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Sphingobium japonicum strain UT26, whose γ-hexachlorocyclohexane-degrading ability has been studied in detail, is a typical aerobic and heterotrophic bacterium that needs organic carbon sources for its growth, and cannot grow on a minimal salt agar medium prepared without adding any organic carbon sources. Here, we isolated a mutant of UT26 with the ability to grow to visible state on such an oligotrophic medium from a transposon-induced mutant library. This high-yield growth under oligotrophic conditions (HYGO) phenotype was CO2-dependent and accompanied with CO2 incorporation. In the HYGO mutant, a transposon was inserted just upstream of the putative Zn-dependent alcohol dehydrogenase (ADH) gene (adhX) so that the adhX gene was constitutively expressed, probably by the transposon-derived promoter. The adhX-deletion mutant (UT26DAX) harbouring a plasmid carrying the adhX gene under the control of a constitutive promoter exhibited the HYGO phenotype. Moreover, the HYGO mutants spontaneously emerged among the UT26-derived hypermutator strain cells, and adhX was highly expressed in these HYGO mutants, while no HYGO mutant appeared among UT26DAX-derived hypermutator strain cells, indicating the necessity of adhX for the HYGO phenotype. His-tagged AdhX that was expressed in Escherichia coli and purified to homogeneity showed ADH activity towards methanol and other alcohols. Mutagenesis analysis of the adhX gene indicated a correlation between the ADH activity and the HYGO phenotype. These results demonstrated that the constitutive expression of an adhX-encoding protein with ADH activity in UT26 leads to the CO2-dependent HYGO phenotype. Identical or nearly identical adhX orthologues were found in other sphingomonad strains, and most of them were located on plasmids, suggesting that the adhX-mediated HYGO phenotype may be an important adaptation strategy to oligotrophic environments among sphingomonads.

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.

Bacterial iron-sulfur (Fe-S) clusters are essential cofactors for many metabolic pathways, and Fe-S cluster-containing proteins (Fe-S proteins) regulate the expression of various important genes. However, biosynthesis of such clusters has remained unknown in genus Burkholderia. Here, we clarified that Burkholderia multivorans ATCC 17616 relies on the ISC system for the biosynthesis of Fe-S clusters, and that the biosynthetic genes are organized as an isc operon, whose first gene encodes IscR, a transcriptional regulatory Fe-S protein. Transcription of the isc operon was repressed and activated under iron-rich and -limiting conditions, respectively, and Fur, an iron-responsive global transcriptional regulator, was indicated to indirectly regulate the expression of isc operon. Further analysis using a ΔiscR mutant in combination with a constitutive expression system of IscR and its derivatives indicated transcriptional repression and activation of isc operon by holo- and apo-forms of IscR, respectively, through their binding to the sequences within an isc promoter-containing (Pisc) fragment. Biochemical analysis using the Pisc fragment suggested that the apo-IscR binding sequence differs from the holo-IscR binding sequence. The results obtained in this study revealed a unique regulatory system for the expression of the ATCC 17616 isc operon that has not been observed in other genera.

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.

In this study, we investigated CIS reaction (clamping-mediated incorporation of single-stranded DNA with concomitant DNA syntheses) of Moloney murine leukaemia virus reverse transcriptase (MMLV-RT), and established a set of conditions with which single-stranded DNA is ligated to a G-tailed model substrate DNA at efficiencies close to 100%. Prior to the CIS reaction, a target blunt-end DNA was 3′ G-tailed by MMLV-RT in the presence of a tailing enhancer, deoxycytidine. In the CIS reaction, the G-tail reacted with a single-stranded DNA carrying a stretch of Cs on its 3′ end (termed as GAO for guide adaptor oligonucleotide), and MMLV-RT performed DNA polymerization, starting from the 3′ overhang, using the GAO as a template. We could append a given nucleotide sequence of as long as 72 nucleotides, which would be sufficient for various NGS-sequencing platforms. The high efficiency and the unique features of this MMLV-RT activity that enables the labelling of each DNA molecule with a unique degenerate sequence as a molecular identifier has many potential uses in biotechnology.

