudomonas putida OCT plasmid are subject to negative and dominant global control depending on the carbon source used and on the physiological status of the cell . We investigated the signals responsible for this control in chemostat cultures under conditions of nutrient or oxygen limitation . Our results show that this global control is not related to the growth rate and responds to two different signals . One signal is the concentration of the carbon source that generates the repressing effect (true catabolite repression control) . The second signal is influenced by the level of expression of the cytochome o ubiquinol oxidase, which in turn depends on factors such as oxygen availability or the carbon source used . Since under carbon limitation conditions the first signal is relieved but the second signal is not, we propose that modulation mediated by the cytochrome o ubiquinol oxidase is not classical catabolite repression control but rather a more general physiological control mechanism . The two signals have an additive, but independent, effect, inhibiting induction of the alkane degradation pathway.

J Bacteriol, 2003 Aug, 185(16), 4755 - 63
In vivo and in vitro evidence that TtgV is the specific regulator of the TtgGHI multidrug and solvent efflux pump of Pseudomonas putida; Rojas A et al.; The TtgGHI efflux pump of Pseudomonas putida DOT-T1E plays a key role in the innate and induced tolerance of this strain to aromatic hydrocarbons and antibiotics . The ttgGHI operon is expressed constitutively from two overlapping promoters in the absence of solvents and at a higher level in their presence, but not in response to antibiotics . Adjacent to the ttgGHI operon is the divergently transcribed ttgVW operon . In TtgV-deficient backgrounds, although not in a TtgW-deficient background, expression of the ttgGHI and ttgVW operons increased fourfold . This suggests that TtgV represses expression from the ttgG promoters and controls its own . TtgW plays no major role in the regulation of expression of these promoters . Primer extension revealed that the divergent ttgG and ttgV promoters overlap, and mobility shift assays indicated that TtgV binds to this region with high affinity . DNaseI footprint assays revealed that TtgV protected four DNA helical turns that include the -10 and -35 boxes of the ttgV and ttgG promoters.

J Bacteriol, 2003 Aug, 185(16), 4707 - 16
Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane; Llamas MA et al.; Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria . Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane . Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain . Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain . The pattern and amount of outer membrane protein in the P . putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane . Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P . putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level . Generation of a proton motive force appeared to be unaffected in these mutants . To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants . These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.

J Biotechnol, 2003 Aug 15, 103(3), 227 - 36
A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida; Park SM et al.; A new variant type of regulatory activator and relevant promoters (designated capR, Pr and Po) involved in the metabolism of phenolic compounds were cloned from Pseudomonas putida KCTC1452 by using PCR . The deduced amino acid sequence of CapR revealed a difference in nine amino acids from the effector binding domain of DmpR . To measure effector specificity, plasmids were constructed in such a way that the expression of luc gene for firefly luciferase or lacZ for beta-galactosidase as a reporter was under the control of capR . When Escherichia coli transformed with the plasmids was exposed to phenol, dramatic increases in the activity of luciferase or beta-galactosidase were observed in a range of 0.01-1 mM . Among various phenolic compounds tested, other effective compounds included catechol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2-chlorophenol, 4-chlorophenol, 2-nitrophenol, resorcinol, and 2, 5-dimethylphenol . The results indicate that CapR has effector specificity different from other related activators, CatR and DmpR . Waste water and soil potentially containing phenolic compounds were also tested by this system and the results were compared with chemical and GC data . The present results indicate that the biosensor consisting of capR and the promoters may be utilized for the development of a phenolic compounds-specific biosensor in monitoring the environmental pollutant.

Mol Microbiol, 2003 Aug, 49(4), 1095 - 108
Co-operative interactions control conjugative transfer of broad host-range plasmid RK2: full effect of minor changes in TrbA operator depends on KorB; Bingle LE et al.; A network of circuits, with KorB and TrbA as key regulators, controls genes for conjugative transfer of broad host range plasmid RK2 . To assess the importance of the TrbA regulon, mutational analysis was applied to the TrbA operator at the trbB promoter and then to other TrbA-regulated promoters in the tra region . All identified TrbA operators are submaximal; in the case of trbBp, a G to A transition that made the operator core a perfect palindrome increased repression by about 50% compared to the wild type . When this change was introduced into the RK2 genome, decreases in transfer frequency of up to three orders of magnitude were observed, with bigger effects when Escherichia coli was the donor compared to Pseudomonas putida . Western blotting showed a significant decrease in Trb protein levels . These effects were much greater than the effect of the mutation on repression by TrbA alone . When KorB was introduced into the reporter system, the effects were closer to those observed in the whole RK2 context . These results indicate that co-operativity, previously observed between TrbA and KorB, allows big changes in transfer gene expression to result from small changes in individual regulator activities.

Biodegradation, 2003 Apr, 14(2), 73 - 82
The determination of the stability constant for the iron(II) complex of the biochelator pyridine-2,6-bis(monothiocarboxylic acid); Brandon MS et al.; Pyridine-2,6-bis(monothiocarboxylic acid), also known as pyridine-2,6-dithiocarboxylic acid (pdtc), is a unique and powerful metal chelator produced by Pseudomonas stutzeri and Pseudomonas putida . The actual physiological roles of pdtc in these pseudomonads are not known with certainty, though it is likely that the compound acts as a siderophore, an antibiotic, or both . The stability constant of Fe(III)(pdtc)2(2-) was determined in previous work to be 10(33.36) . Here we determined that the stability constant of FeII(pdtc)2(2-) is 10(12) . We determined this stability constant through potentiometric and spectrophotometric measurements of a ligand-ligand competition study using 2,6-pyridine dicarboxylic acid as the competitor for iron . Comparing the stability constant for Fe(II)(pdtc)2(2-) to the constant for Fe(III)(pdtc)2(2-) shows that the stability constant of Fe(II)(pdtc)2(2-) is approximately 21 orders of magnitude smaller . This represents a very significant decrease in the binding strength of pdtc toward iron . Thus, if the host cell produces pdtc as a siderophore for sequestering Fe(III), it is likely that a second metabolite or a membrane protein of the host cell is used for reduction of the chelated iron at or near the cell membrane in order to facilitate its release from pdtc for cellular use.

Acta Crystallogr D Biol Crystallogr, 2003 Aug, 59(Pt 8), 1454 - 8 Epub 2003 Jul 23.
Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida; Bell BJ et al.; 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida is a key enzyme in the Entner-Doudoroff pathway which catalyses the cleavage of KDPG via a class I Schiff-base mechanism . The crystal structure of this enzyme has been refined to a crystallographic residual R = 17.1% (R(free) = 21.4%) . The N-terminal helix caps one side of the torus of the (betaalpha)(8)-barrel and the active site is located on the opposite, carboxylic side of the barrel . The Schiff-base-forming Lys145 is coordinated by a sulfate (or phosphate) ion and two solvent water molecules . The interactions that stabilize the trimer are predominantly hydrophobic, with the exception of the cyclically permuted bonds formed between Glu132 OE1 of one molecule and Thr129 OG1 of a symmetry-equivalent molecule . Except for the N-terminal helix, the structure of KDPG aldolase from P . putida closely resembles the structure of the homologous enzyme from Escherichia coli.

Comp Med, 2003 Jun, 53(3), 309 - 12
Postanesthesia death and suspected peracute endotoxic shock due to Pseudomonas putida in a cynomolgous macaque (Macaca fascicularis); Matchett CA et al.; An adult male cynomolgous macaque (Macaca fascicularis) died suddenly after anesthesia for a positron emission tomography scan . Bacteriologic culture of the mucopurulent secretions recovered from the endotracheal tube yielded heavy growth of Pseudomonas putida, a known endotoxin producer . Histologically, the lungs had severe, diffuse perivascular edema and neutrophils marginating to the endothelium . The sudden death and the pathologic findings were consistent with peracute endotoxic shock . Numerous environmental swab specimens of the surgical suite and equipment were submitted for bacteriologic culture, as were swab specimens of endotracheal secretions from a control animal; however, Pseudomonas putida was not isolated from any specimen . The animal in this report may have carried Pseudomonas putida as a commensal in the oropharynx, and the stress of anesthesia may have resulted in increased sensitivity to the endotoxin.

