Utilization of Dimethoate by Wild Type Pseudomonas putida from Polluted Sites in Iran

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

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Effect of Organic Solvents on the Yield of Solvent-Tolerant Pseudomonas putida S12

Applied and Environmental Microbiology ◽

10.1128/aem.65.6.2631-2635.1999 ◽

1999 ◽

Vol 65 (6) ◽

pp. 2631-2635 ◽

Cited By ~ 70

Author(s):

Sonja Isken ◽

Antoine Derks ◽

Petra F. G. Wolffs ◽

Jan A. M. de Bont

Keyword(s):

Growth Rate ◽

Pseudomonas Putida ◽

Organic Solvents ◽

Critical Concentration ◽

Bacterial Membrane ◽

Maximal Growth ◽

Wild Type ◽

Active Efflux ◽

Maximal Growth Rate ◽

Solvent Tolerant

ABSTRACT Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h−1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.

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Characterization of a Pseudomonas putida Rough Variant Evolved in a Mixed-Species Biofilm with Acinetobacter sp. Strain C6

Journal of Bacteriology ◽

10.1128/jb.00041-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4932-4943 ◽

Cited By ~ 50

Author(s):

Susse Kirkelund Hansen ◽

Janus A. J. Haagensen ◽

Morten Gjermansen ◽

Thomas Martini Jørgensen ◽

Tim Tolker-Nielsen ◽

...

Keyword(s):

Natural Selection ◽

Pseudomonas Putida ◽

Wild Type ◽

Low Oxygen ◽

Phenotypic Characteristics ◽

Mixed Species ◽

Evolutionary Forces ◽

Enhanced Production ◽

Oxygen Starvation ◽

Biofilm Development

ABSTRACT Genetic differentiation by natural selection is readily observed among microbial populations, but a more comprehensive understanding of evolutionary forces, genetic causes, and resulting phenotypic advantages is not often sought. Recently, a surface population of Pseudomonas putida bacteria was shown to evolve rapidly by natural selection of better-adapted variants in a mixed-species biofilm consortium (S. K. Hansen, P. B. Rainey, J. A. Haagensen, and S. Molin, Nature 445:533-536, 2007). Adaptation was caused by mutations in a wapH homolog (PP4943) involved in core lipopolysaccharide biosynthesis. Here we investigate further the biofilm physiology and the phenotypic characteristics of the selected P. putida rough colony variants. The coexistence of the P. putida population in a mixed-species biofilm with Acinetobacter sp. strain C6 is dependent on the benzoate excreted from Acinetobacter during the catabolism of benzyl alcohol, the sole carbon source. Examination of biofilm development and the dynamics of the wild-type consortium revealed that the biofilm environment became oxygen limited, possibly with low oxygen concentrations around Acinetobacter microcolonies. In contrast to P. putida wild-type cells, which readily dispersed from the mixed-species biofilm in response to oxygen starvation, the rough variant cells displayed a nondispersal phenotype. However, in monospecies biofilms proliferating on benzoate, the rough variant (like the wild-type population) dispersed in response to oxygen starvation. A key factor explaining this conditional, nondispersal phenotype is likely to be the acquired ability of the rough variant to coaggregate specifically with Acinetobacter cells. We further show that the P. putida rough variant displayed enhanced production of a cellulose-like polymer as a consequence of the mutation in wapH. The resulting phenotypic characteristics of the P. putida rough variant explain its enhanced fitness and ability to form tight structural associations with Acinetobacter microcolonies.

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Peptides’ involvement in Pseudomonas putida biofilm formation

10.5194/biofilms9-38 ◽

2020 ◽

Author(s):

Riho Teras ◽

Hanna Ainelo ◽

Marge Puhm

Keyword(s):

