Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.DNA polymerases are essential for DNA replication and DNA repair. Based on sequence similarities and phylogenetic relationships, DNA polymerases are grouped into six different families (A, B, C, D, X, and Y) (17). In this study, we used a DNA polymerase I (polA) mutant of Escherichia coli as a host for the screening of metagenomic libraries. PolA belongs to family A and contains three different domains: a 5Ј-3Ј exonuclease domain at the N terminus, a central proofreading 3Ј-5Ј exonuclease domain, and a polymerase domain at the C terminus of the enzyme (11). These polymerases are employed as tools in molecular biology, including probe labeling, DNA sequencing, and mutagenic PCR (13). To improve their suitability for such applications, various family A DNA polymerases have been modified; e.g., the Klenow fragment of E. coli DNA polymerase I has been redesigned by the removal of the 5Ј-3Ј exonuclease domain (12). Nevertheless, expanding the known DNA polymerase sequence space and discovery of polymerases with novel properties are required for the development of novel or improved molecular methods and tools (13,20).Metagenomics based on direct isolation of DNA from environmental samples, generation of metagenomic libraries from the isolated DNA, and function-based screening of the constructed libraries has led to identification and characterization of a variety of novel biocatalysts, such as lipases, amylases, amidases, nitrilases, and oxidoreductases (for reviews, see references 6, 7, and 10). In particular, the use of host strains or mutants of host strains that require heterologous complementation for growth under selective conditions has proven to be an efficient strategy to screen complex metagenomic libraries. This approach has been applied to, e.g., the isolation of genes encoding Na ϩ /H ϩ antiporters (14), antibiotic resistance (18), or enzymes involved in poly-3-hydroxybutyrate metabolism (21).In this study, we employed the last-named strategy to recover functional genes encoding DNA polymerases. To our knowledge, this is the first report of identification of polymerases or other DNA-modifying enzymes by function-driven screening of metagenomes. For this purpose, we constructed small-insert and large-insert metagenomic libraries from DNA isolated from glacial ice. The employment of glacial ice samples for metagenomic library construction has not been reported by other researchers. The screening for the targeted genes was based on complementation of a cold-sensitive lethal mutation in the polA gene of E. coli (16).Sample collection and construction of metagenomic glacier ice libraries. G...

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per g of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.Proteolytic enzymes catalyze the hydrolytic cleavage of peptide bonds. These enzymes are present in all living organisms and are essential for cell growth and differentiation. Microorganisms produce a variety of intracellular and/or extracellular proteases. Intracellular microbial proteases are highly specific and are involved in several cellular and metabolic processes such as activation of inactive precursors, maintenance of the cellular protein pool, and sporulation. Extracellular proteases degrade proteins in cell-free environments. The resulting hydrolytic products (small peptides and amino acids) can be transported into the cells and utilized as carbon or nitrogen sources (13, 39). Especially, extracellular proteases are of industrial importance and are used as cleaning agents and food and feed additives.Proteases can be divided into various groups with respect to the functional group present at the active site and the pH profile of the activity. Microbial alkaline proteases, which are defined to be active in a neutral to alkaline pH range (13), possess either a serine center (serine proteases) or are of metal-type (metalloproteases). Extracellular...

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