Abstract
Bacteria can utilize diverse sugars as carbon and energy source, but the regulatory mechanisms directing the choice of the preferred substrate are often poorly understood. Here, we analyzed the role of the YugA protein (now designated GlaR—Galactose–lactose operon Regulatory protein) of the RpiR family as a transcriptional activator of galactose (gal genes) and lactose (lac genes) utilization genes in Lactococcus lactis IL1403. In this bacterium, gal genes forming the Leloir operon are combined with lac genes in a single so‐called gal–lac operon. The first gene of this operon is the lacS gene encoding galactose permease. The glaR gene encoding GlaR lies directly upstream of the gal–lac gene cluster and is transcribed in the same direction. This genetic layout and the presence of glaR homologues in the closest neighborhood to the Leloir or gal–lac operons are highly conserved only among Lactococcus species. Deletion of glaR disabled galactose utilization and abrogated or decreased expression of the gal–lac genes. The GlaR‐dependent regulation of the gal–lac operon depends on its specific binding to a DNA region upstream of the lacS gene activating lacS expression and increasing the expression of the operon genes localized downstream. Notably, expression of lacS‐downstream genes, namely galMKTE, thgA and lacZ, is partially independent of the GlaR‐driven activation likely due to the presence of additional promoters. The glaR transcription itself is not subject to catabolite control protein A (CcpA) carbon catabolite repression (CRR) and is induced by galactose. Up to date, no similar mechanism has been reported in other lactic acid bacteria species. These results reveal a novel regulatory protein and shed new light on the regulation of carbohydrate catabolism in L. lactis IL1403, and by similarity, probably also in other lactococci.
Highlights
Because of the substantial biotechnological relevance of galactose, especially in the dairy industry, where unmetabolized galactose is associated with poor product quality (Baskaran & Sivakumar, 2003; Hutkins, Halambeck, & Morris, 1986; Michel & Martley, 2001), galactose metabolism and its regulation have been thoroughly studied in several Lactic acid bacteria (LAB) species
In several species of the genera Lactobacillus and Streptococcus, the Leloir or gal–lac operons are known to be regulated transcriptionally mainly by repressor proteins belonging to the LacI family, but the regulatory mechanism of the Leloir pathway genes in L. lactis, important dairy industry bacterium, remained unexplored to date
We demonstrate that in L. lactis, the regulation of the gal–lac operon differs from that in other LAB species as it is under a positive control of the RpiR-family transcriptional regulator GlaR
Summary
EMSA was performed using 1 nM of double-stranded DNA fragments (~300 bp) generated by PCR with specific primer pairs (Table 1) multiplying the upstream DNA regions of selected genes (glaR, lacS, galM, galT, thgA, and galE). In the presence of galactose, the expression of most of the Leloir operon genes was significantly lower in L. lactis IL1403ΔglaR, whereas in cellobiose- or glucose-supplemented media, they were expressed at a similar level in both the strains (Figure 3a). Higher transcript levels of the gal–lac operon genes were detected when the wild-type strain was grown in media supplemented with galactose (Gal-M17 or GalC-M17) (Figure 3a). To identify the genomic region to which the GlaR protein binds an in vitro EMSA test was performed with selected upstream regions containing potential promoters of the Leloir operon genes (lacS, galM, galT, thgA, and galE) and of glaR and purified GlaR protein. The transcription of glaR in IL1403 was elevated ca. sevenfold in galactose-containing media compared to its expression in glucose- or cellobiose-supplemented media (Figure 3b), and indicating a possible autoregulation of the glaR gene
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