Control of a family of phosphatase regulatory genes (phr) by the alternate sigma factor sigma-H of Bacillus subtilis - PubMed (original) (raw)

Control of a family of phosphatase regulatory genes (phr) by the alternate sigma factor sigma-H of Bacillus subtilis

R S McQuade et al. J Bacteriol. 2001 Aug.

Abstract

A family of 11 phosphatases can help to modulate the activity of response regulator proteins in Bacillus subtilis. Downstream of seven of the rap (phosphatase) genes are phr genes, encoding secreted peptides that function as phosphatase regulators. By using fusions to lacZ and primer extension analysis, we found that six of the seven phr genes are controlled by the alternate sigma factor sigma-H. These results expand the potential of sigma-H to contribute to the output of several response regulators by controlling expression of inhibitors of phosphatases.

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Figures

FIG. 1

FIG. 1

The family of rap phosphatases and phr peptide regulators. Putative transcriptional start sites, as determined by mapping 5′ ends by primer extension analysis, are indicated by arrows. To analyze expression, DNA fragments (denoted by solid lines underneath the genes) were cloned upstream of lacZ and integrated into the chromosome at amyE. The fragments were amplified by PCR with Vent polymerase and primers with restriction sites (_Eco_RI and _Bam_HI) at the ends. These fragments were subcloned between the _Eco_RI and _Bam_HI sites of pKS2 (23). The resulting plasmids were linearized and transformed into wild-type B. subtilis, selecting for Neor transformants. Transformants were screened for an amylase-deficient phenotype to confirm the plasmid had integrated into the chromosome at the amyE locus. For each phr gene, the fragment end points are indicated relative to the translation start site: phrA, 530 bp upstream, 30 bp downstream; phrE, 275 bp upstream, 73 bp downstream; phrF, 749 bp upstream, 62 bp downstream; phrG, 745 bp upstream, 30 bp downstream; phrI, 465 bp upstream, 37 bp downstream; phrK, 583 bp upstream, 22 bp downstream.

FIG. 2

FIG. 2

Expression of the phr-lacZ transcriptional fusions. β-Galactosidase specific activity was assayed as described previously (13, 24). The time indicated is relative to the transition to stationary phase (_T_0). (A) phrE-lacZ. (B) phrF-lacZ. (C) phrG-lacZ. (D) phrI-lacZ. (E) phrK-lacZ. Solid circles, wild type; open triangles, sigH; solid triangles, spo0A; solid squares, abrB; open squares, spo0A abrB. Strains are indicated in Table 1.

FIG. 3

FIG. 3

Primer extension mapping of the 5′ ends of phr mRNA and sequences of the promoter regions. (A to F) RNA for primer extension was prepared by the Qiagen RNeasy protocol. Approximately 50 μg of RNA was used per primer extension reaction. Each primer extension reaction is shown alongside a sequencing ladder (lanes labeled GATC) produced with the same end-labeled primer. RNA was prepared from wild-type (wt) cells (IRN238 or RSM128) and a sigH mutant (lane H [IRN243 or RSM135]). (A) phrE, primer phrE4 (5′-CCAATTAAAACGGCGGATAAACTGA-3′). (B) phrF, primer phrF5 (5′-GAGCCAGACAAGAGAGTAATAGTTTAGA-3′). (C) phrG, primer RM36 (5′-CTTACACTGTCATACTCTTTCTCGGAC-3′). (D) _phrI_P1, primer phrIPEU (5′-CGTCTTGCTTCAATACTCATACG-3′). (E) _phrI_P2, primer phrIPE (5′-CTATGCCCCTACCCGATCTGCAGC-3′). (F) phrK, primer RM38 (5′-GCTCGTTGCTTCTTCAAAAGCACC-3′). (G) Sigma-H promoters identified by primer extension analysis. Promoters were aligned by the −10 region with spaces added to align the −35 regions. Residues with identity to the sigma-H consensus sequence are indicated in boldface. The transcription start sites (+1) as determined by primer extension are indicated by boldface, lowercase letters. The sigma-A consensus site in _phrE_P1–2 is underlined.

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