SummaryAmino sugars are essential precursor molecules for the biosynthesis of bacterial cell walls. Their synthesis pathway is initiated by glucosamine-6-phosphate (GlcN-6-P) synthase (GlmS) which catalyses the rate limiting reaction. We report here that expression of the Escherichia coli glmS gene is negatively feedback regulated by its product GlcN-6-P at the posttranscriptional level. Initially, we observed that mutants defective for yhbJ, a gene of the rpoN operon, overproduce GlmS. Concomitantly, a glmS mRNA accumulates that is derived from processing of the primary glmUS transcript at the glmU stop codon by RNase E. A transposon mutagenesis screen in the yhbJ mutant identified the small RNA GlmZ (formerly RyiA or SraJ) to be required for glmS mRNA accumulation. GlmZ, which is normally processed, accumulates in its full-length form in the yhbJ mutant. In the wild type, a decrease of the intracellular GlcN-6-P concentration induces accumulation of the glmS transcript in a GlmZ-dependent manner. Concomitantly, GlmZ accumulates in its unprocessed form. Hence, we conclude that the biological function of GlmZ is to positively control the glmS mRNA in response to GlcN-6-P concentrations and that YhbJ negatively regulates GlmZ. As in yhbJ mutants GlcN-6-P has no effect, YhbJ is essential for sensing this metabolite.

In Escherichia coli the glmS gene encoding glucosamine 6-phosphate (GlcN-6-P) synthase GlmS is feedback regulated by GlcN-6-P in a pathway that involves the small RNA GlmZ. Expression of glmS is activated by the unprocessed form of GlmZ, which accumulates when the intracellular GlcN-6-P concentration decreases. GlmZ stabilizes a glmS transcript that derives from processing. Overexpression of a second sRNA, GlmY, also activates glmS expression in an unknown way. Furthermore, mutations in two genes, yhbJ and pcnB, cause accumulation of full-length GlmZ and thereby activate glmS expression. The function of yhbJ is unknown and pcnB encodes poly(A) polymerase PAP-I known to polyadenylate and destabilize RNAs. Here we show that GlmY acts indirectly in a way that depends on GlmZ. When the intracellular GlcN-6-P concentration decreases, GlmY accumulates and causes in turn accumulation of full-length GlmZ, which finally activates glmS expression. In glmZ mutants, GlmY has no effect on glmS, whereas artificially expressed GlmZ can activate glmS expression also in the absence of GlmY. Furthermore, we show that PAP-I acts at the top of this regulatory pathway by polyadenylating and destabilizing GlmY. In pcnB mutants, GlmY accumulates and induces glmS expression by stabilizing full-length GlmZ. Hence, the data reveal a regulatory cascade composed of two sRNAs, which responds to GlcN-6-P and is controlled by polyadenylation.

The Lia system, a cell envelope stress response module of Bacillus subtilis, is comprised of the LiaRS two-component system and a membrane-anchored inhibitor protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all orthologs investigated so far are activated by cell wall antibiotics. In response to envelope stress, the systems in Firmicutes cocci induce the expression of a number of genes that are involved in conferring resistance against its inducers. In contrast, a complete picture of the LiaR regulon of B. subtilis is still missing and no phenotypes could be associated with mutants lacking LiaRS. Here, we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a comprehensive picture of the Lia response of Bacillus subtilis. In addition to the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene affected by LiaR-dependent regulation. The results of detailed follow-up gene expression studies, together with proteomic analysis, demonstrate that the liaIH operon represents the only relevant LiaR target locus in vivo. It encodes a small membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms large oligomeric rings reminiscent of those described for Escherichia coli PspA or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies demonstrated that the gene products of the liaIH operon are involved in protecting the cell against oxidative stress and some cell wall antibiotics. Our data suggest that the LiaFSR system of B. subtilis and, presumably, other Firmicutes bacilli coordinates a phage shock protein-like response.