Genetic Manipulation ofClostridium

Abstract

The genus Clostridium is classified in the family Clostridiaceae of the phylum Firmicutes of Bacteria. The genetic manipulation of clostridia is still in an early stage of development, but significant advances have been made in recent years, and this chapter emphasizes newly developed genetic methods and strategies. Certain physiological properties of clostridia are integral for genetic manipulation and gene expression. Clostridia are classified as proteolytic or saccharolytic depending on their preference for energy-yielding substrates. Conjugal transfer between the same and different species of Clostridium certainly occurs in the environment and in the gastrointestinal tracts of mammals, but it is not used for routine genetic manipulation. This chapter describes the replicons, selective markers, and reporter genes most frequently used for construction of clostridial shuttle vectors. It also describes the latest developments in the field. The most comprehensive and detailed transcriptional analysis including the life cycle of C. acetobutylicum has been performed in the E. T. Papoutsakis laboratory. This study described major cellular regulatory systems; sigma and sporulation factors, including activity assays for major sporulation factors of the canonical sets of genes from their regulons; assessment of expression intensities; and identification of putative histidine kinases that may phosphorylate Spo0A. The availability of genomic sequences provides insights into population genetics and evolution of Clostridium. Ideally, the development of further genetic tools will foster fruitful collaborations within the clostridial community to understand this complex and difficult organism.