Can J Microbiol, 1996 Feb, 42(2), 177 - 82 The repB gene required for production of extracellular enzymes and fluorescent siderophores in Pseudomonas viridiflava is an analog of the gacA gene of Pseudomonas syringae; Liao CH et al.; Two genes, designated repA and repB, are involved in the regulation of the synthesis of extracellular pectate lyase, protease, and alginate in Pseudomonas viridiflava . The repA gene has been shown to encode a protein highly homologous to several bacterial sensors in the two-component regulator family including the LemA of Pseudomonas syringae . In this study, the repB locus, initially identified in a 6.3-kb EcoRI genomic fragment of P . viridiflava, was further characterized . Results obtained from restriction mapping, deletion subclonings, and mini-Mu-LacZ fusions indicated that the repB gene was contained within a 0.8-kb HindIII-PstI region . Sequence analysis of this repB region revealed the presence of an open reading frame, which was predicted to encode a protein similar or identical to the gacA response regulator found in P . syringae and Pseudomonas fluorescens . The repB gene of P . viridiflava also regulated the production of fluorescent siderophores, in addition to the aforementioned extracellular enzymes and alginate . The repB or gacA homologs were detected in the genomes of nine other strains of P . viridiflava, P . fluorescens, and P . syringae included in the study . The data presented here and earlier indicate that the repA/repB gene regulatory system of P . viridiflava is analogous to the lemA/gacA system of P . syringae and P . fluorescens. Appl Environ Microbiol, 1996 Feb, 62(2), 552 - 63 Conservation of the 2,4-diacetylphloroglucinol biosynthesis locus among fluorescent Pseudomonas strains from diverse geographic locations; Keel C et al.; The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (PHL) is a major determinant in the biological control of a range of plant pathogens by many fluorescent Pseudomonas spp . A 4.8-kb chromosomal DNA region from Pseudomonas fluorescens Q2-87, carrying PHL biosynthetic genes, was used as a probe to determine if the PHL biosynthetic locus is conserved within PHL-producing Pseudomonas strains of worldwide origin . The phl gene probe hybridized with the genomic DNA of all 45 PHL-producing Pseudomonas strains tested, including well-characterized biocontrol strains from the United States and Europe and strains isolated from disease-suppressive soils from Switzerland, Washington, Italy, and Ghana . The PHL producers displayed considerable phenotypic and genotypic diversity . Two phenotypically distinct groups were detected . The first produced PHL, pyoluteorin, and hydrogen cyanide and consisted of 13 strains from almost all locations sampled in the United States, Europe, and Africa . The second produced only PHL and HCN and consisted of 32 strains from the U.S . and European soils . Analysis of restriction patterns of genomic DNA obtained after hybridization with the phl gene probe and cluster analysis of restriction patterns of amplified DNA coding for 16S rRNA (ARDRA) and randomly amplified polymorphic DNA (RAPD) markers indicated that the strains that produced both PHL and pyoluteorin were genetically highly similar . In contrast, there was more diversity at the genotypic level in the strains that produced PHL but not pyoluteorin . ARDRA analysis of these strains indicated two clusters which, on the basis of RAPD analysis, split into several subgroups with additional polymorphisms . In general, the occurrence of phenotypically and genotypically similar groups of PHL producers did not correlate with the geographic origin of the isolates, and highly similar strains could be isolated from diverse locations worldwide. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 251 - 7 Construction of a modified mini-Tn5 lacZY non-antibiotic marker cassette: ecological evaluation of a lacZY marked Pseudomonas strain in the sugarbeet rhizosphere; Fedi S et al.; In order to monitor the fate of genetically manipulated fluorescent pseudomonads following release into the environment, a lacZY transposable cassette, lacking antibiotic resistance genes, was constructed using a pUT suicide plasmid delivery system . The resulting plasmid, pUTLacZY, can be easily used to generate lacZY marked pseudomonads without having to use antibiotic resistance determinants . The lacZY transposon generates random, stable transcriptional/translational fusions on integration into the target genome . Pseudomonas fluorescens strain F113 was marked with lacZY and was unaltered with respect to ecological fitness in the rhizosphere . Although lateral gene transfer of the chromosomally integrated lacZY marker could be detected in vitro, it was not detected in rhizosphere microcosms. Microbios, 1996, 88(357), 205 - 12 Drug resistance in Detroit River gram-negative bacilli; Campeau RC et al.; Detroit River Gram-negative bacilli were examined for resistance to agents of interest to public health . The total recoverable population and the lactose-fermenting organisms existed at approximately 10(5) and 10(2) colony forming units per litre, respectively . Lactose-nonfermenting and lactose-fermenting isolates demonstrated resistance to six and four of nine antimicrobial agents, respectively, when tested by a paper disc procedure . Multiple resistance in lactose-nonfermenting organisms included up to five agents . Lactose-fermenting isolates produced multiple resistance to two antibiotics . Only 7% of antibiotic resistance strains were proven to contain plasmids . Biochemical testing indicated that the most common group of resistant bacteria was Pseudomonas fluorescens . Comparison of protein profiles produced by polyacrylamide gel electrophoresis indicated that there was variation between P . fluorescens strains demonstrating the same multiple resistance. J Clin Lab Anal, 1996, 10(4), 167 - 76 Optimization studies of components in enzymatic cholesterol reagents containing cholesterol oxidase from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens; Lolekha PH et al.; Although enzymatic methods for serum cholesterol determination are widely used in clinical laboratories, little is known about the optimization of each component in enzymatic reagents . We investigated the optimal components in the reagents containing cholesterol oxidase isolated from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens . The optimal components in the reagents are: cholesterol oxidase 250 (Nocardia erythropolis), 250 (Streptomyces sp), or 300 (Pseudomonas fluorescens) U/L, cholesterol esterase 200 U/L, peroxidase 10,000 U/L, sodium cholate 3 mmol/L, 4-aminoantipyrine 0.5 mmol/L, phenol 20 mmol/L, Triton X-100 2 mL/L, and phosphate buffer, pH 7.0 . Lower reaction sensitivity and lower cholesterol linearity, < 18.1 mmol/L (700 mg/dL), could be obtained by using lower components than those suggested above . Pseudomonas fluorescens were an improper source for cholesterol oxidase; either Nocardia erythropolis or Streptomyces was suitable cholesterol oxidase . We prefer using Streptomyces sp cholesterol oxidase because of its economical cost and longest reagent stability . Sodium cholate must be included in the enzymatic reagent to prevent turbidity . However, sodium cholate of > 5 mmol/ L will suppress the reaction resulting in low cholesterol linearity. Anal Chem, 1996 Jan 1, 68(1), 192 - 8 Measurements of oxidoreductase-like activity of intact bacterial cells by an amperometric method using a membrane-coated electrode; Ikeda T et al.; The oxidation of D-glucose and nicotinic acid by intact cells of Gluconobacter industrious and Pseudomonas fluorescens, respectively, is successfully measured by an amperometric method using such compounds as Fe(CN)6(3-), p-benzoquinone, and dichlorophenolindophenol as electron acceptors . Analysis of the experimental results reveals that the intact cells behave like oxidoreductases whose kinetics follows a Michaelis-Mententype equation . The catalytic behavior is explained by a model which treats the bacterial cells as bags of enzymes and assumes distribution equilibrium in the concentrations of both the substrate and the electron acceptor between the test solution and the medium within the cells . The catalytic activity can be characterized by three quantities: the maximum reaction rate (vB) and the ratios of the Michaelis constant to the distribution constant for the substrate (Ks,cell/Ks,p) and to that for the electron acceptor (KM,cell/KM,p) . Advanced modification of the model to involve the membrane permeability reveals that the three quantities are effective for explaining the catalytic behavior even when the permeability effect is significant . Thus, the three quantities should be regarded as the parameters which can reflect the permeability effect. Appl Environ Microbiol, 1996 Jan, 62(1), 94 - 9 Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF; Lister DL et al.; A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment . The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF) . Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other . C . acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate . C . acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester . P . fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen . P . fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A . Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine . Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates. Appl Environ Microbiol, 1996 Jan, 62(1), 121 - 7 Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST; Marconi AM et al.; A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340 . Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene . Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment . Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol . The three genes appear to be clustered and are probably encoded by the same DNA strand . In E . coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter. Appl Environ Microbiol, 1996 Jan, 62(1), 100 - 4 Pseudomonas fluorescens adhesion and transport through porous media are affected by lipopolysaccharide composition; Williams V et al.; The objectives of this work were (i) to use transposon mutagenesis to produce mutants of Pseudomonas fluorescens that were altered in adhesion ability and transport through porous media and (ii) to identify the alterations in surface characteristics that were responsible for the changes in attachment . Mutants of P . fluorescens were generated with TnphoA, which enabled identification of mutants that were altered in surface proteins . Transposon mutants were screened for alterations in adhesion ability by attachment assays on hydrophobic polystyrene and water-wettable polystyrene . Four TnphoA mutants with increased adhesion to the hydrophobic surface and decreased adhesion to the water-wettable surface were obtained . Transport of the strains through porous media was evaluated by passing suspensions of each mutant and the parent through columns containing quartz sand and determining the number of cells retained in the columns . The mutants all demonstrated increased adhesion and retention in the columns . Southern analysis demonstrated two types of mutants with separate transposon insertion sites . Polyacrylamide gel electrophoresis of the strains demonstrated that the O antigen on the lipopolysaccharide was either attenuated or absent . Lack of this polysaccharide, and the consequent increased exposure of the lipid moiety of the lipopolysaccharide, is probably responsible for the increase in adhesion to the hydrophobic substrata and retention in the sand column . This work combined with previous studies of attachment of P . fluorescens demonstrates that more than one type of polymer can mediate the adhesion of this organism to nonbiological surfaces. Gene, 1995 Dec 29, 167(1-2), 339 - 40 Sequence of the Leptospira biflexa serovar patoc recA gene; Stamm LV et al.; The nucleotide (nt) sequence of the recA gene of Leptospira biflexa serovar patoc strain Patoc I has been determined . The deduced amino acid (aa) sequence of the RecA protein is 387 aa long with a predicted molecular mass of 42,355 Da . The aa sequence has a high degree of identity to the aa sequences of many bacterial RecA, including Pseudomonas fluorescens, Escherichia coli and Bacillus subtilis . This is the first recA sequence reported for a bacterium in the order Spirochaetales. Carbohydr Res, 1995 Dec 27, 279, 215 - 26 Structures of decasaccharide and tridecasaccharide tetraphosphates isolated by strong alkaline degradation of O-deacylated lipopolysaccharide of Pseudomonas fluorescens strain ATCC 49271; Knirel YA et al.; Mild hydrazinolysis of Pseudomonas fluorescens strain ATCC 49271 lipopolysaccharide (LPS) followed by strong alkaline degradation and purification by anion-exchange HPLC resulted in two phosphorylated oligosaccharides (1 and 2) . On the basis of compositional analysis and 1H, 13C, and 31P NMR spectroscopy, including 2D correlation spectroscopy (COSY), 2D rotating frame NOE spectroscopy (ROESY), and 2D inverse mode H-detected heteronuclear 1H-13C and 1H-31P correlation spectroscopy, the following two structures (1 and 2) could be identified {formula: see text} where Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-octulosonic acid, Non is 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonulosonic acid, and P is phosphate . Decasaccharide 1 and tridecasaccharide 2 represent an incomplete core and the complete core carrying one O-antigen repeating unit, respectively . Both are attached to the lipid A backbone but, due to their degradation protocol, they lack N- and O-acyl substituents, including N- and O-acetyl groups, the 5-N-acetimidoyl group of Non, the 2-N-alanyl group of GalN, and the 7-O-carbamoyl group of Hep as well as diphosphate, triphosphate, and, probably, some of the monophosphate groups that are present in the intact core oligosaccharide. