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PubMed1994

Controlled-expression shuttle vector for pseudomonads based on the trpIBA genes of Pseudomonas putida.

A cloning vector pPS7 (8.5 kb) for Pseudomonas was constructed from pBR322 and the Pseudomonas cryptic low-copy-number pMK1 plasmid. The vector confers resistance to kanamycin (Km) and tetracycline (Tc), contains the par locus of Pseudomonas plasmid pMT2 and a mob site. The new vector was used for construction of controlled-expression vector pPS10 (10.4 kb) based on the trpIBA genes of Pseudomonas putida. This KmR vector contains the trpI gene, encoding activator protein and promoter of trpBA genes (Pba), which are inducible by TrpI and indoleglycerol phosphate (InGP). InGP is an unstable compound, but it accumulates in trpE mutants grown in anthranilate (Anth)-supplemented medium. We show that expression of the Escherichia coli pheA gene, inserted into the pPS10 vector downstream from Pba, increases about 70-fold upon InGP accumulation.

Escherichia coli↗

PubMed1985

Availability of 6-hydroxynicotinic acid for rapid identification of Pseudomonas aeruginosa and Serratia marcescens.

Gas chromatography--mass spectrometry has been used to identify specific metabolites produced by Gram-negative bacteria such as Pseudomonas aeruginosa, Serratia marcescens, Klebsiella pneumoniae and Escherichia coli in a defined medium. 6-Hydroxynicotinic acid was detected in spent culture media of Pseudomonas aeruginosa and Serratia marcescens, but could not be detected in those of Klebsiella pneumoniae and Escherichia coli. The production of 6-hydroxynicotinic acid was recognized by the addition of nicotinic acid in urine with Pseudomonas aeruginosa or Serratia marcescens, but not without the addition of nicotinic acid. Among 10(5) Pseudomonas aeruginosa per 1 ml of urine (criteria for the diagnosis of urinary tract infection), 0.15 microgram of 6-hydroxynicotinic acid was detected in urine at 4 h incubation with nicotinic acid at the optimum pH of 6.9, 38 degrees C. The production of 6-hydroxynicotinic acid was proportional to the number of the bacteria and displayed a time dependency. These results suggest that the availability of 6-hydroxynicotinic acid might make for more rapid identification of bacteria than current methods.

Culture Media↗

PubMed2004

Expression of L-ornithine Ndelta-oxygenase (PvdA) in fluorescent Pseudomonas species: an immunochemical and in silico study.

Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth.

Amino Acid Sequence↗

PubMed2005

Stress-related Pseudomonas genes involved in production of bacteriocin LlpA.

Pseudomonas sp. BW11M1 produces a novel type of bacteriocin that inhibits the growth of Pseudomonas putida GR12-2R3 and some phytopathogenic fluorescent Pseudomonas. A collection of mutants was screened for altered bacteriocin production phenotypes. Strongly reduced bacteriocin production was found to be caused by inactivation of the recA gene or the spoT gene. Conversely, in a recJ mutant, the bacteriocin was constitutively overproduced. The same phenotype was observed for a mutant hit in a gene of unknown function. The predicted gene product belongs to a distinct subgroup of prokaryotic helicase-like proteins within the SWI/SNF family of regulatory proteins. One mutant that also exhibited a bacteriocin overproducer phenotype was deficient in the production of the peptidoglycan-associated lipoprotein OprL. This study shows that various environmental stress response pathways are involved in controlling expression of the Pseudomonas sp. BW11M1 bacteriocin.

Bacterial Outer Membrane Proteins↗

PubMed2004

Characterization of a second functional gene cluster for the catabolism of phenylacetic acid in Pseudomonas sp. strain Y2.

Pseudomonas sp. strain Y2 is a styrene degrading bacterium that mineralises this compound through its oxidation to phenylacetic acid (PAA). We previously identified a complete gene cluster (paa1 cluster) for the degradation of phenylacetate, but, surprisingly, some paa1 deletion mutants were still able to catabolize styrene (STY) suggesting that this strain contained a second catabolic pathway. We report here the characterization of a second and novel paa2 gene cluster comprising 17 genes related to the catabolism of phenylacetate. We have identified a new gene (paaP) that is most likely involved in a transport process. Remarkably, the organization of the paa2 gene cluster is more similar to that of Pseudomonas putida KT2440 than to the paa1 gene cluster. Two new genes of undefined function were located inside the paa2 cluster. Sequence comparison between the paa2 genes and the paa1 and paa clusters of Pseudomonas sp. strain Y2 and P. putida KT2440, respectively, revealed a similar degree of divergence among the three sets of genes. Differences in the gene organization between paa1 and paa2 clusters of Pseudomonas sp. strain Y2 can be explained by an independent evolutionary history, probably associated with the adjacent sty genes. Deletion of either the first (paa1) or the second (paa2) gene cluster did not affect the ability of strain Y2 to grow in phenylacetate, whereas the deletion of both clusters led to the loss of this ability. The co-existence of two functional gene clusters for the degradation of phenylacetic acid in a bacterium has not been reported so far.

