Sporulation Frequency Research Articles

Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells, which was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomic analysis of the whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile.

INDUCTION ENDOSPORE FORMATION OF Bacillus subtilis KM16 AS A PROBIOTIC CANDIDATE BY OPTIMIZATION OF THE GROWTH CONDITIONS

Probiotics are living microorganisms which have beneficial effects. One of the benefits is enhancing immunity. Therefore, probiotics are often supplemented into food, medical, and aquaculture products. B. subtilis KM16 as endospore-producing bacteria have been known to increase probiotic resistance to confront the unstable conditions of the digestive tract. Spore formation of Bacillus is supported by the optimal condition of growth medium and incubation periods. The sufficient availability of carbon, nitrogen, and mineral source in the growth medium, and environmental conditions can support the formation of optimal endospores. This research aimed to determine the suitable sugar composition of a medium and optimum incubation time suitable for endospores formation of B. subtilis KM16. Glucose and maltose were utilized as a carbon source in several concentration as a growth medium for B. subtilis KM 16. The incubation time was 48 and 72 hours. The viable number of vegetative cells was not significantly different for the type of sugar and incubation periods. Based on the glucose concentration, 0.2% glucose exhibited the uppermost number of cells, while 1% glucose concentration caused the decrease of vegetative cells. In this study, 0.2% glucose with 48 hours incubation period showed the highest percentage of sporulation frequencies, up to 48%, but it was not significantly different from the other glucose concentration. The medium that contains 0.2% glucose with 48 hours incubation period is the best medium for the utilization of B. subtilis KM16 cells and spores as probiotic candidates.

The pH-responsive SmrR-SmrT system modulates C. difficile antimicrobial resistance, spore formation, and toxin production.

Clostridioides difficile is an anaerobic gastrointestinal pathogen that spreads through the environment as dormant spores. To survive, replicate, and sporulate in the host intestine, C. difficile must adapt to a variety of conditions in its environment, including changes in pH, the availability of metabolites, host immune factors, and a diverse array of other species. Prior studies showed that changes in intestinal conditions, such as pH, can affect C. difficile toxin production, spore formation, and cell survival. However, little is understood about the specific genes and pathways that facilitate environmental adaptation and lead to changes in C. difficile cell outcomes. In this study, we investigated two genes, CD2505 and CD2506, that are differentially regulated by pH to determine if they impact C. difficile growth and sporulation. Using deletion mutants, we examined the effects of both genes (herein smrR and smrT) on sporulation frequency, toxin production, and antimicrobial resistance. We determined that SmrR is a repressor of smrRT that responds to pH and suppresses sporulation and toxin production through regulation of the SmrT transporter. Further, we showed that SmrT confers resistance to erythromycin and lincomycin, establishing a connection between the regulation of sporulation and antimicrobial resistance.IMPORTANCEClostridioides difficile is a mammalian pathogen that colonizes the large intestine and produces toxins that lead to severe diarrheal disease. C. difficile is a major threat to public health due to its intrinsic resistance to antimicrobials and its ability to form dormant spores that are easily spread from host to host. In this study, we examined the contribution of two genes, smrR and smrT, on sporulation, toxin production, and antimicrobial resistance. Our results indicate that SmrR represses smrT expression, while production of SmrT increases spore and toxin production, as well as resistance to antibiotics.

Functional importance and divergence of plant apurinic/apyrimidinic endonucleases in somatic and meiotic DNA repair.

Apurinic/apyrimidinic (AP) sites are one of the most abundant DNA lesions and are mainly repaired by AP endonucleases (APEs). While most eukaryotic genomes encode two APEs, plants usually possess three APEs, namely APE1L, APE2, and ARP. To date, the biological relevance and functional divergence of plant APEs are unclear. Here, we show that the three plant APEs have ancient origins, with the APE1L clade being plant-specific. In Arabidopsis thaliana, simultaneously mutating APE1L and APE2, but not ARP alone or in combination with either APE1L or APE2, results in clear developmental defects linked to genotoxic stress. Genetic analyses indicated that the three plant APEs have different substrate preferences in vivo. ARP is mainly responsible for AP site repair, while APE1L and APE2 prefer to repair 3'-blocked single-stranded DNA breaks. We further determined that APEs play an important role in DNA repair and the maintenance of genomic integrity in meiotic cells. The ape1l ape2 double mutant exhibited a greatly enhanced frequency of sporulation 1 (SPO11-1)-dependent and SPO11-1-independent double-stranded DNA breaks. The DNA damage response (DDR) was activated in ape1l ape2 to trigger pollen abortion. Our findings suggest functional divergence of plant APEs and reveal important roles of plant APEs during vegetative and reproductive development.

