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The sim Operon Facilitates the Transport and Metabolism of Sucrose Isomers in Lactobacillus casei ATCC 334

John Thompson,^1* Nicholas Jakubovics,^2, Bindu Abraham,^3,¶ Sonja Hess,^3, and Andreas Pikis^1,

Microbial Biochemistry and Genetics Unit,¹ Oral Biofilm Communication Unit,² Oral Infection and Immunity Branch, NIDCR, and the Proteomics and Mass Spectrometry Facility, NIDDK, National Institutes of Health, DHHS, Bethesda, Maryland 20892³

Received 26 December 2007/ Accepted 19 February 2008

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_rs of 50,000 and 17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the 50-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 1.5- and 1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon.

Published ahead of print on 29 February 2008.

Present address: School of Dental Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4BW, England.

^¶ Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892.

Proteome Exploration Laboratory, California Institute of Technology, Pasadena, CA 91125.

Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993.