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12255 - 9 The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5; Sarniguet A et al.; Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol . A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s . Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5 . These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration . A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf-5 . The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum . When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings . Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P . fluorescens . These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P . fluorescens on plant surfaces. J Biol Chem, 1995 Dec 8, 270(49), 29314 - 22 The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain; Fanutti C et al.; Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Piromyces . XYLA and MANA displayed properties typical of endo-beta 1,4-xylanases and mannanases, respectively . Neither enzyme hydrolyzed cellulosic substrates . The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818 base pairs, respectively, coding for proteins of M(r) 68,049 (XYLA) and 68,055 (MANA) . The deduced primary structure of MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp . cellulosa mannanases belonging to glycosyl hydrolase Family 26 . A 40-residue reiterated sequence, which was homologous to duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was located at the C terminus of MANA . XYLA contained two regions that exhibited sequence identity with the catalytic domains of glycosyl hydrolase Family 11 xylanases and were separated by a duplicated 40-residue sequence that exhibited strong homology to the C terminus of MANA . Analysis of truncated derivatives of MANA confirmed that the N-terminal 458-residue sequence constituted the catalytic domain, while the C-terminal domain was not essential for the retention of catalytic activity . Similar deletion analysis of XYLA showed that the C-terminal catalytic domain homologue exhibited catalytic activity, but the corresponding putative N-terminal catalytic domain did not function as a xylanase . Fusion of the reiterated noncatalytic 40-residue sequence conserved in XYLA and MANA to glutathione S-transferase, generated a hybrid protein that did not associate with cellulose, but bound to 97- and 116-kDa polypeptides that are components of the multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively . The role of this domain in the assembly of the enzyme complex is discussed. Poult Sci, 1995 Dec, 74(12), 2041 - 7 Spoilage bacteria of fresh broiler chicken carcasses; Russell SM et al.; Studies were conducted to identify the bacteria responsible for spoilage of fresh broiler chicken carcasses and to characterize the off-odors these bacteria produce . Broiler carcasses were collected from processing plants in the northeast Georgia area, the southeastern U.S., Arkansas, California, and North Carolina . The carcasses were allowed to spoil under controlled conditions at 3 C and spoilage bacteria were isolated . Each spoilage bacterium was separately inoculated into a sterile chicken skin medium, incubated at 25 C for 48 h, and subjectively evaluated for odor . The bacteria isolated from spoiled carcasses that consistently produced off-odors in the chicken skin medium, regardless of the geographical location from which the chickens were obtained, were Shewanella putrefaciens A, B, and D, Pseudomonas fluorescens A, B, and D, and Pseudomonas fragi . These bacteria produced off-odors that resembled "sulfur", "dishrag", "ammonia", "wet dog", "skunk", "dirty socks", "rancid fish", "unspecified bad odor", or a sweet smell resembling "canned corn" . Odors produced by the spoilage bacteria were varied; however, odors most associated with spoiled poultry, such as "dishraggy" odors, were produced by the bacteria that were most consistently isolated, such as S . putrefaciens and the pseudomonads. Biosci Biotechnol Biochem, 1995 Dec, 59(12), 2303 - 4 Pseudomonas fluorescens KKL101, a benzoic acid degrader in a mixed culture that degrades biphenyl and polychlorinated biphenyls; Kikuchi Y et al.; A mixed culture we had isolated, which degrades biphenyl/polychlorinated biphenyls, is composed of two strains, Pseudomonas fluorescens KKL101 and Pseudomonas sp . strain KKS102 . KKS102 produces benzoic acid as a dead-end metabolite in the degradation of biphenyl . In this study we showed that KKL101 grew on benzoic acid as a sole source of carbon . This indicated a role of KKS102 in the growth of KKL101 and KKL101 for the growth of KKS102 in the mixed culture. Mol Ecol, 1995 Dec, 4(6), 755 - 63 Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer; Bailey MJ et al.; A plasmid-free, non-pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes . By site directed homologous recombination a KX cassette {kanamycin resistance (kanr) and catechol 2,3 dioxygenase (xylE)} and a ZY cassette {lactose utilization (lacZY, beta-galactosidase, lactose permease)} were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome . Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro-organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation. Appl Environ Microbiol, 1995 Dec, 61(12), 4209 - 14 Bioconversion of 2,4-diamino-6-nitrotoluene to a novel metabolite under anoxic and aerobic conditions; Gilcrease PC et al.; Under nitrate-reducing, nongrowth conditions, a Pseudomonas fluorescens species reduced 2,4,6-trinitrotoluene to aminodinitrotoluenes, which were then further reduced to diaminonitrotoluenes . 2,4-Diamino-6-nitrotoluene (2,4-DANT) was further transformed to a novel metabolite, 4-N-acetylamino-2-amino-6-nitrotoluene (4-N-AcANT), while 2,6-diamino-4-nitrotoluene (2,6-DANT) was persistent . Efforts to further degrade 2,4-DANT and 2,6-DANT under aerobic, nitrogen-limited conditions were unsuccessful; 2,6-DANT remained persistent, and 2,4-DANT was again transformed to the 4-N-AcANT compound. Appl Environ Microbiol, 1995 Dec, 61(12), 4202 - 8 Survival of, and induced stress resistance in, carbon-starved Pseudomonas fluorescens cells residing in soil; van Overbeek LS et al.; We investigated the survival, cell length, and development of general stress resistance in populations of Pseudomonas fluorescens R2f and its rifampin-resistant mutant, R2f Rpr, following exposure to carbon starvation conditions in liquid cultures and residence in two different soils, Flevo silt loam (FSL) and Ede loamy sand (ELS) . In much the same way as was recently shown for P . putida KT2442, carbon-starved P . fluorescens R2f populations revealed enhanced resistance to otherwise lethal treatments, such as exposure to ethanol, high temperature, osmotic tension, and oxidative stress . A large population of nonculturable P . fluorescens R2f Rpr cells arose shortly after their introduction into ELS soil, whereas the formation of nonculturable cells was not observed in FSL soil . Also, the inoculant cell (based on immunofluorescence) and CFU counts decreased faster in ELS soil than in FSL soil . Introduction of carbon-starved instead of exponential-growth-phase R2f Rpr cells into ELS soil did not affect bacterial survival . The inoculant cell length decreased in soil, and no large differences in cell length in the two soil types were observed . Addition of glucose to ELS soil resulted in a stable cell length of R2f Rpr cells, whereas carbon-starved cells introduced into ELS soil remained small . Exponentially growing R2f Rpr cells developed enhanced resistance to ethanol, high temperature, osmotic tension, and oxidative stress within 1 day in both soils, whereas cells introduced into ELS soil amended with glucose showed decreased resistance . Cells that were carbon starved prior to introduction into ELS soil showed unchanged stress resistance levels upon residence in soil. Eur J Biochem, 1995 Nov 15, 234(1), 225 - 30 The purification and properties of phosphonoacetate hydrolase, a novel carbon-phosphorus bond-cleavage enzyme from Pseudomonas fluorescens 23F; McGrath JW et al.; A novel, inducible, carbon-phosphorus bond-cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown on phosphonoacetate . The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa . Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 37 degrees C, respectively . The purified enzyme had an apparent Km of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3-phosphonopropionate and phosphonoformate . The NH2-terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences. Biochem J, 1995 Nov 15, 312 ( Pt 1), 39 - 48 Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens subsp . cellulosa and Cellvibrio mixtus; Millward-Sadler SJ et al.; To test the hypothesis that selective pressure has led to the retention of cellulose-binding domains (CBDs) by hemicellulase enzymes from aerobic bacteria, four new xylanase (xyn) genes from two cellulolytic soil bacteria, Pseudomonas fluorescens subsp . cellulosa and Cellvibrio mixtus, have been isolated and sequenced . Pseudomonas genes xynE and xynF encoded modular xylanases (XYLE and XYLF) with predicted M(r) values of 68,600 and 65000 respectively . XYLE contained a glycosyl hydrolase family 11 catalytic domain at its N-terminus, followed by three other domains; the second of these exhibited sequence identity with NodB from rhizobia . The C-terminal domain (40 residues) exhibited significant sequence identity with a non-catalytic domain of previously unknown function, conserved in all the cellulases and one of the hemicellulases previously characterized from the pseudomonad, and was shown to function as a CBD when fused to the reporter protein glutathione-S-transferase . XYLF contained a C-terminal glycosyl hydrolase family 10 catalytic domain and a novel CBD at its N-terminus . C . mixtus genes xynA and xynB exhibited substantial sequence identity with xynE and xynF respectively, and encoded modular xylanases with the same molecular architecture and, by inference, the same functional properties . In the absence of extensive cross-hybridization between other multiple cel (cellulase) and xyn genes from P . fluorescens subsp . cellulosa and genomic DNA from C . mixtus, similarity between the two pairs of xylanases may indicate a recent transfer of genes between the two bacteria. Transfusion, 1995 Nov-Dec, 35(11), 911 - 6 Bacteria levels in components prepared from deliberately inoculated whole blood held for 8 or 24 hours at 20 to 24 degrees C; Wagner SJ et al.; BACKGROUND: An increase from 8 to 24 hours in the time that units of whole blood can be held at room temperature after phlebotomy would give blood centers more flexibility in component manufacturing and might allow receipt of many infectious disease test results prior to component preparation . However, the potential for bacterial growth during prolonged holding periods requires further study . STUDY DESIGN AND METHODS: In the Phase I study, 2-unit pools of ABO-identical whole blood were deliberately inoculated on Day 0 with Staphylococcus aureus or Pseudomonas