Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.     

Role of bacterial ribosomes in barotolerance.

The effects of high hydrostatic pressures on protein synthesis by whole cells and cell free preparations of Escherichia coli, Pseudomonas fluorescens, and Pseudomonas bathycetes were determined. Actively growing cells of P. bathycetes and P. fluorescens were less sensitive than were E. coli cells. Protein synthesis by cell free preparations of E. coli and P. fluorescens showed the same extent of inhibition as their respective whole cell preparations, whereas cell free preparations of P. bathycetes showed a marked increase in pressure sensitivity over whole cells. Protein synthesis by hybrid protein synthesizing cell free preparations (the ribosomes from one organism and the S-100 supernatant fraction from another) demonstrated that response to high pressure is dependent on the source of the ribosome employed. A hybrid system containing E. coli ribosomes and P. fluorescens S-100 shows the same sensitivity to pressure as a homologous E. coli system, whereas a hybrid containing P. fluorescens ribosomes and E. coli S-100 shows the greater pressure tolerance characteristic of the P. fluorescens homologous system.     

New alkane-responsive expression vectors for Escherichia coli and pseudomonas

We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB. The expression vectors were tested in several E. coli strains, P. putida GPo12 and P. fluorescens KOB2Delta1 with catechol-2,3-dioxygenase (XylE). Induction factors ranged between 100 and 2700 for pKKPalk in E. coli and pCom8 in Pseudomonas strains, but were clearly lower for pCom8, pCom9, and pCom10 in E. coli. XylE expression levels of more than 10% of total cell protein were obtained for E. coli as well as for Pseudomonas strains.     

Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1

Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37 degrees C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.     

Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens

The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.     

F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens.

Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4).     

Transport of heterologous proteins to the periplasmic space of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> using a variety of native signal sequences

Bacterial expression of recombinant proteins containing disulfide bonds is facilitated by transport of the proteins to the periplasmic space. Several Pseudomonas fluorescens signal sequences have been identified that efficiently direct proteins to the periplasm and provide solubility and yield advantages over the production of proteins fused to the PelB signal sequence in E. coli. For a single chain antibody fragment, the final yield varied from about 1 g/l to 10 g/l when expression in P. fluorescens involved fusion to various P. fluorescens signal sequences.     

The D-glucosaminate dehydratase alpha-subunit from Pseudomonas fluorescens exhibits thioredoxin reductase activity

The complete amino acid sequence of the D-glucosaminate dehydratase (GADH) alpha-subunit from Pseudomonas fluorescens was determined by PCR using genomic DNA from P. fluorescens as a template. The alpha-subunit comprises 320 amino acids and has a molecular mass of about 33.9 kDa. The primary structure of the alpha-subunit demonstrates a high similarity to the structures of thioredoxin reductase (TrxR) from many prokaryotes, especially Pseudomonas aeruginosa (identity 85%, positive 91%), Vibrio cholerae (identity 73%, positive 85%), and Escherichia coli (identity 71%, positive 83%). The purified glucosaminate dehydratase alpha(2)-enzyme exhibited NADPH-dependent TrxR activity, while TrxR from E. coli showed pyridoxal 5'-phosphate (PLP)-dependent GADH activity. The TrxR from E. coli suggests that there are three cofactor binding sites, FAD, NADPH, and PLP in the enzyme and that TrxR catalyzes the FAD- and NADPH-dependent oxidation-reduction reaction and the PLP-dependent alpha,beta-elimination reaction.     

Antigen 43 facilitates formation of multispecies biofilms

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation of E. coli cells in static cultures. Here, we show that Ag43 can be expressed in a functional form on the surface of Pseudomonas fluorescens . Ag43 expression dramatically enhances the biofilm-forming potential of both E. coli and P. fluorescens to abiotic surfaces in simple microtitre well assays and in flow chambers. Importantly, Ag43-expressing E. coli and P. fluorescens cells tagged with Gfp and Rfp were shown to form interwoven biofilms in flow chambers. The three-dimensional structures of the biofilms were analysed by laser-confocal microscopy. Heterogeneous expression of Ag43 induced interspecies cell-to-cell contact that generated multispecies biofilm formation. Our data indicate that this versatile molecular tool can be used for the rational design of multispecies biofilms. More specifically, this novel technology offers opportunities for the design of multispecies consortia in which the concerted action of several bacterial species is required, e.g. waste treatment and degradation of pollutants.     

High-level secretion of Pseudomonas fluorescens type I secretion system-dependent lipase in Serratia marcescens

The type I secretion system-dependent lipase, TliA, of Pseudomonas fluorescens was successfully produced in quantity in Serratia marcescens by coexpressing its cognate ABC transporter, TliDEF. Compared with P. fluorescens and Escherichia coli, S. marcescens showed an outstanding capacity for the secretory production of TliA, which was done with the expression vectors available for use in E. coli, and no growth phase-dependency, which was unlike the typical feature of TOSS-mediated protein secretion. Among the S. marcescens tested, the highest amount of TliA (approximately 2600 units ml(-1)) was achieved by S. marcescens KCTC 2798 containing the expression plasmid pTliDEFA-223. Our results also suggest that strains of Serratia will provide a valuable opportunity for producing other extracellular TOSS-dependent proteins effectively as well as the TliDEF-dependent TliA in this study.     

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T?? values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.     

The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.     

Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of E. coli cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.     

DNA hybridization probe for the Pseudomonas fluorescens group.

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.     

DNA hybridization probe for the Pseudomonas fluorescens group

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.     

Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA

Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.     

Glutamine synthetase of pseudomonads: some biochemical and physicochemical properties.

The glutamine synthetases from several Pseudomonas species were purified to homogeneity, and their properties were compared with those reported for the enzymes from Escherichia coli and other gram-negative bacteria. The glutamine synthetase from Pseudomonas fluorescens was unique because it was nearly precipitated quantitatively as a homogeneous protein during dialysis of partially purified preparations against buffer containing 10 mM imidazole (pH 7.0) and 10 mM MnCl2. The glutamine synthetases from Pseudomonas putida and Pseudomonas aeruginosa were purified by affinity chromatography on Affi-blue gel. Dodecamerous forms of the E. coli and P. fluorescens glutamine synthetases had identical mobilities during polyacrylamide gel electrophoresis. Their dissociated subunits, however, migrated differently and were readily separated by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulfate. This difference in subunit mobilities is not related to the state of adenylylation. Regulation of the Pseudomonas glutamine synthetase activity is mediated by an adenylylation-deadenylylation cyclic cascade system. A sensitive procedure was developed for measuring the average number of adenylylated subunits per enzyme molecule for the glutamine synthetase from P. fluorescens. This method takes advantage of the large differences in transferase activity of the adenylylated and unadenylylated subunits at pH 6.0 and of the fact that the activities of both kinds of subunits are the same at pH 8.45.