Genome-Wide Transcriptional Analysis of the Phosphate Starvation Stimulon ofBacillus subtilis

Abstract

Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P i ) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2,3 cyclic nucleotide 2-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3 or 5 phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.When Bacillus subtilis encounters phosphate starvation stress, it responds by inducing groups of genes that function to restrict the metabolic consequences of the limited supply of this essential nutrient. These groups of genes are collectively referred to as the phosphate (Pho) stimulon. The phosphate stimulon includes at least two well-described regulons, namely, the sigma B ( B ) general stress regulon and the phosphate starvation-specific PhoP regulon. When B. subtilis encounters phosphate starvation, genes of the SigB regulon are induced by the alternative sigma factor, B , and genes of the PhoP regulon are either induced or repressed by activated PhoP (namely, PhoPϳP). The B general stress regulon contains Ͼ100 genes (58, 64). These genes provide a nonspecific response to stress by encoding proteins that protect the DNA, membranes, and proteins from the damaging effects of stress. Proteins induced by B help the cell to survive potentially harmful environmental conditions, such as heat, osmotic, acid, or alkaline shock (6,21,23,26). This protective function is thought to be particularly important in maintaining the viability of nongrowing cells.The PhoP regulon currently consists of 34 members. Six operons (phoPR [56,60], phoB-ydhF [7,14], pstSAC-pstBApstBB [3,67], phoD-tatAD [7,19], resABCDE [10], and tuaABC DEFGH [40,72]) and five monocistronic genes (glpQ [7], phoA [30,31], tatCD [34], ykoL [60], and yttP [62]) are induced and two operons (tagAB and tagDEF (39) are repressed in response to phosphate starvation. phoA and phoB encode alkaline phosphatases (APases) which facilitate the recovery of inorganic phosphate (P i ) from organic sources (11, 30); phoD encodes a phosphodiesterase/APase, putatively involved in cell wall teichoic acid turnover, and is secreted exclus...

Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

“…In phosphate-limited wild-type cells, the relative RNA abundance of 149 genes shows significant changes of at least twofold, among which are members of the PhoPR regulon (see next section): transcripts of 115 genes increase and of 34 genes decrease (see Supplementary Tables S7 and S8, available with the online version of this paper). The number of genes with decreased expression is fewer than found by Allenby et al (2005) who reported decreased expression of 69 genes including genes encoding proteins involved in DNA synthesis, transcription and translation. Among genes downregulated in our study are those involved in teichoic acid, biotin, cobalamin and sulphur (see below) metabolism, but not genes involved in central metabolic processes (Table 1, group B).…”

“…Although the P act of the yycOP operon promoter is unusually low (59 units) for a PhoPR-activated gene (Fig. 2b), transcriptome analysis shows a 26-fold (yycO) and a 13-fold (yycP) induction upon phosphate limitation in a PhoPR-dependent manner while Allenby et al (2005) reported a 3.6-fold induction of yycO in a wild-type strain and a 15-fold induction in a SigB mutant strain. Together these results are consistent with the yycOP operon being a bona fide member of the PhoPR regulon.…”

“…Among genes downregulated in our study are those involved in teichoic acid, biotin, cobalamin and sulphur (see below) metabolism, but not genes involved in central metabolic processes (Table 1, group B). These differences from the study by Allenby et al (2005) are probably due to variations in strain, media [LPDM utilized in this study contains 53 mM phosphate (Müller et al, 1997) while LPDM medium contained 420 mM phosphate in the study by Allenby et al (2005)] and method of analysis (their transcriptome analysis was performed with macroarrays).…”

“…This gene cohort composes the potential PhoPR regulon. Of the upregulated genes, 27 (Table 1, group A, the top 26 genes excluding nasD but including phoPR, which is not included in this table as it is induced only approximately threefold upon phosphate limitation) were previously identified as members of the PhoPR regulon (Hulett, 2002;Prágai & Harwood, 2002;Allenby et al, 2005). Although the P act of the yycOP operon promoter is unusually low (59 units) for a PhoPR-activated gene (Fig.…”

“…To validate the P act profiles established by LCA in this study, we compared the reported expression of several genes that have distinctive profiles (murAA, murAB, cwlS and genes of the WalRK and PhoPR regulons) with the kinetics of P act established in this study. Previous studies show (i) that murAA expression (RNA accumulation) is confined to exponential growth and is turned off when cells enter stationary phase while murAB is expressed (RNA accumulation) during stationary phase (Kock et al, 2004); (ii) that cwlS is expressed (RNA and LacZ accumulation) at significant levels during late exponential growth and early stationary phase (Fukushima et al, 2006); (iii) that expression (RNA accumulation) of WalRK regulon genes yocH, cwlO and ydjM is highest immediately after inoculation into HPDM medium but decreases to a low level thereafter even while cells are growing exponentially (Howell et al, 2003(Howell et al, , 2006 and (iv) that PhoPR regulon genes are induced (RNA and LacZ accumulation) upon phosphate limitation (Allenby et al, 2005;Hulett, 2002 and references therein;Prágai & Harwood, 2002). The P act profiles for these genes determined using transcriptional gfp fusions in this study (Fig.…”