On the Mechanistic Basis of Killer Meiotic Drive in Fungi.

Spore killers are specific genetic elements in fungi that kill sexual spores that do not contain them. A range of studies in the last few years have provided the long-awaited first insights into the molecular mechanistic aspects of spore killing in different fungal models, including both yeast-forming and filamentous Ascomycota. Here we describe these recent advances, focusing on the wtf system in the fission yeast Schizosaccharomyces pombe; the Sk spore killers of Neurospora species; and two spore-killer systems in Podospora anserina, Spok and [Het-s]. The spore killers appear thus far mechanistically unrelated. They can involve large genomic rearrangements but most often rely on the action of just a single gene. Data gathered so far show that the protein domains involved in the killing and resistance processes differ among the systems and are not homologous. The emerging picture sketched by these studies is thus one of great mechanistic and evolutionary diversity of elements that cheat during meiosis and are thereby preferentially inherited over sexual generations.

What's new and notable in bacterial spore killing!

Spores of many species of the orders Bacillales and Clostridiales can be vectors for food spoilage, human diseases and intoxications, and biological warfare. Many agents are used for spore killing, including moist heat in an autoclave, dry heat at elevated temperatures, UV radiation at 254 and more recently 222 and 400 nm, ionizing radiation of various types, high hydrostatic pressures and a host of chemical decontaminants. An alternative strategy is to trigger spore germination, as germinated spores are much easier to kill than the highly resistant dormant spores-the so called "germinate to eradicate" strategy. Factors important to consider in choosing methods for spore killing include the: (1) cost; (2) killing efficacy and kinetics; (3) ability to decontaminate large areas in buildings or outside; and (4) compatibility of killing regimens with the: (i) presence of people; (ii) food quality; (iii) presence of significant amounts of organic matter; and (iv) minimal damage to equipment in the decontamination zone. This review will summarize research on spore killing and point out some common flaws which can make results from spore killing research questionable.

Mechanisms of killing of Bacillus thuringiensis Al Hakam spores in a blast environment with and without iodic acid.

AIMS: To determine the mechanism of killing of spores of Bacillus thuringiensis Al Hakam, a Bacillus anthracis spore surrogate, in a blast environment with or without HIO3 and whether the spores are truly dead. METHODS AND RESULTS: Spores exposed to an aluminium-based blast environment with or without HIO3 with dynamic peak gas phase temperatures near 1000°C persisting for 10's of ms, were killed 97 and 99·99% without and with HIO3 respectively and the spores were truly dead. The survivors of the detonations did not acquire mutations, did not become wet heat sensitive, became sensitive to elevated NaCl but not lack of glucose in recovery media, and many dead spores remained phase bright and retained their Ca-dipicolinic acid. A large fraction of the dead spores could germinate, but most of these germinated spores were dead. CONCLUSIONS: Most spores exposed to a blast environment are truly dead, and HIO3 increases spore death. The likely mechanism of spore killing in these blast environments is damage to some essential spore protein, although spore inner membrane damage could contribute. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that spores of a surrogate for B. anthracis spores are killed in a blast environment without or with HIO3 present, this approach could inactivate up to 99·99% of dry B. anthracis spores, and the spores are likely killed by damage to some essential spore protein.

Effect of Humidity on Sporicidal Activity of Iodine Vapor on Bacillus thuringiensis.

The emergence of Bacillus anthracis as a potential bioterrorism and biological warfare agent points to the need for safe, effective, and economical sporicides for infection prevention and control. This work examined the efficacy of iodine vapor decontamination technologies to inactivate a surrogate for B. anthracis, Bacillus thuringiensis spores on glass materials. 106-107 colony-forming units of spores inoculated onto circular glass cover slips were treated with different concentrations of iodine vapor under various temperature and relative humidity. Only minimal spore killing activity was observed at low humidity. Higher humidity levels, as well as pre-hydration or post-hydration of the spores, increased the rate of inactivation as long as the contact between spores and iodine was maintained in a hydrated environment. Significant sporicidal activity of 3-log and 6-log spore reduction has been observed with 2.1 mg L-1 iodine vapor concentration at 90% relative humidity and 22 °C, with 1 and 24 h of exposure, respectively. The results showed that the relative humidity of the environment is of major importance in regulating the rate at which the spores are inactivated by iodine. The results of this study may provide insight into the parameters of effective decontamination procedures for Bacillus spores using gaseous iodine.

