Mutational analysis of the signal-sensing domain of ResE histidine kinase from Bacillus subtilis

Abstract

The Bacillus subtilis ResD-ResE two-component regulatory system activates genes involved in nitrate respiration in response to oxygen limitation or nitric oxide (NO). The sensor kinase ResE activates the response regulator ResD through phosphorylation, which then binds to the regulatory region of genes involved in anaerobiosis to activate their transcription. ResE is composed of an N-terminal signal input domain and a C-terminal catalytic domain. The N-terminal domain contains two transmembrane subdomains and a large extracytoplasmic loop. It also has a cytoplasmic PAS subdomain between the HAMP linker and C-terminal kinase domain. In an attempt to identify the signal-sensing subdomain of ResE, a series of deletions and amino acid substitutions were generated in the N-terminal domain. The results indicated that cytoplasmic ResE lacking the transmembrane segments and the extracytoplasmic loop retains the ability to sense oxygen limitation and NO, which leads to transcriptional activation of ResDE-dependent genes. This activity was eliminated by the deletion of the PAS subdomain, demonstrating that the PAS subdomain participates in signal reception. The study also raised the possibility that the extracytoplasmic region may serve as a second signal-sensing subdomain. This suggests that the extracytoplasmic region could contribute to amplification of ResE activity leading to the robust activation of genes required for anaerobic metabolism in B. subtilis.

FIG. 1.

Schematic diagram of the putative subdomain organization of ResE and of mutant ResE constructs. The pDG148-derived plasmids carrying resE are listed. All resE expression systems utilize the IPTG-inducible Pspac promoter and the resE SD sequence (E) or the vector-derived SD sequence (V). Numbers in parentheses represent start and end points of amino acid deletions. Solid lines show regions cloned into plasmid pDG148, and dotted lines show deleted regions. The sites and identities of amino acid substitutions in the HAMP subdomain are indicated (X). The top panel shows the conserved histidine-374 (H) and ATPase subdomains in the kinase domain and the two transmembrane subdomains (TM1 and TM2), HAMP, and PAS subdomains in the signal input domain.

FIG. 2.

Production of the wild-type and mutant ResE proteins. (A) resE mutant cells carrying various resE-carrying plasmids shown in Fig. 1 were grown aerobically in 2× YT in the absence (−) or presence (+) of 1 mM IPTG. Equal amounts (6 μg) of total protein from each whole-cell lysate were resolved by 12% SDS-polyacrylamide gel electrophoresis and probed by using anti-ResE antibody. Lane 1, cells carrying pMMN563; lane 2, pMMN525; lane 3, pMMN564; lane 4, pMMN565; lane 5, pAB3; lane 6, pAB4; lane 7, pMMN534; lane 8, pMMN536; lane 9, pMMN537; lane 10, pMMN544; and lane 11, pMMN559. (B) Whole-cell lysate was prepared from aerobic (+) and anaerobic (−) cultures of wild-type JH642 (lane 1) or resE mutants carrying the plasmids pMMN525 (lane 2) and pMMN563 (lane 3). Western analysis was done as described for panel A. (C to E) Whole-cell lysate from IPTG-induced cultures was further separated into cytoplasmic (c) and membrane (m) fractions. Equal protein samples (5 μg from the cytoplasmic fraction and 2.5 μg from the membrane fraction) were loaded onto a 12% SDS-polyacrylamide gel. Western analysis was done by using anti-ResE antibody, anti-Pgm antibody, and anti-QoxA antibody. Pgm and QoxA localize to the cytoplasm and membrane, respectively. Lanes 1, cells carrying pMMN565; lanes 2, pMMN525; lanes 3, pMMN534; and lanes 4, pMMN563. Sizes of molecular mass markers (M) are 108.0, 90.0, 50.7, 35.5, 28.6, and 21.2 kDa.

FIG. 3.

Expression of nasD-lacZ and hmp-lacZ in cells grown under aerobic and anaerobic conditions. resE strains carrying the plasmid pMMN525 (full-length resE with resE SD sequence) (A and D), pMMN563 (full-length resE with vector SD sequence) (B and E), and pMMN565 (cytoplasmic resE with vector SD sequence) (C and F) were grown in 2× YT supplemented with 1% glucose and 0.2% potassium nitrate and in the absence or presence of IPTG. β-Galactosidase activities (β-gal. act.) of nasD-lacZ (A to C) and hmp-lacZ (D to F) were measured at time intervals. ○, aerobic growth in the absence of IPTG; •, aerobic growth in the presence of 1 mM IPTG; ▵, anaerobic growth in the absence of IPTG: ▴, anaerobic growth in the presence of 1 mM IPTG. Time zero indicates the end of the exponential growth.

FIG. 4.

Effect of IPTG concentration on nasD-lacZ expression. resE strains carrying the plasmid pMMN525 (full-length resE with resE SD sequence) (A), pMMN563 (full-length resE with vector SD sequence) (B), pMMN534 (TM2-carrying resE with vector SD sequence) (C), and pMMN565 (cytoplasmic resE with vector SD sequence) (D) were grown as described in the legend of Fig. 3 in the absence of IPTG (○) or in 0.02 mM IPTG (•), 0.05 mM IPTG (▵), 0.2 mM IPTG (▴), or 1 mM IPTG (□). Time zero indicates the end of exponential growth. β-Galactosidase activity (β-gal. act.) is shown in Miller units.

FIG. 5.

Induction of hmp-lacZ with NO. narGH resE strains carrying the plasmid pMMN525 (full-length resE with resE SD) (column 1), pMMN563 (full-length resE with vector SD sequence) (columns 2 and 3), pMMN534 (TM2-carrying resE with vector SD sequence) (column 4), pMMN565 (cytoplasmic resE with vector SD sequence) (column 5), pMMN564 (cytoplasmic resE lacking PAS with vector SD sequence) (column 6), and without plasmid (column 7) were grown anaerobically in 2× YT supplemented with 0.5% glucose, 0.5% pyruvate, and 1 mM IPTG (0.02 mM IPTG for the culture shown in column 2). At the mid-log phase of growth, cells were incubated for 30 min without (open columns) or with (filled columns) 10 μM NO. β-Galactosidase activity (β-gal. act.) is shown in Miller units. The data are the averages of three to five experiments with standard deviations.