Gene 250 (2000) 149–157 www.elsevier.com/locate/gene Expression pattern of Rhizobium etli ccmIEFH genes involved in c-type cytochrome maturation k Josue ´ David Reyes a,1, Maria-Luisa Tabche 1,a, Claudia Morera a, Maria de Lourdes Girard b, David Romero b, Elizaveta Krol a, Juan Miranda a, Mario Sobero ´n a, * a Departamento de Biologı ´a Molecular de Plantas. Instituto de Biotecnologı ´a, U.N.A.M., Apdo Postal 510-3, Cuernavaca, Morelos 62271, Mexico b Programa de Gene ´tica Molecular de Pla ´smidos Bacterianos, Centro de Investigacio ´ n sobre Fijacio ´ n de Nitro ´geno, U.N.A.M. Apdo. Postal 565-A, Cuernavaca, Morelos, Mexico Received 18 January 2000; received in revised form 21 March 2000; accepted 22 March 2000 Received by D.L. Court Abstract In different bacterial species, ccmIEFH genes have been suggested to code for subunits of a bacterial haem-lyase catalyzing the covalent attachment of haem to c-type apoproteins. In Rhizobium etli CE3 there are two copies of ccmIEFH: one in the chromosome and the other located in plasmid pf. However, the null phenotype of chromosomal ccmF mutant indicates that the gene locus of plasmid pf is not functional. Two ccmI chromosomal mutants, previously isolated, produced detectable levels of c- type cytochromes under certain culture conditions in contrast with the ccmF mutant, suggesting that ccmF could be transcribed independently. The transcriptional organization of ccmIEFH operon was established. Two promoters from the chromosomal locus were mapped by primer extension, one located upstream of ccmI and the second located upstream of ccmF. The regulation of the expression of both promoters was studied using appropriate lacZ gene fusions (ccmI–lacZ and ccmEF–lacZ ). The ccmI–lacZ gene fusion was expressed in complex medium, during exponential growth, under microaerobic conditions and in a R. etli mutant that accumulates reducing power, conditions where a higher respiration rate could be limited by c-type cytochrome content. The ccmEF–lacZ fusion was also primarily expressed in complex medium and under microaerophilic conditions. The finding of two independent promoters in this gene locus could suggest that the step catalyzed by CcmFH could be a rate-limiting step for c-type cytochrome assembly under certain culture conditions. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Biogenesis; c-type cytochromes; Regulation; Respiration 1. Introduction of haem to the polypeptide chain, usually via two thioether linkages, formed from vinyl groups of haem and two cystein residues within a CxxCH motif of the The c-type cytochromes are ubiquitous proteins, polypeptide. In Neurospora crassa and Saccharomyces located either on the cytoplasmic membrane or in the cerevisiae, two haem-lyase enzymes, catalyzing the cova- bacterial periplasm, and function as electron carriers lent attachment of the haem moiety, have been between energy transduction complexes during various described, each one specific for c or c 1 cytochromes growth modes (Tho ¨ ny-Meyer, 1997). c-type cyto- (Dumont et al., 1987; Drygas et al., 1989). Homologues chromes are characterized by the covalent attachment of these enzymes have not been found in bacteria, where at least 12 different genes are implicated in the matura- tion of c-type cytochromes ( Tho ¨ny-Meyer et al., 1994; Abbreviations: aa, amino acid(s); bp, base pair(s); nt, nucleotide; ORF, open reading frame; R., Rhizobium; SDS, sodium dodecyl sul- Page et al., 1998). Among these genes, cycHJKL, orga- fate; TMPD, N,N,N∞,N∞-tetramethyl-p-phenylendiamine; wt, wild-type. nized in an operon in some bacterial species, has been k The DNA sequences of the R. etli ccmI, ccmE and ccmF genes suggested to code for a bacterial haem-lyase (Ritz et al., from plasmid pf are available under Accession No. AF176798. 1993, 1995; Delgado et al., 1995; Kereszt et al., 1995). * Corresponding author. Tel.: +52-73-291618; fax: +52-73-172388. In this article, we use the recent nomenclature proposed E-mail address: mario@ibt.unam.mx (M. Sobero ´n) 1 Both authors contributed equally to this work for genes involved in bacterial c-type cytochrome matu- 0378-1119/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S0378-1119(00)00176-1