Bacillus spore wet heat resistance and evidence for the role of an expanded osmoregulatory spore cortex.

UNLABELLED: Previous work reported that decoated Bacillus cereus spores incubated in 4 mol l(-1) CaCl2 are killed at lower temperatures than spores in water. This wet heat sensitization was suggested to support a role for an osmoregulatory peptidoglycan cortex in spore cores' low water content, and their wet heat resistance. Current work has replicated this finding with spores of B. cereus, Bacillus megaterium and Bacillus subtilis. However, this work found that decoated spores apparently killed at 80°C in 4 mol l(-1) CaCl2 : (i) were recovered on plates containing lysozyme; (ii) lost no dipicolinic acid (DPA) and their inner membrane remained impermeable; (iii) released no DPA upon stimulation with nutrient germinants and could not complete germination; and (iv) released DPA relatively normally upon stimulation with dodecylamine. These results indicate that decoated spores treated with 80°C- 4 mol l(-1) CaCl2 are not dead, but some protein(s) essential for spore germination, most likely germinant receptors, are inactivated by this treatment. Thus, the original finding does not support a role for an osmoregulatory cortex in spore wet heat resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus spores' low core water content is a major factor in their wet heat resistance. One suggested mechanism for achieving low spore core water content is osmoregulated expansion of spores' peptidoglycan cortex. Evidence for this mechanism includes a report that decoated Bacillus cereus spores incubated in 4 mol l(-1) CaCl2 exhibit drastically reduced heat resistance. The current work shows that this heat sensitization of decoated spores of three Bacillus species is most likely due to inactivation of some crucial spore germination protein(s), since while treated spores appear dead, their apparent low viability is rescued by triggering spore germination with lysozyme.

Enzyme-driven Bacillus spore coat degradation leading to spore killing.

The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable.

Mechanism of killing of spores of Bacillus anthracis in a high-temperature gas environment, and analysis of DNA damage generated by various decontamination treatments of spores of Bacillus anthracis, Bacillus subtilis and Bacillus thuringiensis.

AIMS: To determine how hydrated Bacillus anthracis spores are killed in a high-temperature gas environment (HTGE), and how spores of several Bacillus species including B. anthracis are killed by UV radiation, dry heat, wet heat and desiccation. METHODS AND RESULTS: Hydrated B. anthracis spores were HTGE treated at c. 220°C for 50 ms, and the treated spores were tested for germination, mutagenesis, rupture and loss of dipicolinic acid. Spores of this and other Bacillus species were also examined for mutagenesis by UV, wet and dry heat and desiccation. There was no rupture of HTGE-treated B. anthracis spores killed 90-99·9%, no mutagenesis, and release of DPA and loss of germination were much slower than spore killing. However, killing of spores of B. anthracis, Bacillus thuringiensis and Bacillus subtilis by UV radiation or dry heat, but not wet heat in water or ethanol, was accompanied by mutagenesis. CONCLUSIONS: It appears likely that HTGE treatment kills B. anthracis spores by damage to spore core proteins. In addition, various killing regimens inactivate spores of a number of Bacillus species by the same mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates how hydrated spores treated in a HTGE such as might be used to destroy biological warfare agent stocks are killed. The work also indicates that mechanisms whereby different agents kill spores are similar with spores of different Bacillus species.

Effects of spore purity on the wet heat resistance of Clostridium perfringens, Bacillus cereus and Bacillus subtilis spores.

