De Gioia L,
( 2002 )
The cold-active lipase of Pseudomonas fragi. Heterologous expression, biochemical characterization and molecular modeling.
PMID : 12084074 : DOI : 10.1046/j.1432-1033.2002.03012.x
A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Structural modifications recurrent in cold-adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme.
( 2001 )
Flagellin gene sequence variation in the genus Pseudomonas.
PMID : 11518318 : DOI : 10.1078/0723-2020-00031
Flagellin gene (fliC) sequences from 18 strains of Pseudomonas sensu stricto representing 8 different species, and 9 representative fliC sequences from other members of the gamma sub-division of proteobacteria, were compared. Analysis was performed on N-terminal, C-terminal and whole fliC sequences. The fliC analyses confirmed the inferred relationship between P. mendocina, P. oleovorans and P. aeruginosa based on 16S rRNA sequence comparisons. In addition, the analyses indicated that P. putida PRS2000 was closely related to P. fluorescens SBW25 and P. fluorescens NCIMB 9046T, but suggested that P. putida PaW8 and P. putida PRS2000 were more closely related to other Pseudomonas spp. than they were to each other. There were a number of inconsistencies in inferred evolutionary relationships between strains, depending on the analysis performed. In particular, whole flagellin gene comparisons often differed from those obtained using N- and C-terminal sequences. However, there were also inconsistencies between the terminal region analyses, suggesting that phylogenetic relationships inferred on the basis of fliC sequence should be treated with caution. Although the central domain of fliC is highly variable between Pseudomonas strains, there was evidence of sequence similarities between the central domains of different Pseudomonas fliC sequences. This indicates the possibility of recombination in the central domain of fliC genes within Pseudomonas species, and between these genes and those from other bacteria.
( 2007 )
Simultaneous detection of Pseudomonas fragi, P. lundensis, and P. putida from meat by use of a multiplex PCR assay targeting the carA gene.
PMID : 17293505 : DOI : 10.1128/AEM.02603-06 PMC : PMC1855653
Species-specific primers and a multiplex PCR assay were developed for the simultaneous identification and differentiation of Pseudomonas fragi, P. lundensis, and P. putida based on the coamplification of different portions of the small subunit of the carbamoyl phosphate synthase gene (carA). The carA multiplex PCR was used to detect the presence of the three Pseudomonas species from beef, chicken, and pork samples and proved to be effective in showing their evolution during the storage of meat.
Ait Tayeb L,
( N/A )
Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates.
PMID : 15950132 : DOI : 10.1016/j.resmic.2005.02.009
Phylogenetic relationships within the genus Pseudomonas were examined by comparing partial (about 1000 nucleotides) rpoB gene sequences. A total of 186 strains belonging to 75 species of Pseudomonas sensu stricto and related species were studied. The phylogenetic resolution of the rpoB tree was approximately three times higher than that of the rrs tree. Ribogroups published earlier correlated well with rpoB sequence clusters. The rpoB sequence database generated by this study was used for identification. A total of 89 isolates (79.5%) were identified to a named species, while 16 isolates (14.3%) corresponded to unnamed species, and 7 isolates (6.2%) had uncertain affiliation. rpoB sequencing is now being used for routine identification of Pseudomonas isolates in our laboratory.
( 2006 )
D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.
PMID : 16325199 : DOI : 10.1016/j.jmb.2005.10.072
The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.
( 2005 )
Mutations in the "lid" region affect chain length specificity and thermostability of a Pseudomonas fragi lipase.
PMID : 15848176 : DOI : 10.1016/j.febslet.2005.03.037
The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chain-length preference profile, increasing the relative activity towards C8 substrates. Moreover, mutations conferred to PFL higher temperature stability. On the other hand, replacement of the serine at position 141 by glycine destabilized the protein.
( 2005 )
Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny.
PMID : 15658983 : DOI : 10.1111/j.1462-2920.2004.00670.x
The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.
( 1992 )
Primary structures of the genes, faoA and faoB, from Pseudomonas fragi B-0771 which encode the two subunits of the HDT multienzyme complex involved in fatty acid beta-oxidation.
