Possible involvement of zinc transporter ZIP13 in myogenic differentiation.

Ehlers-Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.

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.

Clamping-mediated incorporation of single-stranded DNA with concomitant DNA synthesis by Taq polymerase involves nick-translation.

The development and characterization of a new enzyme reaction contribute to advancements in modern biotechnology. Here, we report a novel CIS (clamping-mediated incorporation of single-stranded DNA with concomitant DNA synthesis) reaction catalyzed by Taq polymerase. In the reaction, a single-stranded DNA (ssDNA) with 3' Cs is attached with a preformed 3' G-tail of double-stranded DNA (dsDNA); DNA syntheses starting from both 3' ends result in the incorporation of ssDNA. A 3' G-tail length of 3 nucleotides adequately supports this reaction, indicating that Taq polymerase can clump short Watson-Crick base pairs as short as three pairs and use them to initiate DNA polymerization. The reverse transcriptase from Molony murine leukemia virus catalyzes strand displacement synthesis and produces flapped-end DNA, whereas the reaction by Taq polymerase involves the nick translation. These new reaction properties may be beneficial for the development of new molecular tools applicable in various fields. Apart from its CIS reaction activity, we also report that Taq polymerase has the undesirable characteristic of removing 5' fluorescent labels from dsDNA. This characteristic may have compromised various experiments involving the preparation of fluorescently-labeled dsDNA by PCR for a long time.

Draft genome sequence of Cupriavidus sp. strain TKC, isolated from a γ-hexachlorocyclohexane-degrading community.

Cupriavidus sp. strain TKC was isolated from a microbial community enriched with γ-hexachlorocyclohexane (γ-HCH). This strain did not show γ-HCH-degrading activity but was one of the major members of the community. Here, we present the draft genome sequence of the strain TKC with a size of 7 Mb.

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.

Host range of strand-biased circularizing integrative elements: a new class of mobile DNA elements nesting in Gammaproteobacteria.

BACKGROUND: The strand-biased circularizing integrative elements (SEs) are putatively non-mobilizable integrative elements for transmitting antimicrobial resistance genes. The transposition mode and the prevalence of SEs in prokaryotes remain vague. RESULTS: To corroborate the transposition mode and the prevalence of SEs, hypothetical transposition intermediates of an SE were searched for in genomic DNA fractions of an SE host. Then, the SE core genes were defined based on gene knockout experiments, and the synteny blocks of their distant homologs were searched for in the RefSeq complete genome sequence database using PSI-BLAST. A genomic DNA fractionation experiment revealed that SE copies are present in a double-stranded nicked circular form in vivo. Operonic structure of three conserved coding sequences (intA, tfp, intB) and srap located at the left end of SEs were identified as essential for attL × attR recombination. The synteny blocks of tfp and srap homologs were detected in 3.6% of the replicons of Gammaproteobacteria but not in other taxa, implying that SE movement is host-dependent. SEs have been discovered most frequently in the orders Vibrionales (19% of replicons), Pseudomonadales (18%), Alteromonadales (17%), and Aeromonadales (12%). Genomic comparisons revealed 35 new SE members with identifiable termini. SEs are present at 1 to 2 copies per replicon and have a median length of 15.7 kb. Three newly identified SE members carry antimicrobial resistance genes, like tmexCD-toprJ, mcr-9, and blaGMA-1. Further experiments validated that three new SE members possess the strand-biased attL × attR recombination activity. CONCLUSIONS: This study suggested that transposition intermediates of SEs are double-stranded circular DNA. The main hosts of SEs are a subset of free-living Gammaproteobacteria; this represents a rather narrow host range compared to those of mobile DNA element groups discovered to date. As the host range, genetic organization, and movements are unique among the mobile DNA elements, SEs provide a new model system for host-mobile DNA element coevolution studies.

Reconstruction of a Soil Microbial Network Induced by Stress Temperature.

The microbial community is viewed as a network of diverse microorganisms connected by various interspecific interactions. While the stress gradient hypothesis (SGH) predicts that positive interactions are favored in more stressful environments, the prediction has been less explored in complex microbial communities due to the challenges of identifying interactions. Here, by applying a nonlinear time series analysis to the amplicon-based diversity time series data of the soil microbiota cultured under less stressful (30°C) or more stressful (37°C) temperature conditions, we show how the microbial network responds to temperature stress. While the genera that persisted only under the less stressful condition showed fewer positive effects, the genera that appeared only under the more stressful condition received more positive effects, in agreement with SGH. However, temperature difference also induced reconstruction of the community network, leading to an increased proportion of negative interactions at the whole-community level. The anti-SGH pattern can be explained by the stronger competition caused by increased metabolic rate and population densities. IMPORTANCE By combining amplicon-based diversity survey with recently developed nonlinear analytical tools, we successfully determined the interaction networks of more than 150 natural soil microbial genera under less or more temperature stress and explored the applicability of the stress gradient hypothesis to soil microbiota, shedding new light on the well-known hypothesis.

Algorisms used for in silico finishing of bacterial genomes based on short-read assemblage implemented in GenoFinisher, AceFileViewer, and ShortReadManager.

