Overview of Clostridium BC1The genus Clostridium is a diverse group of anaerobic, gram-positive, rod-shaped, endo-spore forming bacteria. The taxon is very heterogeneous, comprising organisms with considerable variation in genome size (2.5 to 6.5 Mb) and G+C content (24 to 55 mol%). Clostridium sp. BC1 (ATCC No. 53464) (hereafter referred to as BC1), an anaerobic, N2-fixing member of this group that was isolated from coal-cleaning residues, is a potential candidate for ameliorating radionuclide contamination at DOE sites as it has biochemical pathways that can convert water soluble uranyl ion U(VI), to less soluble U(IV). The initial objective of this project is to use a whole genome shotgun sequencing approach to obtain large amounts of BC1 DNA sequence information to discover key functional genes and to understand gene sequence/function relationships in this and related Clostridia/Bacillus species. Sequencing the BC1 genome will also provide an extensive base of knowledge regarding the physiology of this organism and aid in the identification of gene promoter sequences and their modes of regulation, ribosome binding sites, codon usage frequency and other features important for BC1 gene function. This information will eventually help facilitate expression in BC1 of engineered biodegradative pathways from other microorganisms to expand its usefulness as a tool for stabilization of radionuclides in different environments.
Project statusThe chromosome size is determined by PFGE to be 3,815kb.
We have constructed several genomic BC1 libraries and have completed first end sequencing of 2-3 kbp BC1 genomic DNA fragments cloned into plasmid vectors using vector-specific primers. We are currently carrying out second end sequencing of these clones which should provide several hundred kb of additional unique sequence. Clone libraries have also been constructed in vectors we have developed for generating sets of bidirectional nested deletions whose ends are separated by ~400 bp. Fragments in the 10 kbp size range or larger can rapidly be sequenced from ordered sets of nested deletions using universal vector primers. Any gaps remaining can be closed by primer walking on the original clone.
We have obtained over 500kb of unique sequence from end sequencing, and are in the process of performing nested deletions on the clones which appear to have genes of interest. Work is also in progress to construct a physical map of the BC1 genome. The evolutionary relationship of BC1 to different Clostridium and Clostridium-related species was determined by 16S rRNA gene sequence analysis Direct sequencing of PCR amplified 16S rRNA BC1 genes indicates that BC1 is more closely related to C. pasteurianium than to C. acetobutylicum.
Future plansWe plan to clone and express those genes which are probable candidates for involvement in metal reduction. Biochemical studies on these genes will give us a better understanding of how this bacteria reduces heavy metals. This information will be useful for anyone trying to understand the processes involved, and for those trying to engineer microorganisms with improved metal reduction. Other future plans involve performing similar analysis on the mega plasmids from Alcaligenes eutrophus. This bacterium also has the ability to reduce certain heavy metals. Sequence data
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