The default state of the membrane-localized histidine kinase PrrB of Rhodobacter sphaeroides 2.4.1 is in the kinase-positive mode

Abstract

The PrrBA two-component activation system of Rhodobacter sphaeroides plays a major role in the induction of photosynthesis gene expression under oxygen-limiting or anaerobic conditions. The PrrB histidine kinase is composed of two structurally identifiable regions, the conserved C-terminal kinase/phosphatase domain and the N-terminal membrane-spanning domain with six transmembrane helices framing three periplasmic and two cytoplasmic loops. Using a set of PrrB mutants with lesions in the transmembrane domain, we demonstrate that the central portion of the PrrB transmembrane domain including the second periplasmic loop plays an important role in both sensing and signal transduction. Signal transduction via the transmembrane domain is ultimately manifested by controlling the activity of the C-terminal kinase/phosphatase domain. The extent of signal transduction is determined by the ability of the transmembrane domain to sense the strength of the inhibitory signal received from the cbb(3) terminal oxidase (J.-I Oh, and S. Kaplan, EMBO J. 19:4237-4247, 2000). Therefore, the intrinsic ("default") state of PrrB is in the kinase-dominant mode. It is also demonstrated that the extent of prrB gene expression is subject to the negative autoregulation of the PrrBA system.

FIG. 1

Schematic diagram depicting the mutant sites in PrrB (A) and immunoblot analysis of PrrB from the set of PrrB mutants (B). Triangles, insertion sites of a string of five alanines. The membrane portion (gray) indicates the deleted region. Identical amounts of membrane fractions (70 μg of protein) were run on an sodium dodecyl sulfate–12.5% polyacrylamide gel, and PrrB was detected with the anti-His₄ antibody. C1 and C2, negative control PrrB1 with pRK415 and positive control PrrB1 with pPRRB4, respectively. Strains were grown anaerobically with 0.5% (vol/vol) DMSO as the terminal electron acceptor by continuous sparging with a mixture of 95% N₂–5% CO₂ to an optical density at 600 nm of 0.45 to 0.55. See Materials and Methods for additional details about preparation of the membrane fraction.

FIG. 2

Analysis of puf mRNA in R. sphaeroides strains grown anaerobically with DMSO by continuous sparging with a mixture of 95% N₂–5% CO₂ to an optical density at 600 nm of 0.45 to 0.55 or aerobically by sparging with 30% O₂–69% N₂–1% CO₂ to an OD₆₀₀ of 0.4 to 0.5. Approximately 20 μg of total RNA was loaded in each lane. The Northern blots were probed with a labeled 0.47-kb StyI fragment from pUI655 or a 0.5-kb HindIII-PstI fragment from pUCP6.37, which is specific for puf mRNA or processed 23S rRNA (14S), respectively (B). After background correction the values were normalized to the levels of processed 23S rRNA (14S). The normalized values are plotted above the Northern blots. The transcript level in the wild type (2.4.1) with pRK415 grown under anaerobic condition was set at 100 (A). PRRB1, the prrB-negative strain PrrB1. Electrophoresis and Northern blotting were performed at the same time using the same agarose gel and nylon membrane.

FIG. 3

Promoter activities of the prrB gene in R. sphaeroides strains. (A) Schematic diagram of prrB::lacZ transcriptional fusion plasmid pPRRBLAC. (B) Wild-type (2.4.1) and prrA-negative (PRRA2) strains containing pPRRBLAC were grown aerobically by sparging with 30% O₂–69% N₂–1% CO₂ to an optical density at 600 nm of 0.3 or anaerobically in the dark with 0.5% (vol/vol) DMSO. All values provided are the averages of two independent determinations.

FIG. 4

Multiple alignment of the N-terminal transmembrane domain of PrrB homologues. The highly conserved region in the central part is underlined, and the corresponding part is depicted in gray in the schematic model of PrrB. The identical or conservatively substituted residues are indicated by asterisks or colons, respectively. Abbreviations: R.c., R. capsulatus; R.d., Roseobacter denitrificans; PrrB-R.s., PrrB from R. sphaeroides; RegB-R.s., RegB from Rhodovulum sulfidophilum.

FIG. 5

Model for O₂ signaling through the cbb₃-PrrBA signal transduction pathway. The cbb₃ cytochrome c oxidase (cbb₃) and PrrC as well as PrrB are localized in the cytoplasmic membrane. PrrB is thought to exist as a homodimer.