Endospore-forming Bacteria Research Articles

Penicillin-binding proteins exhibit catalytic redundancy during asymmetric cell division in Clostridioides difficile.

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the activity of SEDS family glycosyltransferases that polymerize glycan strands and class B penicillin-binding protein (bPBP) transpeptidases that cross-link them. While most bacteria encode multiple bPBPs that perform specialized roles during specific cellular processes, some bPBPs can play redundant roles that are important for resistance against certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs that drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, is important for synthesizing the asymmetric division septum and spore cortex peptidoglycan during sporulation in the pathogen Clostridioides difficile. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is unexpectedly dispensable for SpoVD to mediate asymmetric division. The dispensability of SpoVD's catalytic activity requires the presence of its SEDS partner, SpoVE, and is facilitated by the catalytic activity of another sporulation-specific bPBP, PBP3. Our data further suggest that PBP3 interacts with components of the asymmetric division machinery, including SpoVD. These findings suggest a possible mechanism by which bPBPs can be functionally redundant in diverse bacteria and facilitate antibiotic resistance.

Ionic liquid-assisted sample preparation mediates sensitive proteomic analysis of Bacillus subtilis spores

Endospore-forming bacteria are ubiquitous. Bacterial endospores are multilayered proteinaceous structures that protects the bacterial genome during stress conditions. They are also responsible for a wide range of critical clinical infections in humans. Precise analysis of spore-forming pathogens remains a major challenge in the field of proteomics because spore structures are highly resistant to conventional solubilizers and denaturing agents, such as sodium dodecyl sulfate and urea. We present an ionic liquid-assisted (i-soln) technique of sample preparation, called pTRUST, which enables shotgun analysis of Bacillus subtilis spores even when the starting materials are in the sub-microgram range. In proteomic analysis, this technique shows 50–2000-fold higher sensitivity than other conventional gel-based or gel-free methods (including one-pot sample processing). Using this technique, we identified 445 proteins with high confidence from trace amounts of highly pure spore preparations, including 52 of the 79 proteins (approximately 70%) previously demonstrated to be localized in spores in the SubtiWiki database and detected through direct protein analysis. Consequently, 393 additional proteins were identified as candidates for spore constitutive proteins. Twenty of these newly identified candidates were produced as green fluorescent protein fusion proteins, and each was evaluated for authenticity as a spore constituent using fluorescence microscopy analysis. The pTRUST method's sensitivity and reliability using the i-soln system, together with hitherto unreported proteins in spores, will enable an array of spore research for biological and clinical applications.

Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt.

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.

Çeşitli Kaynaklardan ve Ticari Gıda Takviyelerinden İzole Edilen Bacillus Türlerinin Bazı Probiyotik Özelliklerinin Karşılaştırmalı Değerlendirilmesi

Bacillus species are gram-positive, aerobic, peritrically flagellated and endospore-forming bacteria. They can be found everywhere in the environment, especially in soil (its common habitat), water, dust or in the air. Probiotics, which have beneficial health effects, constitute an important group of Bacillus species. This study aimed to isolate Bacillus from various sources, identify it molecularly and determine its probiotic properties.For this purpose, eight Bacillus subtilis, Bacillus coagulans and Bacillus clausii strains among 58 isolates from fish intestine, soil, ripened cheese and commercial probiotic supplements were identified and their probiotic properties were characterized. Firstly, Bacillus strains were molecularly identified by 16S rRNA PCR analysis. The growth of Bacillus isolates at various temperatures, salt concentrations, and pH levels, as well as tests for esculin hydrolysis, starch hydrolysis, nitrate reduction, and gas generation from glucose, were all investigated to assess the isolates' physiological and biochemical characteristics. In terms of probiotic potential of Bacillus isolates; tolerance of bile salt, cell surface hydrophobicity, auto aggregation, antibiotic susceptibility tests were conducted. In all analyses, strains obtained from food supplements showed high levels of hydrophobicity and auto-aggregation properties, and the highest values following these strains were observed in Bacillus subtilis strains (F1 and S2) isolated from fish intestines and soil, respectively. All strains showed strong growth features in bile salt conditions. It has been determined that antibiotic sensitivity varies depending on the strain. Overall, high sensitivity to tetracycline has been observed. In summary, this study revealed the potential probiotic properties of Bacillus isolates obtained from different sources. The study also compared these probiotic properties with probiotic Bacillus strains isolated from food supplements.

