Bacillus Subtilis Marburg Research Articles

Genotyping of Bacillus spp. isolate collection from natural samples

The aim of this study was genotyping and identification of collection of 164 Bacillus spp. isolates, from samples of soil, manure, and straw gathered from across Serbia, using Pulse field gel electrophoresis (PFGE) combined with sequencing of tuf gene, one of the housekeeping genes. The PFGE analysis with NotI enzyme was used to determine phylogenetic relationships of isolates and referent strains. Four large groups of Bacillus spp. were distinguishable: cereus, subtilis, pumilus and megaterium and within enormous genetic diversity. Bacillus subtilis Marburg referent strain did not group with rest of the strains from the subtilis group (Bacillus subtilis ATCC6633 and Bacillus atrophaeus ATCC9372). Strains from the cereus group were distinguished and closely grouped together. One representative isolate from each of 21 distinct PFGE groups was identified by sequencing of tuf gene. Eight different species were identified among chosen isolates: B. amyloliquefaciens, B. subtilis, B. pumilus, B. safensis, B. megaterium, B. cereus, B. anthracis and B. thuringiensis. Our results showed that PFGE analysis combined with sequencing of one of the housekeeping genes could be used for characterization of large collections of Bacillus isolates. The determination of tuf gene recommended itself to be an adequate and sufficient analysis for obtaining very clear and unambiguous results, with high resolution of separation of Bacillus species.

Inhibitory effect of prophage SP.BETA. fragments on phage SP10 ribonucleotide reductase function and its multiplication in Bacillus subtilis

Bacteria have evolved various kinds of defense mechanisms against phage infection and multiplication. Analysis of these mechanisms is important for medical and industrial application of phages as well as for their scientific study. Strains of Bacillus subtilis Marburg strain carrying both nonA and nonB mutations are susceptible to the Bacillus phage SP10. The nonB mutation has been shown to have a compromised intrinsic restriction system. The nonA mutation represents the cured state of prophage SPβ whose genome is 135 kb in length and contains 187 ORFs. For this study we investigated the molecular mechanism behind the inhibitory activity of the wild type nonA function against phage SP10 development. The progression of phage-developmental stages was examined in cells harboring wild type nonA, i.e. prophage SPβ. After phage adsorption and DNA injection into host cells, the synthesis of phage specific mRNA proceeded normally. However, phage DNA synthesis was severely inhibited by some effect of wild type nonA. We thus systematically deleted portions of the prophage SPβ region from the B. subtilis genome and the resultant mutant strains were examined as to whether they still retained sufficient wild type nonA functionality to inhibit SP10 phage development. The SPβ region encompassing the bnrdEF gene, which codes for a putative ribonucleotide reductase (RRase), turned out to be responsible for the wild type nonA function. The phage SP10 possesses its own xnrdE gene coding for a putative RRase that complements the temperature-sensitive mutation of the host RRase gene nrdE. This complementation was blocked by an artificially induced transcription from a non-coding strand of the bnrdEF region. It is thus likely that the transcript from the bnrdEF region of SPβ inhibits ribonucleotide reductase function of SP10, resulting in arrest of DNA synthesis during phage SP10 development.

Restriction on Conjugational Transfer of pLS20 inBacillus subtilis168

Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.

Characterization of a Novel Esterase YkoN from Bacillus subtilis Marburg

The product YkoN of the gene of unknown function, ykoN, of Bacillus subtilis Marburg has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and thus YkoN is expected to have a lipase or esterase activity. To characterize the expected enzyme activity the plasmid having a modified ykoN that include the sequence for His(x6) tag at its C-terminus of YkoN, which has 373 amino acid residues, was constructed. His-tagged YkoN protein of 39 kDa was induced in Escherichia coli BL21 (DE3) cells harboring chaperon plasmid pGro7 and purified to near homogeneity by using gel filtration and Ni-agarose. When p-nitrophenyl-esters of different fatty acid chain length were examined, the purified YkoN hydrolyzed the esters of fatty acid with short chain length (4-6 carbon atoms) preferentially. The esters of fatty acid with longer chain (C ≥ 10) were hydrolyzed inefficiently. The activity required no divalent cations and was not affected by addition of EDTA. The optimal pH for the activity was from pH 7.4 to pH 8.6. These results indicate that YkoN is a novel esterase which hydrolyzes the esters of fatty acid with short chain length.

