Pentachlorophenol degradation by Pseudomonas aeruginosa

Five Pseudomonas species were tested for ability to degrade pentachlorophenol (PCP). Pseudomonas aeruginosa completely degraded PCP up to 800 mg/l in 6 days with glucose as co-substrate. With 1000 mg PCP/l, 53% was degraded. NH₄ ⁺ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO₂, with release of Cl^-.The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India.     

Induction and selection of formaldehyde-based resistance inPseudomonas aeruginosa

Summary A formaldehyde resistant (R) phenotype ofPseudomonas aeruginosa was isolated from a formaldehydesensitive (S) parent by sequential treatment with 1,3,5-tris-(ethyl)hexahydro-s-triazine (ET). The resistance of the (R) strain to treatment with ET was approximately 3-fold higher than the parental (S) strain. Two modes of resistance to ET, and simultaneous resistance to formaldehyde, are demonstrated: (1) transient or induced resistance is expressed during shor-term exposure to ET, and this resistance is gradually lost during subsequent growth in the absence of ET, and (2) resistance that results from a stable phenotypic change in the (S) strain following sequential treatment with ET ((R) strain phenotype). The observed activities of three forms of the formaldehyde oxidizing enzyme, formaldehyde dehydrogenase, are strongly correlated with the relative response of the (S) and (R) strains to treatment with ET. The observed resistance of the (R) strain appears to be due to high levels of an NAD⁺-linked, glutathione-dependent form of formaldehyde dehydrogenase as well as a dye-linked formaldehyde dehydrogenase. The transient or induced response of the (R) strain involves an increase in activity of the dye-linked formaldehyde dehydrogenase. The induced response of the (S) strain and an ATCC strain ofP. aeruginosa, however, is correlated with the two forms of the NAD⁺-linked enzyme (glutathione-dependent (EC 1.2.1.1) and independent (EC 1.2.1.46)) with no contribution from the dye-linked enzyme.     

Repeated removal of cadmium and zinc from an industrial effluent by waste biomass Sargassum sp.

Waste biomass Sargassum sp. biosorbed 100% of Cd²⁺ and 99.4% of Zn²⁺ from a 3 and 98 mg l^–1 solution (pH 4.5), respectively, at the end of four serial experiments. Of the five desorbents studied in consecutive adsorption/desorption cycles, CaCl₂ 0.05 M eluted nearly 40% of both metals and decreased the biosorption in only 8% and 17% of Cd²⁺ and Zn²⁺, respectively. Although NaOH desorbent improved the heavy metal uptake from the second cycle onwards, it did not elute metals from the pre-loaded biomass.     

Mechanism of zinc resistance in Pseudomonas putida strain S4

The mechanism of Zn resistance in multiple metal-resistant Pseudomonas putida strain S4 is based on inducible efflux. An ATPase in the strain S4 mediated active extrusion of Zn²⁺, which occurred during the exponential phase of growth. The ATPase activity was inhibited by micromolar concentrations (50 M) of vanadate, suggesting the involvement of a P-type ATPase. The effluxed Zn²⁺ were not ejected out of the cell but stored in the outer membrane and periplasm, which provided the required binding sites. The strain S4, thus, employs a dual strategy of efflux and binding to bring about a proper management of essential ions like Zn.     

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twentyone of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.In memoriam of Professor R. Y. Stanier     

Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.     

Interleukin-1-deficient mice exhibit high sensitivity to gut-derived sepsis caused by Pseudomonas aeruginosa

BACKGROUND: The role of interleukin (IL)-1 in infectious diseases is controversial; some investigators indicated an enhancing effect of IL-1 on host resistance whereas others demonstrated the protective role of IL-1 receptor antagonist in infection. We evaluated the role of endogenous IL-1 in gut-derived sepsis caused by Pseudomonas aeruginosa, by comparing IL-1-deficient mice and wild-type (WT) mice. METHODS: Gut-derived sepsis was induced by intraperitoneal injection of cyclophosphamide after colonization of P. aeruginosa strain D4 in the intestine. RESULTS: The survival rate of IL-1-deficient mice was significantly lower than that of WT mice (P<0.01). Bacterial counts in the liver, mesenteric lymph node and blood were significantly higher in IL-1-deficient mice than in WT mice. Tumor necrosis factor alpha and IL-6 in the liver were significantly higher in IL-1-deficient mice than in WT mice. In vitro, phagocytosis and cytokine production by macrophages were impaired in IL-1-deficient mice compared with WT mice. CONCLUSION: Our results indicate a critical role for IL-1 during gut-derived P. aeruginosa sepsis. The results also suggest that both impairment of cytokine production and phagocytosis by macrophages are caused by IL-1 deficiency and lead to impaired host response.     

