Heavy metals resistant plasmid-mediated utilization of solar by Pseudomonas aeruginosa AA301

Solar-degrading bacteria, Pseudomonas aeruginosa strains, were isolated from Egyptian soil by Mineral Salt Medium (MSM) supplemented with Solar (motor fuel) from different oil-contaminated sites in Sohag province. The strain AA301 of Pseudomonas aeruginosa showed appreciable growth in MSM medium containing high concentrations of Solar ranging from 0.5 to 3% (v/v), with optimum concentration at 1.5%. Solar was used as a sole carbon source and a source of energy by the bacterium. The ability to degrade Solar was found to be associated with a single 60-kb plasmid designated pSOL15. The plasmid-cured variant, which was obtained by culturing in LB broth with kanamycin, lost the plasmid indicative the ability to degrade Solar must depend on this plasmid. The wild type isolate, Pseudomonas aeruginosa AA301 and transformant strain, have maximum growth (OD600 = approximately 2) on Solar, however the plasmid-cured variant did not have any significant growth on Solar. Moreover, resistance to a wide range of heavy metals such as Mn2+, Hg2+, Mg2+, Cd2+, Zn2+, and Ni2+ was also 60-kb plasmid-mediated. Therefore, the strain AA301 could be good candidate for remediation of some heavy metals and oil hydrocarbons in heavily polluted sites.     

Conjugal transfer of plasmid pNB2 to activated sludge bacteria leads to 3-chloroaniline degradation in enrichment cultures

AIMS: The involvement of the aniline-degradative plasmid pNB2 in degradation of 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: Plate matings of a Pseudomonas putida strain containing pNB2 with a mixed bacterial culture derived from activated sludge was carried out. After inoculation of the mating mixtures into batch cultures containing 3-CA, degradation of the compound was observed. A total of five different transconjugant strains could be isolated from one of the batch cultures and two of them were able to degrade 3-CA. These two isolates were identified as Comamonas testosteroni by partial 16S rDNA sequencing. CONCLUSIONS: It can be assumed that pNB2 carries a part of the genes involved in the catabolism of 3-CA, but that completion of the pathway must be provided by chromosomal genes in the host strain. SIGNIFICANCE AND IMPACT OF THE STUDY: pNB2 is a candidate plasmid which can be used in plasmid-mediated bioaugmentation of wastewater bacteria involved in degradation of chlorinated anilines.     

Plasmid-encoded enzymatic degradation of dimethoate

Dimethoate-degrading enzymatic activity in Bacillus licheniformis, Pseudomonas aeruginosa, Aeromonas hydrophila, Proteus mirabilis and Bacillus pumilus was found to be 6.4, 1.760, 4.09, 1.196 and 0.505 units/mg protein, respectively. The Escherichia coli C600 transconjugants of the isolated bacterial strains also exhibited dimethoate-degrading enzymatic activities. The cured derivatives did not show any decrease in the amount of dimethoate substrate and did not harbour plasmid as found in the original and transconjugant strains. Thus, the ability of enzymatic degradation of dimethoate was plasmid-mediated in B. licheniformis, Ps. aeruginosa, A. hydrophila, P. mirabilis and B. pumilus.     

Plasmid-mediated bioaugmentation of activated sludge bacteria in a sequencing batch moving bed reactor using pNB2

AIMS: The applicability of plasmid pNB2 for bioaugmentation of bacteria in model wastewater treatment reactors receiving 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: A setup of three biofilm reactors was studied, all initially inoculated with bacteria from activated sludge. Reactor PB received a Pseudomonas putida pNB2 donor strain not able to degrade 3-CA. Positive control reactor P received a 3-CA degrading Comamonas testosteroni pNB2-transconjugant. The negative control reactor N remained unchanged. Reactor P showed 3-CA degradation from the beginning of the experiment whereas in reactor PB, degradation started after an initial lag period. No degradation was observed in reactor N. PCR analysis showed that the P. putida donor abundance dropped in reactor PB, whereas the plasmid abundance did not, indicating transfer to other bacteria. A number of different 3-CA degrading C. testosteroni strains carrying pNB2 could be isolated from reactor PB. CONCLUSIONS: A successful plasmid-mediated bioaugmentation was achieved with C. testosteroni being the dominant 3-CA degrading pNB2 transconjugant species active in reactor PB. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the potential of gene transfer to contribute to establishment and spread of genetic information in general, particularly emphasizing the spread of xenobiotic degrading potential by dissemination of catabolic genes.     

