Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

An evolutionary comparison of Acinetobacter calcoaceticus trpF with trpF genes of several organisms

The deduced amino acid sequence of Acinetobacter calcoaceticus N-(5'-phosphoribosyl) anthranilate isomerase (PRAI), which is coded by trpF, was compared with TrpF of Caulobacter crescentus, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. Sixty percent of identical or similar amino acids were located in alpha/beta TIM (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. In addition, the analysis of trpF genes presented here supports a model by which fusion between separate trpC and trpF genes arose in some cases by in-frame deletions.     

Extracellular nuclease of Bacillus mesentericus]

Bacillus mesentericus is found to secrete three type of nucleases: alkaline ribonuclease (EC 2.7.7.17), acidic ribonuclease (EC 2.7.7.17) and Ca2+-activated exonucleease (EC 3.1.4.7). These nucleases are purified and characterized. They are similar to those from Bac. subtilis in main biochemical and physico-chemical properties and in their chromatographical behaviour. Studying physiological functions of Bac. mesentericus extracellular nucleases, it is shown that bacteria, which are capable to produce extracellular nucleases, utilize exogenous RNAs and a bit worse, DNAs as a single and additional source of nitrogen or phosphorus. In view of this it is believed that extracellular nucleases participate in bacteria nutrition.     

Insertional Inactivation of trpC in Cloned Bacillus trp Segments: Evidence for a Polar Effect on trpF

Plasmid pUB110 was previously used as a vector to clone fragments of deoxyribonucleic acid that complement the trpC2 mutation in Bacillus subtilis from endonuclease EcoRI digested B. licheniformis, B. pumilus, and B. subtilis cellular deoxyribonucleic acid. Each of several such trp plasmids was subsequently shown to contain a segment of the trp gene cluster on the basis of genetic complementing activity. In the present study, analysis of the Trp enzyme levels in B. subtilis harboring the constructed trp plasmids confirms the genetic constitution of the plasmids. Thus, plasmids that complement mutations in specific trp genes specify the corresponding enzyme activities. The levels of the plasmid-specified Trp enzymes in B. subtilis were generally above the repressed level of the chromosomally specified Trp enzymes and equal to or below the derepressed levels of the chromosomally specified Trp enzymes. Certain cloned trp segments contain a single HindIII-sensitive site. Insertion of HindIII-generated deoxyribonucleic acid fragments into these trp plasmids resulted in inactivation of trpC complementing activity, loss of the trpC-specified enzyme activity, and a 10-fold reduction in the specific activity of the plasmid-specified trpF product. The HindIII insertions had no detectable effect on the level of the trpD product, nor did the insertions detectably alter plasmid-specified complementing activity other than to abolish trpC complementation. Removal of the HindIII insertions was accompanied by recovery of trpC complementing activity and restoration of the trpC-and trpF-determined enzymes to the levels specified by the parent plasmids.     

The structure of the trpE, trpD and 5' trpC genes of Bacillus pumilus

The nucleotide (nt) sequences of the Bacillus pumilus trpE, trpD and 5' portions of trpC genes have been determined. Genetic analysis suggested the presence of an internal promoter upstream from the trpC gene, yet no typical consensus sequences were found. The nt and amino acid sequence homologies between the B. pumilus, Bacillus subtilis and Escherichia coli trp genes are presented.     

Mutation induction by the direct treatment of Bacillus subtilis deoxyribonucleic acid.

A procedure was developed to select for specific mutations obtained by means of transformation with DNA previously exposed to potentially dangerous chemical compounds. The 70% co-transformation of hisB and trpC genes in Bacillus subtilis provided a convenient opportunity to select for new mutations. When purified DNA from wild type bacteria was treated with N(OH) acetyl aminofluorene or Hoechst dye 37 507 and used to transform a recipient bearing of a trpC2 mutaion, a high proportion of the Trp+ transformants had new hisB mutations.     

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.     

Determination of the position of the spo-genes in region 210 of the Bacillus subtilis chromosome

Genes controlling sporulation of Bacillus subtilis SHgW are localized in the region of 210 degrees of Bac. subtilis chromosome between lys genes and the rib operon. The method of constructing deletions of definite size was used in the deletion mapping.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.     

Opsonizing activity of the serum of gnotobiotic guinea pigs contaminated with individual representatives of intestinal microflora]

The influence of contamination of germfree guinea pigs with individual representatives of the intestinal microflora (Bac. mesentericus, Bac. subtilis, S. albus, and S. faecalis) on the formation of the serum opsonic activity was studied. An increase of the opsonic activity to all the microorganisms on the 11th day after a corresponding monocontamination and a stimulating influence of the serum on the intracellular digestion of Bac. mesentericus and Bac. subtilis microbes was noted. As to the pathogenic microorganisms (E. coli 055), S. Faecalis only were capable of stimulating the serum opsonic activity. The results indicated the presence of an association between the microflora composition and the opsonic activity of the animal blood serum. The value of this index also depended on the properties of the phagocytosis object.     

Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus.

We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus. Normalized alignment scores comparing the trpC gene of R. capsulatus with the trpC genes of other bacterial species are reported. An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found.     

Cosmid cloning of five Zymomonas trp genes by complementation of Escherichia coli and Pseudomonas putida trp mutants.

