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.

Temperature limits to deep subseafloor life in the Nankai Trough subduction zone.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.

Culture dependent and independent analyses suggest a low level of sharing of endospore-forming species between mothers and their children.

Spore forming bacteria comprise a large part of the human gut microbiota. However, study of the endospores in gut microbiota is limited due to difficulties of culturing and numerous unknown germination factors. In this study we propose a new method for culture-independent characterization of endospores in stool samples. We have enriched DNA of spore-forming bacterial species from stool samples of 40 mother-child pairs from a previously described mother-child cohort. The samples were exposed to a two-step purification process comprising ethanol and ethidium monoazide (EMA) treatment to first kill vegetative cells and to subsequently eliminate their DNA from the samples. The composition of the ethanol-EMA resistant DNA was characterized by 16S rRNA marker gene sequencing. Operational taxonomic units (OTUs) belonging to the Clostridia class (OTU1: Romboutsia, OTU5: Peptostreptococcaceae and OTU14: Clostridium senso stricto) and one belonging to the Bacillus class (OTU20: Turicibacter) were significantly more abundant in the samples from mothers and children after ethanol-EMA treatment than in those treated with ethanol only. No correlation was observed between ethanol-EMA resistant OTUs detected in children and in their mothers, which indicates that a low level of spore-forming species are shared between mothers and their children. Anaerobic ethanol-resistant bacteria were isolated from all mothers and all children over 1 year of age. Generally, in 70% of the ethanol-treated samples used for anaerobic culturing, 16S rRNA gene sequences of bacterial isolates corresponded to OTUs detected in these samples after EMA treatment. We report a new DNA-based method for the characterization of endospores in gut microbiota. Our method has high degree of correspondence to the culture-based method, although it requires further optimization. Our results also indicate a high turnover of endospores in the gut during the first two years of life, perhaps with a high environmental impact.

Subsurface Endospore-Forming Bacteria Possess Bio-Sealant Properties.

Concrete is a strong and fairly inexpensive building substance, but has several disadvantages like cracking that allows corrosion, thus reducing its lifespan. To mitigate these complications, long-lasting microbial self-healing cement is an alternative that is eco-friendly and also actively repairs cracks. The present paper describes the detailed experimental investigation on compressive strength of cement mortars, mixed with six alkaliphilic bacteria, isolated from subsurface mica mines of high alkalinity. The experiments showed that the addition of alkaliphilic isolates at different cell concentrations (104 and 106 cells/ml) enhanced the compressive strength of cement mortar, because the rapid growth of bacteria at high alkalinity precipitates calcite crystals that lead to filling of pores and densifying the concrete mix. Thus, Bacillus subtilis (SVUNM4) showed the highest compressive strength (28.61%) of cement mortar at 104 cells/ml compared to those of other five alkaliphilic isolates (Brevibacillus sp., SVUNM15-22.1%; P. dendritiformis, SVUNM11-19.9%; B. methylotrophicus, SVUNM9-16%; B. licheniformis, SVUNM14-12.7% and S. maltophilia, SVUNM13-9.6%) and controlled cement mortar as well. This method resulted in the filling of cracks in concrete with calcite (CaCO3), which was observed by scanning electron microscopy (SEM). Our results showed that the alkaliphilic bacterial isolates used in the study are effective in self-healing and repair of concrete cracks.

Exploiting the aerobic endospore-forming bacterial diversity in saline and hypersaline environments for biosurfactant production.

