The subseafloor marine biosphere may be one of the largest reservoirs of microbial biomass on Earth and has recently been the subject of debate in terms of the composition of its microbial ...inhabitants, particularly on sediments from the Peru Margin. A metagenomic analysis was made by using whole-genome amplification and pyrosequencing of sediments from Ocean Drilling Program Site 1229 on the Peru Margin to further explore the microbial diversity and overall community composition within this environment. A total of 61.9 Mb of genetic material was sequenced from sediments at horizons 1, 16, 32, and 50 m below the seafloor. These depths include sediments from both primarily sulfate-reducing methane-generating regions of the sediment column. Many genes of the annotated genes, including those encoding ribosomal proteins, corresponded to those from the Chloroflexi and Euryarchaeota. However, analysis of the 16S small-subunit ribosomal genes suggests that Crenarchaeota are the abundant microbial member. Quantitative PCR confirms that uncultivated Crenarchaeota are indeed a major microbial group in these subsurface samples. These findings show that the marine subsurface is a distinct microbial habitat and is different from environments studied by metagenomics, especially because of the predominance of uncultivated archaeal groups.
Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers ...where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane-carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100-2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.
Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF ...42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.
Standardized procedures must be followed when characterizing, officially describing, and validly naming novel bacteria. For species descriptions, DNA-DNA hybridization still is needed for ...whole-genome comparisons between close relatives, but many established hybridization methods have drawbacks, such as requiring labeled or large amounts of DNA. We evaluated a new technique based on the spectrophotometric method in which renaturation rates are used for calculating the degree of binding, which estimates relatedness. In this new approach, DNA is denatured and reassociated in a real-time PCR thermal cycler and the process monitored fluorimetrically using SYBR Green I dye that selectively binds to double-stranded DNA. We investigated the effects of different parameters on the renaturation rates, such as the quantities of DNA and SYBR Green I used. Then using this technique, we calculated the percent binding for pairs of selected bacterial species representing different taxonomic groups and compared our results with published values. We demonstrated that the SYBR Green I method is useful for describing new species and as a screening tool to quickly identify the relatedness of uncharacterized isolates with similar 16S rRNA gene sequences.
A Gram-negative ultramicrobacterium (designated strain UMB49(T)) was isolated from a 120,000-year-old, 3,042 m deep Greenland glacier ice core using a 0.2 mum filtration enrichment procedure. ...Phylogenetic analysis of the 16S rRNA gene sequence indicated that this strain belonged to the genus Herminiimonas of the family Oxalobacteraceae of the class Betaproteobacteria. Strain UMB49(T) was most closely related to Herminiimonas saxobsidens (99.6 % sequence similarity), Herminiimonas arsenicoxydans (98.4 %), Herminiimonas aquatilis (97.6 %) and Herminiimonas fonticola (97.9 %). Genomic DNA-DNA hybridization showed low levels of relatedness (below 57 %) to H. saxobsidens and H. arsenicoxydans. Cells of strain UMB49(T) were small thin rods with a mean volume of 0.043 mum(3) and possessed 1 or 2 polar and/or 1-3 lateral very long flagella. The original colony pigmentation was brown-purple but after recultivation the colonies were translucent white to tan coloured. Strain UMB49(T) grew aerobically and under microaerophilic conditions. The strain produced catalase and oxidase, but did not reduce nitrate. Sole carbon sources included citrate, succinate, malate, lactate and alanine. The strain produced acid from l-arabinose, d-arabinose, l-xylose, d-xylose and d-ribose. The DNA G+C content was 59.0 mol%. Based on differential characteristics of strain UMB49(T) and recognized Herminiimonas species, it was concluded that strain UMB49(T) represents a novel species of the genus Herminiimonas, for which the name Herminiimonas glaciei sp. nov. is proposed. The type strain is UMB49(T) (=ATCC BAA-1623(T)=DSM 21140(T)).
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