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Three strains isolated by geosmin enrichment from a sand filter in an Australian drinking water treatment works were genome sequenced to identify their taxonomic placement, and a bench-scale batch experiment confirmed their geosmin-degrading capability. Using the average nucleotide identity based on the MUMmer algorithm (ANIm), pairwise digital DNA-DNA hybridization (dDDH), and phylogenomic analyses, the strains were identified as Sphingopyxis species., Competing Interests: The authors declare no conflict of interest.

The genomes of two Methanococcoides spp. that were isolated from marine sediments and are capable of carrying out methanogenesis from choline and other methylotrophic substrates were sequenced. The average nucleotide identity and in silico DNA-DNA hybridization analyses demonstrate that they represent species different from those previously described., (Copyright © 2019 Webster et al.)

Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of Pseudomonas (21%), Burkholderia (7%), and Bacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed Proteobacteria and Firmicutes to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samples (indicated by "JW") was performed, Betaproteobacteria (42.8%) and Gammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera were Marinobacter (15.4%; JW57), Achromobacter (41.6%; JW63), Burkholderia (80.7%; JW76), and Halomonas (66.2%; JW78), all of which were also observed by DGGE. However, the Clostridia (38.5%) and Deltaproteobacteria (11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a Pseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a Mangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a Burkholderia vietnamiensis strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as Pseudomonas.

A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

A new method for detecting chimeras and other anomalies within 16S rRNA sequence records is presented. Using this method, we screened 1,399 sequences from 19 phyla, as defined by the Ribosomal Database Project, release 9, update 22, and found 5.0% to harbor substantial errors. Of these, 64.3% were obvious chimeras, 14.3% were unidentified sequencing errors, and 21.4% were highly degenerate. In all, 11 phyla contained obvious chimeras, accounting for 0.8 to 11% of the records for these phyla. Many chimeras (43.1%) were formed from parental sequences belonging to different phyla. While most comprised two fragments, 13.7% were composed of at least three fragments, often from three different sources. A separate analysis of the Bacteroidetes phylum (2,739 sequences) also revealed 5.8% records to be anomalous, of which 65.4% were apparently chimeric. Overall, we conclude that, as a conservative estimate, 1 in every 20 public database records is likely to be corrupt. Our results support concerns recently expressed over the quality of the public repositories. With 16S rRNA sequence data increasingly playing a dominant role in bacterial systematics and environmental biodiversity studies, it is vital that steps be taken to improve screening of sequences prior to submission. To this end, we have implemented our method as a program with a simple-to-use graphic user interface that is capable of running on a range of computer platforms. The program is called Pintail, is released under the terms of the GNU General Public License open source license, and is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.

Periodontitis is the commonest bacterial disease of humans and is the major cause of adult tooth loss. About half of the oral microflora is unculturable; and 16S rRNA PCR, cloning, and sequencing techniques have demonstrated the high level of species richness of the oral microflora. In the present study, a PCR primer set specific for the genera Porphyromonas and Tannerella was designed and used to analyze the bacterial populations in subgingival plaque samples from inflamed shallow and deep sites in subjects with periodontitis and shallow sites in age- and sex-matched controls. A total of 308 clones were sequenced and found to belong to one of six Porphyromonas or Tannerella species or phylotypes, one of which, Porphyromonas P3, was novel. Tannerella forsythensis was found in significantly higher proportions in patients than in controls. Porphyromonas catoniae and Tannerella phylotype BU063 appeared to be associated with shallow sites. Targeted culture-independent molecular ecology studies have a valuable role to play in the identification of bacterial targets for further investigations of the pathogenesis of bacterial infections.

Phylogenetic analysis of 16S rRNA gene sequences from deep marine sediments identified a deeply branching clade, designated candidate division JS1. Primers for PCR amplification of partial 16S rRNA genes that target the JS1 division were developed and used to detect JS1 sequences in DNA extracted from various sedimentary environments, including, for the first time, coastal marine and brackish sediments.

