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 (PTSCel-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.
Highlights
Lactic acid bacteria (LAB), including Lactococcus lactis, are the focus of intensive research within the field of carbohydrate catabolism and its regulation, important during industrial fermentation processes (Mayo et al 2010)
Escherichia coli cells were grown in Luria-Bertani (LB) medium (Wood 1983) at 37 °C, while L. lactis was cultivated in M17 glucose medium (G-M17) (Terzaghi and Sandine 1975) or in chemically defined medium (CDM) (Sissler et al 1999) supplemented with 1 % glucose (G-CDM), 1 % cellobiose (C-CDM), 1 % lactose (L-CDM), 1 % galactose (GalCDM), 1 % arbutin (A-CDM), 1 % galactose with 1 % cellobiose (GalC-CDM), or 1 % lactose together with an inducing (0.01 %) concentration of cellobiose (LC-CDM)
Expression of celB, bglS, and ptcBA genes is positively regulated by ClaR in the presence of cellobiose Previously, we showed that genes encoding PTSCel-Lac components and bglS are negatively regulated by catabolite control protein A (CcpA) in L. lactis IL1403 (Aleksandrzak-Piekarczyk et al 2011)
Summary
Lactic acid bacteria (LAB), including Lactococcus lactis, are the focus of intensive research within the field of carbohydrate catabolism and its regulation, important during industrial fermentation processes (Mayo et al 2010). Due to the economic importance of lactose fermentation, the metabolism of this sugar is studied extensively in L. lactis These studies mostly focus on two pathways: (i) plasmidlocalized lactose-specific PTS (PTSLac) and (ii) the chromosomally encoded lactose permease-β-galactosidase system (Aleksandrzak-Piekarczyk 2013). We reported on the discovery of a third, alternative lactose uptake system that we found to be operative in L. lactis IL1403 (AleksandrzakPiekarczyk et al 2011) This novel lactose utilization pathway engages chromosomally encoded components of a cellobiosespecific PTS (PTSCel-Lac). The proteins of PTSCel-Lac are encoded in two distinct regions that encompass ptcA, ptcB (one region), and celB (second region), which code for EIIA, EIIB, and EIIC, respectively (Aleksandrzak-Piekarczyk et al 2011) The latter region contains the bglS gene, which encodes a P-sugar-hydrolyzing enzyme with both
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