PelC is a Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of proteins involved in exopolysaccharide transport

Pseudomonas aeruginosa is a gram-negative bacterium, opportunistic pathogen, which causes severe acute or chronic infections, as is the case with cystic fibrosis patients. Chronic infections are frequently accompanied by the development of the bacterial population into a specialized community called biofilm. The pelA-G gene cluster of P. aeruginosa has been shown to be involved in pellicle production and biofilm formation. The pel genes have been proposed to contribute to the formation of the exopolysaccharide-containing pellicle. However, the function and the subcellular localization of the seven different Pel proteins are poorly understood. Based on bioinformatics analysis, we have previously considered that PelF is a putative glycosyltransferase (GT4 family), whereas PelG is a Wzx-like polysaccharide transporter from the PST family. In this study we have further characterized the PelC protein. We have shown that PelC is an outer membrane lipoprotein. The N-terminal signal peptide of the PelC lipoprotein is sufficient to target the protein into the membranes. However, by constructing various PelC hybrid proteins we also proposed that efficient and functional outer membrane insertion of PelC requires not only the signal peptide and the lipid modification, but also requires the C-terminal domain of PelC. Because the gene encoding the outer membrane lipoprotein PelC is part of a putative gene cluster involved in exopolysaccharide biogenesis, we suggest that PelC is a new member of the outer membrane auxiliary (OMA) family of lipoprotein whose Wza, involved in Escherichia coli capsular polysaccharide transport, is an archetype.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

Siderophore-mediated iron uptake in fluorescent Pseudomonas: characterization of the pyoverdine-receptor binding site of three cross-reacting pyoverdines.

Two Pseudomonas fluorescens and one Pseudomonas aeruginosa strains, although producing structurally different pyoverdines, demonstrated highly efficient cross-reactions when tested for pyoverdine-mediated iron uptake. A ferripyoverdine receptor-deficient mutant of the P. aeruginosa strain was unable to use any of the three pyoverdines. Moreover, the three strains presented each a specific outer membrane siderophore-receptor pattern. Thus, the capacity of using heterologous pyoverdines was related not to the presence of supplementary specific ferripyoverdine receptors but to the existence within the respective pyoverdine-peptide chains of a common dipeptide motif which should act as the receptor-binding site for the three pyoverdines. Other pyoverdines sharing the same motif but at another position within the peptide chain were not efficient in iron transport, demonstrating the importance of the spatial position of the binding site.     

Pyrimidine ribonucleoside catabolism in Pseudomonas fluorescens biotype A

The pyrimidine ribonucleosides uridine or cytidine were shown to serve as a source of nitrogen or carbon for the growth of Pseudomonas fluorescens strain A126. After incubation of either pyrimidine ribonucleoside with extracts of this strain, the resultant catabolic products were detected by thin-layer chromatography. It was found that pyrimidine ribonucleoside catabolism in this pseudomonad involved the enzymes nucleoside hydrolase and cytosine deaminase. The specific activities of both these enzymes could be influenced by the nitrogen or carbon source present in the medium.     

S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis

An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.     

Structure,chemistry, and biological activity of pseudophomins A and B,new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens

Pseudophomins A and B are cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, a bacterium with potential application for biocontrol of plant pathogens and weeds. Their chemical structures were established by a combination of spectroscopic data, X-ray crystallography, and selective chemical degradation. This unique chemical degradation allowed the unambiguous determination of the absolute configuration of the amino acid residue Leu-1, due to gamma-lactam formation followed by selective cleavage of the adjacent N(8)-C(7) bond. To the best of our knowledge this is the first application of gamma-lactam formation to the determination of absolute configuration of an adjacent amino acid. Pseudophomin B showed higher antifungal activity against the phytopathogens Phoma lingam/Leptosphaeria maculans and Sclerotinia sclerotiorum than pseudophomin A, and is likely to be the main component responsible for the antifungal activity of EtOAc extracts of strain BRG100. By contrast, pseudophomin A showed stronger inhibition of green foxtail (Setaria viridis) root germination than pseudophomin B.     

