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Conjugative transfer of bacterial plasmids to recipient cells is often mediated by type IV secretion machinery. Experimental investigations into the minimal gene sets required for efficient conjugative transfer suggest that such gene sets are variable, depending on plasmids. We have been analyzing the conjugative transfer of Pseudomonas-derived and IncP-9 plasmids, NAH7 and pWW0, whose conjugation systems belong to the MPFT type. Our deletion analysis and synthetic biology analysis in this study showed that these plasmids require previously uncharacterized genes, mpfK (formerly orf34) and its functional homolog, kikA, respectively, for their efficient conjugative transfer. MpfK was localized in periplasm and had four cysteine residues whose intramolecular or intermolecular disulfide bond formation was suggested to be important for efficient conjugative transfer. The mpfK homologs were specifically carried by many MPFT-type plasmids, including non-IncP-9 plasmids, such as R388 and R751. Intriguingly, the mpfK homologs from the two non-IncP-9 plasmids were not required for conjugation of their plasmids, but were able to complement efficiently the transfer defect of the NAH7 mpfK mutant. Our results suggested the importance of the mpfK homologs for conjugative transfer of MPFT-type plasmids.IMPORTANCE IncP-9 plasmids are important mobile genetic elements for the degradation of various aromatic hydrocarbons. Elucidation of conjugative transfer of such plasmids is expected to greatly contribute to our understanding of its role in the bioremediation of polluted environments. The present study mainly focused on the conjugation system of NAH7, a well-studied and naphthalene-catabolic IncP-9 plasmid. Our analysis showed that the NAH7 conjugation system uniquely requires, in addition to the conserved components of the type IV secretion system (T4SS), a previously uncharacterized periplasmic protein, MpfK, for successful conjugation. Our findings collectively revealed a unique type of T4SS-associated conjugation system in the IncP-9 plasmids.

We report the complete genome sequence of Bradyrhizobium diazoefficiens USDA 122, a nitrogen-fixing soybean symbiont. The genome consists of a 9.1 Mb circular chromosome, and 8,551 coding sequences (CDSs) were predicted on the genome. The sequence will provide insight into the evolution of rhizobial genome, and the symbiotic compatibility with host plants.

NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are well-characterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer ( oriT )-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT ( oriT N ) region was located within a 430-bp fragment, and the strand-specific nicking ( nic ) site and its upstream sequences that were important for efficient conjugation in the oriT N region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriT N region, between two copies of the pWW0 oriT ( oriT W ) region (which is clearly different from the oriT N region), and between the oriT N and oriT W regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriT N -containing plasmid. Such a plasmid was, in the presence of the NAH7Δ nic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system. IMPORTANCE Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations. Degradation genes on such plasmids are often loaded on transposons, which can be inserted into the genomes of the recipient bacterial strains where the transferred plasmids cannot replicate. The aim was to advance detailed molecular knowledge of the determinants of host range for plasmids. This aim is expected to be easily and comprehensively achieved using an experimental strategy in which the oriT region is connected with a plasmid that has a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 oriT -relaxase system has unique properties that are significantly different from other well-studied systems and (ii) the host range of the NAH7 conjugation system is broader than previously thought.

We determined the complete genome sequence of Thalassococcus sp. strain S3, a marine carbazole degrader isolated from Tokyo Bay in Japan that carries genes for aerobic anoxygenic phototrophy. Strain S3 has a 4.7-Mb chromosome that harbors the carbazole-degradative gene cluster and three (96-, 63-, and 46-kb) plasmids.

Here, we present the complete genome sequence of Azoarcus sp. strain DN11, a denitrifying bacterium capable of anaerobic benzene degradation. The DN11 genome is 4,956,835 bp long with a G+C content of 66.3%. Genome analysis suggested the possibility that DN11 utilizes three proposed pathways for anaerobic benzene degradation, namely, methylation, hydroxylation, and carboxylation pathways.

Bacillus licheniformis strain TAB7 degrades short-chain fatty acids responsible for offensive odor in manure and is used as a deodorant in a compost-deodorizing technology. Here, we report the complete genome sequence of strain TAB7, which consists of a 4.37-Mb chromosome and two plasmids (42 kb and 31 kb).

We determined the complete genome sequence of Sphingopyxis macrogoltabida strain 203N, a polyethylene glycol degrader. Because the PacBio assembly (285× coverage) seemed to be full of nucleotide-level mismatches, the Newbler assembly of MiSeq mate-pair and paired-end data was used for finishing and the PacBio assembly was used as a reference. The PacBio assembly carried 414 nucleotide mismatches over 5,953,153 bases of the 203N genome.

Algoriphagus sp. strain M8-2 was isolated from a brackish lake, Lake Sanaru, in Hamamatsu, Japan, as a filterable bacterium through a 0.22-µm-pore-size membrane filter. We report here the complete nucleotide sequence of the M8-2 genome (a 3,882,610-bp chromosome).

The complete genome sequence of Sphingopyxis terrae strain 203-1, which is capable of growing on polyethylene glycol, was determined. The genome consisted of a chromosome with a size of 3.98 Mb and a plasmid with a size of 4,328 bp. The strain was deposited to the National Institute of Technology and Evaluation (Tokyo, Japan) under the number NBRC 111660.

