Metagenomic DNA fragments that affect Escherichia coli mutational pathways.
A multicopy cloning approach was used to search for metagenomic DNA fragments that affect Escherichia coli mutational pathways. Soil metagenomic expression libraries were constructed with DNA samples prepared directly from soil samples collected from the UCLA Botanical Garden. Using frameshift mutator screening, we obtained a total of 26 unique metagenomic fragments that stimulate frameshift rates in an E. coli wild-type host. Mutational enhancer strains such as an ndk-deficient strain and a temperature sensitive mutS strain (mutS60) were used to further verify the mutator phenotype. We found that the presence of multiple copies of certain types of metagenomic DNA sequence repeats cause general genome instability in the wild-type E. coli host and the effect can be suppressed by overproducing a DNA mismatch component MutL. In addition, we identified nine metagenomic mutator genes (designated as smu genes) that encode proteins that have not been linked to mutator phenotypes prior to this study including a putative RNA methyltransferase Smu10A. The strain overproducing Smu10A displays one prominent base substitution hotspot in the rpoB gene, which coincides with the base substitution hotspot we have observed in cells that are partially deficient in the proofreading function carried out by the DNA polymerase III epsilon subunit. Based on the structural conservation of DNA replication/recombination/repair machineries among microorganisms, this approach would allow us to both identify new mutational pathways in E. coli and to find genes involved in DNA replication, recombination or DNA repair from vast unculturable microbes.
Yang
Hanjing
H
Department of Microbiology, Immunology and Molecular Genetics, and the Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
To
Kam H
KH
Aguila
Sharon J
SJ
Miller
Jeffrey H
JH
D000595:Amino Acid Sequence; D044822:Biodiversity; D002140:California; D019295:Computational Biology; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics*; D015723:Gene Library; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011995:Recombination, Genetic; D016415:Sequence Alignment; D012988:Soil Microbiology
D012336:RNA, Ribosomal, 16S