Functional characterization of a wheat plasma membrane Na+/H+ antiporter in yeast

The functional analysis of the sodium exchanger SOS1 from wheat, TaSOS1, was undertaken using Saccharomyces cerevisiae as a heterologous expression system. The TaSOS1 protein, with significant sequence homology to SOS1 sodium exchangers from Arabidopsis and rice, is abundant in roots and leaves, and is induced by salt treatment. TaSOS1 suppressed the salt sensitivity of a yeast strain lacking the major Na⁺ efflux systems by decreasing the cellular Na⁺ content while increasing K⁺ content. Na⁺/H⁺ exchange activity of purified plasma membrane from yeast cells expressing TaSOS1 was higher than controls transformed with empty vector. These results demonstrate that TaSOS1 contributes to plasma membrane Na⁺/H⁺ exchange.     

Genes related to chromate resistance by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon-insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted in the mutants with the PAO1 genome revealed that the genes affected in three mutant strains were oprE (ORF PA0291), rmlA (ORF PA5163), and ftsK (ORF PA2615), respectively. A relationship of these genes with chromate tolerance has not been previously reported. No other phenotypic changes were observed in the oprE mutant but its resistance to chromate was not fully restored by expressing the ChrA protein, which extrudes chromate ions from the cytoplasm to the periplasmic space. These data suggest that OprE participates in the efflux of chromate from the periplasm to the outside. Increased susceptibility of the rmlA mutant to the metals cadmium and mercury and to the anion-superoxide generator paraquat suggests a protective role of LPS against chromate toxicity. A higher susceptibility of the ftsK mutant to compounds affecting DNA structure (ciprofloxacin, tellurite, mitomycin C) suggests a role of FtsK in the recombinational repair of DNA damage caused by chromate. In conclusion, the P. aeruginosa genome contains diverse genes related to its intrinsic resistance to chromate. Systems pertaining to the outer membrane (OprE), the cell wall (LPS), and the cytoplasm (FtsK) were identified in this work as involved in chromate protection mechanisms.     

The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa

Summary The complete nucleotide sequence of the gene coding for one of the carboxymethycellulases (CMCase), expressed by Pseudomonas fluorescens subsp. cellulosa, has been determined. The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon. The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins. Unlike most pseudomonas genes, the CMCase sequence does not have a high G+C (51%) content and there is no marked preference for codons ending in G or C. Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters. A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP). Bal31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase. Subclones of the cellulase gene have been constructed in pUC18 and pUC19. One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2. Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000. This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.Abbreviations CMCase carboxymethylcellulase - PAGE polyacrylamide gel electrophoresis - IPTG isopropyl--D-thiogalactoside - CAT chloramphenicol acetyl transferase     

Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl--D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - Sm^r resistance to streptomycin - Km^r resistance to kanamycin - Ap^r resistance to ampicillin - Tc^r resistance to tetracycline - Cm^r resistance to chloramphenicol - CMCase carboxymethylcellulase     

Evaluation of an Antibiotic-Producing Strain of Pseudomonas fluorescens for Suppression of Plant-Parasitic Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG), produced by some strains of Pseudomonas spp., is involved in suppression of several fungal root pathogens as well as plant-parasitic nematodes. The primary objective of this study was to determine whether Wood1R, a D-genotype strain of DAPG-producing P. fluorescens, suppresses numbers of both sedentary and migratory plant-parasitic nematodes. An experiment was conducted in steam-heated soil and included two seed treatments (with Wood1R and a control without the bacterium) and six plant-nematode combinations which were Meloidogyne incognita on cotton, corn, and soybean; M. arenaria on peanut; Heterodera glycines on soybean; and Paratrichodorus minor on corn. Wood 1R had no effect on final numbers of M. arenaria, P. minor, or H. glycines; however, final numbers of M. incognita were lower when seeds were treated with Wood1R than left untreated, and this reduction was consistent among host plants. Population densities of Wood1R were greater on the roots of corn than on the other crops, and the bacterium was most effective in suppressing M. incognita on corn, with an average reduction of 41%. Despite high population densities of Wood1R on corn, the bacterium was not able to suppress numbers of P. minor. When comparing the suppression of M. incognita on corn in natural and steam-heated soil, egg production by the nematode was suppressed in natural compared to steamed soil, but the presence of Wood1R did not result in additional suppression of the nematodes in the natural soil. These data indicate that P. fluorescens strain Wood1R has the capacity to inhibit some populations of plant-parasitic nematodes. However, consistent suppression of nematodes in natural soils seems unlikely.     

