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Site-specific recombination (SSR) systems are employed for transfer of mobile genetic elements (MGEs), such as lysogenic phages and integrative conjugative elements (ICEs). SSR between attP/I and attB sites is mediated by an integrase (Int) and a recombination directionality factor (RDF). The genome of Bacillus subtilis 168 contains SPβ, an active prophage, skin, a defective prophage, and ICEBs1, an integrative conjugative element. Each of these MGEs harbors the classic SSR unit attL-int-rdf-attR. Here, we demonstrate that these SSR units are all compatible and can substitute for one another. Specifically, when SPβ is turned into a defective prophage by deletion of its SSR unit, introduction of the SSR unit of skin or ICE converts it back to an active prophage. We also identified closely related prophages with distinct SSR units that control developmentally regulated gene rearrangements of kamA (L-lysine 2,3-aminomutase). These results suggest that SSR units are interchangeable components of MGEs.

DOI： 10.1016/j.isci.2019.100805

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Polysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation, Bacillus subtilis, the composition of the spore PS is not known in detail. Here, we have assessed how PS synthesis enzymes produced during the late stages of sporulation affect spore surface properties. Using four methods, bacterial adhesion to hydrocarbons (BATH) assays, India ink staining, transmission electron microscopy (TEM) with ruthenium red staining, and scanning electron microscopy (SEM), we characterized the contributions of four sporulation gene clusters, spsABCDEFGHIJKL, yfnHGF-yfnED, ytdA-ytcABC, and cgeAB-cgeCDE, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in the sps operon result in the production of spores that are more hydrophobic and lack a visible crust, presumably because of reduced PS deposition, while mutations in cgeD and the yfnH-D cluster noticeably expand the PS layer. In addition, yfnH-D mutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype from sps mutant spores was partially rescued by a second mutation inactivating any gene in the yfnHGF operon. While spsI, yfnH, and ytdA are paralogous genes, all encoding glucose-1-phosphate nucleotidyltransferases, each paralog appears to contribute in a distinct manner to the spore PS. Our data are consistent with the possibility that each gene cluster is responsible for the production of its own respective deoxyhexose. In summary, we found that disruptions to the PS layer modify spore surface hydrophobicity and that there are multiple saccharide synthesis pathways involved in spore surface properties.IMPORTANCE Many bacteria are characterized by their ability to form highly resistant spores. The dormant spore state allows these species to survive even the harshest treatments with antimicrobial agents. Spore surface properties are particularly relevant because they influence spore dispersal in various habitats from natural to human-made environments. The spore surface in Bacillus subtilis (crust) is composed of a combination of proteins and polysaccharides. By inactivating the enzymes responsible for the synthesis of spore polysaccharides, we can assess how spore surface properties such as hydrophobicity are modulated by the addition of specific carbohydrates. Our findings indicate that several sporulation gene clusters are responsible for the assembly and allocation of surface polysaccharides. Similar mechanisms could be modulating the dispersal of infectious spore-forming bacteria.

DOI： 10.1128/JB.00321-19

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Rotavirus A (RVA) is the predominant etiological agent of acute gastroenteritis in young children worldwide. Recently, unusual G9P[4] rotavirus strains emerged with high prevalence in many countries. Such intergenogroup reassortant strains highlight the ongoing spread of unusual rotavirus strains throughout Asia. This study was undertaken to determine the whole genome of eleven unusual G9P[4] strains detected in India during 2011-2013, and to compare them with other human and animal global RVAs to understand the exact origin of unusual G9P[4] circulating in India and other countries worldwide. Of these 11 RVAs, four G9P[4] strains were double-reassortants with the G9-VP7 and E6-NSP4 genes on a DS-1-like genetic backbone (G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2). The other strains showed a complex genetic constellation, likely derived from triple reassortment event with the G9-VP7, N1-NSP2 and E6-NSP4 on a DS-1-like genetic backbone (G9-P[4]-I2-R2-C2-M2-A2-N1-T2-E6-H2). Presumably, these unusual G9P[4] strains were generated after several reassortment events between the contemporary co-circulating human rotavirus strains. Moreover, the point mutation S291L at the interaction site between inner and outer capsid proteins of VP6 gene may be important in the rapid spread of this unusual strain. The complex reassortment events within the G9[4] strains may be related to the high prevalence of mixed infections in India as reported in this study and other previous studies.

DOI： 10.1016/j.meegid.2017.07.025

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Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years.

DOI： 10.1371/journal.pntd.0005386

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Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC-state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT-29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. These characteristic changes in VBNC-state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.

