Spore's Inner Membrane Research Articles

New insights into peroxide toxicology: sporulenes help Bacillus subtilis endospores from hydrogen peroxide.

The purpose of the present study was to understand the possible events involved in the toxicity of H2O2 to wild and sporulene-deficient spores of Bacillus subtilis, as H2O2 was previously shown to have deleterious effects. The investigation utilized two strains of B. subtilis, namely the wild-type PY79 (WT) and the sporulene-deficient TB10 (ΔsqhC mutant). Following treatment with 0.05% H2O2 (v/v), spore viability was assessed using a plate count assay, which revealed a significant decrease in cultivability of 80% for the ΔsqhC mutant spores. Possible reasons for the loss of spore viability were investigated with microscopic analysis, dipicholinic acid (DPA) quantification, and propidium iodide (PI) staining. Microscopic examinations revealed the presence of withered and deflated morphologies in spores of ΔsqhC mutants treated with H2O2, indicating a compromised membrane permeability. This was further substantiated by the absence of DPA and a high frequency (50-75%) of PI infiltration. The results of FAME analysis and protein profiling indicated that the potentiation of H2O2-induced cellular responses was manifested in the form of altered spore composition in ΔsqhC B. subtilis. The slowed growth rates of the ΔsqhC mutant and the heightened sporulene biosynthesis pathways in the wild-type strain, both upon exposure to H2O2, suggested a protective function for sporulenes in vegetative cells. Sporulenes serve as a protective layer for the inner membrane of spores, thus assuming a significant role in mitigating the adverse effects of H2O2 in WT B. subtilis. The toxic effects of H2O2 were even more pronounced in the spores of the ΔsqhC mutant, which lacks this protective barrier of sporulenes.

Identification and characterization of new proteins crucial for bacterial spore resistance and germination.

2Duf, named after the presence of a transmembrane (TM) Duf421 domain and a small Duf1657 domain in its sequence, is likely located in the inner membrane (IM) of spores in some Bacillus species carrying a transposon with an operon termed spoVA 2mob. These spores are known for their extreme resistance to wet heat, and 2Duf is believed to be the primary contributor to this trait. In this study, we found that the absence of YetF or YdfS, both Duf421 domain-containing proteins and found only in wild-type (wt) B. subtilis spores with YetF more abundant, leads to decreased resistance to wet heat and agents that can damage spore core components. The IM phospholipid compositions and core water and calcium-dipicolinic acid levels of YetF-deficient spores are similar to those of wt spores, but the deficiency could be restored by ectopic insertion of yetF, and overexpression of YetF increased wt spore resistance to wet heat. In addition, yetF and ydfS spores have decreased germination rates as individuals and populations with germinant receptor-dependent germinants and increased sensitivity to wet heat during germination, potentially due to damage to IM proteins. These data are consistent with a model in which YetF, YdfS and their homologs modify IM structure to reduce IM permeability and stabilize IM proteins against wet heat damage. Multiple yetF homologs are also present in other spore forming Bacilli and Clostridia, and even some asporogenous Firmicutes, but fewer in asporogenous species. The crystal structure of a YetF tetramer lacking the TM helices has been reported and features two distinct globular subdomains in each monomer. Sequence alignment and structure prediction suggest this fold is likely shared by other Duf421-containing proteins, including 2Duf. We have also identified naturally occurring 2duf homologs in some Bacilli and Clostridia species and in wt Bacillus cereus spores, but not in wt B. subtilis. Notably, the genomic organization around the 2duf gene in most of these species is similar to that in spoVA 2mob, suggesting that one of these species was the source of the genes on this operon in the extremely wet heat resistant spore formers.

Organization and dynamics of the SpoVAEa protein and its surrounding inner membrane lipids, upon germination of Bacillus subtilis spores

The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca2+-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the IM complex formed by all GRs termed the germinosome. However, while the GRs’ spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. It seems possible that this movement may be a means of communicating germination signals from the germinosome to the IM SpoVA channel, thus stimulating CaDPA release in germination. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, and the loss of fluorescence of the IM with fluorescent dyes, as well as the appearance of peak SpoVAEa-SGFP2 fluorescent intensity occurred in parallel. These observed events were highly related to spores’ rapid phase darkening, which is considered as due to rapid Ca2+DPA release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40–80 °C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80 °C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.