Cloning and Sequencing of the Gene of Tryptophan-7-Halogenase from Pseudomonas fluorescens Strain CHA0

The gene of tryptophan-7-halogenase from the Pseudomonas fluorescens strain CHA0, a producer of the halogenated antibiotic pyrrolnitrin, has been cloned and sequenced.     

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.     

Gluconic acid: an antifungal agent produced by Pseudomonas species in biological control of take-all

Pseudomonas strain AN5 (Ps. str. AN5), a non-fluorescent Australian bacterial isolate, is an effective biological control (biocontrol) agent of the take-all disease of wheat caused by the fungus Gaeumannomyces graminis var. tritici (Ggt). Ps. str. AN5 controls Ggt by producing an antifungal compound which was purified by thin layer and column chromatography, and identified by NMR and mass spectroscopic analysis to be d-gluconic acid. Commercially bought pure gluconic acid strongly inhibited Ggt. Two different transposon mutants of Ps. str. AN5 which had lost take-all biocontrol did not produce d-gluconic acid. Gluconic acid production was restored, along with take-all biocontrol, when one of these transposon mutants was complemented with the corresponding open reading frame from wild-type genomic DNA. Gluconic acid was detected in the rhizosphere of wheat roots treated with the wild-type Ps. str. AN5, but not in untreated wheat or wheat treated with a transposon mutant strain which had lost biocontrol. The antifungal compounds phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol, produced by other Pseudomonads and previously shown to be effective in suppressing the take-all disease, were not detected in Ps. str. AN5 extracts. These results suggest that d-gluconic acid is the most significant antifungal agent produced by Ps. str. AN5 in biocontrol of take-all on wheat roots.     

Assembly and overexpression of membrane proteins in Escherichia coli

The bacterium Escherichia coli is one of the most popular model systems to study the assembly of membrane proteins of the so-called helix-bundle class. Here, based on this system, we review and discuss what is currently known about the assembly of these membrane proteins. In addition, we will briefly review and discuss how E. coli has been used as a vehicle for the overexpression of membrane proteins.     

l-Lysine-2-monooxygenase production by strains of Pseudomonas fluorescens and P. putida

Twelve strains of Pseudomonas fluorescens and P. putida were grown in a synthetic medium that contained l-lysine as the only source of carbon and nitrogen, and screened for l-lysine-2-monooxygenase production. Best production was by P. putida BKM B-1458 at 30 IU/g wet wt biomass when grown in a shake-flask but 25 IU/g in a 250-l fermenter.     

Functional characterisation of sexual stage specific proteins in Plasmodium falciparum

The various stages of the malaria parasites in the vertebrate host and in the mosquito vector offer numerous candidates for vaccine and drug development. However, the biological complexity of the parasites and the interaction with the immune system of the host continue to frustrate all such efforts thus far. While most of the targets for drug and vaccine design have focused on the asexual stages, the sexual stages of the parasite are critical for transmission and maintenance of parasites among susceptible vertebrate hosts. Sexual stage parasites undergo a series of morphological and biochemical changes during their development, accompanied by a co-ordinated cascade of a distinct expression pattern of sexual stage specific proteins. Mechanisms underlying the developmental switch from asexual parasite to sexual parasite still remain elusive. Methods that can break the malaria transmission cycle thus occupy a central place in the overall malaria control strategies. This paper provides a review of genes expressed in sexually differentiated Plasmodium. In the past few years, a molecular approach based on targeted gene disruption has revealed fascinating biological roles for many of the sexual stage gene products. In addition, we will briefly discuss other functional genomic approaches employed to study not only sexual but also other aspects of host-parasite biology.