DOI： 10.1111/1348-0421.12246

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In developed countries including Japan, malignant tumor (cancer), heart disease and cerebral apoplexy are major causes of death, but infectious diseases are still responsible for a high number of deaths in developing countries, especially among children aged less than 5 years. World Health Statistics published by WHO reports a high percentage of mortality from infectious diseases in children, and many of these diseases may be subject to transmission across borders and could possibly invade Japan. Given this situation, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan initiated Phase I of the Program of Founding Research Centers for Emerging and Reemerging Infectious Disease, which ran from FY 2005 to 2009, and involved 8 Japanese universities and 2 research centers. The program was established for the following purposes: 1) creation of a domestic research structure to promote the accumulation of fundamental knowledge about infectious diseases, 2) establishment of 13 overseas research collaboration centers in 8 countries at high risk of emerging and reemerging infections and at which Japanese researchers are stationed and conduct research in partnership with overseas instructors, 3) development of a network among domestic and overseas research centers, and 4) development of human resources. The program was controlled under MEXT and managed by the RIKEN Center of Research Network for Infectious Diseases (Riken CRNID). Phase II of the program was set up as the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), and has been running in FY 2010-2014. Phase III will start in April 2015, and will be organized by the newly established Japanese governmental organization "Japan Agency for Medical Research and Development (AMED)", the so-called Japanese style NIH. The Collaborative Research Center of Okayama University for Infectious Diseases in India (CRCOUI) was started up in 2007 at the National Institute of Cholera and Enteric Disease, Kolkata, India. Major projects of CRCOUI are concerned with diarrheal diseases such as, 1) active surveillance of diarrheal patients, 2) development of dysentery vaccines, 3) viable but nonculturable (VBNC) Vibrio cholerae, and 4) pathogenic mechanisms of various diarrhogenic microorganisms. This review article outlines project of J-GRID and CRCOUI which the authors carried out collaboratively with NICED staff members.

DOI： 10.4265/bio.20.77

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We tried to discriminate 16 strains of the Bacillus cereus group including B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis strains by the pattern analysis of Random Amplified Polymorphic DNA (RAPD) -PCR. Eight oligonucleotides primers were prepared and the polymorphic patterns of the DNA of each strain were compared with those of others. The primers E and F gave different patterns of RAPD-PCR products in all strains of the B. cereus group, so these primers are effective tools for the discrimination of closely related strains. All eight primers showed different polymorphic patterns of DNA for the four strains of B. cereus isolated from the kitchen of a private home, which verifies the advantage of the RAPD-PCR analysis for the discrimination of isolated strains of B. cereus from the environment.

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The Gram-positive bacterium Bacillus subtilis forms spores when conditions are unsuitable for growth. The spores are encased in a multilayered shell that includes a cortex and a spore coat, and remain viable for long periods in the harsh environment. In the present article, recent progress in our understanding of the outer structure of B. subtilis spores is reviewed in the Japanese language. Although spore coat assembly involves the deposition of at least 70 distinct protein species, the positions of most of such proteins have not been experimentally determined. To this end, the diameters of the protein layers and spores were measured using fluorescence microscopy and then the positions of proteins in the spore coat of B. subtilis spores were estimated. The locations of 16 proteins were determined using this method. One protein was assigned to the cortex, nine to the inner coat, and four to the outer coat. Further, two proteins, CgeA and CotZ, were assigned to a previously unidentified outermost layer. McKenney et al. have also identified the outermost layer using a similar method; the layer was termed the "crust". Immunofluorescence microscopy revealed that the crust is indeed the most external layer of B. subtilis spores. Mutational analysis indicated that all genes in the cotVWXYZ cluster were involved in spore crust synthesis and that CotY and CotZ played critical roles in crust formation.

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To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.

DOI： 10.1128/JB.05310-11

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SpoIIGA is a novel type of membrane-associated aspartic protease that responds to a signal from the forespore by cleaving Pro-σ(E) in the mother cell during sporulation of Bacillus subtilis. Very little is known about how SpoIIGA recognizes Pro-σ(E). By co-expressing proteins in Escherichia coli, it was shown that charge reversal substitutions for acidic residues 24 and 25 of Pro-σ(E), and for basic residues 245 and 284 of SpoIIGA, impaired cleavage. These results are consistent with a model predicting possible electrostatic interactions between these residues; however, no charge reversal substitution for residue 245 or residue 284 of SpoIIGA restored cleavage of Pro-σ(E) with a charge reversal substitution for residue 24 or residue 25. Bacillus subtilis SpoIIGA cleaved Pro-σ(E) orthologs from Bacillus licheniformis and Bacillus halodurans, but not from Bacillus cereus. A triple substitution in the pro-sequence of B. cereus Pro-σ(E) allowed cleavage by B. subtilis SpoIIGA, indicating that residues distal from the cleavage site contribute to substrate specificity. Co-expression of SpoIIGA and Pro-σ(E) orthologs in different combinations suggested that B. licheniformis SpoIIGA has a relatively narrow substrate specificity as compared with B. subtilis SpoIIGA, whereas B. cereus SpoIIGA and B. halodurans SpoIIGA appear to have broader substrate specificity.

