The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H-NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56,613 Da (CaiT), 42,564 Da (CaiA), 59,311 Da (CaiC), 32,329 Da (CaiD) and 21,930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium.

Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis. When cloned into a multicopy plasmid, the 3 region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene.

Anaerobic carnitine metabolism in Escherichia coli was recently shown to involve six genes organized in the cai operon and located at the first minute on the chromosome. The DNA sequence lying at the 5' end of the cai locus was further investigated. It contains four open reading frames organized as an operon. In vivo overexpression of this DNA region revealed four polypeptides with apparent molecular masses of 27, 33, 45 and 6 kDa. These proteins displayed significant amino acid sequence homologies with polypeptides encoded by the fixABCX operons from Azorhizobium caulinodans and Rhizobium meliloti. The four ORFs were thus named fixABCX. The first two gene products were also found to share a high degree of sequence similarity with the subunits beta and alpha, respectively, of mammalian electron transfer flavoproteins, suggesting a role for these proteins in a redox reaction. A singly polycistronic 5 kb mRNA transcript was detected in Northern blots under anaerobic conditions in the presence of DL-carnitine. Expression of a fixA-lacZ transcriptional fusion was induced by L(-)-carnitine and crotonobetaine but not by D(+)-carnitine, gamma-butyrobetaine, glycinebetaine and choline as found previously for the carnitine pathway. Similarly, the fix operon was repressed by glucose and nitrate. Moreover, expression of the fix operon was induced by the global regulatory proteins CRP and FNR and repressed by the histone-like protein H-NS. All these regulatory proteins have been shown also to control expression of carnitine enzymes. Results from Northern blots and lacZ fusion studies indicate a common regulation of expression of fix and cai operons, which implies a physiological linkage between these two loci.

The divergent structural operons caiTABCDE andfixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopylacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in anfnr mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at −41.5 of the caitranscriptional start site, plays a direct role in the solecai activation. In contrast, mutation in CRP site 2, positioned at −69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at −126.5 of fix, might be to reinforce the action of site 2. A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.

SummaryActivation of the two divergent Escherichia coli cai and ®x operons involved in anaerobic carnitine metabolism is co-dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the speci®c carnitinesensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, puri®ed on a heparin column and ran as a 15 kDa protein on SDS±PAGE. DNase I footprinting and interference experiments identi®ed two sites, F1 and F2, with apparently comparable af®nities for the binding of CaiF in the cai± ®x regulatory region. These sites share a common perfect inverted repeat comprising two 11 bp halfsites separated by 13 bp, and centred at À70 and À127 from the ®x transcription start site. They were found to overlap the two low-af®nity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co-operatively to the F1/ CRP2 and F2/CRP3 sites of the intergenic cai±®x region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP±CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the ®x promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N-terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.