PMID : 1607366 : DOI : 10.1093/oxfordjournals.jbchem.a123722
Three enzyme activities involved in fatty acid beta-oxidation, i.e., those of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, are exhibited by one multienzyme complex (HDT) composed of two molecules each of two peptides in Pseudomonas fragi. Using specific antisera against the two subunits of HDT, we isolated the genes encoding the subunits of HDT and designated them "faoA" (for the alpha-subunit) and "faoB" (for the beta-subunit). Their complete nucleotide sequences were determined and it was revealed that faoA and faoB, both with individual putative S.D. sequences at suitable positions, formed a cluster, in that order. The amino acid sequences deduced from the nucleotide sequences of the two genes indicated that the alpha-subunit, encoded by faoA, is a polypeptide of 715 amino acid residues, and that the beta-subunit, encoded by faoB, consists of 390 amino acid residues lacking the first methionine of the primary product encoded by faoB. Immunoblotting of cell lysates prepared from Escherichia coli transformants carrying plasmids which possess the faoA and/or faoB gene with antisera against the subunits of HDT showed that both the faoA and faoB genes were transcribed and translated in E. coli. The overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase were increased in the E. coli cells transformed with the plasmid possessing the faoA gene, suggesting that both the hydratase and dehydrogenase activities may be exhibited by the alpha-subunit of HDT.(ABSTRACT TRUNCATED AT 250 WORDS)
( 2004 )
Structural basis for channelling mechanism of a fatty acid beta-oxidation multienzyme complex.
PMID : 15229654 : DOI : 10.1038/sj.emboj.7600298 PMC : PMC514956 DOI : 10.1038/sj.emboj.7600298 PMC : PMC514956
The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid beta-oxidation cycle. The alpha2beta2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3'-phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3'-phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The alpha-helical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other beta-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.
Nsigue Meilo S,
De Vos P,
( 2011 )
A long-branch attraction artifact reveals an adaptive radiation in pseudomonas.
PMID : 21504889 : DOI : 10.1093/molbev/msr099
A significant proportion of protein-encoding gene phylogenies in bacteria is inconsistent with the species phylogeny. It was usually argued that such inconsistencies resulted from lateral transfers. Here, by further studying the phylogeny of the oprF gene encoding the major surface protein in the bacterial Pseudomonas genus, we found that the incongruent tree topology observed results from a long-branch attraction (LBA) artifact and not from lateral transfers. LBA in the oprF phylogeny could be explained by the faster evolution in a lineage adapted to the rhizosphere, highlighting an unexpected adaptive radiation. We argue that analysis of such artifacts in other inconsistent bacterial phylogenies could be a valuable tool in molecular ecology to highlight cryptic adaptive radiations in microorganisms.
( 2009 )
Closed complex of the D-3-hydroxybutyrate dehydrogenase induced by an enantiomeric competitive inhibitor.
PMID : 19122202 : DOI : 10.1093/jb/mvn186
D-3-Hydroxybutyrate dehydrogenase (HBDH) from Pseudomonas fragi showed a strict stereospecificity to the d-enantiomer of 3-hydroxybutyrate (d-3-HB) as a substrate. The l-enantiomer acts as a competitive inhibitor, with a K(i) value comparable to the K(m) value for d-3-HB. We have determined the crystal structures of the ternary complex of HBDH-NAD(+)-l-3-HB and the binary complex of HBDH-NAD(+). The former structure showed a so-called closed-form conformation, which is considered an active form for catalysis, while the latter stayed mostly in a open-form conformation. The determined structures along with the site-directed mutagenesis confirmed the substrate recognition mechanism that we proposed previously. The hydrogen bonding interaction between Gln196, located in the moving helix, and the carboxyl group of the substrate/inhibitor is important for the stable ternary complex formation. Finally, the crystal structures of the Thr190 mutants, T190S and T190A, indicate that the Thr190 is a key residue for the open-closed conformational change. T190S retained 37% of the activity. In T190A, however, the activity decreased to 0.1% that of the wild-type enzyme. Fixing the position of the hydroxyl group of Thr190 to form hydrogen bonds to the pyrophosphate moiety and the carboxamide of NAD(+) seems to be a significant factor for the open-closed conformational change.
