Moderate high-pressure superdormancy in Bacillus spores: properties of superdormant spores and proteins potentially influencing moderate high-pressure germination

Resistant bacterial spores are a major concern in industrial decontamination processes. An approach known as pressure-mediated germination-inactivation strategy aims to artificially germinate spores by pressure to mitigate their resistance to inactivation processes. The successful implementation of such a germination-inactivation strategy relies on the germination of all spores. However, germination is heterogeneous, with some ‘superdormant’ spores germinating extremely slowly or not at all. The present study investigated potential underlying reasons for moderate high-pressure (150 MPa, 37°C) superdormancy of Bacillus subtilis spores. The water and dipicolinic acid content of superdormant spores was compared to that of the initial dormant spore population. The results suggest that water and dipicolinic acid content are not major drivers of moderate high-pressure superdormancy. Proteomic analysis was used to identify proteins that were quantified at significantly different levels in superdormant spores. Subsequent validation of the germination capacity of deletion mutants revealed that the presence of protein YhcN is required for efficient moderate high-pressure germination and that proteins MinC, cse60, and SspK may also play a role, albeit a minor one. Importance Spore-forming bacteria are ubiquitous in nature, and as a consequence, inevitably enter the food chain or other processing environments. Their presence can lead to significant spoilage or safety related issues. Intensive treatment is usually required to inactivate them; however, this harms important quality attributes. A pressure-mediated germination-inactivation approach can balance the need for effective spore inactivation and retention of sensitive ingredients. However, superdormant spores are the bottleneck preventing the successful and safe implementation of such a strategy. In-depth understanding of moderate high-pressure germination and the underlying causes of superdormancy is necessary to advance the development of mild high pressure-based spore control technologies. The approach used in this work allowed the identification of proteins that have not yet been associated to reduced germination at moderate high-pressure. This research paves the way for further studies on the germination and superdormancy mechanisms in spores, assisting the development of mild spore inactivation strategies.

Omadacycline compared to vancomycin when combined with germinants to disrupt the life cycle of Clostridioides difficile

Clostridioides difficile (C. difficile) infections (CDI) are commonly treated with antibiotics that do not impact the dormant spore form of the pathogen. CDI-directed antibiotics, such as vancomycin and metronidazole, can destroy the vegetative form of C. difficile and protective microbiota. After treatment, spores can germinate into vegetative cells causing clinical disease relapse and further spore shedding. This in vitro study compares the combination of germinants with vancomycin or omadacycline to antibiotics alone in eradicating C. difficile spores and vegetative cells. Among the four strains in this study, omadacycline minimum inhibitory concentrations (0.031-0.125 mg/L) were lower than vancomycin (1-4 mg/L). Omadacycline nor vancomycin in media alone reduced spore counts. In three of the four strains, including the epidemic ribotype 027, spore eradication with germinants was 94.8-97.4% with vancomycin and 99.4-99.8% with omadacycline (p<0.005). In ribotype 012, either antibiotic combined with germinants resulted in 100% spore eradication at 24 hours. The addition of germinants with either antibiotic did not result in significant toxin A or B production, which were below the limit of detection (<1.25 ng/mL) by 48 hours. Limiting the number of spores present in patient GI tracts at the end of therapy may be effective at preventing recurrent CDI and limiting spore shedding in the healthcare environment. These results with germinants warrant safety and efficacy evaluations in animal models.

Metabolic differentiation and intercellular nurturing underpin bacterial endospore formation

Despite intensive research, the role of metabolism in bacterial sporulation remains poorly understood. Here, we demonstrate that Bacillus subtilis sporulation entails a marked metabolic differentiation of the two cells comprising the sporangium: the forespore, which becomes the dormant spore, and the mother cell, which dies as sporulation completes. Our data provide evidence that metabolic precursor biosynthesis becomes restricted to the mother cell and that the forespore becomes reliant on mother cell–derived metabolites for protein synthesis. We further show that arginine is trafficked between the two cells and that proposed proteinaceous channels mediate small-molecule intercellular transport. Thus, sporulation entails the profound metabolic reprogramming of the forespore, which is depleted of key metabolic enzymes and must import metabolites from the mother cell. Together, our results provide a bacterial example analogous to progeny nurturing.

