CotG-Like Modular Proteins Are Common among Spore-Forming Bacilli

Abstract

CotG is an abundant protein initially identified as an outer component of the Bacillus subtilis spore coat. It has an unusual structure characterized by several repeats of positively charged amino acids that are probably the outcome of multiple rounds of gene elongation events in an ancestral minigene. CotG is not highly conserved, and its orthologues are present in only two Bacillus and two Geobacillus species. In B. subtilis, CotG is the target of extensive phosphorylation by a still unidentified enzyme and has a role in the assembly of some outer coat proteins. We report now that most spore-forming bacilli contain a protein not homologous to CotG of B. subtilis but sharing a central "modular" region defined by a pronounced positive charge and randomly coiled tandem repeats. Conservation of the structural features in most spore-forming bacilli suggests a relevant role for the CotG-like protein family in the structure and function of the bacterial endospore. To expand our knowledge on the role of CotG, we dissected the B. subtilis protein by constructing deletion mutants that express specific regions of the protein and observed that they have different roles in the assembly of other coat proteins and in spore germination.

Importance: CotG of B. subtilis is not highly conserved in the Bacillus genus; however, a CotG-like protein with a modular structure and chemical features similar to those of CotG is common in spore-forming bacilli, at least when CotH is also present. The conservation of CotG-like features when CotH is present suggests that the two proteins act together and may have a relevant role in the structure and function of the bacterial endospore. Dissection of the modular composition of CotG of B. subtilis by constructing mutants that express only some of the modules has allowed a first characterization of CotG modules and will be the basis for a more detailed functional analysis.

FIG 1

Model of the CotH-dependent protein interaction network. Shown is a working model of the interactions among the indicated proteins. CotH has a positive effect on the assembly of CotG, CotC, CotU, and CotS (arrows). CotG in turn controls CotB maturation from the immature 46-kDa CotB form to the mature 66-kDa CotB form. The gray line indicates the negative effect of CotG on CotC, CotU, and CotS assembly (17, 20, 30).

FIG 2

Repeats of CotG and CotG-like proteins. The tandem repeats of CotG of B. subtilis (18) and of CotG-like proteins found in the indicated species are shown. B. subtilis CotG modules are boxed. Positively charged amino acids are in red.

FIG 3

cotH transcription in B. cereus and B. licheniformis. (A) Schematic representation of the cotH-cotG locus. Arrows indicate the positions of the synthetic oligonucleotides used for RT-PCR. Dashed arrows indicate the direction of transcription. (B and C) Reverse transcription reactions were performed by using total RNA from sporulating cells of B. cereus (B) or B. licheniformis (C) as a template and were primed with oligonucleotide H1. Amplification reactions were performed by using cDNA as the template and oligonucleotide pair H1/H2 or H1/H3, as indicated. Negative controls (C−) and positive controls (C+) were RNA samples treated and not treated with DNase, respectively. Arrows indicate the amplification products of the expected size, and M indicates the molecular weight marker.

FIG 4

CotG versions. The wild-type protein (CotG) is represented as being composed of three domains: the N- and C-terminal regions of 34 and 35 amino acids, respectively, and a central domain of 126 amino acids organized into tandem repeats (18). CotGΔ lacks the central domain, and CotG-Cterm and CotG-Nterm contain only the C- and N-terminal domains, respectively. All constructs were integrated into the chromosomes of strains carrying either a null mutation in cotG or a double-null mutation in cotG and cotH (indicated in parentheses). The names of strains carrying the different cotG alleles in the two genetic backgrounds are shown.

FIG 5

Effects of CotG on CotB, CotC, and CotU assembly. Western blot analysis of coat proteins extracted from purified spores of the indicated strains was performed. Proteins were fractionated on 15% SDS-PAGE gels, electrotransferred onto a membrane, and incubated with anti-CotB (A) and anti-CotC (B) antibodies. The type of CotG allele expressed in each strain (CotG form) in the presence (+) or in the absence (−) of CotH is also indicated. wt, wild type.

FIG 6

Effects of CotG on CotS assembly. A cotS::gfp fusion was introduced into a wild-type strain (PY79) and into cotH strains expressing wild-type CotG (AZ607), CotGΔ (AZ612), CotG-Cterm (AZ614), and CotG-Nterm (AZ616). Representative fields using phase-contrast microscopy (PC) and fluorescence microscopy (green fluorescent protein [GFP]) are shown. The exposure time was 588 ms in all cases.

FIG 7

Effects of CotG on germination efficiency. Spores derived from strains expressing wild-type CotG (AZ607) (squares), CotGΔ (AZ612) (crosses), CotG-Cterm (AZ614) (circles), and CotG-Nterm (AZ616) (diamonds) in a cotH background were tested for germination efficiency and compared with those of a wild-type strain (PY79) (dashed line). Germination was induced by Asn-GFK and measured as the percent loss of the optical density at 600 nm. Error bars are based on the standard deviations of data from four independent experiments. OD600, optical density at 600 nm.