During a search by computer-aided inspection of two-dimensional (2D) protein gels for sigmaB-dependent general stress proteins exhibiting atypical induction profiles, a protein initially called Hst23 was identified as a product of the yvyD gene of Bacillus subtilis. In addition to the typical sigmaB-dependent, stress- and starvation-inducible pattern, yvyD is also induced in response to amino acid depletion. By primer extension of RNA isolated from the wild-type strain and appropriate mutants carrying mutations in the sigB and/or spo0H gene, two promoters were mapped upstream of the yvyD gene. The sigmaB-dependent promoter drives expression of yvyD under stress conditions and after glucose starvation, whereas a sigmaH-dependent promoter is responsible for yvyD transcription following amino acid limitation. Analysis of Northern blots revealed that yvyD is transcribed monocistronically and confirmed the conclusions drawn from the primer extension experiments. The analysis of the protein synthesis pattern in amino acid-starved wild-type and relA mutant cells showed that the YvyD protein is not synthesized in the relA mutant background. It was concluded that the stringent response plays a role in the activation of sigmaH. The yvyD gene product is homologous to a protein which might modify the activity of sigma54 in gram-negative bacteria. The expression of a sigmaL-dependent (sigmaL is the equivalent of sigma54 in B. subtilis) levD-lacZ fusion is upregulated twofold in a yvyD mutant. This indicates that the yvyD gene product, being a member of both the sigmaB and sigmaH regulons, might negatively regulate the activity of the sigmaL regulon. We conclude that (i) systematic, computer-aided analysis of 2D protein gels is appropriate for the identification of genes regulated by multiple transcription factors and that (ii) YvyD might form a junction between the sigmaB and sigmaH regulons on one side and the sigmaL regulon on the other.