Biomacromolecules, 2003 Jul-Aug, 4(4), 1000 - 12
Role of ionic strength on the relationship of biopolymer conformation, DLVO contributions, and steric interactions to bioadhesion of Pseudomonas putida KT2442; Abu-Lail NI et al.; Biopolymers produced extracellularly by Pseudomonas putida KT2442 were examined via atomic force microscopy (AFM) and single molecule force spectroscopy . Surface biopolymers were probed in solutions with added salt concentrations ranging from that of pure water to 1 M KCl . By studying the physicochemical properties of the polymers over this range of salt concentrations, we observed a transition in the steric and electrostatic properties and in the conformation of the biopolymers that were each directly related to bioadhesion . In low salt solutions, the electrophoretic mobility of the bacterium was negative, and large theoretical energy barriers to adhesion were predicted from soft-particle DLVO theory calculations . The brush layer in low salt solution was extended due to electrostatic repulsion, and therefore, steric repulsion was also high (polymers extended 440 nm from surface in pure water) . The extended polymer brush layer was "soft", characterized by the slope of the compliance region of the AFM approach curves (-0.014 nN/nm) . These properties resulted in low adhesion between biopolymers and the silicon nitride AFM tip . As the salt concentration increased to > or =0.01 M, a transition was observed toward a more rigid and compressed polymer brush layer, and the adhesion forces increased . In 1 M KCl, the polymer brush extended 120 nm from the surface and the rigidity of the outer cell surface was greater (slope of the compliance region = -0.114 nN/nm) . A compressed and more rigid polymer layer, as well as a less negative electrophoretic mobility for the bacterium, resulted in higher adhesion forces between the biopolymers and the AFM tip . Scaling theories for polyelectrolyte brushes were also used to explain the behavior of the biopolymer brush layer as a function of salt concentration.

Biosci Biotechnol Biochem, 2003 Jun, 67(6), 1397 - 400
Molecular cloning of quinohemoprotein alcohol dehydrogenase, ADH IIB, from Pseudomonas putida HK5; Toyama H et al.; Molecular cloning of the gene of quinohemoprotein alcohol dehydrogenase (ADH IIB) from Pseudomonas putida HK5 was done . The gene (qbdA) was 690 amino acids in length, containing a 22-amino acid signal sequence . Another gene (qbdB) upstream of qbdA, probably in the same transcriptional unit, was found . Further upstream, a gene divergently transcribed against qbdBA had identity with NAD-dependent aldehyde dehydrogenase.

Virology, 2003 Jul 5, 311(2), 305 - 15
The complete genomic sequence of lytic bacteriophage gh-1 infecting Pseudomonas putida--evidence for close relationship to the T7 group; Kovalyova IV et al.; The genome of the lytic Pseudomonas putida bacteriophage gh-1 is linear double-stranded DNA containing 37,359 bp with 216-bp direct terminal repeats . Like other members of the T7 group, the gh-1 genome contains regions of high homology to T7 interspersed with nonhomologous regions that contain small open reading frames of unknown function . The genome shares 31 genes in common with other members of the T7 group, including RNA polymerase, and an additional 12 unique putative genes . A major difference between gh-1 and other members of this group is the absence of any open reading frames between the left direct terminal repeat and gene 1 . Sequence analysis of the gh-1 genome also revealed the presence of 10 putative phage promoters with a consensus sequence similar to the promoters of T3 and phiYeO3-12 (consensus: TAAAAACCCTCACTRTGGCHSCM) . P . putida mutants resistant to gh-1 were demonstrated to have an altered lipopolysaccharide structure, indicating that members of this group use lipopolysaccharide as their cellular receptor.

Appl Microbiol Biotechnol, 2003 Jul, 62(1), 83 - 91 Epub 2003 Feb 20.
Characterization and identification of genes essential for dimethyl sulfide utilization in Pseudomonas putida strain DS1; Endoh T et al.; Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle . We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level . Strain DS1 oxidized DMS to dimethyl sulfone (DMSO(2)) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1 . Two of the five DMS-utilization-defective mutants isolated by transposon 5 (Tn 5) mutagenesis had a Tn 5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P . putida strain S-313 . Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD . Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype . Another two mutants had a defect in a gene homologous to pa2354 from P . aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.

Proc Natl Acad Sci U S A, 2003 Jul 22, 100(15), 8692 - 7 Epub 2003 Jun 30.
A multifunctional plasmid-encoded replication initiation protein both recruits and positions an active helicase at the replication origin; Jiang Y et al.; The DnaA replication initiation protein has been shown to be essential for DNA strand opening at the AT-rich region of the replication origin of the Escherichia coli chromosome as well as serving to recruit and position the DnaB replicative helicase at this open region . Homologues of the dnaA gene of E . coli have been found in most bacterial species, and the DnaA protein has been shown to be required for the initiation of replication of both chromosomal and plasmid DNA . For several plasmid elements it has been found that a plasmid-encoded initiation protein is required along with the DnaA protein to bring about opening of the AT-rich region at the replication origin . The broad host range plasmid RK2 encodes two forms of its replication initiation protein (TrfA-33 and TrfA-44) that differ by an additional 98 aa at the N terminus of the larger (TrfA-44) form . Both forms initiate replication of RK2 in E . coli in vitro by a DnaA-dependent mechanism . However, as shown in this study, TrfA-44 specifically interacts with the DnaB replicative helicase of Pseudomonas putida and Pseudomonas aeruginosa and initiates the formation of a prepriming open complex in the absence of DnaA protein . Thus, the TrfA-44 initiation protein has the multifunctional properties of recruiting and positioning an active form of the DnaB helicase at the RK2 replication origin by a DnaA-independent process . This unique property for a replication initiation protein undoubtedly plays an important role in extending the host range of the RK2 antibiotic resistance plasmid.

Anticancer Res, 2003 Mar-Apr, 23(2B), 1181 - 8
Combination efficacy of doxorubicin and adenoviral methioninase gene therapy with prodrug selenomethionine; Gupta A et al.; We have previously demonstrated an enzyme activation prodrug gene therapy strategy using the methionine alpha,gamma-lyase gene (MET) cloned from Pseudomonas putida, in combination with selenomethionine (SeMET) as a prodrug . MET gene transfer via a recombinant adenovirus (Ad-MET) converts the physiologic compound SeMET to highly toxic methylselenol . In this study, we have developed a combination therapy approach using Ad-MET/SeMET gene therapy and doxorubicin (DOX) . The combination significantly delayed the growth of H460, an aggressively-growing human lung cancer cell line, in nude mice . H460 cells were injected intra-dermally in nude mice . Tumor-bearing mice were divided into 12 groups {Control (Ctrl), DOX, SeMET, SeMET + DOX, Ad-Ctrl, Ad-Ctrl + SeMET, Ad-Ctrl + DOX, Ad-Ctrl + SeMET + DOX, Ad-MET, Ad-MET + DOX, Ad-MET + SeMET, and Ad-MET + SeMET + DOX} . DOX (2 mg/kg body weight) was given intra-peritoneally twice at 7-day intervals . SeMET (1 microM/mouse) was given by intra-tumor injection everyday, starting the following day after transfection with adenovirus . Tumor growth in the untreated group showed a 10-fold increase in tumor volume after two weeks . In contrast, the increase was only 2.5-fold in the DOX + Ad-MET/SeMET group . The treatment with DOX alone at the low-dose used showed no effect compared to the control group . There was a 5.8-fold increase in tumor volume in mice treated with Ad-MET/SeMET gene therapy alone . The tumor doubling-time was increased to approximately 10 days with the combination therapy of Ad-MET + SeMET + DOX as opposed to 2-3 days in all other treatment groups.

Bioorg Med Chem, 2003 Jul 17, 11(14), 3083 - 99
Synthesis and activity of 5'-uridinyl dipeptide analogues mimicking the amino terminal peptide chain of nucleoside antibiotic mureidomycin A; Howard NI et al.; A series of 5'-uridinyl dipeptides were synthesised which mimic the amino terminal chain of nucleoside antibiotic mureido omycin A . Aminoacyl-beta-alanyl- and aminoacyl-N-methyl-beta-alanyl- dipeptides were attached either via an ester linkage to the 5'-hydroxyl of uridine, or via an amide linkage to 5'-amino-5'-deoxyuridine . The most active inhibitor of Escherichia coli phospho-MurNAc-pentapeptide translocase (MraY) was 5'-O-(L-Ala-N-methyl-beta-alanyl)-uridine (13l), which also showed 97% enzyme inhibition at 2.35 mM concentration, and showed antibacterial activity at 100 microg/mL concentration against Pseudomonas putida . Both the central N-methyl amide linkage and a 5' uridine ester linkage were required for highest biological activity . Enzyme inhibition was shown to be competitive with Mg(2+) . It is proposed that the primary amino terminus of the inhibitor binds in place of the Mg(2+) cofactor at the MraY active site, positioned via a cis-N-methyl amide linkage.

