Regulon of the N -Acetylglucosamine Utilization Regulator NagR in Bacillus subtilis

Abstract

N-Acetylglucosamine (GlcNAc) is the most abundant carbon-nitrogen biocompound on earth and has been shown to be an important source of nutrients for both catabolic and anabolic purposes in Bacillus species. In this work we show that the GntR family regulator YvoA of Bacillus subtilis serves as a negative transcriptional regulator of GlcNAc catabolism gene expression. YvoA represses transcription by binding a 16-bp sequence upstream of nagP encoding the GlcNAc-specific EIIBC component of the sugar phosphotransferase system involved in GlcNAc transport and phosphorylation, as well as another very similar 16-bp sequence upstream of the nagAB-yvoA locus, wherein nagA codes for N-acetylglucosamine-6-phosphate deacetylase and nagB codes for the glucosamine-6-phosphate (GlcN-6-P) deaminase. In vitro experiments demonstrated that GlcN-6-P acts as an inhibitor of YvoA DNA-binding activity, as occurs for its Streptomyces ortholog, DasR. Interestingly, we observed that the expression of nag genes was still activated upon addition of GlcNAc in a ⌬yvoA mutant background, suggesting the existence of an auxiliary transcriptional control instance. Initial computational prediction of the YvoA regulon showed a distribution of YvoA binding sites limited to nag genes and therefore suggests renaming YvoA to NagR, for N-acetylglucosamine utilization regulator. Whole-transcriptome studies showed significant repercussions of nagR deletion for several major B. subtilis regulators, probably indirectly due to an excess of the crucial molecules acetate, ammonia, and fructose-6-phosphate, resulting from complete hydrolysis of GlcNAc. We discuss a model deduced from NagR-mediated gene expression, which highlights clear connections with pathways for GlcNAc-containing polymer biosynthesis and adaptation to growth under oxygen limitation.

PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

“…The electrophoretic mobility shift assays (EMSA) were performed as previously described (21). Briefly, the 40-bp complementary single-stranded oligonucleotides Binding 1 and Binding 2 or Nbinding 1 and Nbinding 2 (see Table S1 in the supplemental material), respectively, were annealed together.…”

Multiple allosteric effectors control the affinity of DasR for its target sites

“…EMSAs performed with DasR of Saccharopolyspora erythraea (DasR sery ) also revealed that GlcN-6P was able to modulate its DNA-binding capability though in a different way than the simple dissociation of the regulatory protein from its target dre observed for DasR of S. coelicolor (DasR sco ) as, instead, multiple retarded bands were observed upon incubation of DasR sery with GlcN-6P [6]. In Bacilli, the GntR/HutC family NagR (formerly named YvoA [16]) is the DasR orthologue which appears to only control genes for GlcNAc transport and phosphorylation by the PTS (nagP), and the genes for its subsequent deacetylation into GlcN-6P (nagA), and GlcN-6P deamination and isomerization into fructose-6-phosphate (nagB) [17]. The first attempt to identify the allosteric effector of NagR revealed a similar inhibitory effect of GlcN-6P on the DNA-binding ability of NagR as described for DasR sco [17].…”

“…In Bacilli, the GntR/HutC family NagR (formerly named YvoA [16]) is the DasR orthologue which appears to only control genes for GlcNAc transport and phosphorylation by the PTS (nagP), and the genes for its subsequent deacetylation into GlcN-6P (nagA), and GlcN-6P deamination and isomerization into fructose-6-phosphate (nagB) [17]. The first attempt to identify the allosteric effector of NagR revealed a similar inhibitory effect of GlcN-6P on the DNA-binding ability of NagR as described for DasR sco [17]. Later on, footprinting assays and EMSAs performed by Gaug e and colleagues did not show any effect of GlcN-6P and a very minor effect of GlcNAc-6P on the DNA-binding ability of NagR [18].…”

NagR Differentially Regulates the Expression of the glmS and nagAB Genes Required for Amino Sugar Metabolism by Streptococcus mutans

“…When GlcNAc is present in the environment, intracellular GlcNAc-6-P accumulates and activates the expression of genes for GlcNAc and GlcN catabolism by disrupting the interaction between NagC and dre sites (5,(11)(12)(13). In the Gram-positive bacterium Bacillus subtilis, a GntR/HutC-type regulator, designated NagR (formerly YvoA), binds to nonhomologous operator sites, also called dre (14,15), to control the transcription of nagA, nagB, and nagP, which codes for a GlcNAc-specific PTS permease. Unlike in E. coli, though, the allosteric effector(s) that alleviates repression of these target genes by NagR remains undetermined (14,16,17).…”

“…In the Gram-positive bacterium Bacillus subtilis, a GntR/HutC-type regulator, designated NagR (formerly YvoA), binds to nonhomologous operator sites, also called dre (14,15), to control the transcription of nagA, nagB, and nagP, which codes for a GlcNAc-specific PTS permease. Unlike in E. coli, though, the allosteric effector(s) that alleviates repression of these target genes by NagR remains undetermined (14,16,17). Recently, an additional system encoded by ybgBA-gamAP and the GntR-type regulator GamR were discovered in B. subtilis and shown to encode products that regulate and effect the catabolism of GlcN (7,17).…”