DOI： 10.1093/jb/mvr027

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Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.

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Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring- or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.

DOI： 10.1128/JB.01103-09

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The yeeK gene of Bacillus subtilis is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in yabG mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the yeeK gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of yeeK did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in yabG mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.

DOI： 10.1128/JB.01269-08

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The bacterium Bacillus subtilis undergoes endospore formation in response to starvation. sigma factors play a key role in spatiotemporal regulation of gene expression during development. Activation of sigma factors is coordinated by signal transduction between the forespore and the mother cell. sigma(E) is produced as pro-sigma(E), which is activated in the mother cell by cleavage in response to a signal from the forespore. We report that expression of SpoIIR, a putative signaling protein normally made in the forespore, and SpoIIGA, a putative protease, is necessary and sufficient for accurate, rapid, and abundant processing of pro-sigma(E) to sigma(E) in Escherichia coli. Modeling and mutational analyses provide evidence that SpoIIGA is a novel type of aspartic protease whose C-terminal half forms a dimer similar to the human immunodeficiency virus type 1 protease. Previous studies suggest that the N-terminal half of SpoIIGA is membrane-embedded. We found that SpoIIGA expressed in E. coli is membrane-associated and that after detergent treatment SpoIIGA was self-associated. Also, SpoIIGA interacts with SpoIIR. The results support a model in which SpoIIGA forms inactive dimers or oligomers, and interaction of SpoIIR with the N-terminal domain of SpoIIGA on one side of a membrane causes a conformational change that allows formation of active aspartic protease dimer in the C-terminal domain on the other side of the membrane, where it cleaves pro-sigma(E).

DOI： 10.1074/jbc.M708962200

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Endospore formation by Bacillus subtilis involves three differentiating cell types, the predivisional cell, the mother cell, and the forespore. Here we report the program of gene expression in the forespore, which is governed by the RNA polymerase sigma factors sigma(F) and sigma(G) and the DNA-binding proteins RsfA and SpoVT. The sigma(F) factor turns on about 48 genes, including the gene for RsfA, which represses a gene in the sigma(F) regulon, and the gene for sigma(G). The sigma(G) factor newly activates 81 genes, including the gene for SpoVT, which turns on (in nine cases) or stimulates (in 11 cases) the expression of 20 genes that had been turned on by sigma(G) and represses the expression of 27 others. The forespore line of gene expression consists of many genes that contribute to morphogenesis and to the resistance and germination properties of the spore but few that have metabolic functions. Comparative genomics reveals a core of genes in the sigma(F) and sigma(G) regulons that are widely conserved among endospore-forming species but are absent from closely related, but non-spore-forming Listeria spp. Two such partially conserved genes (ykoU and ykoV), which are members of the sigma(G) regulon, are shown to confer dry-heat resistance to dormant spores. The ykoV gene product, a homolog of the non-homologous end-joining protein Ku, is shown to associate with the nucleoid during germination. Extending earlier work on gene expression in the predivisional cell and the mother cell, we present an integrated overview of the entire program of sporulation gene expression.

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We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.

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It is well known that the ykvU-ykvV operon is under the regulation of the sigma(E)-associated RNA polymerase (Esigma(E)). In our study, we observed that ykvV is transcribed together with the upstream ykvU gene by Esigma(E) in the mother cell and monocistronically under Esigma(G) control in the forespore. Interestingly, alternatively expressed ykvV in either the forespore or the mother cell increased the sporulation efficiency in the ykvV background. Studies show that the YkvV protein is a member of the thioredoxin superfamily and also contains a putative Sec-type secretion signal at the N terminus. We observed efficient sporulation in a mutant strain obtained by replacing the putative signal peptide of YkvV with the secretion signal sequence of SleB, indicating that the putative signal sequence is essential for spore formation. These results suggest that YkvV is capable of being transported by the putative Sec-type signal sequence into the space between the double membranes surrounding the forespore. The ability of ykvV expression in either compartment to complement is indeed intriguing and further introduces a new dimension to the genetics of B. subtilis spore formation. Furthermore, electron microscopic observation revealed a defective cortex in the ykvV disruptant. In addition, the expression levels of sigma(K)-directed genes significantly decreased despite normal sigma(G) activity in the ykvV mutant. However, immunoblotting with the anti-sigma(K) antibody showed that pro-sigma(K) was normally processed in the ykvV mutant, indicating that YkvV plays an important role in cortex formation, consistent with recent reports. We therefore propose that ykvV should be renamed spoIVH.

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The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

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To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

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