Base Sequence↗

PubMed2004

Changes in whole cell-derived fatty acids induced by naphthalene in bacteria from genus Pseudomonas.

Fatty acid composition during naphthalene utilization was investigated in three strains of bacteria Pseudomonas vesicularis, Pseudomonas stutzeri and Pseudomonas sp. JS150 that expressed different naphthalene degradation abilities. All strains significantly changed their cellular fatty acid profiles as a response to naphthalene exposure. Since naphthalene was present in the medium P. stutzeri increased ratio of saturated/unsaturated fatty acids from 1.1 to 2.1 and Pseudomonas sp. JS150 from 7.5 to 12.0, respectively. In contrast, this ratio decreased from 2.1 to 1.1 in P. vesicularis under the same growth conditions. The changes comprised also alterations in the percentage of selected groups of fatty acids: iso and anteiso, hydroxy and cyclopropane fatty acids. Our results showed that naphthalene induced in tested strains different changes in fatty acids composition. It may suggest that in the presence of naphthalene microorganisms used different adaptive mechanisms to maintain the cells in appropriate physiological state.

Biodegradation, Environmental↗

PubMed2006

Characterisation of Pseudomonas aeruginosa isolated from freshwater culture systems.

Members of the genus Pseudomonas are important phytopathogens and agents of human infections, while other strains and species exhibit bioremediation and biocontrol activities. Species-specific detection of Pseudomonas species in the environment may help to gain a more complete understanding of the ecological significance of these microorganisms. The objective of present study was comparative analysis of biochemically and PCR based confirmed 10 isolates of Pseudomonas aeruginosa (6 from fish intestine and 4 from pond sediment). PCR-ribotyping and PAGE revealed that there was extensive heterogeneity at the genetic and protein levels. Both genetic and phenotypic heterogeneity were more in the sediment isolates compared to the fish isolates. SDS-PAGE clearly demonstrated the differences between fish and sediment isolates as evident from the higher range of protein profiling. In antibiotic sensitivity test no habitat specific antibiogram was obtained. Zinc adversely affected the DNA of all the isolates to be amplified by PCR as DNA banding pattern was different from normal DNA in stressed DNA. Thus stress, particularly, zinc may interfere monitoring of Pseudomonas by PCR.

Animals↗

PubMed2005

Quantification of the Pseudomonas population in New Zealand soils by fluorogenic PCR assay and culturing techniques.

The genus Pseudomonas contains fast-growing nutritionally versatile bacteria that are able to utilize a wide variety of carbon sources. The ubiquity of the genus has been highlighted by conventional microbiology and the genus is well represented in collections of cultured bacteria. Here we evaluate the Pseudomonas population in New Zealand soils by comparing a culture-independent (real-time PCR combined with fluorescent TaqMan technology) with a culture-dependent (Gould's S1) population estimate. We show that cultivated fluorescent pseudomonads are not numerically dominant and represent a small proportion of <1% of the total Pseudomonas population, and that the total Pseudomonas population itself represents only a small proportion of <1% of the total bacterial population.

Bacteriological Techniques↗

PubMed2026

A novel biocontrol Pseudomonas species with broad-spectrum antagonistic activity against phytopathogens.

