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Characterization of cotJ, a sigma E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores.

ABSTRACT: The outermost protective structure found in endospores of Bacillus subtilis is a thick protein shell known as the coat, which makes a key contribution to the resistance properties of the mature spore and also plays a role in its interaction with compounds able to trigger germination. The coat is organized as a lamellar inner layer and an electron-dense outer layer and has a complex polypeptide composition. Here we report the cloning and characterization of an operon, cotJ, located at about 62 degrees on the B. subtilis genetic map, whose inactivation results in the production of spores with an altered pattern of coat polypeptides. The cotJ operon was identified by screening a random library of lacZ transcriptional fusions for a conditional (inducer-dependent) Lac+ phenotype in cells of a strain in which the structural gene (spoIIGB) for the early-acting, mother-cell-specific transcriptional factor sigma E was placed under the control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible Pspac promoter. Sequence analysis of cloned DNA from the cotJ region complemented by genetic experiments revealed a tricistronic operon preceded by a strong sigma E-like promoter. Expression of an SP beta-borne cotJ-lacZ fusion commences at around h 2 of sporulation, as does expression of other sigma E-dependent genes, and shows an absolute requirement for sigma E. Studies with double-reporter strains bearing a cotJ-gusA fusion and lacZ fusions to other cot genes confirmed that expression of cotJ is initiated during sporulation prior to activation of genes known to encode coat structural proteins (with the sole exception of cotE). An in vitro-constructed insertion-deletion mutation in cotJ resulted in the formation of spores with no detectable morphological or resistance deficiency. However, examination of the profile of electrophoretically separated spore coat proteins from the null mutant revealed a pattern that was essentially identical to that of a wild-type strain in the range of 12 to 65 kDa, except for polypeptides of 17 and 24 kDa, the putative products of the second (cotJB) and third (cotJC) cistrons of the operon, that were missing or reduced in amount in the coat of the mutant. Polypeptides of the same apparent sizes are detected in spores of a cotE null mutant, on which basis we infer that the products of the cotJ operon are required for the normal formation of the inner layers of the coat or are themselves structural components of the coat. Because the onset of cotJ transcription is temporally coincident with the appearance of active sigma E, we speculate that the cotJ-encoded products may be involved in an early state of coat assembly.

SUBMITTER: Henriques AO

PROVIDER: S-EPMC177041 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Characterization of cotJ, a sigma E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores.

Henriques A O AO Beall B W BW Roland K K Moran C P CP

Journal of bacteriology 19950601 12

The outermost protective structure found in endospores of Bacillus subtilis is a thick protein shell known as the coat, which makes a key contribution to the resistance properties of the mature spore and also plays a role in its interaction with compounds able to trigger germination. The coat is organized as a lamellar inner layer and an electron-dense outer layer and has a complex polypeptide composition. Here we report the cloning and characterization of an operon, cotJ, located at about 62 de ...[more]

PMID: 7768848

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Project description:Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid Schaeffer's-glucose (SG) agar plates and 15N metabolically labeled spores prepared in shake flasks containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N:15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. Corroborating the proteomic analyses, electron microscopy analyses show a significantly thinner outer coat layer of the spores cultured on the solid agar medium.

Project description:Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.

Project description:Large-scale shotgun sequencing (RNA-seq) analysis of mRNAs in dormant Bacillus subtilis spores prepared on plates or in liquid generally found the same ∼46 abundant mRNA species, with >250 mRNAs detected at much lower abundances. Knowledge of the amount of phosphate in a single B. subtilis spore allowed calculation of the amount of mRNA in an individual spore as ∼106 nucleotides (nt). Given the levels of abundant spore mRNAs compared to those of other mRNAs, it was calculated that the great majority of low-abundance mRNAs are present in only small fractions of spores in populations. Almost all of the most abundant spore mRNAs are encoded by genes expressed late in sporulation in the developing spore under the control of the forespore-specific RNA polymerase sigma factor, σG, and most of the encoded proteins are in spores. Levels of the most abundant spore mRNAs were also relatively stable for a week at 4°C after spore harvest. RNA-seq analysis of mRNAs in highly purified and less-well-purified spores made in liquid, as well as from spores that were chemically decoated to remove possible contaminating mRNA, indicated that low-abundance mRNAs in spores were not contaminants in purified spore preparations, and several sources of low-abundance mRNAs in spores are suggested. The function of at least the great majority of spore mRNAs seems most likely to be the generation of ribonucleotides for new RNA synthesis by their degradation early in spore revival.IMPORTANCE Previous work indicates that dormant Bacillus subtilis spores have many hundreds of mRNAs, some of which are suggested to play roles in spores' "return to life" or revival. The present work finds only ∼46 mRNAs at ≥1 molecule spore, with others in only fractions of spores in populations, often very small fractions. Less-abundant spore mRNAs are not contaminants in spore preparations, but how spores accumulate them is not clear. Almost all abundant spore mRNAs are synthesized in the developing spore late in its development, most encode proteins in spores, and abundant mRNAs in spores are relatively stable at 4°C. These findings will have a major impact on thinking about the roles that spore mRNAs may play in spore revival.

Dataset Information

Characterization of cotJ, a sigma E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores.

Publications

Characterization of cotJ, a sigma E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores.

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