Bacterial expression of a mitochondrial cytochrome c. Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition.

Saccharomyces cerevisiae iso-1-cytochrome c has been expressed in Escherichia coli by coexpression of the genes encoding the cytochrome (CYC1) and yeast cytochrome c heme lyase (CYC3). Construction of this expression system involved cloning the two genes in parallel into the vector pUC18 to give the plasmid pBPCYC1(wt)/3. Transcription was directed by two promoters, Lac and Trc, that were located upstream from CYC1. Both proteins were expressed in the cytoplasm of E. coli cells harboring the plasmid. Semianaerobic cultures grown in a fermentor produced 15 mg of recombinant iso-1-cytochrome c per liter of culture. Attempts to increase production by addition of IPTG suppressed the number of copies of the CYC1 gene within the population. Wild-type iso-1-cytochrome c expressed with pBPCYC1(wt)/3 in E. coli was compared to the same protein expressed in yeast. At neutral pH, the two proteins exhibit indistinguishable spectroscopic and physical (Tm, Em') characteristics. However, electrospray mass spectrometry revealed that the lysyl residue at position 72 is not trimethylated by E. coli as it is by S. cerevisiae. Interestingly, the pKa of the alkaline transition of the protein expressed in E. coli is approximately 0.6 pKa unit lower than that observed for the cytochrome expressed in yeast (8.5-8.7). 1H NMR spectroscopy of the bacterially expressed cytochrome collected at high pH revealed the presence of a third alkaline conformer that is not observed in the corresponding spectrum of the cytochrome expressed in yeast. These observations suggest that Lys72 can serve as an axial ligand to the heme iron of alkaline iso-1-ferricytochrome c if it is not modified posttranscriptionally to trimethyllysine.

Cloning and sequence analysis of two laccase complementary DNAs from the ligninolytic basidiomycete Trametes versicolor.

Laccases are oxidoreductase enzymes involved in the oxidation of various phenolic compounds. They may play a role in the biodegradation of lignin and in the dechlorination of chlorophenols. The cDNAs encoding laccase LccI and a putative laccase LccIV and the gene for LccI from the white-rot basidiomycete Trametes versicolor were cloned, sequenced and characterized. The genomic DNA of lccI consists of 2128 bp, with the coding region interrupted by 10 introns; the cDNA consists of a 1560 bp open reading frame (ORF). The cDNA of the putative lccIV gene consists of a 1581 bp ORF, with a 794 bp 5' untranslated region. The size of the major transcript for both lccI and lccIV is approximately 2.3 kb. Transcription of lccIV was induced by 2,5-dimethylaniline, whereas the opposite effect was observed for lccI. Laccases I and IV contain highly conserved histidinyl and cysteinyl residues, believed to be involved in binding copper, and share extensive sequence similarity with other laccases produced by both ligninolytic and non-ligninolytic fungi.

Aerobic expression of the cyf gene encoding cytochrome c-553 from Desulfovibrio vulgaris Hildenborough in Escherichia coli.

Monohaem cytochrome c-553 from Desulfovibrio vulgaris Hildenborough is encoded by the cyf gene which could be expressed in Escherichia coli to yield the periplasmic holoform of cytochrome c-553. Covalent haem attachment was shown by labelling with 5-amino[4-14C]laevulinic acid, the immediate precursor for haem biosynthesis. Visible-absorption spectroscopy demonstrated that the haem environment in the recombinant protein did not differ from the native protein. Optimal expression was obtained under aerobic conditions. Furthermore, efficient insertion of haem into a cytochrome c-553-beta-lactamase fusion protein could be demonstrated. We suggest that the varying success in heterologous expression of c-type cytochromes in E. coli may arise as a result of differences in the physical properties of the apoproteins.

Molecular biology of c-type cytochromes from Desulfovibrio vulgaris Hildenborough.

