Nucleotide sequence of sporulation locus spoIIA in Bacillus subtilis

We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.     

Ribosomal Alterations Controlling Alkaline Phosphatase Isozymes in Escherichia coli

Different patterns of isozymes were obtained by starch-gel electrophoresis of alkaline phosphatase from Escherichia coli strains differing only by strA or ram mutations, or both, in the 30S ribosomal subunit. The isozyme spread was reduced in strA and increased in ram strains; this strictly parallels the restriction and enhancement of translational ambiguity produced by these mutations. Streptomycin present during growth had an effect similar to ram on both isozymes and ambiguity. The three isozymes analyzed have different N-terminal residues: aspartic acid, valine, and threonine. Different patterns of isozymes were also obtained in a wild-type strain through the specific action of exogenous arginine. A link between the mechanism of the effect of arginine and that of the ribosome is not obvious. The possibility is discussed that in both cases, although by different mechanisms, N-terminals are formed with different sensitivity to limited degradative attack.     

New types of mutation affecting formation of alkaline phosphatase by Bacillus subtilis in sporulation conditions.

Mutations defining three new loci, sapA, sapB and phoS, were detected by their ability to overcome the phosphatase-negative phenotype of early-blocked asporogenous mutants in sporulation conditions. Synthesis of alkaline phosphatase by Bacillus subtilis is subject to 'vegetative' and 'sporulation' controls. The phoS mutations resulted in constitutive production of alkaline phosphatase and so could be altered in either the 'vegetative' or the 'sporulation' control system. The sapA and sapB mutations only affected alkaline phosphatase formation in sporulation conditions, and were considered to be sporulation specific. They rendered 'sporulation' alkaline phosphatase formation independent of all the spomutations tested, and so independent of the control of the dependent sequences of spo locus expression; as the enzyme was not formed constitutively, it remained subject to some other sporulation control. The sapA and phoS loci were placed between argC4 and metC3 on the genetic map; the sapB locus was located close to purB6. The three loci mapped separately from all known spo loci.     

Ouabain-insensitive salt and water movements in duck red cells. II. The role of chloride in the volume response

This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions.     

Separation of chromosome termini during sporulation of Bacillus subtilis depends on SpoIIIE

Bacillus subtilis undergoes a highly distinctive division during spore formation. It yields two unequal cells, the mother cell and the prespore, and septum formation is completed before the origin-distal 70% of the chromosome has entered the smaller prespore. The mother cell subsequently engulfs the prespore. Two different probes were used to study the behavior of the terminus (ter) region of the chromosome during spore formation. Only one ter region was observed at the time of sporulation division. A second ter region, indicative of chromosome separation, was not distinguishable until engulfment was nearing completion, when one was in the mother cell and the other in the prespore. Separation of the two ter regions depended on the DNA translocase SpoIIIE. It is concluded that SpoIIIE is required during spore formation for chromosome separation as well as for translocation; SpoIIIE is not required for separation during vegetative growth.     

Expression of the sigmaF-directed csfB locus prevents premature appearance of sigmaG activity during sporulation of Bacillus subtilis

During sporulation, σ^G becomes active in the prespore upon the completion of engulfment. We show that the inactivation of the σ^F-directed csfB locus resulted in premature activation of σ^G. CsfB exerted control distinct from but overlapping with that exerted by LonA to prevent inappropriate σ^G activation. The artificial induction of csfB severely compromised spore formation.     

Isolation and characterization of a clone of the spoVE locus of Bacillus subtilis

The Bacillus subtilis spoVE locus was isolated from a lambda clone bank and a 4.7 kbp EcoRV fragment subcloned into the shuttle vector pHV33. The resulting plasmid complemented chromosomal spoVE mutations. Its structure was stable in recE4 strains, but plasmid and chromosomal rearrangements occurred in rec+ strains. New spoVE mutations were obtained by mutagenesis of the plasmid; all the mutations tested mapped within three adjacent HindIII fragments of total length 1140 bp.