Bacterial strains capable of degrading man‐made xenobiotic compounds are good materials to study bacterial evolution towards new metabolic functions. Lindane (γ‐hexachlorocyclohexane, γ‐HCH, or γ‐BHC) is an especially good target compound for the purpose, because it is relatively recalcitrant but can be degraded by a limited range of bacterial strains. A comparison of the complete genome sequences of lindane‐degrading sphingomonad strains clearly demonstrated that (i) lindane‐degrading strains emerged from a number of different ancestral hosts that have recruited lin genes encoding enzymes that are able to channel lindane to central metabolites, (ii) in sphingomonads lin genes have been acquired by horizontal gene transfer mediated by different plasmids and in which IS6100 plays a role in recruitment and distribution of genes, and (iii) IS6100 plays a role in dynamic genome rearrangements providing genetic diversity to different strains and ability to evolve to other states. Lindane‐degrading bacteria whose genomes change so easily and quickly are also fascinating starting materials for tracing the bacterial evolution process experimentally in a relatively short time period. As the origin of the specific lin genes remains a mystery, such genes will be useful probes for exploring the cryptic ‘gene pool’ available to bacteria.

Bacterial strains capable of degrading man-made xenobiotic compounds are thought to have evolved to degrade such compounds within only several decades. Various sphingomonad strains belonging to Alphaproteobacteria were isolated that degrade highly recalcitrant compounds including man-made xenobiotics, indicating the versatility of this bacterial group. However, each strain degrades only a limited set of compounds, and specific genes for the degradation of a specific compound are found only in the genomes of its degraders, suggesting the plasticity of genomes of sphingomonads. Comparison of the complete genome sequences of four γ-hexachlorocyclohexane (γ-HCH)-degrading sphingomonad strains, Sphingobium japonicum UT26, Sphingomonas sp. MM-1, Sphingobium sp. MI1205, and Sphingobium sp. TKS, strongly suggested that the γ-HCH-degrading strains emerged through the recruitment of specific lin genes for the γ-HCH degradation into ancestral strains that had core functions of sphingomonads. Plasmids, most of which are specialized for sphingomonads, and IS6100, the most abundant insertion sequence in the four strains, seem to be involved in the recruitment of the lin genes and the diversification of the lin-flanking regions in the four strains. Here, we show concrete examples and discuss the important roles of such mobile genetic elements for the emergence and evolution of the bacterial strains degrading highly recalcitrant compounds.

γ-Hexachlorocyclohexane (γ-HCH) is a recalcitrant man-made chlorinated pesticide. Here, the complete genome sequences of four γ-HCH-degrading sphingomonad strains, which are most unlikely to have been derived from one ancestral γ-HCH degrader, were compared. Together with several experimental data, we showed that (i) all the four strains carry almost identical linA to linE genes for the conversion of γ-HCH to maleylacetate (designated “specific” lin genes), (ii) considerably different genes are used for the metabolism of maleylacetate in one of the four strains, and (iii) the linKLMN genes for the putative ABC transporter necessary for γ-HCH utilization exhibit structural divergence, which reflects the phylogenetic relationship of their hosts. Replicon organization and location of the lin genes in the four genomes are significantly different with one another, and that most of the specific lin genes are located on multiple sphingomonad-unique plasmids. Copies of IS6100, the most abundant insertion sequence in the four strains, are often located in close proximity to the specific lin genes. Analysis of the footprints of target duplication upon IS6100 transposition and the experimental detection of IS6100 transposition strongly suggested that the IS6100 transposition has caused dynamic genome rearrangements and the diversification of lin-flanking regions in the four strains.

