ClaR--a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403

Abstract

In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-β-glucosidase and P-β-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTS(Cel-Lac)), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism.

Fig. 1

The organization of ClaR domains (a), the claR chromosomal region (b), and nucleotide sequence of the yebE-claR intergenic region (c). The ClaR domains (a) and the genes of the claR chromosomal region (b) are drawn to scale. Stem-loop structures denote rho-independent terminators. Gray-shaded sequences (c) highlight the potential −10/−35 promoter regions upstream of claR. The putative ribosome binding site (RBS) is underlined, and the start and stop codons of claR and yebE, respectively, are shown as black shading. Stars following yebE indicate a rho-independent terminator, the functionality of which was measured in this work

Fig. 2

Growth of the L. lactis IL1403 wild-type strain (gray dots) and its derivatives: IL1403ΔccpA (gray line), IL1403ΔclaR (black dots), and IL1403ΔccpAΔclaR (black line) in CDM containing cellobiose (a), lactose (b), or lactose + cellobiose (c). Sugar concentrations were 1 % except for 0.01 % cellobiose in lactose + cellobiose-supplemented CDM. x-axis = time (in hours), and the y-axis = optical density at 600 nm. Sugar fermentation patterns of the L. lactis IL1403 wild-type strain and its derivatives: IL1403ΔccpA, IL1403ΔclaR, and IL1403ΔccpAΔclaR determined by the API 50CH test (d). The results are shown for 12, 24, and 36 h of incubation; +, good fermentation; +/−, weak fermentation; −, no fermentation (d)