Genetics of l -Sorbose Transport and Metabolism in Lactobacillus casei

Abstract

Genes encoding L-sorbose metabolism of Lactobacillus casei ATCC 393 have been identified on a 6.8-kb chromosomal DNA fragment. Sequence analysis revealed seven complete genes and a partial open reading frame transcribed as two units. The deduced amino acid sequences of the first transcriptional unit (sorRE) showed high similarity to the transcriptional regulator and the L-sorbose-1-phosphate reductase of the sorbose (sor) operon from Klebsiella pneumoniae. The other genes are transcribed as one unit (sorFABCDG) in opposite direction to sorRE. The deduced peptide sequence of sorF showed homology with the D-sorbitol-6-phosphate dehydrogenase encoded in the sor operon from K. pneumoniae and sorABCD to components of the mannose phosphotransferase system (PTS) family but especially to domains EIIA, EIIB, EIIC and EIID of the phosphoenolpyruvate-dependent L-sorbose PTS from K. pneumoniae. Finally, the deduced amino acid sequence of a truncated gene (sorG) located downstream of sorD presented high similarity with ketose-1,6-bisphosphate aldolases. Results of studies on enzyme activities and transcriptional analysis revealed that the two gene clusters, sorRE and sorFABCDG, are induced by L-sorbose and subject to catabolite repression by D-glucose. Data indicating that the catabolite repression is mediated by components of the PTS elements and by CcpA, are presented. Results of sugar uptake assays in L. casei wild-type and sorBC mutant strains indicated that L-sorbose is taken up by L-sorbose-specific enzyme II and that L. casei contains an inducible D-fructose-specific PTS. Results of growth analysis of those strains and a man sorBC double mutant suggested that L-sorbose is probably also transported by the Dmannose PTS. We also present evidence, from studies on a sorR mutant, suggesting that the sorR gene encodes a positive regulator of the two sor operons. Sequence alignment of SorR, SorC (K. pneumoniae), and DeoR (Bacillus subtilis) revealed that they might constitute a new group of transcriptional regulators.In the enteric bacteria Klebsiella pneumoniae and Escherichia coli, the ketose L-sorbose is transported and phosphorylated through a phosphoenolpyruvate-dependent L-sorbose-specific phosphotransferase system (PTS) (36,46,47). The metabolism of L-sorbose involves the formation of D-fructose-6-phosphate, which enters the glycolysis pathway (Fig. 1A). During uptake of a PTS carbohydrate, the common PTS proteins, enzyme I (EI) and HPr, transfer a phosphoryl group from phosphoenolpyruvate to the substrate-specific EII complexes. The L-sorbose PTS EII consists of two membrane-bound proteins, EIIC Sor PTS elements are involved in sugar uptake and also in the regulation of chemotaxis, carbon metabolism, and modulation of gene expression (9,20,27). Many catabolic operons in bacteria are subject to carbon catabolite repression (CR) by rapidly metabolizable carbon sources, especially glucose (31, 37). The main CR mechanism identified in E. coli can be summarized as follows. EIIAB Glc acts as the sensor of extracellular gl...

Molecular analysis of the glucose-specific phosphoenolpyruvate : sugar phosphotransferase system from Lactobacillus casei and its links with the control of sugar metabolism

“…The analysis of a spontaneous 2-DG-resistant mutant (BL30) lacking glucose-specific PTS activity provided the first evidence that the PTS in L. casei is involved in the CCR effect exerted by glucose on the utilization of lactose, ribose and sorbose (Veyrat et al, 1994;Yebra et al, 2000). It has been shown here that the phenotype of this strain is most likely due to a mutation in the IIAB Man protein encoded by manL.…”

“…Previous studies had shown that glucose uptake by the PTS is involved in the regulation of the ribose, lactose and sorbose catabolic pathways in L. casei (Veyrat et al, 1994;Yebra et al, 2000). In order to identify the genes encoding the PTS Man in L. casei, a search for genes encoding putative IID proteins was performed in the draft genomic sequence of L. casei BL23 (Klaenhammer et al, 2002).…”

“…In order to identify the genes encoding the PTS Man in L. casei, a search for genes encoding putative IID proteins was performed in the draft genomic sequence of L. casei BL23 (Klaenhammer et al, 2002). In addition to IID Sor , IID Lev and IID Esu already described in L. casei, (Yebra et al, 2000(Yebra et al, , 2004Mazé et al, 2004) we found 11 ORFs encoding putative IID proteins. An alignment of these proteins showed that one of them (manN) displays an additional 33 aa extension at its C terminus (data not shown), which is characteristic of mannose/glucose PTS transporters of firmicutes (Lortie et al, 2000;Dalet et al, 2001).…”

“…Previous studies had shown that a functional glucose/ mannose PTS is necessary for CCR in L. casei (Veyrat et al, 1994;Yebra et al, 2000). We measured two enzymic activities which are repressed by glucose.…”

Integrative Food-Grade Expression System Based on the Lactose Regulon of Lactobacillus casei

“…However, besides the nisin system, these approaches could not be transferred to species of Lactobacillus. In lactobacilli, the regulation of gene expression has been studied mainly for carbon catabolism pathways, such as those of lactose, xylose, ribose, sorbose, and arginine deiminase (1,2,3,10,18,19,30,34,35,43,49,50). In Lactobacillus casei, the best-characterized sugar transport is the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).…”