J Bacteriol, 2003 Jul, 185(13), 3895 - 904
Expression of the nitroarene dioxygenase genes in Comamonas sp . strain JS765 and Acidovorax sp . strain JS42 is induced by multiple aromatic compounds; Lessner DJ et al.; This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp . strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp . strain JS42 . Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively . NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7 . The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon . Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate . The nitroaromatic compounds appear to be the actual effector molecules . Analysis of beta-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant . Complementation with the closely related regulators NagR (from Ralstonia sp . strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO . The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7 . However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate . NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.

J Biol Inorg Chem, 2003 Sep, 8(7), 733 - 40 Epub 2003 Jun 14.
Xylene monooxygenase, a membrane-spanning non-heme diiron enzyme that hydroxylates hydrocarbons via a substrate radical intermediate; Austin RN et al.; The non-heme diiron enzyme xylene monooxygenase (XylM) has been shown to hydroxylate hydrocarbons via a hydrogen abstraction-carbon radical recombination mechanism (oxygen rebound) . Using the radical clock bicyclo{4.1.0}heptane (norcarane) in a whole-cell assay, and observing the ratio of rearranged 3-(hydroxymethyl)cyclohexene and unrearranged 2-norcaranol products, the lifetime of the substrate radical was determined to be approximately 0.2 ns . The wild-type organism Pseudomonas putida mt-2 and two separate Escherichia coli clones expressing xylMA genes gave similar results . One clone produced the Pseudomonas putida mt-2 XylMA hydroxylase and the other produced Sphingomonas yanoikuyae B1 XylMA hydroxylase . Clones were constructed by inserting genes for xylene monooxygenase and xylene monooxygenase reductase downstream from an IPTG-inducible T7 promoter . Mechanistic investigations using whole-cell assays will facilitate more rapid screening of structure-function relationships and the identification of novel oxygenases . This approach should enable the construction of a picture of the key metalloenzymes and the mechanisms they use in selected parts of the global carbon cycle without requiring the isolation of every protein involved.

Can J Microbiol, 2003 Mar, 49(3), 181 - 8
Biodegradation of cyclic amines by a Pseudomonas strain involves an amine mono-oxygenase; Trigui M et al.; Pseudomonas putida O1G3 catalyzes the degradation of pyrrolidine and piperidine . This strain can use these compounds as the sole source of carbon, nitrogen, and energy . When the cyclic amines were used as the growth substrates, the synthesis of a soluble heme amine mono-oxygenase was induced in this bacteria . This observation was confirmed by spectrophotometric analysis and specific inhibitor . This mono-oxygenase is a NADH-dependent enzyme and catalyzes the cleavage of the C-N bond of the pyrrolidine and piperidine ring by a mechanism similar to a N dealkylation . This reaction could be followed by ring cleavage to form gamma-aminobutyraldehyde oxidized to gamma-aminobutyrate . Further investigations to purify the heme-containing mono-oxygenase are in progress.

Appl Environ Microbiol, 2003 Jun, 69(6), 3650 - 2
Alkylphenol biotransformations catalyzed by 4-ethylphenol methylenehydroxylase; Hopper DJ et al.; 4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol . It was shown to be active with a range of 4-alkylphenols as substrates . 4-n-propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product . The alcohols 1-(4'-hydroxyphenyl)propanol and 1-(4'-hydroxyphenyl)-2-propen-1-ol, produced by the biotransformation of 4-n-propylphenol and chavicol, respectively, were shown to be R(+) enantiomers . 5-Indanol, 6-hydroxytetralin, 4-isopropylphenol, and cyclohexylphenol, with cyclic or branched alkyl groups, gave the corresponding vinyl compounds as their major products.

Appl Environ Microbiol, 2003 Jun, 69(6), 3469 - 75
Secondary metabolites of Flustra foliacea and their influence on bacteria; Peters L et al.; The North Sea bryozoan Flustra foliacea was investigated to determine its secondary metabolite content . Gas chromatography-mass spectrometry analysis of a dichloromethane extract of the bryozoan enabled 11 compounds to be identified . Preparative high-performance liquid chromatography of the extract resulted in the isolation of 10 brominated alkaloids (compounds 1 to 10) and one diterpene (compound 11) . All of these compounds were tested to determine their activities in agar diffusion assays against bacteria derived from marine and terrestrial environments . Compounds 1, 3 to 7, 10, and 11 exhibited significant activities against one or more marine bacterial strains originally isolated from F . foliacea but only weak activities against all of the terrestrial bacteria . By using the biosensors Pseudomonas putida(pKR-C12), P . putida(pAS-C8), and Escherichia coli(pSB403) the antagonistic effect on N-acyl-homoserine lactone-dependent quorum-sensing systems was investigated . Compounds 8 and 10 caused reductions in the signal intensities in these bioassays ranging from 50 to 20% at a concentration of 20 micro g/ml.

Appl Environ Microbiol, 2003 Jun, 69(6), 3263 - 71
Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1; Park W et al.; Hybridization and restriction fragment length polymorphism data (K . G . Stuart-Keil, A . M . Hohnstock, K . P . Drees, J . B . Herrick, and E . L . Madsen, Appl . Environ . Microbiol . 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P . putida NCIB 9816-4 . Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1 . The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes . Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P . putida) and pM3 (antibiotic resistance plasmid of PSEUDOMONAS: spp) . A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA . lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media . This expression did not respond to host exposure to naphthalene . Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media . Thus, conjugation of pCg1 in P . putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.

Appl Environ Microbiol, 2003 Jun, 69(6), 3110 - 8
Repeated introduction of genetically modified Pseudomonas putida WCS358r without intensified effects on the indigenous microflora of field-grown wheat; Viebahn M et al.; To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots . The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively . The level of introduced bacteria in all treatments decreased from 10(7) CFU per g of roots soon after sowing to less than 10(2) CFU per g after harvest 132 days after sowing . The phz and phl genes remained stable in the chromosome of WCS358r . The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999 . The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis . However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected . To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000 . No effect of rotation on the microbial community structure was detected . In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms . Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 684 - 90
Formate-stimulated oxidation of methanol by Pseudomonas putida 9816; Riis V et al.; It has been reported that Pseudomonas putida 9816 is able to grow on methanol, but it does not have methanol dehydrogenase or oxidase activity . To utilize methanol it requires yeast extract . The utilization of methanol can be accelerated by adding formate, which obviously helps oxidize methanol and win biologically useful energy . This pseudo-oxidation is catalyzed by a reverse formaldehyde dismutase . Thus, methanol can be both assimilated and dissimilated . Formate alone cannot replace yeast extract . The strain is auxotrophic.

J Microbiol Methods, 2003 Aug, 54(2), 203 - 11
Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression; Blokpoel MC et al.; Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida {Appl . Envir . Microbiol . 64 (1998) 2240} . We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria . We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene . GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M . smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry . We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M . smegmatis . To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants . We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M . smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase . In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG . Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.

J Bacteriol, 2003 Jun, 185(12), 3575 - 82
Characterization of the second LysR-type regulator in the biphenyl-catabolic gene cluster of Pseudomonas pseudoalcaligenes KF707; Watanabe T et al.; Pseudomonas pseudoalcaligenes KF707 possesses a biphenyl-catabolic (bph) gene cluster consisting of bphR1A1A2-(orf3)-bphA3A4BCX0X1X2X3D . The bphR1 (formerly orf0) gene product, which belongs to the GntR family, is a positive regulator for itself and bphX0X1X2X3D . Further analysis in this study revealed that a second regulator belonging to the LysR family (designated bphR2) is involved in the regulation of the bph genes in KF707 . The bphR2 gene was not located near the bph gene cluster, and its product (BphR2) exhibited a high level of similarity to NahR (the naphthalene- and salicylate-catabolic regulator belonging to the LysR family) in plasmid NAH7 of Pseudomonas putida . A strain containing a disrupted bphR2 gene failed to grow on biphenyl as a sole source of carbon, and the BphD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) activity was significantly reduced compared to that of wild-type strain KF707 . Furthermore, the same strain exhibited extremely low transcription of bphR1, bphA1, bphC, bphX0, and bphD . However, when the bphR2 gene was provided in trans to the bphR2-disrupted strain, the transcription level of these genes was restored . These results indicate that bphR2 regulates the bph genes positively as a second regulator together with BphR1.

