Mutation in yaaT leads to significant inhibition of phosphorelay during sporulation in Bacillus subtilis

Abstract

In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of sigma(H), which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

FIG. 1.

Genetic organization of the yaaT region. The location of the yaaT44 mutation is indicated. aa, amino acids.

FIG. 2.

Effect of yaaT mutation on sporulation. (A) Fluorescence microscopy of wild-type cells and YAAT44 cells stained with FM4-64 6 h after inoculation into DSM. (B) Quantification of morphological stages in the cell population of the yaaT mutant in DSM at T₂₄. Open bars, wild type; solid bars, YAAT44. veg, vegetative cells and stage 0 cells (no asymmetric septa); II, stage II cells with asymmetric septa; III, stage III cells with spore protoplasts (forespores) within the rod-shaped mother cells; IVD and IVB, stage IV cells with forespores becoming phase dark and progressively phase bright, respectively; VIID and VIIB, spore bodies becoming phase dark and phase bright, respectively, with no surrounding rod-shaped mother cells. Altogether, 455 wild-type cells and 406 YAAT44 mutant cells were counted.

FIG. 3.

Expression of the various early sporulation genes in the yaaT mutant. Various strains carrying the lacZ fusions were induced to sporulate, and the β-galactosidase activities were assayed. (A) spoVG-lacZ expression. Symbols: ⋄, VGZ (wild type); ▪, T44VGZ (yaaT). (B) spoIIE-lacZ expression. Symbols: ⋄, IIEZ (wild type); ▪, T44IIEZ (yaaT). (C) spo0A-lacZ expression. Symbols: ⋄, 0AZ (wild type); ▪, T440AZ (yaaT).

FIG. 4.

Western blot analysis of Spo0A protein in the yaaT mutant. The cells were induced to sporulate and collected at different times. Western blots of whole-cell extracts were detected with an antibody that recognizes Spo0A.

FIG. 5.

Expression of spoIIE-lacZ fusion in the yaaT spo0E double mutant. Various strains carrying the lacZ fusions were induced to sporulate, and the β-galactosidase activities were assayed. Symbols: ⋄, IIEZ (wild type); □, 0ESIIEZ (spo0E); ♦, T44IIEZ (yaaT); ▪, T440ESIIEZ (yaaT spo0E).

FIG.6.

Localization of YaaT-GFP fusion protein. (A) Typical phase-contrast (panels a, d, g, j, and m), membrane-stained (FM4-64) (panels b, e, h, k, and n), and GFP fluorescence (panels c, f, i, l, and o) micrographs. Strains carrying YaaT-GFP were observed in the vegetative stage (panels a, b, and c) and at T₀ (panels d, e, and f), T_1.5 (panels g, h, and i), T₃ (panels j, k, and l), and T_4.5 (panels m, n, and o). (B) Western blot analysis of fractionated YaaT-GFP, SpoIIIJ-GFP, and GFP. YAATGFP(φEDTA), JGFP, and TtcGFP cells were grown in DSM at 37°C and collected at different times. Each cell extract was fractionated into soluble and insoluble fractions and examined with an antibody that recognizes GFP. veg, vegetative cells; S, soluble fraction, I, insoluble fraction.

FIG. 7.

Comparison of the amino acid sequences of the YaaT homologues from B. subtilis (YaaT Bsu), Bacillus halodurans (BH0045 Bha), Listeria innocua (lin0206 Lin), Clostridium perfringens (CPE2448 Cpe), Staphylococcus aureus (SA0443 Sau), Thermotoga maritima (TM0772 Tma), Lactococcus lactis (YeaC Lla), Aquifex aeolicus (aq_1527 Aae), Deinococcus radiodurans (DR2511 Dra), and Treponema pallidum (TP0046 Tpa).