CiteULike: vrich culture

Cultivation of the ubiquitous SAR11 marine bacterioplankton clade.

MS Rappé — 2005-10-19T14:56:21-00:00

Nature, Vol. 418, No. 6898. (8 August 2002), pp. 630-633.

The alpha-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation-independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems. This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods. The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems, which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study. Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities into very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 micro m(3), places them among the smallest free-living cells in culture.

Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria.

JC Cho — 2006-11-16T12:41:06-00:00

Appl Environ Microbiol, Vol. 70, No. 1. (January 2004), pp. 432-440.

Forty-four novel strains of Gammaproteobacteria were cultivated from coastal and pelagic regions of the Pacific Ocean using high-throughput culturing methods that rely on dilution to extinction in very low nutrient media. Phylogenetic analysis showed that the isolates fell into five rRNA clades, all of which contained rRNA gene sequences reported previously from seawater environmental gene clone libraries (SAR92, OM60, OM182, BD1-7, and KI89A). Bootstrap analyses of phylogenetic reliability did not support collapsing these five clades into a single clade, and they were therefore named the oligotrophic marine Gammaproteobacteria (OMG) group. Twelve cultures chosen to represent the five clades were successively purified in liquid culture, and their growth characteristics were determined at different temperatures and dissolved organic carbon concentrations. The isolates in the OMG group were physiologically diverse heterotrophs, and their physiological properties generally followed their phylogenetic relationships. None of the isolates in the OMG group formed colonies on low- or high-nutrient agar upon their first isolation from seawater, while 7 of 12 isolates that were propagated for laboratory testing eventually produced colonies on 1/10 R2A agar. The isolates grew relatively slowly in natural seawater media (1.23 to 2.63 day(-1)), and none of them grew in high-nutrient media (>351 mg of C liter(-1)). The isolates were psychro- to mesophilic and obligately oligotrophic; many of them were of ultramicrobial size (<0.1 micro m(3)). This cultivation study revealed that sporadically detected Gammaproteobacteria gene clones from seawater are part of a phylogenetically diverse constellation of organisms mainly composed of oligotrophic and ultramicrobial lineages that are culturable under specific cultivation conditions.

The SAR92 Clade: an Abundant Coastal Clade of Culturable Marine Bacteria Possessing Proteorhodopsin

Ulrich Stingl — 2008-05-10T18:53:03-00:00

Appl. Environ. Microbiol., Vol. 73, No. 7. (1 April 2007), pp. 2290-2296.

Proteorhodopsin (PR) is a protein that is abundant in marine bacterioplankton. PR is hypothesized to be a light-dependent proton pump, thus creating a proton gradient that can be used for energy production without electron transport. Currently, the only culture that has been reported to possesses PR is the highly abundant alphaproteobacterium "Candidatus Pelagibacter ubique" (SAR11 clade), but surprisingly, its growth in batch culture was not enhanced by light. Here, we present the first cultured gammaproteobacterium that possesses a PR gene. Genome sequencing and analysis of HTCC2207 showed that the PR gene is present as a lone transcriptional unit directly followed by an operon containing genes that are presumably involved in the synthesis of retinal, the chromophore of PR. Half-time decay times of different PR intermediates in native HTCC2207 cells ranged between 2 and 15 ms, and the absorbance maximum of PR was determined to be 528 nm. Proteorhodopsin was identified in three additional strains, using a specific PCR assay on other cultured members of the SAR92 clade. Phylogenetic analyses of the PR genes determined that they form a deeply rooting cluster not closely related to any PR genes recovered so far. Fluorescence in situ hybridization and RNA blots showed that the SAR92 clade reaches up to 10% of the total bacterial population in surface waters close to the Oregon coast and decreases over depth and distance from the shore. Although the growth of HTCC2207 is limited by the amount of available carbon that is present in the medium applied, these cultures do not grow at higher rates nor do they have higher growth yields when incubated under light. 10.1128/AEM.02559-06

The small genome of an abundant coastal ocean methylotroph.

Stephen J Giovannoni — 2008-04-10T02:17:16-00:00

Environmental microbiology (3 April 2008)

Summary OM43 is a clade of uncultured beta-proteobacteria that is commonly found in environmental nucleic acid sequences from productive coastal ocean ecosystems, and some freshwater environments, but is rarely detected in ocean gyres. Ecological studies associate OM43 with phytoplankton blooms, and evolutionary relationships indicate that they might be methylotrophs. Here we report on the genome sequence and metabolic properties of the first axenic isolate of the OM43 clade, strain HTCC2181, which was obtained using new procedures for culturing cells in natural seawater. We found that this strain is an obligate methylotroph that cannot oxidize methane but can use the oxidized C1 compounds methanol and formaldehyde as sources of carbon and energy. Its complete genome is 1304 428 bp in length, the smallest yet reported for a free-living cell. The HTCC2181 genome includes genes for xanthorhodopsin and retinal biosynthesis, an auxiliary system for producing transmembrane electrochemical potentials from light. The discovery that HTCC2181 is an extremely simple specialist in C1 metabolism suggests an unanticipated, important role for oxidized C1 compounds as substrates for bacterioplankton productivity in coastal ecosystems.

High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates.

SA Connon — 2006-11-16T12:35:57-00:00

Appl Environ Microbiol, Vol. 68, No. 8. (August 2002), pp. 3878-3885.

Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections. Out of over 40 known prokaryotic phyla, only half have cultured representatives. In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media. In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations-typically 3 orders of magnitude less than common laboratory media. Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-microl aliquots of cultures with densities as low as 10(3) cells/ml. Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years. Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques. Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine Proteobacteria clades known from environmental gene cloning studies. These cultures are related to the clades SAR11 (alpha subclass), OM43 (beta subclass), SAR92 (gamma subclass), and OM60/OM241 (gamma subclass). This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.

Dilution-to-Extinction Culturing of Psychrotolerant Planktonic Bacteria from Permanently Ice-covered Lakes in the McMurdo Dry Valleys, Antarctica

Stingl — 2008-05-04T02:11:13-00:00

Microbial Ecology, Vol. 55, No. 3. (April 2008), pp. 395-405.