Copper Induction of Lactate Oxidase of Lactococcus lactis : a Novel Metal Stress Response

Abstract

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 M copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5 region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

Characterization of the CopR Regulon of Lactococcus lactis IL1403

“…bulgaricus, an oxygen-consuming NADH oxidase serves in eliminating oxygen (14). In a separate report, we recently proposed a similar function for LctO in oxygen removal (3).…”

“…LctO was previously shown to be an NAD-independent lactate oxidase which catalyzes the conversion of lactate to pyruvate with use of molecular oxygen and generation of hydrogen peroxide (3). A Fenton-type reaction with copper and hydrogen peroxide could, of course, generate toxic hydroxyl radicals, but this may be less of a problem than has been surmised.…”

“…2). Similarly, the monocistronic lctO gene features a cop box in the promoter region and is thus under the control of CopR (3).…”

“…The cloned copRZA operon conferred copper resistance to a copper-sensitive Escherichia coli strain, supporting a function of CopA in copper export. The function of LctO has been addressed in a separate report (3), while the elucidation of the function of the remaining genes of the CopR regulon requires further investigation.…”

A Widely Conserved Gene Cluster Required for Lactate Utilization inBacillus subtilisand Its Involvement in Biofilm Formation

“…In some distantly related species, such as Streptococcus spp. and Lactococcus spp., orthologs of lldD (though with limited sequence similarity) were detected and shown to be associated with L-lactate oxidase activity (5,22,35). But only some of these species contain lldD.…”