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Control/Tracking Number : 04-GM-A-4219-ASM
Activity :Abstract
Current Date/Time : 12/19/2003 5:50:47 PM

Attempts to Cultivate RLMO Bacteria Using Site-Specific Water Chemistry Data

T. Manning, II, D. P. Lodge, S. M. Boomer;
Western Oregon University, Monmouth, OR.

Our research focuses on novel filamentous red Green Nonsulfur (R-GNS) bacteria that form splash mats in Yellowstone hot springs (pH 7.5-9, 37-65�C), Red Layer Microbial Observatory (RLMO) sites. Based on 16S rRNA, R-GNS bacteria are 92-95% similar to Roseiflexus castenholzii, a red filamentous phototroph isolated from comparable hot springs in Japan. Despite these similarities, culturing of Yellowstone R-GNS using published media for Roseiflexus has been unsuccessful. One goal of this project was to cultivate Yellowstone R-GNS bacteria for metabolic and genomic studies. Given data that RLMO sites show distinct water chemistry profiles (15 salts and metals), we designed media based on these data, focusing on one RLMO site, Hillside Springs (pH 8, 54�C). We hypothesized that (i) the growth of R-GNS bacteria would depend on site chemistry, and (ii) site-specific media would support more diversity than Roseiflexus medium. Hillside R-GNS samples were inoculated into: (1) site-specific media plus high yeast extract (0.1 g/L), (2) site-specific media plus low yeast (0.01 g/L), (3) Roseiflexus media, (4) and sterilized source water from Hillside. Growth was assessed once a week for four weeks. Microscopic, pigment, DGGE, and 16S rRNA sequence analyses were used to compare diversity enriched by each medium.
Although site-specific media did not support the growth of target R-GNS bacteria, these media enriched for different populations of new Proteobacteria, Gram Positives, and green GNS. Roseiflexus medium enriched for Proteobacteria, Gram Positives, and Nitrospira � but not R-GNS. Sterilized source water supported the most visible growth of red filaments. However, pigment and 16S rRNA analysis of a co-culture derived from these enrichments suggested the filaments were most related to Chloroflexus species 396-1, a Yellowstone green GNS. Although the use of sterilized source water as media appeared to be the most promising, the potential natural impact of source water-based cultivation could be substantial; thus, future cultivation studies should include improved research into the composition of the source water and how to replicate it.

Topic (Complete): Q20 Methods in Environmental Microbiology
Keyword (Complete): water chemistry ; media development ; Yellowstone
Membership and Grant Information (Complete):
ASM Member (or who has submitted an application): : Terrance Manning

Status: Complete

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