Subtractive hybridization and random arbitrarily primed PCR analyses of a benzoate-assimilating bacterium, Desulfotignum balticum.
Subtractive hybridization (SH) and random arbitrarily primed PCR (RAP-PCR) were used to detect genes involved in anaerobic benzoate degradation by Desulfotignum balticum. Through SH, we obtained 121 DNA sequences specific for D. balticum but not for D. phosphitoxidans (a non-benzoate-assimilating species). Furthermore, RAP-PCR analysis showed that a 651-bp DNA fragment, having 55% homology with the solute-binding protein of the ABC transporter system in Methanosarcina barkeri, was expressed when D. balticum was grown on benzoate, but not on pyruvate. By shotgun sequencing of the fosmid clone (38,071 bp) containing the DNA fragment, 33 open reading frames (ORFs) and two incomplete ORFs were annotated, and several genes within this region corresponded to the DNA fragments obtained by SH. An 11.3-kb gene cluster (ORF10-17) revealed through reverse transcription-PCR showed homology with the ABC transporter system and TonB-dependent receptors, both of which are presumably involved in the uptake of siderophore/heme/vitamin B(12), and was expressed in response to growth on benzoate.
Habe
Hiroshi
H
Research Institute for Innovations in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan. hiroshi.habe@aist.go.jp
Kobuna
Akinori
A
Hosoda
Akifumi
A
Kouzuma
Atsushi
A
Yamane
Hisakazu
H
Nojiri
Hideaki
H
Omori
Toshio
T
Watanabe
Kazuya
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism*; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D020564:Deltaproteobacteria / Q000235:genetics* / Q000254:growth & development*; D016680:Genome, Bacterial; D015698:Genomic Library; D008565:Membrane Proteins / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D019289:Pyruvic Acid / Q000378:metabolism; D018038:Sodium Selenite / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001565:Benzoates; D003470:Culture Media; D004269:DNA, Bacterial; D008565:Membrane Proteins; C057616:tonB protein, Bacteria; C044659:molybdate; D008982:Molybdenum; D019289:Pyruvic Acid; D008780:Methyltransferases; D018038:Sodium Selenite