fluorescens . They were then divided in half and stored at 20 to 24 degrees C . Red cells (RBCs) with additive solution, platelet concentrates (PCs), and frozen plasma were prepared after 8 and 24 hours . Bacteria levels in PCs and RBCs were monitored on Day 1; bacteria levels were measured in plasma after thawing . In the Phase II study, the same basic design as in Phase I was used, except that 10 bacterial species were studied, lower inocula were used, and RBCs prepared after a 24-hour room-temperature whole-blood hold were white cell-reduced by filtration . Bacterial growth was monitored during 42-day storage of RBCs (1 - 6 degrees C) and 5-day storage of PCs (20 - 24 degrees C) and after thawing of frozen plasma . RESULTS: For Phase I, significantly higher bacteria levels were observed in RBCs prepared after a prolonged hold (p < 0.05); higher levels were not observed in PCs and thawed plasma units . In Phase II, prior to white cell reduction by filtration, 8 of 10 organisms had significantly higher levels in RBCs prepared after a 24-hour hold than in RBCs prepared after an 8-hour hold, when both were examined on Day 1 (p < 0.05) . For seven of eight organisms examined on Days 1, 21, and 42, filtration (white cell reduction) reduced the bacteria in RBCs prepared from 24-hour whole blood units to those levels found in unfiltered RBCs prepared from whole blood units held at 8 hours . A prolongation of the holding time from 8 to 24 hours resulted in significantly lower bacteria levels (p < 0.05) in PCs early in storage (Days 1, 1 - 2, or 1 - 3) for seven organisms, with no significant difference for two organisms, and a small but significant increase for one organism (Day 3, p < 0.05) . There was no difference in bacteria or endotoxin levels in thawed units of plasma prepared from whole blood after 8- or 24-hour holding times . CONCLUSION: The levels of bacteria present in components after deliberately inoculated whole blood units are held for 8 and 24 hours depended on the organisms tested, the whole-blood holding period, and the blood component assayed; for RBCs, they also depended on whether WBC reduction by filtration was performed. J Bacteriol, 1995 Nov, 177(21), 6230 - 6 A global regulator of secondary metabolite production in Pseudomonas fluorescens Pf-5; Corbell N et al.; Mutations in the apdA (for antibiotic production) gene of the plant root-colonizing bacterium Pseudomonas fluorescens Pf-5 pleiotropically abolish the production of an array of antibiotics, including pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol, as well as the production of tryptophan side chain oxidase, hydrogen cyanide, and an extracellular protease . The lack of production of secondary metabolites by ApdA- mutants was correlated with the loss of inhibition of the phytopathogenic fungus Rhizoctonia solani in culture . Sequencing of the apdA region identified an open reading frame of 2,751 bp . The predicted amino acid sequence of the apdA gene contains conserved domains of the histidine kinases that serve as sensor components of prokaryotic two-component regulatory systems . The apdA nucleotide and predicted amino acid sequences are strikingly similar to the sequences of lemA and repA, genes encoding putative sensor kinases that are required for the pathogenicity of Pseudomonas syringae pv . syringae and Pseudomonas viridiflava, respectively . Introduction of the cloned apdA+ gene restored the wild-type phenotype to both LemA- mutants of P . syringae and ApdA- mutants of Pf-5 . The 101-kDa ApdA protein reacted with an anti-LemA antiserum, further demonstrating the similarity of ApdA to LemA . These results show that apdA encodes a putative sensor kinase component of a classical two-component regulatory system that is required for secondary-metabolite production by P . fluorescens Pf-5. J Appl Bacteriol, 1995 Oct, 79(4), 368 - 78 Some factors affecting the airborne survival of bacteria outdoors; Handley BA et al.; Airborne survival of two pseudomonads and a reference strain of Escherichia coli (strain MRE 162) was studied outdoors using a modified microthread technique . When cells of E . coli were suspended as clusters, survival was much greater than single cells, particularly outdoors . Culture age had a highly significant effect on survival of Pseudomonas maltophila with survival of 24 h cultures being more than 100-fold higher than 48 h cultures . Survival of Pseudomonas fluorescens was variable and depended also upon the method of culture . Survival of E . coli and Ps . maltophila was studied at three locations differing in air quality and was found to be significantly reduced outdoors, particularly when held in direct daylight . Outdoor survival was not significantly different at the three locations but was reduced at increasing temperatures . There was no apparent effect of wind direction or air quality . Results are discussed with reference to the release of genetically-modified micro-organisms. Mol Ecol, 1995 Oct, 4(5), 593 - 603 The phylogenetic distribution of a transposable dioxygenase from the Niagara River watershed; Nakatsu CH et al.; Horizontal gene transfer in the Bacteria has been demonstrated to occur under natural conditions . The ecological impact of gene transfer events depends on the new genetic material being expressed in recipient organisms, and on natural selection processes operating on these recipients . The phylogenetic distribution of cbaAB genes for chlorobenzoate 3,4-(4,5)-dioxygenase, which are carried within Tn5271 on the IncP beta plasmid pBRC60, was investigated using isolates from freshwater microcosms and from the Niagara River watershed . The latter included isolates from surface water, groundwater and bioremediation reactor samples . The cbaAB genes have become integrated, through interspecific transfer, primarily into species of the beta Proteobacteria (44/48 isolates) . Only four isolates, identified as Pseudomonas fluorescens (3/48) and Xanthomonas maltophilia (1/48), belonged to the gamma Proteobacteria, despite the observation that pBRC60 was capable of mobilizing these genes into a wide range of beta and gamma Proteobacteria in the laboratory . The natural host range correlated with the distribution of the meta-ring-fission pathway for metabolism of protocatechuates formed when the cbaAB genes were expressed (45/48 isolates) . We proposed the hypothesis that natural selection has favoured recipients that successfully integrate the activity of the transferred dioxygenase with the conserved meta ring-fission pathway . The hypothesis was tested by transferring a plasmid construct containing the cbaAB genes into type strains representative of the beta and gamma Proteobacteria . The concept of applying mobile catabolic genes to probe the phylogenetic distribution of compatible degradative pathways is discussed. Appl Microbiol Biotechnol, 1995 Oct, 43(5), 794 - 800 Liquid-culture pH, temperature, and carbon (not nitrogen) source regulate phenazine productivity of the take-all biocontrol agent Pseudomonas fluorescens 2-79; Slininger PJ et al.; Strain 2-79 is a biocontrol agent against take-all, an important disease of wheat caused by Gaeumannomyces graminis var . tritici . In the rhizosphere, it produces the antibiotic phenazine 1-carboxylic acid (PCA) as the primary means of disease suppression . One barrier to commercial use of phenazine-producing pseudomonads, like strain 2-79, is the lack of liquid-culture technology for mass production . For instance, there is little published research concerning the impact of liquid-culture secondary metabolism on the biocontrol qualities of the cell harvest, i.e., efficacy, phytotoxicity, and storage survival . Yet it is important to know whether the fermentation process should be designed to enhance or eliminate secondary metabolite accumulation . To enable future exploration of this issue, we identified liquid-culture parameters that could be manipulated to control the phenazine productivity of strain 2-79 . Our results indicated that PCA accumulation was very sensitive to the culture pH and temperature . It was possible to produce large cell populations with either high or low phenazine productivity by choosing to control culture pH at 7 and 8 respectively . Although high cell accumulations were achieved over the broad 25-34 degrees C range studied, high, moderate, or low PCA productivities were observed at 25-27 degrees C, 29-32.5 degrees C, or 34 degrees C respectively . When pH was controlled at 7, specific PCA productions at 25 degrees C could be modulated by the choice of carbon source supplied . PCA accumulation per unit biomass reached 0.31 g/g on glucose, 0.16 g/g on glycerol and xylose, and only 0.09 g/g on fructose.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1995 Sep, 177(18), 5387 - 92 Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities; Schnider U et al.; Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi . The rpoD gene encoding the housekeeping sigma factor sigma 70 of P . fluorescens was sequenced . The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli . Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene . When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0. Chest, 1995 Sep, 108(3), 636 - 41 Machine operator's lung . A hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols; Bernstein DI et al.; Six auto parts manufacturing workers were referred for evaluation of a 6-week history of work-related dyspnea, cough, and fatigue . Two workers also reported fever and weight loss . All six worked in a machining area where a waterbased metalworking fluid was used and recirculated under high pressure, thereby creating an aerosol . Chest radiographs revealed pulmonary interstitial infiltrates in four workers . Lung function tests showed that four workers had decreased diffusing capacity . After removal from the work area, all workers recovered . The metalworking fluid was cultured for bacteria and fungi . Isolates from broth cultures were sonicated to obtain antigen extracts . Serum precipitins to one or more of the microbial isolates were identified in all six workers but not in eight of nine nonexposed control subjects . The most frequent precipitin response (six of six workers) was against antigens of Pseudomonas fluorescens, which was cultured from the metalworking fluid . In all workers, precipitins to at least one other cultured organism were detected; these included Aspergillus niger, Staphylococcus capitas, an acid-fast Rhodococcus sp, and Bacillus pumilus . This represents the first report of hypersensitivity pneumonitis associated with industrial exposure to aerosolized metalworking fluid . Observed precipitin responses to a variety of microbial contaminants in metalworking fluid strongly suggest a causative role for microbial antigens in the induction and elicitation of this manifestation of hypersensitivity pneumonitis. Appl Environ Microbiol, 1995 Sep, 61(9), 3347 - 52 Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay; Jacobsen CS; A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA . A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads . After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components . The MCH was followed by PCR amplification of the specific target DNA . In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg) . This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root . The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined. Biochem J, 1995 Aug 1, 309 ( Pt 3), 749 - 56 The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp . cellulosa is important for the efficient hydrolysis of Avicel; Hall J et al.; A genomic library of Pseudomonas fluorescens subsp . cellulosa DNA, constructed in lambda ZAPII, was screened for carboxymethyl-cellulase activity . The pseudomonad insert from a recombinant phage which displayed elevated cellulase activity in comparison with other cellulase-positive clones present in the library, was excised into pBluescript SK- to generate the plasmid pC48 . The nucleotide sequence of the cellulase gene, designated celE, revealed a single open reading frame of 1710 bp that encoded a polypeptide, defined as endoglucanase E (CelE), of M(r) 59663 . The deduced primary structure of CelE revealed an N-terminal signal peptide followed by a 300-amino-acid sequence that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase Family 5 . Adjacent to the catalytic domain was a 40-residue region that exhibited strong sequence identity to non-catalytic domains located in two other