Artificial activation of both σH and Spo0A in Bacillus subtilis enforced initiation of spore development at the vegetatively growing phase.

When Bacillus subtilis cells face environmental deterioration, such as exhaustion of nutrients and an increase in cell density, they form spores. It is known that phosphorylation of Spo0A and activation of σH are key events at the initiation of sporulation. However, the initiation of sporulation is an extremely complicated process, and the relationship between these two events remains to be elucidated. To determine the minimum requirements for triggering sporulation initiation, we attempted to induce cell sporulation at the log phase, regardless of nutrients and cell density. In rich media such as Luria-Bertani (LB) medium, the cells of B. subtilis do not sporulate efficiently, possibly because of excess nutrition. When the amount of xylose in the LB medium was limited, σH -dependent transcription of the strain, in which sigA was under the control of the xylose-inducible promoter, was induced, and the frequency of sporulation was elevated according to the decreased level of σA. We also employed a fusion of sad67, which codes for an active form of Spo0A, and the IPTG-inducible promoter. The combination of lowered σA expression and activated Spo0A allowed the cells in the log phase to stop growing and rush into spore development. This observation of enforced initiation of sporulation in the mutant strain was detected even in the presence of the wild-type strain, suggesting that only intracellular events initiate and fulfill spore development regardless of extracellular conditions. Under natural sporulation conditions, the amount of σA did not change drastically throughout growth. Mechanisms that sequester σA from the core RNA polymerase and help σH to become active exist, but this has not yet been elucidated.

Identification of serine/threonine kinases that regulate metabolism and sporulation in Clostridium beijerinckii.

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2g/L vs. 16.8g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation.

Nε-Lysine Acetylation of the Histone-Like Protein HBsu Regulates the Process of Sporulation and Affects the Resistance Properties of Bacillus subtilis Spores.

Bacillus subtilis produces dormant, highly resistant endospores in response to extreme environmental stresses or starvation. These spores are capable of persisting in harsh environments for many years, even decades, without essential nutrients. Part of the reason that these spores can survive such extreme conditions is because their chromosomal DNA is well protected from environmental insults. The α/β-type small acid-soluble proteins (SASPs) coat the spore chromosome, which leads to condensation and protection from such insults. The histone-like protein HBsu has been implicated in the packaging of the spore chromosome and is believed to be important in modulating SASP-mediated alterations to the DNA, including supercoiling and stiffness. Previously, we demonstrated that HBsu is acetylated at seven lysine residues, and one physiological function of acetylation is to regulate chromosomal compaction. Here, we investigate if the process of sporulation or the resistance properties of mature spores are influenced by the acetylation state of HBsu. Using our collection of point mutations that mimic the acetylated and unacetylated forms of HBsu, we first determined if acetylation affects the process of sporulation, by determining the overall sporulation frequencies. We found that specific mutations led to decreases in sporulation frequency, suggesting that acetylation of HBsu at some sites, but not all, is required to regulate the process of sporulation. Next, we determined if the spores produced from the mutant strains were more susceptible to heat, ultraviolet (UV) radiation and formaldehyde exposure. We again found that altering acetylation at specific sites led to less resistance to these stresses, suggesting that proper HBsu acetylation is important for chromosomal packaging and protection in the mature spore. Interestingly, the specific acetylation patterns were different for the sporulation process and resistance properties of spores, which is consistent with the notion that a histone-like code exists in bacteria. We propose that specific acetylation patterns of HBsu are required to ensure proper chromosomal arrangement, packaging, and protection during the process of sporulation.

Infection by dsRNA viruses is associated with enhanced sporulation efficiency in Saccharomyces cerevisiae.