Resistance properties and the role of the inner membrane and coat of Bacillus subtilis spores with extreme wet heat resistance.

A protein termed 2Duf greatly increases wet heat resistance of Bacillus subtilis spores. The current work examines the effects of 2Duf on spore resistance to other sporicides, including chemicals that act on or must cross spores' inner membrane (IM), where 2Duf is likely present. The overall aim was to gain a deeper understanding of how 2Duf affects spore resistance, and of spore resistance itself. 2Duf's presence increased spore resistance to chemicals that damage or must cross the IM to kill spores. Spore coat removal decreased 2Duf-spore resistance to chemicals and wet heat, and 2Duf-spores made at higher temperatures were more resistant to wet heat and chemicals. 2Duf-less spores lacking coats and Ca-dipicolinic acid were also extremely sensitive to wet heat and chemicals that transit the IM to kill spores. The new work plus previous results lead to a number of important conclusions as follows. (1) 2Duf may influence spore resistance by decreasing the permeability of and lipid mobility in spores' IM. (2) Since wet heat-killed spores that germinate do not accumulate ATP, wet heat may inactivate some spore IM protein essential in ATP production which is stabilized in a more rigid IM. (3) Both Ca-dipicolinic acid and the spore coat play an important role in the permeability of the spore IM, and thus in many spore resistance properties. The work in this manuscript gives a new insight into mechanisms of spore resistance to chemicals and wet heat, to the understanding of spore wet heat killing, and the role of Ca-dipicolinic acid and the coat in spore resistance.

Inactivation mechanism of slightly acidic electrolyzed water on Bacillus cereus spores

This study aimed to investigate the inactivation mechanism of Bacillus cereus spores by slightly acidic electrolyzed water (SAEW). Spore inactivation efficacy of SAEW at different available chlorine concentrations (ACC, 20, 60 and 100 mg/L), as well as spore structures change, coat damage, mutagenesis, and inner membrane (IM) properties were examined. The viability of treated spores with lysozyme addition and spore germination induced by germinant was also examined. The results showed that SAEW could reach maximal 5.81 CFU/mL log reduction with ACC of 100 mg/L for 20 min treatment. Scanning and transmission electron photomicrographs indicated that SAEW treatment rendered spore surface ruptured, IM damage and core contents loss. No mutants were generated in survivors of SAEW treated-spores. SAEW significantly weakened spore viability in high salt medium, losing its ability to retain pyridine-2,6-dicarboxylic acid (DPA) at 85 °C. SAEW-treated spores germinated with l-alanine or inosine induction were mostly stained with propidium iodide (PI) but could not recover via lysozyme addition. Furthermore, SAEW treatment inhibited spore germination in the induction of germinant (mixture of l-alanine and inosine or dodecylamine). These findings indicated that SAEW inactivated spore primarily by damaging the spore IM.

The effect of high pressure combined with moderate temperature and peptidoglycan fragments on spore inactivation

High pressure processing (HPP) is a promising non-thermal processing method for food production. However, extremely high pressure and temperature are often required to achieve spores inactivation and commercial sterilization using HPP. In this study, the combined treatment of HPP, moderate temperature, and peptidoglycan fragments (PGF) for spore inactivation was investigated. The combined treatment of 200 MPa and 1 mg/mL PGF at 80 °C for 20 min resulted in 8.6 log inactivation of Bacillus subtilis 168 and more than 5 log reductions of Clostridium sporogenes PA3679 spores, respectively. A strong synergistic effect on spore inactivation among HPP, PGF, and temperature was observed. By comparing the effect of the treatment on the fluidity of the inner membrane and structural change of spores using fluorescence assay, a probable inactivation mechanism was proposed. It was concluded that the spores were firstly triggered to enter the Stage I of the germination process by HPP and PGF, and then immediately inactivated by the mild heat. This novel processing method could be an alternative to ensure commercial sterilization in the food industry.