endoglucanases and a xylanase from P . fluorescens . The C-terminal 100 residues of CelE were similar to Type-I cellulose-binding domains (CBDs) . The three domains of the cellulase were joined by linker sequences rich in serine residues . Analysis of the biochemical properties of full-length and truncated derivatives of CelE confirmed that the enzyme comprised an N-terminal catalytic domain and a C-terminal CBD . Analysis of purified CelE revealed that the enzyme had an M(r) of 56000 and an experimentally determined N-terminal sequence identical to residues 40-54 of the deduced primary structure of full-length CelE . The enzyme exhibited an endo mode of action in hydrolysing a range of cellulosic substrates including Avicel and acid-swollen cellulose, but did not attack xylan or any other hemicelluloses . A truncated form of the enzyme, which lacked the C-terminal CBD, displayed the same activity as full-length CelE against soluble cellulose and acid-swollen cellulose, but exhibited substantially lower activity than the full-length cellulase against Avicel . The significance of these data in relation to the role of the CBD is discussed. J Clin Pathol, 1995 Aug, 48(8), 717 - 8 Rate of growth of Pseudomonas fluorescens in donated blood; Gibb AP et al.; AIMS--To examine how delayed refrigeration of blood affects the growth of Pseudomonas fluorescens, one of the two most important causes of sepsis resulting from transfusion of contaminated blood . METHODS--Two donations of whole blood were each divided into three aliquots and inoculated with 5-10 cfu/ml of a P fluorescens strain from a case of transfusion associated sepsis . From each donation, one aliquot was placed at 4 degrees C, one was held at 20 degrees C for six hours prior to refrigeration and the third was held at 20 degrees C for 24 hours prior to refrigeration . Samples were aseptically withdrawn over 17 days and bacterial counts were determined using a pour plate technique . RESULTS--The rate of growth of P fluorescens in blood at 20 degrees C was increased compared with blood at 4 degrees C . At 24 hours the aliquots held at 20 degrees C for six and 24 hours had, respectively, 174 and 29,000 cfu/ml compared with 15 cfu/ml in aliquots held at 4 degrees C . There was no evidence of increased killing of P fluorescens at the higher temperature . CONCLUSIONS--These results suggest that blood for transfusion should be refrigerated as soon as possible after collection. Biochim Biophys Acta, 1995 Jul 19, 1250(2), 149 - 57 A catalytic triad is required by the non-heme haloperoxidases to perform halogenation; Pelletier I et al.; The bacterial non-heme haloperoxidases are highly related to an esterase from Pseudomonas fluorescens, at structural and functional levels . Both types of enzymes displayed brominating activity and esterase activity . The presence of the serine-hydrolase motif Gly-X-Ser-X-Gly, in the esterase as well as in all aligned haloperoxidase sequences, strongly suggested that they belong to the serine-hydrolase family . Sequence alignment with several serine-hydrolases and secondary structure superimposition revealed the striking conservation of structural features characterising the alpha/beta-hydrolase fold structure in all haloperoxidases . These structural predictions allowed us to identify a potential catalytic triad in haloperoxidases, perfectly matching the triad of all aligned serine-hydrolases . The structurally equivalent triad in the chloroperoxidase CPO-P comprised the amino acids Serine 97, Aspartic acid 229 and Histidine 258 . The involvement of this catalytic triad in halogenation was further assessed by inhibition studies and site-directed mutagenesis . Inactivation of CPO-P by PMSF and DEPC strongly suggested that the serine residue from the serine-hydrolase motif and an histidine residue are essential for halogenation, similar to that demonstrated for typical serine-hydrolases . By site-directed mutagenesis of CPO-P, Ser-97 was exchanged against alanine or cysteine, Asp-229 against alanine and His-258 against glutamine . Western blot analysis indicated that each mutant gene was efficiently expressed . Whereas the mutant S97C conserved a very low residual activity, each other mutant S97A, D229A or H258Q was totally inactive . This study gives the direct demonstration of the requirement of a catalytic triad in the halogenation mechanism. J Lipid Res, 1995 Jun, 36(6), 1334 - 44 Lipoprotein lipase enhances removal of chylomicrons and chylomicron remnants by the perfused rat liver; Skottova N et al.; Lipoprotein lipase has been found to efficiently mediate binding of lipoproteins to cell surfaces and to the low density lipoprotein (LDL) receptor-related protein (LRP) under cell culture conditions (Beisiegel et al . 1991 . Proc . Natl . Acad . Sci . USA . 88: 8242-8346) . This supports the previously proposed idea that the lipase could have a role in receptor-mediated uptake of chylomicron remnants in the liver . We have investigated the effects of lipoprotein lipase on the clearance of chylomicrons during perfusions of rat livers . The chylomicrons were doubly labeled in vivo with {14C}retinol (in retinyl esters) and with {3H}oleic acid (in triacylglycerols) and were collected from lymph . In the absence of any lipase the clearance of chylomicron label from the perfusion medium was slow . Addition of lipoprotein lipase caused lipolysis of chylomicron triacylglycerols as evidenced by increased levels of 14C-labeled fatty acids in the perfusate . Simultaneously, the level of {14C}retinyl esters in the perfusate decreased dramatically, indicating core-particle removal . Similar effects were seen with an unrelated lipase from Pseudomonas fluorescens . To discriminate between the effects of lipolysis and a true liganding effect of the lipoprotein lipase protein, the active site inhibitors tetrahydrolipstatinR and hexadecylsulfonylfluoride were used to reduce or totally inhibit the catalytical activity . With lipase covalently inhibited by the latter inhibitor, lipolysis during perfusions was low or absent . Nonetheless, the inhibited enzyme had a clear effect on the removal of chylomicrons by the liver . With 1.2 micrograms of inhibited lipase/ml perfusate, about 70% of the core label had been removed after 15 min as compared to about 20% in perfusions without lipase . With identical amounts of active lipoprotein lipase protein, more than 90% of the label was removed . We conclude that any lipase causing lipolysis of chylomicrons can stimulate their clearance by the liver, but that lipoprotein lipase has an additional effect on the removal, which is not dependent on its catalytic activity. FEBS Lett, 1995 Apr 10, 362(3), 281 - 5 Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven; Jenkins J et al.; Comparison of the recently determined crystal structures Pseudomonas fluorescens subsp . cellulosa family F xylanase, (1-3)-beta-glucanase and (1-3,1-4)-beta-glucanase and the catalytic domain of E . coli beta-galactosidase reveals that they belong to a superfamily of 8-fold beta/alpha-barrels with similar amino acid residues at their active sites . In the three families that these enzymes represent, the nucleophile is a glutamate, which is located close to the carboxy-terminus of beta-strand seven . In addition all three enzymes have the sequence asparagine-glutamate close to the carboxy-terminus of beta-strand four . This glutamate has been identified as the acid/base in the family F xylanases and is essential for catalysis in beta-galactosidase . We suggest that the equivalent residue in the barley glucanases is the acid/base . Analysis of the sequences of family 1 beta-glucosidases and family 5 cellulases shows that these enzymes also belong to this superfamily which we call the 4/7 superfamily. J Wildl Dis, 1995 Apr, 31(2), 166 - 71 Association of Cytophaga psychrophila with mortality among eyed eggs of Atlantic salmon (Salmo salar); Cipriano RC et al.; Although Pseudomonas fluorescens was the predominant bacterium associated with Atlantic salmon (Salmo salar) eggs incubated at the White River National Fish Hatchery (Bethel, Vermont) during January 1992, the fish pathogen Cytophaga psychrophila was isolated only from specific lots of eggs that displayed poor survival (35% eye-up). Appl Environ Microbiol, 1995 Apr, 61(4), 1232 - 9 Effect of impact stress on microbial recovery on an agar surface; Stewart SL et al.; Microbial stress due to the impaction of microorganisms onto an agar collection surface was studied experimentally . The relative recovery rates of aerosolized Pseudomonas fluorescens and Micrococcus luteus were determined as a function of the impaction velocity by using a moving agar slide impactor operating over a flow rate range from 3.8 to 40 liters/min yielding impaction velocities from 24 to 250 m/s . As a reference, the sixth stage of the Andersen Six-Stage Viable Particle Sizing Sampler was used at its operating flow rate of 28.3 liters/min (24 m/s) . At a collection efficiency of close to 100% for the agar slide impactor, an increase in sampling flow rate and, therefore, in impaction velocity produced a significant decline in the percentage of microorganisms recovered . Conversely, when the collection efficiency was less than 100%, greater recovery and lower injury rates occurred . The highest relative rate of recovery (approximately 51% for P . fluorescens and approximately 62% for M . luteus) was obtained on the complete (Trypticase soy agar) medium at 40 and 24 m/s (6.4 and 3.8 liters/min), respectively . M . luteus demonstrated less damage than P . fluorescens, suggesting the hardy nature of the gram-positive strain versus that of the gram-negative microorganism . Comparison of results from the agar slide and Andersen impactors at the same sampling velocity showed that recovery and injury due to collection depends not only on the magnitude of the impaction velocity but also on the degree to which the microorganisms may be embedded in the collection medium.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1995 Apr, 177(8), 1989 - 93 Mechanisms of biodegradation of metal-citrate complexes by Pseudomonas fluorescens; Joshi-Tope G et al.; Biodegradation of metal-citrate complexes by Pseudomonas fluorescens depends on the nature of the complex formed between the metal and citric acid . Bidentate Fe(III)-, Ni-, and Zn-citrate complexes were readily biodegraded, but the tridentate Cd- and Cu-citrate, and U-citrate complexes were not . The biodegradation of Ni- and Zn-citrate commenced after an initial lag period; the former showed only partial (70%) degradation, whereas the latter was completely degraded . Uptake studies with 14C-labeled citric acid and metal-citrate complexes showed that cells grown in medium containing citric acid transported free citric acid at the rate of 28 nmol min-1 and Fe(III)-citrate at the rate of 12.6 nmol min-1 but not Cd-, Cu-, Ni-, U-, and Zn-citrate complexes . However, cells grown in medium containing Ni- or Zn-citrate transported both Ni- and Zn-citrate, suggesting the involvement of a common, inducible transport factor . Cell extracts degraded Fe(III)-, Ni-, U-, and Zn-citrate complexes in the following order: The cell extract did not degrade Cd- or Cu-citrate complexes . These results show that the biodegradation of the U-citrate complex was limited by the lack of transport inside the cell and that the tridentate Cd- and Cu-citrate complexes were neither transported inside the cell nor metabolized by the bacterium. FEMS Microbiol Lett, 1995 Apr 1, 127(3), 267 - 72 Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3; De E et al.; The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl-polyoxyethylene media . Both proteins, characterized by the same size, heat-modifiability and N-terminal sequence were re-incorporated in virtually solvent-free planar lipid bilayers . They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in 1 M NaCl . From experiments of zero-current potential, both porins were determined weakly cation selective . Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3). Appl Environ Microbiol, 1995 Apr, 61(4), 1384 - 90 Application of a strain-specific rRNA oligonucleotide probe targeting Pseudomonas fluorescens Ag1 in a mesocosm study of bacterial release into the environment; Boye M et al.; Sequence analysis of domains 3 and 4 of 23S rRNA from Pseudomonas fluorescens Ag1 was carried out to allow the design of a strain-specific rRNA oligonucleotide probe targeting this strain . The specificity of the probe, Ps-Ag1, was assessed by dot blot analysis and whole-cell hybridization, and it was found to be specific for P . fluorescens Ag1 . The correlation between the ribosomal content of P . fluorescens Ag1 and growth rate was determined during balanced growth conditions with generation times ranging from 1.2 to 31.8 h . Hybridization of the rRNA-targeting probes combined with charged coupled device-enhanced microscopy was used to determine the rRNA content . The total RNA content per cell was determined by staining with acridine orange and charged coupled device-enhanced microscopy . After 2 h under carbon starvation conditions, the rRNA content per cell decreased to 45% of the content of an exponentially growing cell . After 1 day of carbon starvation, the rRNA content had decreased to 20% . When cells were grown at different temperatures, it was found that the rRNA content per cell was only dependent on the substrate in the temperature range from 5 to 30 degrees C . P . fluorescens Ag1 was used in a mesocosm release experiment . The strain could be detected by use of the oligonucleotide probe targeting rRNA for 8 days in the water column and for 10 days on solid surfaces . The standard curve correlating growth rate with rRNA content was used to estimate the physiological activity of P . fluorescens Ag1 in the mesocosm experiment. Biosci Biotechnol Biochem, 1995 Mar, 59(3), 408 - 11 D-glucosaminate aldolase activity of D-glucosaminate dehydratase from Pseudomonas fluorescens and its requirement for Mn2+ ion; Iwamoto R et al.; When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A) . This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-D-gluconate and ammonia: alpha,beta-elimination reaction, B) . The ratio of the activities (A:B) was about 1:4 . However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3-4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB . The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn2+ + pyridoxal 5'-phosphate (PLP)) . When the EDTA-treated enzyme was incubated with (Mn2+ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21% . These results suggested that Mn2+, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme . In the case of the aldolase reaction, Mn2+ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction. Anal Biochem, 1995 Mar 1, 225(2), 286 - 90 Determination of gamma-aminobutyric acid levels in human cerebrospinal fluid using Pseudomonas; Nicholson-Guthrie CS et al.; The principle objective was to demonstrate the efficacy of a bacterial, radioligand, competitive binding method in determining gamma-aminobutyric acid (GABA) levels in human cerebrospinal fluid (CSF) . In a double blind study, CSF GABA concentrations were measured by the bacterial method using a mutant of Pseudomonas fluorescens and by a standard radioligand competitive binding assay using rat brain membranes . Linear regression analysis demonstrated a highly significant correlation (r = 0.84; P < 0.001) between the two methods . In an ancillary study, a similar correlation (r = 0.90; P < 0.001) was found between the bacterial method and HPLC, another standard procedure in determining CSF GABA levels . Furthermore, the mean CSF GABA measurements found with the bacterial method were in agreement with those reported in the literature using the brain membrane method or HPLC . The bacterial method was highly reproducible and reliable with a detection to 5 nM GABA . It was specific for GABA and was not affected by other naturally occurring compounds which are found in higher concentrations in CSF than GABA or which were suspected to interfere with the neurotransmitter in a binding assay . The bacterial radioligand method was shown to be an accurate, sensitive, and rapid assay for CSF GABA. Anal Biochem, 1995 Mar 1, 225(2), 283 - 5 Bacterial assay for quantitative measurement of nanomolar concentrations of a metabolite; Guthrie GD et al.; A mutant of Pseudomonas fluorescens was used to develop a radioligand, competitive binding assay to quantitatively measure gamma-aminobutyric acid . The highly reliable and reproducible assay was sensitive (nM detection), rapid, and easy to perform . Nonspecific activity and scatter were insignificant . Radiolabel was irreversibly fixed by the cells in an energy-dependent reaction . The finding that a bacterium was effective in quantitatively detecting nanomolar amounts of a metabolite suggests that other bacteria or their mutants might be used in competitive binding assays to detect and quantify amino acids or other substances occurring in trace amounts. J Appl Bacteriol, 1995 Mar, 78(3), 216 - 33 Degradation of triglycerides by a pseudomonad isolated from milk: the roles of lipase and esterase studied using recombinant strains over-producing, or specifically deficient in these enzymes; McKay DB et al.; The roles of lipase and esterase in causing hydrolytic spoilage of milk by a highly lipolytic psychrotrophic strain of Pseudomonas fluorescens, LS107d2, has been studied . Strains of LS107d2 have been constructed that over-produce, or are specifically deficient in, a lipase (encoded by lipA) and an esterase (encoded by estA) . Southern blot analysis reveals that LS107d2 contains only one esterase and one lipase (encoded by estA and lipA) and this was confirmed by the phenotypes of mutants on triolein and tributyrin-containing agar . Analysis of broth cultures showed that the lipase is secreted into the culture medium; in contrast, the esterase is not secreted . Free fatty acid (FFA) levels in whole milk cultures of wild-type, over-producing and the mutant strains of LS107d2 have been examined . From these studies it is concluded that esterase is not involved in the accumulation of FFA by hydrolysing short chain fatty acid esters; that the highly lipolytic phenotype of LS107d2 is due solely to a single secreted lipase; and that the main FFA accumulated in milk cultures of LS107d2 are C4, C16, C18 and C18: 1 . Evidence is also presented demonstrating that FFA degradation, as well as production, determines the level of FFA in milk contaminated with lipolytic organisms. Biochim Biophys Acta, 1995 Feb 23, 1243(2), 265 - 9 Specificity of an esterase (XYLD) from Pseudomonas fluorescens subsp . cellulosa; Faulds CB et al.; Activity of an esterase from Pseudomonas fluorescens subsp . cellulosa (XYLD) on an insoluble feruloylated hemicellulose substrate (de-starched wheat bran) was dependent on the source of added endo-xylanase . The esterase exhibited high selectivity for the nature, position of linkage and size of the feruloylated oligosaccharides generated by hydrolysis of the hemicellulose . Increased affinity of XYLD with increasing size of the oligosaccharide substrate suggests that optimal activity is observed on substrates with at least 4 sugars. Biochem J, 1995 Feb 1, 305 ( Pt 3), 1005 - 10 A non-modular endo-beta-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa; Braithwaite KL et al.; Pseudomonas fluorescens subsp . cellulosa when cultured in the presence of carob galactomannan degraded the polysaccharide . To isolate gene(s) from P . fluorescens subsp . cellulosa encoding endo-beta-1,4-mannanase (mannanase) activity, a genomic library of Pseudomonas DNA, constructed in lambda ZAPII, was screened for mannanase-expressing clones using the dye-labelled substrate, azo-carob galactomannan . The nucleotide sequence of the pseudomonad insert from a mannanase-positive clone revealed a single open reading frame of 1257 bp encoding a protein of M(r) 46,938 . The deduced N-terminal sequence of the putative polypeptide conformed to a typical prokaryotic signal peptide . Truncated derivatives of the mannanase, lacking 54 and 16 residues from the N- and C-terminus respectively of the mature form of the enzyme, did not exhibit catalytic activity . Inspection of the primary structure of the mannanase did not reveal any obvious linker sequences or protein motifs characteristic of the non-catalytic domains located in other Pseudomonas plant cell wall hydrolases . These data indicate that the mannanase is non-modulator, comprising a single catalytic domain . Comparison of the mannanase sequence with those in the SWISSPROT database revealed greatest sequence homology with the mannanase from Bacillus sp . Thus the Pseudomonas enzyme belongs to glycosyl hydrolase Family 26, a family containing mannanases and endoglucanases . Analysis of the substrate specificity of the mannanase showed that the enzyme hydrolysed mannan and galactomannan, but displayed little activity towards other polysaccharides located in the plant cell wall . The enzyme had a pH optimum of approx . 7.0, was resistant to proteolysis and had an M(r) of 46,000 when expressed by Escherichia coli. Microbiology, 1995 Feb, 141 ( Pt 2), 459 - 68 A bacterial esterase is homologous with non-haem haloperoxidases and displays brominating activity; Pelletier I et al.; Screening GenBank indicated that an esterase from Pseudomonas fluorescens had high sequence similarity with bacterial non-haem haloperoxides . However, this homology was limited to two distinct domains of the published esterase sequence . As errors in the published sequence were suspected, the esterase gene was sequenced again . The revised sequence displayed between 40 and 50% identical amino acids with the haloperoxidases, but distributed along the whole sequence . In addition to the structural homologies with haloperoxidases, the esterase also displayed functional homology . The recombinant esterase, purified from Escherichia coli cells, was capable of both ester hydrolysis and halogenation, as detected in situ by the formation of bromophenol blue or spectrophotometrically by the bromination of monochlorodimedon . The esterase is thus a bifunctional enzyme . The sequence analysis and the biochemical investigations show that the esterase belongs to the haloperoxidase family . It also possessed, however, a typical feature of serine-hydrolases, namely the consensus motif Gly-X-Ser-X-Gly around the active serine of the catalytic triad . By alignment of the esterase with different serine-hydrolase sequences, it was possible to identify the other two residues of the triad . The triad comprised the residues Ser95, Asp223 and His252 . Interestingly, a structurally equivalent catalytic triad was also identified in the sequences of all bacterial non-haem haloperoxidases, in highly conserved domains . The presence of a catalytic triad in haloperoxidases is expected to be important in the mechanism of halogenation. Eur J Epidemiol, 1995 Feb, 11(1), 1 - 7 Pollution of modern metalworking fluids containing biocides by pathogenic bacteria in France . Reexamination of chemical treatments accuracy; Chazal PM; Pollution by pathogenic bacteria was examined in 150 French metalworking fluid samples . Gram-negative micro-organisms such as Salmonella spp., Shigella spp., and Vibrio spp . as well as Gram-positive cocci were never isolated . Nevertheless opportunistic pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae still contaminated these fluids with an isolation frequency of 17% of samples for each . These two micro-organisms failed to grow or even survive in vitro in sterile cutting fluids protected by biocides . Preliminary growth of other micro-organisms such as Pseudomonas putida or Pseudomonas fluorescens, which are the major part of the indigenous microflora, seemed to be a prerequisite for their growth . These former two Pseudomonas could resist three different classes of biocides and, at least in the case of formaldehyde-releasers, adaptation was followed by biocide deterioration . Resistance magnification was observed in the presence of the three different types of biocides and, in the case of formaldehyde releasers the resistance and deterioration levels were close to those recommended by the manufacturers . This is probably the reason why the preliminary growth of Pseudomonas putida allowed in vitro differed growth of Klebsiella pneumoniae and Pseudomonas aeruginosa . Due to relatively high isolation frequencies of opportunistic pathogens (17% of samples) periodical microbiological examination of cutting fluids should be carried out in order to evaluate risks for human health . Wearing masks and gloves is still recommended, at least in France. J Enzyme Inhib, 1995, 9(4), 309 - 16 Chemical modification of bacterial 4-aminobutyrate aminotransferase by phenylglyoxal; Tunnicliff G; 4-Aminobutyrate aminotransferase (EC 2.6.1.19), obtained from Pseudomonas fluorescens, was irreversibly inhibited by phenylglyoxal, a