Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes : a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge

Author Posting. © Springer, 2008. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Extremophiles 12 (2008): 627-640, doi:10.1007/s00792-008-0167-5.The bacterial and archaeal communities of three deep-sea hydrothermal vent systems located on the Mid-Atlantic Ridge (MAR; Rainbow, Logatchev and Broken Spur) were investigated using an integrated culture-dependent and independent approach. Comparative molecular phylogenetic analyses, using the 16S rRNA gene and the deduced amino acid sequences of the alpha and beta subunits of the ATP citrate lyase encoding genes were carried out on natural microbial communities, on an enrichment culture obtained from the Broken Spur chimney, and on novel chemolithoautotrophic bacteria and reference strains originally isolated from several different deep-sea vents. Our data showed that the three MAR hydrothermal vent chimneys investigated in this study host very different microbial assemblages. The microbial community of the Rainbow chimney was dominated by thermophilic, autotrophic, hydrogen-oxidizing, sulfur- and nitrate reducing Epsilonproteobacteria related to the genus Caminibacter. The detection of sequences related to sulfur-reducing bacteria and archaea (Archaeoglobus) indicated that thermophilic sulfate reduction might also be occurring at this site. The Logatchev bacterial community included several sequences related to mesophilic sulfur-oxidizing bacteria, while the archaeal component of this chimney was dominated by sequences related to the ANME-2 lineage, suggesting that anaerobic oxidation of methane may be occurring at this site. Comparative analyses of the ATP citrate lyase encoding genes from natural microbial communities suggested that Epsilonproteobacteria were the dominant primary producers using the reverse TCA cycle (rTCA) at Rainbow, while Aquificales of the genera Desulfurobacterium and Persephonella were prevalent in the Broken Spur chimney.This research was supported by NSF grants MCB 04-56676 (C.V.), OCE 03-27353 (C.V.), MCB 04-56689 (S.M.S.), a grant from the New Jersey Agricultural Experiment Station to C.V., and a NIH Ph.D. Training Program in Biotechnology Fellowship (NIH NIGMS 5 T32 GM08339) to J.V. M.H. was supported through a postdoctoral scholarship from the Woods Hole Oceanographic Institution

Molecular signatures (unique proteins and conserved indels) that are specific for the epsilon proteobacteria (Campylobacterales)

BACKGROUND: The epsilon proteobacteria, which include many important human pathogens, are presently recognized solely on the basis of their branching in rRNA trees. No unique molecular or biochemical characteristics specific for this group are known. RESULTS: Comparative analyses of proteins in the genomes of Wolinella succinogenes DSM 1740 and Campylobacter jejuni RM1221 against all available sequences have identified a large number of proteins that are unique to various epsilon proteobacteria (Campylobacterales), but whose homologs are not detected in other organisms. Of these proteins, 49 are uniquely found in nearly all sequenced epsilon-proteobacteria (viz. Helicobacter pylori (26695 and J99), H. hepaticus, C. jejuni (NCTC 11168, RM1221, HB93-13, 84-25, CF93-6, 260.94, 11168 and 81-176), C. lari, C. coli, C. upsaliensis, C. fetus, W. succinogenes DSM 1740 and Thiomicrospira denitrificans ATCC 33889), 11 are unique for the Wolinella and Helicobacter species (i.e. Helicobacteraceae family) and many others are specific for either some or all of the species within the Campylobacter genus. The primary sequences of many of these proteins are highly conserved and provide novel resources for diagnostics and therapeutics. We also report four conserved indels (i.e. inserts or deletions) in widely distributed proteins (viz. B subunit of exinuclease ABC, phenylalanyl-tRNA synthetase, RNA polymerase β '-subunit and FtsH protein) that are specific for either all epsilon proteobacteria or different subgroups. In addition, a rare genetic event that caused fusion of the genes for the largest subunits of RNA polymerase (rpoB and rpoC) in Wolinella and Helicobacter is also described. The inter-relationships amongst Campylobacterales as deduced from these molecular signatures are in accordance with the phylogenetic trees based on the 16S rRNA and concatenated sequences for nine conserved proteins. CONCLUSION: These molecular signatures provide novel tools for identifying and circumscribing species from the Campylobacterales order and its subgroups in molecular terms. Although sequence information for these signatures is presently limited to Campylobacterales species, it is likely that many of them will also be found in other epsilon proteobacteria. Functional studies on these proteins and conserved indels should reveal novel biochemical or physiological characteristics that are unique to these groups of epsilon proteobacteria

Next-Generation Pyrosequencing Analysis of Microbial Biofilm Communities on Granular Activated Carbon in Treatment of Oil Sands Process-Affected Water

The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>10(9) gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O(3)/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds

Thermosipho geolei sp nov., a thermophilic bacterium isolated from a continental petroleum reservoir in Western Siberia

