Dormant Spores Research Articles

Application of Microbial Biofilms to Reinstate Lost Soil Microbial Diversity under Global Warming: A Case Study

By the end of the 21st century, global surface temperatures expected to rise by ca. 1.8-3.6° C due to accumulation of CO2 in the atmosphere at higher rates. Excessive use of chemical inputs in modern agriculture is one of the major contributors, because it leads to degrade soil microbial diversity that causes soil carbon loss, and emit C to the atmosphere as CO2. In this context, microbial ameliorators like Biofilm Biofertilizers are studied to reinstate biodiversity, functioning, and sustainability of agroecosystems. The Biofilm Biofertilizers consist of microbes and their exudates having diverse biochemicals; once applied to soil, they break the dormancy of cysts, spores, akinetes, and conidia which reinstate biodiversity while cut short soil restoration in degraded croplands. In the present study, investigated the effect of a Biofilm Biofertilizers having one fungus and two bacteria on microbial diversity in the soils under conventional tea cultivation by refitting to the temperature increase. The soils separately treated with the BFBF and sterilized distilled water in small pots in a plant house under three different temperatures viz. 30° C, 36° C, and 40° C. After one month, the soil samples were collected separately and analyzed for fungal colony, and bacterial colony counts, fungal: bacterial ratio, fungal species richness, and bacterial species richness. The results showed that the application of Biofilm Biofertilizers significantly increased (p<0.05) culturable bacteria, fungi, and their species richness at 40° C over 30° C and 36° C. Therefore, the results conclude that the Biofilm Biofertilizers as a novel biotechnological method to restore degraded agricultural soils in the context of global warming.
 Keywords: Biofilm biofertilizers, Fungal:bacterial ratio, Soil biodiversity

Identification of Functional Spo0A Residues Critical for Sporulation in Clostridioides difficile

Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To survive in the environment and spread to new hosts, C. difficile must form metabolically dormant spores. The formation of spores requires activation of the transcription factor Spo0A, which is the master regulator of sporulation in all endospore-forming bacteria. Though the sporulation initiation pathway has been delineated in the Bacilli, including the model spore-former Bacillus subtilis, the direct regulators of Spo0A in C. difficile remain undefined. C. difficile Spo0A shares highly conserved protein interaction regions with the B. subtilis sporulation proteins Spo0F and Spo0A, although many of the interacting factors present in B. subtilis are not encoded in C. difficile. To determine if comparable Spo0A residues are important for C. difficile sporulation initiation, site-directed mutagenesis was performed at conserved receiver domain residues and the effects on sporulation were examined. Mutation of residues important for homodimerization and interaction with positive and negative regulators of B. subtilis Spo0A and Spo0F impacted C. difficile Spo0A function. The data also demonstrated that mutation of many additional conserved residues altered C. difficile Spo0A activity, even when the corresponding Bacillus interacting proteins are not apparent in the C. difficile genome. Finally, the conserved aspartate residue at position 56 of C. difficile Spo0A was determined to be the phosphorylation site that is necessary for Spo0A activation. The finding that Spo0A interacting motifs maintain functionality suggests that C. difficile Spo0A interacts with yet unidentified proteins that regulate its activity and control spore formation.

Visualization of SpoVAEa Protein Dynamics in Dormant Spores of Bacillus cereus and Dynamic Changes in Their Germinosomes and SpoVAEa during Germination.

