Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

Restriction and modification of bacteriophage SP10 DNA by Bacillus subtilis Marburg 168: stabilization of SP10 DNA in restricting hosts preinfected with a heterologous phage, SP18.

SP10 phage cannot propagate in Bacillus subtilis Marburg 168 containing the wild-type allele of either gene nonA or gene nonB. The latter gene codes for the intrinsic cellular restriction activity. SP10 DNA was degraded in nonB+ derivatives of Marburg 168. The degree of degradation depended upon the previous host in which SP10 was propagated. In the case of SP10 grown in B. subtilis W23 (a nonrestricting, nonmodifying bacterium), 90% of the phage DNA was hydrolyzed to acid solubles, and the residual acid-precipitable material was recovered as 0.5- to 1-megadalton fragments. In contrast, if SP10 was propagated in B. subtilis PS9W7 (a nonA nonB derivative of Marburg 168 that retains modifying activity), 40 to 50% of the input DNA was degraded to acid solubles, and most of the remainder was recovered as 15- to 20-megadalton fragments. In nonA+ nonB cells, SP10 DNA was conserved as unit-length molecules (ca. 80 megadalton). Prior infection of nonB+ cells with SP18 protected superinfecting SP10 DNA, even when rifampin or chloramphenicol was added before the primary infection. The data are discussed in terms of the following conclusions. (i) The nonB gene product of B. subtilis Marburg 168 is required for restriction of SP10 DNA. (ii) Some sites on SP10 DNA are sensitive to both the restricting and modifying activities, whereas other sites are nonmodifiable even though they are sensitive to the restriction enzyme. (iii) In some manner, SP18 antagonizes the action of the nonB gene product.     

Transposition of the Arsenate Resistance Locus of BACILLUS SUBTILIS Strains 23 and 168

Wild-type Bacillus subtilis strains 23 and 168 are resistant to high concentrations of sodium arsenate. The genetic configurations of the arsenate resistance loci of these two related strains of B. subtilis have been characterized. The transformable 168 strain has a single resistance locus which maps between phe and aroD in the terminal third of the genome. In contrast, strain 23 is shown to have its single arsenate resistance locus between purB and thr in the first third of the bacterial chromosome. Moreover, in strain 23 the chromosomal segment equivalent to the phe-linked asa region of 168 strains is missing. DNA isolated from 23 strains is able to transform 168 arsenate-sensitive strains to resistance and the heterologous 23 DNA is found to preferentially establish a new purB linked asa locus in such transformed cells. Thus, the majority of phenotypically arsenate-resistant cells recovered after exposure of competent 168 sensitive mutants to 23 DNA are "heterozygous" and still retain their phe-linked mutated asa locus. The tolerance of several of these heterologously transformed hybrid strains to arsenate suggests that the 168 and 23 asa gene products are similar, and a transposition model for the evolution of arsenate resistance in B. subtilis is proposed.     

Comparative analysis of physical maps of four Bacillus subtilis (natto) genomes

The complete SfiI and I-CeuI physical maps of four Bacillus subtilis (natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure. Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed. No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of the B. subtilis strain Marburg 168. However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SP beta, skin element, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types. The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains.     

The BACILLUS SUBTILIS Genome: Replication-Order and Map-Position Discrepancies—evidence for a Second Origin

Replication order to map position discrepancies have been documented in B. subtilis. The discrepancies were found to occur whenever the genome of B. subtilis was replicated under a variety of physiological conditions and in both 168 and W23 strains. The earliest replicating marker involved in these discrepancies was thr versus purB and aroA versus recA. A detailed linkage analysis of the ura to argA region was consistent with its being a continuous linkage group. This led to the conclusion that an origin for new starts at replication exists between recA and aroA.     

Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50.

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN', produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.     

Pseudo-allelic relationship between non-homologous genes concerned with biosynthesis of polyglycerol phosphate and polyglycerol phosphate teichoic acids in Bacillus subtilis strains 168 and W23

A 60 kbp region of the Bacillus subtilis chromosome encompassing the genes concerned with teichoic acid biosynthesis has been subjected to physical analysis. No homology was detected by Southern hybridization between DNA segments encoding the tag genes of strain 168, concerned with polyglycerol phosphate (poly(groP)) biosynthesis, and the tar genes of strain W23, concerned with polyribitol phosphate (poly-(rboP)) biosynthesis. Analysis of 168/W23 interstrain hybrids that incorporate poly(rboP) instead of poly-(groP) into their cell walls revealed that, in every case, integral substitution of the W23 tar genes for the 168 tag genes had occurred. Interstrain hybrids of the 'W23-like' type have inherited larger segments of W23 DNA than interstrain hybrids of the 'mixed' type. The tag and tar genes are located at equivalent positions on the chromosomes of strains 168 and W23, behaving, in genetic crosses, like an allelic pair. They provide the first example of a pseudo-allelic relationship between non-homologous genes in B. subtilis.     

