Use of low nutrient enrichments to access novel amylase genes in silent diversity of thermophiles

Abstract

By combining low nutrient enrichments and molecular methods, a high diversity of new amylase genes was detected in a neutral sulphide-rich hot spring in Iceland. Enrichments based on hot spring water and low concentrations of starch were used to select slow-growing, starch-degrading microorganisms. Six enrichments had in total 17 bacterial types detected by 16S rRNA analysis, mostly related to the Thermus-Deinococcus group, green non-sulphur bacteria, gram positives, and uncultivated new candidate divisions. No Archaea were found. The apparent 16S rRNA species composition of the enrichments was very different from that of the microbial mat in the same hot spring. DNA samples obtained from 4 enrichments and from hot spring biomass were screened by PCR for amylase genes in glycoside-hydrolase family 13. Degenerate primers, based on conserved amino acid sequences from multiple alignments of family 13, enabled the detection of 18 amylase sequence types in the enrichments, including a-amylases, a-glucosidases, 1,4-a-glucan branching enzymes, cyclomaltodextrin hydrolases, maltogenic amylases and neopullulanases, and unspecified family 13 glycoside-hydrolases. Only one unique neopullulanase sequence, also found in most of the enrichments, was detected in the hot spring biomass DNA. The results suggest that the enrichment method combined with sequence-based screening is an efficient way to access the silent, i.e. not detectable, gene diversity in natural environments.

Thermus islandicus sp. nov., a mixotrophic sulfur-oxidizing bacterium isolated from the Torfajokull geothermal area

“…kawarayensis' (unpublished results). Nearly identical sequences had previously been identified in two hot springs in south-western Iceland (Skirnisdottir et al, 2000;Hjorleifsdottir et al, 2001;Hobel et al, 2004). The aim of this work was to isolate and describe this unknown species of the genus Thermus.…”

A comparison of methods for total community DNA preservation and extraction from various thermal environments

“…As a consequence, the entire Weld of environmental microbiology, from basic ecological research into the organization of microbial communities to bioprospecting for commercially relevant enzymes has changed. Even with recent advances in culturing eVorts [19,38], the majority of microbes in the environment still cannot be cultivated in the laboratory [34]. However, inability to maintain an organism in culture is no longer a major impediment to accessing its genetic diversity.…”

“…Thermal environments have been a particularly rich source of unique organisms [2,13,16,20,43], processes [5], novel enzymes [19,39], and on-going research into the origin and diversity of microbes. Fundamental to any of these studies is maximizing the detectable diversity by optimizing the quality and quantity of DNA examined and minimizing the biases of the methods.…”

Investigation of the Microbial Ecology of Intertidal Hot Springs by Using Diversity Analysis of 16S rRNA 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 and the diversity of chitinase genes promoted by selective growth of chitinolytic microorganisms via in situ enrichments.…”

“…About 100 microbial amino acid sequences of chitinases from the glycoside hydrolase family 18 (4) were retrieved from GenBank and Pfam (18) and used by multiple alignments as template for the design of degenerate PCR probes as described by Hobel et al (7). All chitinases belonged to group A of the catalytic domains from family 18 of the glycoside hydrolases, which appear to be most prevalent in nature (13).…”