Cancer Gene Ther, 2003 Jun, 10(6), 445 - 50
Methioninase gene therapy with selenomethionine induces apoptosis in bcl-2-overproducing lung cancer cells; Yamamoto N et al.; We have previously shown that the toxic pro-oxidant methylselenol is released from selenomethionine (SeMET) by cancer cells transformed with the adenoviral methionine alpha,gamma-lyase (methioninase, MET) gene cloned from Pseudomonas putida . Methylselenol damaged the mitochondria via oxidative stress, and caused cytochrome c release into the cytosol thereby activating caspase enzymes and thereby apoptosis . However, gene therapy strategies are less effective if tumor cells overexpress the antiapoptotic mitochondrial protein bcl-2 . In this study, we investigated whether rAdMET/SeMET was effective against bcl-2-overproducing A549 lung cancer cells . We established two clones of the human lung cancer A549 cell line that show moderate and high expression levels of bcl-2, respectively, compared to the parent cell line, which has very low bcl-2 expression . Staurosporine-induced apoptosis was inhibited in the bcl-2-overproducing clones as well as in the parental cell line . In contrast to staurosporine, apoptosis was induced in the bcl-2-overproducing clones as well as the parental cell line by AdMET/SeMET . Apoptosis in the rAdMET-SeMET-treated cells was determined by fragmentation of nuclei, and release of cytochrome c from mitochondria to the cytosol . A strong bystander effect of AdMET/SeMET was observed on A549 cells as well as the bcl-2-overproducing clones . rAdMET/SeMET prodrug gene therapy is therefore a promising novel strategy effective against bcl-2 overexpression, which has blocked other gene therapy strategies.

Appl Microbiol Biotechnol, 2003 Jun, 61(5-6), 528 - 35 Epub 2003 Mar 27.
Functional analyses of genes involved in the metabolism of ferulic acid in Pseudomonas putida KT2440; Plaggenborg R et al.; Pseudomonas putida KT2440 is a physiologically extremely versatile non-pathogenic bacterium that is applied as a "biosafety strain" in biotechnological processes, as authorized by the USA National Institute of Health . Analysis of the P . putida KT2440 whole-genome sequence revealed the genetic organization of the genes fcs, ech, and vdh, which are essential for ferulic acid conversion to vanillic acid via vanillin . To confirm the physiological function of these structural genes as feruloyl-CoA synthetase (Fcs), enoyl-CoA hydratase/aldolase (Ech), and vanillin dehydrogenase (Vdh), respectively, they were cloned and expressed in Escherichia coli . Recombinant strains harboring fcs and ech were able to transform ferulic acid to vanillin . The enzyme activities of Fcs and Vdh were determined in protein extracts of these cells . The essential involvement of fcs, ech and vdh in the catabolism of ferulic acid in P . putida KT2440 was proven by separately inactivating each gene by insertion of Omega-elements . The corresponding mutant strains KT2440 fcsOmegaKm, KT2440 echOmegaKm, and KT2440 vdhOmegaKm were not able to grow on ferulic acid . The potential application of P . putida KT2440 and the mutant strains in biotechnological vanillin production process is discussed.

Environ Technol, 2003 Apr, 24(4), 411 - 8
Kinetic studies of attachment and detachment of microbial cells from soil; Ahn IS et al.; A mathematical model was developed to describe the kinetics of cell attachment and detachment from soil . Soil-column experiments were performed to evaluate the model parameters . Pseudomonas putida G7 capable of degrading naphthalene was used as a model microorganism . A sediment sample taken from an uncontaminated area near a coal tar waste site in upstate New York, USA was used as a test soil . The kinetics of cell attachment and detachment from the model soil could be described by the developed first-order model . The equilibrium constant of attachment (11.4 ml g(-1)), the rate coefficient of cell attachment (0.299 ml g(-1) min(-1) and the rate coefficient of cell detachment (0.0263 min(-1)) were determined from the soil-column experiment . The equilibrium constant of attachment determined in this study (11.4 ml g(-1)) was within the range of those reported in the literature for bacterial attachment to soil (0.55 to 12.6 ml g(-1)) . The kinetic model succesfully predicted the data of batch experiment for cell attachment and detachment soil.

J Biol Chem, 2003 Jul 25, 278(30), 27695 - 702 Epub 2003 May 16.
Recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of alpha subunit carboxyl-terminal domain on an UP-like element; Macchi R et al.; The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting . A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein . Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively . The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro . The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78 . The addition of IHF resulted in perfect position symmetry of the two alphaCTDs . These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter . In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.

J Bacteriol, 2003 Jun, 185(11), 3379 - 83
Sigma 54 levels and physiological control of the Pseudomonas putida Pu promoter; Jurado P et al.; The cellular levels of the alternative sigma factor sigma(54) of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins . The levels of sigma(54) were also monitored in P . putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the sigma(54)-dependent Pu promoter of the TOL plasmid . Our results show that approximately 80 +/- 26 molecules of sigma(54) exist per cell . Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of sigma(54) determine adaptation and differentiation when cells face starvation, sigma(54) in P . putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed . The number of sigma(54) molecules per cell in P . putida is barely above the predicted number of sigma(54)-dependent promoters . These figures impose a framework on the mechanism by which Pu (and other sigma(54)-dependent systems) may become amenable to physiological control.

Extremophiles, 2003 Oct, 7(5), 371 - 6 Epub 2003 May 13.
Comparative genomic analysis of solvent extrusion pumps in Pseudomonas strains exhibiting different degrees of solvent tolerance; Segura A et al.; Organic solvents are inherently toxic for microorganisms . Their effects depend not only on the nature of the compound, but also on the intrinsic tolerance of the bacterial species and strains . Three efflux pumps belonging to the RND (resistance-nodulation-cell division) family of multidrug extrusion pumps are the main factor involved in the high intrinsic tolerance to toluene of Pseudomonas putida DOT-T1E . We have analyzed the tolerance to toluene shocks {0.1% and 0.3% (v/v)} of a number of strains belonging to different species of the genus Pseudomonas upon growth in the absence and in the presence of sublethal concentrations of toluene . The strains can be grouped in three categories: (1) highly resistant strains, in which almost 100% of the cells precultured in the presence of sublethal concentrations of toluene withstood a 0.3% (v/v) toluene shock, (2) moderately resistant strains, in which only a fraction (10(-4)-1) of the cells withstood a 0.1% (v/v) toluene shock, but fewer than 1 in 10(7) cells survived a sudden 0.3% (v/v) toluene shock regardless of the growth conditions, and (3) sensitive strains, in which regardless of the growth conditions fewer than 10(-5) cells survived a 0.1% (v/v) toluene shock . We also studied the number and type of efflux pumps in different strains in comparison with the P . putida DOT-T1E strain.

Biochemistry, 2003 May 20, 42(19), 5649 - 56
A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple; Pochapsky SS et al.; The camphor hydroxylase cytochrome P450(cam) (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida . CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys(4)Fe(2)S(2) ferredoxin putidaredoxin (Pdx) . Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101 . Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential (1)H, (15)N, and (13)C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx . NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes . A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover . A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer.

Anal Bioanal Chem, 2003 May, 376(1), 26 - 32 Epub 2003 Mar 29.
Biosorption of cadmium, copper, lead and zinc by inactive biomass of Pseudomonas Putida; Pardo R et al.; The accumulation of Cd(II), Cu(II), Pb(II) and Zn(II) at mg L(-1) concentration levels by inactive freeze-dried biomass of Pseudomonas Putida has been investigated . These metals could be efficiently removed from diluted aqueous solutions . A contact time of 10 min was sufficient to reach equilibrium . The pH has a strong effect on metal biosorption and the optimal pH values were 6.0, 5.0-6.0, 6.0-6.5 and 7.0-7.5 for Cd(II), Cu(II), Pb(II) and Zn(II) respectively . Under these conditions there was 80% removal for all metals studied . The process of biosorption can be described by a Langmuir-type adsorption model . This model accounts for 98% of the data variance . The K(A) and q(max) parameters for each metal are strongly correlated (at confidence levels greater than 98%) with the metal acidity, quantified by the constant of the corresponding M(OH)(+) complex, thus confirming previous assertions by other authors.

J Biol Chem, 2003 Jul 25, 278(30), 28229 - 36 Epub 2003 May 06.
Origin of the different pH activity profile in two homologous ketosteroid isomerases; Yun YS et al.; Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles . TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not . Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT . To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels . PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8 . The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT . The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity . This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes . Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.