Variation in 16S-23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates

The genome of the Bacillus subtilis 168-type strain contains 10 ribosomal RNA (rRNA) operons. In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnI-H-G and rrnJ-W, lack a trinucleotide signature region. Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168-type strain and B. subtilis 168-like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence. However, PCR of the ISR segment for five other B. subtilis 168 isolates generated only a 114 bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains. Additional genetic variability between the seven B. subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis. The 168-type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern. Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067. Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding. These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B. subtilis subgroup 168. It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.     

Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168.

A set of nine reference strains bringing convenient markers in the genetic background of Bacillus subtilis Marburg 168 has been prepared to allow rapid mapping of new markers.     

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.     

Tracing the domestication of a biofilm-forming bacterium

Over the course of more than a century of laboratory experimentation, Bacillus subtilis has become "domesticated," losing its ability to carry out many behaviors characteristic of its wild ancestors. One such characteristic is the ability to form architecturally complex communities, referred to as biofilms. Previous work has shown that the laboratory strain 168 forms markedly attenuated biofilms compared with the wild strain NCIB3610 (3610), even after repair of a mutation in sfp (a gene involved in surfactin production) previously known to impair biofilm formation. Here, we show that in addition to the sfp mutation, mutations in epsC, swrA, and degQ are necessary and sufficient to explain the inability of the laboratory strain to produce robust biofilms. Finally, we show that the architecture of the biofilm is markedly influenced by a large plasmid present in 3610 but not 168 and that the effect of the plasmid can be attributed to a gene we designate rapP. When rapP is introduced into 168 together with wild-type alleles of sfp, epsC, swrA, and degQ, the resulting repaired laboratory strain forms biofilms that are as robust as and essentially indistinguishable in architecture from those of the wild strain, 3610. Thus, domestication of B. subtilis involved the accumulation of four mutations and the loss of a plasmid-borne gene.     

The Saccharomyces cerevisiae W303-K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt

Saccharomyces cerevisiae strain W303 is a widely used model organism. However, little is known about its genetic origins, as it was created in the 1970s from crossing yeast strains of uncertain genealogy. To obtain insights into its ancestry and physiology, we sequenced the genome of its variant W303-K6001, a yeast model of ageing research. The combination of two next-generation sequencing (NGS) technologies (Illumina and Roche/454 sequencing) yielded an 11.8 Mb genome assembly at an N50 contig length of 262 kb. Although sequencing was substantially more precise and sensitive than whole-genome tiling arrays, both NGS platforms produced a number of false positives. At a 378× average coverage, only 74 per cent of called differences to the S288c reference genome were confirmed by both techniques. The consensus W303-K6001 genome differs in 8133 positions from S288c, predicting altered amino acid sequence in 799 proteins, including factors of ageing and stress resistance. The W303-K6001 (85.4%) genome is virtually identical (less than equal to 0.5 variations per kb) to S288c, and thus originates in the same ancestor. Non-S288c regions distribute unequally over the genome, with chromosome XVI the most (99.6%) and chromosome XI the least (54.5%) S288c-like. Several of these clusters are shared with Σ1278B, another widely used S288c-related model, indicating that these strains share a second ancestor. Thus, the W303-K6001 genome pictures details of complex genetic relationships between the model strains that date back to the early days of experimental yeast genetics. Moreover, this study underlines the necessity of combining multiple NGS and genome-assembling techniques for achieving accurate variant calling in genomic studies.     

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.     

Genetic and nucleotide sequence homologies in bacillus genomes

Summary The extent of divergence in the organization of the aromatic amino acid cluster among the heterogenetic strains of Bacillus subtilis has been examined by hybridizations to a trp homolog from B. pumilus and bymarker survivals after restriction. The trp operon in the W23, 3610 and 168M genomes exhibit variations in the location of the EcoRI and HindIII cleavage sites consistent with the relative transforming activity of the surviving genes and the history of the strains.     

