Cloning of the RNase H genes from a metagenomic DNA library: identification of a new type 1 RNase H without a typical active-site motif.
OBJECTIVE
The study aimed to combine a metagenomics approach with complementary genetics to identify novel bacterial genes with orthologous functions, with the identification of novel RNase H genes as a test case.


RESULTS
A metagenomic DNA library was prepared from leaf-and-branch compost and used to screen for the RNase H genes by their abilities to complement the temperature-sensitive growth phenotype of the rnhA mutant Escherichia coli strain MIC3001. Determination of the nucleotide sequences of the cloned DNA fragments allowed us to identify 12 different genes encoding type 1 RNases H. Eleven of them encode novel RNases H, which show 40-72% amino acid sequence identities to those available from database. One of them lacks a typical DEDD/E active-site motif, which is almost fully conserved in various RNases H.


CONCLUSIONS
Functional screening of environmental DNA without cultivation of microbes is a useful procedure to isolate novel RNase H genes.


CONCLUSIONS
One of the identified RNase H genes had no sequence similarity to a previously assumed conserved motif, suggesting multiple catalytic mechanisms exist. This test case illustrates that metagenomics combined with complementary genetics can identify novel genes that are orthologous without sequence similarity to those from cultivated bacteria.
Kanaya
E
E
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan. kanaya@mls.eng.osaka-u.ac.jp
Sakabe
T
T
Nguyen
N T
NT
Koikeda
S
S
Koga
Y
Y
Takano
K
K
Kanaya
S
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D054892:Metagenome; D056186:Metagenomics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016914:Ribonuclease H / Q000737:chemistry* / Q000145:classification / Q000235:genetics*; D016415:Sequence Alignment
D004269:DNA, Bacterial; D016914:Ribonuclease H