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Strain Lep1, isolated from a bacterial consortium capable of aerobic degradation of 4-methylquinoline (4-MQ), was chosen for further characterization as it was the only member of the consortium able to grow on 4-MQ in pure culture. Lep1 was identified as a Sphingomonas sp. based on phylogenetic analysis of 16S rDNA. Furthermore, the presence of sphingolipids and 2-hydroxy fatty acids in the membrane, and a 63% G + C ratio supports the placement of Lep1 in this genus. Additional genetic, physiological, and ecological characterization of bacteria such as Lep1 will allow for the potential exploitation of degradative strains for purposes of bioremediation of contaminated soils.

An efficient system for electroporation of the methylotrophic bacteria Hyphomicrobium facilis, Hyphomicrobium denitrificans, Methylobacillus glycogenes, Methylobacterium extorquens, and Methylophilus methylotrophus is described. It could be demonstrated that vectors based on the broad-host-range plasmid pBBR1 could be transferred into these strains. Plasmid pBBR1KAN (3.9 kb), a kanamycin-resistant derivative of pBBR1, was suitable for transformation experiments in these methylotrophic bacteria. Transformation efficiencies up to 10(4) transformants/microgram plasmid pBBR1KAN were obtained. The broad-host-range plasmid pLA2917 was transferred into Hyphomicrobium species by a triparental mating. However, this plasmid was integrated into the genome of Hyphomicrobium spp. Plasmids pLA2917, pKT231, pSUP2021, pRZ705, and phage DNA could not be transferred in Hyphomicrobium spp. by electroporation under the conditions applied.

Thirteen resin acid degrading bacteria enriched on abietic or dehydroabietic acids were isolated from waste water from the aerated stabilization basin of a bleached kraft pulp mill. Standard biochemical tests were used to characterize each isolate. Each isolate was tested for its ability to degrade six abietane- and pimarane-type resin acids. Resin acid concentrations were determined by high pressure liquid chromatography and UV absorbance. Cluster analysis based on phenotypic characteristics identified two distinct clusters of degraders that differed in their ability to utilize carbohydrates as carbon sources. Fatty acid methyl ester analysis of representative isolates from each cluster identified A19-6a and D11-13 as Comamonas and Alcaligenes species, respectively. To determine genotypic relatedness, enterobacterial repetitive intergenic consensus sequences were used to amplify genomic DNA fragments from 10 isolates. These results supported the phenotypic analysis for all isolates tested except A19-5 and A19-6b. These two organisms were clustered closely together based on phenotype but had distinctly different banding patterns, suggesting that they are not related genotypically. All isolates degraded a subset of the six resin acid congeners. Isolates A19-3, A19-6a, A19-6b, and D11-37 were the most effective at degrading all six congeners.

Six well-known strains of halotolerant bacteria, including two strains previously identified only as NRCC 41227 and Ba1, have been compared using 125 phenotypic characters and DNA-DNA hybridization. Although these strains represent some of the most heavily studied salt-tolerant bacteria, they have never been taxonomically compared. The data presented show that these bacteria form a relatively homogeneous group related at the genus level. The taxonomic comparison showed that these six organisms represented four distinct species all related above the 65% Jaccard coefficient level. In addition to two previously identified bacterial species, Halomonas elongata (ATCC 33173T) and Halomonas halodurans (ATCC 29686T), the strains included in this study represent two previously unnamed Halomonas species. These two new taxa have been assigned the names Halomonas israelensis (ATCC 43985T) and Halomonas canadensis (NRCC 41227T = ATCC 43984). DNA-DNA hybridization show that these two species are related to the type species H. elongata at 54.9 and 48.9%, respectively.

In this study, we isolated and characterized biphenyl (BP) and polychlorinated biphenyl (PCB) degrading bacterial strains found in PCB-contaminated soil from an auto manufacturing plant located in Syracuse, New York. Twenty-one BP and PCB-degrading bacteria were randomly selected to form a representative sample of the bacterial population present at the site. Of the 21 bacteria, 13 were identified as Comamonas testosteroni, constituting about 60% of the bacterial population examined. Other PCB degraders identified were Acidovorax facilis, Alcaligenes xylosoxydans, Bacillus sphericus, Hydrogenophaga pseudoflava, Pseudomonas avanae, and Rhodococcus fascians. Owing to the abundance of C. testosteroni at this site, only these isolates were further characterized for their PCB congener degradation profile, 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and genetic relatedness by polymerase chain reaction (PCR) analysis. The PCB congener degradation pattern revealed a high degree of variability among the C. testosteroni isolates. The majority of the C. testosteroni isolates tested could degrade more than 95% of the PCB congeners up to pentachlorinated biphenyl. Only four isolates could degrade more than 80% of hexachlorobiphenyl. All 12 isolates of C. testosteroni tested were able to attack 2,3,4,5,6,3',4'-heptachlorobiphenyl, indicating involvement of biphenyl 2,3-dioxygenase, while 2,3,5,6,2',3',6'-heptachlorobiphenyl was attacked by 6 strains, suggesting an oxidation reaction mediated by 3,4-dioxygenase. 2,3-Dihydroxybiphenyl 1,2-dioxygenase activity was also found to vary among the C. testosteroni isolates tested in this study. Eleven strains showed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity specific for 2,3-dihydroxybiphenyl, whereas isolate BW 169 could metabolize both 2,3-dihydroxybiphenyl and 4-methylcatechol, and isolate BW74 had the ability to metabolize all three substrates (2,3-dihydroxybiphenyl, 4-chlorocatechol, and 4-methylcatechol).(ABSTRACT TRUNCATED AT 250 WORDS)

The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53,095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.

An inoculant strain of Rhizobium leguminosarum biovar trifolii containing a Tn5 marked symbiotic plasmid transferred this plasmid by conjugation to Sphingobacterium multivorum, an organism that can be found in soil. The transconjugant bacteria nodulated the roots of white clover (Trifolium repens) seedlings but did not fix atmospheric nitrogen. Microscopic examination revealed abnormal nodule structures. Bacteria isolated from the nodules were shown to be closely related to the recipient S. multivorum and Southern blots of genomic digests probed with nodA DNA confirmed that the transconjugants contained symbiotic genes. This is the first report of the spontaneous transfer, by conjugation, of a symbiotic plasmid from R. leguminosarum biovar trifolii to S. multivorum.

In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.