Pseudomonas Putida ◽

Mutant Strain ◽

Type Strain ◽

Deletion Mutant ◽

Wild Type Strain ◽

Positive Impact ◽

Proteinase K ◽

Wild Type ◽

Positive Effect ◽

Positively Charged

Pseudomonas putida rapidly forms a biofilm, after which its biomass usually disperses to half its initial amount. We have observed different biofilm dynamics of P. putida in a complex medium LB and a minimal medium M9+glc+CAA and inquired about the importance of extracellular factors for the formation of P. putida biofilm. The proteinaceous component of LB increases the biomass of P. putida biofilm. Supplementation of M9 with tryptone but not CAA increased the biofilm biomass. Proteinase K treatment of LB medium reduced the biomass of P. putida biofilm. At the same time, growth rate or maximum OD of planktic bacteria in used media did not correlate with biofilm biomass of the same media. Thus, peptides appeared to have a positive effect on the biofilm as an extracellular factor and not as a source of C and N. We replaced tryptone in M9 medium with positively charged poly-L-lysine (MW. 1000-5000 Da), negatively charged poly-L-glutaminic acid (MW. 1500-5500 Da) or neutral poly-LD-alanine (MW. 3000-7000). Poly-lysine and poly-glutamic acid had a slight positive effect on the biomass of P. putida wild type strain PSm biofilm and poly-alanine did not affect the biofilm. We have previously shown that overexpression of fis in P. putida strain F15 increases biofilm biomass by increasing the lapA expression, the main adhesin gene of biofilm. Using media similar to that used for the wild-type strain for strain F15, we ascertained that only poly-lysine out of these three polypeptides restored the positive effect of fis-overexpression on the biofilm biomass. At the same time, the positive impact of fis-overexpression was absent in lapA deletion mutant strain, but not in lapF deletion mutant strain. In conclusion, the formation of P. putida biofilm depends on polypeptides in the environment. The enhancing effect of positively charged polypeptides appears to be evident in the presence of LapA, a key factor for P. putida biofilm.

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Catalase activity and the survival of Pseudomonas putida, a root colonizer, upon treatment with peracetic acid

Canadian Journal of Microbiology ◽

10.1139/w01-002 ◽

2001 ◽

Vol 47 (3) ◽

pp. 222-228 ◽

Cited By ~ 6

Author(s):

Anne J Anderson ◽

Charles D Miller

Keyword(s):

Hydrogen Peroxide ◽

Pseudomonas Putida ◽

Peracetic Acid ◽

Catalase Activity ◽

Specific Activity ◽

Active Oxygen Species ◽

Oxygen Species ◽

Deficient Mutant ◽

Wild Type ◽

Cell Extracts

Peracetic acid is used as a sterilant in several industrial settings. Cells of a plant-colonizing bacterium, Pseudomonas putida in liquid suspension, were more sensitive to killing by peracetic acid when they lacked a major catalase activity, catalase A. Low doses of peracetic acid induced promoter activity of the gene encoding catalase A and increased total catalase specific activity in cell extracts. Microbes present in native agricultural soils rapidly degraded the active oxygen species present in peracetic acid. The simultaneous release of oxygen was consistent with a role for catalase in degrading the hydrogen peroxide that is part of the peracetic acid-equilibrium mixture. Amendment of sterilized soils with wild-type P. putida restored the rate of degradation of peracetic acid to a higher level than was observed in the soils amended with the catalase A-deficient mutant. The association of the bacteria with the plant roots resulted in protection of the wild-type as well as the catalase-deficient mutant from killing by peracetic acid. No differential recovery of the wild-type and catalase A mutant of P. putida was observed from roots after the growth matrix containing the plants was flushed with peracetic acid.Key words: Pseudomonas putida (Pp), activated oxygen species (AOS), hydrogen peroxide, luciferase, colonization.

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Site-Directed Mutagenesis Identifies a Molecular Switch Involved in Copper Sensing by the Histidine Kinase CinS in Pseudomonas putida KT2440

Journal of Bacteriology ◽

10.1128/jb.00551-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5304-5311 ◽

Cited By ~ 13

Author(s):

Davide Quaranta ◽

Megan M. McEvoy ◽

Christopher Rensing

Keyword(s):

Pseudomonas Putida ◽

Histidine Kinase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Copper Binding ◽

Wild Type ◽

Two Component System ◽

Pseudomonas Putida Kt2440 ◽

Component System ◽

Two Component

ABSTRACT In the presence of copper, Pseudomonas putida activates transcription of cinAQ via the two-component system CinS-CinR. The CinS-CinR TCS was responsive to 0.5 μM copper and was specifically activated only by copper and silver. Modeling studies of CinS identified a potential copper binding site containing H37 and H147. CinS mutants with H37R and H147R mutations had an almost 10-fold reduced copper-dependent induction of cinAQ compared to the wild type.

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Competitiveness in root colonization by Pseudomonas putida requires the rpoS gene

Canadian Journal of Microbiology ◽

10.1139/w00-123 ◽

2001 ◽

Vol 47 (1) ◽

pp. 41-48 ◽

Cited By ~ 6

Author(s):

Charles D Miller ◽

Young-Cheol Kim ◽

Anne J Anderson

Keyword(s):

Pseudomonas Putida ◽

Culture Medium ◽

Wild Type Strain ◽

Root Colonization ◽

Plant Root ◽

Wild Type ◽

Rpos Gene ◽

Activated Oxygen ◽

Increased Sensitivity ◽

Dose Dependent

The rpoS gene in Pseudomonas putida was essential for plant root colonization under competitive conditions from other microbes. The RpoS- mutant survived less well than the wild-type strain in culture medium, and unlike the wild-type, failed to colonize the roots in a peat matrix containing an established diverse microflora. The RpoS-deficient P. putida isolate was generated by insertion of a glucuronidase-npt cassette into the rpoS gene. The RpoS- mutant had dose-dependent increased sensitivity to oxidative stress and produced Mn-superoxide dismutase activity earlier than the parent. While extracts from wild-type P. putida stationary-phase cells contained three isozymes of catalase (CatA, CatB, and CatC), the σ38-deficient P. putida lacked CatB. These results are consistent with previous findings that CatB is induced in stationary-phase.Key words: catalase, starvation, activated oxygen species.