Conjugative transfer of bacterial plasmid is one of the major mechanisms of horizontal gene transfer, which is mediated by direct contact between donor and recipient cells. Gene expression of a conjugative plasmid is tightly regulated mostly by plasmid-encoded transcriptional regulators, but it remains obscure how differently plasmid genes are expressed in each cell during the conjugation event. Here, we report a comprehensive analysis of gene expression during conjugative transfer of plasmid RP4, which is transferred between isogenic strains of Pseudomonas putida KT2440 at very high frequency. To discriminate the expression changes in the donor and recipient cells, we took advantage of conjugation in the presence of rifampicin (Rif). Within 10 min of mating, we successfully detected transient transcription of plasmid genes in the resultant transconjugant cells. This phenomenon known as zygotic induction is likely attributed to derepression of multiple RP4-encoded repressors. Interestingly, we also observed that the traJIH operon encoding relaxase and its auxiliary proteins were upregulated specifically in the donor cells. Identification of the 5′ end of the zygotically induced traJ mRNA confirmed that the transcription start site of traJ was located 24-nt upstream of the nick site in the origin of transfer (oriT) as previously reported. Since the traJ promoter is encoded on the region to be transferred first, the relaxase may be expressed in the donor cell after regeneration of the oriT-flanking region, which in itself is likely to displace the autogenous repressors around oriT. This study provides new insights into the regulation of plasmid transfer processes.

We determined the complete genome sequence of Erythrobacter sp. strain KY5, a bacterium isolated from Tokyo Bay and capable of degrading carbazole., We determined the complete genome sequence of Erythrobacter sp. strain KY5, a bacterium isolated from Tokyo Bay and capable of degrading carbazole. The genome consists of a 3.3-Mb circular chromosome that carries the gene clusters involved in carbazole degradation and biosynthesis of the photosynthetic apparatus of aerobic anoxygenic phototrophic bacteria.

Plasmid vector and allelic exchange mutagenesis systems were established for the genetic analysis of a phenanthrene-degrading mycobacterial strain, Mycobacterium sp. EPa45. Successful application of these systems revealed the necessity of the EPa45 phdI gene for the degradation of 1-hydroxy-2-naphthoate, which has been proposed to be an intermediate product in the degradation pathway of phenanthrene.

Metagenomes contain the DNA from many microorganisms, both culturable and non-culturable, and are a potential resource of novel genes. In this study, a 5.2-Gb metagenomic DNA library was constructed from a soil sample (artificially polluted with four aromatic compounds, i.e., biphenyl, phenanthrene, carbazole, and 3-chlorobenzoate) in Escherichia coli by using a broad-host-range cosmid vector. The resultant library was introduced into naphthalene-degrading Pseudomonas putida-derived strains having deficiencies in their naphthalene dioxygenase components, and indigo-forming clones on the indole-containing agar plates were screened. Cosmids isolated from 29 positive clones were classified by their various properties (original screening hosts, hosts showing indigo-forming activity, and digestion patterns with restriction enzymes), and six representative cosmids were chosen. Sequencing and in vitro transposon mutagenesis of the six cosmids resulted in the identification of genes encoding putative class B and D flavoprotein monooxygenases, a multicomponent hydroxylase, and a reductase that were responsible for the indigo-forming activity in the host cells. Among them, the genes encoding the multicomponent hydroxylase were demonstrated to be involved in phenol degradation. Furthermore, two genes encoding ring-cleavage dioxygenases were also found adjacent to the genes responsible for the indigo formation, and their functions were experimentally confirmed.

