Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A of Bacillus subtilis

Abstract

The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the Cterminal half of Spo0A that is highly conserved among species of endospore-forming Bacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.The Spo0A protein of Bacillus subtilis is a member of the phosphorylation-activated response regulator family of twocomponent signal transduction proteins (3,16,35,42) and is required in a signal transduction pathway that controls the initiation of sporulation in response to nutrient limitation (10, 47). Spo0A functions as both a repressor and activator of gene transcription during the transition from exponential growth to the stationary phase (43) and during the early stages of sporulation (39,45,47). Phosphorylated Spo0A represses the transcription of a key transition state regulator, AbrB, by binding to the promoter of the abrB gene (43,45). In addition, phosphorylated Spo0A is required for the activation of transcription of key sporulation-specific genes, which include spoIIA (47), spoIIE (53), and spoIIG (6, 7). Spo0A has been shown to bind to specific sequences in the DNA upstream of the promoters it positively regulates and downstream of the promoters it negatively regulates (41). The Spo0A recognition sequence in DNA is referred to as the 0A box and consists of the 7-bp sequence 5Ј-TGTCGAA-3Ј (6, 43).The response regulator family of two-component signal transduction proteins is characterized by a conserved aminoterminal phosphoacceptor domain and unique carboxyl-terminal effector domains (3, 35, 42) (see Fig. 1A). Response regulators that function as transcription factors, i.e., OmpR (38), UhpA (28), and others (3, 35, 42), generally consist of two domains: the phosphoacceptor domain and an effector domain containing DNA-binding and...

Molecular insights into the initiation of sporulation in Gram-positive bacteria: new technologies for an old phenomenon

“…Perhaps surprisingly, only Glu213, Arg214 and Arg217 make direct contact to the DNA bases, to impart sequence‐specificity on the interaction, and just three of the seven bases (positions 2, 4 and 5) in the canonical ‘0A’ box, 5′‐TGTCGAA‐3′, are contacted directly by these three amino acids. Mutation in Spo0A at Glu213 and Arg214 abolishes DNA‐binding and transcription activation and repression [46,47]. This structure validates the findings that mutation of ‘0A’ boxes at various promoters at positions 4 and 5 that increase Spo0A activity restore the consensus sequence whereas mutations away from the consensus at positions 4 and 5 reduce Spo0A activity [21].…”

Evolutionary Analysis for the Functional Divergence of the Spo0A Protein: The Key Sporulation Control Element

“…The R206G/A mutant lost both functions [Hatt et al, 2000]. The mode of evolution for Spo0A has maintained these residues in all species; there could therefore be Spo0A-boosted activities, depending on the type of mutation induced.…”

“…The region shown in italics corresponds to the CRII sequence; this area is fundamental in recognising the 0A box [Hatt et al, 2000;Lewis et al, 2000a]; the residues highlighted in bold were identified as being traces for this area of the protein. The functional role of several residues identified in this region have already been studied; residues Y190, R203 and H210 are important in determining the conformational characteristics of the protein and residues E205, R206 and R209 are fundamental in interaction with the target DNA molecule [Zhao et al, 2002].…”

Molecular insights into the initiation of sporulation in Gram-positive bacteria: new technologies for an old phenomenon

“…Mutation in Spo0A at Glu213 and Arg214 abolishes DNA-binding and transcription activation and repression [46,47]. Perhaps surprisingly, only Glu213, Arg214 and Arg217 make direct contact to the DNA bases, to impart sequence-specificity on the interaction, and just three of the seven bases (positions 2, 4 and 5) in the canonical Ô0AÕ box, 5 0 -TGTCGAA-3 0 , are contacted directly by these three amino acids.…”