Bacterial and fungal diseases cause significant losses in horticultural crops, and biocontrol using beneficial microorganisms offers a sustainable alternative to chemical pesticides. In this study, a novel Pseudomonas strain D3 was isolated from Actinidiae rhizosphere. D3 exhibited strong antibacterial activity in LB medium but showed no activity against fungi or oomycetes. However, when cultured in KIDO medium, it demonstrated potent antifungal activity. Phylogenetic analysis based on 16S rRNA gene showed that D3 was most closely related to Pseudomonas mosselii CIP_105259T, while whole-genome sequencing revealed ANI values below 95% with eight known P. mosselii strains. Digital DNA-DNA hybridization (dDDH) further confirmed its genomic distinctiveness, with the highest dDDH value (58.2%) against the type strain P. mosselii DSM 17497T, well below the 70% species delineation threshold, supporting D3 as a novel Pseudomonas species. Functional validation via targeted gene knockout revealed a dichotomy in the antagonistic mechanisms of D3. Knockout of individual biosynthetic gene clusters (BGCs) only partially reduced antibacterial activity against Pseudomonas syringae pv. actinidiae, indicating that multiple BGCs contribute to this activity in a partially redundant manner. In contrast, disruption of a specific lipopeptide synthase cluster completely abolished antifungal activity against Valsa mali. LC-MS/MS analysis confirmed that this lipopeptide was produced exclusively in KIDO medium, consistent with the observed medium-dependent antifungal activity. Detached leaf and twig assays showed that D3 provides strong preventive biocontrol against both pathogens. Collectively, strain D3 employs a dual biocontrol mechanism, combining antibacterial activity mediated by multiple BGCs with lipopeptide-dependent antifungal activity, positioning it as a promising agent for sustainable disease management in horticultural crops.

Pseudomonas↗

PubMed2005

Plasmid host-range: restrictions to F replication in Pseudomonas.

Host-range, a fundamental property of a bacterial plasmid, is primarily determined by the plasmid replication system. To investigate the basis of the restricted host-range of the well-studied F-plasmid of Escherichia coli, we characterized in vitro the interactions of the host DnaA initiation protein and DnaB helicase from Pseudomonas aeruginosa and Pseudomonas putida with the replication origin, oriS, and initiation protein, RepE, of the RepFIA replicon. The results presented here show that a pre-priming complex can form at the F-origin with the replication proteins from the non-native hosts in the presence of RepE. However, RepE cannot form a stable complex with DnaB of P. aeruginosa or P. putida but does stably interact with E. coli DnaB. This unstable association may affect the ability of F to replicate in Pseudomonas. In addition, replication studies in vivo suggest that inefficient expression of the RepE initiation protein from its native promoter in Pseudomonas is a factor in restricting its host-range. This, however, is not the only barrier to F replication, as mini-F derivatives with an alternative promoter for RepE expression do not replicate in P. putida and are not stably maintained in P. aeruginosa.

Adenosine Triphosphatases↗

PubMed2005

Metabolic activation of bladder procarcinogens, 2-aminofluorene, 4-aminobiphenyl, and benzidine by Pseudomonas aeruginosa and other human endogenous bacteria.

Pseudomonas aeruginosa, an opportunistic pathogen of the human urinary tract, and other selected human endogenous bacteria were investigated for metabolic activation of the bladder procarcinogens, 2-aminofluorene (2-AF), 4-aminobiphenyl (4-AB), and benzidine (Bz). The cell-free extracts of Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Staphylococcus saprophyticus, Klebsiella pneumoniae, and intestinal anaerobes, Bacteroides fragilis, Clostridium perfringens, and Eubacterium aerofaciens produced increased histidine revertant frequencies with the tester strain Salmonella typhimurium TA98 in the Ames Salmonella mutagenicity assay. In addition, the cell-free extracts of Pseudomonas aeruginosa, Bacteroides fragilis, and Eubacterium aerofaciens each showed the presence of a cytochrome P450 absorption peak in the carbon monoxide (CO) difference spectrum. This was not demonstratable for the other bacteria. Our findings indicate that human endogenous bacteria, which are opportunistic pathogens of the urinary bladder, can metabolically activate the bladder procarcinogens 2-AF, 4-AB, and Bz into mutagens. The metabolic activation by Pseudomonas aeruginosa, Bacteroides fragilis, and Eubacterium aerofaciens is mediated by a cytochrome P450 enzyme. For those organisms that induced metabolic activation but did not show a P450 absorption peak with the cell-free extracts, other oxidative enzymes may be involved.

Aminobiphenyl Compounds↗

PubMed2000

[Postsurgical meningitis caused by Pseudomonas aeruginosa: study of 15 cases and review of the literature].

OBJECTIVE: Pseudomonas aeruginosa meningitis is a rare condition which is usually associated with pathology in the ORL field, neurosurgery or local neurologic manipulations. The characteristics, epidemiology, and course of this entity were determined. METHODS: Fifteen episodes of nosocomial postsurgical Pseudomonas aeruginosa meningitis occurred between 1989 and 1996 were retrospectively analyzed. RESULTS: A previous cranioencephalic trauma was recorded in 46.6% of patients. The portals of entry included: intraventricular catheter (IC) (12 cases), CSF fistula (2 cases), and craniotomy (1 case). In five occasions (41.6%) the microorganism was also recovered from the intraventricular catheter. Once culture results were available, therapy with active drugs against Pseudomonas was instituted and in 7 occasions was accompanied by the removal of IC. Eight patients eventually cured and two patients relapsed. The absence of cure was significantly associated with non-removal of the IC (p < 0.01). The infection resulted in death in 26.6% of patients. CONCLUSIONS: Postsurgical Pseudomonas aeruginosa meningitis is an entity of growing relevance. It is associated with relevant morbi-mortality. Catheter removal is essential to obtain a favorable outcome.

