Enzymes produced by halotolerant spore-forming gram-positive bacterial strains isolated from a resting habitat (Restinga de Jurubatiba) in Rio de Janeiro, Brazil: focus on proteases.

The screening for hydrolases-producing, halotolerant, and spore-forming gram-positive bacteria from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides, a plant found in the Restinga de Jurubatiba located at the northern region of Rio de Janeiro State, Brazil, resulted in the isolation of 22 strains. These strains were identified as Halobacillus blutaparonensis (n = 2), Oceanobacillus picturae (n = 5), and Oceanobacillus iheyensis (n = 15), and all showed the ability to produce different extracellular enzymes. A total of 20 isolates (90.9 %) showed activity for protease, 5 (22.7 %) for phytase, 3 (13.6 %) for cellulase, and 2 (9.1 %) for amylase. Some bacterial strains were capable of producing three (13.6 %) or two (9.1 %) distinct hydrolytic enzymes. However, no bacterial strain with ability to produce esterase and DNase was observed. The isolate designated M9, belonging to the species H. blutaparonensis, was the best producer of protease and also yielded amylase and phytase. This strain was chosen for further studies regarding its protease activity. The M9 strain produced similar amounts of protease when grown either without or with different NaCl concentrations (from 0.5 to 10 %). A simple inspection of the cell-free culture supernatant by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of three major alkaline proteases of 40, 50, and 70 kDa, which were fully inhibited by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-phenylalanine chloromethyl ketone (TPCK) (two classical serine protease inhibitors). The secreted proteases were detected in a wide range of temperature (from 4 to 45 °C) and their hydrolytic activities were stimulated by NaCl (up to 10 %). The serine proteases produced by the M9 strain cleaved gelatin, casein, albumin, and hemoglobin, however, in different extensions. Collectively, these results suggest the potential use of the M9 strain in biotechnological and/or industrial processes.

Effects of short chain fatty acid producing bacteria on epigenetic regulation of FFAR3 in type 2 diabetes and obesity.

The human gut microbiota and microbial influences on lipid and glucose metabolism, satiety, and chronic low-grade inflammation are known to be involved in metabolic syndrome. Fermentation end products, especially short chain fatty acids, are believed to engage the epigenetic regulation of inflammatory reactions via FFARs (free fatty acid receptor) and other short chain fatty acid receptors. We studied a potential interaction of the microbiota with epigenetic regulation in obese and type 2 diabetes patients compared to a lean control group over a four month intervention period. Intervention comprised a GLP-1 agonist (glucagon-like peptide 1) for type 2 diabetics and nutritional counseling for both intervention groups. Microbiota was analyzed for abundance, butyryl-CoA:acetate CoA-transferase gene and for diversity by polymerase chain reaction and 454 high-throughput sequencing. Epigenetic methylation of the promoter region of FFAR3 and LINE1 (long interspersed nuclear element 1) was analyzed using bisulfite conversion and pyrosequencing. The diversity of the microbiota as well as the abundance of Faecalibacterium prausnitzii were significantly lower in obese and type 2 diabetic patients compared to lean individuals. Results from Clostridium cluster IV and Clostridium cluster XIVa showed a decreasing trend in type 2 diabetics in comparison to the butyryl-CoA:acetate CoA-transferase gene and according to melt curve analysis. During intervention no significant changes were observed in either intervention group. The analysis of five CpGs in the promoter region of FFAR3 showed a significant lower methylation in obese and type 2 diabetics with an increase in obese patients over the intervention period. These results disclosed a significant correlation between a higher body mass index and lower methylation of FFAR3. LINE-1, a marker of global methylation, indicated no significant differences between the three groups or the time points, although methylation of type 2 diabetics tended to increase over time. Our results provide evidence that a different composition of gut microbiota in obesity and type 2 diabetes affect the epigenetic regulation of genes. Interactions between the microbiota and epigenetic regulation may involve not only short chain fatty acids binding to FFARs. Therefore dietary interventions influencing microbial composition may be considered as an option in the engagement against metabolic syndrome.

Thermophilic lignocellulose deconstruction.

Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. 'free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective.

Characterization of a metal-dependent D-psicose 3-epimerase from a novel strain, Desmospora sp. 8437.