The novel isolate reduced elemental sulfur ana cystine. but not thiosulfate sulfate, to hydrogen sulfide, The G+C content of the genomic DNA was 30.0 mol%, As determined by 16S rDNA sequence analysis, this organism Three strictly anaerobic, thermophilic bacteria (SL31(T), SL30 and MLM39636) were isolated from a deep continental oil reservoir in Western Siberia (Russia), Following the mid-exponential phase of growth, the non-motile rod-shaped organisms were surrounded by a sheath-like structure, As DNA-DNA hybridizations showed that these strains were highly related genomically, only strain SL31T was studied in detail, The temperature range for growth of strain SL31T was between 45 and 75 degreesC, with optimum growth at 70 degreesC, its optimum ph and NaCl concentration for growth were pH 7.5 and 20-30 g l(-1), respectively. The novel isolate reduced elemental sulfur and cystine, but not thiosulfate or belonged to the genus Thermosipho. DNA-DNA hybridization levels between strain SL31T and type strains of the previously described species of Thermosipho were less than 10%, On the basis of physiological and molecular properties, it is proposed that this organism should be placed in a new species, Thermosipho geolei sp, nov, The novel organism represents the first species of the genus Thermosipho that has been isolated from a petroleum reservoir, The type strain is SL31(T) (= DSM 13256(T) = JCM 10986(T))

Petrotoga olearia sp nov and Petrotoga sibirica sp nov., two thermophilic bacteria isolated from a continental petroleum reservoir in Western Siberia

Strictly anaerobic, thermophilic bacteria (strains SL24(T), SL25(T), SL27, SL29 and SL32) were isolated from a deep, continental oil reservoir in Western Siberia (Russia). These motile, rod-shaped organisms were surrounded by a sheath-like structure, a feature characteristic of the Thermotogales. On the basis of partial 16S rDNA sequences (500 nucleotides), strains SL25(T), SL27, SL29 and SL32 were identical. Therefore, only strains SL24(T) and SL25(T) were studied in detail. The optimum temperature for growth of both strains was 55degreesC. Their optimum pH for growth was 7.5 and their optimum NaCl concentration was between 20 and 30 g l(-1). The novel isolates reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C contents of the genomic DNA of strains SL24(T) and SL25(T) were respectively 35 and 33 mol%. Phylogenetically, both strains are most closely related to Petrotoga miotherma, there being 98.9-99.4% similarity between their 16S rDNA sequences. Phenotypic properties and DNA-DNA hybridization experiments indicate that the strains belong to two novel species, for which the names Petrotoga olearia (type strain SL24(T)=DSM 13574(T)=JCM 11234(T)) and Petrotoga sibirica (type strain SL25(T)=DSM 13575(T)=JCM 11235(T)) are proposed

Nautilia lithotrophica gen. nov., sp nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent

A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525(T), was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13degrees N). This organism grew in the temperature range 37-68 degreesC, the optimum being 53 degreesC, and in the pH range 6.4-7.4, the optimum being 6.8-7.0. The NaCl range for growth was 0.8-5.0%, the optimum being 3.0%. Strain 525(T) grew lithoautotrophically with H-2 as energy source, S-o as electron acceptor and CO2 as carbon source. Alternatively, strain 525(T) was able to use formate as an energy source. The G+C content of the genomic DNA was 34.7 mol %. Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525(T) in the c-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives. On the basis of phenotypic and phylogenetic differences between strain 525(T) and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen. nov., sp. nov. is proposed. The type strain is strain 525(T) (= DSM 13520(T))

Caminibacter profundus sp nov., a novel thermophile of Nautiliales ord. nov within the class `Epsilonproteobacteria', isolated from a deep-sea hydrothermal vent

A novel moderately thermophilic, microaerobic to anaerobic, chemolithoautotrophic bacterium, designated strain CRT, was isolated from a deep-sea hydrothermal vent site at 36degreesN on the Mid-Atlantic Ridge. Cells were Gram-negative, non-motile rods. The organism grew at 45-65 degreesC and pH 6.5-7.4,with optimum growth at 55 degreesC and pH 6.9-7.1. The NaCl range for growth was 5-50 g l(-1) (optimum 30 g l(-1)). Strain CRT was an obligate chemolithoautotroph, growing with H-2 as energy source, sulfur, nitrate or oxygen as electron acceptors and CO2 as carbon source. Hydrogen sulfide and ammonium were the respective products of sulfur and nitrate reduction. The G+C; content of the genomic DNA was 32.1 mol%. Based on 16S rRNA gene sequence analysis, this organism was most closely related to Caminibacter hydrogeniphilus (94.9% similarity). On the basis of phenotypic and phylogenetic data, it is proposed that the isolate represents a novel species, Caminibacter profundus sp. nov. The type strain is CRT (= DSM 15016(T)= JCM 11957(T)). The phylogenetic data also correlate well with the significant phenotypic differences between the lineage encompassing the genera Nautilia and Caminibacter and other members of the class `Epsilonproteobacteria'. The lineage encompassing the genera Nautilia and Caminibacter is therefore proposed as a new order, Nautiliales ord. nov., represented by a single family, Nautiliaceae fam. nov

