Alternative lactose catabolic pathway in Lactococcus lactis IL1403

Abstract

In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 beta-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-beta-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-beta-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-beta-glucosidase, it also exhibits phospho-beta-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.

FIG. 1.

Growth of L. lactis IL1403 in CDM supplemented with 1% lactose (black line), 0.01% cellobiose (dashed line), and 1% lactose with 0.01% cellobiose (gray line). O.D._{660 nm}, optical density at 660 nm.

FIG. 2.

Growth of IBB550 (ccpA mutant) (open circles) and L. lactis IL1403 (black squares) in CDM containing 1% glucose (A), 1% cellobiose (B), 1% lactose with 0.01% cellobiose (C), or 1% lactose (D).

FIG. 3.

Schematic representation of the proposed mechanism of lactose- and cellobiose-inducible lactose metabolism in L. lactis IL1403. C, PTS β-glucosides-specific component, which also shows affinity for lactose (in IL1403, after induction by cellobiose; in IBB550, without induction) and is repressed by CcpA. A and B, components of the PTS.