J Biol Chem, 2003 Jul 25, 278(30), 27483 - 94 Epub 2003 May 02.
Gene cluster of Arthrobacter ilicis Ru61a involved in the degradation of quinaldine to anthranilate: characterization and functional expression of the quinaldine 4-oxidase qoxLMS genes; Parschat K et al.; A genetic analysis of the anthranilate pathway of quinaldine degradation was performed . A 23-kb region of DNA from Arthrobacter ilicis Ru61a was cloned into the cosmid pVK100 . Although Escherichia coli clones containing the recombinant cosmid did not transform quinaldine, cosmids harboring the 23-kb region, or a 10.8-kb stretch of this region, conferred to Pseudomonas putida KT2440 the ability to cometabolically convert quinaldine to anthranilate . The 10.8-kb fragment thus contains the genes coding for quinaldine 4-oxidase (Qox), 1H-4-oxoquinaldine 3-monooxygenase, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, and N-acetylanthranilate amidase . The qoxLMS genes coding for the molybdopterin cytosine dinucleotide-(MCD-), FeSI-, FeSII-, and FAD-containing Qox were inserted into the expression vector pJB653, generating pKP1 . Qox is the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host, P . putida KT2440 pKP1; the catalytic properties and the UV-visible and EPR spectra of Qox purified from P . putida KT2440 pKP1 were essentially like those of wild-type Qox . This provides a starting point for the construction of protein variants of Qox by site-directed mutagenesis . Downstream of the qoxLMS genes, a putative gene whose deduced amino acid sequence showed 37% similarity to the cofactor-inserting chaperone XdhC was located . Additional open reading frames identified on the 23-kb segment may encode further enzymes (a glutamyl tRNA synthetase, an esterase, two short-chain dehydrogenases/reductases, an ATPase belonging to the AAA family, a 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase-like protein, and an enzyme of the mandelate racemase group) and hypothetical proteins involved in transcriptional regulation, and metabolite transport.

Biosci Biotechnol Biochem, 2003 Feb, 67(2), 438 - 41
Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from pseudomonas putida E23; Chowdhury EK et al.; The structural gene for NAD+-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins . The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase . Transformed E . coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield . Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.

Prikl Biokhim Mikrobiol, 2003 Mar-Apr, 39(2), 199 - 201
{Reversed-phase high performance liquid chromatography of the products of microbiological degradation of toluene }; Zelenkova NF et al.; Conditions have been selected for a reversed-phase high-performance liquid chromatographic assay of intermediate products formed in the course of utilization of toluene by Pseudomonas putida . The composition of products indicates that degradation of toluene by strain BS590-P proceeds primarily through the formation of benzoate and catechol . This is followed by degradation of catechol via ortho-cleavage . In strain BS3701-P, toluene oxidation involves both the side chain and the aromatic ring.

Biotechnol Bioeng, 2003 Jun 30, 82(7), 833 - 42
Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale; Buhler B et al.; Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means . Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to the complexity of oxygenase based biocatalysts and demanding process implementation . We have combined recombinant whole-cell catalysis in a two-liquid phase system with fed-batch cultivation in an optimized medium and developed an industrially feasible process for the kinetically controlled and complex multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde using the xylene monooxygenase of Pseudomonas putida mt-2 in Escherichia coli . Successful scale up to 30 L working volume using downscaled industrial equipment allowed a productivity of 31 g L(-1) d(-1) and a product concentration of 37 g L(-1) . These performance characteristics meet present industry requirements . Product purification resulted in the recovery of 469 g of 3,4-dimethyl- benzaldehyde at a purity of 97% and an overall yield of 65% . This process illustrates the general feasibility of industrial biocatalytic oxyfunctionalization .

Water Res, 2003 Feb, 37(3), 561 - 8
Role of cell surface components on Cu2+ adsorption by Pseudomonas putida 5-x isolated from electroplating effluent; Wang L et al.; A gram-negative bacterium Pseudomonas putida 5-x with high Cu2+ accumulating capability was isolated from electroplating effluent in Kwun Tong, Hong Kong . The pretreated cells without superficial layer-capsule, isolated cell envelopes and the separated peptidoglycan layer materials were obtained from fresh P . putida 5-x cells, their Cu2+ adsorption capacities and properties were compared with that of the fresh cells . Pretreatment by 0.1 mol L(-1) HCl enhanced Cu2+ adsorption capacity due to the degradation of cell superficial layer-capsule of P . putida 5-x cells . Isolated cell envelopes possessed five times more Cu2+ adsorption capacity than that of fresh intact cell . The Cu2+ adsorption of separated peptidoglycan layer materials indicated that the peptidoglycan layer only played 10-15% part of the Cu2+ adsorption capacity, and implied other cell surface components such as outer membrane or inner membrane might play an important role in such high Cu2+ binding of the cell envelopes . The adsorption process of fresh cells, pretreated cells and cell envelopes of P . putida 5-x could be described with Freundlich isotherm, while the adsorption of Cu2+ by separated peptidoglycan layer materials was better described with Langmuir isotherm.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Mar, 38(3), 521 - 31
Ni2+ removal and recovery from electroplating effluent by Pseudomonas putida 5-x cell biomass; Wang L et al.; Ni2+ and Cu2+ are the major heavy metal ions in electroplating wastewater of Hong Kong . In the present study, Pseudomonas putida 5-x cell biomass was used to remove Ni2+ from electroplating effluent . Ni2+ adsorption capacity of P . putida 5-x cell biomass cultured in sulphate-limiting medium was found to be minimum in early logarithmic growth phase, and maximum of 28.1 mg g(-1) in late stationary growth phase . Pretreated cells by 0.1 mol L(-1) HCl could greatly enhance the Ni2+ adsorption capacity of cell biomass from 28.1 to 36.7 mg g(-1) and had no significant effect on biomass loss . The adsorption process of P . putida 5-x fresh cells and pretreated cell all could be expressed with Freundlich isotherm . TEM analyses indicated that acidic pretreatment degraded the superficial layer-capsule outside of the fresh cell to improve the adsorption capacity of cell to Ni2+ . The Ni2+ bound by P . putida 5-x cell biomass could be efficiently recovered using 0.1 mol L(-1) HCl, and the cell biomass could be reused at least five cycles for Ni2+ removal and recovery with 93% above removal efficiency and 98% above recovery rate . Owing to the Cu2+ presented in electroplating wastewater inhibiting Ni2+ adsorption process by P . putida 5-x cell biomass, two-stage biosorption processes should be designed to remove and recover Cu2+ and Ni2+ sequentially from electroplating effluent.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 579 - 85 Epub 2003 Apr 10.
Metabolism of benzyl alcohol via catechol ortho-pathway in methylnaphthalene-degrading Pseudomonas putida CSV86; Basu A et al.; Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways . In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes . However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol . Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway . Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons . Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid . The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway . CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.