Involvement of YkoN in production of a new phospholipid in Bacillus subtilis Marburg

To understand the physiological role of the ykoN gene of Bacillus subtilis, the gene was expressed from the IPTG-inducible spac promoter in B. subtilis cells. When lipid composition of the cells induced for ykoN expression was examined, a new spot of phospholipid was found on the thin layer chromatogram. Induction of ykoN in Escherichia coli cells produced a similar new spot. The new spot was produced in mutant E. coli cells lacking cardiolipin or phosphatidylethanolamine, but not in the cells lacking phosphatidylglycerol. The result suggests an involvement of phosphatidylglycerol in production of the new spot. Examination of the lipid composition during the stages of B. subtilis growth revealed that the new phospholipid was produced four hours after cessation of logarithmic growth, consistent with an increase in the promoter activity of ykoN in the late stage in sporulation. YkoN has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and shows a lipolytic activity (Matsuura et al., in press). The mutant YkoN (S205A) in which the central serine residue of the motif is replaced with alanine produced the new phospholipid in the amount as small as only 1/10 of the wild type, suggesting that the lipolytic activity of YkoN is involved in the new phospholipid production.

Cardiolipin Enrichment in Spore Membranes and Its Involvement in Germination of Bacillus subtilis Marburg

Examination of the lipid composition of spore membranes of Bacillus subtilis Marburg, extracted after treatment of spores with dithiothreitol/urea and NaOH followed by lysozyme digestion, revealed that the spore membranes had significantly higher cardiolipin (CL) content than the membranes of exponentially growing cells. Analysis of the membranes of coat-defective, cotE::cat and gerE::cat mutant spores, which are susceptible to lysozyme digestion without chemical treatment, confirmed that spore membranes contain a high level of CL. After addition of the germinants L-alanine or AGFK (a combination of asparagine, glucose, fructose, and KCl), the turbidity of wild type spore suspensions decreased to 50% within 30 min. Suspensions of spores with only trace amounts of CL, however, showed no decrease in turbidity when L-alanine was added and the initial decrease in turbidity with AGFK was slight (14% after 60 min). These results indicate that CL is involved in an early step of germination, related to the functioning of germinant receptors. This is the first conspicuous in vivo evidence that CL in bacterial membranes has a specific role, in which it cannot be replaced by other anionic phospholipids.

Difference in Transcription Levels of cap Genes for γ-Polyglutamic Acid Production between Bacillus subtilis IFO16449 and Marburg 168

In a strain carrying capB-lacZ fusion of Bacillus subtilis IFO16449, which produces a large amount of gamma-polyglutamic acid (PGA), beta-galactosidase activity was enhanced by about five times with the addition of L-glutamic acid. This increase was also confirmed by Northern blot analysis. On the other hand, the activity was not detected in a strain carrying capB-lacZ fusion of B. subtilis Marburg 168. However, when the cap genes (capBCA and ywtC) were fused to the IPTG-inducible spac promoter, B. subtilis Marburg 168 produced PGA. These results suggest that the inability of B. subtilis Marburg 168 to produce PGA is due to defective expression of the cap genes.

Acquisition of certain streptomycin-resistant (str) mutations enhances antibiotic production in bacteria.

Physiological differentiation (including antibiotic production) in microorganisms usually starts when cells encounter adverse environmental conditions and is frequently accompanied by an increase in the accumulation of intracellular ppGpp. We have found that the acquisition of certain streptomycin-resistant (str) mutations enables cells to overproduce antibiotics, demonstrating an increase in productivity 5- to 50-fold greater than that of wild-type strains. The frequency of such antibiotic-overproducing strains among the str mutants was shown to range from 3 to 46%, as examined with several strains of the genera Streptomyces, Bacillus, and Pseudomonas. Analysis of str mutants from Bacillus subtilis Marburg 168 revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12, changing Lys-56 (corresponding to Lys-43 in Escherichia coli) to Asn, Arg, Thr, or Gln. Antibiotic productivity increased in a hierarchical manner depending upon which amino acid residue replaced Lys at this position. The strA1 mutation, a genetic marker frequently used for mapping, had no effect on antibiotic productivity even though it was found to result in an amino acid alteration of Lys-56 to Ile. Gene replacement experiments with the str alleles demonstrated unambiguously that the str mutation is responsible for the antibiotic overproductivity observed. These results offer a rational approach for improving the production of antibiotic (secondary metabolism) from microorganisms.