Upon starvation diploid cells of the facultative sexual yeast Saccharomyces cerevisiae undergo sporulation, forming four metabolically quiescent and robust haploid spores encased in a degradable ascus. All endosymbionts, whether they provide net benefits or costs, utilize host resources; in yeast, this should induce an earlier onset of sporulation. Here, we tested whether the presence of endosymbiotic dsRNA viruses (M satellite and L‐A helper) correspond with higher sporulation rate of their host, S. cerevisiae. We find that S. cerevisiae hosting both the M and L‐A viruses (so‐called “killer yeasts”) have significantly higher sporulation efficiency than those without. We also found that the removal of the M virus did not reduce sporulation frequency, possibly because the L‐A virus still utilizes host resources with and without the M virus. Our findings indicate that either virulent resource use by endosymbionts induces sporulation, or that viruses are spread more frequently to sporulating strains. Further exploration is required to distinguish cause from effect.

Defence against Bremia lactucae conferred by the resistance gene Dm7 in lettuce is broken by treatment with dichloroisonicotinic acid

AbstractThe effect of inducers of systemic acquired resistance, dichloroisonicotinic acid (DCINA) and acibenzolar‐S‐methyl (BION), on compatible interactions between Bremia lactucae and lettuce were examined using a detached cotyledon infection assay. Treatment with both activators caused a reduction in sporulation on susceptible cultivars Cobham Green challenged with isolate CL9W and Diana inoculated with isolate Tv, with DCINA being more effective than BION on an equimolar basis. Unexpectedly, treatment with both compounds suppressed the resistance conferred by the Dm7 gene in cv. Diana challenged by isolate CL9W (A7). The frequency of sporulation was greatly increased by DCINA in the incompatible interaction. The suppression of defence was associated with a delay in the onset of the Dm7‐based hypersensitive reaction as indicated by the extended viability of penetrated epidermal cells, and reductions in both the accumulation of the phytoalexin lettucenin A and the deposition of autofluorescent phenolics such as syringaldehyde on plant and oomycete cell walls. The analysis of DCINA homologues indicated that 2‐chloroisonicotinic acid was as effective as the dichloro‐derivative in suppressing resistance in cv. Diana, whereas the absence of the carboxyl group rendered 2,6‐dichloropyridine inactive. Infection of cotyledons by Botrytis cinerea was also found to be enhanced by DCINA treatment. Based on our results, we discuss the possibility that DCINA reduces Dm7 transcription through an epigenetic mechanism, as is supported by bioinformatic analyses of the resistance gene, and that it suppresses jasmonate‐dependent resistance to B. cinerea.

Extracellular DNA, cell surface proteins and c-di-GMP promote biofilm formation in Clostridioides difficile

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.

Characteristic analysis of the fermentation and sporulation properties of the traditional sake yeast strain Hiroshima no.6.

General sake yeasts (e.g., Kyokai no.7, K7) show high fermentation ability and low sporulation frequency. Former is related to stress-response defect due to the loss-of-function of MSN4 and RIM15. Later is mainly caused by low IME1 expression, leading to difficulty in breeding and genetic analysis. Sake yeast Hiroshima no.6 (H6), which had been applied for sake fermentation, has sporulation ability. However, its detailed properties have not been unveiled. Here we present that the fermentation ability of H6 is suitable for sake brewing, and the precursor of dimethyl trisulfide in sake from H6 is low. MSN4 but not RIM15 of H6 has the same mutation as K7. Our phylogenetic analysis indicated that H6 is closely related to the K7 group. Unlike K7, H6 showed normal sporulation frequency in a partially RIM15-dependent manner, and IME1 in H6 was expressed. H6 possesses excellent properties as a partner strain for breeding by crossing.

Metabolic Engineering of Histidine Kinases in Clostridium beijerinckii for Enhanced Butanol Production.