( 2017 )
Spoilage potential characterization of Shewanella and Pseudomonas isolated from spoiled large yellow croaker (Pseudosciaena crocea).
PMID : 27747903 : DOI : 10.1111/lam.12687
Ten strains were isolated from a spoiled large yellow croaker (Pseudosciaena crocea). All of them were able to grow aerobically from 4 to 30�XC, and reduce trimethylamine-N-oxide to trimethylamine (TMA) and produce H2 S except SB01, PF05 and PF07. Biochemical characterization and phylogenetic analysis of 16S rRNA gene showed that eight H2 S-producing isolates were closely related to Shewanella baltica, and two isolates PF05 and PF07 were identified as Pseudomonas fluorescens and Pseudomonas fragi respectively. However, of the eight Shewanella, seven isolates cluster with S. baltica and one with Shewanella glacialipiscicola based on the analysis of the gyrB gene. Shewanella baltica also had the ability to produce biogenic amines, while two Pseudomonas had high activities of proteinase and lipase, and failed to produce TMA and biogenic amines. In spoilage potential evaluation, the TVB-N value of S. baltica was significantly higher than that of Pseudomonas in sterile fish juice, although its growth was slower than Pseudomonas. Therefore, this work demonstrated that S. baltica was able to cause rapid and strong spoilage and was therefore identified as a specific spoilage organism in refrigerated P. crocea. Members of the bacterial genera Shewanella and Pseudomonas are widely known to be responsible for the specific spoilers in iced fish. Ten strains isolated from spoiled large yellow croaker (Pseudosciaena crocea) were identified as Shewanella baltica and Pseudomonas spp. S. baltica was demonstrated as the predominant spoiler in the refrigerated P. crocea due to its high metabolic activities. This work has generated baseline information for a better understanding of the role of various spoilage bacteria in chilled marine fish and for the control of contamination and growth of main spoilage bacteria to extend the shelf life of marine fish.
( 1989 )
Specificity of endoproteinase Asp-N (Pseudomonas fragi): cleavage at glutamyl residues in two proteins.
PMID : 2669754 : DOI : 10.1016/0006-291x(89)90848-6
Endoproteinase Asp-N, a metalloprotease from a mutant strain of Pseudomonas fragi, has been reported to specifically cleave on the N-terminal side of aspartyl and cysteic acid residues. We utilized this enzyme to generate fragments for determining the amino acid sequence of the D-aspartyl/L-isoaspartyl methyltransferase isozyme I from human erythrocytes. Surprisingly, we identified cleavage sites for this enzyme at the N-terminal side of several glutamyl residues in addition to the expected cleavage sites at aspartyl residues. The ability of this enzyme to cleave polypeptides at both glutamyl and aspartyl residues was confirmed by mapping additional sites on erythrocyte carbonic anhydrase I. These results indicate that a more appropriate name for this enzyme may be Endoproteinase Asp/Glu-N.
Lavilla Lerma L,
Casado Muñoz Mdel C,
( 2014 )
Antibiotic multiresistance analysis of mesophilic and psychrotrophic Pseudomonas spp. isolated from goat and lamb slaughterhouse surfaces throughout the meat production process.
PMID : 25172860 : DOI : 10.1128/AEM.01998-14 PMC : PMC4249046
The aim of this study was to investigate the phenotypic and genotypic antibiotic resistance profiles of pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which were representative of areas that are possible sources of meat contamination. Mesophilic (85 isolates) and psychrotrophic (37 isolates) pseudomonads identified at the species level generally were resistant to sulfamethoxazole, erythromycin, amoxicillin, ampicillin, chloramphenicol, trimethoprim, rifampin, and ceftazidime (especially mesophiles), as well as colistin and tetracycline (especially psychrotrophes). However, they generally were sensitive to ciprofloxacin, gentamicin, imipenem, and kanamycin regardless of species identity. Worryingly, in the present study, we found multidrug resistance (MDR) to up to 13 antibiotics, which was related to intrinsic and acquired resistance mechanisms. Furthermore, a link between various antimicrobial resistance genes was shown for beta-lactams and tetracycline, trimethoprim, and sulfonamides. The distribution and resistome-based analysis of MDR pseudomonads in different slaughterhouse zones indicated that the main sources of the identical or related pseudomonad strains were the animals (feet and wool) and the slaughterhouse environment, being disseminated from the beginning, or entrance environment, to the environment of the finished meat products. Those facts must be taken into consideration to avoid cross-contamination with the subsequent flow of mobile resistance determinants throughout all slaughterhouse zones and then to humans and the environment by the application of adequate practices of hygiene and disinfection measures, including those for animal wool and feet and also the entrance environment.