ABSTRACTBacillus cereus spores, like most Bacillus spores, can survive for years and germinate when their surroundings become suitable, and germination proteins play an important role in the initiation of germination. Because germinated spores lose the extreme resistance of dormant spores, information on the function of germination proteins could be useful in developing new strategies to control B. cereus spores. Prior work has shown that (i) the channel protein SpoVAEa exhibits high-frequency movement in the outer leaflet of the inner membrane (IM) in dormant B. subtilis spores and (ii) the formation of the foci termed germinosomes between two germination proteins, the germinant receptor GerR and the scaffold protein GerD, in developing B. cereus spores is slower than foci formation by GerR and GerD individually. However, the movement dynamics of SpoVAEa in B. cereus spores, and the behavior of the germinosome upon B. cereus spore germination, are not known. In this study, we found that SpoVAEa fluorescent foci in dormant B. cereus spores move on the IM, but slower than in B. subtilis spores, and they likely co-localize transiently with GerD-mScarlet-I in the germinosome. Our results further indicate that (i) the expression of GerR-SGFP2 and SpoVAEa-SGFP2 with GerD-mScarlet-I from a plasmid leads to more heterogeneity and lower efficiency of spore germination in B. cereus, and (ii) germinosome foci observed by Fluorescence resonance energy transfer (FRET) between GerR-SGFP2 and GerD-mScarlet-I can be lost soon after the spore-phase transition. However, this is not always the case, as some GerR-SGFP2 and GerD-mScarlet-I foci continued to exist, co-localize, and even show a weak FRET signal. These data highlight the heterogeneous behavior of spore germination protein complexes and indicate that some complexes may persist beyond the initiation of germination.IMPORTANCE Bacillus cereus is commonly present in soil and infects humans via contaminated food. In this study, we used B. cereus spores to investigate the movement of the spore-specific inner membrane (IM) channel protein SpoVAEa, the interaction between SpoVAEa and the germinosome scaffold protein GerD, and the dynamics of germinosomes with GerR and GerD in spore germination. Our results expand upon observations of interactions between specific B. cereus spore germination proteins, in particular the GerR germinant receptor A, B, and C subunits and GerD, as well as those between SpoVAEa and GerD. The approaches used in this work could also be used to examine the interactions between GerD and SpoVAEa and other germination proteins in spores of other Bacillus species.

The SpoVA membrane complex is required for dipicolinic acid import during sporulation and export during germination.

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth in response to nutrients. The small molecule dipicolinic acid (DPA) plays a central role in both the stress resistance of the dormant spore and its exit from dormancy during germination. The spoVA locus is required for DPA import during sporulation and has been implicated in its export during germination, but the molecular bases are unclear. Here, we define the minimal set of proteins encoded in the Bacillus subtilis spoVA operon required for DPA import and demonstrate that these proteins form a membrane complex. Structural modeling of these components combined with mutagenesis and in vivo analysis reveal that the C and Eb subunits form a membrane channel, while the D subunit functions as a cytoplasmic plug. We show that point mutations that impair the interactions between D and the C-Eb membrane complex reduce the efficiency of DPA import during sporulation and reciprocally accelerate DPA release during germination. Our data support a model in which DPA transport into spores involves cycles of unplugging and then replugging the C-Eb membrane channel, while nutrient detection during germination triggers DPA release by unplugging it.

Reviving the view: evidence that macromolecule synthesis fuels bacterial spore germination.

The Gram positive bacterium Bacillus subtilis and its relatives are capable of forming a durable dormant long-lasting spore. Although spores can remain dormant for years, they possess the remarkable capacity to rapidly resume life and convert into actively growing cells. This cellular transition initiates with a most enigmatic irreversible event, termed germination, lasting only for a few minutes. Germination is typified by a morphological conversion that culminates in loss of spore resilient properties. Yet, the molecular events occurring during this brief critical phase are largely unknown. The current widely accepted view considers germination to occur without the need for any macromolecule synthesis; however, accumulating data from our laboratory and others, highlighted here, provide evidence that both transcription and translation occur during germination and are required for its execution. We further underline numerous overlooked studies, conducted mainly during the 1960s-1970s,reinforcing this notion. We propose to revisit the fascinating process of spore germination and redefine it as a pathway involving macromolecule synthesis. We expect our perspective to shed new light on the awakening process of a variety of spore-forming environmental, commensal, and pathogenic bacteria and possibly be applicable to additional organisms displaying a quiescent life form.

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.

Marine Actinomycetes: A Review

The marine environment is a prolific resource for the isolation of less exploited microorganisms, and as a matter of fact in the sea, untapped habitats exist with unique characteristics. This is partly caused by the lack of effort spent in exploring marine actinomycetes, whereas terrestrial actinomycetes have been, until recently, a successful source of novel bioactive metabolites. Furthermore, scepticism regarding the existence of indigenous population of marine actinomycetes arises from the fact that the terrestrial bacteria produce resistant spores that are known to be transported from land into sea, where they remain available but dormant for many years. Thus, it has been frequently assumed that actinomycetes isolated from marine samples are merely of terrestrial origin. It may be better to seek evidence instead that marine derived actinomycetes are metabolically active or capable of growing in the marine environment. Other useful characteristics include the display of specific marine adaptations, the formation of associations with plants or invertebrates and the demonstration of a role in nutrient cycling, all of which provide an indication that these bacteria are not merely present as dormant spores. When coupled with phylogenetic novelty, evidence for any of the above traits provides a strong case that the specific population under study can be considered as marine. As marine organisms live in a significantly different environment from those of the terrestrial organisms, it is reasonable to expect that their metabolites will differ considerably. Although actinomycetes can be readily isolated from marine sediments using appropriate selective isolation techniques, it is only through the use of molecular phylogenetics, a clear picture of the diversity, distributions and taxonomic uniqueness of certain populations can be obtained. Once recognized as unique, this population can then be selected as the focal point for intensive natural product studies. Clearly, actinomycetes have the potential to continue to play a major role in microbial drug discovery, and the recent observations on significant population of marine-adapted actinomycetes occur in ocean sediments, could certainly emphasize the potential importance of these strains in future drug discovery efforts.

