Bacillus subtilis NhaC, an Na ⁺ /H ⁺ Antiporter, Influences Expression of the phoPR Operon and Production of Alkaline Phosphatases

Abstract

When Bacillus subtilis is subjected to phosphate starvation, genes of the Pho regulon are either induced or repressed. Among those induced are genes encoding alkaline phosphatases (APases). A set of isogenic mutants, with a β-galactosidase gene transcriptionally fused to the inactivated target gene, was used to identify genes that influence the operation of the Pho regulon. One such gene was nhaC (previously yheL). In the absence of NhaC, growth and APase production were enhanced, while the production of other non-Pho-regulon secretory proteins (proteases and α-amylase) did not change. The influence of NhaC on growth, APase synthesis, and its own expression was dependent on the external Na+ concentration. Other monovalent cations such as Li+ or K+ had no effect. We propose a role for NhaC in the uptake of Na+. nhaC appears to be encoded by a monocistronic operon and, contrary to previous reports, is not in the same transcriptional unit as yheK, the gene immediately upstream. The increase in APase production was dependent on an active PhoR, the sensor kinase of the two-component system primarily responsible for controlling the Pho regulon. Transcriptional fusions showed that the phoPR operon and both phoA (encoding APaseA) and phoB (encoding APaseB) were hyperinduced in the absence of NhaC and repressed when this protein was overproduced. This suggests that NhaC effects APase production via phoPR

Transcriptional Regulation of thephoPROperon inBacillus subtilis

“…lacZ transcriptional reporter studies indicated that the phoPR operon exhibits constitutive low-level expression during exponential growth (i.e., P i -replete conditions) and is induced in response to phosphate starvation (32): 4 h after the transition from exponential to stationary phase, the expression of phoPR was threefold higher than that in the exponential phase (17,30). To confirm these data and to determine the size of phoPR transcript(s), Northern blot analyses were performed on RNA extracted from B. subtilis 168 at various times during growth and phosphate starvation-induced stationary phase (Fig.…”

“…Plasmid and chromosomal DNA extraction, restriction endonuclease digestion, agarose gel electrophoresis, transformation of E. coli cells, PCR, and bioinformatical analyses were carried out as described previously (30,33). Enzymes, molecular size markers, and deoxynucleotides were purchased from Roche Diagnostics, Ltd. (Lewes, United Kingdom), and from Amersham Pharmacia Biotech, Ltd. (Little Chalfont, United Kingdom).…”

“…The PhoP response regulator is activated by its cognate sensor kinase, PhoR. Phosphorylated PhoP (PhoPϳP) induces the expression of the phoPR operon about threefold from a low constitutive level of expression (17,30,32) and is required for the induction or repression of other members of the Pho regulon (15).…”

Purification and Properties of Alkaline Phosphatase fromBacillus Cereus

“…Alkaline phosphatases have been identified in a wide variety of organisms, including bacteria (Escherichia coli, Bacillus species, Mycobacterium smegmatis, Thermotoga maritime, Haloarcula marismortui) and humans (3,15,12,13,17,21,22). Although the actual purpose of the enzyme is still not fully understood, the simple hypothesis, that it is a means for the bacteria to generate free phosphate groups for uptake and use (20).…”

Functional Analysis of Conserved Polar Residues in Vc-NhaD, Na+/H+ Antiporter of Vibrio cholerae

“…The genome of pathogenic Vibrio cholerae encodes as many as six putative structural genes of Na ϩ /H ϩ antiporters (24). The physiological basis for such apparent redundancy could be in part explained by recent findings of additional, predominantly regulatory functions of certain bacterial Na ϩ /H ϩ antiporters affecting processes such as antibiotic efflux (25), the transcription of the Pho operon (26), the initiation of sporulation (27), and endospore germination (28). Studying Na ϩ /H ϩ antiport in V. cholerae, we have cloned a new Na ϩ /H ϩ antiporter from V. cholerae, Vc-NhaD, and expressed it in its functional form in the ⌬NhaA⌬NhaB strain of E. coli (29).…”