Killing of bacterial spores by dodecylamine and its effects on spore inner membrane properties.

The objective of this study was to determine the effects of Ca-dipicolinic acid (CaDPA), cortex-lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. Bacillus subtilis spores, wild-type, CaDPA-less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine-treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild-type and CaDPA-less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores±CaDPA, with up to 99% killing with 1moll-1 dodecylamine in 4h at 45°C with spores at ~108 ml-1 . Dodecylamine killing of decoated wild type and CLE-less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA-less spores, with ≥99% killing in 5min. Propidium iodide stained intact spores±CaDPA minimally, decoated CaDPA-replete spores or dodecylamine-killed CLE-less spores peripherally, and cores of decoated CaDPA-less spores and dodecylamine-killed intact spores with CLEs. The IM of some decoated CaDPA-less spores was greatly reorganized. Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. This work gives new information on killing bacterial spores by dodecylamine, and how spore IM's relative impermeability is maintained.

High pressure CO2 reduces the wet heat resistance of Bacillus subtilis spores by perturbing the inner membrane

Spores of wild-type Bacillus subtilis PS533 were treated by wet heat at 75 °C for 30 min, and high pressure CO2 (HPCD) at 6.5 MPa and 30 °C or 75 °C for 30 min. The spores were analyzed for wet heat resistance (85 °C, 90 °C, 95 °C) and typical germination events including DPA release and cortex hydrolysis, inner membrane permeability, and germination triggered by nutrient (L-valine and AGFK) or non-nutrient (dodecylamine and high pressure at 150 MPa or 550 MPa) germinants. The results showed that (i) HPCD-treated spores exhibited reduced wet heat resistance compared to the untreated or wet heat-treated spores; (ii) HPCD-treated spores did not undergo typical germination events such as DPA release or cortex hydrolysis compared to normally germinated spores; (iii) HPCD-treated spores released more metal ions and exhibited decreased ability to maintain DPA, indicating that the permeability of inner membrane of HPCD-treated spores was increased; (iv) HPCD-treated spores exhibited reduced germination rate when triggered by L-valine or 150 MPa, but increased germination rate when triggered by dodecylamine or 550 MPa, suggesting that the fluidity of the inner membrane of HPCD-treated spores might be increased. These results indicated that HPCD could reduce the wet heat resistance of spores, and this resistance decrease was probably due to the modification of the inner membrane caused by HPCD. Industrial relevanceThe extremely high wet heat resistance of spores makes them a significant problem in the thermal processing of foods. Thus, it of great interest to develop a process to reduce the wet heat resistance of spores. In this work, we found that HPCD can significantly reduce the wet heat resistance of B. subtilis spores, and this was achieved by perturbing the inner membrane of spores. These results can improve our understanding of the inactivation mechanism of spores by HPCD, and also provide an alternative approach for spore inactivation in foods.

The Synergistic Effect of High Pressure CO2 and Nisin on Inactivation of Bacillus subtilis Spores in Aqueous Solutions.

The inactivation effects of high pressure CO2 + nisin (simultaneous treatment of HPCD and nisin, HPCD + nisin), HPCD→nisin (HPCD was followed by nisin), and nisin→HPCD (nisin was followed by HPCD) treatments on Bacillus subtilis spores in aqueous solutions were compared. The spores were treated by HPCD at 6.5 or 20 MPa, 84–86°C and 0–30 min, and the concentration of nisin was 0.02%. Treated spores were examined for the viability, the permeability of inner membrane (IM) using flow cytometry method and pyridine-2, 6-dicarboxylic acid (DPA) release, and structural damage by transmission electron microscopy. A synergistic effect of HPCD + nisin treatment on inactivation of the spores was found, and the inactivation efficiency of the spores was HPCD + nisin > HPCD→nisin or nisin→HPCD. Moreover, HPCD + nisin caused higher IM permeability and DPA release of the spores than HPCD. A possible action mode of nisin-enhanced inactivation of the spores was suggested as that HPCD firstly damaged the coat and cortex of spores, and nisin penetrated into and acted on the IM of spores, which increased the damage to the IM of spores, and resulted in higher inactivation of the spores.