Optimization of Bacillus velezensis S26 sporulation for enhanced biocontrol of gray mold and anthracnose in postharvest strawberries

The growing demand for organic and pesticide-free food has prompt the adoption of biological strategies for controlling postharvest diseases. While strawberries are appreciated for their sensory characteristics and nutritional value, the pseudofruit are highly susceptible to fungal infections, leading to potential contamination with chemical residues. Bacillus spp. are endospore-forming bacteria that offer an alternative for managing various plant diseases. The present study aimed to investigate the effectiveness of both Bacillus velezensis S26 endospores and endospore-rich inoculant in controlling anthracnose and gray mold in strawberries. Initially, the sporulation process was fine-tuned, followed by the utilization of vegetative cells and endospores for in vitro assays. Subsequently, in vivo assays were performed using an endospore-rich inoculant on strawberries. The optimized sporulation conditions led to the production of 10.3 log10 spores mL−1 after 16 h of cultivation (sporulation yield of 96%). Both the vegetative cells and endospores of B. velezensis S26 inhibited mycelial growth and conidial germination of Botrytis cinerea and Colletotrichum spp., which are the causal agents of gray mold and anthracnose, respectively. While the cell viability of endospores decreased after storage, fresh and stored inoculants proved effective in controlling anthracnose and gray mold during storage. Consequently, the endospores of B. velezensis S26 demonstrated their viability and antagonistic potential against postharvest diseases in strawberries, thereby contributing to an extended bacterial shelf life.

Proteome profiling of Paenibacillus sonchi genomovar Riograndensis SBR5T under conventional and alternative nitrogen fixation

Paenibacillus sonchi SBR5T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N2-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N2-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo‑nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N2-fixation by Fe-only nitrogenase. SignificanceIn this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.

Multifactorial resistance of Bacillus subtilis spores to low-pressure plasma sterilization.

Common sterilization techniques for labile and sensitive materials have far-reaching applications in medical, pharmaceutical, and industrial fields. Heat inactivation, chemical treatment, and radiation are established methods to inactivate microorganisms, but pose a threat to humans and the environment and can damage susceptible materials or products. Recent studies have demonstrated that cold low-pressure plasma (LPP) treatment is an efficient alternative to common sterilization methods, as LPP's levels of radicals, ions, (V)UV-radiation, and exposure to an electromagnetic field can be modulated using different process gases, such as oxygen, nitrogen, argon, or synthetic (ambient) air. To further investigate the effects of LPP, spores of the Gram-positive model organism Bacillus subtilis were tested for their LPP susceptibility including wild-type spores and isogenic spores lacking DNA-repair mechanisms such as non-homologous end-joining (NHEJ) or abasic endonucleases, and protective proteins like α/β-type small acid-soluble spore proteins (SASP), coat proteins, and catalase. These studies aimed to learn how spores resist LPP damage by examining the roles of key spore proteins and DNA-repair mechanisms. As expected, LPP treatment decreased spore survival, and survival after potential DNA damage generated by LPP involved efficient DNA repair following spore germination, spore DNA protection by α/β-type SASP, and catalase breakdown of hydrogen peroxide that can generate oxygen radicals. Depending on the LPP composition and treatment time, LPP treatment offers another method to efficiently inactivate spore-forming bacteria.IMPORTANCESurface-associated contamination by endospore-forming bacteria poses a major challenge in sterilization, since the omnipresence of these highly resistant spores throughout nature makes contamination unavoidable, especially in unprocessed foods. Common bactericidal agents such as heat, UV and γ radiation, and toxic chemicals such as strong oxidizers: (i) are often not sufficient to completely inactivate spores; (ii) can pose risks to the applicant; or (iii) can cause unintended damage to the materials to be sterilized. Cold low-pressure plasma (LPP) has been proposed as an additional method for spore eradication. However, efficient use of LPP in decontamination requires understanding of spores' mechanisms of resistance to and protection against LPP.

Diversification of division mechanisms in endospore-forming bacteria revealed by analyses of peptidoglycan synthesis in Clostridioides difficile

The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions.