Sulfate reducing bacteria of the genus Desulfovibrio harbor a wide variety of redox proteins. Three different c-type cytochromes, cytochrome c-553, cytochrome c3 and the high molecular mass cytochrome have been isolated from these bacteria. The high molecular mass cytochrome is part of an operon that encodes a transmembrane protein complex that mediates electron transfer across the cytoplasmic membrane. The physiological function of the other two cytochromes is less clear. They are encoded by monocistronic genes and their redox partners can thus not be identified by gene sequencing. Expression of genes for c-type cytochromes in a foreign host are complicated due to the requirement for covalent heme insertion. Cytochrome c-553 is readily expressed in Escherichia coli in functional form, but cytochrome c3 and the high molecular mass cytochrome are for reasons that are presently not clear.

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.

Biochemical and spectroscopic characterization of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough expressed in Desulfovibrio desulfuricans G200.

The gene of high molecular weight, multiheme cytochrome c (Hmc) from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has been overexpressed in Desulfovibrio desulfuricans G200. The recombinant protein has been purified. Its molecular weight (65,600), amino acid composition, and NH2-terminal sequence were found to be identical to those of the wild-type protein. The recombinant protein has been spectroscopically characterized (optical spectrum, EPR, circular dichroism) and compared to the wild-type protein. We have found 16 hemes per molecule by iron analysis and the pyridine hemochrome test. Both high- and low-spin features were observed in the EPR spectrum. A detailed spin quantitation analysis indicates 1 or 2 high-spin hemes and 14 or 15 low-spin hemes per molecule. The redox potentials of the hemes determined by voltammetric techniques gave an average of three different values, 0, -100, and -250 mV (versus NHE), for the wild-type and the recombinant cytochrome. The low potential values are similar to the values observed for the bis(histidinyl) coordinated hemes of cytochrome c3. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc has shown that the latter contains four domains, three of which are complete c3-like domains, while the fourth represents an incomplete c3-like domain which may contain His-Met coordinated hemes. These data are in agreement with the detailed study of the number and types of hemes reported in this paper.

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes.

Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli.

Plasmid pJRDC800-1, containing the cyc gene encoding cytochrome c3 from Desulfovibrio vulgaris subsp. vulgaris Hildenborough, was transferred by conjugation from Escherichia coli DH5 alpha to Desulfovibrio desulfuricans G200. The G200 strain produced an acidic cytochrome c3 (pI = 5.8), which could be readily separated from the Hildenborough cytochrome c3 (pI = 10.5). The latter was indistinguishable from cytochrome c3 produced by D. vulgaris subsp. vulgaris Hildenborough with respect to a number of chemical and physical criteria.

Expression of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein.

The expression of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) was examined in Escherichia coli transformed with either of two plasmids, pJ8 and pJ81. The former has an 840 bp insert of D. vulgaris DNA, containing the structural gene for cytochrome c3 (387 bp) and its promoter region. Plasmid pJ81 was generated from pJ8 by deoxyoligonucleotide-directed mutagenesis to direct the synthesis of a protein with an altered signal peptidase cleavage site [Ala(-1)----Asp(-1)]. Synthesis of the 14 kDa precursor, which was partly processed to the 12 kDa mature protein, was observed in cells of E. coli TG2(pJ8) by SDS gel electrophoresis and Western blotting. Analysis of spheroplasts revealed that the processed polypeptide was present in the periplasm while the precursor was found only in the membrane/cytoplasmic fraction. No processing was observed in E. coli TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site. No insertion of haem into the E. coli product could be detected in E. coli TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis. The sensitivity of the cytochrome c3 synthesized in E. coli TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed. The results indicate that E. coli is capable of synthesizing and exporting the cytochrome c3 polypeptide, but fails to insert the haems.

Prolapse of invaginated colon through the anus in golden hamsters (Mesocricetus auratus).

Six cases of invagination of the colon with prolapse through the rectum were reported in the Golden Syrian hamster. The anatomy of the colon and its enveloping mesentery in the hamster are thought to be predisposing factors in the process of invagination. The mesenteric fold incorporates the spleen in this animal and acts as a ligament stopping further invagination. The gross pathology associated with the process of invagination was described. Observations with respect to the pathogenesis were included, emphasizing the point that peristalsis and not the initiating intestinal irritant is the major causal factor.