Investigating the Inactivation Mechanism of Bacillus subtilis Spores by High Pressure CO2.

The objective of this study was to investigate the inactivation mechanism of Bacillus subtilis spores by high pressure CO2 (HPCD) processing. The spores of B. subtilis were subjected to heat at 0.1 MPa or HPCD at 6.5-20 MPa, and 64-86°C for 0-120 min. The germination, the permeability of inner membrane (IM) and cortex, the release of pyridine-2, 6-dicarboxylic acid (DPA), and changes in the morphological and internal structures of spores were investigated. The HPCD-treated spores did not lose heat resistance and their DPA release was lower than the inactivation, suggesting that spores did not germinate during HPCD. The flow cytometry analysis suggested that the permeability of the IM and cortex of HPCD-treated spores was increased. Furthermore, the DPA of the HPCD-treated spores were released in parallel with their inactivation and the fluorescence photomicrographs showed that these treated spores were stained by propidium iodide, ensuring that the permeability of IM of spores was increased by HPCD. The scanning electron microscopy photomicrographs showed that spores were crushed into debris or exhibited a hollowness on the surface, and the transmission electron microscopy photomicrographs exhibited an enlarged core, ruptured and indistinguishable IM and a loss of core materials in the HPCD-treated spores, indicating that HPCD damaged the structures of the spores. These findings suggested that HPCD inactivated B. subtilis spores by directly damaging the structure of the spores, rather than inducing germination of the spores.

Bacillus subtilis Spore Inner Membrane Proteome.

The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation, these spore formers can survive very harsh physical and chemical conditions. Yet, they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics-based membrane protein predictions, ca. one-third could be predicted to be membrane-localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were identified. In addition to previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology, unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets.

Structure-function analysis of the Bacillus megaterium GerUD spore germinant receptor protein.

Germination of Bacillus spores is triggered by the interaction of germinant molecules with specialized receptor proteins localized to the spore inner membrane. Germinant receptors (GRs) are comprised typically of three interacting protein subunits, each of which is essential for receptor function. At least some GRs appear to have a fourth component, referred to as a D-subunit protein. A number of D-subunit proteins were shown previously to be capable of modulating the activity of associated GRs. Here, we investigate the topology and structure–function relationships of the Bacillus megaterium QM B1551 GerUD protein, which is associated with the GerU GR. The presented data demonstrate that GerUD can be subjected to relatively extensive structural modifications while retaining function. Indeed, the presence of either of the two transmembrane spanning domains is sufficient to modulate an efficient GerU-mediated germinative response. The precise function of D-subunit proteins has yet to be established, although they may act as molecular chaperones within the spore inner-membrane environment.

Structural and Functional Analysis of the GerD Spore Germination Protein of Bacillus Species

Spore germination in Bacillus species represents an excellent model system with which to study the molecular mechanisms underlying the nutritional control of growth and development. Binding of specific chemical nutrients to their cognate receptors located in the spore inner membrane triggers the germination process that leads to a resumption of metabolism in spore outgrowth. Recent studies suggest that the inner membrane GerD lipoprotein plays a critical role in the receptor-mediated activation of downstream germination events. The 121-residue core polypeptide of GerD (GerD60-180) from Geobacillus stearothermophilus forms a stable α-helical trimer in aqueous solution. The 2.3-Å-resolution crystal structure of the trimer reveals a neatly twisted superhelical rope, with unusual supercoiling induced by parallel triple-helix interactions. The overall geometry comprises three interleaved hydrophobic screws of interacting helices linked by short turns that have not been seen before. Using complementation analysis in a series of Bacillus subtilis gerD mutants, we demonstrated that alterations in the GerD trimer structure have profound effects on nutrient germination. This important structure–function relationship of trimeric GerD is supported by our identification of a dominant negative gerD mutation in B. subtilis. These results and those of others lead us to propose that GerD mediates clustering of germination proteins in the inner membrane of dormant spores and thus promotes the rapid and cooperative germination response to nutrients.