Dispersal and Distribution of Thermophilic Endospores in Deep-Sea Ecosystems

The distance-decay relationship is a central concept in biogeography and spatial biodiversity, describing how two distinct entities decrease in similarity as the distance between them increases. The decay of community similarity with geographical distance is driven by multiple factors, such as gene drift, environmental selection, and the accumulation of mutations over time. While the distance-decay relationship has been recognized for several decades, there are certain circumstances where the biogeographical patterns of certain species and communities cannot be predicted by this relationship. The example addressed in this project is the case of thermophilic endospore-forming bacteria found in permanently cold deep ocean sediments. Thermophilic endospores (thermospores) are routinely found on the deep ocean floor, a permanently cold environment that does not support their metabolic activity (Hubert et al. 2009). Thermospores are metabolically dormant states developed by some thermophilic bacteria having optimal growth temperatures between 40°C and 70°C. There is evidence that these heat-loving bacteria originate from the deep subsurface and are transported upward to the deep ocean via geological features of the oceanic crust including geofluid fluxes in high-temperature axial systems and natural hydrocarbon seeps (Gittins et al. 2022). Due to the ability of endospores to stay viable for thousands of years and resist a wide range of physicochemical stressors, they can disperse over long distances while remaining unaffected by changing factors such as selection, drift, or mutation (Gittins et al. 2022; Fig. 1). Consequently, these thermospores have the potential to challenge the distance-decay relationship and exhibit unique biogeographical patterns. This work quantifies endospores in sediment cores at and around hydrocarbon seeps using cores from deep-sea Scotian Slope sediments. Given the challenge of quantifying specific groups of endospores (e.g., thermophiles but not mesophiles), high-temperature germination assays that allow tracking of an exponential increase in signal as spores in marine sediment samples germinate and grow can be used. Distinct exponential increases can be attributed to different populations of germinated thermospores in the post-germination growth phase and are being monitored through measurements of sulfate reduction rates (using radiolabelled 35SO4), strain-specific quantitative PCR (qPCR), and fluorescence in-situ hybridization (FISH) (Rezende et al. 2017). These measurements revealed growth dynamics enabling an estimation of the initial cell numbers using exponential functions. These approaches are being applied to samples obtained via push coring using a remotely operated vehicle at different distances from deep sea hydrocarbon seeps, to test for the presence of abundance gradients of different thermospores. Current results already show a difference in thermospore abundance between sites at different distances from the seep. Quantitative tracking of the dispersal of thermospores by oceanic currents in the deep sea provides an excellent opportunity to investigate their distribution and potential to colonize more remote habitats, improving the understanding of microbial biodiversity and biogeography on our planet. By investigating thermospores and their interactions with the geophysical features of the deep sea and deep subsurface, this study aims to challenge the canonical idea that speciation occurs with distance and presents new perspectives on the theme of microbial biogeography. The evidence supporting the subsurface origin of thermophilic endospores, along with the proposed model for their dispersal and distribution, will contribute toward improving an integrated understanding of ecology and geology and their intersection in the microbial realm.

Elevated bacterial endospores associated with thermogenic hydrocarbon seeps in deep sea sediments.

