The Bacillus subtilis spoIIIG gene codes for a sigma factor termed sigma G which directs transcription of genes expressed only in the forespore compartment of the sporulating cell. Use of spoIIIG-lacZ transcriptional fusions showed that spoIIIG is cotranscribed with the spoIIG operon beginning at t0.5-1 of sporulation. However, this large mRNA produced little if any sigma G, and transferring the spoIIIG gene without the spoIIG promoter into the amyE locus resulted in a Spo+ phenotype. Significant translation of spoIIIG began at t2.5-3 with use of an mRNA whose 5' end is just upstream of the spoIIIG coding sequence. Synthesis of this spoIIIG-specific mRNA was not abolished by a deletion in spoIIIG itself. Similar results were obtained when a spoIIIG-lacZ translational fusion lacking the spoIIG promoter was integrated at the amyE locus. These data suggest that synthesis of sigma G is dependent neither on transcription from the spoIIG promoter nor on sigma G itself but can be due to another transcription factor. This transcription factor may be sigma F, the product of the spoIIAC locus, since a spoIIAC mutation blocked spoIIIG expression, and sequences upstream of the 5' end of the spoIIIG-specific mRNA agree well with the recognition sequence for sigma F. RNA polymerase containing sigma F (E sigma F) initiated transcription in vitro on a spoIIIG template at the 5' end found in vivo, as did E sigma G. However, E sigma F showed a greater than 20-fold preference for spoIIIG over a known sigma G-dependent gene compared with the activity of E sigma G.
The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K ؉ (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.Spores of various Bacillus species can remain dormant for long periods of time but can rapidly "return to life" through the process of spore germination (18,19,20,28). Spore germination is normally triggered by the stereospecific binding of specific low-mol...
The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor ␣/-and ␥-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (B. cereus T and B. thuringiensis subsp. Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.Significant effort has been expended in recent years to develop rapid techniques for identification of spores of Bacillus species, in particular those of B. anthracis. Some of these techniques have utilized mass spectrometry to identify biomarkers characteristic of spores of these organisms. In the latter analyses the biomarkers have most often been released in situ for laser desorption by treating spores with acetonitrile:water:trifluoroacetic acid (TFA), and species of 1.5 to 10 kDa in molecular size have been characterized by using matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF [MS]) (4,5,8,13,20). Most of these desorbed biomarkers were found to be secondary metabolites (9, 13) synthesized by the microorganism during growth and could be used to differentiate between different Bacillus spore species. Unfortunately, it has been noticed that these secondary metabolites can quantitatively (8) and qualitatively (13) change with culture conditions, which makes them not very reliable biomarkers for spore identification. However, much higher levels of biomarkers were detected when spores, suspended in acetonitrile:water:TFA, were also subjected to corona plasma discharge or sonication (8,20). Some of these biomarkers were identified as members of the small, acid-soluble spore protein (SASP) family (2,20).The SASP (6 to 10 kDa) are of two types, ␣/-type and ␥-type, named after the predominant protein(s) of these types in B. subtilis spores (3, 23). Both types of SASP are synthesized only in the developing spore late in sporulation, and they comprise 8 to 15% of total spore protein and even more of the spore's soluble protein. The ␣/-type SASP are products of a multigene family with 4 to 7 genes in both Bacillus and Clostridium species. While all of these genes (termed ssp) are expressed in parallel, in spores of Bacillus species two proteins make up Ն80% of the ␣/-type SASP pool, which is ϳ50% of the total SASP. Although the ␣/-type SASP exhibit no sequence similarity to other proteins or protein motifs in available databases, these proteins are extremely similar in amino acid sequence both within and across species. However, even among closely related species there are significan...
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