Nonrhizobial Methylobacterium spp. inhabit the phyllosphere of a wide variety of plants. We report here the complete genome sequence of Methylobacterium sp. AMS5, which was isolated from a soybean stem. The information is useful for understanding the molecular mechanisms of the interaction between nonrhizobial Methylobacterium spp. and plants.

Here, we report the complete genome sequence of a γ-hexachlorocyclohexane (γ-HCH) degrader, Sphingobium sp. strain TKS, which was isolated from a γ-HCH-degrading microbial community. The genome of TKS consists of two chromosomes and nine plasmids. The lin genes for conversion of γ-HCH to β-ketoadipate are dispersed on chromosome 1 and three out of the nine plasmids.

A polychlorinated biphenyl (PCB)/biphenyl degradation gene cluster in Acidovorax sp. strain KKS102, which is very similar to that in Tn 4371 from Cupriavidus oxalaticus A5, was transferred to several proteobacterial strains by conjugation. The mobilized DNA fragment consisted of 61,807 bp and carried genes for mating-pair formation ( mpf ), DNA transfer ( dtr ), integrase ( int ), and replication-partition proteins ( rep - parAB ). In the transconjugants, transferred DNA was integrated at ATTGCATCAG or similar sequences. The circular-form integrative and conjugative element (ICE) was detected by PCR, and quantitative PCR analyses revealed that, in KKS102 cells, the ratio of the circular form to the integrated form was very low (approximately 10 −5 ). The circular form was not detected in a mutant of the int gene, which was located at the extreme left and transcribed in the inward direction, and the level of int transcriptional activity was much higher in the circular form than in the integrated form. These findings clearly demonstrated that the genes for PCB/biphenyl degradation in KKS102 cells are located on an ICE, which was named ICE KKS102 4677 . Comparisons of similar ICE-like elements collected from the public database suggested that those of beta- and gammaproteobacteria were distinguishable from other ICE-like elements, including those in alphaproteobacteria, with respect to the gene composition and gene organization.

Sphingopyxis macrogoltabida strain 203, the type strain of the species, grew on polyethylene glycol (PEG) and has been deposited to the stock culture at the Biological Resource Center, National Institute of Technology and Evaluation (NITE), under the number NBRC 15033. Here, we report the complete genome sequence of strain NBRC 15033. Unfortunately, genes for PEG degradation were missing.

Strain EY-1 was isolated from a microbial consortium growing on a random polymer of ethylene oxide and propylene oxide. Strain EY-1 grew on polyethylene glycol and polypropylene glycol and identified as Sphingopyxis macrogoltabida . Here, we report the complete genome sequence of Sphingopyxis macrogoltabida EY-1. The genome of strain EY-1 is comprised of a 4.76-Mb circular chromosome, and five plasmids. The whole finishing was conducted in silico , with aids of computational tools GenoFinisher and AceFileViewer. Strain EY-1 is available from Biological Resource Center, National Institute of Technology and Evaluation (Tokyo, Japan) (NITE).

The phenanthrene-degrading Burkholderia sp. HB-1 was isolated from a phenanthrene-enrichment culture seeded with a pristine farm soil sample. We report the complete genome sequence of HB-1, which has been deposited to the stock culture (NBRC 110738) at Biological Resource Center, National Institute of Technology and Evaluation (NITE), Tokyo, Japan. The genome of strain HB-1 comprises two circular chromosomes of 4.1 Mb and 3.1 Mb. The finishing was facilitated by the computational tools GenoFinisher, AceFileViewer, and ShortReadManager.

Rhodovulum sulfidophilum DSM 2351 is the nonsulfur photosynthetic bacterium that efficiently releases nucleic acids into the extracellular milieu, which leads to flocculation. In this study, we determined the complete genome sequence of R. sulfidophilum DSM 2351, which will provide new insights into the mechanism of its unique nucleic acid release.

Sulfurospirillum strains UCH001 and UCH003 were isolated from anaerobic cis -1,2-dichloroethene-dechlorinating microbial consortia derived from groundwater in Japan. Here, we report the complete genome sequences of strains UCH001 and UCH003.

The α-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, γ-hexachlorocyclohexane (γ-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of γ-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS 6100 composite transposon containing two copies of linB , and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other α-proteobacterial strains but not to any of the β- or γ-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for γ-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in γ-HCH degradation.

Sphingomonas paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of γ-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF , that is essential for the utilization of γ-HCH in UT26. A gene named linEb , whose deduced product showed significant identity to LinE (53%), was located close to linF . LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for γ-HCH utilization.

To improve the capabilities of microorganisms relevant for biodegradation, we developed a new genetic approach and applied it to the bph operon ( bphEGF [orf4] A1A2A3CD [orf1] A4R ) of Pseudomonas sp. strain KKS102 to enhance its biphenyl- and polychlorinated biphenyl (PCB)-degrading activity. A native promoter of the bph operon, which was under control, was replaced through homologous recombination by a series of promoters that had constitutive activity. By testing a series of promoters with various strengths, we were able to obtain strains that have enhanced degradation activity for biphenyl and PCBs. This strategy removes the rate-limiting factor associated with transcription and has the potential to improve the degradation activity of a wide variety of microorganisms involved in biodegradation.