( 2014 )
Tracking the blue: a MLST approach to characterise the Pseudomonas fluorescens group.
PMID : 24387861 : DOI : 10.1016/j.fm.2013.11.012
The Pseudomonas fluorescens group comprises several closely related species that are involved in food contamination and spoilage. Specifically, the interest in P. fluorescens as a spoiler of dairy products increased after the cases of "blue mozzarella" that occurred in Italy in 2010. A Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise 136 isolates (reference strains and food borne isolates) at strain level, to reveal the genetic relationships among them and to disclose any possible genetic clustering of phenotypic markers involved in food spoilage (protease, lipase, lecithinase activities and pigmented or fluorescent molecule production). The production of dark blue diffusible pigment was evaluated on several bacterial culture media and directly on mozzarella cheese. The MLST scheme provided precise genotyping at the strain level, and the population analyses of the concatenated sequences allowed major taxa to be defined. This approach was revealed to be suitable for tracking the strains according to their origin, such as dairy plants or food matrices. The genetic analysis revealed the presence of a connection between the blue pigment production and a specific phylogenetic cluster. The development of the online database specific to the P. fluorescens group (http://pubmlst.org/pfluorescens) will facilitate the application of the scheme and the sharing of the data.
( 1994 )
Transcription of the faoAB operon which encodes the HDT multienzyme complex involved in fatty acid beta-oxidation in Pseudomonas fragi B-0771.
PMID : 8206878 : DOI : 10.1093/oxfordjournals.jbchem.a124330
Transcription of the two clustered genes, faoA and faoB, for the multienzyme complex for fatty acid beta-oxidation (HDT) from the Gram-negative bacterium, Pseudomonas fragi, was investigated. Northern blot hybridization and primer extension analysis indicated that these genes comprised an operon, giving a 3.6 kb mRNA. Transcription of the 3.6 kb faoAB mRNA was induced by palmitic acid. Using the lacZ gene as a reporter gene for a promoter activity search, the essential region for induction of the faoAB gene transcription by palmitic acid was mapped at the 331 bp region just upstream of the transcription initiation site. Deletion of the 136 bp SalI-NheI fragment just downstream of the transcription initiation site caused the constitutive expression of the faoA-lacZ fusion protein in P. fragi, suggesting that there is a regulatory element interacting with the transcription repressor.
( 1995 )
PMID : 7674963 : DOI : 10.1016/0076-6879(95)48053-6
( 1967 )
Purification and characterization of the lipase of Pseudomonas fragi.
PMID : 6052627 : DOI : 10.1099/00221287-48-3-317
( 1980 )
Substrate specificity of a proteolytic enzyme isolated from a mutant of Pseudomonas fragi.
PMID : 7188696 :
Previous studies have described the isolation of mutationally altered proteases in Pseudomonas fragi (Noreau, J., and Drapeau, G.R. (1979) J. Bacteriol, 140, 911-916. In the present study, it is shown that one of these proteases cleaves specifically the peptide bonds on the NH2-terminal side of either aspartic acid or cysteic acid residues in oxidized ribonuclease. With myoglobin as the substrate, a similar specificity was observed except that only four out of the six aspartyl bonds present were hydrolyzed.
( 1965 )
The extracellular nature of the lipase of Pseudomonas fragi.
PMID : 5881339 : DOI : 10.1016/0005-2760(65)90079-2
( 1988 )
Cloning, sequencing and expression of the lipase gene from Pseudomonas fragi IFO-12049 in E. coli.
