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 ami