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PubMed1999

Glutamine-enriched total parenteral nutrition preserves respiratory immunity and improves survival to a Pseudomonas Pneumonia.

BACKGROUND: Addition of 2% glutamine (GLN), a specific lymphocyte fuel, prevents deleterious effects of TPN on gut-associated lymphoid tissue and IgA while preserving IgA-mediated upper respiratory immunity to influenza virus. We examined whether a 2% GLN-enhanced TPN solution preserves respiratory immunity to a lethal and clinically relevant pneumonia challenge. MATERIALS AND METHODS: Male ICR mice were randomized to chow (n = 20), TPN (n = 20), or an isonitrogenous, isocaloric TPN-2% GLN solution (n = 17). All groups were immunized 10 days before surgery with Pseudomonas polysaccharide-containing liposomes (LIP) to confer immunity except for a nonimmune chow-fed LIP control group (n = 21) which received LIP without Pseudomonas. Mice received 5 days of diet and then were given an LD90 dose of 1.2 x 10(8) intratracheal Pseudomonas bacteria, and mortality was recorded. RESULTS: Immunization reduced mortality compared with LIP alone. TPN impaired immunity and reduced survival while GLN maintained immunization effectiveness. CONCLUSIONS: Pseudomonas immunization reduces mortality to Pseudomonas pneumonia, but this immunity is lost with TPN. Addition of 2% GLN to TPN preserves immunity in the respiratory tract and reduces mortality to a lethal bacterial challenge compared with standard TPN.

Animals↗

PubMed1992

The structural genes encoding CO dehydrogenase subunits (cox L, M and S) in Pseudomonas carboxydovorans OM5 reside on plasmid pHCG3 and are, with the exception of Streptomyces thermoautotrophicus, conserved in carboxydotrophic bacteria.

Employing deoxyoligonucleotide probes and Southern hybridizations, we have examined in carboxydotrophic bacteria the localization on the genome of genes encoding the large, medium and small subunits of CO dehydrogenase (coxL, M and S, respectively). In Pseudomonas carboxydovorans OM5 coxL, M and S were identified on the plasmid pHCG3; they were absent on the chromosome. This was evident from positive hybridizations with plasmid DNA of the wild-type strain OM5 and the absence of hybridizations with chromosomal DNA from the plasmid cured mutant strain OM5-12. The genes coxL, M and S were found on plasmids in all other plasmid-containing carboxydotrophic bacteria e.g. Alcaligenes carboxydus, Azomonas B1, Pseudomonas carboxydoflava, Pseudomonas carboxydovorans OM2 and OM4. Cox L, M and S could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, Pseudomonas carboxydohydrogena, and Pseudomonas carboxydovorans OM3. These results essentially confirm and extend former reports that cox genes are rather conserved among carboxydotrophic bacteria of distinct taxonomic position. However, Streptomyces thermoautotrophicus is an noteworthy exception since none of the three cox genes could be detected. This refers to a new type of CO dehydrogenase and is in accord with results indicating that the S. thermoautotrophicus CO dehydrogenase has an unusual electron acceptor specificity and some other properties setting it apart from the 'classical' CO dehydrogenases.

Alcaligenes↗

PubMed1994

Nucleotide sequence analysis and potential environmental distribution of a ferric pseudobactin receptor gene of Pseudomonas sp. strain M114.

The nucleotide sequence of the Pseudomonas sp. strain M114 pbuA gene, encoding the outer membrane receptor for ferric pseudobactin M114, has been determined. The region sequenced spans 2788 bases of plasmid pCUP3, within which the receptor gene had previously been localised. A single open reading frame, potentially encoding 826 amino acids and including a leader peptide of 44 amino acids, is evident and is followed by an inverted repeat segment, which may act as a transcriptional terminator. A 20 bp region of DNA, having significant homology with the E. coli Fur-binding consensus sequence, is located upstream of the open reading frame. PbuA displays characteristics in common with other outer membrane proteins and displays strong homology with the TonB boxes of both E. coli and Pseudomonas receptors. More extensive homologies were found with the PupA receptor of P. putida WCS358 and the FhuE and BtuB receptors of E. coli. It is suggested that areas exhibiting the least homology between these receptors may represent ferric siderophore-specific recognition sites of the PbuA protein. The deduced amino acid sequence of pbuA was compared with that of pupX, encoding the outer membrane receptor for ferric pseudobactin B10, of Pseudomonas sp. strain B 10. A direct alignment of the two proteins gave an identity score of 92.5%. The distribution of PbuA-like receptors among Pseudomonas isolates was investigated by DNA-DNA hybridisation analysis. The results suggest that a PbuA-like receptor may be widely distributed among Pseudomonas rhizosphere isolates.