reagent that specifically modifies arginyl residues . The inactivation appeared to be the result of a simple, bimolecular reaction since no evidence of a reversible complex between inhibitor and enzyme emerged . The second-order rate constant was 0.221 +/- 0.077 M-1 sec-1 . The concentration of either substrate had no effect on the inhibition, but an increase in the concentration of pyridoxal 5'-phosphate reduced the rate of inactivation by phenylglyoxal . The data are consistent with the modification of amino acid residues at the cofactor binding site on the enzyme. Teratog Carcinog Mutagen, 1995, 15(3), 103 - 8 Evaluation of cytogenetic effects of a naturally occurring non-ice-nucleation Pseudomonas fluorescens strain in Chinese hamster ovary (CHO) cells; Caruso P et al.; One of the main methods for eliminating ice-nucleation-active (INA+) bacteria the micro-organisms responsible for frost injuries to plants at mild freezing temperatures, is the use, as competitors, of other naturally occurring non-nucleating strains (non-INA) . In the present article we investigated the cytogenetic effects of a naturally occurring non-INA strain of Pseudomonas fluorescens (MS 1640 R3), evaluating the induction of chromosomal aberrations and sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells in the absence and presence of rat S9 metabolism . The results obtained did not show any increase in either chromosomal aberrations or SCEs, both in the absence and presence of rat S9 metabolism when used as i) intact bacteria cells, ii) sonicated bacteria (i.e., potential endotoxins), or iii) metabolic bacterial products (i.e., potential exotoxins) released in the growth medium. Appl Environ Microbiol, 1995 Jan, 61(1), 397 - 8 Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces; Eriksson PV et al.; An inhibition enzyme-linked immunosorbent assay was developed for Pseudomonas fluorescens enumeration of meat surfaces . The assay detected contamination levels as low as 3 x 10(5) bacteria per ml and could be completed within 4 h . It could be used as a framework for a test system for quantifying P . fluorescens spoilage in meat products. J Appl Bacteriol, 1995 Jan, 78(1), 71 - 81 A numerical taxonomic study of fluorescent Pseudomonas strains isolated from natural mineral waters; Elomari M et al.; Forty-six strains of fluorescent pseudomonads, isolated from natural mineral waters, together with 12 strains from clinical material and 44 reference strains, were phenotypically classified by 281 characteristics . The data were processed by the Dice similarity coefficient and unweighted pair group algorithm with arithmetic averages . Eight clusters were defined at the 62% similarity level . Clusters I, II and IV were further divided into nine subclusters . Virtually all the mineral water strains fall into three groups: Ib (eight strains), IIa (14 strains) and V (16 strains) . Subclusters Ib and IIa included natural mineral water strains only . Cluster V contained 13 mineral water strains and three culture collection strains of Pseudomonas fluorescens biovar III . DNA/DNA hybridization studies are needed to define the taxonomic status of these three groups within the genus Pseudomonas. Rev Latinoam Microbiol, 1995 Jan-Mar, 37(1), 1 - 6 {Pseudomonas fluorescens: production of pyoverdine in human blood at 4 degrees C and cytotoxic effect of the pigment}; Pajaro MC et al.; Pseudomonas fluorescens PAB strain produced pyoverdine in a synthetic medium, this pigment was purified by solvent extraction and ion exchange, then sterilized by filtration . Where cytotoxic effect on human leukocytes was assayed, death and lysis was observed . Sublytic doses decreased leukocytes phagocytosis and chemotaxis . Bacteria grew and produced pigment in blood stored a 4 degrees C, with a pyoverdine production of 0.13 mg/ml of serum after 5 days of incubation. Antonie Van Leeuwenhoek, 1995, 67(2), 201 - 10 Cloning of genes required for hypersensitivity and pathogenicity in Pseudomonas syringae pv . aptata; Minardi P; A genomic library of Pseudomonas syringae pv . aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31 . The 13.4 kb EcoRI fragment of the cosmid pHIR11, containing the hrp (hypersensitive response and pathogenicity) gene cluster of the closely related bacterium Pseudomonas syringae pv . syringae strain 61, was used as a probe to identify a homologous hrp gene cluster in P . syringae pv . aptata . Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium, Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco . Southern blot analysis of EcoRI-digested genomic DNA of P . syringae pv . aptata showed hybridizing bands of 12 kb and 4.4 kb . Only a 12 kb fragment hybridized in digests of the cosmids . Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome of P . syringae pv . aptata . Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce . The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kb Bg/II fragment of pHIR11 . These results indicate that P . syringae pv . aptata harbours hrp genes that are similar to, but arranged differently from, homologous hrp genes of P . syringae pv . syringae. Mol Microbiol, 1995 Jan, 15(2), 297 - 306 Iron-responsive gene expression in Pseudomonas fluorescens M114: cloning and characterization of a transcription-activating factor, PbrA; Sexton R et al.; In response to iron limitation . Pseudomonas fluorescens M114 induces a number of genes including an iron-scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease . A Tn5lacZ-induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron-regulated genes . A cosmid clone was identified which complements this mutation . This clone is capable of activating a number of iron-regulated promoter fusion constructs from P . fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli . A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions . DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseudobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the sigma 70 sigma factor family, including fecl required for expression of the ferric dicitrate outer-membrane receptor protein of E . coli . Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated. Mikrobiol Z, 1995 Jan-Feb, 57(1), 95 - 101 {The destruction of mono- and polycyclic aromatic hydrocarbons by cultures of Pseudomonas fluorescens 1-D biovar II and Bacillus subtilis 2-D}; Dumans'ka TU; Cenosis of microorganisms, coal resin destructors, is selected by the percolation method . A microbiological analysis of bacterial associations able to destruction of aromatic hydrocarbons composing the coal resin is carried out . Pure cultures are isolated as the most active destructors of these substrates . Destructive cultures are identified as Pseudomonas fluorescens 1-D biovar II and Bacillus subtilis 2-D . Efficiency of the microbiological refinement of coal-resin-contaminated soil by the isolated pure cultures and by their mixture is compared with self-refinement of soil . It is shown that complete refinement of soil from coal resin with contamination of 1 g per 1 kg of soil was attained after 160 days using the mixture of isolated destructive cultures P . fluorescens 1-D biovar II and B . subtilis 2-D . Usage of only P . fluorescens 1-D biovar II provided destruction of 87% of contaminating substances for the same period, whereas self-purification of soil by the natural cenosis of microorganisms made the purification level of 42%. Can J Microbiol, 1995, 41 Suppl 1, 170 - 9 Substrate specificities of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13; Schirmer A et al.; The isolation of poly(3-hydroxyoctanoic acid)- and poly(6-hydroxyhexanoic acid)-degrading bacteria yielded 28 strains with abilities to degrade various polymers . The most versatile strains hydrolyzed five different polyesters comprising short chain length and medium chain length poly(hydroxyalkanoates) . The new isolates together with previously isolated poly(hydroxyalkanoate)-degrading bacteria were classified into 11 groups with respect to their polymer-degrading specificities . All PHA depolymerases studied so far have been characterized by the lipase consensus sequence Gly-X-Ser-X-Gly in their amino acid sequence, which is a known sequence for serine hydrolases . When we replaced the central residue, Ser-172, in the corresponding sequence Gly-Ile-Ser-Ser-Gly of the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, with alanine the enzyme lost its activity completely . This result of the mutational experiment indicates that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases. Gene, 1994 Dec 2, 150(1), 199 - 200 Sequence of the cobA gene encoding S-adenosyl-L-methionine: uroporhyrinogen III methyltransferase of Pseudomonas fluorescens; De Mot R et al.; Sequence analysis of the region downstream of oprF, from Pseudomonas fluorescens OE 28.3, revealed the presence of cobA homologue encoding a putative S-adenosyl-L-methionine: uroporhyrinogen III methyltransferase . A similar gene organization exists in P . aeruginosa. J Bacteriol, 1994 Dec, 176(24), 7468 - 75 Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae; Rich JJ et al.; Mutational analysis of the bean-pathogenic Pseudomonas syringae pv . syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase . The analysis of a collection of spontaneous mutants of P . syringae pv . syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin . The gacA gene originally was identified as a regulator of extracellular antibiotic production by Pseudomonas fluorescens, and the predicted GacA protein is a member of the FixJ family of bacterial response regulators . The sequence of the putative B728a GacA protein revealed 92% identity with the P . fluorescens GacA protein . An insertional mutation within the P . syringae pv . syringae gacA gene abrogated lesion formation on beans, production of extracellular protease, and production of the toxin syringomycin, the same phenotypes affected by a lemA mutation . DNA sequence analysis identified the P . syringae pv . syringae uvrC gene immediately downstream of the gacA gene, an arrangement conserved in P . fluorescens and Escherichia coli . The gacA insertional mutant was sensitive to UV, presumably because of polarity on transcription of the downstream uvrC gene . Southwestern (DNA-protein) analysis revealed that the lemA and gacA genes were required for the full expression of a DNA binding activity. Protein Sci, 1994 Dec, 3(12), 2245 - 53 Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin; van Berkel WJ et al.; The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts . Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds . Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution . The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD . The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes . Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2) . The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1) . The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme . Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized . It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity. FEMS Microbiol Lett, 1994 Nov 15, 124(1), 23 - 7 Ethylbenzene degradation by Pseudomonas fluorescens strain CA-4; Corkery DM et al.; Pseudomonas fluorescens strain CA-4 is a bioreactor isolate capable of ethylbenzene degradation . Transposon mutagenesis and enzyme assays have been performed which allow us to propose the ethylbenzene degradative pathway in operation in this strain . Ethylbenzene is initially converted to 2-phenylethanol . This is degraded to phenylacetaldehyde and then to phenylacetic acid . The major inducer of the pathway is ethylbenzene itself . The pathway is regulated by the presence of non-aromatic carbon sources . Oxidation of ethylbenzene is repressed by glutamate, but not by citrate or glucose . A clone from a chromosomal library has been found to complement a mutant deficient in the ability to convert ethylbenzene to 2-phenylethanol. Structure, 1994 Nov 15, 2(11), 1107 - 16 Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites; Harris GW et al.; BACKGROUND: Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G . Family F enzymes show no sequence similarity with any known structure and their architecture is unknown . Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases . RESULTS: We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp . cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20 . The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine . As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal . We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base . Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E . Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates . CONCLUSIONS: The architecture of P . fluorescens subsp . cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel . Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates. J Bacteriol, 1994 Nov, 176(22), 7065 - 73 Molecular characterization of the extracellular poly(3-hydroxyoctanoic acid) {P(3HO)} depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product; Schirmer A et al.; phaZPfi, the gene encoding the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, was cloned, sequenced, and characterized . It comprises 837 bp and is transcribed as a monocistronic message of about 950 bp from a putative sigma 70-like promoter 32 bp upstream of the ATG start codon . The deduced protein of 278 amino acids reveals a typical leader peptide at its N terminus . When expressed in Escherichia coli, the mature depolymerase started with Ala-23, whereas the mature enzyme purified from P . fluorescens GK13 started with both Leu-34 and Arg-35 determining proteins of 26,687 and 26,573 Da, respectively . The depolymerase is a strongly hydrophobic protein and includes the lipase consensus sequence Gly-X-Ser-X-Gly, which is known for serine hydrolases . Replacement of the central residue, Ser-172, in the corresponding sequence (Gly-Ile-Ser-Ser-Gly) of PhaZPfl with alanine resulted in complete loss of enzyme activity, indicating that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases. Microbiology, 1994 Nov, 140 ( Pt 11), 3125 - 30 Growth temperature regulates the induction of beta-lactamase in Pseudomonas fluorescens through modulation of the outer membrane permeation of a beta-lactam-inducing antibiotic; Orange N; The psychrotrophic bacterium, Pseudomonas fluorescens strain MFO, is more sensitive to the beta-lactam mezlocillin at a low growth temperature (i.e . 8 degrees C) than at a higher growth temperature (28 degrees C) . An early effect of this antibiotic at all temperatures is bacterial filamentation, but this occurs later at 8 degrees C than at 28 degrees C, which suggests a lower permeability of the cell envelopes to mezlocillin at low growth temperature . beta-Lactamase production is later induced by mezlocillin, but the level of this induction also depends on the growth temperature, the overall induction being much less efficient at 8 degrees C . It is hypothesized that the periplasmic concentration of the drug might be too low at 8 degrees C to allow efficient beta-lactamase induction; this hypothesis was confirmed by the demonstration that beta-lactamase production is drastically enhanced in cells cultivated at 8 degrees C permeabilized for 10 min by Na-EDTA . In addition, induction kinetic curves display a marked dependence upon growth temperature . A rapid saturation was evident when mezlocillin concentrations were increased at 8 degrees C; this was not seen at 28 degrees C at up to 1000 micrograms mezlocillin ml-1 . The results are discussed in terms of two different routes of drug permeation, depending on the growth temperature. Biosci Biotechnol Biochem, 1994 Nov, 58(11), 2099 - 101 DNA sequence of proline permease gene from Pseudomonas fluorescens and predicted structure of proline permease; Hosoya H et al.; The proline permease gene of Pseudomonas fluorescens has been isolated from the promoter region isolated by using a promoter probe (transposon Tn5-B21) . By DNA sequencing of 2222 bp the primary structure of permease (494 aa) was deduced . The DNA sequence is 71.0% and 70.7% identical and the amino acid sequence is 75.8% and 76.0% similar to those of Escherichia coli and Salmonella typhimurium, respectively. Mol Ecol, 1994 Oct, 3(5), 479 - 87 Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA; Laguerre G et al.; A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described . Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized . Amplified rDNA was digested with 13 different restriction endonucleases . The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes . All type strains belonging to different species were differentiated . The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level. J Bacteriol, 1994 Oct, 176(19), 5912 - 8 Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens; Huang Z et al.; A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670 . The enzyme requires no cofactors and contains no prosthetic groups . Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution . The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid . This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction . The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity . Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity. Int J Food Microbiol, 1994 Oct, 23(2), 215 - 9 Malate and glucose in milk incubated with psychrotrophic bacteria; Matias P et al.; L-Malate consumption by the natural microflora of milk and a strain of Pseudomonas fluorescens was followed during storage of milk at 7 degrees C . Use of milk malate by somatic cells from mastitic milk was investigated and found to be insignificant . L-Malate seems to be a potential indicator for the growth of psychrotrophic bacteria in refrigerated milk at levels between 10(5) and 10(7) CFU/ml. Ecotoxicol Environ Saf, 1994 Oct, 29(1), 86 - 92 Effect of mixtures of chlorophenols, surfactants, and aniline on growth of Pseudomonas fluorescens; Torslov J et al.; Industrial wastewater often contains a mixture of chemical substances and knowledge of joint action of toxicants is therefore important when the toxicity of an effluent is evaluated or reduction of the toxicity is needed . In this study, the joint action of three pairs of toxicants, selected on the basis of their expected different modes of toxic actions, was tested for inhibition of the growth of the bacteria Pseudomonas fluorescens: pentachlorophenol and aniline, the surfactants nonylphenolethoxylate and tetrapropylenebenzenesulfonate, and pentachlorophenol and 2, 4-dichlorophenol . The joint effect of pentachlorophenol and aniline did not differ significantly from additivity, whereas less than additive responses were observed for mixtures of nonylphenolethoxylate and tetrapropylenbenzenesulfonate . A more than additive response was observed for mixtures of pentachlorophenol and 2,4-dichlorophenol at some concentration levels, while at others additive responses were found . It was concluded that joint actions other than additivity may occur between commonly known toxic substances, and that the modes of toxic action of the substances studied can explain the different types of joint action observed in this study . Further, test strategy followed proved useful in the evaluation of the joint toxicity of binary mixtures. J Biol Chem, 1994 Sep 30, 269(39), 24304 - 9 Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli . A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase; Yanagisawa T et al.; To elucidate the mode of action of pseudomonic acid, we have compared the deduced amino acid sequences of isoleucyl-tRNA synthetases (ILeRS) from wild-type Escherichia coli strain MC4100, a pseudomonic acid-resistant mutant (strain PS102) of MC4100, and a pseudomonic acid-producing strain, Pseudomonas fluorescens . Compared with the wild-type enzyme, the deduced amino acid sequence of E . coli mutant ileS gene in strain PS102 shows a single amino acid substitution of leucine for phenylalanine at residue 594 of the IleRS . This mutational alteration in IleRS of an E . coli pseudomonic acid-resistant mutant resides in a region of the enzyme in close proximity to one of the consensus sequences of class I aminoacyl-tRNA synthetases, the KMSKS sequence between residues 602 and 606 of the E . coli IleRS . DNA sequence of the cloned ileS gene predicts that the P . fluorescens IleRS consists of 943 amino acids with 54% identity with the E . coli IleRS . The P . fluorescens ileS gene and the wild type and PS102 alleles of E . coli ileS were cloned into an expression vector, pEXPCR, and the sensitivities of E . coli DH5 alpha cells harboring each of these plasmids were compared . The cells harboring the P . fluorescens ileS were found to be most resistant to pseudomonic acid, while the transformants expressing the PS102 IleRS were more resistant than those containing the wild-type E . coli IleRS . IleRS purified from the wild-type E . coli was specifically cleaved by trypsin between Lys605 and Ser606 in the region of K602MSKS606 . The protection of the IleRS from the trypsin digestion was found with pseudomonic acid or ATP, but not with isoleucine or tRNA(1Ile) . Based on these results, we propose that pseudomonic acid binds to IleRS in the vicinity of the KMSKS sequence that is an ATP-binding subsite, and that pseudomonic acid is a bifunctional inhibitor with characteristics of both isoleucine and ATP, for example, an analog of isoleucyladenylate. Gene, 1994 Sep 15, 147(1), 81 - 3 The chitinase encoding Tn7-based chiA gene endows Pseudomonas fluorescens with the capacity to control plant pathogens in soil; Koby S et al.; A transposon system based on Tn7 carrying the chiA gene (encoding chitinase) of Serratia marcescens was established . The transposon was introduced by conjugation into Pseudomonas fluorescens as a stable integrate expressing and secreting the active chitinase enzyme . The resulting strain was shown to control the plant pathogen Rhizoctonia solani in soil. FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 13 - 8 Lipase and acidic phosphatase from the psychrotrophic bacterium Pseudomonas fluorescens: two enzymes whose synthesis is regulated by the growth temperature; Burini JF et al.; In the psychrotrophic bacterium Pseudomonas fluorescens, the production of several enzymes that otherwise differ in their cell location, growth phase production and inducibility appeared to be similarly regulated by the growth temperature . In order to determine the level of this regulation, the expression of the apo and lip genes encoding two of these enzymes, the acidic phosphatase and lipase, respectively, was studied at different temperatures . Both genes were optimally expressed at 17.5 degrees C, i.e., at the optimal temperature of production of the enzymes; however, the low level of activity at the highest temperature could be due to an additional post-transcriptional control. Eur J Biochem, 1994 Sep 15, 224(3), 845 - 52 Purification and properties of zinc-metallophospholipase C from Pseudomonas fluorescens; Crevel I et al.; Phospholipase C produced by Pseudomonas fluorescens, isolated as a laboratory contaminant, has been purified to apparent homogeneity by ammonium sulphate fractionation, anion-exchange and size-exclusion chromatographies . The apparent molecular mass of the purified polypeptide was 39.5 kDa . Purified preparations of phospholipase C were used to characterize its enzymic properties and to obtain amino acid sequence of the N-terminus of the molecule . The P . fluorescens phospholipase C hydrolysed PtdEtn, PtdCho and PtdSer (PtdEtn > PtdCho >> PtdSer) and was relatively thermostable . The enzyme was inactivated in the presence of chelating agent o-phenanthroline and the activity restored after addition of zinc . Properties of this enzyme and in particular the requirement for zinc ions for the activity, revealed similarity with the well characterised Bacillus cereus phospholipase C . Similarities with other bacterial and mammalian enzymes reported to be related to the B . cereus type are discussed. Microbiology, 1994 Sep, 140 ( Pt 9), 2315 - 31 Phenotypic and genotypic diversity of fluorescent pseudomonads isolated from field-grown sugar beet; Rainey PB et al.; A sample of 30 fluorescent pseudomonads isolated from the phyllosphere of sugar beet throughout a single growing season and shown to be closely related on the basis of fatty acid methyl ester (FAME) analysis was subjected to detailed phenotypic and genotypic characterization . Phenotypic traits were assessed on the basis of biochemical properties, assimilation of sole carbon sources, FAME analysis, organic pyrolysate content (MS-pyrolysis), and total cellular protein profiles . With the exception of total cellular protein profiles, numerical analysis of the data revealed two main clusters, each of which was divided into several subclusters . Numerical analysis of total cellular protein data failed to differentiate isolates into two main clusters, but