Whole Genome Complete Resequencing of Bacillus subtilis Natto by Combining Long Reads with High-Quality Short Reads

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food “natto.” The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome.     

DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23.

Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).     

Effect of EcoRI restrictase on the transforming DNA of different bacilli]

The transforming activity of DNA from Bacillus subtilis 168, Bac. subtilis W23, Bac. subtilis NRS and Bac. aterrimus after the EcoRI restrictase treatment was studied. The auxotrophic strains of Bac. subtilis 168 were used as recipients in bacterial transformation. The transforming activity for different markers decreased up to 0.001--6 per cent from the initial level for different Bacilli DNAs. In some cases the sensitivity of the same marker from different Bacilli differed in more than 50 times. Bac. subtilis and Bac. aterrimus have demonstrated the maximal differences. Such differences can be used for the identification of close related Bacilli.     

Peptidoglycan synthesis by partly autolyzed cells of Bacillus subtilis W23.

Partly autolyzed, osmotically stabilized cells of Bacillus subtilis W23 synthesized peptidoglycan from the exogenously supplied nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramyl pentapeptide. Freshly harvested cells did not synthesize peptidoglycan. The peptidoglycan formed was entirely hydrolyzed by N-acetylmuramoylhydrolase, and its synthesis was inhibited by the antibiotics bacitracin, vancomycin, and tunicamycin. Peptidoglycan formation was optimal at 37 degrees C and pH 8.5, and the specific activity of 7.0 nmol of N-acetylglucosamine incorporated per mg of membrane protein per h at pH 7.5 was probably decreased by the action of endogenous wall autolysins. No cross-linked peptidoglycan was formed. In addition, a lysozyme-resistant polymer was also formed from UDP-N-acetylglucosamine alone. Peptidoglycan synthesis was inhibited by trypsin and p-chloromercuribenzenesulfonic acid, and we conclude that it occurred at the outer surface of the membrane. Although phospho-N-acetylmuramyl pentapeptide translocase activity was detected on the outside surface of the membrane, no transphosphorylation mechanism was observed for the translocation of UDP-N-acetylglucosamine. Peptidoglycan was similarly formed with partly autolyzed preparations of B. subtilis NCIB 3610, B. subtilis 168, B. megaterium KM, and B. licheniformis ATCC 9945. Intact protoplasts of B. subtilis W23 did not synthesize peptidoglycan from externally supplied nucleotides although the lipid intermediate was formed which was inhibited by tunicamycin and bacitracin. It was therefore considered that the lipid cycle had been completed, and the absence of peptidoglycan synthesis was believed to be due to the presence of lysozyme adhering to the protoplast membrane. The significance of these results and similar observations for teichoic acid synthesis (Bertram et al., J. Bacteriol. 148:406-412, 1981) is discussed in relation to the translocation of bacterial cell wall polymers.     

Comparative analysis of the development of swarming communities of Bacillus subtilis 168 and a natural wild type: critical effects of surfactin and the composition of the medium

The natural wild-type Bacillus subtilis strain 3610 swarms rapidly on the synthetic B medium in symmetrical concentric waves of branched dendritic patterns. In a comparison of the behavior of the laboratory strain 168 (trp) on different media with that of 3610, strain 168 (trp), which does not produce surfactin, displayed less swarming activity, both qualitatively (pattern formation) and in speed of colonization. On E and B media, 168 failed to swarm; however, with the latter, swarming was arrested at an early stage of development, with filamentous cells and rafts of cells (characteristic of dendrites of 3610) associated with bud-like structures surrounding the central inoculum. In contrast, strain 168 apparently swarmed efficiently on Luria-Bertani (LB) agar, colonizing the entire plate in 24 h. However, analysis of the intermediate stages of development of swarms on LB medium demonstrated that, in comparison with strain 3610, initiation of swarming of 168 (trp) was delayed and the greatly reduced rate of expansion of the swarm was uncoordinated, with some regions advancing faster than others. Moreover, while early stages of swarming in 3610 are accompanied by the formation of large numbers of dendrites whose rapid advance involves packs of cells at the tips, strain 168 advanced more slowly as a continuous front. When sfp+ was inserted into the chromosome of 168 (trp) to reestablish surfactin production, many features observed with 3610 on LB medium were now visible with 168. However, swarming of 168 (sfp+) still showed some reduced speed and a distinctive pattern compared to swarming of 3610. The results are discussed in terms of the possible role of surfactin in the swarming process and the different modes of swarming on LB medium.