Microbiology, 2003 Apr, 149(Pt 4), 991 - 1000
A CysB-regulated and sigma54-dependent regulator, SfnR, is essential for dimethyl sulfone metabolism of Pseudomonas putida strain DS1; Endoh T et al.; Pseudomonas putida strain DS1 utilizes dimethyl sulfide (DMS) as a sulfur source, and desulfurizes it via dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO(2)) and methanesulfonate (MSA) . Its Tn5 mutant, Dfi74J, no longer utilized DMS, DMSO and DMSO(2), but could oxidize DMS to DMSO(2), suggesting that the conversion of DMSO(2) to MSA was interrupted in the mutant . Sequencing of the Tn5 flanking region of Dfi74J demonstrated that a gene, sfnR (designated for dimethyl sulfone utilization), encoding a transcriptional regulator containing an ATP-dependent sigma(54)-association domain and a DNA-binding domain, was disrupted . sfnR is part of an operon with two other genes, sfnE and sfnC, located immediately upstream of sfnR and in the same orientation . The genes encode NADH-dependent FMN reductase (SfnE) and FMNH(2)-dependent monooxygenase (SfnC) . Complementation of Dfi74J with an sfnR-expressing plasmid led to restoration of its growth on DMS, DMSO and DMSO(2) . An rpoN-defective mutant of strain DS1, which lacks the sigma(54) factor, grew on MSA, but not on DMS, DMSO and DMSO(2), indicating that SfnR controls expression of gene(s) involved in DMSO(2) metabolism by interaction with sigma(54)-RNA polymerase . Northern hybridization and a reporter gene assay with an sfn-lacZ transcriptional fusion elucidated that expression of the sfnECR operon was induced under sulfate limitation and was dependent on a LysR-type transcriptional regulator, CysB . This is believed to be the first report that a sigma(54)-dependent transcriptional regulator induced under sulfate limitation is involved in sulfur assimilation.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 272 - 7
The active site structure of quinohemoprotein amine dehydrogenase inhibited by p-nitrophenylhydrazine; Satoh A et al.; Quinohemoprotein amine dehydrogenase (QH-AmDH) catalyzes the oxidative deamination of aliphatic and aromatic amines . The enzyme from Pseudomonas putida has an alpha beta gamma heterotrimeric structure with two heme c groups in the largest alpha subunit, and a novel quinone cofactor {cysteine tryptophylquinone (CTQ)} and hitherto unknown internal cross-bridges in the smallest gamma subunit . The crystal structure of the enzyme in the complex with the inhibitor {p-nitrophenylhydrazine (pNPH)} has been determined at a 2.0 A resolution.(1) The hydrazone of the cofactor with the inhibitor was nicely modeled into the omit electron density map, identifying the C6 carbonyl group as the reactive site of the cofactor . The Asp33 gamma is unambiguously determined as the catalytic base to abstract the alpha-proton from a substrate, because N beta atom of the inhibitor corresponding to the C alpha atom of the substrate amine is neighbored to Asp33 gamma . The bound inhibitor is completely enclosed in the active site pocket formed by the residues from the beta- and gamma-subunits . The cofactor-inhibitor adduct may be predominantly in the hydrazone with the azo form as a minor component . The binding of the inhibitor causes minor but important conformational changes in the residues surrounding the active site . The inhibitor may have access to the active site pocket through the water-filled crevice between the beta- and gamma-subunits.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 110 - 5
The quinohaemoprotein lupanine hydroxylase from Pseudomonas putida; Hopper DJ et al.; Lupanine hydroxylase catalyses the first reaction in the catabolism of the alkaloid lupanine by Pseudomonas putida . It dehydrogenates the substrate, which can then be hydrated . It is a monomeric protein of M(r) 72,000 and contains a covalently bound haem and a molecule of PQQ . The gene for this enzyme has been cloned and sequenced and the derived protein sequence has a 26 amino acid signal sequence at the N-terminal for translocation of the protein to the periplasm . Many of the features seen in the sequence of lupanine hydroxylase are common with other quinoproteins including the W-motifs that are characteristic of the eight-bladed propeller structure of methanol dehydrogenase . However, the unusual disulfide bridge between adjacent cysteines that is present in some PQQ-containing enzymes is absent in lupanine hydroxylase . The C-terminal domain contains characteristics of a cytochrome c and overall the sequence shows similarities with that of the quinohaemoprotein, alcohol dehydrogenase from Comamonas testosteroni . The gene coding for lupanine hydroxylase has been successfully expressed in Escherichia coli and a procedure has been developed to renature and reactivate the enzyme, which was found to be associated with the inclusion bodies . Reactivation required addition of PQQ and was dependent on calcium ions.

Appl Environ Microbiol, 2003 Apr, 69(4), 2209 - 16
Effect of temperature, pH, and initial cell number on luxCDABE and nah gene expression during naphthalene and salicylate catabolism in the bioreporter organism Pseudomonas putida RB1353; Dorn JG et al.; One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses . For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory . Therefore, this study was conducted to determine what additional factors influence the luminescent response . Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P . putida RB1353 and the maximum degradation rate (V(max)) during salicylate and naphthalene catabolism . Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V(max) during salicylate and naphthalene catabolism, respectively . Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism . Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V(max) during salicylate and naphthalene catabolism . Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude . The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1 degrees C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude . Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products . For example, even though lux gene expression was occurring at approximately 35 degrees C, the luciferase enzyme was inactive . Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 647 - 51
Aerobic degradation of a mixture of azo dyes in a packed bed reactor having bacteria-coated laterite pebbles; Senan RC et al.; A microbial consortium capable of aerobic degradation of a mixture of azo dyes consisting of two isolated strains (RRL,TVM) and one known strain of Pseudomonas putida (MTCC 1194) was immobilized on laterite stones . The amount of bacterial biomass attached to the laterite stones was 8.64 g per 100 g of the stone on a dry weight basis . The packed bed reactor was filled with these stones and had a total capacity of 850 mL and a void volume of 210 mL . The feed consisted of an equal mixture of seven azo dyes both in water as well as in a simulated textile effluent, at a pH of 9.0 and a salinity of 900 mg/L . The dye concentrations of influent were 25, 50, and 100 microg/mL.The residence time was varied between 0.78 and 6.23 h . It was found that at the lowest residence time 23.55, 45.73, and 79.95 microg of dye was degraded per hour at an initial dye concentration of 25, 50, and 100 microg, respectively . The pH was reduced from 9.0 to 7.0 . Simulated textile effluent containing 50 microg/mL dye was degraded by 61.7% . Analysis of degradation products by TLC and HPLC showed that the dye mixture was degraded to nontoxic smaller molecules . The bacteria-coated pebbles were stable, there was no washout even after 2 months, and the reactor was found to be suitable for the aerobic degradation of azo dyes.

J Bacteriol, 2003 Apr, 185(8), 2451 - 6
Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633; McLeish MJ et al.; The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source . The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment . Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon . Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD . All were transcribed in the opposite direction from the genes of the mdlABC operon . Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD) . The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.

Eur J Biochem, 2003 Apr, 270(7), 1567 - 77
Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P . putida 86-1 deltaqor pUF1 and analysis of the Qor proteins; Frerichs-Deeken U et al.; The availability of a system for the functional expression of genes coding for molybdenum hydroxylases is a prerequisite for the construction of enzyme variants by mutagenesis . For the expression cloning of quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86--that contains the molybdopterin cytosine dinucleotide molybdenum cofactor (Mo-MCD), two distinct {2Fe-2S} clusters and FAD--the qorMSL genes were inserted into the broad host range vector, pJB653, generating pUF1 . P . putida KT2440 and P . putida 86-1 deltaqor were used as recipients for pUF1 . Whereas Qor from the wild-type strain showed a specific activity of 19-23 U x mg(-1), the specific activity of Qor purified from P . putida KT2440 pUF1 was only 0.8-2.5 U x mg(-1), and its apparent k(cat) (quinoline) was about ninefold lower than that of wild-type Qor . The apparent Km values for quinoline were similar for both proteins . UV/visible and EPR spectroscopy indicated the presence of the full set of {2Fe-2S} clusters and FAD in Qor from P . putida KT2440 pUF1, however, the very low intensity of the Mo(V)-rapid signal, that occurs in the presence of quinoline, as well as metal analysis indicated a deficiency of the molybdenum center . In contrast, the metal content, and the spectroscopic and catalytic properties of Qor produced by P . putida 86-1 deltaqor pUF1 were essentially like those of wild-type Qor . Release of CMP upon acidic hydrolysis of the Qor proteins suggested the presence of the MCD form of the pyranopterin cofactor; the CMP contents of the three enzymes were similar.

Eur J Biochem, 2003 Apr, 270(7), 1393 - 8
In situ proton NMR analysis of alpha-alkynoate biotransformations . From 'invisible' substrates to detectable metabolites; Brecker L et al.; Only 2% of the known natural products with acetylenic bonds are alpha-alkynoates . Their polarized, conjugated triple bond is an optimal target for an enzymic hydration . Therefore they are good substrates for the enzymes involved in metabolism of acetylenic compounds, resulting in products that are suitable for bacterial growth . We isolated a Pseudomonas putida strain growing on 2-butynedioate as well as on propynoate, and determined the metabolic pathways of these two alpha-alkynoates . The triple bonds in both compounds were initially hydrated and 2-ketobutandioate as well as 3-ketopropanoate were formed . These two beta-keto acids were decarboxylated resulting in pyruvate and acetaldehyde, respectively . Pyruvate was further hydrolysed mainly to acetate and formate, whereas minor amounts were reduced to lactate . In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate . Analyses of these metabolic processes were performed by in situ 1H-NMR spectroscopy in 1H2O, although the substrates, propynoate and 2-butynedioate, carried only one or even no detectable protons, respectively . However, while protons from the solvent are incorporated in the course of the pathway, the metabolites can be detected and identified . Therefore a detailed determination of the metabolic process is possible.

Ann Chim, 2003 Jan-Feb, 93(1-2), 35 - 43
Pseudomonas putida cell biosensor operating in n-hexane to determine benzene in hydrophobic matrices; Campanella L et al.; An immobilised Pseudomonas putida cell based biosensor, able to work directly in organic solvent (n-hexane), was designed and built . The response, in n-hexane, to benzene and some of its derivatives was studied . The proposed biosensor was found to be suitable for determining benzene in hydrophobic matrices.