Microbial degradation of quinoline and methylquinolines.

Several bacterial cultures were isolated that are able to degrade quinoline and to transform or to degrade methylquinolines. The degradation of quinoline by strains of Pseudomonas aeruginosa QP and P. putida QP produced hydroxyquinolines, a transient pink compound, and other undetermined products. The quinoline-degrading strains of P. aeruginosa QP and P. putida QP hydroxylated a limited number of methylquinolines but could not degrade them, nor could they transform 2-methylquinoline, isoquinoline, or pyridine. Another pseudomonad, Pseudomonas sp. strain MQP, was isolated that could degrade 2-methylquinoline. P. aeruginosa QP was able to degrade or to transform quinoline and a few methylquinolines in a complex heterocyclic nitrogen-containing fraction of a shale oil. All of the quinoline- and methylquinoline-degrading strains have multiple plasmids including a common 250-kilobase plasmid. The 225-, 250-, and 320-kilobase plasmids of the P. aeruginosa QP strain all contained genes involved in quinoline metabolism.     

Microbial degradation of quinoline and methylquinolines

Several bacterial cultures were isolated that are able to degrade quinoline and to transform or to degrade methylquinolines. The degradation of quinoline by strains of Pseudomonas aeruginosa QP and P. putida QP produced hydroxyquinolines, a transient pink compound, and other undetermined products. The quinoline-degrading strains of P. aeruginosa QP and P. putida QP hydroxylated a limited number of methylquinolines but could not degrade them, nor could they transform 2-methylquinoline, isoquinoline, or pyridine. Another pseudomonad, Pseudomonas sp. strain MQP, was isolated that could degrade 2-methylquinoline. P. aeruginosa QP was able to degrade or to transform quinoline and a few methylquinolines in a complex heterocyclic nitrogen-containing fraction of a shale oil. All of the quinoline- and methylquinoline-degrading strains have multiple plasmids including a common 250-kilobase plasmid. The 225-, 250-, and 320-kilobase plasmids of the P. aeruginosa QP strain all contained genes involved in quinoline metabolism.     

Kelthane degradation by genetically engineered Pseudomonas aeruginosa BS827 in a soil ecosystem

The aim of this study was to study the degradation of kelthane by Pseudomonas aeruginosa BS827, which carried the plasmid pBS3. This plasmid encodes naphthalene oxidation. The strain was able to survive in the presence of kelthane and to retain its degradative ability. Kelthane also stabilized the biodegradative plasmid that was preserved by 70 to 100% of the cell population. Cells deficient in Nah or Sal characters were less effective in degrading kelthane, whereas plasmid-free cells lost this ability completely. Evidently, the degradative activity of P. aeruginosa BS827 was conditioned by plasmid determinants coupled with genes of the plasmid pBS3 Nah region.     

Kelthane degradation by genetically engineered Pseudomonas aeruginosa BS827 in a soil ecosystem.

The aim of this study was to study the degradation of kelthane by Pseudomonas aeruginosa BS827, which carried the plasmid pBS3. This plasmid encodes naphthalene oxidation. The strain was able to survive in the presence of kelthane and to retain its degradative ability. Kelthane also stabilized the biodegradative plasmid that was preserved by 70 to 100% of the cell population. Cells deficient in Nah or Sal characters were less effective in degrading kelthane, whereas plasmid-free cells lost this ability completely. Evidently, the degradative activity of P. aeruginosa BS827 was conditioned by plasmid determinants coupled with genes of the plasmid pBS3 Nah region.     

Recruitment of a chromosomally encoded maleylacetate reductase for degradation of 2,4-dichlorophenoxyacetic acid by plasmid pJP4.

When Pseudomonas aeruginosa PAO1c or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes enzymes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not grow on TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6- and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1.     

Inducible UV repair potential of Pseudomonas aeruginosa PAO

Pseudomonas aeruginosa PAO lacks UV-inducible Weigle reactivation and Weigle mutagenesis of UV-damaged bacteriophages. This lack of UV-inducible, error-prone DNA repair appears to be due to the absence of efficiently expressed umuDC-like genes in this species. When the P. aeruginosa recA gene is introduced into a recA(Def) mutant of Escherichia coli K12, the P. aeruginosa recA gene product is capable of mediating UV-induced mutagenesis, indicating that it could participate in a recA-lexA-like regulatory network and function in inducible DNA repair pathways if such existed in P. aeruginosa. The presence of the IncP9, UV-resistance plasmid R2 in RecA+ strains of P. aeruginosa PAO allows UV-inducible, mutagenic DNA repair of UV-irradiated bacteriophages. R2 also greatly stimulates the ability of UV radiation to induce mutagenesis of the bacterial chromosome. When R2 is introduced into P. aeruginosa strains containing either the recA908 or recA102 mutation, plasmid-mediated UV resistance and Weigle reactivation are not observed. These observations suggest that the increased protection afforded to P. aeruginosa by R2 is derived from a RecA-mediated, DNA-damage-inducible, error-prone DNA repair system which complements the lack of a chromosomally encoded umuDC-like operon.     