Clostridium beijerinckii, a promising industrial microorganism for butanol production, suffers from low butanol titer and lack of high-efficiency genetical engineering toolkit. A few histidine kinases (HKs) responsible for Spo0A phosphorylation have been demonstrated as functionally important components in regulating butanol biosynthesis in solventogenic clostridia such as C. acetobutylicum, but no study about HKs has been conducted in C. beijerinckii. In this study, six annotated but uncharacterized candidate HK genes sharing partial homologies (no less than 30%) with those in C. acetobutylicum were selected based on sequence alignment. The encoding region of these HK genes were deleted with CRISPR-Cas9n-based genome editing technology. The deletion of cbei2073 and cbei4484 resulted in significant change in butanol biosynthesis, with butanol production increased by 40.8 and 17.3% (13.8 g/L and 11.5 g/L vs. 9.8 g/L), respectively, compared to the wild-type. Faster butanol production rates were observed, with butanol productivity greatly increased by 40.0 and 20.0%, respectively, indicating these two HKs are important in regulating cellular metabolism in C. beijerinckii. In addition, the sporulation frequencies of two HKs inactivated strains decreased by 96.9 and 77.4%, respectively. The other four HK-deletion (including cbei2087, cbei2435, cbei4925, and cbei1553) mutant strains showed few phenotypic changes compared with the wild-type. This study demonstrated the role of HKs on sporulation and solventogenesis in C. beijerinckii, and provided a novel engineering strategy of HKs for improving metabolite production. The hyper-butanol-producing strains generated in this study have great potentials in industrial biobutanol production.

Structure-Function Studies of the Bacillus subtilis Ric Proteins Identify the Fe-S Cluster-Ligating Residues and Their Roles in Development and RNA Processing.

In Bacillus subtilis, the RicA (YmcA), RicF (YlbF), and RicT (YaaT) proteins accelerate the phosphorylation of the transcription factor Spo0A, contributing to genetic competence, sporulation, and biofilm formation, and are also essential for the correct maturation of several protein-encoding and riboswitch RNAs. These proteins form a stable complex (RicAFT) that carries two [4Fe-4S]+2 clusters. We show here that the complex is a 1:1:1 heterotrimer, and we present the X-ray crystal structures of a RicAF heterotetramer and of a RicA dimer. We also demonstrate that one of the Fe-S clusters (cluster 1) is ligated by cysteine residues donated exclusively by RicT and can be retained when the RicT monomer is purified by itself. Cluster 2 is ligated by C167 from RicT, by C134 and C146 located near the C terminus of RicF, and by C141 at the C terminus of RicA. These findings imply the following novel arrangement: adjacent RicT residues C166 and 167 ligate clusters 1 and 2, respectively, while cluster 2 is ligated by cysteine residues from RicT, RicA, and RicF. Thus, the two clusters must lie close to one another and at the interface of the RicAFT protomers. We also show that the cluster-ligating cysteine residues, and therefore most likely both Fe-S clusters, are essential for cggR-gapA mRNA maturation, for the regulation of ricF transcript stability, and for several Ric-associated developmental phenotypes, including competence for transformation, biofilm formation, and sporulation. Finally, we present evidence that RicAFT, RicAF, and RicA and the RicT monomer may play distinct regulatory roles in vivoIMPORTANCE The RicA, RicF, and RicT proteins are widely conserved among the firmicute bacteria and play multiple roles in Bacillus subtilis Among the phenotypes associated with the inactivation of these proteins are the inability to be genetically transformed or to form biofilms, a decrease in sporulation frequency, and changes in the stability and maturation of multiple RNA species. Despite their importance, the molecular mechanisms of Ric protein activities have not been elucidated and the roles of the two iron-sulfur clusters on the complex of the three proteins are not understood. To unravel the mechanisms of Ric action, molecular characterization of the complex and of its constituent proteins is essential. This report represents a major step toward understanding the structures of the Ric proteins, the arrangement and roles of the Fe-S clusters, and the phenotypes associated with Ric mutations.

The whole genome insight on condition-specific redox activity and arsenopyrite interaction promoting As-mobilization by strain Lysinibacillus sp. B2A1.

A gram-positive spore former, Lysinibacillus sp. B2A1 was isolated from a high arsenic containing groundwater of Beimen2A well, Chianan Plain area, Southwestern Taiwan. Noteworthy, in the subsurface-mimicking anoxic incubation with a Na-lactate amendment system, this isolate could interact with arsenic-source mineral arsenopyrite and enhance arsenic mobilization. Further, the isolate showed elevated levels of arsenic resistance, 200 mM and 7.5 mM for arsenate and arsenite, respectively. Lysinibacillus sp. B2A1 demonstrated condition-specific redox activities including salient oxic oxidation of arsenite and anoxic reduction of arsenate. The elevated rate of As(III) oxidation (Vmax = 0.13 μM min-1 per 106 cells, Km = 15.3 μM) under oxic conditions was potent. Correlating with stout persistence in an arsenic-rich niche, remarkably, the lesser toxic effects of arsenic ions on bacterial sporulation frequency and germination highlight this strain's ability to thrive under catastrophic conditions. Moreover, the whole genome analysis elucidated diverse metal redox/resistance genes that included a potential arsenite S-adenosylmethyltransferase capable of mitigating arsenite toxicity. Owing to its arsenic resistance, conditional redox activities and ability to interact with arsenic minerals leading to arsenic mobilization, the presence of such spore-forming strains could be a decisive indication towards arsenic mobilization in subsurface aquifers having a high concentration of soluble arsenic or its source minerals.

