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.     

Restriction and modification of SP10 phage by BsuM of Bacillus subtilis Marburg

Bacillus subtilis Marburg has only one intrinsic restriction and modification system BsuM that recognizes the CTCGAG (XhoI site) sequence. It consists of two operons, BsuMM operon for two cytosine DNA methyltransferases, and BsuMR operon for a restriction nuclease and two associated proteins of unknown function. In this communication, we analyzed the BsuM system by utilizing phage SP10 that possesses more than twenty BsuM target sequences on the phage genome. SP10 phages grown in the restriction and modification-deficient strain could not make plaques on the restriction-proficient BsuMR(+) indicator strain. An enforced expression of the wild type BsuMM operon in the BsuMR(+) indicator strain, however, allowed more than thousand times more plaques. DNA extracted from SP10 phages, thus, propagated became more but not completely refractory to XhoI digestion in vitro. Thus, the SP10 phage genome DNA is able to be nearly full-methylated but some BsuM sites are considered to be unmethylated.     

DNA methyltransferases affecting the sequence 5''CCGG

B. subtilis phage SPbeta and Moraxella sp. code for DNA methyltransferases which methylate both cytosines of the sequence 5'CCGG. Experiments using a B. subtilis strain whose DNA is sensitive to HpaII and resistant to MspI degradation, indicated that methylation of the outer C of this sequence provides protection against the restriction enzyme MspI.     

Characterization of Bacillus subtilis bacteriophages

Brodetsky, Anna M. (University of California, Los Angeles), and W. R. Romig. Characterization of Bacillus subtilis bacteriophages. J. Bacteriol. 90:1655-1663. 1965.-A group of six phages, SP5, SP6, SP7, SP8, SP9, and SP13, which use the Marburg strain of Bacillus subtilis as host was characterized. These phages, referred to as group 1, were examined for the following properties: host range, plaque morphology, stability, adsorption kinetics, one-step growth characteristics, calcium requirements, serum neutralization, thermal inactivation, and inactivation by ultraviolet irradiation. Five unrelated B. subtilis phages, SP3, SP10, PBS1, SP alpha, and SP beta, were included in the studies. When first isolated, none of the group 1 phages was able to replicate efficiently on B. subtilis SB19, a mutant of the "transforming" B. subtilis 168. Host range mutants capable of growth in SB19 were isolated for all of the group 1 phages except SP13, and are designated the "star" phages (SP5* through SP9*). For characterization, SB19 was used as host for the star phages, and another B. subtilis mutant, 168B, was host for SP13.     

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.     

Transformation of Bacillus subtilis in α-Amylase Productivity by Deoxyribonucleic Acid from B. subtilis var. amylosacchariticus

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.     

Bacillus subtilis bacteriophage SPbeta: localization of the prophage attachment site, and specialized transduction.

The attachment site for the prophage of SPbeta lies between ilvA and kauA on the chromosome of Bacillus subtilis strain 168. Specialized transduction of citK and kauA can be carried out by certain lysates of SPbeta.     

Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis

The gene coding for alpha-amylase from Bacillus amyloliquefaciens was isolated by direct shotgun cloning using B. subtilis as a host. The genome of B. amyloliquefaciens was partially digested with the restriction endonuclease MboI and 2- to 5-kb fragments were isolated and joined to plasmid pUB110. Competent B. subtilis amylase-negative cells were transformed with the hybrid plasmids and kanamycin-resistant transformants were screened for the production of alpha-amylase. One of the transformants producing high amounts of alpha-amylase was characterized further. The alpha-amylase gene was shown to be present in a 2.3-kb insert. The alpha-amylase production of the transformed B. subtilis could be prevented by inserting lambda DNA fragments into unique sites of EcoRI, HindIII and KpnI in the insert. Foreign DNA inserted into a unique ClaI site failed to affect the alpha-amylase production. The amount of alpha-amylase activity produced by this transformed B. subtilis was about 2500-fold higher than that for the wild-type B. subtilis Marburg strain, and about 5 times higher than the activity produced by the donor B. amyloliquefaciens strain. Virtually all of the alpha-amylase was secreted into the culture medium. The secreted alpha-amylase was shown to be indistinguishable from that of B. amyloliquefaciens as based on immunological and biochemical criteria.     