Iron and heme play very important roles in various metabolic functions in bacteria, and their intracellular homeostasis is maintained because high concentrations of free forms of these molecules greatly facilitate the Fenton reaction-mediated production of large amounts of reactive oxygen species that severely damage various biomolecules. The ferric uptake regulator (Fur) from Burkholderia multivorans ATCC 17616 is an iron-responsive global transcriptional regulator, and its fur deletant exhibits pleiotropic phenotypes. In this study, we found that the phenotypes of the fur deletant were suppressed by an additional mutation in hemP . The transcription of hemP was negatively regulated by Fur under iron-replete conditions and was constitutive in the fur deletant. Growth of a hemP deletant was severely impaired in a medium containing hemin as the sole iron source, demonstrating the important role of HemP in hemin utilization. HemP was required as a transcriptional activator that specifically binds the promoter-containing region upstream of a Fur-repressive hmuRSTUV operon, which encodes the proteins for hemin uptake. A hmuR deletant was still able to grow using hemin as the sole iron source, albeit at a rate clearly lower than that of the wild-type strain. These results strongly suggested (i) the involvement of HmuR in hemin uptake and (ii) the presence in ATCC 17616 of at least part of other unknown hemin uptake systems whose expression depends on the HemP function. Our in vitro analysis also indicated high-affinity binding of HemP to hemin, and such a property might modulate transcriptional activation of the hmu operon. IMPORTANCE Although the hmuRSTUV genes for the utilization of hemin as a sole iron source have been identified in a few Burkholderia strains, the regulatory expression of these genes has remained unknown. Our analysis in this study using B. multivorans ATCC 17616 showed that its HemP protein is required for expression of the hmuRSTUV operon, and the role of HemP in betaproteobacterial species was elucidated for the first time, to our knowledge, in this study. The HemP protein was also found to have two additional properties that have not been reported for functional homologues in other species; one is that HemP is able to bind to the promoter-containing region of the hmu operon to directly activate its transcription, and the other is that HemP is also required for the expression of an unknown hemin uptake system.

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.

To identify bacterial genetic determinants for fitness in a soil environment, signature-tagged mutagenesis (STM) was applied to a soil bacterium, Burkholderia multivorans ATCC 17616. This strain was randomly mutagenized by each of 36 different signature-tagged plasposons, and 36 mutants with different tags were grouped as a set. A total of 192 sets consisting of 6912 independent mutants were each inoculated into soil and incubated. Two-step STM screening based on quantitative real-time PCR of total DNAs extracted from the resulting soil samples using the tag-specific primers led to the selection of 39 mutant candidates that exhibited a reduction in relative competitive fitness during incubation in the soil, and 32 plasposon-insertion sites were determined. Among them, mutants having plasposon insertion in fur, deaD or hrpA exhibited reduced fitness during incubation in soil when compared with the control strain. The deficiency in the soil fitness of the fur mutant was recovered by the introduction of the wild-type fur gene, indicating that the fur gene is one of the genetic determinants for fitness in the soil.

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.

The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.

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.

Here, we report the complete genome sequence of a γ-hexachlorocyclohexane (γ-HCH)-degrading bacterium, Sphingobium sp. strain MI1205. The genome of MI1205 consists of two chromosomes and four plasmids with sizes of 33 to 292 kb. All the lin genes for γ-HCH metabolism are dispersed on the four plasmids.

Activity-based screening techniques were applied to clone a gene encoding γ-hexachlorocyclohexane (γ-HCH) dehydrochlorinase with its flanking regions from a cosmid-based library of DNA that was extracted from a γ-HCH-added suspension of HCH-contaminated soil. A total of 11 cosmid clones showing the γ-HCH dehydrochlorinase activity were obtained through the screenings, indicating the validity of this approach. All the clones had a linA gene identical to known one, but its flanking regions showed some structural variations with known ones, suggesting high likelihood of genetic divergence in the linA-flanking regions.

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.

Cyanobacterial phytochrome-class photosensors are recently emerging optogenetic tools. We isolated Fischerella sp. strain NIES-3754 from hotspring at Suwa-shrine, Suwa, Nagano, Japan. We determined complete genome sequence of the NIES-3754 strain, which is composed of one chromosome and two putative replicons (total 5,826,863bp containing no gaps). We identified photosensor genes of 5 phytochromes and 9 cyanobacteriochromes, which will facilitate optogenetics of thermophile.

Burkholderia multivorans ATCC 17616 was originally isolated from a soil sample, and it carries three chromosomes. To identify traits of likely adaptive significance for colonization of soil, an in vivo expression technology system for ATCC 17616 was constructed using the promoterless and tandemly arranged dapB and lacZ genes as the reporters, and this system was applied to identify the genomic loci of ATCC 17616 that were induced in sterilized soil. Our screening of a library consisting of dapB-lacZ-inserted clones resulted in the isolation of 713 clones in which the insertion sites of genome were putatively transcribed in the soil but not in laboratory media. All insertion sites in the genome were determined by high-throughput sequencing using genomic DNA as the templates, and subsequent analysis led to a reliable list of a total of 116 genomic loci as the B. multivorans ATCC 17616 loci induced in a soil environment (mls). These 116 mls carried the genes for energy acquisition from various substances, as well as genes for cell-envelope integrity and the niche adaptation. The distribution of these loci was biased to the second chromosome, suggesting the importance of this replicon as a source of adaptive traits enhancing survival of this organism in natural environments.

