Chromosomal insertions of Tn917 in Bacillus subtilis

Vandeyar, Mark A.; Zahler, Stanley A.

doi:10.1128/jb.167.2.530-534.1986

Cited by 106 publications

(48 citation statements)

References 13 publications

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“…In agreement with this result, examination of the restriction map of a previously isolated Tn917 insertion (nic-82::Tn917 [25,34] Comparison of amino acid sequences of the nifS gene product from B. subtilis (Bs) and from Anabaena strain PCC7120 (An). TheAnabaena sequence was taken from reference 22, and the B. subtilis sequence is from Fig.…”

Section: Discussionmentioning

confidence: 77%

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis

Sun

Setlow

1993

J Bacteriol

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NAD plays an important role in cellular metabolism, as it functions as a cofactor in numerous oxidation-reduction reactions. NAD is synthesized from quinolinic acid (Qa) via a pathway which is similar in all organisms which have been examined (Fig. 1). On the other hand, the pathway for Qa formation differs in different organisms (7). In Escherichia coli and Salmonella typhimurium, as well as in many other eubacteria, Qa is synthesized from L-aspartate and dihydroxyacetone phosphate by a Qa synthetase complex (7,33). This complex consists of two enzymes, L-aspartate oxidase and Qa synthetase A. Aspartate oxidase catalyzes oxidation of L-aspartate to iminoaspartate, which is subsequently condensed with dihydroxyacetone phosphate by Qa synthetase A to form Qa (Fig. 1). In E. coli, the latter two enzymes are encoded by the unlinked genes nadB and nadA4, respectively, both of which have been cloned and sequenced (6). Qa is then converted to nicotinic acid mononucleotide (NaMN) by Qa phosphoribosyltransferase, which is coded for by the nadC gene (23). NaMN then reacts with ATP in a reaction catalyzed by the nadD gene product to give nicotinic acid adenine dinucleotide, which is then amidated by the nadE gene product to form NAD. Cells can also convert exogenous nicotinic acid (Nic) to NaMN by Nic phosphoribosyltransferase. When either nadA, nadB, or nadC is inactivated, cells become dependent on Nic for growth.The NAD biosynthetic pathway in Bacillus subtilis has been thought to be the same as in E. coli (7), although the regulation of some of the B. subtilis enzymes, for example, Qa phosphoribosyltransferase, may be rather different than in E. coli (28). Nic-requiring auxotrophic mutants have been isolated in B. subtilis, and the mutations (termed nic) were mapped near the spoOB-pheBA region (2,10,13 The chromosomal DNA library was used to transform strain PS1589 to chloramphenicol resistance. Strain PS1589 carries the divIVB1 mutation, which is known to be closely linked to the site of nic mutations (16,26). Several different Cmr transformants were found to have the cat gene closely linked to divIVB1. Three-factor crosses showed that in one of the Cmr transformants, strain PS1591, the order of markers was divIVB-spoOB-cat.

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Section: Discussionmentioning

confidence: 77%

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis

Sun

Setlow

1993

J Bacteriol

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“…We initially used mutant spoVS8-9 and then confirmed the analysis in mutants spoVS11-4 and spoVS9-4. We found 34% linkage to one of the insertions, zdi-82::Tn917 (RL340), which had been mapped to 140Њ on the B. subtilis chromosome (34). Three other markers in the same region of the chromosome also showed linkage to spoVS8-9: a silent insertion near spoIIG (RL215, 135Њ) exhibited 15% linkage; a marker in the gene for cotE (RL48, 150Њ) showed 90% linkage; and a marker in the gene for cotC (RL52, 168Њ) showed 26% linkage.…”

Section: Methodsmentioning

confidence: 99%

“…For mapping the spoVS mutations, we used PBS1-mediated generalized transduction (11) to determine the linkage of the spoVS mutations to a mapped set of cryptic Tn917 insertions (34). We initially used mutant spoVS8-9 and then confirmed the analysis in mutants spoVS11-4 and spoVS9-4.…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of sporulation gene spoVS from Bacillus subtilis

