In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80°C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90°C, and a Chloroflexus mat is formed at 65 to 70°C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter ؊1 ) below 70°C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter ؊1 ) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.Sulfide-rich hot springs with neutral or alkaline pH are relatively rare in most geothermal areas in the world. However, these types of hot springs are rather common in Iceland due to high ground water level and climatic conditions, i.e., from melting snow and rain. Bacteria that thrive in such springs often form long streamers or mats, but the appearance of the mats and the types of bacteria in them seem to vary depending on the sulfide concentration, pH, temperature, and other chemical and physical factors (6,7,10,11,12,20,33). Many Icelandic hot springs have sulfide concentrations as high as 30 mg liter Ϫ1 and, under such conditions, thick bacterial mats which can be spectacularly white or bright yellow from precipitated sulfur are formed.The diversity of many microbial ecosystems has now been studied with different molecular methods, such as analysis of small-subunit (SSU) rRNA by sequencing, denaturing gradient gel electrophoresis, or restriction fragment length polymorphism analyses. These studies show that the diversity of microbial ecosystems is typically 100 to 1,000 times greater than that shown by cultivation alone (14,15,22,23,30,31). The sequencing of rRNA genes from environmental samples is very informative, since it provides information on both the phylogenetic relationship and the population structure of the microbial community. With increased understanding of the role and importance of microbes in many ecosystems, the benefit of microbial diversity studies is being recognized. The practical value of these methods is already widespread, as they can be used to study the performance of wastewater treatment plants (27), monitor changes upo...

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The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na(+) and Ca(2+) ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.