gerT, a newly discovered germination gene under the control of the sporulation transcription factor sigmaK in Bacillus subtilis

Abstract

We report the identification of a gene, herein designated gerT (formerly yozR), that is involved in germination by spores of Bacillus subtilis. The gerT gene is induced late in sporulation under the positive control of the transcription factor sigma(K) and under the negative control of the DNA-binding protein GerE. The gerT gene product (GerT) is a component of the spore coat, and its incorporation into the coat takes place in two stages. GerT initially assembles into foci, which then spread around the developing spore in a process that is dependent on the morphogenetic protein CotE. Mutant spores lacking GerT respond poorly to multiple germinants and are impaired at an early stage of germination.

FIG. 1.

The gerT chromosomal region. (A) gerT is located in a convergent orientation with yojB and overlaps the hypothetical open reading frame yojC. Regions deleted in Δ1, Δ2, and Δ3 and the germination phenotypes of these deletion mutants are depicted. The black bar indicates the region used to complement gerT at the amyE locus. (B) The gerT coding gene and promoter regions are depicted. The putative −10 and −35 regions are boxed, and the consensus σ^K binding sequences and conserved spacing are shown above. Highly conserved residues are shown in large, bold letters. Perfect matches to the ideal ribosome binding site (RBS) are underlined. The boxed residues ATG and TAA are the projected start and stop codons for gerT. (C) The hypothetical yojC coding region is shown. Dotted underlining represents regions of overlap with the gerT and yojB coding sequences. Annotated start and stop codons are boxed.

FIG. 2.

gerT is turned on late during sporulation under the control of σ^K and is repressed by GerE. Accumulation of β-galactosidase was measured in strains harboring lacZ fused to the first six codons of gerT and upstream regulatory regions. Samples were collected at the indicated times after suspension of growing cultures in Sterlini-Mandelstam medium. Expression of gerT-lacZ was monitored in the wild type (gray boxes; strain CF174) and in spoIVCB (part of the coding gene for σ^K; black boxes; strain CF183) and gerE (white boxes; strain CF184) mutant cells.

FIG. 3.

GerT is required for efficient germination. (A) Purified spores of the wild type (white boxes; strain PY79), from a gerT mutant (containing gerTΔ3) (gray boxes; strain CF164), and from a strain with a mutated gerT gene and containing a copy of wild-type gerT at the amyE locus (black boxes; strain CF167) were germinated with either l-alanine (left panel) or AGFK (right panel). Optical density at 600 nm (OD₆₀₀) was measured as it decreased over time. (B) Wild-type (triangles; strain AHB83) and gerT mutant (squares; strain CF205) strains were induced to express the genes of the Photorhabdus luminescens luciferase operon during sporulation, and spores were purified. l-Alanine (black) or water (white) was then added to spore suspensions, and luciferase activity was monitored over time using a luminescence counter.

FIG. 4.

gerT mutant spores are impaired in release of DPA during germination and in responding to DPA as a germinant. (A) Purified spores of the wild type (black; strain PS832), a gerT mutant (gray; strain CF280), and a gerT mutant harboring a copy of gerT at the amyE locus (white; strain CF292) were germinated with l-alanine. Samples were collected by centrifugation at the indicated times and assayed for DPA. (B) Wild-type (squares; strain PS832), gerA mutant (diamonds, left-hand panel; strain CF224), and gerT mutant (triangles, right-hand panel; strain CF280) spores were germinated in l-alanine (white) or Ca²⁺-DPA (black). Samples were collected at the indicated times and then tested for heat resistance.

FIG. 5.

GerT-GFP localizes to the coat in a two-step process. Wild-type (A to F; strain CF172) and cotE mutant cells (G to L; strain CF177) harboring gerT-gfp were grown in Difco sporulation medium. The cells were visualized by phase-contrast microscopy (PC), and localization of GerT-GFP was visualized by fluorescence microscopy at the indicated times after the start of sporulation. White arrowheads in panels I and L indicate spores that have been released from the mother cells.