Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro

Abstract

o$/0-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the a/$-type wild-type SASP, SspCwt. One mutation (SSpCTYr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other o/I0-type SASP. The other mutations changed residues conserved in all a/0-type SASP.In one (SspCAIa), residue 52 (Gly) was changed to Ala; in the second (SSpCGIn), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspCwt and SSpCTYr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major a/l/-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAIa had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SSpCAla and SspCwt. Strikingly, neither SspCAla nor SSpCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of a/,-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.Approximately 5 to 12% of the protein of dormant spores of Bacillus and Clostridium species is a group of small, acid-soluble proteins (SASP) of the ot/r type (25). These small proteins (60 to 72 residues) are coded for by a multigene family of at least seven members (termed ssp genes) in Bacillus species. The a/a-type SASP are synthesized in parallel only in the developing forespore during stage III of sporulation and are rapidly degraded during spore germination. While all ssp genes appear to be expressed, generally two (coding for SASP-ot and -1 in Bacillus subtilis) are expressed at high levels, with others expressed at lower levels. One function of a(/a-type SASP is to provide, by their degradation, amino acids for protein synthesis and metabolism early in spore germination. These proteins also have a second role in the resistance of spores to UV light (26). A variety of studies, both in vivo and in vitro, have shown that oa/,-type SASP play a major role in determining the structure of spore DNA, undoubtedly by binding to the DNA and changing its conformation from the B form to A form, with an attendant change in DNA photochemistry which results in spore UV resistance (5,13,(16)(17)(18)(19)23).That a/,8-type SASP are DNA structural proteins is consistent with the extreme conservation of their primary sequence, both within and across species (10,25). Within the Bacillus line of gram-positive spore formers, the sequences of 18 different a/,3-type SASP can be aligned without introduction of gaps; these sequences have 26 identical residues as well ...

Synthesis and characterization of a 29‐amino acid residue DNA‐binding peptide derived from α/β‐type small, acid‐soluble spore proteins (SASP) of bacteria

“…First, it has a net charge of 2+, compared with 2-for the amino-terminal portion of the molecule (calculated for the consensus sequence at pH 7.0). Second, it conrains the site of the most functionally disruptive sitedirected mutation yet produced: SspC ")~ has agly ~ ala substitution at position 403 and lacks all of the signature properties of c~/,6-type SASP -conferring UV resistance on spores [15], promotion of plasmid negative supercoiling [15], protection of DNA against DNase I digestion [2,15], and alteration of DNA photochemistry in vitro [17,18]. For such a chemically minor change to have such profound functional consequences it seems most probable that the change directly disrupts the protein/DNA interface.…”

“…These included SASP-(z from Clostridium b~ermentans and mutant derivatives of SspC from B. subtilia: SspCt~e(L29Y), SspC~"(K57Q), and SspC~J"(GS2A)~. The DNA-bindins characteristics of SspO )' are indistinguishable from those of wild.type SspC whereas SspC ~ lacks DNA binding properties almost entirely both ir~ rive and in vitro [2,15]. SspC ~o" represents an intermediat~ ca~: it binds to poly(dG).…”

“…SspC ~o" represents an intermediat~ ca~: it binds to poly(dG). poly(dC), but not to other DNAs [15].…”

“…-10 -5 1 5 10 15 20 25 30 (gl-6) 35 40 45 50 55 60 I l I I I I I I [ I I 1 I I We have now embarked on structural studies aimed at elucidating the mechanism of action of this novel family of DNA binding proteins. The availability of cloned genes coding for o./,8-type SASP provides sufficient protein, as well as the opportunity to perform site-directed mutagenesis [14,15], Analysis of several such mutants [15] as well as the analysis of conserved o/fl-type SASP sequence features ( Fig. 1) and comparison of these with the distantly related HBs protein of Bacillus stearothermophilus has allowed us tentatively to identify the earboxyl-terminal part of the v./,6-type SASP as containing the region which contacts DNA directly [16].…”

N-terminal Amino Acid Residues Mediate Protein-Protein Interactions between DNA-bound α/β-Type Small, Acid-soluble Spore Proteins from Bacillus Species

“…To explore this question in more detail, a number of N-terminal variants of SspC, a minor ␣/␤-type SASP from B. subtilis, were produced and their DNA binding properties analyzed. SspC was chosen for this study because it is easily overexpressed and purified from both B. subtilis spores and E. coli, and its interaction with and effects upon DNA have been studied extensively both in vitro and in vivo (5,9,10). Furthermore, SspC binds to DNA with a higher affinity than many other ␣/␤-type SASP, and also has one of the longest N termini of all identified ␣/␤-type SASP (4, 15).…”