Characterization of a novel member of the DegS-DegU regulon affected by salt stress in Bacillus subtilis

Abstract

As a soil bacterium also found in estuarine and marine habitats, Bacillus subtilis has evolved various sensing and adaptation systems in order to face salt stress conditions. Among these regulatory mechanisms is the DegS-DegU signal transduction system, which was previously shown to be stimulated by high salt concentrations. A search for promoters regulated in response to salt stress led to the identification of wapA, encoding a wall-associated protein, which is strongly expressed at low salt concentrations and almost completely repressed in the presence of 0.7 M disodium succinate. Repression of wapA transcription by salt stress was shown to require the phosphorylated form of DegU. Moreover, DegU-mediated repression of wapA occurred only in high-salt medium. Alignment between the control region of wapA and other DegU-regulated promoters allowed the identification of a putative DegU target sequence, AGAAN(11)TTCAG. Mutation/deletion analyses of the wapA promoter region confirmed the role of the putative DegU control site in repression of wapA transcription at high salt concentrations and revealed a second site of repression located downstream from the transcription start site. Since residual negative control was observed at this second site in the absence of DegU, it seems likely that an additional repressor acts on the wapA control region to further downregulate wapA transcription under salt stress conditions.

FIG. 1

Effect of salt stress on β-galactosidase expression driven from a wapA′-lacZ transcriptional fusion integrated at the amyE locus. B. subtilis strain QB4950 was grown in MB medium (1% tryptone, 0.5% yeast extract, 50 μg of tryptophan per ml, 1 mM MgSO₄) containing 100 mM disodium succinate (□) or 0.7 M disodium succinate (▪). The corresponding growth curves are shown with open (high salt) and closed (low salt) triangles. OD, optical density at 600 nm.

FIG. 2

Effects of degU mutations on the expression of wapA′-lacZ under salt stress conditions. B. subtilis QB4871, QB4883, and QB4955 were grown in high-salt MB medium. Symbols: ⧫, QB4871 (wild type); □, QB4883 (degU::erm); ○, QB4955 (degU146).

FIG. 3

Determination of the transcription start site of wapA by primer extension analysis using an oligonucleotide complementary to the DNA sequence from positions +59 to +33 relative to the wapA start codon. The products of a sequencing reaction generated with the same oligonucleotide were run in parallel. mRNA was isolated from low-salt (lanes 1 and 2) and high-salt (lanes 3 and 4) cell cultures of the wild-type strain 168 and the degU strain QB4487, respectively. Equivalent amounts of total RNA were used in all primer extension experiments.

FIG. 4

Comparison of nucleotide sequences located upstream of genes reported to be regulated by DegU. Numbers preceding the sequences indicate positions of the leftmost nucleotide relative to the transcriptional start site; those in parentheses correspond to positions relative to the translational start site. The yxjJI operon was sequenced by Glaser et al. (10) in the framework of the genome sequencing project, and its transcription was shown to be repressed by DegU (4a).

FIG. 5

Localization of random mutations leading to high-salt-resistant expression of wapA. The mutations indicated above the nucleotide sequence conferred specific derepression under salt stress conditions, while mutations that exhibited a nonspecific increase of expression in both low- and high-salt media are indicated below the sequence. Asterisks mark nucleotides modified by site-directed mutagenesis. The black-boxed sequences correspond to the conserved motif found upstream of genes known to be regulated by DegU, also referred to as the upstream site or site I. The dots under nucleotides in the upstream region indicate a potential dyad symmetry. The gray boxes highlight the 6-bp direct repeats encompassing the second focus of mutations (site II).

FIG. 6

Schematic representation of the wapA regulatory region together with the different constructs designed to alter or delete each one or both of the putative sites involved in repression by salt stress. Plasmid pWP259 contains the entire wild-type 5′ regulatory region of wapA transcriptionally fused to lacZ. In pWP280, sequences located upstream of the two clusters of random mutations have been removed. Plasmid pWP263 is a pWP259 derivative that lacks the downstream site (site II). Plasmid pWP267 harbors the TT_−22/−23AA site-directed mutation, pWP259.15 carries the A₋₃₈G mutation isolated in site I by random mutagenesis, and pWP265 combines deletion of site II and mutation A₋₃₈G in site I. Each plasmid was integrated at the chromosomal amyE locus of the wild-type, degU, and degU32(Hy) strains. The corresponding β-galactosidase activities were determined in high-salt liquid cultures as described for Table 3 and are shown at the right. The data relative to the wapA[TT_−22/−23AA] mutant obtained by site-directed mutagenesis are shown for easier comparison.