BACKGROUND: Biosurfactants are surface-active biomolecules with great applicability in the food, pharmaceutical and oil industries. Endospore-forming bacteria, which survive for long periods in harsh environments, are described as biosurfactant producers. Although the ubiquity of endospore-forming bacteria in saline and hypersaline environments is well known, studies on the diversity of the endospore-forming and biosurfactant-producing bacterial genera/species in these habitats are underrepresented. METHODS: In this study, the structure of endospore-forming bacterial communities in sediment/mud samples from Vermelha Lagoon, Massambaba, Dois Rios and Abraão Beaches (saline environments), as well as the Praia Seca salterns (hypersaline environments) was determined via denaturing gradient gel electrophoresis. Bacterial strains were isolated from these environmental samples and further identified using 16S rRNA gene sequencing. Strains presenting emulsification values higher than 30 % were grouped via BOX-PCR, and the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20 % NaCl to test their emulsifying activities in these extreme conditions. Mass spectrometry analysis was used to demonstrate the presence of surfactin. RESULTS: A diverse endospore-forming bacterial community was observed in all environments. The 110 bacterial strains isolated from these environmental samples were molecularly identified as belonging to the genera Bacillus, Thalassobacillus, Halobacillus, Paenibacillus, Fictibacillus and Paenisporosarcina. Fifty-two strains showed emulsification values of at least 30%, and they were grouped into 18 BOX groups. The stability of the emulsification values varied when the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20% NaCl. The presence of surfactin was demonstrated in one of the most promising strains. CONCLUSION: The environments studied can harbor endospore-forming bacteria capable of producing biosurfactants with biotechnological applications. Various endospore-forming bacterial genera/species are presented for the first time as biosurfactant producers.

Production of autoinducer-2 by aerobic endospore-forming bacteria isolated from the West African fermented foods.

Autoinducer-2 (AI-2) is a quorum-sensing (QS) molecule which mediates interspecies signaling and affects various bacterial behaviors in food fermentation. Biosynthesis of AI-2 is controlled by S-ribosylhomocysteine lyase encoded by the luxS gene. The objective of this study was to investigate production of AI-2 by aerobic endospore-forming bacteria (AEB) isolated from the West African alkaline fermented seed products Mantchoua and Maari. The study included 13 AEB strains of Bacillus subtilis, B. cereus, B. altitudinis, B. amyloliquefaciens, B. licheniformis, B. aryabhattai, B. safensis, Lysinibacillus macroides and Paenibacillus polymyxa. All the tested strains harbored the luxS gene and all strains except for P. polymyxa B314 were able to produce AI-2 during incubation in laboratory medium. Production of AI-2 by AEB was growth phase dependent, showing maximum activity at the late exponential phase. AI-2 was depleted from the culture medium at the beginning of the stationary growth phase, indicating that the tested AEB possess a functional AI-2 receptor that internalizes AI-2. This study provides the evidences of QS system in Bacillus spp. and L. macroides and new knowledge of AI-2 production by AEB. This knowledge contributes to the development of QS-based strategies for better control of alkaline fermentation.

Sporulation in Bacteria: Beyond the Standard Model.

Endospore formation follows a complex, highly regulated developmental pathway that occurs in a broad range of Firmicutes. Although Bacillus subtilis has served as a powerful model system to study the morphological, biochemical, and genetic determinants of sporulation, fundamental aspects of the program remain mysterious for other genera. For example, it is entirely unknown how most lineages within the Firmicutes regulate entry into sporulation. Additionally, little is known about how the sporulation pathway has evolved novel spore forms and reproductive schemes. Here, we describe endospore and internal offspring development in diverse Firmicutes and outline progress in characterizing these programs. Moreover, comparative genomics studies are identifying highly conserved sporulation genes, and predictions of sporulation potential in new isolates and uncultured bacteria can be made from these data. One surprising outcome of these comparative studies is that core regulatory and some structural aspects of the program appear to be universally conserved. This suggests that a robust and sophisticated developmental framework was already in place in the last common ancestor of all extant Firmicutes that produce internal offspring or endospores. The study of sporulation in model systems beyond B. subtilis will continue to provide key information on the flexibility of the program and provide insights into how changes in this developmental course may confer advantages to cells in diverse environments.

Paenibacillus hodogayensis sp. nov., capable of degrading the polysaccharide produced by Sphaerotilus natans.

Sphaerotilus natans is a sheathed bacterium often found in activated sludge that has a bulking problem. A bacterial strain that is able to degrade the extracellular polysaccharide produced by S. natans was isolated. The isolate was a spore-forming, aerobic, rod-shaped bacterium. The Gram reaction was variable or negative. The optimum growth temperature was 30 degrees C and the optimum pH was 8. The G+C content of the DNA was 55 mol%. The major cellular fatty acid and respiratory quinone were anteiso-C(15 : 0) and MK-7, respectively. Phylogenetic analysis based on the 16S rRNA gene indicated that the isolate was a member of the genus Paenibacillus. The nearest relative, with a similarity of 94.2 %, was Paenibacillus koleovorans, a bacterium capable of degrading the sheath of S. natans. The phenotypic characteristics of the isolate were apparently different from those of related species in the genus Paenibacillus. It is proposed that the isolate be designated Paenibacillus hodogayensis sp. nov. The type strain is SG(T) (=JCM 12520(T)=KCTC 3919(T)).