Effect of DNA repair deficiency on mutational specificity in the lacZ gene of Escherichia coli

When the cells of Bacillus subtilis Marburg strains were grown in the presence of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) at a non-toxic concentration, they acquired resistance with regard to both lethality (enhancement of survival) and mutagenicity (reduction of mutation frequency) to subsequent challenge with MNNG at higher concentrations. Thus, like Escherichia coli cells (Samson and Cairns, 1977), B. subtilis cells respond adaptively to MNNG. Protein synthesis was necessary for the adaptation, and an activity to destroy specifically O6-methylguanine residues in methylated DNA was present in the extracts of the adapted cells. Therefore, this response is attributable to an error-free DNA-repair system induced by the exposure to MNNG. The adaptive response was observed with the cells carrying uvrA, polA or recA mutation, indicating this to be independent of the excision repair or recA-dependent repair.For the acquisition of the resistance to MNNG, methylating or ethylating agents were effective, but neither N-butyl-N-nitrosorea (BNU) nor N-propyl-N′-nitro-N-nitrosoguanidine (PNNG) was effective. The cells pre-treated with MNNG became resistant to the challenge with alkynitrosoureas or N-alkyl-N′-nitro-N-nitrosoguanidine, but not to the challenge with methyl methanesulphonate (MMS) or dimethyl sulphate (DMS).Thus, the target of the induced DNA-repair activity is likely to be O-alkylated bases in DNA. Though the phenomenology of the adaptive response seems similar to that in E. coli, the following two lines of evidence indicate that the underlying mechanisms may not be identical: (1) in B. subtilis, the cells of polA strains are adapted both with regard to lethality and to mutagenicity; (2) in B. subtilis, MMS or DMS acts only as the inducer of the response.

Preparation of antimicrobial cellulose derivatives.

Epoxy-activated cellulose (CGE) was reacted with hexamethylenediamine (HMDA) and polyethyleneimine (PEI, Mw of 6.0 × 104) to give two aminated celluloses (HMDA- and PEI-celluloses) which were further utilized to prepare antimicrobial cellulose derivatives. Ampicillin (AMP) was immobilized on HMDA-cellulose with glutaraldehyde, and heavy metalions such as Ag (I), Hg (II), Cu (II), Cd (II) and Zn (II) were adsorbed on PEI-cellulose, and their antimicrobial activity were analyzed. The minimal inhibitory concentration (MIC) of AMP immobilized on cellulose was 68 μmol/L for Escherichia coli HB101 (E. coli) and less than 4 μmol/L for Bacillus subtilis Marburg 168 (B. subtilis) on the broth dilution method. On the other hand, PEI-cellulose bearing Ag (I), Hg (II), Cu (II) or Zn (II) ion showed higher antimicrobial activity for E. coli than that for B. subtilis. These effects were considered to be due to the liberation of antimicrobial agents from cellulose derivatives.

Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168

The levels of two aspartokinase isozymes, a lysine-sensitive enzyme and an aspartokinase that is inhibited synergistically by lysine plus threonine, differ strikingly in different strains of Bacillus subtilis. In derivatives of B. subtilis 168 growing in minimal medium, the predominant isozyme is the lysine-sensitive aspartokinase. In B. subtilis ATCC 6051, the Marburg strain, the level of the lysine-sensitive aspartokinase is much lower during growth in minimal medium, and the major aspartokinase activity is the lysine-plus-threonine-sensitive isozyme. Molecular cloning and nucleotide sequence determination of the genes for the lysine-sensitive isozymes from the two B. subtilis strains and their upstream control regions showed these genes to be identical. Evidence that the lysine-sensitive aspartokinase, referred to as aspartokinase II, is distinct from the threonine-plus-lysine-sensitive aspartokinase comes from the observation that disruption of the aspartokinase II gene by recombinational insertion had no effect on the latter. Mutants were obtained from the aspartokinase II-negative strain that also lacked the threonine-plus-lysine-sensitive aspartokinase, which will be referred to as aspartokinase III. Aspartokinase II could be selectively restored to these mutants by transformation with plasmids carrying the aspartokinase II gene. Study of the growth properties of the various mutant strains showed that the loss of either aspartokinase II or aspartokinase III had no effect on growth in minimal medium but that the loss of both enzymes interfered with growth unless the medium was supplemented with the three major end products of the aspartate pathway. It appears, therefore, that aspartokinase I alone cannot provide adequate supplies of precursors for the synthesis of lysine, threonine, and methionine by exponentially growing cells.

Nucleotide sequence of the chromosomal gene coding for the aminoglycoside 6-adenylyltransferase from Bacillus subtilis Marburg 168

Gene aadK of Bacillus subtilis is 855 bp long and codes for aminoglycoside 6-adenylyltransferase.