Cloning and expression of bacteriophage SP02 DNAZ polymerase gene L in Bacillus subtilis, using the Staphylococcus aureus plasmid pC194

HindIII restriction endonuclease fragments of DNA from temperate Bacillus subtilis bacteriophage SP02 were cloned in B. subtilis by using the plasmid pC194. Three hybrid plasmids which permit growth of the mutant SP02 susL244 in suppressor-negative bacteria were isolated. SP02 gene L is thought to code for a DNA polymerase essential for autonomous replication of SP02 DNA. Extracts of bacteria carrying one of these hybrid plasmids, pC194-96, had 10- to 30-fold increased DNA polymerase activity. The plasmid-induced DNA polymerase activity differed from that of the known B. subtilis DNA polymerases in several respects. The results of the experiments support the idea that phage SP02 codes for a new DNA polymerase.     

Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02.

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.     

Alkaline phosphatase possessing alkaline phosphodiesterase activity and other phosphodiesterases in Bacillus subtilis.

In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase) and alkaline phosphodiesterase (APDase). Revertants recovered the two activities. APases with APDase activity were purified from the membrane fraction of B. subtilis 6160-BC6 and from the culture fluid of an APase-secreting B. subtilis mutant strain, RAN 1. In addition to these major APases with APDase activity, at least two kinds of phosphodiesterase (PDase) without phosphatase activity were found in the cytoplasmic supernatants of RAN 1 and an APase-less B. subtilis mutant strain, SP25. Another minor APase with a molecular weight of about 80,000, which had almost no PDase activity, was isolated from the membrane fraction of strain 6160-BC6. Enzyme distribution in subcellular fractions from various strains cultured in high- and low-phosphate media was analyzed. The PDases did not cross-react with rabbit antiserum against the RAN 1 APase with APDase activity. The main component of the PDases had a molecular weight of about 80,000 and was most active at pH 8.0. These results suggest that APase with APDase activity is different from PDases detected in cytoplasmic supernatants and that phoP is the structural gene for the phosphate-repressible APase with APDase activity.     

Ethidium Bromide-Resistant Mutant of Bacillus subtilis

An ethidium bromide-resistant mutant (EB8) derived from a Marburg strain of Bacillus subtilis was found to be conditionally resistant to 10 mug of ethidium bromide per ml. Expression of resistance is complete only during vegetative growth at incubation temperatures above 30 C in complex medium or minimal medium supplemented with Casamino Acids. Strain EB8 is cross-resistant to acriflavine and proflavine. The ethidium bromide resistance marker is co-transduced with hisA1 at a frequency of 6% and is located to the right of hisA1 on the B. subtilis chromosome as it is usually represented on the map. Incorporation of [5-(3)H] uridine by strain EB8 showed that ribonucleic acid synthesis in both whole cells and protoplasts is ethidium bromide-resistant.     

A viable Bacillus subtilis strain without functional extracytoplasmic function sigma genes

We constructed a Bacillus subtilis Marburg strain that harbors deletion mutations in all seven extracytoplasmic function (ECF) sigma genes. The strain shows wild-type growth at 37 degrees C both in a complex and in a synthetic medium and exhibits wild-type sporulation. ECF sigma genes of B. subtilis are dispensable as long as no stress is imposed, although they seem to be required for quick response to stresses.     

Syringopeptin SP25A-mediated killing of gram-positive bacteria and the role of teichoic acid d-alanylation

The Pseudomonas syringae syringopeptins are cationic cyclic lipodepsipeptides that inhibit fungi and bacteria. The homolog syringopeptin (SP)25A was strongly inhibitory to several Gram-positive bacteria with minimum inhibitory concentrations ranging between 1.95 and 7.8 microg mL(-1). In contrast, it was not inhibitory to several Gram-negative bacteria. At 5 and 10 microg mL(-1), SP25A rapidly inhibited the growth of Bacillus subtilis and was bacteriocidal. Teichoic acid D-alanylation dltB- and dltD-defective mutant strains of B. subtilis were more susceptible to SP25A compared with the parental wild-type strain. The degree of susceptibility of the parent strain, but not the dltB and dltD mutant strains, increased at alkaline pH (9.0). In contrast, the parental and mutant strains had the same susceptibilities to syringopeptins SP22A and SP508A at pH 7.0 and 9.0. These results suggest that the cell wall anionic teichoic acids modulate SP25A action against B. subtilis, and they provide an explanation for the selective inhibition of Gram-positive bacteria by SP25A.     