Introduction and approach Bacterial endospore distributions in marine sediments are influenced by geological conduits providing routes for subsurface to surface microbial dispersal. To examine this phenomenon in more detail, endospore abundance was determined by quantifying the biomarker 2,6-pyridine dicarboxylic acid (dipicolinic acid or DPA) in 16 deep sea sediment cores from hydrocarbon prospective areas in the NW Atlantic Ocean. DPA is specific to endospore-forming bacteria from the phylum Firmicutes and constitutes a significant percentage of endospore dry weight. DPA is therefore a potential biomarker for sediment dwelling endospores and geological conduits. Piston cores (10), gravity cores (3) and box cores (3) were collected during two expeditions to the Scotian Slope in the NW Atlantic Ocean off the east coast of Canada aboard the CCGS Hudson in 2016 and 2018 (Campbell (2016), Campbell and Normandeau (2018), Campbell and MacDonald. (2016)). Sampling sites were 1970 to 2791 m water depth, with piston cores (n=3) ranging from 344 to 953 cmbsf and gravity cores (n=10) ranging from 43 to 739 cmbsf, box coring captured the top 25 cmbsf. To address the efficacy of DPA biomarker analysis as a tool for hydrocarbon seep location we established a modified Tb3+ chelation method (Lomstein and Jørgensen (2012), Rattray (2021)). Sediment samples were extracted using acid hydrolysis, chelated with Tb3+ and analysed using HPLC fluorescence, measuring at 270 nm emission and 545 nm excitation. DPA concentrations were converted to Endospore numbers were calculated using 2.24 fmol DPA per endospore (Fichtel 2007), a conversion factor routinely used in other studies (Braun 2017, Gittins 2022, Heuer 2020, Lomstein 2012, Rattray 2022, Wörmer 2019, Lomstein and Jørgensen 2012). DPA concentrations were compared with measurements of over 250 different gaseous and liquid hydrocarbon compounds used to assess for the presence of thermogenic hydrocarbons. Results and discussion Samples and locations were assessed as being thermogenic hydrocarbon gas positive (stations 16-41, 18-07) or thermogenic hydrocarbon negative based on the abundance of C1-C5 hydrocarbons in sediments sampled from the same cores. Station 18-14 contained hydrocarbons from biogenic origin. Station 18-06 is the only site with higher endospore abundance but that was determined to be hydrocarbon negative. Deep water Scotian Slope sediment cores show high endospore abundance correlates with thermogenic hydrocarbon seeps (Fig. 1). Cores from locations lacking evidence for thermogenic hydrocarbons generally contained significantly lower endospore abundances, with the notable exception of site 18-06. This potential paleoenvironmental hydrocarbon seep site highlights the utility of a DPA proxy for potentially identifying ancient hydrocarbon seeps and investigating past geological systems. The association of high endospore abundances with thermogenic hydrocarbons and the quantity of gas expulsion points to an interesting new biological tool for understanding hydrocarbon seepage in the deep biosphere, based on DPA assays in marine sediments.

Genome analysis of Tepidibacter sp. SWIR-1, an anaerobic endospore-forming bacterium isolated from a deep-sea hydrothermal vent.

Tepidibacter sp. SWIR-1, a putative new species isolated from deep-sea hydrothermal vent field on the Southwest Indian Ridge (SWIR), is an anaerobic, mesophilic and endospore-forming bacterium belonging to the family Peptostreptococcaceae. In this study, we present the complete genome sequence of strain SWIR-1, consists of a single circular chromosome comprising 4,122,966 nucleotides with 29.25% G+C content and a circular plasmid comprising 38,843 nucleotides with 29.46% G+C content. In total, 3861 protein coding genes, 104 tRNA genes and 46 rRNA genes were obtained. SWIR-1 genome contains numerous genes related to sporulation and germination. Compared with the other three Tepidibacter species, SWIR-1 contained more spore germination receptor proteins. In addition, SWIR-1 contained more genes involved in chemotaxis and two-component systems than other Tepidibacter species. These results indicated that SWIR-1 has developed versatile adaptability to the Southwest Indian Ridge hydrothermal vent environment. The genome of strain SWIR-1 will be helpful for further understanding adaptive strategies used by bacteria dwelling in the deep-sea hydrothermal vent environments of different oceans.

Oxytetracycline-resistant Paenibacillus larvae identified in commercial beekeeping operations in Saskatchewan using pooled honey sampling.

American foulbrood (AFB) is an infectious disease of honey bee brood caused by the endospore-forming bacterium Paenibacillus larvae. P. larvae spores are resilient in the environment, thus colonies with clinical signs of AFB are often destroyed by burning to eradicate the causative agent. To prevent outbreaks of AFB, oxytetracycline metaphylaxis is widely used in North America, resulting in sustained selective pressure for oxytetracycline resistance in P. larvae. To determine if antimicrobial resistance (AMR) is present among P. larvae isolates from commercial beekeeping operations in Saskatchewan, Canada, we performed antimicrobial susceptibility testing of 718 P. larvae samples cultured from pooled, extracted honey collected from 52 beekeepers over a 2-y period, 2019 and 2020. We found that 65 of 718 (9%) P. larvae samples collected from 8 beekeepers were resistant to oxytetracycline with minimum inhibitory concentration (MIC) values of 64-256 µg/mL. Eight of 718 (1%) samples from 4 beekeepers had intermediate resistance to oxytetracycline (MIC: 4-8 µg/mL). Susceptibility testing for tylosin and lincomycin indicated that P. larvae in Saskatchewan continue to be susceptible to these antimicrobials (tylosin MIC: <1 µg/mL, lincomycin MIC: ≤2 µg/mL). Most oxytetracycline-resistant P. larvae samples were identified in northeastern Saskatchewan. Whole-genome sequence analysis identified the P. larvae-specific plasmid pMA67 with tetracycline-resistance gene tet(L) in 9 of 11 oxytetracycline-resistant P. larvae isolates sequenced. Our results highlight the advantage of using pooled, extracted honey as a surveillance tool for monitoring AMR in P. larvae.

