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Applied and Environmental Microbiology, September 2002, p. 4593-4603, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4593-4603.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse Chloroflexaceae-Related Bacteria in Hot Spring Microbial Mats{dagger}

Ulrich Nübel,1,2* Mary M. Bateson,1 Verona Vandieken,2 Andrea Wieland,3 Michael Kühl,3 and David M. Ward1

Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana,1 Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany,2 Marine Biological Laboratory, University of Copenhagen, Helsingør, Denmark3

Received 11 February 2002/ Accepted 5 June 2002

We investigated the diversity, distribution, and phenotypes of uncultivated Chloroflexaceae-related bacteria in photosynthetic microbial mats of an alkaline hot spring (Mushroom Spring, Yellowstone National Park). By applying a directed PCR approach, molecular cloning, and sequence analysis of 16S rRNA genes, an unexpectedly large phylogenetic diversity among these bacteria was detected. Oligonucleotide probes were designed to target 16S rRNAs from organisms affiliated with the genus Chloroflexus or with the type C cluster, a group of previously discovered Chloroflexaceae relatives of this mat community. The application of peroxidase-labeled probes in conjunction with tyramide signal amplification enabled the identification of these organisms within the microbial mats by fluorescence in situ hybridization (FISH) and the investigation of their morphology, abundance, and small-scale distribution. FISH was combined with oxygen microelectrode measurements, microscope spectrometry, and microautoradiography to examine their microenvironment, pigmentation, and carbon source usage. Abundant type C-related, filamentous bacteria were found to flourish within the cyanobacterium-dominated, highly oxygenated top layers and to predominate numerically in deeper orange-colored zones of the investigated microbial mats, correlating with the distribution of bacteriochlorophyll a. Chloroflexus sp. filaments were rare at 60°C but were more abundant at 70°C, where they were confined to the upper millimeter of the mat. Both type C organisms and Chloroflexus spp. were observed to assimilate radiolabeled acetate under in situ conditions.

* Corresponding author. Mailing address: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1B, 38124 Braunschweig, Germany. Phone: 49-531-2616-105. Fax: 49-531-2616-418. E-mail: unuebel{at}dsmz.de. {dagger}Journal Series no. 2002-49, Montana Agricultural Experiment Station, Montana State University—Bozeman.

Applied and Environmental Microbiology, September 2002, p. 4593-4603, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4593-4603.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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