Culture-dependent and culture-independent approaches were used to determine the relationship between the dehalogenase gene pool in bacteria enriched and isolated on 2,2-dichloropropionic acid (22DCPA) and the environmental metagene pool (the collective gene pool of both the culturable and uncultured microbes) from which they were isolated. The dehalogenases in the pure-cultures isolates, which were able to degrade 22DCPA, were similar to previously described group I and II dehalogenases. Significantly, the majority of the dehalogenases isolated from activated sludge by degenerate PCR with primers specific for alpha-halocarboxylic acid dehalogenases were not closely related to the dehalogenases in any isolate. Furthermore, the dehalogenases found in the pure cultures predominated in the enrichments but were a minor component of the community used to inoculate the batch cultures. Phylogenetic analysis of the dehalogenase sequences isolated by degenerate PCR showed that the diversity of the group II deh gene was greater than that of the group I deh gene. Direct plating of the activated sludge onto minimal media supplemented with 22DCPA resulted in biomass and DNA from which dehalogenases were amplified. Analysis of the sequences revealed that they were much more closely related to the sequences found in the community used to start the enrichments. However, no pure cultures were obtained with this isolation method, and thus no pure cultures were available for identification. In this study we examined the link between genes found in pure cultures with the metagene pool from which they were isolated. The results show that there is a large bias introduced by culturing, not just in the bacteria isolated but also the degradative genes that they contain. Moreover, our findings serve as a caveat for studies involving the culturing of pure cultures of bacteria and conclusions which are drawn from analysis of these organisms.

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.

Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families. The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehR(I), was isolated from strain PP3 by using the TOL plasmid pWW0. DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehR(I) were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase. A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively. The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout. Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients. In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria. Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P. putida PaW340. Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P. putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing. Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes. The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.

River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors. In this study the bacterial diversity of River Taff epilithon in South Wales was investigated. A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones. Members of the Cytophaga-Flexibacter-Bacteroides division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria), gamma-Proteobacteria, gram-positive bacteria, Cyanobacteria, beta-Proteobacteria, delta-Proteobacteria, and the Prosthecobacter group. This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376. The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division. CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b. The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced. They affiliated with the RDP-designated groups Cytophaga, Sphingobacterium, Lewinella, and Cytophaga aurantiaca. CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use. Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance.

Recently developed molecular methods have made it possible to characterize mixed microflora in their entirety, including the substantial numbers of bacteria which do not grow on artificial culture media. In a previous study, molecular analysis of the microflora associated with acute oral infections resulted in the identification of three phylotypes, PUS3.42, PUS9.170, and PUS9.180, representing as-yet-uncultured organisms. The aim of this study was to design and validate specific PCR primers for these phylotypes and to determine their incidences in samples collected from healthy and diseased periodontal tissues. Two specific reverse primers were devised for each phylotype, and these were used in duplex PCRs with universal forward and reverse primers. All three phylotypes were detected in periodontal sites; PUS9.170, related to oral asaccharolytic Eubacterium spp., was significantly associated with disease. This study demonstrates the possibility of using unculturable, and therefore uncharacterized, organisms as markers of disease.

Dehalogenases are key enzymes in the metabolism of halo-organic compounds. This paper describes a systematic approach to the isolation and molecular analysis of two families of bacterial alpha-halocarboxylic acid (alphaHA) dehalogenase genes, called group I and group II deh genes. The two families are evolutionarily unrelated and together represent almost all of the alphaHA deh genes described to date. We report the design and evaluation of degenerate PCR primer pairs for the separate amplification and isolation of group I and II deh genes. Amino acid sequences derived from 10 of 11 group I deh partial gene products of new and previously reported bacterial isolates showed conservation of five residues previously identified as essential for activity. The exception, DehD from a Rhizobium sp., had only two of these five residues. Group II deh gene sequences were amplified from 54 newly isolated strains, and seven of these sequences were cloned and fully characterized. Group II dehalogenases were stereoselective, dechlorinating L- but not D-2-chloropropionic acid, and derived amino acid sequences for all of the genes except dehII degrees P11 showed conservation of previously identified essential residues. Molecular analysis of the two deh families highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations. Group I deh genes included two putative cryptic or silent genes, dehI degrees PP3 and dehI degrees 17a, produced by different organisms. Group II deh genes included two cryptic genes and an active gene, dehIIPP3, that can be switched off and on. All alphaHA-degrading bacteria so far described were Proteobacteria, a result that may be explained by limitations either in the host range for deh genes or in isolation methods.

We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

The microflora associated with three dentoalveolar abscesses was determined by cultural and molecular methods. 16S rRNA genes were randomly amplified by means of conserved eubacterial primers and cloned. Restriction fragment length polymorphism analysis of the clones and amplified genes encoding 16S rRNA from the cultured bacteria was used to detect putative unculturable bacteria. Clones representative of five predominant groups of uncultured organisms were sequenced. Two were identified as Porphyromonas gingivalis and Prevotella oris, and one was found to be closely related to Peptostreptococcus micros. The remaining two clones did not correspond to known, previously sequenced organisms. One was related to Zoogloea ramigera, a species of aerobic waterborne organisms, while the other was distantly related to the genus Prevotella. This study has demonstrated the possibility of the characterization of microflora associated with human infection by molecular methods without the inherent biases of culture.