The Bactericidal Activity of a Novel Aneurinibacillus aneurinilyticus Isolate Effectively Controls Foodborne Pathogens Campylobacter jejuni and Listeria monocytogenes

Human zoonotic infection with Campylobacter is a major cause of gastroenteritis in the United States and worldwide. Listeria monocytogenes causes a potentially fatal infection in humans and is often attributed to contaminated food. Genetic typing has demonstrated that Campylobacter infection is often associated with the consumption of contaminated poultry products, with Campylobacter often colonizing the poultry gastrointestinal tract, while listeriosis is commonly associated with the consumption of contaminated ready-to-eat (RTE) foods. In this study, a strain of endospore-forming bacterium (strain NH) that is bactericidal towards the human food pathogens Campylobacter jejuni and Listeria monocytogenes was identified and characterized. Transwell experiments demonstrated that the bactericidal effect on both C. jejuni and L. monocytogenes is due to secretions from the spore former. These foodborne pathogens consistently exhibited 7 log reductions in growth when exposed to the NH cell-free culture filtrate. Sequencing of the 16s rRNA gene V4 variable region and analysis of the full-length 16s rRNA gene sequence from the WGS indicated that strain NH belongs to the species Aneurinibacillus aneurinilyticus. A microplate bioassay demonstrated that a bactericidal substance that is sensitive to protease could be collected from cell-free filtrates by salting out with ammonium sulfate. Gel filtration chromatography indicated a native molecular weight for the bactericidal protein of ca. 50 kDa, consistent with a class III bacteriocin. The active protein bound strongly to a cation-exchange resin and with an isoelectric point of ten, suggesting a positively charged protein. Both cation-exchange chromatography and isoelectric focusing indicated the enrichment of an 11 kDa protein on SDS-PAGE. This protein was identified through mass spectroscopy as the flgM protein, an anti-sigma factor. Analysis of whole genome sequencing (WGS) of the strain NH genome indicated the presence of a number of non-conservative amino acid substitutions in the flgM-gene-derived amino acid sequence of strain NH and A. aneurinilyticus compared to other members of the Aneurinibacillus genus. Further investigation is needed to determine whether these substitutions are correlated with the bactericidal activity. The identified strain may be useful as a feed additive for the pre-harvest control of Campylobacter jejuni in poultry.

Sporolactobacillus mangiferae sp. nov., a spore-forming lactic acid bacterium isolated from tree bark in Thailand.

A Gram-stain-positive, facultatively anaerobic and endospore-forming rod-shaped bacterium, designed strain CPB3-1T, was isolated from tree bark. This homofermentative strain produced dl-lactic acid from glucose. It grew at 20-45 °C, pH 4.0-9.5 and in 0-3.0 % (w/v) NaCl. It contained meso-diaminopimelic acid in cell-wall peptidoglycan and had menaquinone with seven isoprene units (MK-7) as the predominant component. The major fatty acid was anteiso-C17 : 0. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid and an unknown lipid. Based on the results of 16S rRNA gene sequence analysis, strain CPB3-1T belonged to the genus Sporolactobacillus and was closely related to Sporolactobacillus kofuensis DSM 11701T and Sporolactobacillus spathodeae BK117-1T (both 96.7 % similarity), Sporolactobacillus inulinus NRIC 1133T and Sporolactobacillus terrae DSM 11697T (both 96.6 % similarity), and Sporolactobacillus shoreicorticis MK21-7T, Sporolactobacillus laevolacticus DSM 442T, Sporolactobacillus shoreae BK92T and Sporolactobacillus pectinivorans GD201205T (all 95.8-96.5 % similarity). The draft genome of strain CPB3-1T contained 2 930 919 bps with 3117 coding genes. The DNA G+C content was 45.1 mol%. The digital DNA-DNA hybridization values between strain CPB3-1T and closely related type strains were 19.2-24.0 %. The average nucleotide identity (84.0-87.6 %) and average amino acid identity (66.5-76.3 %) values were lower than the cut-off values for species delineation. Strain CPB3-1T was clearly distinguished from related Sporolactobacillus species based on its phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence similarity and the results of draft genome analysis. Therefore, the strain represents a novel species of the genus Sporolactobacillus, for which the name Sporolactobacillus mangiferae sp. nov. is proposed. The type strain is CPB3-1T (=JCM 35082T=TISTR 10004T).