The rare sugar d-psicose is an ideal sucrose substitute for food products, due to having 70% of the relative sweetness but 0.3% of the energy of sucrose. It also shows important physiological functions. d-Tagatose 3-epimerase (DTEase) family enzymes can produce d-psicose from d-fructose. In this paper, a new member of the DTEase family of enzymes was characterized from Desmospora sp. 8437 (GenBank accession no. WP_009711885 ) and was named Desmospora sp. d-psicose 3-epimerase (DPEase) due to its highest substrate specificity toward d-psicose. Desmospora sp. DPEase was strictly metal-dependent and displayed maximum activity in the presence of Co(2+). The optimum pH and temperature were 7.5 and 60 °C, respectively. The enzyme was relatively thermostable below 50 °C, but easily lost initial activity when preincubated at 60 °C. The thermostability property was almost not affected by the addition of Co(2+). Desmospora sp. DPEase had relatively high catalysis efficiency for the substrates d-psicose and d-fructose, which were measured to be 327 and 116 mM(-1) min(-1), respectively. The equilibrium ratio between d-psicose and d-fructose of Desmospora sp. DPEase was 30:70. The enzyme could produce 142.5 g/L d-psicose from 500 g/L of d-fructose, suggesting that the enzyme is a potential d-psicose producer for industrial production.

Stage 0 sporulation gene A as a molecular marker to study diversity of endospore-forming Firmicutes.

In this study, we developed and validated a culture-independent method for diversity surveys to specifically detect endospore-forming Firmicutes. The global transcription regulator of sporulation (spo0A) was identified as a gene marker for endospore-forming Firmicutes. To enable phylogenetic classification, we designed a set of primers amplifying a 602 bp fragment of spo0A that we evaluated in pure cultures and environmental samples. The amplification was positive for 35 strains from 11 genera, yet negative for strains from Alicyclobacillus and Sulfobacillus. We also evaluated various DNA extraction methods because endospores often result in reduced yields. Our results demonstrate that procedures utilizing increased physical force improve DNA extraction. An optimized DNA extraction method on biomass pre-extracted from the environmental sample source (indirect DNA extraction) followed by amplification with the aforementioned primers for spo0A was then tested in sediments from two different sources. Specifically, we validated our culture-independent diversity survey methodology on a set of 8338 environmental spo0A sequences obtained from the sediments of Lakes Geneva (Switzerland) and Baikal (Russia). The phylogenetic affiliation of the environmental sequences revealed a substantial number of new clades within endospore-formers. This novel culture-independent approach provides a significant experimental improvement that enables exploration of the diversity of endospore-forming Firmicutes.

Quantification of endospore-forming firmicutes by quantitative PCR with the functional gene spo0A.

Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 10(4) cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.

[Isolation and functional characterization of lipase from the thermophilic alkali-tolerant bacterium Thermosyntropha lipolytica].

As a result of sequencing the genome of the termophilic alkali-tolerant lipolytic bacterium Thermosyntropha lipolytica, the gene encoding a lipase secreted into the medium was identified. The recombinant enzyme was expressed in Escherichia coli. It was isolated, purified, and functionally characterized. The lipase exhibited hydrolytic activity toward para-nitrophenyl esters of various chain lengths, as well as triglycerides, including vegetable oils. The optimal reaction conditions were achieved at temperatures from 70 to 80 degrees C and pH 8.0. Enzyme saved more than 80% of its activity in the presence of 10% methanol. This new thermostable lipase may be a promising biocatalyst for organic synthesis; it may find application in the food and detergent industry and biodiesel production.

Screening of anaerobic activities in sediments of an acidic environment: Tinto River.

The Tinto River (Huelva, Spain) is a natural acidic rock drainage environment produced by the bio-oxidation of metallic sulfides from the Iberian Pyritic Belt. A geomicrobiological model of the different microbial cycles operating in the sediments was recently developed through molecular biological methods, suggesting the presence of iron reducers, methanogens, nitrate reducers and hydrogen producers. In this study, we used a combination of molecular biological methods and targeted enrichment incubations to validate this model and prove the existence of those potential anaerobic activities in the acidic sediments of Tinto River. Methanogenic, sulfate-reducing, denitrifying and hydrogen-producing enrichments were all positive at pH between 5 and 7. Methanogenic enrichments revealed the presence of methanogenic archaea belonging to the genera Methanosarcina and Methanobrevibacter. Enrichments for sulfate-reducing microorganisms were dominated by Desulfotomaculum spp. Denitrifying enrichments showed a broad diversity of bacteria belonging to the genera Paenibacillus, Bacillus, Sedimentibacter, Lysinibacillus, Delftia, Alcaligenes, Clostridium and Desulfitobacterium. Hydrogen-producing enrichments were dominated by Clostridium spp. These enrichments confirm the presence of anaerobic activities in the acidic sediments of the Tinto River that are normally assumed to take place exclusively at neutral pH.

Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB).

Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.

Characterization of Halanaerocella petrolearia gen. nov., sp. nov., a new anaerobic moderately halophilic fermentative bacterium isolated from a deep subsurface hypersaline oil reservoir : New taxa: Firmicutes (Class Clostridia, Order Halanaerobiales, Halobacteroidaceae, Halobacteroides).

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47°C with an optimum at 40-45°C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H(2), and CO(2). The predominant cellular fatty acids were non-branched fatty acids C(16:1), C(16:0), and C(14:0). The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).

Robinsoniella peoriensis bacteremia in a patient with pancreatic cancer.

Robinsoniella peoriensis was recently identified as a Gram-positive, spore-forming, anaerobic rod originally isolated from swine manure storage pits. We describe here a case of R. peoriensis bacteremia in a 42-year-old male with pancreatic cancer. The identification was confirmed by unique phenotypic profiles and partial 16S rRNA gene sequences.

PCR detection of thermophilic spore-forming bacteria involved in canned food spoilage.

Thermophilic bacteria that form highly heat-resistant spores constitute an important group of spoilage bacteria of low-acid canned food. A PCR assay was developed in order to rapidly trace these bacteria. Three PCR primer pairs were designed from rRNA gene sequences. These primers were evaluated for the specificity and the sensitivity of detection. Two primer pairs allowed detection at the species level of Geobacillus stearothermophilus and Moorella thermoacetica/thermoautrophica. The other pair allowed group-specific detection of anaerobic thermophilic bacteria of the genera Thermoanaerobacterium, Thermoanaerobacter, Caldanerobium and Caldanaerobacter. After a single enrichment step, these PCR assays allowed the detection of 28 thermophiles from 34 cans of spoiled low-acid food. In addition, 13 ingredients were screened for the presence of these bacteria. This PCR assay serves as a detection method for strains able to spoil low-acid canned food treated at 55°C. It will lead to better reactivity in the canning industry. Raw materials and ingredients might be qualified not only for quantitative spore contamination, but also for qualitative contamination by highly heat-resistant spores.

Gracilibacillus ureilyticus sp. nov., a halotolerant bacterium from a saline-alkaline soil.

A Gram-stain-positive, halotolerant, neutrophilic, rod-shaped bacterium, strain MF38(T), was isolated from a saline-alkaline soil in China and subjected to a polyphasic taxonomic characterization. The isolate grew in the presence of 0-15 % (w/v) NaCl and at pH 6.5-8.5; optimum growth was observed with 3.0 % (w/v) NaCl and at pH 7.0. Chemotaxonomic analysis showed menaquinone MK-7 as the predominant respiratory quinone and anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(15 : 0), C(17 : 0) and C(16 : 0) as major fatty acids. The genomic DNA G+C content was 35.3 mol%. 16S rRNA gene sequence similarities of strain MF38(T) with type strains of described Gracilibacillus species ranged from 95.3 to 97.7 %. Strain MF38(T) exhibited the closest phylogenetic affinity to the type strain of Gracilibacillus dipsosauri, with 97.7 % 16S rRNA gene sequence similarity. The DNA-DNA reassociation between strain MF38(T) and G. dipsosauri DSM 11125(T) was 45 %. On the basis of phenotypic and genotypic data, strain MF38(T) represents a novel species of the genus Gracilibacillus, for which the name Gracilibacillus ureilyticus sp. nov. (type strain MF38(T) =CGMCC 1.7727(T) =JCM 15711(T)) is proposed.

Paenibacillus pocheonensis sp. nov., a facultative anaerobe isolated from soil of a ginseng field.