Microbiology, 2003 Mar, 149(Pt 3), 795 - 805
Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively; Choi EN et al.; Pseudomonas putida F1 can assimilate benzene, toluene and ethylbenzene using the toluene degradation pathway, and can also utilize p-cymene via p-cumate using the p-cymene and p-cumate catabolic pathways . In the present study, P . putida F1 strains were isolated that were adapted to assimilate new substrates such as n-propylbenzene, n-butylbenzene, cumene and biphenyl, and the molecular mechanisms of genetic adaptation to an expanded range of aromatic hydrocarbons were determined . Nucleotide sequence analyses showed that the selected strains have mutations in the cymR gene but not in todF gene . The impairment of the repressor CymR by mutation led to the constitutive expression of CmtE, a meta-cleavage product hydrolase from the cmt operon . This study also showed that CmtE has a broad range of substrates and can hydrolyse meta-cleavage products formed from biphenyl and other new growth substrates via the toluene degradation pathway . However, the artificially constructed strain P . putida F1(cymR : : Tc(r)) and a recombinant P . putida F1, which expressed CmtE constitutively, could not grow on the new substrates . The adapted strains possess the tod operon, which is induced by new growth substrates that are poor inducers of wild-type P . putida F1 . When the todS gene from the adapted strains was introduced in a trans manner to P . putida F1(cymR : : Tc(r)), the resulting recombinant strains were able to grow on biphenyl and other new substrates . This finding indicates that the TodS sensor was altered to recognize these substrates and this conclusion was confirmed by nucleotide sequence analyses . Amino acid substitutions were found in the regions corresponding to the receiver domain and the second PAS domain and their boundaries in the TodS protein . These results showed that P . putida F1 adapted strains capable of growth on n-propylbenzene, n-butylbenzene, cumene and biphenyl possess mutations to employ CmtE and to induce the tod catabolic operon by the new growth substrates.

Environ Toxicol Chem, 2003 Mar, 22(3), 510 - 7
Biodegradation during contaminant transport in porous media: 6 . Impact of sorption on coupled degradation-transport behavior; Famisan GB et al.; Bioavailability is one of the critical factors influencing the biodegradation and bioremediation of organic compounds . The bioavailability of many organic contaminants is controlled in part by the nature, magnitude, and rate of sorption/desorption processes . This study investigates the impact of sorption and associated retardation on the bioavailability and biodegradation of aromatic hydrocarbons during transport in porous media . Miscible-displacement experiments were conducted using naphthalene and 2-naphthol as the model sorbing compounds and salicylate, a degradation product of naphthalene, as a nonsorbing reference compound . Two porous media were used, one (Eustis soil, FL, USA) with moderate sorption capacity and one (quartz sand) with no measurable sorption of the compounds . The porous media were sterilized and inoculated with Pseudomonas putida RB1353, an organism that degrades naphthalene and its derivatives . The biodegradation and transport of all three substrates in quartz sand were significantly influenced by microbial lag, the effects of which were observed within two to three pore volumes (3-4.5 h) . This was also true for salicylate transport in the Eustis soil system . Conversely, biodegradation lag effects were not observed for naphthalene or 2-naphthol in the Eustis soil system . In addition, the masses of naphthalene and 2-naphthol degraded were significantly greater for the Eustis soil system compared to the quartz sand system . As noted previously, naphthalene and 2-naphthol were sorbed by the Eustis soil but not the quartz sand, while salicylate was not sorbed by either media . These results indicate that the increased residence time associated with sorption of naphthalene and 2-naphthol by Eustis soil enhanced overall biodegradation and obviated the impact of lag on observed transport behavior.

Appl Environ Microbiol, 2003 Mar, 69(3), 1372 - 6
Expression of chlorocatechol 1,2-dioxygenase and chlorocatechol 2,3-dioxygenase genes in chlorobenzene-contaminated subsurface samples; Alfreider A et al.; In order to evaluate the in situ degradative capabilities of microorganisms in an underground reactor facility housing two flowthrough columns filled with aquifer soil, we examined the distribution and phylogeny of gene transcripts encoding enzymes capable of catalyzing the cleavage of the chlorinated aromatic ring during transformation of the main pollutant, chlorobenzene . Initial biostimulation of the autochthonous bacteria in the originally anaerobic reactor columns was achieved by injecting nitrate and oxygen in the form of H(2)O(2) . Two broad-range primer pairs were used for reverse transcriptase PCR (RT-PCR) of partial subunit genes of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase from RNA directly extracted from different groundwater and aquifer samples . Samples retrieved from the lowermost sections of the reactor columns, which were operated in upflow mode, were positive for the presence of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase mRNA . On the other hand, chlorocatechol 1,2-dioxygenase RT-PCR products were detected in a larger part of each reactor column, up to a zone 5.5 m above the bottom . Phylogenetic analyses of these chlorocatechol 1,2-dioxygenase sequences clearly separated them into two main clusters, one of which was closely affiliated with the broad-spectrum chlorocatechol 1,2-dioxygenase from Pseudomonas chlororaphis RW71 . Analysis of sequences obtained from RT-PCR products amplified with catechol 2,3-dioxygenase primers revealed that their closest relative was the chlorocatechol 2,3-dioxygenase gene cbzE from Pseudomonas putida GJ31 (A . E . Mars, J . Kingma, S . R . Kaschabek, W . Reineke, and D . B . Janssen, J . Bacteriol . 181:1309-1318, 1999), with sequence similarities between 97.8 and 99.0%.

Can J Microbiol, 2002 Dec, 48(12), 1076 - 81
Control of pyrimidine formation in Pseudomonas putida ATCC 17536; Santiago MF et al.; The regulation of de novo pyrimidine biosynthesis in Pseudomonas putida ATCC 17536 by pyrimidines was explored . The pathway enzyme activities were higher in glucose-grown cells than in succinate-grown cells, indicating catabolite repression by succinate . In P . putida cells grown on succinate as a carbon source, only aspartate transcarbamoylase activity was greatly diminished by uracil supplementation . When glucose was the carbon source, orotic acid supplementation significantly decreased orotate phosphoribosyltransferase and orotidine 5'-monophosphate (OMP) decarboxylase activities . Uracil auxotrophs, deficient for dihydroorotase activity or with reduced phosphoribosyltransferase activity, were isolated . After pyrimidine limitation of both auxotrophs, the greatest derepression of enzyme activity was observed for OMP decarboxylase independent of carbon source . Orotic acid induced both phosphoribosyltransferase and decarboxylase activities in glucose-grown cells of the dihydroorotase-deficient strain . Regulation at the transcriptional level of de novo pyrimidine biosynthetic enzyme synthesis in P . putida ATCC 17536 was observed, which contrasts with previous observations.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 214 - 7
Functions of malonate decarboxylase subunits from Pseudomonas putida; Chohnan S et al.; Malonate decarboxylase from Pseudomonasputida is composed of five subunits, alpha, beta, gamma, delta, and epsilon . Two subunits, delta and epsilon, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively . Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coi formed inclusion bodies . To resolve this problem, we used a coexpression system with GroEL/ES from E . coli, and obtained active recombinant subunits . Enzymatic analysis of the purified recombinant subunits showed that the alpha subunit was an acetyl-S-ACP:malonate ACP transferase and that the betagamma-subunit complex was a malonyl-S-ACP decarboxylase.

Chem Biol Interact, 2003 Feb 1, 143-144, 211 - 8
Crystal structure of glutathione-independent formaldehyde dehydrogenase; Tanaka N et al.; Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase (ADH) family . The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as co-substrate) in typical ADHs, is tightly but not covalently bound to the protein and acts as a cofactor . Such enzymes with tightly bound NAD(P)(H) acting as a cofactor are called nicotinoproteins . The structural basis of the tightly bound cofactor of PFDH is unknown . The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65 A resolution . The 170-kDa-homotetrameric PFDH molecule shows 222-point group symmetry . Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed.

J Bacteriol, 2003 Mar, 185(5), 1730 - 3
Mechanism of cis-trans isomerization of unsaturated fatty acids in Pseudomonas putida; von Wallbrunn A et al.; We studied the pattern of the cis-trans isomerization of unsaturated fatty acids in cells of Pseudomonas putida S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond . Direct evidence that isomerization does not include a transient saturation of the double bond was obtained . In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome c type.