PMID : 3060375 : DOI : 10.1016/0014-5793(88)80980-3
The lipase gene from Pseudomonas fragi IFO-12049 was isolated using the expression library and the primary structure of lipase deduced from the nucleotide sequence was determined. It is composed of 277 amino acid residues and a protein of Mr 29,966, which was close to the value of the lipase expressed in E. coli.
( 1986 )
Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi.
PMID : 3800995 : DOI : 10.1016/s0006-291x(86)80352-7
The gene coding for the lipase of Pseudomonas fragi was cloned into Escherichia coli JM83 by inserting Sau3A-generated DNA fragments into the BamH I site of pUC9. The plasmid isolated, pKKO, was restriction mapped and the position of the lipase gene on the 2.0 kb insert was pinpointed by subcloning. DNA sequencing revealed that the open reading frame comprises 405 nucleotides and gives a preprotein of 135 amino acids with a predicted Mr of 14643. By comparing the putative lipase amino acid sequence with porcine pancreatic, rat lingual and Staphylococcus hyicus lipases the amino acid sequence around the reactive serine was found to be common among the types of lipase which have been reported.
Assur Sanghai Z,
( 2018 )
Structure-based analysis of CysZ-mediated cellular uptake of sulfate.
PMID : 29792261 : DOI : 10.7554/eLife.27829 PMC : PMC5967866
Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.
( 2013 )
Evaluation of oprI and oprL genes as molecular markers for the genus Pseudomonas and their use in studying the biodiversity of a small Belgian River.
PMID : 23246592 : DOI : 10.1016/j.resmic.2012.12.001
A multiplex PCR based on oprI and oprL, coding for the outer membrane lipoprotein I and the peptidoglycan-associated lipoprotein OprL, respectively, was developed for the detection of Pseudomonas strains from a bacterial collection isolated from a small river. To study the diversity of these Pseudomonas isolates, an oprI-oprL gene sequence database of 94 Pseudomonas type strains was constructed. Phylogenetic analysis of the concatenated oprI and oprL gene sequences of the Pseudomonas type strains showed that they were largely congruent with the classification based on the MLSA approach based on 16S rRNA, gyrB, rpoB and rpoD gene sequences of Mulet et al. in 2010. Identification of the isolates demonstrated a high diversity of Pseudomonas isolates at the source of the river located in a forest of which most isolates belonged to the Pseudomonas fluorescens lineage. On the other hand, the Pseudomonas population isolated at an anthropized site at the mouth of the river, receiving waste water from both households and industry, was very different and contained many Pseudomonas aeruginosa isolates.
( 1997 )
The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins.
PMID : 9393697 : DOI : 10.1128/jb.179.23.7331-7342.1997 PMC : PMC179683
The psychrotrophic bacterium Pseudomonas fragi was subjected to cold shocks from 30 or 20 to 5 degrees C. The downshifts were followed by a lag phase before growth resumed at a characteristic 5 degrees C growth rate. The analysis of protein patterns by two-dimentional gel electrophoresis revealed overexpression of 25 or 17 proteins and underexpression of 12 proteins following the 30- or 20-to-5 degrees C shift, respectively. The two downshifts shared similar variations of synthesis of 20 proteins. The kinetic analysis distinguished the induced proteins into cold shock proteins (Csps), which were rapidly but transiently overexpressed, and cold acclimation proteins (Caps), which were more or less rapidly induced but still overexpressed several hours after the downshifts. Among the cold-induced proteins, four low-molecular-mass proteins, two of them previously characterized as Caps (CapA and CapB), and heat acclimation proteins (Haps) as well as heat shock proteins (Hsps) for the two others (TapA and TapB) displayed higher levels of induction. Partial amino acid sequences, obtained by microsequencing, were used to design primers to amplify by PCR the four genes and then determine their nucleotide sequences. A BamHI-EcoRI restriction fragment of 1.9 kb, containing the complete coding sequence for capB, was cloned and sequenced. The four peptides belong to the family of small nucleic acid-binding proteins as CspA, the major Escherichia coli Csp. They are likely to play a major role in the adaptative response of P. fragi to environmental temperature changes.