A Gram-positive, aerobic or facultatively anaerobic, rod-shaped, spore-forming bacterium, strain Gsoil 1138(T), was isolated from soil of a ginseng field in Pocheon Province, South Korea, and was characterized in order to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, strain Gsoil 1138(T) was shown to belong to the family Paenibacillaceae and was most closely related to the type strains of Paenibacillus chondroitinus (98.2 % similarity) and Paenibacillus alginolyticus (96.5 %). Levels of 16S rRNA gene sequence similarity between strain Gsoil 1138(T) and the type strains of other recognized species of the genus Paenibacillus were below 96.5 %. The G+C content of the genomic DNA of strain Gsoil 1138(T) was 52.1+/-0.2 mol% (mean+/-sd of three determinations). Phenotypic and chemotaxonomic data (MK-7 as the major menaquinone and anteiso-C(15 : 0) and iso-C(16 : 0) as the predominant fatty acids) supported the affiliation of strain Gsoil 1138(T) to the genus Paenibacillus. The results of DNA-DNA hybridization experiments and physiological and biochemical tests allowed strain Gsoil 1138(T) to be distinguished genotypically and phenotypically from recognized species of the genus Paenibacillus. Strain Gsoil 1138(T) is therefore considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus pocheonensis sp. nov. is proposed. The type strain is Gsoil 1138(T) (=KCTC 13941(T)=LMG 23404(T)).

Proteocatella sphenisci gen. nov., sp. nov., a psychrotolerant, spore-forming anaerobe isolated from penguin guano.

A novel, obligately anaerobic, psychrotolerant bacterium, designated strain PPP2T, was isolated from guano of the Magellanic penguin (Spheniscus magellanicus) in Chilean Patagonia. Cells were Gram-stain-positive, spore-forming, straight rods (0.7-0.8x3.0-5.0 microm) that were motile by means of peritrichous flagella. Growth was observed at pH 6.7-9.7 (optimum pH 8.3) and 2-37 degrees C (optimum 29 degrees C). Growth was observed between 0 and 4% (w/v) NaCl with optimum growth at 0.5% (w/v). Strain PPP2T was a catalase-negative chemo-organoheterotroph that was capable of fermentative metabolism. Peptone, bacto-tryptone, Casamino acids, oxalate, starch, chitin and yeast extract were utilized as substrates. The major metabolic products were acetate, butyrate and ethanol. Strain PPP2T was resistant to ampicillin, but sensitive to tetracycline, chloramphenicol, rifampicin, kanamycin, vancomycin and gentamicin. The DNA G+C content of strain PPP2T was 39.5 mol%. Phylogenetic analysis revealed that strain PPP2T was related most closely to Clostridium sticklandii SR (approximately 90% 16S rRNA gene sequence similarity). On the basis of phylogenetic analysis and phenotypic characteristics, strain PPP2T is considered to represent a novel species of a new genus, for which the name Proteocatella sphenisci gen. nov., sp. nov. is proposed. The type strain of Proteocatella sphenisci is PPP2T (=ATCC BAA-755T=JCM 12175T=CIP 108034T).

Tellurite resistance and reduction by a Paenibacillus sp. isolated from heavy metal-contaminated sediment.

A gram-positive bacterium (designated as strain TeW) that is highly resistant to tellurite was isolated from sediment. The bacterium can grow in the presence of up to 2,000 micromol/L of potassium tellurite (K2TeO3). Reduction of K2TeO3 to tellurium was indicated by the blackening of the growth medium. No lag in growth was observed when cells unexposed to tellurite were transferred to the growth medium containing K2TeO3, indicating that resistance to tellurite was not inducible. Up to 50 and 90% of the metalloid oxyanion tellurite (TeO(3)(2-)) was removed from the medium by strain TeW during growth in nonstatic (shaking) and static (without shaking) conditions, respectively. The bacterium was identified as a Paenibacillus sp. according to its morphology, physiology, and 16S rDNA sequence homology.

Effects of chelating reagents on colonial appearance of Paenibacillus alvei isolated from canine oral cavity.

A bacterial strain isolated from the oral cavity of a healthy dog revealed an unusual colony formation in nebular appearance on agar plates. The isolated bacterial strain was Gram-positive, spore-forming rod with peritrichous flagella, and grown under aerobic conditions, but unable to grow at 45 degrees C. The strain was tentatively classified as Paenibacillus alvei according to the biochemical properties and the 16S rRNA gene sequence. The isolate exhibits collective locomotion on solid agar plates. The bacterial motility was inhibited with EDTA and was restored by adding magnesium. We concluded that magnesium ion is essential for collective locomotion of P. alvei. This suggests that EDTA is useful for inhibition of biofilm formation.

Desmospora activa gen. nov., sp. nov., a thermoactinomycete isolated from sputum of a patient with suspected pulmonary tuberculosis, and emended description of the family Thermoactinomycetaceae Matsuo et al. 2006.

