CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

Abstract

CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons in Bacillus subtilis. Some of the most strongly repressed targets of CodY, the nupNOPQ (formerly, yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of the nupN gene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6-to 8-fold nupN repression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site to nupN regulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites.Bacillus subtilis cells are able to take up purine and pyrimidine nucleosides, allowing these compounds to serve as substrates for nucleotide biosynthesis via salvage pathways and as sources of carbon and nitrogen (40). Although two major transporters for pyrimidine and purine nucleosides, NupC and NupG, respectively, have been described in B. subtilis, mutants lacking NupC or NupG retain considerable ability to take up pyrimidine or purine nucleosides (19, 33). Thus, additional transporters for both pyrimidine and purine nucleosides remain to be identified (2,19,33).The B. subtilis nupNOPQ (formerly, yufNOPQ) genes appear to encode the components of an ABC transport system of unknown specificity. The first gene of this apparent operon, nupN, codes for an apparent lipoprotein that could serve as the substrate-binding, specificity-determining subunit of the transporter. The putative product of nupN is homologous (43 to 50% identity) to several proteins that are involved in purine and pyrimidine nucleoside transport in Treponema pallidum, Streptococcus mutans, and Lactococcus lactis (9,26,41). The nupO and nupPQ genes appear to encode the ATP-binding protein and integral membrane proteins typical of ABC transporters, respectively.The nupNOPQ genes were previously shown to be among the genes that are most highly repressed by CodY as detected by DNA microarray analysis and lacZ fusions (29). CodY is a global transcriptional regulator that controls expression of more than 100 genes and operons in B. subtilis.Many of the CodY-regulated genes are involved in nitrogen or carbon metabolism (14,29,(37)(38)(39). The main role of CodY in B. subtilis appears to be establishing the temporal hierarchy of utilization of various nitrogen (and carbon) compounds under cond...

Interplay of CodY and ScoC in the Regulation of Major Extracellular Protease Genes of Bacillus subtilis

“…1B). In general, nonspecific binding of CodY in gel shift experiments is observed only at 400 to 800 nM CodY (45,50). Confirming the specificity of interaction, DNase I footprinting experiments showed that CodY protected a single region of DNA, located at positions Ϫ7 to ϩ30 with respect to the aprE transcription start point (Fig.…”

Direct Regulation of GTP Homeostasis by (p)ppGpp: A Critical Component of Viability and Stress Resistance

“…First, most known mechanisms regulate the de novo pathway; (p)ppGpp blocks both de novo and salvage GTP biosynthesis, globally regulating GTP production from all sources. Second, transcriptional feedback mechanisms that regulate GTP biosynthesis genes (Ebbole and Zalkin, 1989; Belitsky and Sonenshein, 2011) may not prevent accumulation of GTP as rapidly as direct enzymatic inhibition by (p)ppGpp. Third, previously known mechanisms of GTP homeostasis involve only precursors and intermediates of purine biosynthesis; pppGpp is an off-pathway product synthesized from GTP to provide a negative feedback loop (Figure 3B).…”

Lowering GTP Level Increases Survival of Amino Acid Starvation but Slows Growth Rate for Bacillus subtilis Cells Lacking (p)ppGpp

“…One possible downstream effector is the transcriptional regulator CodY. CodY is activated by GTP and branched-chain amino acids, represses stationary-phase and guanosine transporter genes, and induces expression of guaB (20,22,35,36). Therefore, it is possible that the effect of guanosine/GTP level on growth rate requires CodY activity.…”