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AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.

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AIMS: This study investigated the influence of water chemistry on copper solvation (cuprosolvency) by pure culture biofilms of heterotrophic bacteria isolated from copper plumbing. METHODS AND RESULTS: Heterotrophic bacteria isolated from copper plumbing biofilms including Acidovorax delafieldii, Flavobacterium sp., Corynebacterium sp., Pseudomonas sp. and Stenotrophomonas maltophilia were used in laboratory coupon experiments to assess their potential for cuprosolvency. Sterile copper coupons were exposed to pure cultures of bacteria to allow biofilm formation and suspended in drinking waters with different chemical compositions. Sterile coupons not exposed to bacteria were used as controls. After 5 days of incubation, copper release and biofilm accumulation was quantified. The results demonstrated that cuprosolvency in the control experiments was influenced by water pH, total organic carbon (TOC) and conductivity. Cuprosolvency in the presence of biofilms correlated with the chemical composition of the water supplies particularly pH, Langeliers Index, chloride, alkalinity, TOC and soluble phosphate concentrations. CONCLUSIONS: The results suggest water quality may influence cuprosolvency by biofilms present within copper plumbing pipes. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for water chemistry to influence cuprosolvency by biofilms may contribute to the sporadic nature of copper corrosion problems in distribution systems.

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Two new transposon-based tagging vectors have been constructed using the gfp marker gene under control of either constitutive or inducible promoters. The two vectors, along with the established pUTminiTn5gfp were used to tag a diesel-degrading Pseudomonas strain. Tagged strains were obtained that were not affected in terms of their growth or ability to use diesel as a carbon source. The transposon tags were stably maintained in the strains without selection and provided visible fluorescence as colonies or single cells in suspension. Tagging did not impede the survival of tagged Pseudomonas aeruginosa GP41B strains in diesel-contaminated soil microcosms. The tagged strains were easily recovered from the microcosms after a 3-month period. The tagging of bacteria with gfp using either native or introduced constitutive/inducible promoters is an effective and easy way to monitor their survival in soil.

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Petroleum hydrocarbons are widespread environmental pollutants. Although biodegradation of petroleum hydrocarbons has been the subject of numerous investigations, information on their toxicity to microorganisms in soil is limited, with virtually no work conducted on soil algae. We carried out a screening experiment for total petroleum hydrocarbons (TPH) and their toxicity to soil algal populations, microbial biomass, and soil enzymes (dehydrogenase and urease) in a long-term TPH-polluted site with reference to an adjacent unpolluted site. Microbial biomass, soil enzyme activity, and microalgae declined in medium to high-level (5,200-21,430 mg kg(-1) soil) TPH-polluted soils, whereas low-level (<2,120 mg kg(-1) soil) pollution stimulated the algal populations and showed no effect on microbial biomass and enzymes. However, inhibition of all the tested parameters was more severe in soil considered to have medium-level pollution than in soils that were highly polluted. This result could not be explained by chemical analysis alone. Of particular interest was an observed shift in the species composition of algae in polluted soils with elimination of sensitive species in the medium to high polluted soils. Also, an algal growth inhibition test carried out using aqueous eluates prepared from polluted soils supported these results. Given the sensitivity of algae to synthetic pollutants, alteration in the algal species composition can serve as a useful bioindicator of pollution. The results of this experiment suggest that chemical analysis alone is not adequate for toxicological estimations and should be used in conjunction with bioassays. Furthermore, changes in species composition of algae proved to be more sensitive than microbial biomass and soil enzyme activity measurements.

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A set of vectors containing a mutated gfp gene was constructed for use with Gram-negative bacteria other than Escherichia coli. These constructs were: pTn3gfp for making random promoter probe gfp insertions into cloned DNA in E. coli for subsequent introduction into host strains; pUTmini-Tn5gfp for making random promoter probe gfp insertions directly into host strains; p519gfp and p519nfp, broad host range mob+ plasmids containing gfp expressed from a lac and an npt2 promoter, respectively.

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pTIM3 is a suicide plasmid vector for the delivery of a transposition defective derivative of Tn5, expressing inducible mercury resistance (HgR) and catechol 2,3-dioxygenase (C23O) activity, to a range of gram-negative microorganisms. pTIM3 was constructed by a four-stage process from pNMM1, a derivative of pSUP5011 containing a modified Tn5 where antibiotic-resistance determinants have been replaced by the mer operon of Tn501 and tdnC (encoding a C23O). In pTIM3, tdnC is fused to merD, to bring C23O expression under the control of the mer operon. pTIM3 was introduced into Acinetobacter calcoaceticus, Pseudomonas putida, Burkholderia cepacia, and Alcaligenes sp., and isolates were examined for loss of the vector, which is not stably maintained outside the Enterobacteriaceae, but retention of the Tn5 derivative. Transposition results from the action of a vector-encoded Tn5 transposase (Tnp) on the ends of IS50L and IS50R retained in the Tn5 construct. Between 10 and 20% of the isolates contained a single copy of the defective transposon in the chromosome. A single-copy isolate of each species was assayed for inducibility of C23O activity using HgCl2 as inducer. The increase in C230 activity was linear with increasing HgCl2 concentration and ranged between 10- and 20-fold at 20 micrograms/ml. HgR and C23O+ phenotypes were found to be stably maintained in each of the isolates following 40 generations of nonselective growth. A novel aspect of this marker gene system is that isolates can be recovered on nonselective medium and then sprayed with a catechol/HgCl2 mixture for rapid colorimetric detection. The system can be applied to the tracking of genetically modified microorganisms in the environment and has the advantage that their detection may be achieved cheaply and without the use of sophisticated equipment.