Pseudomonas aeruginosa MTB-1 does not degrade gamma-hexachlorocyclohexane (γ-HCH), but this bacterium persistently coexists with a γ-HCH-degrading strain, Sphingomonas sp. MM-1, in a microbial community enriched by the technical formulation of HCH. Here we report the complete MTB-1 genome sequence, with a 6.6-Mb circular chromosome.

Pseudomonas sp. strain TKP does not degrade γ-hexachlorocyclohexane (γ-HCH), but it persistently coexists with the γ-HCH-degrading Sphingobium sp. strain TKS in a mixed culture enriched by γ-HCH. Here, we report the complete genome sequence of strain TKP, which consists of one circular chromosome with a size of 7 Mb.

Ralstonia pickettii strain DTP0602 utilizes 2,4,6-trichlorophenol as its sole carbon and energy source. Here, we report the complete genome sequence of strain DTP0602, which comprises three chromosomes and no plasmids. We also found that the two had gene clusters responsible for the degradation of 2,4,6-trichlorophenol are located on the 2.9-Mb chromosome 2.

γ-Hexachlorocyclohexane (γ-HCH) is a man-made chlorinated insecticide that has caused serious environmental problems. Here, we report the complete genome sequence of the γ-HCH-degrading bacterium Sphingomonas sp. strain MM-1, which consists of one chromosome and five plasmids. All the specific lin genes that are almost identical to those of Sphingobium japonicum UT26 for the conversion of γ-HCH to β-ketoadipate are dispersed on four out of the five plasmids.

Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles and is the only microorganism that has been isolated from sarcoid lesions. We report the complete genome sequence of P. acnes , which was isolated from a Japanese patient with sarcoidosis.

We report the complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl-degrading strain isolated from a soil sample in Tokyo. The genome contains a single circular 5,196,935-bp chromosome and no plasmids.

Bacterial class II (Tn3-like) transposons generally carry the genes for their transposition (tnpA, tnpR, and res) and one or more phenotypic traits between their terminal inverted repeats (IRs), which have sizes of less than 50 bp (Fig. 1A and B) (23). These transposons move by a two-step and replicative mechanism (6, 23). In the first step, the tnpA product (transposase) acts at the IRs to generate a cointegrate of the donor and target molecules connected by two directly repeated copies of the transposon, one at each junction. In the second step, the cointegrate resolves at the resolution (res) sites by means of the tnpR product (resolvase). A 5-bp duplication of the target sequence is generated upon transposition. The transposases of the class II transposons are able to catalyze their transposition even when the tnpA gene and cognate IRs are located on separate molecules (6, 23). Several class II transposons have been reported to play an important role in the wide dissemination of various catabolic gene clusters, such as toluene-xylenes, naphthalene, and carbazole (17, 29-31). To date, three major groups (Tn3, Tn21, and Tn4651) of class II transposons have been characterized in detail with respect to their structural and functional aspects (Fig. ​(Fig.1A1A). FIG. 1. Structures of class II transposons, their IR sequences, and IS1071 derivatives. (A) Schematic structures of the class II transposons. The sizes are arbitrary. The black and white arrowheads indicate the terminal IR sequences of IS1071 and those of the ... IS1071 is a 3.2-kb insertion sequence (IS) that was originally identified in a chlorobenzoate-catabolic transposon, Tn5271, from Comamonas testosteroni BR60 (20). On the basis of structural features of its 110-bp IRs and 2,913-bp tnpA gene, IS1071 has been considered to belong to the class II transposons (7, 20). However, this IS element shows the uniqueness in its long (110-bp) IRs and its lack of the resolution function. The identification of many IS1071 sequences in close proximity to various xenobiotic-degrading genes on self-transmissible plasmids from environmental bacteria, e.g., Pseudomonas (18), Comamonas (2, 13), and Wautersia (3), indicates that IS1071 must have been involved in the recruitment of catabolic genes to these plasmids and in the dissemination of these genes among various host strains. We have also identified a haloacetate-catabolic IS1071-composite transposon, TnHad1, on an IncP-1β plasmid, pUO1, from Delftia acidovorans strain B (Fig. ​(Fig.1A)1A) (24, 25). TnHad1 is located within a defective class II transposon, TnHad2, which is a Tn21-related transposon that lacks the tnpA and tnpR genes (Fig. ​(Fig.1A)1A) (24). We have previously reported that the two intact copies and one truncated copy of IS1071 in TnHad2 might have been incorporated into an ancestor of TnHad2 (24). However, no clear transposition events of the TnHad1-specified IS1071 element were observed. No functional analysis of IS1071 has been carried out since its discovery more than a decade ago. Our functional analysis of IS1071 in this study has indicated that (i) efficient transposition of IS1071 occurred in two specific host strains, (ii) IS1071 had the functional features of the class II transposons, and (iii) almost the entire region of the 110-bp IR was required for transposition.

Sphingobium japonicum strain UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in γ-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.