Heat resistance of spores of Clostridium perfringens 8238 (Hobbs Serotype 2), Bacillus cereus NCTC 11143 (4810/72), and Bacillus subtilis PS533, an isogenic derivative of strain PS832 (a 168 strain) was determined in ground beef at 95 °C. Spore purification was by centrifugation and washing with sterile distilled water (dH2O), followed by sonication and then Histodenz centrifugation for B. subtilis and C. perfringens, and centrifugation and washing with sterile dH2O followed by Histodenz centrifugation for B. cereus. Bags containing inoculated beef samples were submerged in a temperature-controlled water bath and held at 95 °C for predetermined lengths of time. Surviving spore populations were enumerated by plating on mannitol egg yolk polymyxin agar (MYP) agar plates for B. cereus and B. subtilis, and on tryptose-sulfite-cycloserine agar (TSC) agar plates for C. perfringens. Survivor curves were fitted to linear, linear with tail, and Weibull models using the USDA Integrated Pathogen Modeling Program (IPMP) 2013 software. The Weibull model provided a relatively better fit to the data since the root mean square error (RMSE), mean square error (MSE), sum of squared errors (SSE), and Akaike information criterion (AIC) values were lower than the values obtained using the linear or the linear with tail models. Additionally, the Weibull model accurately predicted the observed D-values at 95 °C for the three spore-formers since the accuracy factor (Af) values ranged from 1.03 to 1.08 and the bias factor (Bf) values were either 1.00 or 1.01. Times at 95 °C to achieve a 3-log reduction decreased from 206 min for C. perfringens spores purified with water washes alone to 191 min with water washes followed by sonication and Histodenz centrifugation, from 7.9 min for B. cereus spores purified with water washes alone to 1.4 min with water washes followed by Histodenz centrifugation, and from 20.6 min for B. subtilis spores purified with water washes alone to 6.7 min for water washes followed by sonication and Histodenz centrifugation. Thermal-death-time values reported in this study will assist food processors to design thermal processes to guard against bacterial spores in cooked foods. In addition, clearly spore purity is an additional factor in spore wet heat resistance, although the cause of this effect is not clear.

Evaluation of vaporized hydrogen peroxide fumigation as a method for the bio-decontamination of the high efficiency particulate air filter unit.

OBJECTIVE: To evaluate the performance of vaporized hydrogen peroxide (VHP) for the bio-decontamination of the high efficiency particulate air (HEPA) filter unit. METHODS: Self-made or commercially available bioindicators were placed at designated locations in the HEPA filter unit under VHP fumigation. The spores on coupons were then extracted by 0.5 h submergence in eluent followed by 200- time violent knocks. RESULTS: Due to the presence of HEPA filter in the box, spore recovery from coupons placed at the bottom of the filter downstream was significantly higher than that from coupons placed at the other locations. The gap of decontamination efficiency between the top and the bottom of the filter downstream became narrower with the exposure time extended. The decontamination efficiency of the bottom of the filter downstream only improved gently with the injection rate of H2O2 increased and the decontamination efficiency decreased instead when the injection rate exceeded 2.5 g/min. The commercially available bioindicators were competent to indicate the disinfection efficiency of VHP for the HEPA filter unit. CONCLUSION: This assay developed can detect all 16 ß-lactams demanded by the European Union (EU). The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.

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.

The spore killers, fungal meiotic driver elements.

During meiosis, both alleles of any given gene should have equal chances of being inherited by the progeny. There are a number of reasons why, however, this is not the case, with one of the most intriguing instances presenting itself as the phenomenon of meiotic drive. Genes that are capable of driving can manipulate the ratio of alleles among viable meiotic products so that they are inherited in more than half of them. In many cases, this effect is achieved by direct antagonistic interactions, where the driving allele inhibits or otherwise eliminates the alternative allele. In ascomycete fungi, meiotic products are packaged directly into ascospores; thus, the effect of meiotic drive has been given the nefarious moniker, "spore killing." In recent years, many of the known spore killers have been elevated from mysterious phenotypes to well-described systems at genetic, genomic, and molecular levels. In this review, we describe the known diversity of spore killers and synthesize the varied pieces of data from each system into broader trends regarding genome architecture, mechanisms of resistance, the role of transposable elements, their effect on population dynamics, speciation and gene flow, and finally how they may be developed as synthetic drivers. We propose that spore killing is common, but that it is under-observed because of a lack of studies on natural populations. We encourage researchers to seek new spore killers to build on the knowledge that these remarkable genetic elements can teach us about meiotic drive, genomic conflict, and evolution more broadly.

Novel spore lytic enzyme from a Bacillus phage leading to spore killing.

