Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of twocomponent signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa 3 or caa 3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration in B. subtilis.

The Pho regulon of Bacillus subtilis is controlled by three two-component signal-transduction systems: PhoP/PhoR, ResD/ResE, and the phosphorelay leading to the phosphorylation of SpoOA. Two of these systems act as positive regulators, while the third is involved in negative regulation of the Pho regulon. Under phosphate-starvation-induction conditions, the response regulator (RR) PhoP, and the histidine protein kinase (HK) PhoR, are involved in the induction of Pho-regulon genes including the phoPR operon and genes encoding the major vegetative alkaline phosphatases, phoA and phoB. ResD (the RR) and ResE (the HK) are positive regulators of both aerobic and anaerobic respiration in B. subtilis. Current data suggest that they are also positive regulators of the Pho regulon, as is the transition-state regulatory protein AbrB. Data presented reveal that ResDE and AbrB are involved in activation of the Pho regulon through separate regulatory pathways. SpoOA approximately P (RR) exerts a negative effect on the Pho regulon through its repression of AbrB, and possibly through repression of ResDE. Both pathways converge to regulate transcription of the phoPR operon.

SummaryThe Bacillus subtilis ResD-ResE two-component system is responsible for the regulation of a number of genes involved in cytochrome c biogenesis and haem A biosynthesis, and it is required for anaerobic respiration in this organism. We reported previously that the operon encoding these regulatory proteins, the resABCDE operon, is induced under several conditions, one of which is phosphate starvation. We report here that this transcription requires the PhoP-PhoR two-component system, whereas other induction conditions do not. The PhoPϳP response regulator directly binds to and is essential for transcriptional activation of the resABCDE operon as well as being involved in repression of the internal resDE promoter during phosphate-limited growth. The concentration of ResD in various phoP mutant strains corroborates the role of PhoP in the production of ResD. These interactions result in a regulatory network that ties together the cellular functions of respiration/energy production and phosphate starvation. Significantly, this represents the first evidence for direct involvement of one two-component system in transcription of a second two-component system.

The phoPR operon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of the Bacillus subtilis Pho regulon in response to an extracellular phosphate deficiency. Induction of phoPR upon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on the phoPR promoter. Primer extension analyses, using RNA from JH642 or isogenic sigE or sigB mutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of the phoPR operon represents the sum of five promoters, each responding to a specific growth phase and environmental controls. The temporal expression of the phoPR promoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenic sigE or sigB mutants. In vitro transcription studies using reconstituted E A , E B , and E E holoenzymes identified P A4 and P A3 as E A promoters and P E2 as an E E promoter. Phosphorylated PhoP (PhoPϳP) enhanced transcription from each of these promoters. E B was sufficient for in vitro transcription of the P B1 promoter. P 5 was active only in a sigB mutant strain. These studies are the first to report a role for PhoPϳP in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoPϳP at an E E promoter. Information concerning P B1 and P 5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.Inorganic phosphate (P i ) is the limiting nutrient for biological growth in the soil, the natural habitat of Bacillus subtilis. To thrive in this environment where P i levels are often 2 to 3 orders of magnitude lower than levels of other required ions (29), B. subtilis has evolved complex regulatory systems for utilization of this limiting nutrient. At least three global regulatory systems are responsible for changes in gene expression upon phosphate deprivation. One set of genes is controlled either positively or negatively by the PhoP-PhoR two-component regulators, genes referred to as the Pho regulon genes (for review, see reference 12). Other genes that are induced upon phosphate limitation are dependent on SigB (1), an alternative stress sigma factor. A third class of genes is expressed under phosphate-limiting growth conditions that are independent of either SigB or PhoP-PhoR (1). The regulatory coordination between these three sets of genes is unclear, although up-regulation of certain Pho regulon genes has been reported in a sigB mutant strain (12, 33).Pho regulon genes are the most extensively studied set of phosphate-regulated genes in B. subtilis. Identification of genes of known function that are directly regulated by PhoP-PhoR provides insight into one strategy B. subtilis may use to deal with conditions of limiting phosphate. A high-affinity P i tra...