Plasmid-mediated Molluscicide Bayluscide Degradation by Bacterial Strain Isolated from Schistosome Vector Snail Bulinus truncates

Pseudomonas spp. strain Bal1 was isolated from Schistosome vector snails Bulinus Truncates. Strain Bal1 was identified as Pseudomonas putida using partial sequence of 16s rRNA. This strain was able to utilize a Bayluscide as a sole source of carbon and nitrogen. The degradation of Bayluscide by Bal1 strain is mediated by pBal1 (110 Kb) plasmid. The loss of the plasmid resulted irreversibly in a derivative strain that was unable to degrade Bayluscide. The transfer of these plasmids from wild-type strain Bal1 to Bal1M derivative restored completely its capability to degrade the molluscicide. It is proposed that pBal1 is a conjugative plasmid and is involved in the Bayluscide degradation. The effect of bacterial degradation upon toxicity was tested, and it was shown that the molluscicidal action of Bayluscide was significantly reduced by bacterial action.     

TOL is a broad-host-range plasmid.

We readily isolated insertions of the carbenicillin resistance element Tn401 into the TOL plasmid in Pseudomonas putida. Hybrid TOL::Tn401 plasmids stably express the Cbr phenotype in Pseudomonas aeruginosa and Escherichia coli. Whereas the replicative and conjugative functions are expressed in both hosts, the ability to grow on m-toluate is only expressed in the Pseudomonas species.     

长链烷烃降解菌的降解特性

对长链烷烃降解菌的降解能力和摄取模式进行了研究。评价14株烃降解菌利用中长链烃生长的能力,发现只有少数烃降解菌能够获得良好生长,其中Mycobacterium fortuitum514,Pseudomonas aeruginosa1785和Pseudomonas marginata766等3株菌能够高效降解C20到C33的长链烷烃。辛烷不能支持这些长链烷烃降解菌的生长,说明其烃氧化酶与Pseudomonas oleovorans的OCT质粒编码的单氧酶不同。此外,M.fortuitum不产胞外表面活性剂,而P.aeruginosa和P.marginata则是表面活性剂产生菌,然而三者在以烃为碳源生长时均显示出很高的细胞表面疏水性。根据生长现象分析3株菌采用了不同的烷烃摄取模式。     

Some evidences for the involvement of plasmid in diuron herbicide degradation

Pseudomonas sp. strain Bk8 was isolated from field soil contaminated with different urea-herbicides. This strain is a plasmid (pBkB)-harbouring organism capable of complete degradation of diuron herbicide. Plasmid-cured strain Bk8M was obtained by treatment of Pseudomonas sp. Bk8 with Mitomycin C. This cured strain is capable of only partial degradation of diuron side chain and accumulated a phenolic compound in the medium during growing on diuron as a sole source of carbon and energy. Conjugation experiment was carried out using Bk8M as a recipient and Bk8 as a donor of pBk8 plasmid. The transconjugant was able to degrade a diuron without accumulation of phenolic compound. It was proposed that plasmid pBk8 is self-transmissible and involved in the degradation of diuron aromatic ring but it is not connected with the transformation of diuron into diuron phenol compound.     

Mineralization of methyl tert-butyl ether and other gasoline oxygenates by Pseudomonads using short n-alkanes as growth source