Several types of environmental bacteria that can aerobically degrade various aromatic compounds have been identified. The catabolic genes in these bacteria have generally been found to form operons, which promote efficient and complete degradation. However, little is known about the degradation pathways in bacteria that are difficult to culture in the laboratory. By functionally screening a metagenomic library created from activated sludge, we had earlier identified 91 fosmid clones carrying genes for extradiol dioxygenase (EDO), a key enzyme in the degradation of aromatic compounds. In this study, we analyzed 38 of these fosmids for the presence and organization of novel genes for aromatics degradation. Only two of the metagenomic clones contained complete degradation pathways similar to those found in known aromatic compound-utilizing bacteria. The rest of the clones contained only subsets of the pathway genes, with novel gene arrangements. A circular 36.7-kb DNA form was assembled from the sequences of clones carrying genes belonging to a novel EDO subfamily. This plasmid-like DNA form, designated pSKYE1, possessed genes for DNA replication and stable maintenance as well as a small set of genes for phenol degradation; the encoded enzymes, phenol hydroxylase and EDO, are capable of the detoxification of aromatic compounds. This gene set was found in 20 of the 38 analyzed clones, suggesting that this 'detoxification apparatus' may be widespread in the environment.

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.

The fur (ferric uptake regulator) gene of Burkholderia multivorans ATCC 17616 was identified by transposon mutagenesis analysis. The fur deletion mutant of strain ATCC 17616 (i) constitutively produced siderophores, (ii) was more sensitive to reactive oxygen species (ROS) than the wild-type strain, (iii) showed lower superoxide dismutase and catalase activities than the wild-type strain, (iv) was unable to grow on M9 minimal agar plates containing several substrates that can be used as sole carbon sources by the wild-type strain, and (v) was hypersensitive to nitrite and nitric oxide under microaerobic and aerobic conditions, respectively. These results clearly indicate that the Fur protein in strain ATCC 17616 plays pleiotropic roles in iron homeostasis, removal and/or resistance to ROS and nitrosative stress, and energy metabolism. Furthermore, employment of an in vivo Fur titration assay system led to the isolation from the ATCC 17616 genome of 13 Fur-binding DNA regions, and a subsequent electrophoretic mobility-shift assay confirmed the direct binding of Fur protein to all of these DNA regions. Transcriptional analysis of the genes located just downstream of the Fur-binding sites demonstrated that Fur acts as a repressor for these genes. Nine of the 13 regions were presumed to be involved in the acquisition and utilization of iron.

Recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (TOL) plasmids and their residing transposons. To elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pWW53, a TOL plasmid from Pseudomonas putida MT53. pWW53 was found to belong to the IncP-7 incompatibility group that play important roles in the catabolism of several xenobiotics. pWW53 carried two distinct transposase-resolvase gene clusters (tnpAR modules), five short terminal inverted repeats (IRs), and three site-specific resolution (res) sites that are all typical of class II transposons. This organization of pWW53 suggested the four possible transposable regions, Tn4657 to Tn4660. The largest 86 kb region (Tn4657) spanned the three other regions, and Tn4657 and Tn4660 (62 kb) covered all of the 36 xyl genes for toluene catabolism. Our subsequent transposition experiments clarified that the three transposons, Tn4657 to Tn4659, indeed exhibit their transposability, and that pWW53 also generated another 37 kb toluene-catabolic transposon, Tn4656, which carried the two separated and inversely oriented segments of pWW53: the tnpRA-IR module of Tn4658 and a part of xyl gene clusters on Tn4657. The Tn4658 transposase was able to mediate the transposition of Tn4658, Tn4657, and Tn4656, while the Tn4659 transposase catalyzed only the transposition of Tn4659. Tn4656 was formed by the Tn4658 resolvase-mediated site-specific inversion between the two inversely oriented res sites on pWW53. These findings and comparison with other catabolic plasmids clearly indicate multiple copies of transposition-related genes and sites on one plasmid and their recombination activities contribute greatly to the diversification of plasmid structures as well as wide dissemination of the evolutionary common gene clusters in various plasmids.

Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway.

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.

Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 utilizes the important insecticide gamma-hexachlorocyclohexane as a sole source of carbon and energy. In previous studies, we isolated and characterized six structural genes (linA to linF) and one regulatory gene (linR) of UT26 for the degradation of gamma-hexachlorocyclohexane to beta-ketoadipate. Our analysis in this study indicated that the UT26 genome consists of three large circular replicons of 3.6 Mb, 670 kb, and 185 kb. The 3.6 Mb and the 670 kb replicons had one and two copies, respectively, of the 16S ribosomal RNA gene, and these replicons were designated as chromosomes (Chr) I and II, respectively. Chr I was indicated to be a main chromosome carrying the dnaA gene. The first three lin genes, linA to linC, for conversion of gamma-hexachlorocyclohexane to 2,5-dichlorohydroquinone, were dispersed on Chr I. The 185 kb plasmid, pCHQ1, carried the linRED operon for the conversion of 2,5-dichlorohydroquinone to maleylacetate and was conjugatively transferred to another sphingomonad strain. The linF gene encoding maleylacetate reductase was located on Chr II. These results indicated that the genes for the complete gamma-hexachlorocyclohexane degradation are dispersed on the three large replicons of UT26.

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 explore the diverse photoreceptors of cyanobacteria, we isolated Nostoc sp. strain NIES-3756 from soil at Mimomi-Park, Chiba, Japan, and determined its complete genome sequence. The Genome consists of one chromosome and two plasmids (total 6,987,571 bp containing no gaps). The NIES-3756 strain carries 7 phytochrome and 12 cyanobacteriochrome genes, which will facilitate the studies of phytochrome-based bioengineering.

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.

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.

The bph genes involved in PCB/biphenyl degradation in Pseudomonas sp. KKS102 are clustered as bphEGFA1A2A3BCDA4R. The bph genes are inducibly expressed in the presence of biphenyl. In order to understand the induction more fully, the inducer of bph gene expression was investigated. To identify the inducer molecule, we constructed four deletion mutants of the structural genes and analyzed the inducibility of the bphE gene in each mutant strain. In the wild-type cell and the bphD deletion mutant, the levels of the bphE transcript were enhanced in the presence of biphenyl. On the other hand, in the bphA, bphB, and bphC deletion mutants, levels of the bphE transcript were not enhanced in the presence of biphenyl. These results demonstrated that the series of reactions catalyzed by biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), and 2, 3-dihydroxybiphenyl dioxygenase (BphC) are necessary to convert biphenyl to the inducer. It is known that these reactions convert biphenyl to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), and it was found that the expression of the bph genes was induced by purified HOPDA. These results clearly indicate that HOPDA is the inducer of the bph genes in KKS102.

Bacterial capabilities to degrade various recalcitrant compounds are often encoded on mobile genetic elements (MGEs) such as transposons, plasmids, and integrative and conjugative elements (ICEs). The movement of the transposons and consequently induced rearrangements of genome in a cell and the intercellular transfer of the latter two MGEs greatly facilitate rapid adaptation and evolution of the host cells and wide dissemination of catabolic genes and gene clusters in a variety of phylogenetically distinct environmental bacteria. This chapter summarizes how MGEs participate in the emergence of degradative bacteria based on the earlier and recent findings and analysis of catabolic MGEs in pseudomonads.

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.