Nucleotide sequence homology of the tetracycline-resistance determinant naturally maintained in Bacillus subtilis Marburg 168 chromosome and the tetracycline-resistance gene of B. subtilis plasmid pNS1981

The nucleotide sequence (1579 bp) of tetracycline-resistance determinant and flanking regions of the cloned 5.1 kb DNA fragment from Bacillus subtilis GSY908 chromosome (Sakaguchi, R. and Shishido, K. (1988) Biochim. Biophys. Acta 949, 49–57) were determined and compared with those of the B. subtilis tetracycline-resistance plasmid pNS1981. The tetracycline-resistance structural ( tet) genes of the B. subtilis GSY908 chromosome ( tet BS908) and pNS1981 ( tet pNS1981) were found to be highly homologous (80% identical). Both tet genes were composed of 1374 bp and 458 amino-acid residues initiating from a GTG codon preceded by a ribosome-binding site (RBS-2). Upstream from tet BS908 there exists a short open reading frame (20 amino acids) initiating from a ATG codon preceded by its own RBS (RBS-1). This leader sequence was also highly homologous to that of tet pNS1981 except for a deletion of one bp between the RBS-1 and the ATG codon.

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation.

The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.

The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg.

Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied. In a restriction-deficient but modification-proficient mutant of B. subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E. coli (10(4) clones per microgram target DNA). Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb). In the restriction-proficient B. subtilis strain, the class of large inserts was underrepresented. Transformation of B. subtilis with E. coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways. First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host. The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb). Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid. In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0-16 kb), and no structural instability was observed. It is concluded that for shotgun cloning in B. subtilis, the use of restriction-deficient strains is highly preferable. Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction. It is postulated that AsuII sites are additional target sites for BsuM restriction.

Purification and characterization of chromosomal streptomycin adenylyltransferase from derivatives ofBacillus subtilisMarburg 168

Chromosomal streptomycin(Sm) adenylyltransferase was detected in Sm-susceptible derivatives of Bacillus subtilis Marburg 168. The enzyme was purified from one of the strains and its enzymological properties were investigated. The isoelectric point and mr of the enzyme were 6.0 and 35 000, respectively. The optimum pH was 8.0 and the optimum temperature for the inactivation of Sm was 35°C.

Purification and characterization of chromosomal streptomycin adenylyltransferase from derivatives of Bacillus subtilis Marburg 168

Purification and characterization of chromosomal streptomycin adenylyltransferase from derivatives of Bacillus subtilis Marburg 168

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.

The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites

We present the sequence of a 2 kb fragment of the Bacillus subtilis Marburg genome containing sacB, the structural gene of levansucrase, a secreted enzyme inducible by sucrose. The peptide sequence deduced for the secreted enzyme is very similar to that directly determined by Delfour (1981) for levansucrase of the non-Marburg strain BS5. The peptide sequence is preceded by a 29 amino acid signal peptide. Codon usage in sacB is rather different from that in the sequenced genes of other secreted enzymes in B. subtilis, especially alpha-amylase. Genetic evidence has shown that the sacB promotor is rather far from the beginning of sacB (200 bp or more). The 200 bp region preceding sacB shows some of the features of an attenuator. A preliminary discussion of the putative workings and roles of this attenuator-like structure is proposed. sacRc mutations, which allow constitutive expression of levansucrase, have been located within the 450 bp upstream of sacB. It is shown that sacRc and sacR+ alleles control in cis the expression of the adjacent sacB gene.

Effect of 5-azacytidine on DNA methylation and on the enzymes of de novo pyrimidine biosynthesis in Bacillus subtilis Marburg strain.

In Bacillus subtilis, 5-azacytidine, an analog of cytidine, causes a time- and dose-dependent growth inhibition. Methyl donors are unable to overcome azacytidine-induced inhibition while pyrimidine nucleosides, except orotidine, can revert this inhibition totally. On the other hand, pyrimidine bases, except uracil, are unable to restore growth in azacytidine-treated cells. Uracil, at a high concentration, can revert growth inhibition only inefficiently. However, a considerable relief of growth inhibition by uracil occurs in the presence of a ribose donor. In azacytidine-treated B. subtilis cells methylation of bases in DNA is not affected either quantitatively or qualitatively and DNA methyltransferase activity remains unaltered as compared to the untreated cells, apparently due to the absence of azacytidine incorporation into the DNA. The inability of B. subtilis cytidine kinase to phosphorylate azacytidine is the probable reason for this non-incorporation. Analysis of the enzymes of de novo pyrimidine biosynthesis has shown that orotidine monophosphate pyrophosphorylase is specifically repressed by azacytidine treatment.