The yvyD gene of Bacillus subtilis is under dual control of sigmaB and sigmaH

Abstract

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.

FIG. 1

Schematic representation of the yvyD region in the chromosome of B. subtilis. The yvyD gene is located between the fli operon and the secA gene. A rho-independent terminator is located upstream and downstream of yvyD (7). The locations of the primer pair P1-P2, used for construction of plasmid pKD1, and the primer pair P3-P4, used for the construction of plasmid pKD2, are indicated. The BglII site within yvyD was used for the construction of the yvyD mutant by the insertion of a kanamycin resistance cassette in plasmid pKD1, resulting in plasmid pKD11.

FIG. 2

Multiple, partial alignment (approximately 100 amino acids at the N terminus) of YvyD with proteins functioning as ς⁵⁴ modulating factors from gram-negative bacteria. Bs, B. subtilis; Ac, Acinetobacter calcoaceticus (orf2) (10); Ae, Alcaligenes eutrophus (Ralstonia eutropha) (orf130) (55); Av, Azotobacter vinelandii (second open reading frame) (32); Bj, Bradyrhizobium japonicum (orf203) (27); Ec, E. coli (orfII) (25); Kp, K. pneumoniae (orf95) (33); Pp, Pseudomonas putida (orf102) (26); St, Salmonella typhimurium (36); Tf, Thiobacillus ferrooxidans (orf3) (3); C, consensus sequence.

FIG. 3

Determination of the transcriptional start sites of yvyD. (A) Primer extension analysis of RNA. The B. subtilis wild-type strain IS58, the sigB mutant strain BGH1, and the sigH mutant strain BKD2 were exposed at an OD₅₀₀ of 0.5 to various stresses as described in Materials and Methods. For RNA isolation, bacteria were harvested before exposure (C, control) and 6 min after exposure to the different stressors (H, heat [48°C]; S, salt [4% NaCl]; E, ethanol [4% ethanol]). In cases of nutrient starvation bacteria were harvested at transient phase (glucose depletion [GT₀]) or 30 min after entry into stationary phase (deprived of glucose [GS], amino acids [AS], or nitrogen [NS]). A total of 5 μg of each RNA preparation was used for each primer extension reaction. Lanes T, G, C, and A show the sequencing ladder obtained with the same primer as that used for primer extension. (B) DNA sequence of the yvyD promoter region. Potential start sites are indicated by asterisks. The ς^B- and ς^H-dependent promoters are printed in bold. The consensus sequence for ς^B-dependent promoters is taken from the work of Hecker et al. (20), and the consensus sequence for ς^H-dependent promoters is derived from the work of Haldenwang (17). The ribosomal binding site is underlined, and the translational start codon is shown in bold.

FIG. 4

Northern blot analysis of stress-inducible yvyD transcription. Total RNA was isolated from B. subtilis IS58, BGH1, BKD2, and BKD3 after exposure to various stress conditions as described in the legend to Fig. 3. A total of 5 μg of RNA was applied in each lane. The locations of RNA molecular size markers and the size of the yvyD transcript are marked. C, control; S, salt; E, ethanol; H, heat; GT₀, glucose depletion at transient phase; GS, glucose depletion at stationary phase.

FIG. 5

Sections of 2D protein gels in the YvyD region. Strains BR16 (wild type) and BR17 (relA) were grown to an OD₅₀₀ of 0.5. For panels B and C an artificial depletion of amino acids was generated by the addition of dl-norvaline to a final concentration of 0.05%. Labelling of cells with [³⁵S]methionine (5 μCi/ml) was performed after 15 min of incubation with dl-norvaline for 3 min. (A) Control (strain BR16, exponentially growing cells, labelling at an OD₅₀₀ of 0.5, and no dl-norvaline addition); (B) strain BR16 after dl-norvaline treatment; (C) strain BR17 after dl-norvaline treatment. The locations of the proteins ClpP, SodM, GreA, AhpC, and RsbW and of the unidentified proteins a, b, c, d, and e are included as references. Note in panel C that the YvyD protein is no longer induced in the relA mutant and that the protein RplJ is not subjected to the stringent response.

FIG. 6

Northern blot analysis of stress induction of yvyD transcription in a relA mutant. Total RNA was isolated from B. subtilis BR16 and BR17 before (C, control) and after depletion of amino acids (AT₀, transient phase; AS, 30 min after entry into stationary phase). A total of 2.5 μg of RNA was applied in each lane. The locations of RNA molecular size markers and the size of the yvyD transcript are marked. Note that induction of yvyD transcription does not occur in the relA mutant strain.

FIG. 7

β-Galactosidase synthesis originating from a levD-lacZ fusion in the wild type and the yvyD mutant. Strains BKD11 (levD-lacZ) and BKD12 (levD-lacZ yvyD) were grown in minimal medium containing either 0.2% glucose (repressing conditions) (BKD11 [■] and BKD12 [▴]) or 0.2% fructose (inducing conditions) (BKD11 [•] and BKD12 [▾]) as the carbon source. At the times indicated, samples were removed and assayed for β-galactosidase activity (corresponding open symbols).