1995

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We report the identification and characterization of an additional sporulation gene from Bacillus subtilis called spoVS, which is induced early in sporulation under the control of H . We show that spoVS is an 86-codon-long open reading frame and is capable of encoding a protein of 8,796 Da which exhibits little similarity to other proteins in the databases. Null mutations in spoVS have two contrasting phenotypes. In otherwise wild-type cells they block sporulation at stage V, impairing the development of heat resistance and coat assembly. However, the presence of a spoVS mutation in a spoIIB spoVG double mutant (which is blocked at the stage [II] of polar septation) acts as a partial suppressor, allowing sporulation to advance to a late stage. The implications of the contrasting phenotypes are discussed in the context of the formation and maturation of the polar septum.During the developmental pathway of sporulation in Bacillus subtilis, cells undergo a series of elaborate morphological transformations (18,25), commencing with the formation of an asymmetrically positioned septum. The sporulation septum partitions the developing cell (or sporangium) into two cells of unequal size, known as the forespore (the smaller cell) and the mother cell. Following septum formation, the membrane of the mother cell migrates around the forespore, engulfing the smaller cell and pinching it off as a free protoplast within the mother cell. Further events in spore formation include the deposition of cortex and primordial cell wall between the membranes that separate the engulfed forespore from the surrounding mother cell, dehydration of the forespore cytoplasm (which will become the spore core), and assembly around the developing spore of a thick protein coat. Finally, upon maturation, the spore is released from the sporangium by lysis of the mother cell.Here we are concerned with events involved in the formation and maturation of the sporulation septum, which differs in two important respects from the septum of vegetative cells undergoing binary fission. First, sporulation septum placement is polar, while in binary fission, the septum is positioned medially. Second, shortly after the formation of the sporulation septum, the layer of cell wall between the membranes of the septum is observed to undergo dissolution (13). This degradation starts in the middle of the septum and proceeds towards but does not reach the layer of cell wall that surrounds the sporangium. Thus, the resulting products of polar septation are retained side by side within the sporangium. In contrast, during binary fission, autolysis begins at the cell wall and proceeds towards the middle of the septum. Rather than degrading the wall, this process splits the wall in two along its length, releasing the daughter cells from each other.Mutations in several sporulation genes that cause cells to be blocked at substages of the process of septum formation and maturation have been identified. These can be classified into early-acting mutations, which allow the sporangium ...

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“…Strain 1A616 nic-82::Tn917 trpC2 SPPc2 (40) was obtained from the Bacillus Genetic Stock Center. Strain 1S93 spoIIIFS90 lys-J (originally obtained from the Bacillus Genetic Stock Center) was provided by P. Setlow.…”

Section: Methodsmentioning

confidence: 99%

Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis

Popham

Stragier

1991

J Bacteriol

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Mutation of the spoVB gene in Bacillus subtilis causes the production of spores containing a defective cortex and unable to acquire heat resistance. The spoVB locus is highly linked to another spo locus, spoIIIF, characterized by a single mutation (I. L. Lamont and J. Mandelstam, J. Gen. Microbiol. 130:1253-1261. A 18-kb DNA region overlapping the spoIIIF-spoVB region was cloned in successive steps starting from a Tn917 insertion in the nic locus. The exact location of the spollIF and spoVB loci was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spollIF and spoVB are a single monocistronic locus encoding a 518-amino-acid polypeptide with features of an integral membrane protein. The precise location of the spoIIIF590 and spoVB91 mutations in that unique open reading frame was determined, and both mutations were sequenced. A null mutation was engineered in the spoIIIF-spoVB locus and led to a typical spoVB phenotype, identical to the phenotype created by either spoIIIF590 or spoVB9l, suggesting that the original spollIF mutant contained a secondary mutation arresting sporulation at an earlier stage. A transcriptional spoVB-lacZ fusion was constructed, and its expression was found to be directly dependent on RNA polymerase containing SE. A null mutation of spoVB had no effect on expression of sspB and cotA, members of the 7G-and rK-controlled regulons respectively, while expression of cotC, a member of the latest known mother cell regulon, was delayed and strongly reduced. These results are consistent with SpoVB being involved in cortex biosynthesis and affecting only indirectly expression of late sporulation genes.Sporulation in bacilli involves a succession of coordinated steps in which two cellular compartments within a sporangium supply different components to the spore (23). The smaller compartment, the forespore, produces the smaller DNA-binding proteins which confer UV resistance to the spore (25, 33) and apparently the inner peptidoglycan layer surrounding the spore, the germ cell wall (38). The larger compartment, the mother cell, provides the coat proteins which form the outermost layers of the spore (3) and some activities required for synthesis of the cortex (38), a thick outer layer of peptidoglycan. The cortex seems to be involved in attaining or maintaining the dehydrated state of the spore protoplasm, a condition required for heat resistance (12), while the coat proteins provide a barrier to agents such as lysozyme (15,43). The successive production of these spore components is programmed by a series of regulatory genes which appear to activate their respective steps in response to morphological signals of completion of the preceding steps (36).A large number of mutations which block the sporulation process at various morphological steps have been isolated in Bacillus subtilis (28). Analysis of these mutations has shown that many of them are in regulatory genes, presumably because mutation of a factor which has pleiotropic effects on ...

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Chromosomal insertions of Tn917 in Bacillus subtilis

Cited by 106 publications

References 13 publications

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis

Identification and characterization of sporulation gene spoVS from Bacillus subtilis

Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis

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