Cloning and nucleotide sequence determination of the Clostridium pasteurianum ferredoxin gene.

Abstract

We have constructed a library of Clostridium pasteurianum DNA cloned in the plasmid pBR322. Based on the known amino acid sequence for C. pasteurianum ferredoxin, a 64-fold degenerate heptadecanucleotide pool was synthesized. This mixed probe hybridized to two clones which were shown to contain >6 kilobase pairs of the same genomic DNA. Sequence analysis of a common Sau3Al 0.6-kilobasepair fragment revealed that it contains the information for the apoferredoxin structural gene. According to the DNA sequence, the only post-translational processing of this small apoprotein is the hydrolysis of the initiator methionine. Putative transcription and translation start and stop signals are present within the sequence.Non-heme iron-sulfur proteins function as electron carriers and redox enzymes in a variety of organisms. The Holm and Ibers (2). Furthermore, the Fd apoprotein from a variety of bacteria has been sequenced, demonstrating the small size (less than 6000 Da) and highly conserved nature of this protein (3).Although much is known about the ferredoxins and other iron-sulfur proteins, the mechanism by which an Fe:S cluster forms in vivo is not well understood. We have been developing clostridial Fd as a model for the biosynthesis of an apoprotein and its conversion to a functional Fe:S clustercontaining form. Early attempts to produce Fd in vitro led to the development of fractionated protein synthesizing systems from several Gram-positive organisms (4-6). However, synthesis of apoFd in the fractionated systems was not detected, probably due to the relatively low amounts of Fd mRNA or gene copies required to direct it. Some progress was made in the partial purification of Fd mRNA from Clostridium pasteurianum, but the amount and purity obtained were not sufficient for biosynthesis studies (7). We have therefore set out to clone the C. pasteurianum Fd gene to direct apoFd synthesis in our in vitro systems. Based on the known protein sequence (3), we have synthesized a mixed oligonucleotide probe pool and used it to identify clones containing the C. pasteurianum Fd gene. Although at least two clostridial genes have been cloned by complementation of a deficiency in the host strain (8, 9), this work presents the sequence of a clostridial gene. MATERIALS AND METHODSMaterials. Restriction enzymes, T4 polynucleotide kinase, DNA polymerase I, and the large fragment of DNA polymerase I (Klenow fragment) were purchased from Bethesda Research Laboratories and used according to manufacturer's instructions. Proteinase K, RNases A and T1, and calf intestinal alkaline phosphatase were supplied by Boehringer Mannheim. The phosphatase was purified by G-75 gel filtration and stored in 50% glycerol at -20°C. Chemicals were of reagent-grade quality.Genomic DNA. C. pasteurianum (American Type Culture Collection, no. 6013) soil spores were revived in potato medium and 2-liter cultures were grown in synthetic medium (10). Escherichia coli HB101 was grown in LB medium and supplemented with antibiotics as noted. Genomic DNA was...

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli

“…Genes involved in amino acid biosynthesis (64), glutamine synthetase (57), alcohol dehydrogenase (62), xylanase (65), endoglucanase (64), and cellobiase (64) have all been cloned from a gene library for C. acetobutylicum P262 by direct selection for expression in E. coli. These results would suggest that E. coli-like promoters and ribosome binding sites are widespread in C. acetobutylicum, as has been reported for a number of genes from other gram-positive bacteria (21,38,40). Indeed, such sequences have been identified in the glnA (30), cbgA and cbgR (the present study) and adhl (61) genes from C. acetobutylicum, but in the case of the adhl gene, a vector promoter was necessary for expression in E. coli.…”

“…Putative E. coli-like promoter and ribosome binding sites are underlined. The dashed underlining identifies a 46-bp extended promoter sequence similar to a consensus sequence characteristic of many gram-positive bacterial promoters (21). The position of TnS insertions in the sequence and their allele numbers are shown by V (Lac') and V (Lac-).…”

“…Indeed, such sequences have been identified in the glnA (30), cbgA and cbgR (the present study) and adhl (61) genes from C. acetobutylicum, but in the case of the adhl gene, a vector promoter was necessary for expression in E. coli. However, there are reports of other Clostridium genes that are not expressed in E. coli (19,21) despite the presence of the appropriate transcriptional and translational signals. The explanation proposed for this is the existence of extreme differences in codon usage between C. acetobutylicum and E. coli (19).…”

Molecular analysis of the mannitol operon of Clostridium acetobutylicum encoding a phosphotransferase system and a putative PTS-modulated regulator

“…6). The corresponding promoter sequence (5h-TTGAAA-17 bp-TATCAT) showed reasonable homology to the consensus suggested for Gram-positive bacteria and clostridia (Graves & Rabinowitz, 1986 ;Young et al, 1989) and partially overlapped the CRE2 DNA sequence found in this position (Fig. 6).…”

Expression of Bacillus amyloliquefaciens amylase and Vibrio alginolyticus protease A fusion genes

“…alginolyticus proA gene carried on pBVP100 did not produce active Pro A. This was presumably due to differences in promoter consensus requirements between Gram-positive and Gram-negative bacteria (Graves & Rabinowitz, 1986).…”