Interactions Between Clostridioides difficile and Fecal Microbiota in in Vitro Batch Model: Growth, Sporulation, and Microbiota Changes.

Disturbance in gut microbiota is crucial for the development of Clostridioides difficile infection (CDI). Different mechanisms through which gut microbiota influences C. difficile colonization are known. However, C. difficile could also affect gut microbiota balance as previously demonstrated by cultivation of fecal microbiota in C. difficile conditioned medium. In current study, the interactions of C. difficile cells with gut microbiota were addressed. Three different strains (ribotypes 027, 014/020, and 010) were co-cultivated with two types of fecal microbiota (healthy and dysbiotic) using in vitro batch model. While all strains showed higher sporulation frequency in the presence of dysbiotic fecal microbiota, the growth was strain dependent. C. difficile either proliferated to comparable levels in the presence of dysbiotic and healthy fecal microbiota or grew better in co-culture with dysbiotic microbiota. In co-cultures with any C. difficile strain fecal microbiota showed decreased richness and diversity. Dysbiotic fecal microbiota was more affected after co-culture with C. difficile than healthy microbiota. Altogether, 62 OTUs were significantly changed in co-cultures of dysbiotic microbiota/C. difficile and 45 OTUs in co-cultures of healthy microbiota/C. difficile. However, the majority of significantly changed OTUs in both types of microbiota belonged to the phylum Firmicutes with Lachnospiraceae and Ruminococcaceae origin.

Morphometric and Internally Transcribed Spacer Gene Sequence-Based Characterization of <i>Alternaria burnsii</i>

Alternaria burnsii , the pathogen responsible for cumin blight, was collected from diseased plant samples from North Gujarat. AB-01 showed maximum growth at 28 ± 1°C. Conidia ranged from 44.92 to 63.28 μm in length and 10.84 to 24.36 μm in width whereas beak length of conidia ranged from 20.34 to 47.85 μm. The isolate AB-01 showed the highest sporulation frequency (1.24 x 10 5 /ml), but the highest percentage of disease intensity was observed in AB-08 (31.4). Internally transcribed spacer gene sequence based phylogenetic grouping using MEGA5.6 and the factorial analysis using DARwin5 reflect the presence of two distinct groups. The fast growing isolates that show high pathogenicity are present in group-I of the dendrogram whereas the members of group-II show grey black colony colour, light brown colony margin, plain irregular growth pattern, and comparatively larger beak length of conidia.

Genome engineering of Clostridium difficile using the CRISPR-Cas9 system

ObjectivesClostridium difficile is a notorious pathogenic species that can cause severe gastrointestinal infections in humans and animals. C. difficile infection (CDI) results in thousands of deaths worldwide every year. The elucidation of related mechanisms of CDI and exploration of potential therapeutic strategies are largely delayed due to the lack of efficient genetic engineering tools for C. difficile strains. MethodsPlasmids carrying the CRISPR-Cas9 system were constructed and transformed into C. difficile through conjugation. Mutants were identified using colony PCR with primers annealing to the regions flanking the target gene deletion/integration locus. Heat-survival assay was used to compare the sporulation frequency between the mutant with spo0A deletion and the wild type strain. The fluorescence in the mutant with the insertion of the green fluorescent protein (GFP) gene was inspected under a fluorescent microscope. ResultsAn efficient genome editing tool was developed for C. difficile based on the CRISPR-Cas9 system. With this tool, spo0A was deleted with a 100% mutation efficiency. Conversely, an anaerobic GFP gene was successfully inserted into the C. difficile chromosome (with a mutation efficiency of 80%). ConclusionsThe developed CRISPR-Cas9-based genome engineering tool will facilitate functional genomic studies in C. difficile as well as the elucidation of mechanisms related to host–bacteria interaction and pathogenesis of CDI. This will be highly beneficial for the development of innovative strategies for CDI diagnostics and therapies.

Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis.

In this study, a sporulation-specific gene (tentatively named cwlC) involved in mother cell lysis in Bacillus thuringiensis was characterized. The encoded CwlC protein consists of an N-terminal N-acetylmuramoyl-l-alanine amidase (MurNAc-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from Escherichia coli were able to directly bind to and digest the B. thuringiensis cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is an N-acetylmuramoyl-l-alanine amidase. Results of transcriptional analyses indicated that cwlC is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σK). Deletion of cwlC completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase for B. thuringiensis mother cell lysis during sporulation. Engineered B. thuringiensis strains targeting cwlC, which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.IMPORTANCE Mother cell lysis has been well studied in Bacillus subtilis, which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis in Bacillus thuringiensis CwlC of B. thuringiensis only shows 9 and 21% sequence identity with known B. subtilis mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ between B. subtilis and B. thuringiensis The cwlC gene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.

Is interspecific gene flow and speciation in peatmosses (Sphagnum) constrained by phylogenetic relationship and life-history traits?

Peatmosses are interesting for studies of speciation processes not only because of their frequent hybridization and recent diversification, but also their phenotypic diversity, ecological importance and ancient history. Diverse and widespread hybridization has been widely documented in the genus, but little is known about what factors underlie this phenomenon. We hypothesize that these factors include phylogenetic distance and variation in life-history traits of parental species. We summarize current knowledge about the occurrence of hybridization in peatmosses and explore how it is associated with phylogenetic distance and life-history trait variation of parental species. Possibly as much as one out of five (or more) peatmoss species hybridize, mostly producing allopolyploid hybrids. Parents of admixed haploids are more closely related to each other than parents of allopolyploids. Hybridization seems to be most frequent in 1) monoicous and polyoicous species exhibiting 2) relatively high sporulation frequency, 3) producing relatively small spores, as well as 4) growing in poor habitats. Surprisingly, neither phylogenetic proximity nor life-history trait variation explain patterns of hybridization in peatmosses, and other likely explanations for patterns observed are discussed.

Clinical, epidemiological and microbiological characteristics of relapse and re-infection in Clostridium difficile infection

Recurrent diarrhea is a common complication of Clostridium difficile infection (CDI). Recurrent CDI (r-CDI) may be produced by the persistence of spores (relapse) or by the acquisition of a new strain (reinfection). In this study, we analyze epidemiological, clinical, microbiological and laboratory data from patients with r-CDI, relapse, and reinfection-CDI over 5 years and compared with a control group (non r-CDI). Among 60 patients with r-CDI, 36 patients had stool samples collected from two or more episodes, which were molecularly analyzed. Based on ribotyping, 63.9% of the samples were relapse, and 36.1% reinfection. In a multivariable logistic regression analysis, previous antibiotic exposure was found to be a risk factor for r-CDI (OR: 2.23; 95% CI: 1.0–4.9; p = 0.04). Patients with relapse had previous antibiotic exposure more frequently than did patients with reinfection (p = 0.03), and patients with reinfection suffered more frequently from chronic liver disease (p = 0.02) than did relapse patients. Relapse patients compared with the control group had a higher percentage of previous antibiotic exposure, although the difference was statistically no significant (73.9% vs. 91.3 p = 0.06). No significant differences for the selected variables were observed between the reinfection and control groups, although we observed a higher percentage of patients with chronic liver disease (30.8% vs 13.3%; p = 0.08). All isolates were sensitive to metronidazole and vancomycin. No significant differences in antibiotic susceptibility were found between the different groups. Sporulation and germination frequency of r-CDI were higher than non r-CDI (p = 0.02 and p < 0.01, respectively). Nevertheless, there were statistically not significant differences between the relapse and reinfection groups. Both frequencies were compared between the first and second episode of CDI for the relapse and reinfection groups, but differences were not observed to be statistically significant. In conclusion, our study showed that the recurrence of CDI was associated with antibiotic use and sporulation/germination frequency, regardless of relapse or reinfection. The use of antibiotics would produce a dysbiosis and favor the persistence of the C. difficile spores and relapse. A possible alteration of the intestinal microbiota and the bile salts produced by chronic liver disease could favor reinfection.