A library of Zymomonas mobilis genomic DNA was constructed in the broad-host-range cosmid pLAFR1. The library was mobilized into a variety of Escherichia coli and Pseudomonas putida trp mutants by using the helper plasmid pRK2013. Five Z. mobilis trp genes were identified by the ability to complement the trp mutants. The trpF, trpB, and trpA genes were on one cosmid, while the trpD and trpC genes were on two separate cosmids. The organization of the Z. mobilis trp genes seems to be similar to the organization found in Rhizobium spp., Acinetobacter calcoaceticus, and Pseudomonas acidovorans. The trpF, trpB, and trpA genes appeared to be linked, but they were not closely associated with trpD or trpC genes.     

The induction of auxotrophic mutations for riboflavin by the integration of plasmid pLRS33 into the chromosome of Bacillus subtilis

The transformation of Bacillus subtilis Lys- strains with plasmid pLRS33 containing pBR322 and the Bac. subtilis chromosomal fragment carrying the genes for lysin biosynthesis and the riboflavin operon regulatory operator region (ribO) leads to the appearance of Rib- mutants. It was shown that these mutants contained long deletions covering a great portion of the riboflavin operon.     

Nature of the mutations that determine the ability of Bacillus subtilis A-50 to sporulate at high glucose concentrations in the medium]

The ability of Bacillus subtilis A-50 to sporulate in the medium containing high glucose concentrations is caused by at least two mutation types: pts mutations and cat (or tgl) mutations, both of them affecting differently the level of alkaline proteinase synthesis. The decrease of the level of enzyme activity in the case of pts mutation (gluR3 mutant) occurs at the expense of glucose transport disturbance. The mutation cat (tgl) (mutant gluR5) causes the increase in enzyme synthesis at the expense of catabolic resistance to glucose of genes controlling alkaline proteinase synthesis and the spore formation in Bac. subtilis A-50. cat5(gluR5) and pts3(gluR3) mutations are located on the chromosome of Bac. subtilis in the region metD and argC respectively. The over-synthesis of alkaline proteinase characteristic of Bac. subtilis A-50 is controlled by the polygenic system, as the level of alkaline proteinase synthesis in argA+ transformants makes up 25% of the level of activity of the original strain. The productivity of Bac. subtilis A-50 can be enhanced by introducing an additional cat mutation.     

Genome polymorphism in dissociative variants of Bacillus subtilis (mesentericus) 76. Identification of dissociants using genome fingerprinting

DNA fingerprinting procedure with M13 repeat probe as we have shown earlier makes it possible to apply a new approach in theoretical and applied fields of microbiology and bacteriology. In this work, using the method described we have revealed genomic polymorphism of dissociative variants of Bac. subtilis (mesentericus) 76. The data obtained may be referred as strong evidence that bacterial dissociation do has genetic nature.     

Restriction-modification systems in Bacillus strains related to Bacillus subtilis

127 strains of bacilli sensitive to different phages of Bacillus subtilis were isolated from the soil of Moscow and its country-side. In 6 strains, restriction and modification systems were discovered which differed from these previously described for Bac. subtilis BsuR system. Two strains has identical restriction-modification systems, and one strain possessed two different systems. Using DNA from all 6 strains, it was possible to transform competent cells of Bac. subtilis RUB834. Two of these 6 strains could serve as recipients in transformation and transfection experiments.     

Study of catalase and proteolytic activities of different variants of Bacillus mesentericus]

Catalase and proteolytic activity of the culures and morphological variants of Bacillus mesentericus fuscus, Bac. mesentericus vulgatus were studied. The variants were obtained as a result of prolonged cultivation of the stock strains in the potato mash under the layer of vaseline oil. The level of catalase activity varies in different morphological variants of the same culture, changes with age and depends on the storage conditions. The catalase activity in the rough, smooth and papillar variants that were freshly isolated from the potato mash was 1.5=2.5 times lower than that in the variants long kept on the agar medium. The quantitative indexes of the proteolytic activity of different variants also varied.     

Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis

Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade+, Trp+, or Ade+ Trp+ with DNA in protoplast lysates of B. subtilis AC819 (hisH, tet-1, rpsL, smo-1). The cotransfer ratio of purB to trpC was constant at 7-9% (Ade+ Trp+/Trp+) or 3% (Ade+ Trp+/Ade+) at protoplast concentrations of 2.7 x 10(3) to approximately 2.7 x 10(6) per ml. The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7 x 10(3) protoplasts per ml is about 100 times lower than 3.2 x 10(5) competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations. Similar results were obtained with the cotransfer ratio of purA to trpC. The transformation requires several Com proteins including ComK.     

The combined action of N-methyl-N'-nitro-N-nitrosoguanidine and UV light on Bacillus subtilis

The mutagenic interaction between ultraviolet irradiation and the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine was studied in repair-competent and excision-deficient strains of Bacillus subtilis. Pre-exposure to low doses of MNNG with following treatment by low and intermediate doses of UV light increase the resistance of Bac. subtilis to UV radiation (antagonistic effect). Probably pre-exposition with MNNG leads to induction of enzymes reparation, UV damages being controlled with adaptive response genes.