Population genomics of Bacillus anthracis from an anthrax hyperendemic area reveals transmission processes across spatial scales and unexpected within-host diversity.

Genomic sequencing has revolutionized our understanding of bacterial disease epidemiology, but remains underutilized for zoonotic pathogens in remote endemic settings. Anthrax, caused by the spore-forming bacterium Bacillus anthracis , remains a threat to human and animal health and rural livelihoods in low- and middle-income countries. While the global genomic diversity of B. anthracis has been well-characterized, there is limited information on how its populations are genetically structured at the scale at which transmission occurs, critical for understanding the pathogen’s evolution and transmission dynamics. Using a uniquely rich dataset, we quantified genome-wide SNPs among 73 B. anthracis isolates derived from 33 livestock carcasses sampled over 1 year throughout the Ngorongoro Conservation Area, Tanzania, a region hyperendemic for anthrax. Genome-wide SNPs distinguished 22 unique B. anthracis genotypes (i.e. SNP profiles) within the study area. However, phylogeographical structure was lacking, as identical SNP profiles were found throughout the study area, likely the result of the long and variable periods of spore dormancy and long-distance livestock movements. Significantly, divergent genotypes were obtained from spatio-temporally linked cases and even individual carcasses. The high number of SNPs distinguishing isolates from the same host is unlikely to have arisen during infection, as supported by our simulation models. This points to an unexpectedly wide transmission bottleneck for B. anthracis , with an inoculum comprising multiple variants being the norm. Our work highlights that inferring transmission patterns of B. anthracis from genomic data will require analytical approaches that account for extended and variable environmental persistence, as well as co-infection.

Application of yeast spores as β-glucan particles

β-Glucan exhibits various beneficial biological activities including immunostimulation. This polysaccharide is present in the cell wall of microorganisms including the yeast Saccharomyces cerevisiae. Although vegetative yeast cells are used as dietary supplements, β-glucan in the vegetative cell wall is covered with mannan. During nutrient starvation, yeast cells differentiate into dormant cell spores. β-Glucan is also present in the spore wall, but spores have been poorly studied as application tools. The spore wall consists of four layers and β-glucan forms the third layer from the outside. However, unlike vegetative cells, the β-glucan layer can be exposed to the cell surface in spores by genetic manipulation. For example, chs3Δ mutant spores exhibit such a spore wall configuration. Dectin-1 is the primary receptor for β-glucan in mammals and, its activation induces phagocytosis and proinflammatory cytokine secretions. We found that chs3Δ spores are phagocytosed in mammalian cells in a manner dependent on dectin-1 and induce higher levels of TNF-α and IL-6 than vegetative cells. Our results demonstrate that chs3Δ spores are β-glucan particles with immunostimulatory activities. Thus, they would be attractive materials for application in the food industry.

Inactivation of vegetative cells, germinated spores, and dormant spores of Bacillus atrophaeus by pulsed electric field with fixed energy input

AbstractIncreasing the electric energy input during pulsed electric field (PEF) treatment is a straightforward technique to boost the efficiency of microbial inactivation. However, drawbacks such as electrode corrosion, arc formation, and high cost are also involved. This study aims to investigate the impact of medium conductivity (), electric field strength (), and the number of pulses () on the inactivation of vegetative cells, germinated spores, and dormant spores of Bacillus atrophaeus with fixed energy input. Our results indicated that (1) vegetative cells were susceptible to PEF treatment, and the relative importance of , , and was ; (2) germinated spores were more resistant to PEF treatment than vegetative cells, and the relative importance of , , and was ; and (3) similar to the thermal resistance property, dormant spores were also highly resistant to PEF treatment and were not significantly inactivated in our experiment setting. It is concluded that the degree of PEF resistance of cell at different physiological states may be related to its structure and size. This study added to the body of knowledge on selecting PEF treatment settings and gave insight into the scaling up of PEF applications in the food processing industry.Practical ApplicationsIn the food processing industry, eliminating contaminants such as bacterial spores is essential, and PEF processing is a common nonthermal technique. While the traditional method of increasing the electric energy input to increase microbial inactivation efficiency is beneficial, it is not cost‐effective. The results of this investigation demonstrated that decreasing spore resistance through germination followed by parameter‐optimized PEF treatments is a practical method for improving pasteurization efficiency. This novel technique might be used in the food sector.

