Sequencing and characterization of a gene cluster encoding the enzymes for L-rhamnose metabolism in Escherichia coli

Abstract

The sequencing of the EcoRI-HindIII fragment complementing mutations in the structural genes of the L-rhamnose regulon of Escherichia coli has permitted identification of the open reading frames corresponding to rhaB, rhaA, and rhaD. The deduced amino acid sequences gave a 425-amino-acid polypeptide corresponding to rhamnulose kinase for rhaB, a 400-amino-acid polypeptide corresponding to rhamnose isomerase for rhaA, and a 274-amino-acid polypeptide corresponding to rhamnulose-1-phosphate aldolase for rhaD. Transcriptional fusions of the three putative promoter regions to lacZ showed that only the rhaB leader region acted as a promoter, as indicated by the high 13-galactosidase activity induced by rhamnose, while no significant activity from the rhaA and rhaD constructions was detected. The rhaB transcription start site was mapped to -24 relative to the start of translation. Mutations in the catabolic genes were used to show that L-rhamnose may directly induce rhaBAD transcription.L-Rhamnose, a methylpentose, is metabolized in Escherichia coli by a set of enzymes encoded by genes constituting the rhamnose regulon, which maps at 88.4 min in the chromosome (2). Four structural genes have been described: rhaA, encoding rhamnose isomerase; rhaB, encoding rhamnulose kinase; rhaD, encoding rhamnulose-1-phosphate aldolase (32); and rhaT, encoding the rhamnose transport system (17). The rhaT gene has been mapped in the rha locus, separated from rhaA, rhaB, and rhaD by the regulatory operon rhaC, which has been found to be formed by two partially overlapping genes, rhaR and rhaS (40). The gene order of the region, counterclockwise, is glpK... sodArhaT-rhaR-rhaS-rhaB-rhaA-rhaD.In E. coli, rhaT, encoding the transporter (17, 39), and rhaR and rhaS, governing expression (40, 41), have been sequenced and extensively analyzed. In this species, another methylpentose, L-fucose, is metabolized by a parallel metabolic pathway integrated by a set of specific enzymes encoded by the fuc gene cluster, which has been located at 60.2 min (23) and completely sequenced (11,25). In Salmonella typhimurium LT2, rhaB, for rhamnulose kinase; rhaC2, one of the regulatory genes (31); and rhaT, encoding the transporter (39) have also been sequenced.Here we present a sequence analysis of three structural genes of the rhamnose pathway, some experiments involving their expression, and a comparison with the corresponding gene sequences of the L-fucose system. MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. Cells were grown aerobically as described previously (7) on LB or minimal medium. For growth on minimal medium, L-rhamnose, glucose, or L-fucose was added to 0.2%. When indicated, X-Gal (5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside) was added to 40 ,ug/ml. Ampicillin was used at 100 p,g/ml, kanamycin was used at 30 ,ug/ml, and streptomycin was used at 25 ,ug/ml. For primer extension analysis, the * Corresponding author. strains were grown in M10 medium (33) contain...

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