Biodegradation of 8-anilino-1-naphthalenesulfonic acid by Pseudomonas aeruginosa

Pseudomonas aeruginosa, isolated from soil near tannery effluent was able to degrade 8-anilino-1-naphthalenesulfonic acid (ANSA), a sulfonated aromatic amine. The organism degraded this amine up to a concentration of 1,200 mg l⁻¹ using glucose and ammonium nitrate as carbon and nitrogen sources respectively. The degradation started when the organism reached its late exponential growth phase. Salicylic acid and β-ketoadipic acid were identified as intermediate compounds using HPLC and GC–MS and provide evidence for ortho pathway reactions. Further proof for the pathway is obtained from the dioxygenase activity of the strain growing exponentially in medium with ANSA and glucose.     

Biosorption of thorium (IV) by a Pseudomonas biomass

Lyophilized biomass of a Pseudomonas soilisolate adsorbed thorium (IV) (430 mg g^–1 dry wt) optimally at pH 4, with 91% of equilibrium loading being reached in 1 min. Equilibrium metal sorption showing conformity to Langmuir isotherm model suggested a monolayered thorium binding. Thorium binding remained unaffected or slightly affected (< 20% inhibition) in presence of equimolar (430 M) concentration of several interfering ions except Fe³⁺ (40% inhibition). More than 90% of loaded thorium could be recovered using 1 M CaCO₃, though mineral acids and Na₂CO₃ were also effective.     

Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322 bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.     

Mutants of Pseudomonas aeruginosa unable to inactivate allantoinase and NADP-dependent glutamate dehydrogenase

Both allantoinase and NADP-GDH in Pseudomonas aeruginosa were inactivated when cells reached the stationary phase of growth. Mutants unable to inactivate these enzymes were isolated. Results with recombinants showed that the mutation is not located in the structural genes of these enzymes but in an independent gene involved in the inactivation.Abbreviations NADP-GDH NADP-dependent glutamate dehydrogenase - Ani^- mutant allantoinase non-inactivating mutant - GOGAT glutamate synthase     

Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2

Zinc concentrations ranging between 0.1 and 1 mm only slightly reduced maximal growth of wild-type Pseudomonas aeruginosa 7NSK2 in iron-limiting casamino acid medium, but had a clear negative effect on the growth of mutant MPFM1 (pyoverdin negative) and especially mutant KMPCH (pyoverdin and pyochelin negative). Production of pyoverdin by wild-type strain 7NSK2 was significantly increased in the presence of 0.5 mm zinc and could not be repressed by iron even at a concentration of 100 m. Siderophore detection via isoelectrofocusing revealed that mutant KMPCH did not produce any siderophores, while mutant MPFM1 overproduced a siderophore with an acidic isoelectric point, most likely pyochelin. Pyochelin production by MPFM1 was stimulated by the presence of zinc in a similar way as pyoverdin for the wild-type. Analysis of outer membrane proteins revealed that three iron regulated outer membrane proteins (IROMPs) (90, 85 and 75 kDa) were induced by iron deficiency in the wild-type, while mutants were found to have altered IROMP profiles. Zinc specifically enhanced the production of a 85 kDa IROMP in 7NSK2, a 75 kDa IROMP in MPFM1 and a 90 kDa IROMP in KMPCH.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Recognition of mucin components by Pseudomonas aeruginosa

Pseudomonas aeruginosa remains one of the most important bacterial pathogens in lung diseases and especially in Cystic fibrosis. This unusual predilection is best explained by the existence of defects in host defense mechanisms as resulting from the genetic lesion and the presence of a specific colonization niche within the lungs. The niche has been identified as the mucus layer wherein mucin glycoproteins provide a substrate for binding and allows the persistence of this organism in this milieu by a number of possible mechanisms. While this organism is capable of binding to non CF mucins, it is perhaps a combination of factors e.g. increased binding and decreased mucociliary clearance that is responsible for this marked state of colonization in CF. The organism uses chiefly proteins of its flagellar apparatus to initiate this binding and recognizes a variety of oligosaccharides that have been identified in mucins. Among these are both, neutral oligosaccharides and several forms of acidic oligosaccharides derived from the Lewis antigens. There are more than likely a larger repertoire of receptors than those identified and certainly more adhesins present than those currently known. However, the information gathered to date provides an excellent example of the specificity of bacterial interactions with mucins that will certainly be expanded as we study more pulmonary pathogens.     