Unique relationships between phages and endospore-forming hosts.

As part of their survival strategy under harsh environmental conditions, endospore-forming bacteria can trigger a sporulation developmental program. Although the regulatory cascades that precisely control the transformation of vegetative bacteria into mother cells and resilient spores have been described in detail, less is known about how bacteriophages that prey on endospore-formers exploit sporulation. Herein, we argue that phages infecting these bacteria have evolved several specific molecular mechanisms, not yet known in other bacteria, that manifest from the phage-driven alliance to negative effects on the host. We anticipate that the relationships between phages and endospore-formers outlined here will inspire studies on phage ecology and evolution, and could facilitate important advances in the development of phage therapies against pathogenic spore-formers.

Paenibacillus melissococcoides sp. nov., isolated from a honey bee colony affected by European foulbrood disease.

A novel, facultatively anaerobic, Gram-stain-positive, motile, endospore-forming bacterium of the genus Paenibacillus , designated strain 2.1T, was isolated from a colony of Apis mellifera affected by European foulbrood disease in Switzerland. The rod-shaped cells of strain 2.1T were 2.2–6.5 µm long and 0.7–1.1 µm wide. Colonies of strain 2.1T were orange-pigmented under oxic growth conditions on solid basal medium at 35–37 °C. Strain 2.1T showed catalase and cytochrome c oxidase activity. Its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and phospholipid. The only respiratory quinone was menaquinone 7, and the major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0, iso-C17 : 0 and palmitic acid (C16 : 0), which is consistent with other members of the genus Paenibacillus . The G+C content of the genomic DNA of strain 2.1T was 53.3 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence similarity showed that strain 2.1T was closely related to Paenibacillus dendritiformis LMG 21716T (99.7 % similarity) and Paenibacillus thiaminolyticus DSM 7262T (98.8 %). The whole-genome average nucleotide identity between strain 2.1T and the type strains of P. dendritiformis and P. thiaminolyticus was 92 and 91 %, respectively, and thus lower than the 95 % threshold value for delineation of genomic prokaryotic species. Based on the results of phylogenetic, genomic, phenotypic and chemotaxonomic analyses we propose the name Paenibacillus melissococcoides sp. nov. for this novel Paenibacillus species. The type strain is 2.1T (=CCOS 2000T=DSM 113619T=LMG 32539T).

Paenibacillus mangrovi sp. nov., a novel endophytic bacterium isolated from bark of Kandelia candel.

A endospore-forming bacterium, designated strain KQZ6P-2T, was isolated from surface-sterilized bark of the mangrove plant Kandelia candel, collected from Maowei Sea Mangrove Nature Reserve in Guangxi Zhuang Autonomous Region, China. Strain KQZ6P-2T was able to grow at NaCl concentrations in the range of 0-3 % (w/v) with optimum growth at 0-1 % (w/v) NaCl. Growth occurred at 20-42 °C (optimal growth at 30-37 °C) and pH 5.5-6.5 (optimal growth at pH 6.5). The 16S rRNA gene sequence similarity between strain KQZ6P-2T and its closest phylogenetic neighbour Paenibacillus chibensis JCM 9905T was 98.2 %. Phylogenetic analyses using 16S rRNA gene sequences showed that strain KQZ6P-2T formed a distinct lineage with Paenibacillus chibensis JCM 9905T. The draft genome of strain KQZ6P-2T was 5 937 633 bp in size and its DNA G+C content was 47.2mol%. Comparative genome analysis revealed that the average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values among strain KQZ6P-2T and its related species were below the cut-off levels of 95, 70 and 95.5%, respec-tively. The cell-wall peptidoglycan of strain KQZ6P-2T contained meso-diaminopimelic acid as the diagnostic diamino acid. Major cellular fatty acids were anteiso-C15:0 and C16:0. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, four unidentified phospholipids, an unidentified aminolipid and five unidentified lipids. Based on phylogenetic, phenotypic and chemotaxonomic data, strain KQZ6P-2T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus mangrovi sp. nov. is proposed. The type strain is KQZ6P-2T (=MCCC 1K07172T =JCM 34931T).