Roles of LysM and LytM domains in resuscitation-promoting factor (Rpf) activity and Rpf-mediated peptidoglycan cleavage and dormant spore reactivation

Bacterial dormancy can take many forms, including formation of Bacillus endospores, Streptomyces exospores, and metabolically latent Mycobacterium cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of rpf mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in Streptomyces, Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes.

Basidiospores from Wood-Decay Fungi Transform Laccase Substrates in the Absence of Glucose and Nitrogen Supplements

Preparations of bacterial endospores and fungal conidia are applied in biocontrols, biocatalyses, and lignocellulose fermentations. The biocatalytic abilities of basidiospores from mushrooms of the order Agaricales are unknown. To assess their potential in colonizing recalcitrant substrates solely with their inherent resources, spores of the white-rot fungi Stropharia rugoso-annulata (Stru) and Kuehneromyces mutabilis (Kmt, Strophariaceae) were analyzed for surface-bound and internal total carbohydrates, phenols, proteins, minerals, and oxidoreductases to estimate their chemistry and the preconditions to transform the laccase substrates guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) independent of external glucose and nitrogen. Surfaces of Stru/Kmt spores released (mg kg−1) hexoses, 7300/9700; phenols, >62/220; proteins, 21/168; and laccases, 42/0–0.15 µmol ABTS•+ kg−1 min−1 that mimicked oxidative activities of the resting spores. Milled-spore extracts contained pentoses, 96,600/6750; hexoses, 160,000/15,130; phenols, 452/767; protein, 12,600/924; true laccase, 688/0.30; and enzyme-protein-activating transition metals such as Cu in concentrations typical of wheat grains. Independent of external N and C supply, spores (<1‰) germinated in bideionized water, supported by their surface resources. Kmt spores germinated, too, at comparable rates in N-free solutions of glucose and the not immediately metabolizable ABTS and guaiacol. The release of proteins and oxidoreductase(s) by Kmt spores starting upon germination was higher in guaiacol-incubated idiophase- than in glucose-incubated trophophase-spores and led to the 3–4-fold formation of guaiacol polymerizates and ABTS•+. Constitutive aromatic ring-cleaving dioxygenases in the dormant spore that could be involved in the intrinsic metabolization of guaiacol were not detected. It is concluded that intrinsic resources enable (germinating) spores to release the highly efficient laccases of basidiomycetes and to transform aromatic compounds in the absence of sugar amendments. Spores show therefore plant seed-like autonomy in nutrient modification and acquisition during the early stages of the colonization of inert substrates.

SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis

ABSTRACT When Bacillus subtilis spores detect nutrients, they exit dormancy through the processes of germination and outgrowth. A key step in germination is the activation of two functionally redundant cell wall hydrolases (SleB and CwlJ) that degrade the specialized cortex peptidoglycan that surrounds the spore. How these enzymes are regulated remains poorly understood. To identify additional factors that affect their activity, we used transposon sequencing to screen for synthetic germination defects in spores lacking SleB or CwlJ. Other than the previously characterized protein YpeB, no additional factors were found to be specifically required for SleB activity. In contrast, our screen identified SafA and YlxY (renamed SwsB) in addition to the known factors GerQ and CotE as proteins required for CwlJ function. SafA is a member of the spore’s proteinaceous coat and we show that, like GerQ and CotE, it is required for accumulation and retention of CwlJ in the dormant spore. SwsB is broadly conserved among spore formers, and we show that it is required for CwlJ to efficiently degrade the cortex during germination. Intriguingly, SwsB resembles polysaccharide deacetylases, and its putative catalytic residues are required for its role in germination. However, we find no chemical signature of its activity on the spore cortex or in vitro. While the precise, mechanistic role of SwsB remains unknown, we explore and discuss potential activities. IMPORTANCE Spore formation in Bacillus subtilis has been studied for over half a century, and virtually every step in this developmental process has been characterized in molecular detail. In contrast, how spores exit dormancy remains less well understood. A key step in germination is the degradation of the specialized cell wall surrounding the spore called the cortex. Two enzymes (SleB and CwlJ) specifically target this protective layer, but how they are regulated and whether additional factors promote their activity are unknown. Here, we identified the coat protein SafA and a conserved but uncharacterized protein YlxY as additional factors required for CwlJ-dependent degradation of the cortex. Our analysis provides a more complete picture of this essential step in the exit from dormancy.

