Frequency of the Insertion Sequence IS4Bsu1 among Bacillus subtilis Strains Isolated from Fermented Soybean Foods in Southeast Asia

Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome. In contrast, none of 49 B. subtilis strains of non-food origin contained IS4Bsu1. Frequent occurrence of this mobile DNA element in the soybean-fermenting B. subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.     

Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6

Probiotics and Antimicrobial Proteins - The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi....     

Comparative Studies of Bacillus Cereus Strains Isolated From Various Foods and Food Poisoning Outbreaks

Certain properties of 22 Bacillus cereus strains isolated from different foods and food poisoning episodes were investigated in order to evaluate possible différences between strains isolated from diarrhoeal and vomiting type food poisoning outbreaks. None of the strains isolated from vomiting type episodes produced acid from salicin and mannose, whereas 80 and 40 % of the strains from diarrhoeal type outbreaks were positive, respectively. No association between the antibiotic sensitivity pattern or the fatty acid composition and the source of a strain could be found, although some strains differed from the general pattern of B. cereus in some instances. No significant differences in the production of the skin factor between strains isolated from the two types of outbreaks were found either. The findings of this study support the observation that the food environment itself essentially affects the enterotoxin formation of B. cereus.     

Probiotic Potential of Bacillus Strains Isolated from an Acidic Fermented Food Idli

Present study is intended to assess the probiotic properties of Bacillus spp. isolated from idli batter, a traditional fermented food of Southern India and Sri Lanka. A total of 32 isolates were screened for potential pathogenic behaviour through haemolysis assay, DNase activity and antibiotics sensitivity. Two of the isolates were found to be potentially safe and identified as Bacillus spp. These strains were characterized for in vitro probiotic attributes and antioxidant activity. Both the strains showed strong acid and bile tolerance, transit tolerance, lysozyme tolerance, cell surface hydrophobicity, auto-aggregation, co-aggregation, biofilm formation potential and adhesion to human colon adenocarcinoma (HT 29) cell line demonstrating potential probiotic ability. These strains also exhibited considerable cholesterol binding, thermostability, β-galactosidase production, proteolytic, amylolytic and lipolytic activity. Cell-free supernatant inhibited the biofilm formation by Pseudomonas aeruginosa (KT266804) to 90%. Intact cells showed significant DPPH (41%), hydroxyl (31%), radical scavenging activity and lipid peroxidation inhibition (20.38%), while cell-free extracts exhibited significant superoxide anion radical scavenging activity (16.25%). Results revealed that isolates could be potential probiotic candidate after further assessment of in vivo probiotic properties and safety evaluation and could be utilised as starter cultures in functional foods.

     

Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.     

Genome Sequencing and Analysis of BCG Vaccine Strains

Background

Although the Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) has been available for more than 75 years, one third of the world''s population is still infected with Mycobacterium tuberculosis and approximately 2 million people die of TB every year. To reduce this immense TB burden, a clearer understanding of the functional genes underlying the action of BCG and the development of new vaccines are urgently needed.

Methods and Findings

Comparative genomic analysis of 19 M. tuberculosis complex strains showed that BCG strains underwent repeated human manipulation, had higher region of deletion rates than those of natural M. tuberculosis strains, and lost several essential components such as T-cell epitopes. A total of 188 BCG strain T-cell epitopes were lost to various degrees. The non-virulent BCG Tokyo strain, which has the largest number of T-cell epitopes (359), lost 124. Here we propose that BCG strain protection variability results from different epitopes. This study is the first to present BCG as a model organism for genetics research. BCG strains have a very well-documented history and now detailed genome information. Genome comparison revealed the selection process of BCG strains under human manipulation (1908–1966).

Conclusions

Our results revealed the cause of BCG vaccine strain protection variability at the genome level and supported the hypothesis that the restoration of lost BCG Tokyo epitopes is a useful future vaccine development strategy. Furthermore, these detailed BCG vaccine genome investigation results will be useful in microbial genetics, microbial engineering and other research fields.     

Analysis of Multiple Ethyl Methanesulfonate-Mutagenized Caenorhabditis elegans Strains by Whole-Genome Sequencing

