Regulation of four genes encoding small, acid-soluble spore proteins in Bacillus subtilis

Abstract

Three genes (sspH, sspL, and tlp) encoding new, minor small, acid-soluble proteins (SASP) unique to spores of Bacillus subtilis are expressed only in the forespore compartment during sporulation of this organism. The sspH and sspL genes are monocistronic, whereas tlp is the second gene in an operon with a second small orf, which we have termed sspN. The sspH and sspL genes are recognized primarily by the forespore-specific sigma factor for RNA polymerase, σ^G; the sspN-tlp operon is recognized equally well by σ^G and the other forespore-specific σ factor, σ^F. Sequences centered 10 and 35 nt upstream of the 5′-ends of sspH, sspL, and sspN mRNAs all show homology to −10 and −35 sequences recognized by σ^F and σ^G, which are generally quite similar. Mutations disrupting the sspH, sspL, sspN-tlp, or tlp loci cause a loss of the appropriate SASP from spores, but have no discernible effect on sporulation, spore properties, or spore germination.

Introduction

Spores of Bacillus species have several properties, including dormancy and resistance to a variety of treatments, that distinguish them from their growing cell counterparts (Gerhardt and Marquis, 1989, Setlow, 1994). Among factors determining the unique properties of the dormant spore, two major factors are the presence of unique structural features and gene products. Among the unique gene products present in the spore are the proteins that make up the spore coat, a structure that is involved in spore resistance to enzymes, some chemicals and mechanical disruption (Setlow, 1993), and the small, acid-soluble proteins (SASP) present largely in the central region of the spore, the spore core (Bagyan et al., 1998b, Setlow, 1994). There are three major SASP in spores of B. subtilis, one γ-type SASP and two α/β-type SASP; together, these proteins comprise 8–15% of the protein in spores of Bacillus species (Setlow, 1988). The only known function of a spore's γ-type SASP is to be degraded early in spore germination, thus providing amino acids for protein synthesis during this period of development, and α/β-type SASP also serve this function (Setlow, 1988, Setlow, 1995). However, α/β-type SASP are also DNA-binding proteins that saturate the spore's chromosome, and protect the spore's DNA from a variety of types of damage; this DNA protection by α/β-type SASP is a significant component of spore resistance to a variety of treatments, including heat, oxidizing agents and UV radiation (Setlow, 1995).

In addition to their three major SASP, spores of B. subtilis also contain a large number of minor SASP (Bagyan et al., 1998b). Two of these (SspC and SspD) are minor α/β-type SASP, and one other, termed SspF, exhibits some degree of sequence homology to α/β-type SASP (Setlow, 1993). However, B. subtilis spores contain at least nine other minor SASP that exhibit no sequence relatedness to α/β- (or γ)-type SASP (Bagyan et al., 1998b). These latter proteins appear unique to spores and are of interest for a variety of reasons. First, by analogy with the important functions of α/β-type SASP in spore resistance, these new SASP might also play a key role in some spore property. Second, the genes encoding a number of these new SASP were not identified as ORFs in an analysis of the complete sequence of the B. subtilis genome (Kunst et al., 1997). Consequently, analysis of the expression and function of these genes will help to complete the analysis of the B. subtilis genomic sequence. Finally, since these new SASP appear to be unique to spores, their coding genes seem likely to be expressed only during sporulation. Consequently, analysis of the regulation of these new genes (termed ssp) will increase our knowledge of regulation of gene expression during sporulation.

To date, analysis of two genes (sspG and sspJ) that code for new minor SASP has shown that both genes are transcribed only in sporulation, with sspG transcribed in the mother cell compartment of the sporulating cell by RNA polymerase using the mother cell-specific sigma factor σ^K, while sspJ is transcribed in the forespore compartment under control of the forespore-specific σ^G (Bagyan et al., 1998b). Analysis of the sporulation, spore properties and spore germination of mutant strains lacking SspG or SspJ revealed no major defect other than a slight decrease in the rate of outgrowth of sspJ spores (Bagyan et al., 1998b). The latter result was somewhat disappointing, but perhaps these particular minor SASP are not essential for spores, or loss of either of these proteins gives a phenotype only when combined with loss of one or more other minor SASP. Indeed, a mutation removing one of the two major α/β-type SASP has no effect on spore properties, and only when both proteins are lost is a major phenotype observed (Setlow, 1988, Setlow, 1995). In this communication, we analyse the regulation of expression of the sspH, sspL and tlp genes (Bagyan et al., 1998b) and examine the function of the respective gene products.