Bacterial spores maintain metabolic dormancy and have high resistance to external pressure. Germination requires degradation of the spore cortex and the participation of germination-specific cortex-lytic enzymes (GSLEs). Previously reported GSLEs have been identified in bacteria and facilitate germination. In this study, we have characterized a novel spore lytic enzyme, Ply67, from Bacillus pumilus phage vB_BpuM_BpSp. Ply67 had a similar cortex-lytic activity to GSLEs but disrupted the inner membranes (IMs) of spores, leading to spore killing rather than germination. The amino acid sequence of the complete protein, Ply67FL, exhibited 40% homology to the GSLE SleB. Domain prediction showed that Ply67FL was composed of three domains: a signal peptide, N-terminal domain protein and C-terminal domain protein. Ply67FL rapidly caused E. coli cells lysis when it was expressed in E. coli. The protein containing the C-terminal domain protein, Ply67C, could kill B. pumilus spores. The protein containing the N-terminal domain protein, Ply67N, could combine with the decoated B. pumilus spores, indicating that N-terminal was the binding domain and C-terminal was the hydrolase domain. The protein lacking the signal peptide but containing the N-terminal and C-terminal domain proteins, Ply67, had activity against spores of various Bacillus species. The surface of spores treated with Ply67 shrank and the permeability barrier was disrupted, and the inner contents leaked out. Immunoelectron microscopic observation showed that Ply67 was mainly acted on the spore cortex. Overall, Ply67 is a novel spore lytic enzyme that differs from other GSLEs not only in amino acid sequence but also in activity against spores, and Ply67 might have the potential to kill spores of pathogenic Bacillus species, e.g., B. cereus and B. anthracis.

Identification of rfk-1, a Meiotic Driver Undergoing RNA Editing in Neurospora.

Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1's MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.

A Meiotic Drive Element in the Maize Pathogen Fusarium verticillioides Is Located Within a 102 kb Region of Chromosome V.

Fusarium verticillioides is an agriculturally important fungus because of its association with maize and its propensity to contaminate grain with toxic compounds. Some isolates of the fungus harbor a meiotic drive element known as Spore killer (Sk(K)) that causes nearly all surviving meiotic progeny from an Sk(K) × Spore killer-susceptible (Sk(S)) cross to inherit the Sk(K) allele. Sk(K) has been mapped to chromosome V but the genetic element responsible for meiotic drive has yet to be identified. In this study, we used cleaved amplified polymorphic sequence markers to genotype individual progeny from an Sk(K) × Sk(S) mapping population. We also sequenced the genomes of three progeny from the mapping population to determine their single nucleotide polymorphisms. These techniques allowed us to refine the location of Sk(K) to a contiguous 102 kb interval of chromosome V, herein referred to as the Sk region. Relative to Sk(S) genotypes, Sk(K) genotypes have one extra gene within this region for a total of 42 genes. The additional gene in Sk(K) genotypes, herein named SKC1 for Spore Killer Candidate 1, is the most highly expressed gene from the Sk region during early stages of sexual development. The Sk region also has three hyper-variable regions, the longest of which includes SKC1 The possibility that SKC1, or another gene from the Sk region, is an essential component of meiotic drive and spore killing is discussed.

Sterilization of hydrogen peroxide resistant bacterial spores with stabilized chlorine dioxide.

Bacillus pumilus SAFR-032 spores isolated from a clean room environment are known to exhibit enhanced resistance to peroxide, desiccation, UV radiation and chemical disinfection than other spore-forming bacteria. The survival of B. pumilus SAFR-032 spores to standard clean room sterilization practices requires development of more stringent disinfection agents. Here, we report the effects of a stabilized chlorine dioxide-based biocidal agent against spores of B. pumilus SAFR-032 and Bacillus subtilis ATCC 6051. Viability was determined via CFU measurement after exposure. Chlorine dioxide demonstrated efficacy towards sterilization of spores of B. pumilus SAFR-032 equivalent or better than exposure to hydrogen peroxide. These results indicate efficacy of chlorine dioxide delivered through a stabilized chlorine dioxide product as a means of sterilization of peroxide- and UV-resistant spores.