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores' return to life within minutes in the process of germination. This process requires a number of spore-specific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores' IM through which the spore core's huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. (i) How do nutrient germinants penetrate through spores' outer layers to access GRs in the IM? (ii) What happens during the highly variable and often long lag period between the exposure of spores to nutrient germinants and the commitment of spores to germinate? (iii) What do GRs and GerD do, and how do these proteins interact? (iv) What is the structure of the SpoVA channel in spores' IM, and how is this channel gated? (v) What is the precise state of the spore IM, which has a number of novel properties even though its lipid composition is very similar to that of growing cells? (vi) How is CLE activity regulated such that these enzymes act only when germination has been initiated? (vii) And finally, how does the germination of spores of clostridia compare with that of spores of bacilli?

Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: A molecular rotor/FLIM study

We utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.

Topology and Accessibility of Germination Proteins in the Bacillus subtilis Spore Inner Membrane

Access to a membrane-impermeant biotinylation reagent as well as protease sensitivity was used to determine germination proteins' topology in the inner membrane (IM) of decoated dormant spores and intact germinated Bacillus subtilis spores. The proteins examined were four nutrient germinant receptor (GR) subunits, the GerD protein, essential for normal GR-dependent spore germination, the SpoVAD protein, essential for dipicolinic acid movement across the IM, the SleB cortex-lytic enzyme, and the YpeB protein, essential for SleB assembly in spores, as well as green fluorescent protein (GFP) in the spore core. GerD and SpoVAD as well as GFP in the spore were not biotinylated in decoated dormant spores. However, GR subunits, SleB, and YpeB were biotinylated 4 to 36% in decoated dormant spores, although these levels were not increased by higher biotinylation reagent concentrations or longer reaction times. In contrast, the germination proteins were largely biotinylated in germinated spores, although GFP was not. All of the germination proteins in the germinated spore's IM, but not spore core GFP, were largely sensitive to an exogenous protease. These results, coupled with predicted or experimentally determined structural data, indicate that (i) these germination proteins are at least partially and in some cases completely on the outer surface of the spore's IM and (ii) there is significant reorganization of these germination proteins' structure or environment in the IM during spore germination.

Localization of the GerD spore germination protein in the Bacillus subtilis spore

The GerD protein of Bacillus subtilis is required for efficient spore germination in l-alanine, and for germination in the alternative germinant combination of amino acids plus sugars. Only germination via nutrient receptors is affected in the mutant. The GerD protein is predicted to be a lipoprotein that is produced in the forespore compartment of the sporulating cell. Using antibody raised against the GerD protein, Western blots of proteins from spore fractions revealed that, as might be expected, the protein was detected in the inner membrane of spores, but it was also present at a high level in spore integuments (comprising coat, cortex and germ cell wall layers), and to some extent in the soluble fraction. It is likely that the GerD protein in the outer layers of dormant spores is located in the germ cell wall, as it was detected in coat-defective spores, and in the cell wall fraction of cells that were outgrowing from spores. Which of the multiple locations of GerD is important for its function is not known, but the inner membrane association would be appropriate for any interaction with germinant receptor proteins or SleB cortex lytic enzyme. Substitution of alanine for cysteine in the conserved cleavage site of the predicted prelipoprotein signal sequence of GerD resulted in mutant spores that lacked the GerD protein entirely.

