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Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012. [ABSTRACT FROM PUBLISHER]

This study examined the association between peak exercise systolic blood pressure and other exercise test parameters and the long-term (19-year) survival of 625 patients with myocardial infarction who were original participants of the National Exercise and Heart Disease Project, a 3-year (1976 to 1979) multicenter randomized exercise clinical trial. Results show that low peak exercise systolic blood pressure (< or =140 mm Hg) was associated with increased mortality throughout the 19 years of follow-up, and men with this finding obtained no survival benefit from participating in an exercise program. [ABSTRACT FROM AUTHOR]

The diagnosis of intravascular large B-cell lymphoma (IVLBCL) is often challenging owing to its nonspecific clinical manifestations and imaging findings. Herein, we present a rare case of IVLBCL in which seizure was the initial symptom, and unilateral hemispheric hyperperfusion on arterial spin labeling (ASL) was the only abnormal finding observed on brain magnetic resonance imaging (MRI). A 68-year-old male with a history of hypertension and type 2 diabetes was transferred to the emergency room owing to the sudden onset of altered consciousness and abnormal behavior. Upon arrival, the patient was disoriented and confused, and cerebrospinal fluid analysis revealed pleocytosis and elevated protein level. Even after the administration of acyclovir and antiepileptic drugs, his consciousness remained impaired, with repeated transient right hemiparesis indicating a focal seizure. The initial and follow-up MRI scans showed no obvious abnormalities in diffusion-weighted imaging (DWI), T2-weighted imaging, or susceptibility-weighted imaging (SWI); however, ASL revealed markedly increased blood flow to the left hemisphere. Subsequently, the rapid elevation of serum lactate dehydrogenase (LDH) and soluble interleukin-2 receptor (sIL-2R) levels after admission led to the diagnosis of IVLBCL by random skin biopsy and bone marrow examination. Despite the initiation of chemotherapy, the patient developed tumor lysis syndrome and succumbed to multiple organ failure. This case underscores the importance of considering IVLBCL in adult patients with refractory seizures and highlights the potential utility of ASL on MRI for early diagnosis., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Imamura et al.)

The Bacillus subtilis spore is composed of a core, containing chromosomal DNA, surrounded by a cortex layer made of peptidoglycan, and a coat composed of concentric proteinaceous layers. A polysaccharide layer is added to the spore surface, and likely anchored to the crust, the coat outermost layer. However, the identity of the coat protein(s) to which the spore polysaccharides (SPS) are attached is uncertain. First, we showed that the crust proteins CotVWXYZ and CgeA were all contained in the peeled SPS layer obtained from a strain missing CotE, the outer coat morphogenetic protein, suggesting that the SPS is indeed bound to at least one of the spore surface proteins. Second, CgeA is known to be located at the most downstream position in the crust assembly pathway. An analysis of truncated variants of CgeA suggested that its N-terminal half is required for localization to the spore surface, while its C-terminal half is necessary for SPS addition. Third, an amino acid substitution strategy revealed that SPS was anchored at threonine 112 (T112), which constitutes a probable O-glycosylation site on CgeA. Our results indicated that CgeA is a glycoprotein required to initiate SPS assembly and serves as an anchor protein linking the crust and SPS layers., (© 2023 John Wiley & Sons Ltd.)

Site-specific recombination (SSR) systems are employed in many genetic mobile elements, including temperate phages, for their integration and excision. Recently, they have also been used as tools for applications in fields ranging from basic to synthetic biology. SPβ is a temperate phage of the Siphoviridae family found in the laboratory standard Bacillus subtilis strain 168. SPβ encodes a serine-type recombinase, SprA, and recombination directionality factor (RDF), SprB. SprA catalyzes recombination between the attachment site of the phage, attP, and that of the host, attB, to integrate phage genome into the attB site of the host genome and generate attL and attR at both ends of the prophage genome. SprB works in conjunction with SprA and switches from attB/attP to attL/R recombination, which leads to excision of the prophage. In the present study, we took advantage of this highly efficient recombination system to develop a site-specific integration and excision plasmid vector, named pSSβ. It was constructed using pUC plasmid and the SSR system components, attP, sprA and sprB of SPβ. pSSβ was integrated into the attB site with a significantly high efficiency, and the resulting pSSβ integrated strain also easily eliminated pSSβ itself from the host genome by the induction of SprB expression with xylose. This report presents two applications using pSSβ that are particularly suitable for gene complementation experiments and for a curing system of SPβ prophage, that may serve as a model system for the removal of prophages in other bacteria.

Cholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred. IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

Antimicrobial peptides play an important role in host defense against Vibrio cholerae Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS , regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carR r and carR s alleles, respectively, and replaced the carR s allele of a sensitive strain with the carR r allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarR s protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR -regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca 2+ but increased with a rise in pH. The downregulation of almEFG in CarR s strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains., (Copyright © 2020 American Society for Microbiology.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2017. Published by Elsevier B.V.)

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.

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., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

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.

To clarify the influence of moisture on the structural changes of heated nano materials, in situ high temperature transmission electron microscopy (TEM) has been carried out using a conventional analytical TEM combined with a gas injection-specimen heating holder. Air with high moisture content, above 94% relative humidity (RH), from a humidifier was directly injected onto the heated platinum catalyst dispersed on carbon black (Pt/CB), and the morphological changes of the specimens were observed at high magnification dynamically. The result of the experiment was compared with a result obtained from an experiment using air with a low moisture content, 34% RH. Active movement of the Pt particles, leading agglomeration and grain growth, occurred prior to degradation of the CB support at high moisture content. In contrast, the degradation of the CB support leading agglomeration and grain growth of the Pt particles occurred before the displacement of the Pt particles on the CB supports in a low humidity environment.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

Background: This study examined whether a supervised exercise program improved 19-year survival in 30- to 64-year-old male myocardial infarction patients., Methods and Results: The men (n=651) were participants in the National Exercise and Heart Disease Project, a 3-year multicenter randomized clinical trial conducted in the United States (1976-1979). The treatment group (n=315) exercised for 8 weeks in a laboratory. Thereafter, they jogged, cycled, or swam in a gymnasium/pool setting guided by an individualized target heart rate. Participants in the control group (n=319) were to maintain normal routines but not participate in any regular exercise program. Participants were followed up until their death or December 31, 1995. Cox proportional hazards analysis revealed the all-cause mortality risk estimates (95% CIs) in the exercise group compared with controls to be 0.69 (0.39 to 1.25) after an average follow-up of 3 years, 0.84 (0.55 to 1.28) after 5 years, 0.95 (0.71 to 1.29) after 10 years, 1.02 (0.79 to 1.32) after 15 years, and 1.09 (0.87 to 1. 36) after 19 years. Cardiovascular disease (CVD) mortality risk estimates (95% CI) for the same follow-up periods were 0.73 (0.37 to 1.43), 0.98 (0.60 to 1.61), 1.21 (0.79 to 1.60), 1.14 (0.84 to 1.54), and 1.16 (0.88 to 1.52). However, each 1-MET increase in work capacity from baseline to the end of the original trial resulted in consistent reductions in all-cause and CVD mortality risk at each follow-up period, regardless of initial work-capacity level., Conclusions: These findings indicate exercise-program participation resulted in nonsignificantly reduced mortality risks early in the follow-up period. Benefits diminished as time since participation increased, which suggests that the protective mechanisms associated with the program may be short term. Contamination between groups over time could also explain the diminished effects, because increased work capacity provided survival benefits up to 19 years.