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.     

The Bacillus subtilis spoIIA locus is expressed at two times during sporulation

Abstract The Bacillus subtilis spoIIA and spoIIAB genes were fused to the Escherichia coli lacZ gene on a novel integrational plasmid vector. The constructs were integrated into the B. subtilis chromosome, and used to show that the spoIIA locus was expressed at two times during sporulation.     

Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis

We have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations. The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000. Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA. A novel form of complementation analysis, based on derivatives of bacteriophage phi 105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations. The spoVA locus is the largest polycistronic sporulation operon yet characterized.     

Effects of transition mutations in the regulatory locus spoIIA on the incidence of sporulation in Bacillus subtilis

We have determined the changes in DNA sequence corresponding to three mutations in the promoter-proximal open reading frame of spoIIA, a locus that regulates sporulation in Bacillus subtilis. All three mutations prevent the synthesis of two sporulation-associated enzymes, but they differ in their effects on spore incidence. We now find that mutation spo-42, which allows spores to be produced at a low incidence, is a transition that changes Gly95 to Asp in the protein encoded by the open reading frame. Mutation spo-69, which blocks sporulation entirely, consists of two transitions: these change Gly62 to Asp and Ala1 16 to Thr. Mutation sas-1, which partially suppresses spo-69, is also a transition: this changes residue 62 (which had become Asp as a result of the spo-69 mutation) to Asn.     

Isolation and characterization of a recombinant plasmid carrying a functional part of the Bacillus subtilis spoIIA locus

Plasmid pHM2 consists of a 3.3 kb insert of Bacillus subtilis DNA in the chimeric plasmid pHV33, and can replicate in Escherichia coli and B. subtilis. In B. subtilis, pHM2 complements the defects resulting from mutations spo-42, spo-50, spo-69 and sas-1 in the spoIIA locus. This complementation can occur in recE4 strains where recombination of the plasmid with the chromosome is prevented, and the chromosome retains the mutant allele. Thus the plasmid carries a functional part of the spoIIA locus; it does not contain the complete locus as it cannot complement several other spoIIA mutations. It is likely that the locus is complex, containing at least two genes.     

Cloning of the Bacillus subtilis spoIIA and spoV A loci in phage phi 105DI:1t

A 6.95 kb HindIII-generated DNA fragment from Bacillus subtilis 168 was inserted into the DNA of phage phi 105DI:1t. The recombinant phage (phi 105DS1) contained DNA of 33.8 kb as compared with 35.2 kb for phi 105DI:1t and 39.2 kb for the wild-type phage. In the presence of helper phage, phi 105DS1 complemented both spoIIA and spoV A mutations in B. subtilis.     

Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of tet(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in tet(L) mRNA stability. Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a B. subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5' end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other B. subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.     

The Products of the spoVA Operon Are Involved in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis

Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.     

Variety of sporulation phenotypes resulting from mutations in a single regulatory locus, spoIIA, in Bacillus subtilis

Closely linked mutations in either of the two putative genes of the sporulation locus spoIIA can affect, in quite diverse ways, spore incidence, the production of alkaline phosphatase and DNAase, and the stability of the cells in sporulation medium. It is concluded that the locus has a regulatory function affecting the activation or induction of at least two, and possibly more, sporulation-associated operons.     

Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon

A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in L-alanine-triggered germination, but not in germination with Ca2+-dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca2+-DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.     

Itoic Acid Synthesis in Bacillus subtilis

Under conditions of iron deficiency, strains of Bacillus subtilis produced 2,3-dihydroxybenzoic acid (DHB), 2,3-dihydroxybenzolyglycine (DHBG), or both of these compounds. DHB(G) production [production of DHB(G) refers to the production of DHB, or DHBG, or both] was proportional to the amount of iron present and occurred logarithmically, paralleling growth. Supplementation of media with more than 150 mug of iron per liter at zero-time inhibited DHB accumulation completely. In the presence of DHB, lower levels of iron inhibited DHB(G) production, so that the actual inhibitor of synthesis may involve the Fe(3+):[DHB(G)](3) complex. The strains producing DHBG also produced coproporphyrin III during iron-deficient growth, whereas a strain producing DHB did not produce coproporphyrin III under these conditions. Accumulation of DHB(G) was influenced by the levels of aromatic amino acids and anthranilic acid in the medium. In vivo experiments with strain B-1471 demonstrated that DHB was coupled to added glycine to form DHBG. Metabolism of DHB(G) was observed in two of the strains studied.