A novel Gram-positive, aerobic, catalase-positive, filamentous micro-organism, designated strain IMMIB L-1269(T), originating from sputum was characterized using phenotypic and molecular taxonomic methods. It showed cell-wall chemotype III, phospholipid type PII (with phosphatidylethanolamine as the diagnostic phospholipid) and contained an unsaturated menaquinone with seven isoprene units (MK-7) as the predominant isoprenoid quinone. It synthesized long-chain cellular fatty acids of the straight-chain saturated, monounsaturated and iso- and anteiso-branched types (with iso-C(15 : 0), C(16 : 0) and iso-C(17 : 0) predominating) and possessed a DNA G+C content of 49.3 mol%. On the basis of its morphological, biochemical and chemical characteristics, strain IMMIB L-1269(T) did not conform to any presently recognized taxon. Comparative analyses based on 16S rRNA gene sequences confirmed the distinctiveness of the isolate, as it displayed sequence-divergence values greater than 7.7 % with respect to recognized Gram-positive taxa. Phylogenetic treeing analysis served to reinforce the view that strain IMMIB L-1269(T) was distinct from recognized taxa, as it formed a relatively long subline branching within a 16S rRNA gene sequence cluster that encompassed the genera Thermoactinomyces, Laceyella, Mechercharimyces, Thermoflavimicrobium, Planifilum, Seinonella and Shimazuella of the family Thermoactinomycetaceae. On the basis of phenotypic and molecular phylogenetic evidence, strain IMMIB L-1269(T) represents a novel genus and species, for which the name Desmospora activa gen. nov., sp. nov. is proposed. The type strain of Desmospora activa is strain IMMIB L-1269(T) (=DSM 45169(T) =CCUG 55916(T)). An emended description of the family Thermoactinomycetaceae is also given.

Robinsoniella peoriensis gen. nov., sp. nov., isolated from a swine-manure storage pit and a human clinical source.

A polyphasic taxonomic study was performed on six strains of an unknown Gram-positive, non-motile, spore-forming, short oval to rod-shaped bacterium isolated from a swine-manure storage pit. In addition to these strains, an isolate deposited in the Culture Collection of the University of Göteborg (Sweden) was found to be biochemically related to the manure strains. The major end products of metabolism included acetate and succinate but not butyrate. Comparative 16S rRNA gene sequencing confirmed that all these isolates were closely related to each other and formed a hitherto unknown lineage within the clostridial rRNA XIVa cluster of organisms. On the basis of phylogenetic, biochemical and phenotypic evidence, it is proposed that the unknown bacterium represents a novel genus and species, for which the name Robinsoniella peoriensis gen. nov., sp. nov. is proposed. The type strain of Robinsoniella peoriensis is PPC31T (=CCUG 48729T =NRRL B-23985T).

The effects of multiple infections on the expression and evolution of virulence in a Daphnia-endoparasite system.

Multiple infections of a host by different strains of the same microparasite are common in nature. Although numerous models have been developed in an attempt to predict the evolutionary effects of intrahost competition, tests of the assumptions of these models are rare and the outcome is diverse. In the present study we examined the outcome of mixed-isolate infections in individual hosts, using a single clone of the waterflea Daphnia magna and three isolates of its semelparous endoparasite Pasteuria ramosa. We exposed individual Daphnia to single- and mixed-isolate infection treatments, both simultaneously and sequentially. Virulence was assessed by monitoring host mortality and fecundity, and parasite spore production was used as a measure of parasite fitness. Consistent with most assumptions, in multiply infected hosts we found that the virulence of mixed infections resembled that of the more virulent competitor, both in simultaneous multiple infections and in sequential multiple infections in which the virulent isolate was first to infect. The more virulent competitor also produced the vast majority of transmission stages. Only when the less virulent isolate was first to infect, the intrahost contest resembled scramble competition, whereby both isolates suffered by producing fewer transmission stages. Surprisingly, mixed-isolate infections resulted in lower fecundity-costs for the hosts, suggesting that parasite competition comes with an advantage for the host relative to single infections. Finally, spore production correlated positively with time-to-host-death. Thus, early-killing of more competitive isolates produces less transmission stages than less virulent, inferior isolates. Our results are consistent with the idea that less virulent parasite lines may be replaced by more virulent strains under conditions with high rates of multiple infections.