Deferribacter abyssi sp nov., an anaerobic thermophile from deep-sea hydrothermal vents of the Mid-Atlantic Ridge

Two strains of thermophilic, anaerobic, chemolithoautotrophic bacteria, designated JR(T) and DR, were isolated from hydrothermal samples collected on the Mid-Atlantic Ridge from the Rainbow (36degrees 16' N, 33degrees 54' W) and Menez Gwen (37degrees 50' N, 31degrees 50' W) vent fields, respectively. Cells of both isolates were short, straight- to vibrio-shaped, motile rods with one polar flagellum, and were Gram-negative and non-sporulating. Strain JRT was characterized in detail. It was found to grow optimally at pH 6.5-6.7, at 60 degreesC and in the presence of 30 g NaCl l(-1). Strain JR(T) could use molecular hydrogen, acetate, succinate, pyruvate and proteinaceous compounds as electron donors, and elemental sulfur, nitrate or Fe(III) as electron acceptors. No fermentation of organic substrates occurred. The G + C content of the DNA of strain JRT was 30.8 mol%. Strain DR (= DSM 14927) possessed the same morphology and pH, temperature and salinity optima and ranges, and used the same electron acceptors as strain JR(T). On the basis of their 16S rDNA sequences (1517 nucleotides), strains JRT and DR were identical and distantly related to Deferribacter thermophilus and Deferribacter desulfuricans (95.3 and 95.2 % sequence similarity, respectively). Based on their phenotypic and phylogenetic characteristics, it is proposed that both strains are members of a new species of the genus Deferribacter, for which the name Deferribacter abyssi (type strain JRT = DSM 14873(T) = JCM 11955(T)) is proposed

Vulcanithermus mediatlanticus gen. nov., sp nov., a novel member of the family Thermaceae from a deep-sea hot vent

A novel thermophilic, microaerophilic, facultatively chemolithoheterotrophic bacterium designated strain TRT was isolated from a sample of a deep-sea hydrothermal chimney collected at the Rainbow vent field on the Mid-Atlantic Ridge (36degrees14'N). Gram-negative, non-spore-forming, non-motile rods occurred singly or in pairs. The organism grew in the temperature range 37-80 degreesC with an optimum at 70 degreesC and at pH 5.5-8.4 with an optimum around 6.7. The NaCl range for growth was 10-50 g l(-1) with an optimum of 30 g l(-1). Strain TRT grew chemoorganoheterotrophically with carbohydrates, proteinaceous substrates, organic acids and alcohols using oxygen or nitrate as electron acceptors. The isolate was able to grow at oxygen concentrations from 0.5 to 21 %. Oxygen concentrations that promoted fastest growth ranged from 4 to 8 % under agitation. The novel isolate was able to grow lithoheterotrophically with molecular hydrogen as the energy source. The G+C content of the genomic DNA was 68.4 mol%. Phylogenetic analysis of the 16S rDNA sequence placed strain TRT within the phylum Deinococcus-Thermus of the Bacteria. On the basis of phenotypic and phylogenetic data, it is proposed that this isolate should be described as a member of a novel species of a new genus as Vulcanithermus mediatlanticus gen. nov., sp. nov. The type strain is TRT (= DSM 14978(T) = VKM B-2292(T) = JCM 11956(T))

Oceanithermus profundus gen. nov., sp nov., a thermophilic, microaerophilic, facultatively chemolithoheterotrophic bacterium from a deep-sea hydrothermal vent

A novel moderately thermophilic, organotrophic, microaerophilic, facultatively chemolithotrophic bacterium, designated strain 506 T, was isolated from a deep-sea hydrothermal vent site at 13degreesN in the East Pacific Rise. Cells were Gram-negative, non-motile rods. The organism grew in the temperature range 40-68degreesC, with an optimum at 60degreesC, and in the pH range 5.5-8.4, with an optimum around pH 7.5. The NaCl concentration for growth was in the range 10-50 g l(-1), with an optimum at 30 g l(-1). Strain 506 T grew chemoorganoheterotrophically with carbohydrates, proteinaceous substrates, organic acids and alcohols using oxygen or nitrate as electron acceptor. Alternatively, strain 506(T) was able to grow lithoheterotrophically with molecular hydrogen as the energy source. The G + C content of the genomic DNA was 62.9 mol%. Phylogenetic analysis of the 16S rDNA sequence placed strain 506 T in the family Thermaceae. On the basis of phenotypic and phylogenetic data, strain 506(T) (= DSM 14977(T) = VKM B-2274(T)) is proposed as the type strain of a novel species in a new genus, Oceanithermus profundus gen. nov., sp. nov