Novel self-transmissible plasmids were exogenously captured from environmental samples by triparental matings with pBBR1MCS-2 as a mobilizable plasmid and Pseudomonas resinovorans as a recipient. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. The whole nucleotide sequences of six plasmids were determined, including one IncP-1 plasmid (pSN1104-59), four PromA-like plasmids (pSN1104-11, pSN1104-34, pSN0729-62, and pSN0729-70), and one novel plasmid (pSN1216-29), whose incompatibility group has not been previously identified. No previously known antibiotic resistance genes were found in these plasmids. In-depth phylogenetic analyses showed that the PromA-like plasmids belong to subgroups of PromA (designated as PromAγ and PromAδ) different from previously proposed subgroups PromAα and PromAβ. Twenty-four genes were identified as backbone genes by comparisons with other PromA plasmids. The nucleotide sequences of pSN1216-29 share high identity with those found in clinical isolates. A minireplicon of pSN1216-29 was successfully constructed from repA encoding a replication initiation protein and oriV. All the captured plasmids were found to have a broad host range and could be transferred to and replicated in different classes of Proteobacteria. Notably, repA and oriV of pSN1216-29 showed high similarity with one of two replication systems of pSRC119-A/C, known as a plasmid with multidrug resistance genes found in Salmonella enterica serovar Senftenberg. Our findings suggest that these “cryptic” but broad-host-range plasmids may be important for spreading several genes as “vehicles” in a wider range of bacteria in natural environments.

Cupriavidus necator NH9, a 3-chlorobenzoate (3-CB)-degrading bacterium, was isolated from soil in Japan. In this study, the complete genome sequence of NH9 was obtained via PacBio long-read sequencing to better understand the genetic components contributing to the strain's ability to degrade aromatic compounds, including 3-CB. The genome of NH9 comprised two circular chromosomes (4.3 and 3.4 Mb) and two circular plasmids (427 and 77 kb) containing 7,290 coding sequences, 15 rRNA and 68 tRNA genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the protein-coding sequences in NH9 revealed a capacity to completely degrade benzoate, 2-, 3-, or 4-hydroxybenzoate, 2,3-, 2,5-, or 3,4-dihydroxybenzoate, benzoylformate, and benzonitrile. To validate the identification of NH9, phylogenetic analyses (16S rRNA sequence-based tree and multilocus sequence analysis) and whole-genome sequence analyses (average nucleotide identity, percentage of conserved proteins, and tetra-nucleotide analyses) were performed, confirming that NH9 is a C . necator strain. Over the course of our investigation, we noticed inconsistencies in the classification of several strains that were supposed to belong to the two closely-related genera Cupriavidus and Ralstonia . As a result of whole-genome sequence analysis of 46 Cupriavidus strains and 104 Ralstonia strains, we propose that the taxonomic classification of 41 of the 150 strains should be changed. Our results provide a clear delineation of the two genera based on genome sequences, thus allowing taxonomic identification of strains belonging to these two genera.

bThe enzymes LinB UT and LinB MI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of ␤-hexachlorocyclohexane (␤-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB MI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB MI were categorized into three groups based on the efficiency of the first-step (from ␤-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB MI and its seven point mutants indicated how each mutated residue contributed to the first-and second-step conversions by LinB MI . The dynamics simulation analyses of wild-type LinB MI and LinB UT revealed that the entrance of the substrate access tunnel of LinB UT was more flexible than that of LinB MI , which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.H exachlorocyclohexane (HCH) is a six-chlorine-substituted cyclohexane. One of its isomers, the ␥ isomer, has insecticidal properties and has been widely used as an insecticide around the world (1). Although the use of ␥-HCH has been prohibited in most countries due to its toxicity and long persistence, the largescale production, widespread use, and dumping of the other noninsecticidal isomers (␣-, ␤-, and ␦-HCHs) in past decades still continue to create problems with HCH contamination in soil and groundwater (2). ␤-HCH in particular is a persistent and problematic isomer of HCH.Several ␤-HCH-degrading bacteria whose ␤-HCH-degrading enzymes can be utilized for bioremediation have been identified (3-5). LinB MI and LinB UT are haloalkane dehalogenases isolated from Sphingobium sp. MI1205 and Sphingobium japonicum UT26, respectively, that can cleave the carbon-halogen bond in ␤-HCH. Haloalkane dehalogenases belong to the ␣/␤-hydrolase family, and their catalytic mechanism consists of the following steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond in the substrate and formation of an intermediate covalently bound to the nucleophile, (iii) hydrolysis of the alkyl-enzyme intermediate, and (iv) release of halide ion and alcohol (6). LinB MI and LinB UT share 98% sequence identity, with only 7 different amino acid residues (at positions 81, 112, 134, 135, 138, 247, and 253) out of 296 residues, but these enzymes exhibit different enzymatic properties (Fig. 1). LinB MI catalyzes the two-step dehalogenation and converts ␤-HCH to 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and further to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) (7) in the manner of LinB2 from Sphingomonas sp. BHC-A (8) and LinB from Sphingobium indicum B90A (9), whereas LinB UT catalyzes only the firs...

CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9, activates the transcription of chlorocatechol-degradative enzymes. To activate the transcription, CbnR needs to bind not only to the cbnA promoter but also to the inducer. In this study, the transcriptional activity and DNA-binding activity of twenty-five mutants of CbnR were analyzed. Of the 17 mutants of the DNA-binding domain, 11 mutants lost their ability to activate transcription. While most mutants without transcriptional activation did not show DNA-binding activity, Asn17Ala, Gln29Ala, and Pro30Ala retained DNA-binding activity, suggesting that transcriptional activation by CbnR requires more than its binding to promoter DNA. Of the 8 mutants of the regulatory domain, 6 mutants changed their responses to the inducer, when compared with wild-type CbnR. Interestingly, Arg199Ala and Val246Ala induced constitutive expression of the cbnA promoter without the inducer, suggesting that these mutations brought about a conformational change mimicking that induced by the inducer molecule.