Staphylolytic enzyme from Pseudomonas aeruginosa: characterization and immunocytochemical localization

Staphylolytic enzyme, a specific peptidase produced by Pseudomonas aeruginosa, has been characterized by using immunochemical procedures. Lytic activity was detected in the extracellular medium of Pseudomonas cultures at the beginning of the stationary growth phase. No activity was detected in bacterial cells. However, lytic protein antigen was present in periplasmic and cytoplasmic fractions, suggesting that staphylolytic enzyme is synthesized as an inactive precursor which becomes active during translocation to the extracellular broth. Results obtained in immunolocalization experiments indicate the presence of the precursor in the outer part of cells. The export pathway of staphylolytic enzyme through the periplasmic space is proposed.Abbreviations DCE dialyzed crude extract - CFU colonies forming units - LU lytic unit     

Desulfurization of light gas oil by a novel recombinant strain from Pseudomonas aeruginosa

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa NCIMB 9571 by using a transposon vector. Resting cells of the recombinant strain, PAR41, desulfurized 63 mg sulfur l^–1 of light gas oil (LGO) containing 360 mg S l^–1. The desulfurization activity for LGO by the resting cells of strain PAR41 grown with n-tetradecane (50% v/v) was much higher (1018-fold) than in glucose-grown cells. P. aeruginosa NCIMB 9571 is able to take up water-insoluble compounds from an oil phase which is enhanced by n-alkane.     

The stereoisomers of pyochelin, a siderophore of Pseudomonas aeruginosa

The Pseudomonas aeruginosa siderophore pyochelin is obtained from the bacterial culture medium as a mixture of two epimers. Chromatically isolated pure stereoisomers equilibrate readily in most solvents. Experiments will be reported which allow to isolate one of the isomers in pure form and which shed some additional light on the epimerization reaction.     

Annexin II is a novel receptor for Pseudomonas aeruginosa

Infections with Pseudomonas aeruginosa (P. aeruginosa) are critical in ventilated and poly-traumatized patients. Most important, these bacteria cause frequent and chronic pulmonary infections in patients with cystic fibrosis. Therefore, identification of molecular mechanisms that mediate the infection of mammalian cells with P. aeruginosa is urgently required. Here, we aimed to identify novel receptors that are involved in internalization of P. aeruginosa into mammalian epithelial cells. Employing SDS-PAGE purification and mass spectrometry we demonstrate that annexin II specifically binds to P. aeruginosa. The significance of the interaction of annexin II with P. aeruginosa for the infection of mammalian cells is indicated by the finding that neutralization of the ligands on P. aeruginosa by incubation of the bacteria with recombinant, soluble annexin II prevents internalization of P. aeruginosa into human epithelial cells.     

Adaptation to phenylacetamide as a growth substrate by an acetanilide-utilizing mutant of Pseudomonas aeruginosa

Mutants able to utilize phenylacetamide as sole nitrogen source were isolated from the acetanilide (N-phenylacetamide)-utilizing Pseudomonas aeruginosa mutant strain AI3 and from its parent strain L10. Growth properties of the mutants (Ph strains) on amide media and the physicochemical properties of their amidases in cell free extracts indicated that their phenylacetamidase activities were attributable to alterations in their amidases. Differences in amide hydrolase specificities between the AI3-and the L10-Ph mutants were observed. The AI3 group had a high level of activity towards 4-nitrophenylacetamide, activity towards phenylacetyl-4-nitroaniline but, unlike strain AI3, no activity towards acetyl-4-nitroaniline; the L10 group had a low activity towards 4-nitrophenylacetamide, no activity towards phenylacetyl-4-nitroaniline but retained the low level of activity towards acetyl-4-nitroaniline exhibited by strain L10. Confirmation of the association between these altered specificition of alterations in amidases was obtained from analysis of the properties of phenylacetamidases purified from an AI3-Ph mutant (pH5) and an L 10-Ph mutant (Ph14). The original mutation in the amidase gene of strain AI3 appeared responsible for the differences between the two groups of Ph mutants and the binding interactions with acetanilide that it determined were eliminated in AI3-Ph mutants.