Amino Acid Sequence↗

PubMed1994

Novel narrow-host-range vectors for direct cloning of foreign DNA in Pseudomonas.

Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in a Pseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly in Pseudomonas, DNA fragments from Agrobacterium, Pseudomonas, and Rhizobium. A clone containing Agrobacterium and KmR gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of five Pseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA in Pseudomonas.

Base Sequence↗

PubMed1995

Septic shock due to Pseudomonas aeruginosa in a previously healthy woman.

Pseudomonas aeruginosa septicemia rarely occurs in non-immunocompromised adults. We present a case of septic shock following Pseudomonas aeruginosa pneumonia in a previously healthy 48-year-old woman. The onset was sudden, with back pain, pyrexia and shock. Chest radiographs revealed pneumonia, and Pseudomonas aeruginosa was identified from blood and sputum cultures. Therapy with dopamine, piperacillin and fluid replacement led to a prompt recovery. Laboratory tests failed to reveal any immunological deficits. Including this case, only five cases of Pseudomonas aeruginosa septicemia in patients though to be non-immunocompromised have been reported. Two remarkable features of this type of Pseudomonas infection are apparent: i) it commonly develops from pneumonia and ii) it has a better prognosis than that in immunocompromised hosts.

Community-Acquired Infections↗

PubMed1985

Use of transposon mutants to assess the role of exoenzyme S in chronic pulmonary disease due to Pseudomonas aeruginosa.

Among the potential virulence factors produced by Pseudomonas aeruginosa there are two distinct ADP-ribosyl transferases, exotoxin A and exoenzyme S. The role of exoenzyme S in Pseudomonas aeruginosa infection was studied using the rat chronic pulmonary infection model. Pseudomonas aeruginosa strain DG1 and an isogenic mutant of DG1 differing only in its capacity to produce exoenzyme S were employed in the study. Both Pseudomonas aeruginosa strains tested established a chronic pulmonary infection in this model and organisms recovered from lung homogenates were phenotypically unaltered with respect to exoenzyme S production in vitro. The extent of the observed pathology was markedly greater with the strain producing exoenzyme S, indicating that exoenzyme S may play a role in the progressive pathology observed in chronic lung disease due to Pseudomonas aeruginosa.

ADP Ribose Transferases↗

PubMed1999

Identification and molecular characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199.

The gene loci ehyA and ehyB, which are involved in the bioconversion of eugenol to coniferyl alcohol by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a eugenol hydroxylase that represents an enzyme of the flavocytochrome c class. These genes were localized downstream of the eugenol catabolism genes vanA and vanB, encoding vanillate-O-demethylase, on an EcoRI fragment (E230) that has recently been cloned from a Pseudomonas sp. strain HR199 genomic library. The gene encoding the cytochrome c subunit (ehyA) was identified on a subfragment (K18) of E230 by complementation of a nitrosoguanidine-induced, eugenol-negative mutant of strain HR199. The nucleotide sequences of fragment K18 and adjacent regions were determined, revealing open reading frames of 354 and 1,554 bp that represent ehyA and ehyB, respectively. These genes are most probably organized in one operon together with a third open reading frame (ORF2) of 687 bp that was located between ehyA and ehyB. The deduced amino acid sequences of ehyA and ehyB exhibited up to 29 and 55% amino acid identity to the corresponding subunits of p-cresol methylhydroxylase from Pseudomonas putida. Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for a eugenol dehydrogenase of Pseudomonas fluorescens E118 corresponded well with appropriate regions of ehyA and ehyB. The sequence of ORF2 and the deduced amino acid sequence exhibited no significant similarities to any DNA or amino acid sequence from the databases. The eugenol hydroxylase genes were amplified by PCR, cloned in pBluescript SK(-), and functionally expressed in Escherichia coli. Transfer of a DNA fragment comprising ehyA and ehyB to various strains of Pseudomonas species that were unable to utilize eugenol as a carbon source conferred to these bacteria the ability to grow on this substrate.

Amino Acid Sequence↗

PubMed2002

Diversity of Pseudomonas spp. isolated from rice rhizosphere populations grown along a salinity gradient.