In these days, for bacterial genome sequence determination, ultralong reads with homopolymeric troubles are used in combinations with short reads, resulting in genomic sequences with possible incorrect uniformity of repeat sequences. We have been determining complete bacterial genomic sequences based on NGS short reads and Newbler assemblage by utilizing functions implemented in 3 software GenoFinisher, AceFileViewer, and ShortReadManager without conducting additional experiments for gap closing, proving the concept that NGS short reads enclose enough information to determine complete genome sequences. Although some manual in silico tasks are to be conducted, they will ultimately be solved in a single pipeline. In this review, we describe the tools and implemented ideas that have enabled complete sequence determination solely based on short reads, which would be useful for establishing the basis for the future development of a short-read-based assembler that enables complete and accurate genome sequence determination at a lower cost.

Genome evolution related to γ-hexachlorocyclohexane metabolic function in the soil microbial population.

γ-Hexachlorocyclohexane (γ-HCH)-degrading strain, Sphingobium sp. TA15, was newly isolated from an experimental field soil from which the archetypal γ-HCH-degrading strain, S. japonicum UT26, was isolated previously. Comparison of the complete genome sequences of these 2 strains revealed that TA15 shares the same basic genome backbone with UT26, but also has the variable regions that are presumed to have changed either from UT26 or from a putative common ancestor. Organization and localization of lin genes of TA15 were different from those of UT26. It was inferred that transposition of IS6100 had played a crucial role in these genome rearrangements. The accumulation of toxic dead-end products in TA15 was lower than in UT26, suggesting that TA15 utilizes γ-HCH more effectively than UT26. These results suggested that genome evolution related to the γ-HCH metabolic function in the soil microbial population is ongoing.

Microbial communities developing within bulk sediments under fish carcasses on a tidal flat.

Animal carcasses are often brought into tidal flats where they are at the boundary between terrestrial and marine ecosystems. Since these carcasses act as microhabitats with large amounts of energy and nutrients, they likely develop unique bacterial assemblages in the ambient sediment, which in turn may stimulate colonization of other organisms such as protozoans. However, little is known about the microbial assemblages colonized in sediment around animal carcasses in the tidal zone. Herein we examined the bacterial and ciliophoran assemblages developed in association with fish carcasses by incubating the carcasses in the Higashiyachi tidal flat (Sendai, Japan). We collected sediment samples at 2, 9, and 42 days of incubation and analyzed the bacterial and ciliophoran assemblages by 16S and 18S rRNA gene amplicon sequencing. We observed significant differences in the composition and relative abundance of bacterial and ciliophoran operational taxonomic units (OTUs) between the sediments with and without the carcasses. Our analyses suggest that these unique assemblages were created through the direct effects of the carcass and indirect effects through interactions between bacteria and ciliophorans. These results also suggest that animal carcasses developed a temporally unique microbial food web in the sediments close to the carcasses, although it disappeared for several weeks.

Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family.

Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and α4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (ΔT m = -5 to 6 °C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear ß-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties.

Transcriptome Analysis of Zygotic Induction During Conjugative Transfer of Plasmid RP4.

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.

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.

Long-term changes and effect of pterygium size on corneal topographic irregularity after recurrent pterygium surgery.

This retrospective observational study compared long-term topographic changes after recurrent- and primary-pterygium surgery depending on pterygium size. Patients who underwent recurrent-pterygium excision between 2002-2013 and age, sex, and pterygium size-matched controls who underwent primary-pterygium surgery were included (33 eyes of 33 patients in each group). Pterygium size was graded per advancing edge position: <1/3 of corneal diameter (grade 1), outside the pupil (grade 2), and within the pupillary area (grade 3). Surface asymmetry index (SAI), surface regularity index (SRI) in corneal topography, and uncorrected and best-spectacle-corrected visual acuity were compared before and 1, 3, 6, and 12 months postoperatively. Three, 17, and 13 eyes had grades 1, 2, and 3, respectively. In grade 2, the SAI and SRI were respectively significantly larger at all observation points (p = 0.01, 0.03, 0.02, 0.02, and 0.004) and before and 6 and 12 months postoperatively (p = 0.02, 0.04, and 0.03) in recurrent pterygium. In grade 3, the SAI was significantly larger before and 1, 3, and 12 months postoperatively (p = 0.04, 0.01, 0.01, and 0.02) and the SRI was significantly larger before and 12 months postoperatively (p < 0.001, 0.02) in recurrent pterygium. Corneal irregularity persisted 12 months after recurrent-pterygium surgery compared with that in same-size primary pterygium.

Expression of an alcohol dehydrogenase gene in a heterotrophic bacterium induces carbon dioxide-dependent high-yield growth under oligotrophic conditions.

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.

Special Issue: Microbial Degradation of Xenobiotics.

Xenobiotics are released into the environment by human activities, and they often cause problems such as environmental pollution, since most such compounds cannot be readily degraded, and have harmful effects on human beings and the natural ecosystem [...].

Properties and efficient scrap-and-build repairing of mechanically sheared 3' DNA ends.

Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3'-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3' termini (about 20% of the total) was not repaired after the removal of 3' phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3'- > 5' exonuclease activity of exonuclease III (3'- > 5' exonuclease, insensitive to the 3' phosphate group) was counterbalanced by the 5'- > 3' polymerase activity of T4DP. This combined reaction, termed "SB-repairing" (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es.

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.

Lessons from the genomes of lindane-degrading sphingomonads.

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.

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.