ThesimOperon Facilitates the Transport and Metabolism of Sucrose Isomers inLactobacillus caseiATCC 334

Abstract

Inspection of the genome sequence of Lactobacillus casei ATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells of L. casei ATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides with M r s of ϳ50,000 and ϳ17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the ϳ50-kDa protein as an NAD ؉ -and metal ion-dependent phospho-␣-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-␣-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising the sucrose isomer metabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth of L. casei ATCC 334 on sucrose isomers. Internal simA and simF gene probes hybridized to ϳ1.5-and ϳ1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon.Comparative genomics and phylogenetic analyses of the lactic acid bacteria (LAB) have provided evidence that the order Lactobacillales comprises the families Lactobacillaceae, Streptococcaceae, Enterococcaceae, and Leuconostocaceae (17, 25; http://www.ncbi.nlm.nih.gov/Taxonomy/). These generally fastidious gram-positive organisms are used extensively for the fermentation of dairy, meat, and vegetable products. The industrial importance of LAB, and Lactobacillus casei strains in particular, results from the capacity of these microorganisms to metabolize carbohydrates rapidly (and primarily) to lactic acid. Prior to their homolactic fermentation via the glycolytic pathway, many carbohydrates are accumulated simultaneously with phosphorylation via sugar-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs) (7,24,29). The multicomponent PEP-dependent PTSs comprise membrane-localized, sugar-specific transporters (IICB enzymes) that may be fused or associated with a third protein (EIIA), as well as two general cytoplasmic proteins (EI and HPr). Collectively, these interactive proteins constitute a fivestage phosphorelay that, via transfer of the high-energy phosphoryl moiety from PEP, catalyzes the simultaneous phosphorylation and translocation of sugars through the cytoplasmic membrane. Biochemical and physiological studies performed during the past 25 years have established the presence of a variety of sugar PEP-dependent PTSs in L. casei, including PTSs for glucose (41, 45), galactose (2, 6), lactose (4,5,8,9), sorbose (46), and pentitols (15, 16). Significantly, in the past decade, the development ...

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