Genetic manipulation of the obligate chemolithoautotrophic bacterium Thiobacillus denitrificans

Journal Article · Fri Jul 15 04:00:00 EDT 2011 · Microbial Systems Biology: Methods and Protocols, Methods in Molecular Biology series

OSTI ID:1051280

Beller, H R; Legler, T C; Kane, S R

Chemolithoautotrophic bacteria can be of industrial and environmental importance, but they present a challenge for systems biology studies, as their central metabolism deviates from that of model organisms and there is a much less extensive experimental basis for their gene annotation than for typical organoheterotrophs. For microbes with sequenced genomes but unconventional metabolism, the ability to create knockout mutations can be a powerful tool for functional genomics and thereby render an organism more amenable to systems biology approaches. In this chapter, we describe a genetic system for Thiobacillus denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. Insertion mutations are generated by in vitro transposition, the mutated genes are amplified by the PCR, and the amplicons are introduced into T. denitrificans by electroporation. Use of a complementation vector, pTL2, based on the IncP plasmid pRR10 is also addressed.

• Research Organization: Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, CA (US)
• Sponsoring Organization: Earth Sciences Division
• DOE Contract Number: AC02-05CH11231
• OSTI ID: 1051280
• Report Number(s): LBNL-5097E
• Journal Information: Microbial Systems Biology: Methods and Protocols, Methods in Molecular Biology series, Journal Name: Microbial Systems Biology: Methods and Protocols, Methods in Molecular Biology series Vol. 881
• Country of Publication: United States
• Language: English

Related Subjects

54 ENVIRONMENTAL SCIENCES
58 GEOSCIENCES
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOLOGY
FUNCTIONALS
GENES
GENETICS
IN VITRO
METABOLISM
MUTATIONS
PLASMIDS
RECOMBINATION