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Abstract:
Using a DNA probe originally designed for detection of sigma factor genes in C. acetobutylicum, a 4722-bp fragment of clostridial chromosomal DNA was cloned and sequenced. Three complete open reading frames (ORFs) and a fourth truncated ORF at the 5' end of the fragment could be identified. The deduced amino acid sequence of the truncated ORF showed significant similarity to the kdpC gene of Escherichia coli and was therefore designed kdpC'. The following ORF did not reveal any homology to other known genes and proteins and was thus marked orfX. The deduced amino acid sequences of the last two ORFs showed high similarity to the proteins KdpD and KdpE of E. coli and the clostridial genes were named accordingly. KdpD and KdpE of C. acetobutylicum revealed significant homology to sensor histidine kinases and response regulators. This is the first complete two component signal transduction system identified in C. acetobutylicum. Putative transcription termination structures could be identified downstream of orfX as well as downstream of kdpE. By primer extension analysis, a transcription start site was determined 236 bp upstream of kdpD. The two genes kdpD and kdpE formed an operon as revealed by reverse transcriptase-polymerase chain reaction (RT-PCR). In vitro transcription/translation experiments resulted in formation of KdpD and KdpE; orfX was not expressed. Using wobbled oligonucleotides corresponding to conserved regions of KdpD and KdpE the presence of several additional putative sensor kinases and response regulators in C. acetobutylicum could be revealed by Southern hybridization experiments. (C) 1996 Academic Press.