Paenibacillus phyllosphaerae sp. nov., a xylanolytic bacterium isolated from the phyllosphere of Phoenix dactylifera.

A bacterial strain, designated PALXIL04(T), was isolated from the phyllosphere of Phoenix dactylifera. Phylogenetic analysis placed the isolate within the genus Paenibacillus with the closest relatives being Paenibacillus curdlanolyticus and Paenibacillus kobensis. DNA-DNA hybridization measurements showed low DNA relatedness (15-20 %) between the isolate and its closest relatives. Cells were Gram-variable, facultatively anaerobic, motile, sporulating rods. Catalase and oxidase were produced by the organism. Cellulose, starch, aesculin and xylan were hydrolysed. Growth was supported by many carbohydrates as the carbon source. MK-7 was the predominant menaquinone and anteiso-C(15 : 0) the major fatty acid. The G+C content of the DNA was 50.7 mol%. Phylogenetic, DNA-DNA relatedness and phenotypic analyses indicated that strain PALXIL04(T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillus phyllosphaerae sp. nov. is proposed. The type strain is PALXIL04(T) (=LMG 22192(T)=CECT 5862(T)).

Paenibacillus koleovorans sp. nov., able to grow on the sheath of Sphaerotilus natans.

Two bacterial strains that are able to grow specifically on the sheath of a sheathed filamentous bacterium, Sphaerotilus natans, were isolated from soil samples. The sheath-degrading organisms, designated strains TB(T) and TK, are facultatively anaerobic and form endospores. The Gram reaction was negative at all stages of cultivation. The optimum growth temperature and pH were 30 degrees C and pH 7. The DNA G+C content was 54.0-55.8 mol%. MK-7 was the predominant menaquinone and anteiso-C15:0 was the major fatty acid. Phylogenetic analysis based on the 16S rDNA sequences revealed that the isolates were closely related to Paenibacillus chondroitinus, Paenibacillus alginolyticus, Paenibacillus koreensis, Paenibacillus validus, Paenibacillus larvae subsp. larvae and P. larvae subsp. pulvifaciens. The sequences were found to contain consensus sequences characteristic of all Paenibacillus species. The isolates were able to lyse and utilize the purified sheath of S. natans as the sole carbon and energy source. Acid was not produced from common carbon sources, allowing easy distinction from other members of Paenibacillus. It is concluded that the two strains represent a novel Paenibacillus species, for which the name Paenibacillus koleovorans sp. nov. is proposed. The type strain is strain TB(T) (= JCM 11186T = IAM 14926T = KCTC 13912T).

Thermovenabulum ferriorganovorum gen. nov., sp. nov., a novel thermophilic, anaerobic, endospore-forming bacterium.

A thermophilic, anaerobic, spore-forming bacterium (strain Z-9801T) was isolated from a terrestrial hydrothermal source in the Uzon caldera on the Kamchatka peninsula. Cells of strain Z-9801T were straight, sometimes branched rods, 0.5-0.6 microm in diameter and 1.5-7.0 microm in length, with peritrichous flagella. The temperature range for growth was 45-76 degrees C, with an optimum at 63-65 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.7-6.9. The substrates utilized by strain Z-9801T included peptone, yeast extract, beef extract, Casamino acids, starch, pyruvate, melibiose, sucrose, fructose, maltose, xylose and ribose. The fermentation products from melibiose were ethanol, acetate, H2 and CO2. Strain Z-9801T used H2 in the presence of Fe(III) and an organic electron donor. Strain Z-9801T reduced Fe(III), Mn(IV), nitrate, fumarate, sulfite, thiosulfate, elemental sulfur and 9,10-anthraquinone 2,6-disulfonate. The G+C content of strain Z-9801T DNA was 36 mol%. 16S rDNA sequence analysis revealed that the isolated organism forms a separate branch within the Bacillus/Clostridium group. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain Z-9801T (= DSM 14006T = UNIQEM 210T) should be assigned to a novel species of a new genus, Thermovenabulum ferriorganovorum gen. nov., sp. nov.