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During sporulation in Bacillus subtilis, a group of mother cell-specific proteins guides the assembly of the coat, a multiprotein structure that protects the spore and influences many of its environmental interactions. SafA and CotE behave as party hubs, governing assembly of the inner and outer coat layers. Targeting of coat proteins to the developing spore is followed by encasement. Encasement by SafA and CotE requires E, a region of 11 amino acids in the encasement protein SpoVID, with which CotE interacts directly. Here, we identified two single alanine substitutions in E that prevent binding of SafA, but not of CotE, to SpoVID, and block encasement. The substitutions result in the accumulation of SafA, CotE and their dependent proteins at the mother cell proximal spore pole, phenocopying a spoVID null mutant and suggesting that mislocalized SafA acts as an attractor for the rest of the coat. The requirement for E in SafA binding is bypassed by a peptide with the sequence of E provided in trans. We suggest that E allows binding of SafA to a second region in SpoVID, enabling CotE to interact with E and SpoVID to function as a non-competitive hub during spore encasement.

The coat is the outermost layer of spores of many Bacillus species, and plays a key role in these spores' resistance. The Bacillus subtilis spore coat contains > 70 proteins in four distinct layers: the basement layer, inner coat, outer coat and crust. In this issue of Molecular Microbiology, McKenney and Eichenberger study the dynamics of spore coat assembly using GFP-fusions to 41 B. subtilis coat proteins. A key finding in the work is that formation of the spore coat is initiated by the apparently simultaneous assembly of foci of proteins from all four coat layers on the developing spore just as forespore engulfment by the mother cell begins. The expansion of these foci before completion of forespore engulfment then sets up the scaffold to which coat proteins added later in sporulation are added. This study provides new understanding of the mechanism of the assembly of a multi-protein, multi-lamellar structure.

Bacterial spores such as Bacillus atrophaeus are one of the most resistant life forms known and are extremely resistant to chemical and environmental factors in the dormant state. During germination, as bacterial spores progress towards the vegetative state, they become susceptible to anti-sporal agents. B. atrophaeus spores were exposed to the non-nutritive germinant dodecylamine (DDA), a cationic surfactant that can also be used as a killing agent, for up to 60 min, or to the nutrient germinant l-alanine. In kinetic studies, 99% of the spores were killed within 5 min of exposure to DDA. Atomic force microscopy (AFM) can be used as a sensitive tool to assess how the structure of the spore coat changes upon exposure to germinants or killing agents. Changes in cell height and roughness over time of exposure to DDA were examined using AFM. DDA caused the spore height to decrease by >50%, which may have been due to a partial breakdown of the spore coat. Treatment of B. atrophaeus with the nutrient germinant resulted in a decrease in height of spores after 2 h of incubation, from 0.7 ± 0.1 µm to 0.3 ± 0.2 µm. However, treatment with l-alanine did not change the surface roughness of the spores, indicating that the changes that occur during germination take place underneath the spore coat. We propose that exposure to DDA at high concentrations causes pores to form in the coat layer, killing B. atrophaeus without the need to fully germinate spores. Published 2009 by John Wiley & Sons, Ltd.

Summary Spore formation in Bacillus subtilis involves the formation of a thick, proteinaceous shell or coat that is assembled around a specialized membrane known as the outer forespore membrane. Here we present evidence that the assembling coat is tethered to the outer forespore membrane by a 26-amino-acid peptide called SpoVM, which is believed to form an amphipathic helix. We show that proper localization of SpoVM is dependent on SpoIVA, a morphogenetic protein that forms the basement layer of the spore coat, and conversely, that proper localization of SpoIVA is dependent on SpoVM. Genetic, biochemical and cytological evidence indicates that this mutual dependence is mediated in part by contact between an amino acid side-chain located near the extreme C-terminus of SpoIVA and an amino acid side-chain on the hydrophilic face of the SpoVM helix. Evidence is also presented that SpoVM adheres to the outer forespore membrane via hydrophobic, amino acid side-chains on the hydrophobic face of the helix. The results suggest that the SpoVM helix is oriented parallel to the membrane with the hydrophobic face buried in the lipid bilayer.

