Transgenic Arabidopsis thaliana plants expressing bacterial γ-hexachlorocyclohexane dehydrochlorinase LinA.

BACKGROUND: γ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation. RESULTS: We generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h. CONCLUSION: The transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.

Three distinct metabolic phases of polychlorinated biphenyls/biphenyl degrader Acidovorax sp. KKS102 in nutrient broth.

Acidovorax sp. KKS102 is a beta-proteobacterium capable of degrading polychlorinated biphenyls (PCBs). In this study, we examined its growth in liquid nutrient broth supplemented with different carbon sources. KKS102 had at least 3 distinct metabolic phases designated as metabolic phases 1-3, with phase 2 having 2 sub-phases. For example, succinate, fumarate, and glutamate, known to repress the PCB/biphenyl catabolic operon in KKS102, were utilized in phase 1, while acetate, arabinose, and glycerol in phase 2, and glucose and mannose in phase 3. We also showed that the BphQ response regulator mediating catabolite control in KKS102, whose expression level increased moderately through the growth, plays important roles in carbon metabolism in phases 2 and 3. Our study elucidates the hierarchical growth of KKS102 in nutrient-rich media. This insight is crucial for studies exploiting microbial biodegradation capabilities and advancing studies for catabolite regulation mechanisms.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate - TraD and TraD - T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.

Degradation of DDT by γ-hexachlorocyclohexane dehydrochlorinase LinA.

1,1,1-Trichloro-2,2-bis(4-chlorophenyl)-ethane (DDT) is the first synthetic insecticide and one of the most widely used pesticides. The use of DDT has been banned, but it remains one of the most notorious environmental pollutants around the world. In this study, we found that γ-hexachlorocyclohexane (γ-HCH) dehydrochlorinase LinA from a γ-HCH-degrading bacterium, Sphingobium japonicum UT26, converts DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl)-ethylene (DDE). Because of the weak DDT degradation activity of LinA, we could not detect such activity in UT26 cells expressing LinA constitutively. However, the linA-deletion mutant of UT26 harboring a plasmid for the expression of LinA, in which LinA was expressed at a higher level than UT26, showed the DDT degradation activity. This outcome highlights the potential for constructing DDT-degrading sphingomonad cells through elevated LinA expression.

Ligand-displaying Escherichia coli cells and minicells for programmable delivery of toxic payloads via type IV secretion systems.

IMPORTANCE: The rapid emergence of drug-resistant bacteria and current low rate of antibiotic discovery emphasize the urgent need for alternative antibacterial strategies. We engineered Escherichia coli to conjugatively transfer plasmids to specific E. coli and Pseudomonas aeruginosa recipient cells through the surface display of cognate nanobody/antigen (Nb/Ag) pairs. We further engineered mobilizable plasmids to carry CRISPR/Cas9 systems (pCrispr) for the selective killing of recipient cells harboring CRISPR/Cas9 target sequences. In the assembled programmed delivery system (PDS), Nb-displaying E. coli donors with different conjugation systems and mobilizable pCrispr plasmids suppressed the growth of Ag-displaying recipient cells to significantly greater extents than unpaired recipients. We also showed that anucleate minicells armed with conjugation machines and pCrispr plasmids were highly effective in killing E. coli recipients. Together, our findings suggest that bacteria or minicells armed with PDSs may prove highly effective as an adjunct or alternative to antibiotics for antimicrobial intervention.

Ligand-Displaying E. coli Cells and Minicells for Programmable Delivery of Toxic Payloads via Type IV Secretion Systems.