Comparison of the properties of Bacillus subtilis spores made in liquid or on agar plates

To compare the properties of the spores of Bacillus subtilis prepared in liquid and on plates. The spores of B. subtilis were prepared at 37 degrees C using a nutrient exhaustion medium either in liquid or on agar plates. The levels of core water, dipicolinic acid (DPA) and small, acid-soluble spore proteins (SASP) were essentially identical in spores made in liquid or on plates. Spores prepared in liquid were killed approximately threefold more rapidly at 90 degrees C in water than the spores prepared on plates, and the spores prepared in liquid were more sensitive to nitrous acid and a diluted stable superoxidized water. Spores prepared in liquid also germinated more rapidly with several agents than those prepared on plates. Pellets of spores prepared on plates were darker than spores prepared in liquid, and spores prepared in liquid had more readily extracted coat protein. However, there were no major differences in the relative levels of individual coat proteins or the cross-linking of the coat protein GerQ in the two types of spores, although the inner membrane of spores prepared on plates had a higher ratio of anteiso- to iso-fatty acids. The preparation in liquid yielded spores with some different properties than those made on agar plates. Spores made in liquid had lower resistance to heat and several chemicals, and germinated more readily with several agents. There were also differences in the composition of the inner membrane of spores prepared under these two conditions. However, there were no major differences in the levels of DPA, core water, SASP and individual coat proteins or the cross-linking of a coat protein in spores made in liquid and on plates. This work demonstrates that the preparation method can affect the resistance and germination properties of bacterial spores, even if an identical medium and temperature are used. Evidence was also obtained consistent with the role of the inner membrane in spore resistance and germination, and that some factor in addition to core water, DPA and SASP content plays a role in spore resistance to wet heat.

The Clostridium botulinum GerAB germination protein is located in the inner membrane of spores

Clostridium botulinum dormant spores germinate in presence of l-alanine via a specific receptor composed of GerAA, GerAB and GerAC proteins. In Bacillus subtilis spores, GerAA and GerAC proteins were located in the inner membrane of the spore. We studied the location of the GerAB protein in C. botulinum spore fractions by Western-blot analysis, using an antipeptidic antibody. The protein GerAB was in vitro translated and used to confirm the specificity of the antibodies. GerAB was not present in a coat and spore outer membrane fraction but was present in a fraction of decoated spores containing inner membrane. These results strongly suggest that the protein GerAB is located in the inner membrane of the spore.

Analysis of factors that influence the sensitivity of spores of Bacillus subtilis to DNA damaging chemicals

To elucidate factors influencing the sensitivity of Bacillus subtilis spores to DNA damaging chemicals. Wild-type spores of B. subtilis made at lower temperatures were more sensitive to the DNA damaging chemicals formaldehyde and nitrous acid than were spores made at higher temperatures, but this was not the case with the DNA alkylating agents ethylmethanesulphonate and methylmethanesulphonate. Spores lacking most DNA protective alpha/beta-type small, acid-soluble proteins (termed alpha-beta- spores) made at lower temperatures were also more sensitive to killing through DNA damage by hydrogen peroxide than were alpha-beta- spores made at higher temperatures. The spore coat, whose composition varies significantly with sporulation temperature, played only a minor role in spore resistance to these DNA damaging agents. Spores made at lower temperatures exhibited higher permeability to the methylamine and germinated more rapidly with the surfactant dodecylamine than did spores made at higher temperatures. Treatment of spores with the oxidizing agent cumene hydroperoxide sensitized the surviving spores to all these DNA damaging agents. The fatty acid composition of the inner membrane of spores made at different temperatures differed significantly, but levels of unsaturated fatty acids in the inner membrane did not influence spore resistance to DNA damaging agents or the sensitization to such agents by prior treatment with cumene hydroperoxide. The higher rates of methylamine permeation across the inner membrane of spores made at lower temperatures and the greater sensitivity of wild-type spores made at lower temperatures to formaldehyde and nitrous acid and of alpha-beta- spores made at lower temperatures to hydrogen peroxide, all agents that must pass through the spore's inner membrane to damage DNA in the spore core, suggest that the permeability of the inner membrane is a significant factor influencing spore sensitivity to these agents. The sensitization of spores to DNA damaging chemicals by pretreatment with an oxidizing agent, a treatment that increases the permeability of the spore's inner membrane, and the more rapid dodecylamine germination of spores made at lower temperatures are consistent with this suggestion. The results in this communication provide new insight into the factors that influence the resistance of spores of Bacillus species to chemicals that kill spores by damaging spore DNA.