Bacterial spores are surrounded by a multilayered proteinaceous shell called the coat. In Bacillus subtilis, a coat protein called CotE guides the assembly of a major subset of coat proteins. To understand how CotE carries out its role in coat morphogenesis, we subjected its gene to mutagenesis and studied the effects of altered versions of CotE on coat formation. We identified regions within the C-terminal 28 amino acids that direct the deposition of the coat proteins CotA, CotB, CotG, CotSA, CotS and 35 kDa and 49 kDa proteins likely to be the spore proteins CotR (formerly known as YvdO) and YaaH respectively. The timing and genetic dependency of CotR accumulation are consistent with control of its gene by sigmaK and GerE. In addition, we identified a 35-amino-acid internal region involved in targeting of CotE to the forespore. Finally, we found that sequences within this 35-amino-acid region as well as within an 18-amino-acid stretch in the N-terminus of CotE direct the formation of CotE multimers, most probably homooligomers. These results suggest that: (i) most interactions between CotE and the coat proteins assembled under CotE control take place at the CotE C-terminus; (ii) an internal region of CotE connects it with the forespore surface; and (iii) interactions between CotE molecules depend on residues within an 18-amino-acid region in the N-terminal half of CotE.

The effect of three rapid decompression methods to clear the mechanism of inactivation of heat tolerant spores of Bacillus stearothermophilus IFO 12550 was investigated. Pressurization of the spores at 200 MPa and 75°C for 60 min caused a kill of 104 CFU/mL by the link-motion system but the nonrotational rod valve and E.G. seal methods gave a kill of about 103 CFU/mL. Sterilization was due to the physical breakdown of spore coat, and was induced by its physical permeability of water at high pressure and temperature. Rapid decompression by the link-motion system at 200 MPa decreased the D-value of the spores from 3000 min (100°C, one atmosphere) to 6 min, 11 min, and 17 min at 95, 85, and 75°C, respectively.

Galactosamine-6-phosphate was identified as a component of the coat of the Bacillus megaterium QM B1551 spore. It was one of the main constituents of the outermost layer of the spore coat, but it was absent from the other integuments including the cortex. These findings suggest that galactosamine-6-phosphate comprises the phosphorus-containing skeleton structure of the spore coat.

SUMMARY A number of factors of possible significance in elucidating the mechanism of inactivation of spores exposed to the alkylating agent, ethylene oxide (ETO), were investigated in this study. The sterilant system consisted of a mixture of ethylene oxide (12%) end dichlorodifluoro-methane (88%). Spores were exposed to ETO end suspended in distilled water overnight to determine if the ETO treatment would effect release of protein, RNA, DNA, or DPA. Only DPA release was effected, it being appreciably greeter from exposed than unexposed spores. Pretreatment of spores with agents postulated to effect the intactness of the spore coat was noted to alter ETO resistance characteristics. The resulting survivor curve exhibited en unusually long death lag period followed by an increased first-order death rate. Such an alteration, however, could be counteracted by increasing the time between pretreatment end ETO exposure. A comparison of lyophilized vegetative cells, germinated spores, and heat-activated spores revealed that the ETO resistance of germinated spores was closer to that of heat-activated spores than to that of vegetative cells. To obtain a logarithmic death rate for heat-activated spores, it was necessary to precondition at about 98% R.H. prior to the normal preconditioning et 33% R.H. Vegetative cells end germinated spores, however, did not require preconditioning other then et 33% R.H. The effect of increasing time of ETO exposure on germination end postgerminative end vegetative development of spores in a rich medium end in a deficient medium was evaluated. No significant change in rate or efficiency of germination was observed for either medium. However, the postgerminative phase of development of treated spores was notably lengthened over that of untreated spores in either medium. Treated spores which were incubated in the rich medium entered the vegetative phase of growth before those incubated in the deficient medium, this difference becoming more pronounced with increasing periods of exposure.