DNA base composition, susceptibility to bacteriophages and interspecific transformation as criteria for classification in the genus Bacillus

With 25 strains belonging to 12 species of the genus Bacillus, the base composition of DNA, the susceptibility to bacteriophages, and the ability to transform Bacillus subtilis strain Marburg were studied. Analyses of phage DNAs were also performed. The results were as follows: (1) The DNA base compositions were not uniform even among strains belonging to one taxonomic species. (2) The DNAs extracted from B. natto, B. megaterium and B. polymyxa could transform genetic traits of B. subtilis Marburg although the frequencies were not equal. (3) The host ranges of some temperate bacteriophages were correlated with the taxonomical data. On these bases, the phylogenetic relatedness of B. subtilis to B. megaterium was discussed.     

Determination of DNA sequences containing methylcytosine in Bacillus subtilis Marburg.

The methylcytosine-containing sequences in the DNA of Bacillus subtilis 168 Marburg (restriction-modification type BsuM) were determined by three different methods: (i) examination of in vivo-methylated DNA by restriction enzyme digestion and, whenever possible, analysis for methylcytosine at the 5' end; (ii) methylation in vitro of unmethylated DNA with B. subtilis DNA methyltransferase and determination of the methylated sites; and (iii) the methylatability of unmethylated DNA by B. subtilis methyltransferase after potential sites have been destroyed by digestion with restriction endonucleases. The results obtained by these methods, taken together, show that methylcytosine was present only within the sequence 5'-TCGA-3'. The presence of methylcytosine at the 5' end of the DNA fragments generated by restriction endonuclease AsuII digestion and the fact that in vivo-methylated DNA could not be digested by the enzyme XhoI showed that the recognition sequences of these two enzymes contained methylcytosine. As these two enzymes recognized a similar sequence containing a 5' pyrimidine (Py) and a 3' purine (Pu), 5'-PyTCGAPu-3', the possibility that methylcytosine is present in the complementary sequences 5'-TTCGAG-3' and 5'-CTCGAA-3' was postulated. This was verified by the methylation in vitro, with B. subtilis enzyme, of a 2.6-kilobase fragment of lambda DNA containing two such sites and devoid of AsuII or XhoI recognition sequences. By analyzing the methylatable sites, it was found that in one of the two PyTCGAPu sequences, cytosine was methylated in vitro in both DNA strands. It is concluded that the sequence 5'-PyTCGAPu-3' is methylated by the DNA methyltransferase (of cytosine) of B. subtilis Marburg.     

Mixed Infections of Bacillus subtilis Involving Bacteriophages SP82 and β22

Progeny yields and the synthesis of nucleic acids have been investigated in two strains of Bacillus subtilis mixedly infected with two unrelated phages, SP82 and beta22. When B. subtilis strain 168 was the host, the first phage added dominated the infection; when B. subtilis strain SB11 was the host, beta22 produced progeny even when added to cells 5 min after infection with SP82. Dominance in these mixed infections could be correlated with qualitative and quantitative differences in the synthesis of phage-specific RNAs.     

Genome organization of Sp beta c2 bacteriophage carrying the thyP3 gene

Thymine auxotrophs of Bacillus subtilis strains lysogenic for temperate bacteriophage SP beta c2 were transformed to prototrophy by DNA from related phage phi 3T. During transformation, the phi 3T-encoded thymidylate synthetase gene, thyP3, became integrated into the extreme right end of the SP beta c2 prophage near the bacterial citK gene. Upon heat induction, the transformed B. subtilis cells released SP beta c2T phages that could lysogenize thymine auxotrophs and convert them to prototrophy. Comparison of restriction endonuclease fragments of DNAs from SP beta c2 and SP beta c2T phages revealed that the latter contained a large region of deletion and substitution near the center of the chromosome. This region included the phage attachment site on the SP beta c2 genome.