Dormancy-to-death transition in yeast spores occurs due to gradual loss of gene-expressing ability

ABSTRACTDormancy is colloquially considered as extending lifespan by being still. Starved yeasts form dormant spores that wake-up (germinate) when nutrients reappear but cannot germinate (die) after some time. What sets their lifespans and how they age are open questions because what processes occur - and by how much - within each dormant spore remains unclear. With single-cell-level measurements, we discovered how dormant yeast spores age and die: spores have a quantifiable gene-expressing ability during dormancy that decreases over days to months until it vanishes, causing death. Specifically, each spore has a different probability of germinating that decreases because its ability to - without nutrients - express genes decreases, as revealed by a synthetic circuit that forces GFP expression during dormancy. Decreasing amounts of molecules required for gene expression - including RNA polymerases - decreases gene-expressing ability which then decreases chances of germinating. Spores gradually lose these molecules because they are produced too slowly compared to their degradations, causing gene-expressing ability to eventually vanish and, thus, death. Our work provides a systems-level view of dormancy-to-death transition.Short summaryThis study identifies systems-level quantities that decay during dormancy in Saccharomyces cerevisiae spores and thereby reveals the meaning of ageing for dormant yeast spores and shows that they die when their gene-expressing ability is irreversibly lost.HighlightsFor a given glucose concentration, a dormant yeast spore has a well-defined probability of germinating (“germination ability”).A spore’s germination ability positively correlates with its RNAP I-III levels and the gene-expression (GFP) level it can realize when the expression is forced without nutrients.Ageing during dormancy means gradual decreases in germination ability, RNAP levels, and GFP-level realizable when expression is forced.Spores die after sufficiently losing gene-expressing ability and drugs that inhibit gene expression during dormancy shorten spores’ lifespans (e.g., from months to a day).

From root to tips: sporulation evolution and specialization inBacillus subtilisand the intestinal pathogenClostridioides difficile

Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of a highly resistant, dormant spore. Spores allow environmental persistence, dissemination and for pathogens, are infection vehicles. In both the modelBacillus subtilis, an aerobic species, and in the intestinal pathogenClostridioides difficile, an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation. Using the genes involved in the process inB. subtilisandC. difficile, and estimating their gain-loss dynamics in a comprehensive bacterial macro-evolutionary framework we show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of theB. subtilisgroup and within the Peptostreptococcaceae family, which includesC. difficile. We also show that early and late sporulation regulons have been co-evolving and that sporulation genes entail greater innovation inB. subtiliswith many Bacilli-lineage restricted genes. In contrast,C. difficilemore often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota and an adjustment of sporulation to this particular ecosystem.

Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency inBacillus anthracisSterne

ABSTRACTBacillus anthracisis a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth ofB. anthracisunder iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficientin vitrosporulation ofB. anthracisrequires petrobactin, that the petrobactin biosynthesis operon (asbAto-F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, thein vitrocontributions of petrobactin to sporulation as well as growth imply that petrobactin may be required forB. anthracistransmission via the spore during natural infections, in addition to its key known functions during active anthrax infections.IMPORTANCEBacillus anthraciscauses the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron.B. anthracisrequires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.

The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile uses the soluble pseudoprotease, CspC, to detect bile salt germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile salt germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile salt-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since the mechanism by which C. difficile spores sense co-germinants is unknown, our study provides the first evidence that CspC senses distinct classes of co-germinants in addition to bile salts. Since we observed that specific residues control CspC’s responsiveness to these different signals, CspC is critical for regulating C. difficile germination in response to multiple environmental signals.