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.

As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy. The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain PP3. DEH recombined with target plasmid DNAs at high frequencies (e.g. 3.8 x 10(-4) per RP4.5 plasmid transferred). The regulated expression of dehI was detected in P. putida, Pseudomonas aeruginosa, and Escherichia coli strains containing derivative plasmids of RP4.5 and pWW0 recombined with DEH. Movement of DEH from the unstable RP4 derivatives pNJ5000 and pMR5 resulted in the insertion of DEH into the chromosome of RecA+ strains of P. putida but not in RecA+ nor RecA- strains of E. coli. Rescue of DEH from the chromosome of P. putida KT2441 onto plasmid RP4 involved recombination at a frequency (2.7 x 10(-4) per RP4 plasmid transferred) comparable to that observed in strain PP3. The DEH element was not classified as a conventional transposon because it did not move as a discrete DNA fragment: dehI-containing inserts in plasmid DNA targets varied in size between 6 and 13 kb. In addition, DEH exhibited a marked preference for insertion into a specific site on the plasmid pWW0, but its transposition, independent of host recombinational systems, remains to be demonstrated. However, the transposonlike characteristics of DEH included the conservation of restriction endonuclease sites, high-frequency recombination with different target replicons (plasmid and chromosomal DNA), and promiscuous insertion into plasmid RP4-based replicons. Therefore, it is proposed that DEH is an unusual mobile genetic element.

Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII. The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location. From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned. Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P. putida and Escherichia coli. The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI). Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA). Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters. Expression of dehI in P. putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent. The intact DEH element transferred to P. putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA. This indicated that the cloned DEH element contained functions associated with recombination.

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.

Mutant strains of Pseudomonas putida PP3 capable of utilizing monochloroacetate (MCA) and dichloroacetate (DCA) as the sole sources of carbon and energy were isolated from chemostat cultures. The mutants differed from the parent strain in that they could grow on products of MCA and DCA dehalogenation (catalyzed by inducible dehalogenases I and II) and were resistant to growth inhibition by the two substrates. The growth inhibition of strain PP3 by MCA, DCA, and other halogenated alkanoic acids was studied. Sensitivity to dehalogenase substrates was related to the expression of the dehalogenase genes. For example, mutants producing elevated levels of one or both of the dehalogenases were sensitive to 2-monochloropropionate and 2-monochlorobutanoate at concentrations which did not affect the growth of strain PP3. P. putida PP1, the parent of strain PP3, was resistant to the inhibitory effects of MCA and DCA. Spontaneous mutants of strain PP3, also resistant to MCA and DCA, were selected at high frequency, and four different classes of these strains were distinguished on the basis of dehalogenase phenotype. All dehalogenase-producing mutants were inducible; no constitutive mutant has yet been isolated. Most of the resistant mutants examined did not produce one or both of the dehalogenase, and over half of those tested failed to revert back to the parental (strain PP3) phenotype, indicating that the observed mutations involved high-frequency deletion of DNA base sequences affecting expression of genes encoding dehalogenases and associated permease(s).

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.

Plasmid pJP4 permits its host bacterium, strain JMP134, to degrade and utilize as sole sources of carbon and energy 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981). Mutagenesis of pJP4 by transposons Tn5 and Tn1771 enabled localization of five genes for enzymes involved in these catabolic pathways. Four of the genes, tfdB, tfdC, tfdD, and tfdE, encoded 2,4-dichlorophenol hydroxylase, dichlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and chlorodienelactone hydrolase, respectively. No function has been assigned to the fifth gene, tfdF, although it may encode a trans-chlorodiene-lactone isomerase. Inactivation of genes tfdC, tfdD, and tfdE, which encode the transformation of dichlorocatechol to chloromaleylacetic acid, prevented host strain JMP134 from degrading both 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid, which indicates that the pathways for these two substrates utilize common enzymes for the dissimilation of chlorocatechols. Studies with cloned catabolic genes from pJP4 indicated that whereas all essential steps in the degradation of 2,4-dichlorophenoxyacetic acid are plasmid encoded, the conversion of 3-chlorobenzoate to chlorocatechol is specified by chromosomal genes.