Biodegradation of methyl tert-butyl ether (MTBE) by cometabolism has shown to produce recalcitrant metabolic intermediates that often accumulate. In this work, a consortium containing Pseudomonads was studied for its ability to fully degrade oxygenates by cometabolism. This consortium mineralized MTBE and TBA with C3-C7 n-alkanes. The highest degradation rates for MTBE (75 +/- 5 mg g(protein) (-1) h(-1)) and TBA (86.9 +/- 7.3 mg g(protein) (-1) h(-1)) were obtained with n-pentane and n-propane, respectively. When incubated with radiolabeled MTBE and n-pentane, it converted more than 96% of the added MTBE to (14)C-CO(2). Furthermore, the consortium degraded tert-amyl methyl ether, tert-butyl alcohol (TBA), tert-amyl alcohol, ethyl tert-butyl ether (ETBE) when n-pentane was used as growth source. Three Pseudomonads were isolated but only two showed independent MTBE degradation activity. The maximum degradation rates were 101 and 182 mg g(protein) (-1) h(-1) for Pseudomonas aeruginosa and Pseudomonas citronellolis, respectively. The highest specific affinity (a degrees (MTBE)) value of 4.39 l g(protein) (-1) h(-1) was obtained for Pseudomonas aeruginosa and complete mineralization was attained with a MTBE: n-pentane ratio (w/w) of 0.7. This is the first time that Pseudomonads have been reported to fully mineralize MTBE by cometabolic degradation.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Multiple genes coding for octopine-degrading enzymes in Agrobacterium.

Most biotype 2 strains of Agrobacterium tumefaciens and A. radiobacter which utilize nopaline also degrade octopine. In all such strains studied, the ability to degrade octopine did not appear to be transferred to plasmidless recipient cells under conditions of plasmid transfer in which the ability to utilize nopaline was transferred. An octopine-degrading mutant was isolated in a strain cured of its plasmid, suggesting that genes of octopine degradation may have a chromosomal location in some strains. In strains in which octopine utilization is coded by plasmid genes, octopine degradation was always inducible, whereas in strains which degrade both octopine and nopaline, octopine utilization was constitutive although nopaline degradation was inducible. When plasmids coding for octopine-utilizing ability were transformed into a strain containing either a nopaline- or null-type plasmid, transformants able to degrade octopine were either not observed or were unstable upon purification. All of these data suggest that plasmids associated with virulence are incompatible with one another, and therefore imply that the major groups of plasmids associated with virulence have a common origin.     

The branched-chain dodecylbenzene sulfonate degradation pathway of Pseudomonas aeruginosa W51D involves a novel route for degradation of the surfactant lateral alkyl chain.

Pseudomonas aeruginosa W51D is able to grow by using branched-chain dodecylbenzene sulfonates (B-DBS) or the terpenic alcohol citronellol as a sole source of carbon. A mutant derived from this strain (W51M1) is unable to degrade citronellol but still grows on B-DBS, showing that the citronellol degradation route is not the main pathway involved in the degradation of the surfactant alkyl moiety. The structures of the main B-DBS isomers and of some intermediates were identified by gas chromatography-mass spectrometric analysis, and a possible catabolic route is proposed.     

Isolation and characterization of Pseudomonas putida R-prime plasmids

A number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient. For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved. Physical analysis of one R-prime showed that 3.5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.     

Identification of small RNAs involved in nitrogen fixation in Anabaena sp. PCC 7120 based on RNA-seq under steady state conditions

The aim of the present study was to investigate the tolerance of five new Achromobacter and Pseudomonas strains to kerosene and to establish if the production of several secondary metabolites increases or not when these bacteria were grown in the presence of kerosene. The biodegradation of kerosene by isolated bacteria was also investigated in this study. Five Proteobacteria were isolated from different samples polluted with petroleum and petroleum products. Based on their morphological, biochemical, and molecular characteristics, isolated bacteria were identified as Achromobacter spanius IBBPo18 and IBBPo21, Pseudomonas putida IBBPo19, and Pseudomonas aeruginosa IBBPo20 and IBBPo22. All these bacteria were able to tolerate and degrade kerosene. Higher tolerance to kerosene and degradation rates were observed for P. aeruginosa IBBPo20 and IBBPo22, compared with that observed for A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19. All these bacteria were able to produce several secondary metabolites, such as surfactants and pigments. Glycolipid surfactants produced by P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 have a very good emulsification activity, and their activity increased when they were grown in the presence of kerosene. The production of rhamnolipid surfactants by P. aeruginosa IBBPo20 and IBBPo22 was confirmed by detection of rhlAB gene involved in their biosynthesis. Pyocyanin and pyoverdin pigments were produced only by P. aeruginosa IBBPo20 and IBBPo22, while carotenoid pigments were produced by all the isolated bacteria. Significant changes in pigments production were observed when P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 were grown in the presence of kerosene. Due to their ability to tolerate and degrade kerosene, and also to produce several secondary metabolites, the isolated bacteria could be used in the bioremediation of kerosene-polluted environments.