Fur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes, such as an inability to efficiently use several carbon sources, as well as high sensitivity to hydrogen peroxide (H(2)O(2)), paraquat (a superoxide-producing compound) and nitric oxide (NO). To gain more insight into the pleiotropic role of the Fur protein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 fur mutant that restored tolerance to NO were isolated and characterized in this study. The microarray-based comparative genomic analysis and subsequent sequencing analysis indicated that such suppressor mutants had a 2 bp deletion in the oxyR gene, whose orthologues encode H(2)O(2)-responsive transcriptional regulators in other bacterial species. The suppressor mutants and the reconstructed fur-oxyR double-deletion mutant showed indistinguishable phenotypes in that they were all (i) more resistant than the fur mutant to H(2)O(2), superoxide, NO and streptonigrin (an iron-activated antibiotic) and (ii) able to use carbon sources that cannot efficiently support the growth of the fur mutant. These results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant. The fur-oxyR double mutants were found to overexpress the KatG (catalase/peroxidase) and AhpC1 and AhpD (alkyl hydroperoxide reductase subunits C and D) proteins, and their enzymic activities to remove reactive oxygen and nitrogen species were suggested to be responsible for the suppression of phenotypes caused by the fur mutation.

A γ-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of γ-HCH led to the detection of five γ-HCH metabolites, γ-pentachlorocyclohexene, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol, 2,5-dichlorohydroquinone, 1,2,4-trichlorobenzene, and 2,5-dichlorophenol, strongly suggesting that MM-1 has the lin genes for γ-HCH degradation originally identified in the well-studied γ-HCH-degrading strain Sphingobium japonicum UT26. Southern blot, PCR amplification, and sequencing analyses indicated that MM-1 has seven lin genes for the conversion of γ-HCH to β-ketoadipate (six structural genes, linA to linF, and one regulatory gene, linR). MM-1 carried four plasmids, of 200, 50, 40, and 30 kb. Southern blot analysis revealed that all seven lin genes were dispersed across three of the four plasmids, and that IS6100, often found close to the lin genes, was present on all four plasmids.

The complete genome sequencing of a γ-hexachlorocyclohexane-degrading strain, Sphingobium japonicum UT26, revealed that the genome consists of two circular chromosomes [with sizes of 3.5 Mb (Chr1) and 682kb (Chr2)], a 191-kb large plasmid (pCHQ1), and two small plasmids with sizes of 32 and 5kb. The lin genes are dispersed on Chr1, Chr2, and pCHQ1. Comparison of the UT26 genome with those of other sphingomonad strains demonstrated that the "specific"lin genes for conversion of γ-HCH to β-ketoadipate (linA, linB, linC, linRED, and linF) are located on the DNA regions unique to the UT26 genome, suggesting the acquisition of these lin genes by horizontal transfer events. On the other hand, linGHIJ and linKLMN are located on the regions conserved in the genomes of sphingomonads, suggesting that the linGHIJ-encoded β-ketoadipate pathway and the LinKLMN-type ABC transporter system are involved in core functions of sphingomonads. Based on these results, we propose a hypothesis that UT26 was created by recruiting the specific lin genes into a strain having core functions of sphingomonads. Most of the specific lin genes in UT26 are associated with IS6100. Our analysis of spontaneous linA-, linC-, and linRED-deletion mutants of UT26 revealed the involvement of IS6100 in their deduced genome rearrangements. These facts strongly suggest that IS6100 plays important roles both in the dissemination of the specific lin genes and in the genome rearrangements.

Pseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivation media exhibited alginate lyase activity to depolymerize the alginate molecules having homopolymeric and heteropolymeric forms of mannuronate and guluronate so as to mainly convert into the dimer to tetramer. A DNA fragment encoding a portion of alginate lyase was amplified from AR06 genomic DNA by PCR using a set of degenerated primers, and then the whole alginate lyase gene, named alyA, and its flanking regions were obtained from a cosmid library of AR06 genomic DNA. The alyA mutant of AR06 showed (1) the loss of alginate depolymerization activity on alginate agar plate and (2) significant growth defects in alginate minimal medium; these defects were complemented by the introduction of the alyA gene. Furthermore, zymography and biochemical analyses revealed that three extracellular protein bands of AR06 had alginate lyase activities and that all three protein bands were derived from the nascent alyA gene product. These results clearly indicated that the alyA gene greatly contributes to the assimilation of alginate in AR06. The transcription of the alyA gene was induced by the presence of alginate in minimal medium, but its obvious induction was not observed in rich medium even in the presence of alginate.

gamma-Hexachlorocyclohexane (gamma-HCH, also called gamma-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of gamma-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. gamma-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the gamma-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the gamma-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.