Parasitism of Meloidogyne incognita eggs by Native Fungi of Assam

In the current investigation, four fungi viz., Paecilomyces niphetodes, Acremonium falciforme, Fusarium oxysporium, and F. solani were capable of parasitizing Meloidogyne incognita eggs. The high percentage of egg parasitism was recorded by P. niphetodes. Microscopic examinations showed that the hyphae of P. niphetode, A. falcifrome, F. oxysporum and F. solani were closely attached to the egg surface of the parasitized eggs, made perforations, penetrated, ramified, and completely fed upon the internal content of the eggs. The parasitization of the second stage juvenile that emerged from the eggs by P. niphetodes and A. falciforme was seen. Whereas, A. falcifrome, F. oxysporum, and F. solani established as dormant spores inside or outside of M. incognita eggs.

Expression of SARS-CoV-2 Spike Protein Receptor Binding Domain on Recombinant B. subtilis on Spore Surface: A Potential COVID-19 Oral Vaccine Candidate.

Various types of vaccines, such as mRNA, adenovirus, and inactivated virus by injection, have been developed to prevent SARS-CoV-2 infection. Although some of them have already been approved under the COVID-19 pandemic, various drawbacks, including severe side effects and the requirement for sub-zero temperature storage, may hinder their applications. Bacillus subtilis (B. subtilis) is generally recognized as a safe and endotoxin-free Gram-positive bacterium that has been extensively employed as a host for the expression of recombinant proteins. Its dormant spores are extraordinarily resistant to the harsh environment in the gastrointestinal tract. This feature makes it an ideal carrier for oral administration in resisting this acidic environment and for release in the intestine. In this study, an engineered B. subtilis spore expressing the SARS-CoV-2 spike protein receptor binding domain (sRBD) on the spore surface was developed. In a pilot test, no adverse health event was observed in either mice or healthy human volunteers after three oral courses of B. subtilis spores. Significant increases in neutralizing antibody against sRBD, in both mice and human volunteers, after oral administration were also found. These findings may enable the further clinical developments of B. subtilis spores as an oral vaccine candidate against COVID-19 in the future.

Efficient inactivation of intracellular bacteria in dormant amoeba spores by FeP

Waterborne pathogens and related diseases are a severe public health threat worldwide. Recent studies suggest that microbial interactions among infectious agents can significantly disrupt the disinfection processes, and current disinfection methods cannot inactivate intracellular pathogens effectively, posing an emerging threat to the safety of drinking water. This study developed a novel strategy, the FeP/persulfate (PS) system, to effectively inactivate intracellular bacteria within the amoeba spore. We found that the sulfate radical (SO4•−) produced by the FeP/PS system can be quickly converted into hydroxyl radicals (•OH), and •OH can penetrate the amoeba spores and inactivate the bacteria hidden inside amoeba spores. Therefore, this study proposes a novel technique to overcome the protective effects of microbial interactions and provides a new direction to inactivate intracellular pathogens efficiently.

Enhanced Cr(VI) adsorption using chemically modified dormant Aspergillus niger spores: Process and mechanisms

Adsorption materials based on Aspergillus niger spores were synthesized with chemically modified surfaces, and the CTAB surface modification adsorbent was the best. Zeta potential was in the range of pH 1 to pH 4.38, and CTAB-modified spores (CMS) surface had a positive charge, leading to adsorption of negatively charged chromic acid ions. The biosorption capacity of CMS was also markedly increased. The maximum biosorption capacity reached 177 mg/g following CTAB modification. XPS and FTIR confirmed the importance of secondary amine in the reduction adsorption process. Secondary amine (-NH-) transfer electrons to unoccupied orbit of the Cr(VI) for the reduction of hexavalent chromium. At the same time, chromium(III) is coordinated with OC-O functional group and (NH+/N+) and adsorbed on the modified spores. The fungal biological adsorbent can adsorb and desorb hexavalent chromium for five consecutive times without a clear decrease.