nevertheless grouped isolates into six subclusters . On the basis of biochemical and carbon source assimilation profiles, 19 isolates were identified as Pseudomonas fluorescens biovar V, eight isolates as P . fluorescens biovar III and three isolates as P . syringae pathovar syringae . In general, all methods of phenotypic analysis grouped isolates according to time of sampling and leaf type . Genome analysis was undertaken by pulsed-field gel electrophoresis (PFGE) of PacI, SpeI, SwaI and XbaI macrorestriction fragments and revealed the presence of eight distinct genomic (clonal) groups . These groups correlated closely with the clusters generated by numerical analysis of phenotypic data, but there was no correlation between macrorestriction fragment profile and isolate identification; in fact the variation in macrorestriction fragment patterns within P . fluorescens biovars was as great as the variation detected between biovars, and between P . fluorescens and P . syringae . Statistical evaluation of macrorestriction fragment patterns revealed two examples of recent strain divergence: one was due to the presence of a 400 kbp plasmid within one isolate of a collection of nine otherwise genomically identical isolates, and the other was observed between two phenotypically similar isolates sampled 220 d apart . Genetic variation was expressed in terms of nucleotide diversity (pi) and pairwise comparisons yielded values ranging from 0.0029 to 0.1517 . The mean intrapopulation genetic variation was high (0.0993), but limited genetic variation was detected among isolates sampled on each occasion . Taken together this suggests a population comprised of a variety of apparently distantly related clones (genomic groups), each adapted to local conditions . Genome sizes were estimated from the sum of SpeI restriction fragments and ranged from 4.2 to 5.5 Mbp . Examination of the distribution of XbaI, SpeI, SwaI and PacI restriction endonuclease sites showed that the distribution of SpeI sites differed significantly from the expected (random) distribution. Mikrobiologiia, 1994 Sep-Oct, 63(5), 831 - 9 {Characteristics of Pseudomonas fluorescens lipopolysaccharide}; Veremeichenko SN et al.; A lipopolysaccharide was isolated from Pseudomonas fluorescens IMV 1152 (biovar I) . The fractions of the structural moieties of lipopolysaccharide macromolecule were extracted and studied separately . 3-hydroxydecanoic, 3-hydroxydodecanoic and 2-hydroxydodecanoic fatty acids were identified in lipid A, total quantity of those was more than 80% . In acid hydrolysate of lipid A the glucosamine and phosphoethanolamine were found . The rhamnose, arabinose, glucose, glucosamine and phosphorus were identified as the components of core oligosaccharide fractions . A trisaccharide repeating unit of the O-cpecific polysaccharide chain consists of the residues of aminosugars: 4-acet-amido-4,6-dideoxy-D-galactose, 2-acetamido-2,6-dideoxy-D-glucose and 2-acetamido-2,6-dideoxy-L-glucose . The studied lipopolysaccharide resembled the structure of lipid A and core oligosaccharide with lipopolysaccharide from type strain of Pseudomonas fluorescens IMV 4125 (ATCC 13525), whereas their O-chains were different. Plasmid, 1994 Sep, 32(2), 219 - 21 A plasmid responsible for malonate assimilation in Pseudomonas fluorescens; Kim YS et al.; A novel, broad-host-range 60-kb R-plasmid, which encodes for malonate assimilation, was isolated from Pseudomonas fluorescens and was designated pPSF1 . Pseudomonas, which can utilize malonate as a sole carbon source, was unable to grow on malonate medium upon curing with mitomycin C, indicating loss of plasmid pPSF1 . Furthermore, Escherichia coli transformed with pPSF1 was able to grow on malonate medium as a sole carbon source . Malonate decarboxylase, a key enzyme in malonate assimilation, was detected in transformed E . coli grown on malonate . pPSF1 also encodes resistance to several antibiotics such as ampicillin, kanamycin, and streptomycin and is transmissible between E . coli and Pseudomonas by conjugation. J Nat Prod, 1994 Sep, 57(9), 1200 - 5 (+)-(S)-dihydroaeruginoic acid, an inhibitor of Septoria tritici and other phytopathogenic fungi and bacteria, produced by Pseudomonas fluorescens; Carmi R et al.; Three antibiotics were isolated from a CH2Cl2 extract of the liquid culture of Pseudomonas fluorescens strain PFM2 . Two of the antibiotics were identified as 2,4-diacetylphloroglucinol and pyoluterin . The structure elucidation, absolute stereochemistry, synthesis, and biological activities of the new antibiotic (+)-(S)- dihydroaeruginoic acid {1} are reported. Biosci Biotechnol Biochem, 1994 Sep, 58(9), 1564 - 8 Purification and characterization of an alkaline lipase from Pseudomonas fluorescens AK102; Kojima Y et al.; An extracellular, novel alkaline lipase produced by Pseudomonas fluorescens AK102 was purified by ultrafiltration, ammonium sulfate precipitation, and DEAE-Toyopearl 650M and Phenyl-Toyopearl 650M column chromatographies . The purified enzyme was homogeneous on SDS-PAGE . The molecular weight was estimated to be about 33,000 by SDS-PAGE . The isoelectric point was pH 4.0 by isoelectric focusing . The pH stability was 4 to 10 and the optimum pH was 8 to 10 . The optimum temperature was 55 degrees C and the enzyme was stable below 50 degrees C . The enzyme unspecifically liberated short chain to long chain fatty acids from p-nitrophenyl esters, methyl esters, and triglycerides . In the presence of an anionic surfactant, the enzyme was characteristically stable . These results suggested that the enzyme can be used as a home laundry product ingredient. Biochemistry, 1994 Aug 23, 33(33), 10161 - 70 Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate . Evidence for a proton channel and a new binding mode of the flavin ring; Schreuder HA et al.; The crystal structures of wild-type p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with the substrate analogues 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate have been determined at 2.3-, 2.5-, and 2.8-A resolution, respectively . In addition, the crystal structure of a Tyr222Ala mutant, complexed with 2-hydroxy-4-aminobenzoate, has been determined at 2.7-A resolution . The structures have been refined to R factors between 14.5% and 15.8% for data between 8.0 A and the high-resolution limit . The differences between these complexes and the wild-type enzyme-substrate complex are all concentrated in the active site region . Binding of substrate analogues bearing a 4-amino group (4-aminobenzoate and 2-hydroxy-4-aminobenzoate) leads to binding of a water molecule next to the active site Tyr385 . As a result, a continuous hydrogen-bonding network is present between the 4-amino group of the substrate analogue and the side chain of His72 . It is likely that this hydrogen-bonding network is transiently present during normal catalysis, where it may or may not function as a proton channel assisting the deprotonation of the 4-hydroxyl group of the normal substrate upon binding to the active site . Binding of substrate analogues bearing a hydroxyl group at the 2-position (2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate) leads to displacement of the flavin ring from the active site . The flavin is no longer in the active site (the "in" conformation) but is in the cleft leading to the active site instead (the "out" conformation) . It is proposed that movement of the FAD out of the active site may provide an entrance for the substrate to enter the active site and an exit for the product to leave. Appl Environ Microbiol, 1994 Aug, 60(8), 2759 - 65 Effect of cadmium and zinc on attachment and detachment interactions of Pseudomonas fluorescens H2 with glass; McEldowney S; The physiological and physicochemical bases for the effect of 5, 10, 50, or 100 micrograms of Cd and Zn ml-1 on the attachment and detachment interactions of Pseudomonas fluorescens H2 with glass substrata were determined . Attachment and detachment varied with the type and concentration of metal and the time at which cells were exposed to the metal . The largely inhibitory effect of the metals on bacterial motility and physiological activity did not directly influence attachment . The amount of Cd or Zn accumulated by the cells increased with metal concentration and was greater for free than for attached cells . The hydrophobicity and negative and positive charges of the bacterial surfaces (measured by hydrophobic and electrostatic interaction chromatography) were increased by cell exposure to the metals, particularly after Cd treatment . Cells exposed to Cd prior to attachment showed increased adhesion . Zinc-treated cells did not . There was a positive correlation between adhesion and Cd concentration in the attachment solution . No such relationship existed for Zn . P . fluorescens H2 exposed to Cd prior to attachment desorbed similarly to untreated controls . Zinc pretreatment resulted in decreased desorption . Cells attached in 5 or 10 micrograms of Cd or Zn ml-1 detached less than those attached in 50 or 100 micrograms of Cd or Zn ml-1 . The presence of Cd or Zn during detachment had little effect on desorption . The dominant influence of Cd and Zn on attachment and detachment appears to be through modification of the bacterial surface . In natural ecosystems, heavy metals may influence the distribution of bacteria between the solid and liquid phases. J Bacteriol, 1994 Aug, 176(16), 5033 - 43 Phylogenetic analysis of sequences from diverse bacteria with homology to the Escherichia coli rho gene; Opperman T et al.; Genes from Pseudomonas fluorescens, Chromatium vinosum, Micrococcus luteus, Deinococcus radiodurans, and Thermotoga maritima with homology to the Escherichia coli rho gene were cloned and sequenced, and their sequences were compared with other available sequences . The species for all of the compared sequences are members of five bacterial phyla, including Thermotogales, the most deeply diverged phylum . This suggests that a rho-like gene is ubiquitous in the Bacteria and was present in their common ancestor . The comparative analysis revealed that the Rho homologs are highly conserved, exhibiting a minimum identity of 50% of their amino acid residues in pairwise comparisons . The ATP-binding domain had a particularly high degree of conservation, consisting of some blocks with sequences of residues that are very similar to segments of the alpha and beta subunits of F1-ATPase and of other blocks with sequences that are unique to Rho . The RNA-binding domain is more diverged than the ATP-binding domain . However, one of its most highly conserved segments includes a RNP1-like sequence, which is known to be involved in RNA binding . Overall, the degree of similarity is lowest in the first 50 residues (the first half of the RNA-binding domain), in the putative connector region between the RNA-binding and the ATP-binding domains, and in the last 50 residues of the polypeptide . Since functionally defective mutants for E . coli Rho exist in all three of these segments, they represent important parts of Rho that have undergone adaptive evolution. J Bacteriol, 1994 Aug, 176(15), 4635 - 41 Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid; Ramos-Gonzalez MI et al.; The Pseudomonas putida TOL plasmid pWW0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer) . Transconjugants are recipient cells that have received DNA from donor cells, whereas retrotransconjugants are donor bacteria that have received DNA from a recipient . The TOL plasmid pWW0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrated into the chromosome of other P . putida strains, a process that appears to involve a single conjugational event . The rate of retrotransfer (as well as of direct transfer) of the chromosomal marker is influenced by the location of the kanamycin marker on the chromosome and ranges from 10(-3) to less than 10(-8) retrotransconjugants per donor (transconjugants per recipient) . The mobilized DNA is incorporated into the chromosome of the retrotransconjugants (transconjugants) in a process that seems to occur through recombination of highly homologous flanking regions . No interspecific mobilization of the chromosomal marker in matings involving P . putida and the closely related Pseudomonas fluorescens, which belongs to rRNA group I, was observed. Can J Microbiol, 1994 Jul, 40(7), 541 - 7 Identification of variability of ribosomal DNA spacer from Pseudomonas soil isolates; Gill S et al.; The polymerase chain reaction was used to am