Investigation of the Microbial Ecology of Intertidal Hot Springs by Using Diversity Analysis of 16S rRNA and Chitinase Genes

Abstract

The microbial diversity of intertidal hot springs on the seashore of northwest Iceland was examined by combining directed in situ enrichments, artificial support colonization, and mat sampling. Analysis of 16S rRNA genes revealed the presence of clones related to both marine and terrestrial, thermophilic, mesophilic, and psychrophilic microorganisms scattered among 11 bacterial divisions. No archaea were found. The species composition of the enrichments was affected by the length of the hot periods experienced at low tide and was very different from those found in the biomass. A total of 36 chitinase genes were detected by molecular screening of the samples with degenerate primers for glycoside hydrolase family 18. The chitinase gene diversity was at least twofold higher in the enrichment samples than in the controls, indicating that a much higher diversity of hydrolytic genes can be accessed with this approach.The coastal hot springs located on the Reykjanes peninsula in northwest Iceland can be regarded as one of the most extreme microbial environments on Earth. A series of freshwater hot springs are located on the seashore, from the tidal zone to about 100 m off the coast. The geothermal system is essentially supplied with deep fresh and slightly alkaline groundwater. It is also low in sulfide and slightly mixed with seawater in the hot spring outlets (chloride, Յ600 g/ml). The temperature ranges from 45 to 95°C in different springs, and many are covered by the sea at high tides (9). The tides are as high as 4 m in this area, and therefore the organisms living in those hot springs can be subject to almost 100°C fluctuations twice per day, in addition to severe changes in salinity, light, and oxygen concentration. This type of habitat is quite rare, and no studies have been conducted on the unculturable microflora in these environments until now. Furthermore, the effects of drastic temperature and salinity fluctuations remain so far unknown.Chitin is an abundant polysaccharide in the marine environment. Information on microbial chitinases has been prominently restricted to a few culturable microorganisms belonging to ␤-and ␥-proteobacteria, gram-positive bacteria, and the domain Archaea (3,5,6,21). Yet, the use of chitinase genes as molecular markers has demonstrated the presence of a large pool of uncultured chitinolytic microorganisms in marine and soil environments (3,13,14,24).In this report, we used molecular diversity methods, combined with in situ enrichments and artificial support colonization, to investigate the apparent microbial diversity of 16S rRNA genes and chitinase genes. 16S rRNA gene analysis of natural samples has shown that the number of microbial species present can differ drastically in occurrence and that organisms that are below 0.1% can hardly be detected (2). However, combining cultivation methods and direct molecular techniques enables access to more diversity than does either method alone (7,16). By applying different sampling methods we analyzed both the diversity of 16S rRNA genes...

Microbial Community Structures of Novel Icelandic Hot Spring Systems Revealed by PhyloChip G3 Analysis

“…A previously favored technique to describe the microbial composition of hot springs at a molecular level was the cloning and Sanger sequencing method (Marteinsson et al, 2001a(Marteinsson et al, , 2001bHobel et al, 2005;Kvist et al, 2007). Many publications show the limitations of this technique, in particular, its low sensitivity due to the small amount of clones sequenced per sample (La Duc et al, 2009;Hazen et al, 2010).…”

Function-specific response to depletion of microbial diversity

“…Chitinase genes were amplified with the primer pair ChiA_F2 (5 0 -CGTGGACATCGACTGGGARTWY CC-3 0 ) and ChiA_R2 (5 0 -CCCAGGCGCCGTAGARRT CRTARSWCA-3 0 ) (Hobel et al, 2005). Products were obtained in a two-step PCR.…”

Bacterial Chitin Hydrolysis in Two Lakes with Contrasting Trophic Statuses

“…Family 19 chitinases were formerly thought to be restricted to plant origin but have since also been found in various Streptomyces species and other bacteria (4,49,57). However, the most information on bacterial diversity and distribution in diverse environments is available for family 18 group A chitinases (11,24,25,36,38,55).…”