Whole-genome sequencing (WGS) of organisms displaying a specific mutant phenotype is a powerful approach to identify the genetic determinants of a plethora of biological processes. We have previously validated the feasibility of this approach by identifying a point-mutated locus responsible for a specific phenotype, observed in an ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans strain. Here we describe the genome-wide mutational profile of 17 EMS-mutagenized genomes as assessed with a bioinformatic pipeline, called MAQGene. Surprisingly, we find that while outcrossing mutagenized strains does reduce the total number of mutations, a striking mutational load is still observed even in outcrossed strains. Such genetic complexity has to be taken into account when establishing a causative relationship between genotype and phenotype. Even though unintentional, the 17 sequenced strains described here provide a resource of allelic variants in almost 1000 genes, including 62 premature stop codons, which represent candidate knockout alleles that will be of further use for the C. elegans community to study gene function.INDUCING molecular lesions in a genome is an effective approach to interrogate the genome for its functional elements. Molecular lesions can be induced using a variety of methods. Because of their efficiency and their ability to generate alleles with various different alterations in gene activity (e.g., amorphic, antimorphic, hypomorphic, and hypermorphic), chemical mutagens, such as ethyl methanesulfonate (EMS), are frequently used in genetic mutant screens (Anderson 1995). However, due to mutagen efficiency, a mutant animal selected for a single-locus phenotype invariably contains EMS-induced “background mutations” in its genome. Experimenters try to minimize the potential impact of background mutations through outcrossing to animals with a wild-type genome. Yet no full snapshots of genome sequences right after EMS mutagenesis and after outcrossing have so far been provided to illustrate the extent of background mutations and the extent to which they can indeed be eliminated.Another caveat of using base-changing chemical mutagens is the relative difficulty associated with identifying the phenotype-causing molecular lesion. In multicellular genetic model organisms, mutant identification involves time-consuming positional cloning approaches, usually involving breeding with genetically marked strains that allow pinpointing of the location of a molecular lesion. Even with rapid, SNP-based mapping approaches in animals with short generation times, such as Caenorhabditis elegans, substantial time hurdles, particularly in the final, fine-mapping stages, still exist. Conceptually similar problems in defining the location of a molecular lesion are encountered by human geneticists who attempt to identify disease-causing genetic lesions.Whole-genome sequencing (WGS) is beginning to emerge as an efficient and cost-effective tool to shortcut time-consuming mapping and positional cloning efforts (Hobert 2010). The sequencing of an entire genome and its ensuing comparison to a wild-type reference genome can potentially directly pinpoint the molecular lesion that results in the mutant phenotype the animal has been selected for. Proof-of-concept studies in bacteria, yeast, plants, worms, and flies have validated the applicability of this approach (Sarin et al. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009; Irvine et al. 2009; Flowers et al. 2010).Present-day deep sequencing platforms used for WGS generate relatively short sequence reads, thereby posing the bioinformatic challenge to align those reads to a reference genome. We previously described a software pipeline, MAQGene, which is based on the standard alignment program MAQ (Li et al. 2008) and facilitates this bioinformatic step by providing the end user with an extensively curated list of sequence variants from a WGS run of a mutated genome compared to a reference genome (Bigelow et al. 2009). This pipeline can be used for well-annotated, assembled genomes, such as C. elegans or Drosophila. In this article, we describe that this pipeline can identify not only point mutations but also deletions. We then use this pipeline to analyze a total of 17 EMS-mutagenized genomes. We find that EMS-mutagenized genomes carry a significant mutational load including presumptive loss-of-function alleles in several protein-coding genes that can lead to synthetic genetic interactions, one of which we describe here in more detail. We show that outcrossing to wild-type animals can lighten the mutational load; however, a substantial number of sequence variants are also introduced during outcrossing. Even though background mutations uncovered by WGS may complicate the interpretation of mutant phenotypes, they do provide a potentially useful source for functional studies of the affected genes.     

Comparative Genomic Analysis of Pathogenic Factors of Pectobacterium Species Isolated in South Korea Using Whole-Genome Sequencing

In this study, we conducted whole-genome sequencing with six species of Pectobacterium composed of seven strains, JR1.1, {"type":"entrez-nucleotide","attrs":{"text":"BP201601.1","term_id":"52051741","term_text":"BP201601.1"}}BP201601.1, JK2.1, HNP201719, MYP201603, PZ1, and HC, for the analysis of pathogenic factors associated with the genome of Pectobacterium. The genome sizes ranged from 4,724,337 bp to 5,208,618 bp, with the GC content ranging from 50.4% to 52.3%. The average nucleotide identity was 98% among the two Pectobacterium species and ranged from 88% to 96% among the remaining six species. A similar distribution was observed in the carbohydrate-active enzymes (CAZymes) class and extracellular plant cell wall degrading enzymes (PCWDEs). HC showed the highest number of enzymes in CAZymes and the lowest number in the extracellular PCWDEs. Six strains showed four subsets, and HC demonstrated three subsets, except hasDEF, in type I secretion system, while the type II secretion system of the seven strains was conserved. Components of human pathogens, such as Salmonella pathogenicity island 1 type type III secretion system (T3SS) and effectors, were identified in PZ1; T3SSa was not identified in HC. Two putative effectors, including hrpK, were identified in seven strains along with dspEF. We also identified 13 structural genes, six regulator genes, and five accessory genes in the type VI secretion system (T6SS) gene cluster of six Pectobacterium species, along with the loss of T6SS in PZ1. HC had two subsets, and JK2.1 had three subsets of T6SS. With the GxSxG motif, the phospholipase A gene did locate among tssID and duf4123 genes in the T6SSa cluster of all strains. Important domains were identified in the vgrG/paar islands, including duf4123, duf2235, vrr-nuc, and duf3396.     