Near-Complete Whole-Genome Sequence of Paenibacillus sp. nov. Strain J5C2022, a Sucretolerant and Endospore-Forming Bacterium Isolated from Highly Concentrated Sugar Brine.

Here, we present a 7.62-Mbp genome sequence of Paenibacillus sp. nov. strain J5C2022, a Gram-positive facultatively anaerobic bacterium that was isolated from 4-month-old fruit pickle brine and sequenced using the Illumina platform.

Genome-wide mapping of GlnR-binding sites reveals the global regulatory role of GlnR in controlling the metabolism of nitrogen and carbon in Paenibacillus polymyxa WLY78

BackgroundPaenibacillus polymyxa WLY78 is a Gram-positive, endospore-forming and N2-fixing bacterium. Our previous study has demonstrated that GlnR acts as both an activator and a repressor to regulate the transcription of the nif (nitrogen fixation) operon (nifBHDKENXhesAnifV) according to nitrogen availability, which is achieved by binding to the two GlnR-binding sites located in the nif promoter region. However, further study on the GlnR-mediated global regulation in this bacterium is still needed.ResultsIn this study, global identification of the genes directly under GlnR control is determined by using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assays (EMSA). Our results reveal that GlnR directly regulates the transcription of 17 genes/operons, including a nif operon, 14 nitrogen metabolism genes/operons (glnRA, amtBglnK, glnA1, glnK1, glnQHMP, nasA, nasD1, nasD2EF, gcvH, ansZ, pucR, oppABC, appABCDF and dppABC) and 2 carbon metabolism genes (ldh3 and maeA1). Except for the glnRA and nif operon, the other 15 genes/operons are newly identified targets of GlnR. Furthermore, genome-wide transcription analyses reveal that GlnR not only directly regulates the expression of these 17 genes/operons, but also indirectly controls the expression of some other genes/operons involved in nitrogen fixation and the metabolisms of nitrogen and carbon.ConclusionThis study provides a GlnR-mediated regulation network of nitrogen fixation and the metabolisms of nitrogen and carbon.

Radiobacillus kanasensis sp. nov., a halotolerant bacterium isolated from woodland soil.

A novel Gram-positive, aerobic, rod-shaped, non-motile, endospore-forming salt-tolerant bacterium strain (80T), was isolated from woodland soil collected near Kanas lake in the Altay region of Xinjiang, PR China. The strain grew at 15-45 °C, pH6.0-9.0 and with 0-14 % (w/v) NaCl. The complete genome size of the novel strain was 4 031 766 bp including a circle chromosome and a circle plasmid. The genomic DNA G+C content was 38.99 mol %. Phylogenetic analysis based on 16S rRNA gene sequence and genome showed that strain 80T has the highest similarity to Radiobacillus deserti TKL69T. However, the novel strain showed an average nucleotide identity value of 78.65 % (lower than 95 %) and a digital DNA-DNA hybridization value of 22.30 % with R. deserti TKL69T based on the genome sequences. The major fatty acids were anteiso-C15 : 0, iso-C15 : 0, anteiso-C17:0 and C16 : 1 ω7c alcohol. The only respiratory quinone was MK-7. The cell wall peptidoglycan was meso-diaminopimelic acid. Diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid, one unidentified aminophospholipid and two unidentified glycolipids were identified as the major polar lipids. The phylogenetic, phenotypic and chemotaxonomic analyses showed that strain 80T represents a novel species of the genus Radiobacillus and the name Radiobacillus kanasensis sp. nov. is proposed. The type strain is 80T (=GDMCC 1.2844T=JCM 35077T).