The
copYAZ
Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans
Authors: Singh Kamna , Senadheera Dilani B. , Lévesque Céline M. , Cvitkovitch Dennis G. 
Published: May 2015

In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the
copYABZ
operon for copper homeostasis, where
copA
and
copB
encode copper-transporting P-type ATPases, whereas
copY
and
copZ
regulate the expression of the
cop
operon.
Streptococcus mutans
is a biofilm-forming oral pathogen that harbors a putative copper-transporting
copYAZ
operon. Here, we characterized the role of
copYAZ
operon in the physiology of
S. mutans
and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in
S. mutans
cells by generating oxidative stress and disrupting their membrane potential. Deletion of the
copYAZ
operon in
S. mutans
strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of
S. mutans
to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the
gtfB
,
gtfC
,
gtfD
,
gbpB
, and
gbpC
genes, whose products have roles in maintaining the structural and/or functional integrity of the
S. mutans
biofilm. Furthermore, supplementation with copper or loss of
copYAZ
resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the Δ
copYAZ
strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of
S. mutans
. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the
copYAZ
-null mutant. Taking these results collectively, we have established the function of the
S. mutans
CopYAZ system in copper export and have further expanded knowledge on the importance of copper homeostasis and the CopYAZ system in modulating streptococcal physiology, including stress tolerance, membrane potential, genetic competence, and biofilm formation.

IMPORTANCE
S. mutans
is best known for its role in the initiation and progression of human dental caries, one of the most common chronic diseases worldwide.
S. mutans
is also implicated in bacterial endocarditis, a life-threatening inflammation of the heart valve. The core virulence factors of
S. mutans
include its ability to produce and sustain acidic conditions and to form a polysaccharide-encased biofilm that provides protection against environmental insults. Here, we demonstrate that the addition of copper and/or deletion of
copYAZ
(the copper homeostasis system) have serious implications in modulating biofilm formation, stress tolerance, and genetic transformation in
S. mutans
. Manipulating the pathways affected by copper and the
copYAZ
system may help to develop potential therapeutics to prevent
S. mutans
infection in and beyond the oral cavity.