Genome Sequence of Lysinibacillus boronitolerans F1182, Isolated from a Traditional Korean Fermented Soybean Product

Lysinibacillus is a Gram-positive, rod-shaped, and round-spore-forming bacterial genus of the family Bacillaceae. We analyzed the genome sequence of Lysinibacillus boronitolerans F1182, isolated from a traditional Korean fermented soybean product. The genome sequence contained 4.46 Mbp with a G+C content of 37.5%. This is the first report of an L. boronitolerans genome.     

Comparative Genome Analysis of Helicobacter pylori Strains

DNA macroarrays were used to characterize 17 Helicobacter pylori strains isolated in four geographic regions of Russia (Moscow, St. Petersburg, Kazan, and Novosibirsk). Of all genes, 1272 (81%) proved to occur in all strains and to constitute a functional core of the genome, and 293 (18.7%) were strain-specific and greatly varied among the H. pylori strains. Most (71%) of the latter had unknown functions; the remainder included restriction–modification genes (3–9%), transposition genes (2–4%), and genes coding for outer membrane proteins (2–4%). The Russian H. pylori strains did not differ in genome organization or in the number and distribution of strain-specific genes from strains isolated in other countries.     

Draft Genome Sequence of Staphylococcus vitulinus F1028, a Strain Isolated from a Block of Fermented Soybean

Staphylococcus vitulinus is a coagulase-negative staphylococcus in the family Staphylococcaceae. This report describes the draft genome sequence of S. vitulinus F1028, which was isolated from a traditional Korean soybean food (meju). This 2.56-Mbp genome sequence is the first S. vitulinus genome of a strain isolated from a fermented soybean product.     

枯草芽胞杆菌基因组混组方法

基因组混组作为一种育种方法,通过循环原生质体融合等手段,使得不同菌株来源的基因组能够得到充分重组,增加将正向突变整合到同一重组子中的机会。使用4株带有4种不同标记的枯草芽胞杆菌亲本为初始菌株,通过循环转化、循环转导或循环原生质体融合的手段进行基因组混组,统计后代中非亲本类型占整个群体的比例,以衡量基因组混组的效果。分别经过5轮循环原生质体融合、循环转化或者循环转导,结果显示,重组程度较高者在后代群体中的比例较低,带有4种标记的后代未出现,带有3种标记的后代最高分别为4.53×10?4、1.64×10?4、4.47×10?3,明显低于文献报道的天蓝色链霉菌中同样实验的结果:带4种和3种标记的后代分别占2.5%、17%。对比上述实验的结果和文献报道的天蓝色链霉菌、乳杆菌基因组混组的结果,并结合计算机模拟循环融合过程,分析后认为:要达到较充分的基因组混组,需要有能够实现微生物细胞间高频重组的操作技术作为基础,重组频率应该不低于10?3～10?2数量级。     

Site-Specific Deoxyribonucleases in Bacillus subtilis and Other Bacillus Strains

We systematically studied site-specific deoxyribonucleases in Bacillus strains and detected deoxyribonuclease activities in 20 of 62 strains tested.     

Experimental Evolution of Enhanced Growth by Bacillus subtilis at Low Atmospheric Pressure: Genomic Changes Revealed by Whole-Genome Sequencing

Knowledge of how microorganisms respond and adapt to low-pressure (LP) environments is limited. Previously, Bacillus subtilis strain WN624 was grown at the near-inhibitory LP of 5 kPa for 1,000 generations and strain WN1106, which exhibited increased relative fitness at 5 kPa, was isolated. Genomic sequence differences between ancestral strain WN624 and LP-evolved strain WN1106 were identified using whole-genome sequencing. LP-evolved strain WN1106 carried amino acid-altering mutations in the coding sequences of only seven genes (fliI, parC, ytoI, bacD, resD, walK, and yvlD) and a single 9-nucleotide in-frame deletion in the rnjB gene that encodes RNase J2, a component of the RNA degradosome. By using a collection of frozen stocks of the LP-evolved culture taken at 50-generation intervals, it was determined that (i) the fitness increase at LP occurred rapidly, while (ii) mutation acquisition exhibited complex kinetics. A knockout mutant of rnjB was shown to increase the competitive fitness of B. subtilis at both LP and standard atmospheric pressure.     

Isolation and Characterization of Thymineless Mutants from Ultraviolet-Sensitive Bacillus subtilis Strains

Thymineless mutants of Bacillus subtilis 168 ind, hcr-9 were isolated by using trimethoprim. These and other Thy⁻ strains differed drastically from Thy⁺ ones in their patterns of [³H]thymidine uptake and growth in trimethoprim-containing medium. Transformation was negligible between most mutants derived from the ultraviolet-sensitive strain 168 ind, hcr-9 but significant between 168 ind, thy and these mutants. The latter and these new mutants all grow in the presence of trimethoprim plus thymidine or thymine and fail to grow if thymine or thymidine is omitted.