Phylogenetic analysis of the bacterial communities in marine sediments

Abstract

For the phylogenetic analysis of microbial communities present in environmental samples microbial DNA can be extracted from the sample, 16S rDNA can be amplified with suitable primers and the PCR, and clonal libraries can be constructed. We report a protocol that can be used for efficient cell lysis and recovery of DNA from marine sediments. Key steps in this procedure include the use of a bead mill homogenizer for matrix disruption and uniform cell lysis and then purification of the released DNA by agarose gel electrophoresis. For sediments collected from two sites in Puget Sound, over 96% of the cells present were lysed. Our method yields high-molecular-weight DNA that is suitable for molecular studies, including amplification of 16S rRNA genes. The DNA yield was 47 g per g (dry weight) for sediments collected from creosote-contaminated Eagle Harbor, Wash. Primers were selected for the PCR amplification of (eu)bacterial 16S rDNA that contained linkers with unique 8-base restriction sites for directional cloning. Examination of 22 16S rDNA clones showed that the surficial sediments in Eagle Harbor contained a phylogenetically diverse population of organisms from the Bacteria domain (G.

An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments

“…Likewise, Bacteroidetes, Betaproteobacteria and Gammaproteobacteria were observed throughout the core. Although these functionally diverse groups have often been found in 16S rRNA gene clone libraries from marine sediments [16,[45][46][47][48], and detected by FISH [2,4,5,7], their abundance in the deeper parts of sandy sediments has not been quantified previously. Desulfosarcinales were also abundantly identified throughout the sandy core.…”

Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hills, Eastern Antarctica

“…Unfortunately, only limited comparisons can so far be made between clone library data because of the lack of samples that have been analysed. Clone library studies based on marine sediments include a small clone library analysis of coastal sediment of Puget Sound, USA (Gray and Herwig, 1996), deep sea sediment and an extensive clone library derived from Arctic Ocean sediment (Ravenschlag et al, 1999). Some other 16S rDNA clone libraries are based on enrichment cultures from marine sediment (Phelps et al, 1998).…”

Bacterial diversity in organically-enriched fish farm sediments

“…Clone library construction has previously been used to investigate the microbial diversity of various sediments (Bowman et al, 1993(Bowman et al, , 1997Gray & Herwig, 1996;Ravenschlag et al, 1999;Bowman & McCuaig, 2003). These studies have provided much information on the phylotypes that inhabit sediments and the massive bacterial diversity that sediments harbour.…”