Bacterial type IV secretion systems (T4SSs) are highly versatile macromolecular translocators and offer great potential for deployment as delivery systems for therapeutic intervention. One major T4SS subfamily, the conjugation machines, are well-adapted for delivery of DNA cargoes of interest to other bacteria or eukaryotic cells, but generally exhibit modest transfer frequencies and lack specificity for target cells. Here, we tested the efficacy of a surface-displayed nanobody/antigen (Nb/Ag) pairing system to enhance the conjugative transfer of IncN (pKM101), IncF (F/pOX38), or IncP (RP4) plasmids, or of mobilizable plasmids including those encoding CRISPR/Cas9 systems (pCrispr), to targeted recipient cells. Escherichia coli donors displaying Nb's transferred plasmids to E. coli and Pseudomonas aeruginosa recipients displaying the cognate Ag's at significantly higher frequencies than to recipients lacking Ag's. Nb/Ag pairing functionally substituted for the surface adhesin activities of F-encoded TraN and pKM101-encoded Pep, although not conjugative pili or VirB5-like adhesins. Nb/Ag pairing further elevated the killing effects accompanying delivery of pCrispr plasmids to E. coli and P. aeruginosa transconjugants bearing CRISPR/Cas9 target sequences. Finally, we determined that anucleate E. coli minicells, which are clinically safer delivery vectors than intact cells, transferred self-transmissible and mobilizable plasmids to E. coli and P. aeruginosa cells. Minicell-mediated mobilization of pCrispr plasmids to E. coli recipients elicited significant killing of transconjugants, although Nb/Ag pairing did not enhance conjugation frequencies or killing. Together, our findings establish the potential for deployment of bacteria or minicells as Programmed Delivery Systems (PDSs) for suppression of targeted bacterial species in infection settings. IMPORTANCE: The rapid emergence of drug-resistant bacteria and current low rate of antibiotic discovery emphasize an urgent need for alternative antibacterial strategies. We engineered Escherichia coli to conjugatively transfer plasmids to specific E. coli and Pseudomonas aeruginosa recipient cells through surface display of cognate nanobody/antigen (Nb/Ag) pairs. We further engineered mobilizable plasmids to carry CRISPR/Cas9 systems (pCrispr) for selective killing of recipient cells harboring CRISPR/Cas9 target sequences. In the assembled Programmed Delivery System (PDS), Nb-displaying E. coli donors with different conjugation systems and mobilizable pCrispr plasmids suppressed growth of Ag-displaying recipient cells to significantly greater extents than unpaired recipients. We also showed that anucleate minicells armed with conjugation machines and pCrispr plasmids were highly effective in killing of E. coli recipients. Together, our findings suggest that bacteria or minicells armed with PDSs may prove highly effective as an adjunct or alternative to antibiotics for antimicrobial intervention.

IncFV plasmid pED208: Sequence analysis and evidence for translocation of maintenance/leading region proteins through diverse type IV secretion systems.

Two phylogenetically distantly-related IncF plasmids, F and pED208, serve as important models for mechanistic and structural studies of F-like type IV secretion systems (T4SSFs) and F pili. Here, we present the pED208 sequence and compare it to F and pUMNF18, the closest match to pED208 in the NCBI database. As expected, gene content of the three cargo regions varies extensively, although the maintenance/leading regions (MLRs) and transfer (Tra) regions also carry novel genes or motifs with predicted modulatory effects on plasmid stability, dissemination and host range. By use of a Cre recombinase assay for translocation (CRAfT), we recently reported that pED208-carrying donors translocate several products of the MLR (ParA, ParB1, ParB2, SSB, PsiB, PsiA) intercellularly through the T4SSF. Here, we extend these findings by reporting that pED208-carrying donors translocate 10 additional MLR proteins during conjugation. In contrast, two F plasmid-encoded toxin components of toxin-antitoxin (TA) modules, CcdB and SrnB, were not translocated at detectable levels through the T4SSF. Remarkably, most or all of the pED208-encoded MLR proteins and CcdB and SrnB were translocated through heterologous T4SSs encoded by IncN and IncP plasmids pKM101 and RP4, respectively. Together, our sequence analyses underscore the genomic diversity of the F plasmid superfamily, and our experimental data demonstrate the promiscuous nature of conjugation machines for protein translocation. Our findings raise intriguing questions about the nature of T4SS translocation signals and of the biological and evolutionary consequences of conjugative protein transfer.

Contributions of F-specific subunits to the F plasmid-encoded type IV secretion system and F pilus.

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSF s). Assembly of T4SSF s and associated F pili requires 10 VirB/VirD4-like Tra subunits and eight or more F-specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SSF s undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F-specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SSF structures by in situ cryoET. Mutant phenotypes supported the assignment of F-specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SSF -associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure-driven model in which F-specific subunits contribute to distinct steps of T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.

Monoblastic leukemia (M5a) with chronic basophilic leukemia in a cat.

A cat was presented with depression and anorexia. The complete blood cell count (CBC) revealed non-regenerative anemia (PCV, 8.5%), marked thrombocytopenia (2,400/µl), and leukocytosis (32,090/µl). In the peripheral blood, proliferation of blast cells (85%; 27,276/µl) and basophils (7.7%; 2,460/µl) was observed. Bone marrow aspirate showed hyperplasia with 8.8% blasts and 90.2% basophils of all nucleated cells. The blast cells were negative for myeloperoxidase staining and positive for alpha-naphthol butyrate esterase staining, indicating the agranular blasts are monoblasts. Thus, acute monoblastic leukemia (M5a) with chronic basophilic leukemia was diagnosed. Basophils accounted for more than 40% of the bone marrow, and we diagnosed secondary basophilic leukemia. Secondary basophilic leukemia should be included in the differential list when abnormal basophil increases are observed in feline bone marrow.

Protein Transfer through an F Plasmid-Encoded Type IV Secretion System Suppresses the Mating-Induced SOS Response.

Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence genes. Here, we report that the pED208-encoded T4SS (TrapED208) translocates not only this F plasmid but several plasmid-encoded proteins, including ParA, ParB1, single-stranded DNA-binding protein SSB, ParB2, PsiB, and PsiA, to recipient cells. Conjugative protein translocation through the TrapED208 T4SS required engagement of the pED208 relaxosome with the TraD substrate receptor or coupling protein. T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells. Transfer of pED208 deleted of psiB or ssb, which, respectively, encode the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, elicited a significantly stronger SOS response than pED208 or mutant plasmids deleted of psiA, parA, parB1, or parB2. Conversely, translocation of PsiB or SSB, but not PsiA, through the TrapED208 T4SS suppressed the mating-induced SOS response. Our findings expand the repertoire of known substrates of conjugation systems to include proteins with functions associated with plasmid maintenance. Furthermore, for this and other F-encoded Tra systems, docking of the DNA substrate with the TraD receptor appears to serve as a critical activating signal for protein translocation. Finally, the observed effects of PsiB and SSB on suppression of the mating-induced SOS response establishes a novel biological function for conjugative protein translocation and suggests the potential for interbacterial protein translocation to manifest in diverse outcomes influencing bacterial communication, physiology, and evolution. IMPORTANCE Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or "mating" channels, can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly. The repertoire of translocated proteins includes the plasmidic SOS inhibitor protein PsiB, single-stranded DNA-binding protein SSB, and several partitioning proteins. We demonstrate that intercellular transmission of PsiB and SSB suppresses the SOS response, which is triggered in recipient cells upon acquisition of the single-stranded DNA transfer intermediate during mating. Our findings identify a new biological function for conjugative protein translocation in mitigating potentially deleterious consequences to plasmid and genome integrity resulting from SOS-induced recombination and mutation events.

A transcriptional regulator, IscR, of Burkholderia multivorans acts as both repressor and activator for transcription of iron-sulfur cluster-biosynthetic isc operon.

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 IncP-9 Catabolic Plasmids Requires a Previously Uncharacterized Gene, mpfK, Whose Homologs Are Conserved in Various MPFT-Type Plasmids.

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.

Suppression of substrate inhibition in phenanthrene-degrading Mycobacterium by co-cultivation with a non-degrading Burkholderia strain.

In natural environments contaminated by recalcitrant organic pollutants, efficient biodegradation of such pollutants has been suggested to occur through the cooperation of different bacterial species. A phenanthrene-degrading bacterial consortium, MixEPa4, from polluted soil was previously shown to include a phenanthrene-degrading strain, Mycobacterium sp. EPa45, and a non-polycyclic aromatic hydrocarbon (PAH)-degrading strain, Burkholderia sp. Bcrs1W. In this study, we show that addition of phenanthrene to rich liquid medium resulted in the transient growth arrest of EPa45 during its degradation of phenanthrene. RNA-sequencing analysis of the growth-arrested cells showed the phenanthrene-dependent induction of genes that were predicted to be involved in the catabolism of this compound, and many other cell systems, such as a ferric iron-uptake, were up-regulated, implying iron deficiency of the cells. This negative effect of phenanthrene became much more apparent when using phenanthrene-containing minimal agar medium; colony formation of EPa45 on such agar was significantly inhibited in the presence of phenanthrene and its intermediate degradation products. However, growth inhibition was suppressed by the co-residence of viable Bcrs1W cells. Various Gram-negative bacterial strains, including the three other strains from MixEPa4, also exhibited varying degrees of suppression of the growth inhibition effect on EPa45, strongly suggesting that this effect is not strain-specific. Growth inhibition of EPa45 was also observed by other PAHs, biphenyl and naphthalene, and these two compounds and phenanthrene also inhibited the growth of another mycobacterial strain, M. vanbaalenii PYR-1, that can use them as carbon sources. These phenomena of growth inhibition were also suppressed by Bcrs1W. Our findings suggest that, in natural environments, various non-PAH-degrading bacterial strains play potentially important roles in the facilitation of PAH degradation by the co-residing mycobacteria.

Establishment of plasmid vector and allelic exchange mutagenesis systems in a mycobacterial strain that is able to degrade polycyclic aromatic hydrocarbon.

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.

Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase.

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 (oriTN) 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 oriTN 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 oriTN region, between two copies of the pWW0 oriT (oriTW) region (which is clearly different from the oriTN region), and between the oriTN and oriTW 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 oriTN-containing plasmid. Such a plasmid was, in the presence of the NAH7Δnic 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.

Comparison of the complete genome sequences of four γ-hexachlorocyclohexane-degrading bacterial strains: insights into the evolution of bacteria able to degrade a recalcitrant man-made pesticide.

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.

Complete Genome Sequence of a γ-Hexachlorocyclohexane-Degrading Bacterium, Sphingobium sp. Strain MI1205.

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

Complete Genome Sequence of a γ-Hexachlorocyclohexane Degrader, Sphingobium sp. Strain TKS, Isolated from a γ-Hexachlorocyclohexane-Degrading Microbial Community.

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

Complete Genome Sequence of Pseudomonas sp. Strain TKP, Isolated from a γ-Hexachlorocyclohexane-Degrading Mixed Culture.

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.