Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes.
Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening and enzyme activity-based screening. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon-induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.
Uchiyama
Taku
T
Laboratory of Applied Microbiology, Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan.
Abe
Takashi
T
Ikemura
Toshimichi
T
Watanabe
Kazuya
K
D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D002469:Cell Separation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D030541:Databases, Genetic; D004777:Environment; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D016678:Genome; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D019151:Peptide Library; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry
D018076:DNA, Complementary; D019151:Peptide Library; D049452:Green Fluorescent Proteins