Evaluation of a new fluorimetric DNA-DNA hybridization method.

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.

Herminiimonas glaciei sp. nov., a novel ultramicrobacterium from 3042 m deep Greenland glacial ice.

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)).

Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core.

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 microm3) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5 degrees C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-microm, 0.2-microm, and even 0.1-microm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-microm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.

Rhodoglobus vestalii gen. nov., sp. nov., a novel psychrophilic organism isolated from an Antarctic Dry Valley lake.

A novel, psychrophilic, gram-positive bacterium (designated strain LV3T) from a lake near the McMurdo Ice Shelf, Antarctica, has been isolated and characterized. This organism formed red-pigmented colonies, had an optimal growth temperature of 18 degrees C and grew on a variety of media between -2 and 21 degrees C. Scanning electron micrographs of strain LV3T that showed small rods with unusual bulbous protuberances during all phases of growth were of particular interest. The G + C content of the genomic DNA was approximately 62 mol%. The cell walls contained ornithine as the diamino acid. The major fatty acids were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. Cells grown at -2 degrees C contained significant amounts of anteiso-C15:1. The major menaquinones found in strain LV3T were MK-11 and MK-12. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain LV3T was a member of the family Microbacteriaceae and related to, but distinct from, organisms belonging to the genera Agreia, Leifsonia and Subtercola. In addition, alignments of 16S rRNA sequences showed that the sequence of strain LV3T contained a 13 bp insertion that was found in only a few related sequences. Based on the low growth temperature, unusual cell shape, distinct 16S rRNA gene sequence and structure and cell-wall amino acid and menaquinone compositions, Rhodoglobus vestalii gen. nov., sp. nov. is proposed, with the type strain LV3T (= ATCC BAA-534T = CIP 107482T).

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core.

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.