The mechanism of insertion of a segment of heterologous DNA into the chromosome of Bacillus subtilis

An Escherichia coli plasmid, p1949, that is derived from pMB9 and pC194, and unable to replicate in Bacillus subtilis, can give rise to stable CmR transformants of the latter species if it is inserted into the bacterial chromosome. A purified segment of the B. subtilis chromosome, with transforming activity against pheA1 and nic-38 recipients, was used to direct the insertion of p1949 into the B. subtilis chromosome. Insertion of the ligated DNA segments occurred in the region of the chromosome from which the purified phe-nic segment was derived. Many of the properties of the resulting CmR transformants of B. subtilis are consistent with the occurrence of a Campbell recombination mechanism leading to integration. However, certain of these properties are more easily explained if it is proposed that integration occurs by a reciprocal recombination event involving a linear ligation product. Evidence is presented suggesting that the inserted sequences may be tandemly duplicated. This may effectively vitiate the use of p1949 as a convenient means for complementation analysis of recessive mutations in B. subtilis.     

Developmentally regulated phosphoproteins associated with chromosome complexes in yeast

Previous studies on our laboratory have shown that nuclear DNA from Saccharomyces cerevisiae can be isolated in the form of fast-sedimenting chromosome complexes (FSCC). In cycling cells, three FSCC forms, denoted g1 and g2, can be distinguished by their characteristic sedimentation velocities and are found correspondingly in cells in G1, S and G2 of the cell cycle, respectively. A fourth form, denoted go, is found exclusively in stationary-phase and nitrogen-starved cells and defines the non-cycling state, Go. We used the differing sedimentation velocities of the FSCC of proliferating and non-cycling cells as a way to isolate and examine their associated proteins. We report here a two-dimensional polyacrylamide gel electrophoresis analysis of [35S]methionine and 32PO4-labelled proteins extracted from FSCC isolated from cycling cells (g1, r and g2, collectively denoted 'cycling FSCC'), and nutritionally arrested cells (go FSCC). Among the 120 35S-labelled FSCC-associated polypeptides detected, 25 were unique to go FSCC and 7 were unique to cycling FSCC. Among the 84 32P-labelled FSCC-associated polypeptides detected, 52 were unique to go FSCC and 7 were unique to cycling FSCC. Comigrating 35S and 32P-labelled polypeptides were matched in 34 of the 84 phosphorylated polypeptides, and 21 of these showed the same specificity of association to either cycling or go FSCC. This analysis demonstrates that there are major differences in the proteins associated with FSCC from cycling and nutritionally arrested cells, and indicates that a relationship exists between the growth state of the yeast cell, protein phosphorylation and chromosome-complex structure.     

Industrial applications of a cloned neutral protease gene in Bacillus subtilis

Summary Genes for the -amylase and neutral protease were cloned from an industrial Bacillus isolate, Bsl, onto two separate plasmids and introduced into a B. subtilis strain. Both plasmids were stably maintained in this strain. Analysis of the extracellular proteins showed that the plasmidcarrying strain produced predominantly the Bsl -amylase and neutral protease with few contaminating B. subtilis exoenzymes. The presence of high levels of protease enabled the strain to produce considerably more -amylase when grown on a complex industrial medium rich in protein.     

Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid.

A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation. This is the first demonstration of a spo locus in cloned B. subtilis deoxyribonucleic acid.     

Expression of the cloned xylanases from an alkalophilic,thermophilic Bacillus in Bacillus subtilis

A recombinant plasmid construct, pLPX6.5, harbouring a 6.5 kb Hind III fragment of genomic DNA, from an alkalophilic, thermophilic Bacillus NCIM 59 and coding for xylanase activity, was electroporatically transformed into Bacillus subtilis MI 111. The expression of the recombinant xylanases was confirmed by cross-reactivity with antibodies raised against purified xylanase II (M _r 15,800) from NCIM 59. However, as there were different xylan hydrolysis products from NCIM 59 and the host B. subtilis, the two xylanases appear to have different modes of action. Xylanase expression in the transformants was 6-fold higher than in the host. There was no significant enhancement in the expression of recombinant xylanases by adding xylan to the growth medium.The authors are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune-411008, India