Environ Microbiol, 2003 Mar, 5(3), 163 - 73
Evolution of a chlorobenzene degradative pathway among bacteria in a contaminated groundwater mediated by a genomic island in Ralstonia; Muller TA et al.; The genetic structure of two Ralstonia spp., strain JS705 and strain JS745, isolated from the same groundwater aquifer, was characterized with respect to the degradation capacities for toluene and chlorobenzene degradation . Cosmid library construction, cloning, DNA sequencing and mating experiments indicated that the genes for chlorobenzene degradation in strain JS705 were a mosaic of the clc genes, previously described for Pseudomonas sp . strain B13, and a 5 kb fragment identical to strain JS745 . The 5 kb fragment identical to both JS705 and JS745 was flanked in JS705 by one complete and one incomplete insertion (IS) element . This suggested involvement of the IS element in mobilizing the genes from JS745 to JS705, although insertional activity of the IS element in its present configuration could not be demonstrated . The complete genetic structure for chlorobenzene degradation in strain JS705 resided on a genomic island very similar to the clc element (Ravatn, R., Studer, S., Springael, D., Zehnder, A.J., van der Meer, J.R . 1998 . Chromosomal integration, tandem amplification, and deamplification in Pseudomonas putida F1 of a 105-kilobase genetic element containing the chlorocatechol degradative genes from Pseudomonas sp . strain B13 . J Bacteriol 180: 4360-4369) . The unique reconstruction of formation of a metabolic pathway through the activity of IS elements and a genomic island in the chlorobenzene-degrading strain JS705 demonstrated how pathway evolution can occur under natural conditions in a few 'steps'.

Mol Microbiol, 2003 Feb, 47(4), 993 - 1006
Identification of a novel Gsp-related pathway required for secretion of the manganese-oxidizing factor of Pseudomonas putida strain GB-1; De Vrind J et al.; The manganese-oxidizing factor of Pseudomonas putida strain GB-1 is associated with the outer membrane . One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp) . The gsp genes are called xcp in Pseudomonas species . In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor . In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1) . We show that xcmT1 is part of a two-gene operon that includes an xcpS homologue (designated xcmS) . No other xcp-like genes are present in the regions flanking the xcmT1/xcmS cluster . We also characterized the site of transposon insertion of another transport mutant of P . putida GB-1 . This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin-related protein . This xcmX is clustered with two other xcpT-related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side . The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium . A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P . putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well-described 'regular'xcp/gsp clusters . They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking . The results suggest a novel Xcp-related system for the transport of manganese-oxidizing enzymes to the cell surface.

Ecotoxicol Environ Saf, 2002 Oct, 53(2), 323 - 9
Microbial toxicity tests and chemical analysis as monitoring parameters at composting of creosote-contaminated soil; Ahtiainen J et al.; Traditionally, chemical analyses are used in the assessment of contaminated soil and in monitoring the efficiency of soil remediation processes . We investigated if chemical analysis could be supported and even partly replaced by biological toxicity tests . In two case studies creosote-contaminated soil was composted outdoors in 5- and 100-m3 windrows . Degradation of polyaromatic hydrocarbons (PAHs) was followed by chemical analysis and toxicity tests . Polyaromatic hydrocarbons were quantified and identified by HPLC . Because the soil was also contaminated by copper-, chromium-, and arsenic-containing fungicides, these elements were analyzed by atomic absorption spectrometry . The toxicity of soil samples was assessed by a soil-contact modification of the luminescent bacteria (Vibrio fischeri) test and in the other case also by enzyme synthesis inhibition (Toxi-ChromoPad test, Escherichia coli) . The toxicity of soil water extracts was measured by the standard luminescent bacteria (V . fischeri) test and bacterial (Pseudomonas putida) growth inhibition test . After the first 4 months of the composting period the total amount of PAHs was reduced in all windrows, and in particular, the loss of two- and some three-ring compounds was high, almost 90% . Toxicity decreased concurrently with the decrease in PAH concentration during composting, but after 4 months, one of the piles inoculated with mycobacteria and containing more three- and four-ring compounds was found to be more toxic than at the beginning . After the next summer, total PAH content was further reduced but some four-ring or heavier compounds were demonstrated to be poorly degraded . The toxicity was also reduced to the same level as in the control pile . The total PAH content and the toxicity were both reduced significantly during 5 months of composting.

J Bacteriol, 2003 Feb, 185(4), 1261 - 5
Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor, FpvI; Redly GA et al.; A search of the pvd pyoverdine biosynthesis locus of Pseudomonas aeruginosa identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in Escherichia coli and Pseudomonas putida . Deletion of this gene, dubbed fpvI, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and fpvA gene expression, while the cloned gene stimulated fpvA expression . A Fur-binding site was identified immediately upstream of fpvI, consistent with the observed iron-regulated expression of fpvI and fpvA.

Indian J Exp Biol, 2001 Jun, 39(6), 590 - 3
Removal of copper by Pseudomonas putida strain S4 isolated from copper mines; Saxena D et al.; A bacterial strain, Pseudomonas putida S4, was isolated from smelter drainage of copper mines . The strain exhibited resistance to several heavy metals, like aluminium (Al), zinc (Zn), nickel (Ni), cobalt (Co) besides copper (Cu) . Strain S4 could accumulate Cu from the Cu-supplemented growth medium . In the present study, we have demonstrated the Cu2+ removal capacity of this strain from various samples such as mine effluent, low-grade ore and ore-tailings, collected from the mining site . Moreover, approximately 80% of the accumulated Cu2+ could be recovered from the loaded biomass by a simple desorption procedure.

Environ Microbiol, 2002 Dec, 4(12), 856 - 71
Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida; Greated A et al.; The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas . It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653 . Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames . Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition . All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions . The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins . pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids . In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events . Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified . Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds . The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.

Environ Microbiol, 2002 Dec, 4(12), 842 - 55
Detection of multiple extracytoplasmic function (ECF) sigma factors in the genome of Pseudomonas putida KT2440 and their counterparts in Pseudomonas aeruginosa PA01; Martinez-Bueno MA et al.; Pseudomonas putida KT2440 is highly successful in colonizing a variety habitats, including aquatic and edaphic niches . In accordance with this ability and with the need to adapt to changing environmental conditions, P . putida has developed sophisticated mechanisms of transcriptional regulation . We analysed, at the genome level, the repertoire of sigma factors in P . putida KT2440 and identified 24 sigma factors, 19 of which corresponded to the subfamily of extracytoplasmic function (ECF) sigma factors . We detected 13 ECF sigma factors that showed similarity to the Escherichia coli FecI sigma factor, which is involved in iron acquisition . In 11 cases, a fecR-like gene was found adjacent to the fecI-like gene and, in 10 cases, a gene encoding an iron receptor lies in the vicinity of the fecI/fecR cluster . This may explain the ability of P . putida KT2440 to grow under low iron availability conditions . Five fecI/fecR/iron receptor gene clusters from P . putida were also identified in the human pathogen Pseudomonas aeruginosa.

Environ Microbiol, 2002 Dec, 4(12), 824 - 41
Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440; Jimenez JI et al.; Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes) . Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified . Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P . putida KT2440 . Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P . putida KT2440 . The global view on the mineralization of aromatic compounds by P . putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.

Environ Microbiol, 2002 Dec, 4(12), 819 - 23
The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis; Stjepandic D et al.; As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource . To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations . Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced . This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring co-linearity of the eventual sequence assembly with the actual genome . A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.

Environ Microbiol, 2002 Dec, 4(12), 809 - 18
Global features of the Pseudomonas putida KT2440 genome sequence; Weinel C et al.; The compositional bias of the G+C, di- and tetranucleotide contents in the 6 181 862 bp Pseudomonas putida KT2440 genome was analysed in sliding windows of 4000 bp in steps of 1000 bp . The genome has a low GC skew (mean 0.066) between the leading and lagging strand . The values of GC contents (mean 61.6%) and of dinucleotide relative abundance exhibit skewed Gaussian distributions . The variance of tetranucleotide frequencies, which increases linearly with increasing GC content, shows two overlapping Gaussian distributions of genome sections with low (minor fraction) or high variance (major fraction) . Eighty per cent of the chromosome shares similar GC contents and oligonucleotide bias, but 105 islands of 4000 bp or more show atypical GC contents and/or oligonucleotide signature . Almost all islands provide added value to the metabolic proficiency of P . putida as a saprophytic omnivore . Major features are the uptake and degradation of organic chemicals, ion transport and the synthesis and secretion of secondary metabolites . Other islands endow P . putida with determinants of resistance and defenceor with constituents and appendages of the cell wall . A total of 29 islands carry the signature of mobile elements such as phage, transposons, insertion sequence (IS) elements and group II introns, indicating recent acquisition by horizontal gene transfer . The largest gene carries the most unusual sequence that encodes a multirepeat threonine-rich surface adhesion protein . Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.

Environ Microbiol, 2002 Dec, 4(12), 799 - 808
Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440; Nelson KE et al.; Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes . The bacterium also has considerable potential for biotechnological applications . Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems . Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent . Analysis of the genome gives insight into the non-pathogenic nature of P . putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

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