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Assimilation of Nitrogen from Nitrite and Trinitrotoluene in Pseudomonas putida JLR11

Journal of Bacteriology ◽

10.1128/jb.187.1.396-399.2005 ◽

2005 ◽

Vol 187 (1) ◽

pp. 396-399 ◽

Cited By ~ 40

Author(s):

Antonio Caballero ◽

Abraham Esteve-Núñez ◽

Gerben J. Zylstra ◽

Juan L. Ramos

Keyword(s):

Pseudomonas Putida ◽

Parental Strain ◽

Glutamate Synthase ◽

Regulatory Proteins ◽

Wild Type ◽

Nitrate And Nitrite

ABSTRACT Pseudomonas putida JLR11 releases nitrogen from the 2,4,6-trinitrotoluene (TNT) ring as nitrite or ammonium. These processes can occur simultaneously, as shown by the observation that a nasB mutant impaired in the reduction of nitrite to ammonium grew at a slower rate than the parental strain. Nitrogen from TNT is assimilated via the glutamine syntethase-glutamate synthase (GS-GOGAT) pathway, as evidenced by the inability of GOGAT mutants to use TNT. This pathway is also used to assimilate ammonium from reduced nitrate and nitrite. Three mutants that had insertions in ntrC, nasT, and cnmA, which encode regulatory proteins, failed to grow on nitrite but grew on TNT, although slower than the wild type.

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Mechanism of Chloride Elimination from 3-Chloro- and 2,4-Dichloro-cis,cis-Muconate: New Insight Obtained from Analysis of Muconate Cycloisomerase Variant CatB-K169A

Journal of Bacteriology ◽

10.1128/jb.183.15.4551-4561.2001 ◽

2001 ◽

Vol 183 (15) ◽

pp. 4551-4561 ◽

Cited By ~ 20

Author(s):

Ursula Kaulmann ◽

Stefan R. Kaschabek ◽

Michael Schlömann

Keyword(s):

Pseudomonas Putida ◽

Wild Type ◽

Wild Type Enzyme ◽

Significant Rate ◽

Chloride Elimination ◽

Muconate Cycloisomerase

ABSTRACT Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate tocis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation ofcis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such ascis,cis-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-muconate. Interestingly,cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.

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Identification of Bacterial Proteins Mediating the Interactions Between Pseudomonas putida UW4 and Brassica napus (Canola)

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-6-0686 ◽

2009 ◽

Vol 22 (6) ◽

pp. 686-694 ◽

Cited By ~ 26

Author(s):

Zhenyu Cheng ◽

Jin Duan ◽

Youai Hao ◽

Brendan J. McConkey ◽

Bernard R. Glick

Keyword(s):

Pseudomonas Putida ◽

Root Exudates ◽

Translational Regulation ◽

Cell Envelope ◽

Effective Means ◽

Wild Type ◽

Bacterial Strains ◽

Uncharacterized Protein ◽

Altered Expression ◽

Bacterial Interactions

The influence of canola root exudates on the proteome of Pseudomonas putida UW4 and the mutant strain P. putida UW4/AcdS–, which lacks a functional 1-aminocyclopropane-1-carboxylate deaminase gene, was examined using two-dimensional difference in-gel electrophoresis. Seventy-two proteins with significantly altered expression levels in the presence of canola root exudates were identified by mass spectrometry. Many of these proteins are involved in nutrient transport and utilization, cell envelope synthesis, and transcriptional or translational regulation and, hence, may play important roles in plant–bacterial interactions. Four proteins showing large changes in expression in response to canola root exudates in both the wild-type and mutant strains of P. putida UW4 (i.e., outer membrane protein F, peptide deformylase, transcription regulator Fis family protein, and a previously uncharacterized protein) were both overexpressed and disrupted in P. putida UW4 in an effort to better understand their functions. Functional studies of these modified strains revealed significantly enhanced or inhibited plant-growth-promoting abilities compared with the wild-type P. putida UW4, in agreement with the suggested involvement of three of these four proteins in plant–bacterial interactions. The work reported here suggests strategies to both identify potential antibacterial agents and develop bacterial strains that might be useful adjuncts to agriculture. This approach may be an effective means of identifying key proteins mediating the interactions of bacteria with their rhizosphere environment.

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