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.

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.

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.

[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.

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.

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.

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.

A strain of Lactobacillus plantarum affects segmented filamentous bacteria in the intestine of immunosuppressed mice.

Segmented filamentous bacteria (SFB) are present in the gastrointestinal tract of mice from weaning until the maturation of the immune system. Probiotic bacteria also have an effect on host immunity. To study the relationships established between these bacteria, samples from a mouse model fed with Lactobacillus plantarum under different immunological conditions were analysed. SFB populations were measured by a newly designed group-specific quantitative PCR assay. The results confirmed the presence of the probiotic in the intestine and an expansion of SFB in the ileum of immunocompromised mice, which was abolished upon administration of L. plantarum, an effect not described to date.

Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms.

Bacterial contamination of raw milk can originate from different sources: air, milking equipment, feed, soil, faeces and grass. It is hypothesized that differences in feeding and housing strategies of cows may influence the microbial quality of milk. This assumption was investigated through comparison of the aerobic spore-forming flora in milk from organic and conventional dairy farms. Laboratory pasteurized milk samples from five conventional and five organic dairy farms, sampled in late summer/autumn and in winter, were plated on a standard medium and two differential media, one screening for phospholipolytic and the other for proteolytic activity of bacteria. Almost 930 isolates were obtained of which 898 could be screened via fatty acid methyl ester analysis. Representative isolates were further analysed using 16S rRNA gene sequencing and (GTG)(5)-PCR. The majority of aerobic spore-formers in milk belonged to the genus Bacillus and showed at least 97% 16S rRNA gene sequence similarity with type strains of Bacillus licheniformis, Bacillus pumilus, Bacillus circulans, Bacillus subtilis and with type strains of species belonging to the Bacillus cereus group. About 7% of all isolates may belong to possibly new spore-forming taxa. Although the overall diversity of aerobic spore-forming bacteria in milk from organic vs. conventional dairy farms was highly similar, some differences between both were observed: (i) a relatively higher number of thermotolerant organisms in milk from conventional dairy farms compared to organic farms (41.2% vs. 25.9%), and (ii) a relatively higher number of B. cereus group organisms in milk from organic (81.3%) and Ureibacillus thermosphaericus in milk from conventional (85.7%) dairy farms. One of these differences, the higher occurrence of B. cereus group organisms in milk from organic dairy farms, may be linked to differences in housing strategy between the two types of dairy farming. However, no plausible clarification was found for the relatively higher number of thermotolerant organisms and the higher occurrence of U. thermosphaericus in milk from conventional dairy farms. Possibly this is due to differences in feeding strategy but no decisive indications were found to support this assumption.

Differential expression of nifH and anfH genes in Paenibacillus durus analysed by reverse transcriptase-PCR and denaturing gradient gel electrophoresis.

AIMS: The aim of this study was to develop an approach based on a reverse transcriptase (RT)-PCR/denaturing gradient gel electrophoresis (DGGE) for the detection of the functional genes nifH and anfH in Paenibacillus durus. METHODS AND RESULTS: Two sets of primers were employed to study the expression of the nitrogen fixation genes in a pure-culture system of P. durus grown in media with increasing concentrations of ammonium (NH(4)(+)), tungsten (W) or molybdenum (Mo). The results obtained indicate that the expression of nitrogenase genes from P. durus can take place in the presence of relatively high levels of fixed nitrogen. It was also observed that the addition of 20 micromol l(-1) molybdenum and 2 mmol l(-1) tungstate did not interfere in the mRNA levels of nifH and anfH genes. CONCLUSIONS: Our results demonstrate the presence and transcription of nifH and anfH in P. durus under a variety of growth conditions. A specific set of primers was designed for the detection of the alternative system for nitrogen fixation in P. durus. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR/DGGE system enables the rapid gathering of incremental data about the regulation of conventional and alternative nitrogenase genes in P. durus strains.

Nucleotide sequence of the sspE genes coding for gamma-type small, acid-soluble spore proteins from the round-spore-forming bacteria Bacillus aminovorans, Sporosarcina halophila and S. ureae.

The single sspE genes coding for gamma-type small, acid-soluble spore proteins (SASP) of three round-spore-forming bacteria, Bacillus aminovorans, Sporosarcina halophila and S. ureae, have been cloned and sequenced. While the deduced amino acid sequences of these three gamma-type SASP show clear homology to those from six Bacillus species that do not form round spores, there are no residues conserved completely among the 9 sequences known. In addition, the 139 residue B. aminovorans protein is 35 residues larger than any other while the 60 residue S. halophila protein is one of the smallest. These data suggest that the sspE genes have been under little selective pressure in recent evolutionary time.

Gene prediction using the Self-Organizing Map: automatic generation of multiple gene models.

BACKGROUND: Many current gene prediction methods use only one model to represent protein-coding regions in a genome, and so are less likely to predict the location of genes that have an atypical sequence composition. It is likely that future improvements in gene finding will involve the development of methods that can adequately deal with intra-genomic compositional variation. RESULTS: This work explores a new approach to gene-prediction, based on the Self-Organizing Map, which has the ability to automatically identify multiple gene models within a genome. The current implementation, named RescueNet, uses relative synonymous codon usage as the indicator of protein-coding potential. CONCLUSIONS: While its raw accuracy rate can be less than other methods, RescueNet consistently identifies some genes that other methods do not, and should therefore be of interest to gene-prediction software developers and genome annotation teams alike. RescueNet is recommended for use in conjunction with, or as a complement to, other gene prediction methods.

Identification by in situ hybridization of segmented filamentous bacteria in the intestine of diarrheic rainbow trout (Oncorhynchus mykiss).

Nonculturable segmented filamentous bacteria (SFB) have been described in the gut of rats, mice and chickens, and 16S rRNA sequences for these organisms are available. These organisms, peripherically related to Clostridium phylogenetic group I, have been provisionally named 'Candidatus Arthromitus'. This work reports the observation of similar bacteria in the intestinal content of the distal intestine, preferentially, in the adult rainbow trout (Oncorhynchus mykiss) that exhibited episodic acute diarrhea, usually during the summer. Abdominal distension, intestinal fluid-mucus content and epithelium detachment were observed in trout. The demonstration that the observed microorganisms are bacteria and belong in the 'Candidatus Arthromitus' group was achieved by in situ hybridization with, respectively, a eubacterial probe and an oligonucleotide probe designed to react specifically with SFB 16S rRNA (encoded by the rrs gene) sequences. The sequenced rrs gene was compared with published sequences and found to be closely related to (although distinct from) other SFB sequences. Implication of these bacteria in trout diarrheic illness remains hypothetical.

Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences.

The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of "Sporolactobacillus dextrus" and Bacillus coagulans. Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type strain of S. inulinus. The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accommodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus.

Molecular characterisation of bacterial contamination in semi-final gelatine extracts, using denaturing gradient gel electrophoresis.

Contamination of gelatine may affect the safety and/or quality of its applications. Characterisation of bacterial isolates from semi-final gelatine batches revealed thermotolerant, aerobic, endosporeforming contaminants. In this paper, bacterial contamination in gelatine batches is analysed without previous isolation, by means of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA sequences. V9 and V6-V8 regions of the 16S rDNA gene were found more suitable for this purpose than V1 or V3 regions. Bacillus fumarioli, Bacillus licheniformis, members of the 'Bacillus cereus group', Bacillus subtilis, Bacillus shackletonii, Brevibacillus borstelensis and Brevibacillus agri were detected.

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.

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.