Along the coastline of Tamil Nadu, five sites were chosen to assess the diversity of Pseudomonas populations isolated from rice (Oryza sativa) cultivated along a salinity gradient. One of these sites was under organic farming while the other four were under inorganic farming. A total of 256 Pseudomonas strains isolated from these five sites were analyzed using both phenotypic (substrate utilization patterns and antibiotic resistance assay) and genotypic (PCR-RFLP of 16S rDNA) characteristics. The results derived from this study indicate that soil salinity affects rhizosphere Pseudomonas populations. It was observed that increasing salinity led to decreasing diversity. Fluorescent pseudomonads were the dominant species found in the non-saline site, while in the saline sites they were replaced by salt-tolerant species, in particular Pseudomonas alcaligenes and P. pseudoalcaligenes. An interesting observation was the increase in diversity found in the saline site under organic farming. Organic farming was found to be capable of mitigating the harmful effects of saline stress to a large extent, and restoring the Pseudomonas diversity, thereby making it comparable with the diversity encountered in the non-saline site.

Adaptation, Physiological↗

PubMed2003

Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by Pseudomonas stutzeri strain JJ.

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.

Anaerobiosis↗

PubMed2005

Effect of dissolved oxygen regime on growth dynamics of Pseudomonas spp during benzene degradation.

We investigated the effect of different oxygen regimes on growth patterns of Pseudomonas spp. during benzene degradation in microcosm batch studies. Benzene degradation was induced by limiting oxygen available for microbial activity, which consists of three initial-dissolved oxygen (DO) levels of oxic, hypoxic, and anoxic conditions. Batch experiments were performed for cell growth and benzene degradation by inoculating three strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) in mineral salt medium containing aqueous benzene. Results showed that all strains were capable to grow and degrade benzene under all oxygen regimes but in a different manner. The highest cell growth of P. aeruginosa and P. fluorescens was achieved under oxic and anoxic condition, respectively, but there was no substantial difference on benzene degradation between the oxygen treatments with about 25% reduction for both strains. P. putida showed a facultative process for both cell growth and benzene degradation. This reveals that care should be taken in selection of microorganisms with regard to environmental studies since they exhibit different responses for given environmental conditions such as DO levels.

Benzene↗

PubMed2001

Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones.

Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.

Burkholderia cepacia↗

PubMed2004

A polyhydroxybutyrate-producing Pseudomonas sp. isolated from Antarctic environments with high stress resistance.

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.

Antarctic Regions↗

PubMed2006

Different photoresponses of Staphylococcus aureus and Pseudomonas aeruginosa to 514, 532, and 633 nm low level lasers in vitro.

Low Level Light irradiation (LLLI) may proliferate cell growth at certain conditions during a set of photochemical reactions called biostimulation. However, phototoxic inhibitory reactions after irradiating natural or artificially inoculated cells are possible. The purpose of this study was to determine these effects on Pseudomonas aeruginosa and Staphylococcus aureus. Ar ion laser at 514 nm was used to determine the effect of various energy densities of green light on these bacteria. The most effective energy densities of Ar ion laser were chosen to irradiate both bacteria with He-Ne (633 nm) and SHG Nd:YAG (532 nm) lasers to compare the effect of red and green lights on the growth. Irradiation of Pseudomonas aeruginosa for both He-Ne and SHG Nd:YAG lasers in the presence of toluidine blue O or safranine O as photosensitizers was also studied. All energy densities of Ar ion laser showed a proliferative effect on Pseudomonas aeruginosa and inhibitory effect on Staphylococcus aureus. Similarly, SHG Nd:YAG and He-Ne lasers with chosen energy densities were again proliferating for Pseudomonas aeruginosa and inhibitory for Staphylococcus aureus and SHG Nd:YAG was more effective than He-Ne in both cases. Irradiation of Pseudomonas aeruginosa in the presence of both photosensitizers led to the decrease of the cell population compared to the control.

Argon↗

PubMed1986

Morphologic and microbiologic features of Pseudomonas aeruginosa pneumonia in normal hamsters.

A model of pneumonia due to Pseudomonas aeruginosa was produced in hamsters by an intratracheal bolus instillation of microorganisms. Sequential lung changes from 4 hr through 11 days were studied by morphologic and microbiologic methods. Hamsters inoculated with greater than 10(6) pseudomonads survived but consistently had histologic evidence of mild bronchopneumonia 24 hr postinoculation, whereas a severe bronchopneumonia and a 100% mortality were elicited with a 10(8) inoculum of organisms in 0.5 ml phosphate-buffered saline (PBS). An inoculum of 10(7) pseudomonads/0.5 ml PBS was then used to define the changes in the bacterial population in Pseudomonas pneumonia and to obtain serial histopathologic observations. Quantitative lung cultures obtained within 1 hr postinoculation demonstrated a mean of 10(6) colony forming units per lung, and none of the hamsters were bacteremic. However, by 24 hr bacterial counts had increased and all animals were bacteremic. Bacterial proliferation continued through 48 hr; however, the number of bacteremic animals had decreased. By 72 hr, bacterial counts had decreased with total Pseudomonas clearance noted by 120 hr. A striking polymorphonuclear leukocyte-rich alveolar exudate was present by 12 hr. Pseudomonas "vasculitis" was evident by 24 hr. The evolution of this vascular lesion correlated with the bacteremic state of the hamsters. By 11 days, resolution of the pneumonic process was seen. The macroscopic and microscopic features of this hamster model of Pseudomonas pneumonia are very similar to those reported in infected patients.