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The survival of selected naturally occurring and genetically engineered bacteria in a fully functional laboratory-scale activated-sludge unit (ASU) was investigated. The effect of the presence of 3-chlorobenzoate (3CB) on the survival of Pseudomonas putida UWC1, with or without a chimeric plasmid, pD10, which encodes 3CB catabolism, was determined. P. putida UWC1(pD10) did not enhance 3CB breakdown in the ASU, even following inoculation at a high concentration (3 x 10(8) CFU/ml). The emergence of a natural, 3CB-degrading population appeared to have a detrimental effect on the survival of strain UWC1 in the ASU. The fate of two 3CB-utilizing bacteria, derived from activated-sludge microflora, was studied in experiments in which these strains were inoculated into the ASU. Both strains, AS2, an unmanipulated natural isolate which flocculated readily in liquid media, and P. putida ASR2.8, a transconjugant containing the recombinant plasmid pD10, survived for long periods in the ASU and enhanced 3CB breakdown at 15 degrees C. The results reported in this paper illustrate the importance of choosing strains which are well adapted to environmental conditions if the use of microbial inoculants for the breakdown of target pollutants is to be successful.

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A restriction endonuclease map was derived for the aromatic amine and m-toluate catabolic plasmid pTDN1 present in Pseudomonas putida UCC22, a derivative of P. putida mt-2. The plasmid is 79 +/- 1 kbp in size and can be divided into a restriction-site-deficient region of 51 +/- 1 kbp and a restriction-site-profuse region of 28 kbp which begins and ends with directly repeating sequences of at least 2 kbp in length. A mutant plasmid isolated after growth of the host on benzoate had lost the restriction-profuse region by a straightforward recombinational loss retaining one copy of the direct repeat. Analysis of clones, deletion and Tn5 insertion mutants strongly suggested that the meta-cleavage pathway of pTDN1 was situated in the region readily deleted. The catechol 2,3-dioxygenase (C23O) gene of pTDN1 showed no hybridization or restriction homology to previously described C23O genes of TOL plasmids pWW0 and pWW15. In addition, there was little homology between intact pTDN1, pWW0 and pWW15, suggesting the presence of a unique meta-cleavage pathway. We also demonstrated that pTDN1 did not originate from P. putida mt-2 chromosome.

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The possibility of the accidental or deliberate release of genetically engineered microorganisms into the environment has accentuated the need to study their survival in, and effect on, natural habitats. In this study, Pseudomonas putida UWC1 harboring a non-self-transmissible plasmid, pD10, encoding the breakdown of 3-chlorobenzoate was shown to survive in a fully functioning laboratory-scale activated-sludge unit (ASU) for more than 8 weeks. The ASU maintained a healthy, diverse protozoal population throughout the experiment, and the introduced strain did not adversely affect the functioning of the unit. Although plasmid pD10 was stably maintained in the host bacterium, the introduced strain did not enhance the degradation of 3-chlorobenzoate in the ASU. When reisolated from the ASU, derivatives of strain UWC1 (pD10) were identified which were able to transfer plasmid pD10 to a recipient strain, P. putida PaW340, indicating the in situ transfer of mobilizing plasmids from the indigenous population to the introduced strain. Results from plate filter matings showed that bacteria present in the activated-sludge population could act as recipients for plasmid pD10 and actively expressed genes carried on the plasmid. Some of these activated-sludge transconjugants gave higher rates of 3-chlorobenzoate breakdown than did strain UWC1(pD10) in batch culture.

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Pseudomonas putida mt-2 (ATCC 33015) carrying the TOL plasmid pWW0 could adapt to growth on the aromatic amines aniline and m- and p-toluidine. In strain UCC2, a derivative adapted to rapid growth on these compounds, they were oxidatively deaminated to catechol or 4-methylcatechol, which in turn were dissimilated by a meta-cleavage pathway. The aniline/toluidine oxygenase and the meta-cleavage pathway enzymes were inducible by aromatic amines. Evidence is presented that in strain UCC2, plasmid pWW0 has undergone deletion of its catabolic genes, and that it is a novel plasmid, pTDN1, which is involved in the catabolism of aniline and m- and p-toluidine. The meta-cleavage pathway genes which are carried by pTDN1 were shown not to have originated in pWW0.

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