Summary Acidovorax sp. (formally Pseudomonas sp.) strain KKS102 carries a bph operon for the degradation of PCB/biphenyl. Transcription from the pE promoter for the bph operon was found to be under catabolite control, i.e. the promoter activity was at a lower level when succinate, fumarate or acetate was added to the culture. Some mutations in the immediate upstream region of the pE promoter resulted in catabolite-insensitive and constitutively low promoter activity, suggesting that a transcriptional activator was involved in catabolite control. A genetic screen for a pE promoter activator identified two tandemly arranged genes, bphP and bphQ, that encoded proteins homologous to the sensor kinases and response regulators, respectively, of two-component regulatory system. In the bphPQ double mutant, pE promoter activity was weak and catabolite-insensitive, and a supply of the bphQ gene alone led to the restoration of the catabolite response. The mechanism of catabolite repression in KKS102 is explained in terms of inhibition of activation by BphQ. The genes highly similar to bphQ were found from several β-proteobacteria, such as Burkholderia cenocepacia J2315, B. multivorans ATCC17616, B. xenovorans LB400 and Ralstonia solanacearum RS1085.

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis -derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS 401 , IS 402 , IS 406 , IS 407 , and IS 408) , three novel IS elements, IS Bmu1 , IS Bmu2 , and IS Bmu3 . The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To reveal the distribution and organization of the genes for fundamental cell functions on the genome of this bacterium, the dnaA and dnaK gene regions of ATCC 17616 were cloned and characterized. The gene organization of the dnaA region was rnpA-rmpH-dnaA-dnaN-gyrB with a single consensus DnaA-binding box (TTATCCACA) between the rmpH and dnaA genes. This intergenic region, however, did not work as an autonomously replicating sequence in ATCC 17616. On the other hand, the gene organization of the dnaK region was grpE-orf1 (gene for thioredoxin homologue)-dnaK-dnaJ-pabB (gene for p-aminobenzoate synthetase component homologue). A putative heat-shock promoter that showed good homology to the sigma32-dependent promoter consensus sequence in Escherichia coli was found upstream of the grpE gene, suggesting that these five genes constitute an operon. In M9 succinate minimal medium the dnaJ mutant grew more slowly than the wild-type strain, indicating that this operon is functional. Pulsed-field gel electrophoresis and Southern blot analyses indicated that both the dnaA and dnaK gene regions exist as single copies on the 3.4 Mb chromosome.

The bph genes in Pseudomonas sp. KKS102, which are involved in the degradation of polychlorinated biphenyl/biphenyl, are induced in the presence of biphenyl. In this study our goal was to understand the regulatory mechanisms involved in the inducible expression. The bph genes (bphEGF(orf4)A1A2A3BCD(orf1)A4R) constitute an operon, and its expression is strongly dependent on the pE promoter located upstream of the bphE gene. A bphS gene, whose deduced amino acid sequence showed homology with the GntR family transcriptional repressors, was identified at the upstream region of the bphE gene. Disruption of the bphS gene resulted in constitutive expression of bph genes, suggesting that the bphS gene product negatively regulated the pE promoter. The gel retardation and DNase footprinting analyses demonstrated specific binding of BphS to the pE promoter region and identified four BphS binding sites that were located within and immediately downstream of the -10 box of the pE promoter. The four binding sites were functional in repression because their respective elimination resulted in derepression of the pE promoter. The binding of BphS was abolished in the presence of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, an intermediate compound in the biphenyl degradation pathway. We concluded that the negative regulator BphS plays a central role in the regulation of bph gene expression through its action at the pE promoter.

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.