Animals↗

PubMedDate not supplied

Molecular cloning of the gamma-glutamyltranspeptidase gene from a Pseudomonas strain.

gamma-Glutamyltranspeptidase (GGT) was purified from a Pseudomonas sp. strain A14. The purified enzyme was found to be composed of two nonidentical subunits with molecular weights of 39,000 and 22,000 and had a pI of > 8.6. The partial N-terminal amino acid sequences of both subunits and some proteolytic fragments were determined. Using mixed oligonucleotides designed from the partial amino acid sequences as hybridization probes, one cosmid clone which contained the GGT gene was isolated from a Pseudomonas sp. strain A14 cosmid genome library, and the DNA sequence of the GGT gene was determined. The nucleotide sequence and the protein sequence analysis revealed that GGT was synthesized as a precursor protein of 575 amino acids and then processed to mature enzyme, presumably after removal of a signal peptide. Comparison of the predicted amino acid sequence of Pseudomonas GGT with published results for Escherichia coli K-12 and rat kidney GGTs shows that the protein sequence of Pseudomonas GGT is 51% and 33% identical to the E. coli and rat GGT sequences, respectively. Higher similarity is observed among the small subunits, which have been thought to have a binding site for the gamma-glutamyl residue. Expression of the cloned Pseudomonas GGT gene in E. coli was subjected to Western blot analysis using antibody raised against the purified GGT. This suggested that processing of the precursor protein to its subunits is temperature-dependent, because the amount of mature GGT protein was increased when the culture was performed at low temperature.

Amino Acid Sequence↗

PubMed1966

Microbial growth on C1 compounds. The role of folat in the metabolism of Pseudomonas AM1.

1. The growth of Pseudomonas AM1 is much more sensitive to inhibition by sulphanilamide when methanol, rather than succinate, acts as the sole carbon and energy source; a sulphanilamide concentration of 1mm, which causes almost complete inhibition of growth on methanol, has little effect in a succinate medium. 2. Similar results have been obtained with sulphadiazine and sulphathiazole. Sulphanilic acid has little effect. 3. A similar differential sensitivity to sulphanilamide is shown by Protaminobacter ruber and Pseudomonas extorquens when grown on methanol media as compared with succinate. 4. Sulphanilamide inhibited the growth of Pseudomonas oxalaticus on formate, oxalate and succinate media to about the same extent. 5. Inhibition of growth of Pseudomonas AM1 by sulphanilamide is accompanied by an accumulation of glycine in the cells. 6. Inhibition of growth by sulphanilamide can be reversed by p-aminobenzoic acid. 7. Microbiological assays of the folate content of Pseudomonas AM1 have been performed after growth on both methanol and succinate, and the results are discussed in terms of differences in metabolism.

Aminobenzoates↗

PubMed1976

Some spectral and steady-state kinetic properties of Pseudomonas cytochrome oxidase.

Some spectra of Pseudomonas cytochrome oxidase are reported, both for comparison with those of other workers and to illustrate the differences between the ascorbate- and dithionite-reduced forms of the enzyme. A spectrum of the reduced enzyme-CO complex, prepared in the absence of added reductants by incubation under CO, is also included. Ultracentrifugation studies yielded a value for the sedimentation coefficient (s20,w) of 7.5S, and an isoelectric point of pH6.9 was determined by isoelectric focusing. Steady-state kinetic constants of the electron donors, quinol, sodium ascorbate, reduced Pseudomonas azurin and Pseudomonas ferrocytochrome c551 were investigated giving Km values of 30mM, 4mM, 49muM and 5.6muM respectively. The two protein substrates were observed to be subject to product inhibition and the Ki for oxidized Pseudomonas azurin was evaluated at 4.9muM. Steady-state kinetics were also used to investigate the effects of the oxidation products of dithionite on the oxidase and nitrite reductase activities of Pseudomonas cytochrome oxidase. These experiments showed that whereas the oxidase activity was inhibited, the nitrite reductase activity was slightly enhanced.

Ascorbic Acid↗

PubMed1979

Some magnetic properties of Pseudomonas cytochrome oxidase.

The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.

Ascorbic Acid↗