Adolescent↗

PubMed1975

Anti-Pseudomonas heat-stable opsonins in acute lymphoblastic leukemia of childhood.

Heat-stable opsonic activity against Pseudomonas aeruginosa and Staphylococcus epidermidis was measured in sera of 33 children with acute lymphoblastic leukemia at selected times during treatment of their disease. Compared to adults, opsonization of P. aeruginosa was normal in children tested at the time of diagnosis and before chemotherapy. Immediately after achievement of remission, opsonic activity against Pseudomonas was significantly decreased (P smaller than 0.05) compared with pretreatment activity. Activity usually returned to normal and remained so during long-term remission maintenance therapy. In children studied just prior to death from unremitting leukemia, however, anti-Pseudomonas opsonic activity was significantly decreased when compared with that of a group of children before any leukemic treatment (p smaller than 0.005). Anti-S. EPIDERMIDIS OPSONIC ACTIVITY SHOWED NO CHANGES DURING THE PATIENT'S COURSE. Decreased serum opsonic activity may significantly contribute to the increased incidence of severe Pseudomonas infections in patients with acute lymphoblastic leukemia.

Acute Disease↗

PubMed1976

Studies of introital colonization in women with recurrent urinary infections. V. The inhibitory activity of normal vaginal fluid on Proteus mirabilis and Pseudomonas aeruginosa.

Normal vaginal fluid from premenopausal volunteers was inoculated with 10 strains of Proteus mirabilis and 14 strains of Pseudomonas aeruginosa at pH's of 4.3, 4.6 and 4.9. All bacteria were killed at pH 4.3. Nine of 10 strains of Proteus mirabilis and 12 of 14 Pseudomonas aeruginosa were killed at pH 4.6. Only 4 of 14 strains of Pseudomonas aeruginosa were killed at pH 4.9, while 8 of 10 strains of Proteus mirabilis were killed at the same pH. We conclude that in comparison to the common 0 group strains of Escherichia coli, vaginal fluid is more bactericidal to Proteus mirabilis and Pseudomonas aeruginosa and that these observations may help explain the relative infrequency of bacteriuria owing to the organisms.

Escherichia coli↗

PubMed2001

Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads.

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.

Environmental Monitoring↗

PubMed1999

The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri.

The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.

Alcaligenes↗

PubMed1997

Infectious pseudomonas subglottic stenosis occurring in a patient with acquired immunodeficiency syndrome.

Pseudomonas aeruginosa is emerging as an increasingly common opportunistic infective agent in the immunocompromised human immunodeficiency virus (HIV) positive patient (Kielhofner et al., 1992). Improvements in the prevention and treatment of opportunistic infections in HIV and acquired immunodeficiency syndrome (AIDS) has led to longer life expectancy (Graham et al., 1992), and this has changed the incidence of Pseudomonas aeruginosa infection in this population (Baron and Hollander, 1993). We present a case of a patient with AIDS who developed a fulminant Pseudomonas aeruginosa stenosing subglottic infection. We are unaware of any previous reports of this particular manifestation of Pseudomonas aeruginosa infection.

AIDS-Related Opportunistic Infections↗

PubMed1984

Persistent isolation of an unusual Pseudomonas species from a phenolic disinfectant system.

A well-characterized unusual Pseudomonas species contaminated the piped disinfectant system of a newly-opened laminar air flow intensive care facility. This organism was frequently isolated (10(4)-10(6) cfu/ml) from phenolic diluted 1:256 in the system, and could also be recovered (0.01-0.2 cfu/ml) from undiluted phenolic. During the 20-month period when this unusual Pseudomonas was present, none of the severely compromised, granulocytopenic oncology patients treated in the intensive care facility were either colonized or infected with this Pseudomonas sp. Eradication of the organism from the system proved difficult and was accomplished by removing a contaminated reservoir of diluted phenolic disinfectant followed by transient cleansing of the system with very high concentrations (84,000 ppm) of chlorine. This experience demonstrates that phenolics should be added to the list of disinfectants which can harbor Pseudomonas spp. in the clinical setting.

Air Microbiology↗