Dodecylamine rapidly kills of spores of multiple Firmicute species: properties of the killed spores and the mechanism of the killing.

Previous work showed that Bacillus subtilis dormant spore killing and germination by dodecylamine take place by different mechanisms. This new work aimed to optimize killing of B. subtilis and other Firmicutes spores and to determine the mechanism of the killing. Spores of seven Firmicute species were killed rapidly by dodecylamine under optimal conditions and more slowly by decylamine or tetradecylamine. The killed spores were not recovered by additions to recovery media, and some of the killed spores subsequently germinated, all indicating that dodecylamine-killed spores truly are dead. Spores of two species treated with dodecylamine were more sensitive to killing by a subsequent heat treatment, and spore killing of at least one species was faster with chemically decoated spores. The cores of dodecylamine-killed spores were stained by the nucleic acid stain propidium iodide, and dodecylamine-killed wild-type and germination-deficient spores released their stores of phosphate-containing small molecules. This work indicates that dodecylamine is likely a universal sporicide for Firmicute species, and it kills spores by damaging their inner membrane, with attendant loss of this membrane as a permeability barrier. There is a significant need for agents that can effectively kill spores of a number of Firmicute species, especially in wide area decontamination. Dodecylamine appears to be a universal sporicide with a novel mechanism of action, and this or some comparable molecule could be useful in wide area spore decontamination.

Dormant spores sense amino acids through the B subunits of their germination receptors

Bacteria from the orders Bacillales and Clostridiales differentiate into stress-resistant spores that can remain dormant for years, yet rapidly germinate upon nutrient sensing. How spores monitor nutrients is poorly understood but in most cases requires putative membrane receptors. The prototypical receptor from Bacillus subtilis consists of three proteins (GerAA, GerAB, GerAC) required for germination in response to L-alanine. GerAB belongs to the Amino Acid-Polyamine-Organocation superfamily of transporters. Using evolutionary co-variation analysis, we provide evidence that GerAB adopts a structure similar to an L-alanine transporter from this superfamily. We show that mutations in gerAB predicted to disrupt the ligand-binding pocket impair germination, while mutations predicted to function in L-alanine recognition enable spores to respond to L-leucine or L-serine. Finally, substitutions of bulkier residues at these positions cause constitutive germination. These data suggest that GerAB is the L-alanine sensor and that B subunits in this broadly conserved family function in nutrient detection.

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus.

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores.

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins–hydrophobins (HFBs)–that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.

Genetic Evidence for Signal Transduction within the Bacillus subtilis GerA Germinant Receptor.

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to l-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA, gerAB, and gerAC. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA*) that carries a mutation in gerAA and show that it constitutively activates germination even in the presence of a wild-type copy of gerA. Using an enrichment strategy to screen for suppressors of gerA*, we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one (gerAB[E105K]) reduces the GerA complex's ability to respond to l-alanine, while another (gerAB[F259S]) disrupts the germinant signal downstream of l-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway. IMPORTANCE Endospore formers are a broad group of bacteria that can enter dormancy upon starvation and exit dormancy upon sensing the return of nutrients. How dormant spores sense and respond to these nutrients is poorly understood. Here, we identify a key step in the signal transduction pathway that is activated after spores detect the amino acid l-alanine. We present a model that provides a more complete picture of this process that is critical for allowing dormant spores to germinate and resume growth.

Timing and duration of light exposure during conidia development determine tolerance to ultraviolet radiation.

Metarhizium is an important genus of soil-inhabiting fungi that are used for the biological control of insects. The efficiency of biocontrol is dependent on the maintenance of inoculum viability under adverse field conditions such as solar ultraviolet (UV) radiation. Therefore, increasing the tolerance of Metarhizium to UV radiation is necessary. It was previously established that, in mycelium, exposure to visible light increases tolerance to UV radiation. Similarly, growth under visible light for 14 days induces the production of tolerant conidia. However, a study evaluating if and how visible light affects conidia and their relationship with UV radiation was never performed. Here, we report that a relatively short and timed exposure to light around the time of conidiation is sufficient to induce the production of conidia with increased photoreactivating capacity and UV tolerance in Metarhizium acridum. Conidia produced by this method retain their characteristic higher tolerance even after many days of being transferred to the dark. Furthermore, we show that mature conidia of M. acridum and Metarhizium brunneum can still answer to light and regulate UV tolerance, suggesting that gene expression is possible even in dormant spores. Being able to respond to light in the dormant stages of development is certainly an advantage conferring improved environmental persistence to Metarhizium.