Cloning and expression of the hydrogenase gene from <i>Clostridium butyricum</i> in <i>Escherichia coli</i>

Karube, Isao; Urano, Naoto; Yamada, Takashi; Hirochika, Hirohiko; Sakaguchi, Kenji

doi:10.1016/0014-5793(83)80689-9

Cited by 37 publications

(16 citation statements)

References 14 publications

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“…Similar findings were recently reported for the nifH genes of C. pasteurianum (7). Surprisingly, all attempts to express ben in E. coli were unsuccessful, although heterologous expression of the cellulase genes from C. thermocellum and a hydrogenase gene from C. butyricum has been reported (11,20). One is forced to conclude that the lack of expression of bcn in E. coli is due to incompatible codon usage and the large size of the gene.…”

Section: Resultssupporting

confidence: 76%

Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene

Garnier¹,

Cole²

1986

J Bacteriol

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The bacteriocinogenic plasmid pIP404 from Clostridium perfringens was isolated and cloned in Eschenichia coli, and its physical map was deduced. Expression of the bcn gene, encoding bacteriocin BCN5, is inducible by UV irradiation of C. perfiringens and thus resembles the SOS-regulated bacteriocin genes of enteric bacteria.The location of bcn on pIP4W4 was established by a dot-blot procedure, using specific hybridization probes to analyze mRNA samples from induced and uninduced cultures. From the nucleotide sequence of its gene, the molecular weight of BCN5 was deduced to be 96,591, and a protein of this size was secreted by bacteriocinproducing cultures of C. perfiringens. The primary structure of the protein suggests that it may function as an ionophore, since a hydrophobic domain, resembling those of the ionophoric colicins, is present at the COOH terminus. No bacteriocin activity could be detected in E. coli harboring plasmids bearing the bcn gene, even when the transcriptional and translational signals were replaced by those of lacZ. A possible explanation may be found in the unusual codon usage of the adenine-thymine-rich bcn gene, as this shows a preference for codons with a high adenine-plus-thymine content, especially in the wobble position. Many of the frequently used codons correspond to those recognized by minor tRNA species in E. coli. Consequently, bcn expression might be limnited by tRNA availability in this bacterium.

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Section: Resultssupporting

confidence: 76%

Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene

Garnier¹,

Cole²

1986

J Bacteriol

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“…Strains and plasmids used in this study are listed in Table 1. R. opacus strain MR11, originally isolated by D. Siebert, is the type strain (Aggag & Schlegel, 1973;Klatte et al, 1994). The derivative strains MR2246 and MR1463 were described (Sensfuss et al, 1986;Reh & Schlegel, 1981) and studied in this laboratory.…”

Section: Methodsmentioning

confidence: 99%

Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

et al. 1997

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The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus M R l l into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7-4 kbp Asnl fragment of the linear plasmid pHG2Ol via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -Wand ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hoxF, -U, -Y and -H encode the subunits a, 7, 6 and B, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the / 3 subunit. Finally, ORF7 encodes a protein which has similarities to CAMP-and cGMP-binding protein kinases, but its function is not known. ORFI, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus M R l l indicated that the hydrogenase genes are under control of a like promoter located at the right end of ORFI and are even transcribed under heterotrophic conditions a t a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHYl harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.Keywords : Rhodococcus opacus MR11, NAD-reducing hydrogenase, box genes INTRODUCTIONRhodococcus opacus strain M R l l (formerly Nocardia opaca 1 b) is a Gram-positive, facultative chemolithoautotrophic bacterium which can grow on carbon dioxide and gaseous H, as the sole carbon and energy sources. Physiologically, it belongs to the knallgas bacteria, a group which is composed of phylogenetically diverse organisms. For the activation of H,, all knallgas bacteria contain hydrogenases, which are of two basic types : the membrane-bound hydrogenases (MBHs) , which are coupled to the electron transfer chain and are The GenBank accession number for the nucleotide sequence reported in this paper is U70364.not capable of reducing NAD, and the soluble hydrogenases (SHs), which are localized in the cytoplasm and catalyse the transfer of electrons directly to NAD (Aragno & Schlegel, 1992;Schneider et al., 1984a;Zaborosch et al., 1989; Schink & Schlegel, 1979). The MBHs belong to the more common group of NiFehydrogenases consisting of one large and one small subunit, whereas the SHs belong to a family of less abundant multimeric NiFe-hydrogenases (Friedrich & Schwartz, 1993 ;Wu & Mandrand, 1993). The majority of the knallgas bacteria contain only the MBH, but a few, ...

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“…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.…”

mentioning

confidence: 99%

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

Graves¹,

Mullenbach²,

Rabinowitz³

1985

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Cloning and expression of the hydrogenase gene from Clostridium butyricum in Escherichia coli

Cited by 37 publications

References 14 publications

Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene

Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene

Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

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

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