CcpA-Dependent Carbohydrate Catabolite Repression Regulates Galactose Metabolism in Streptococcus oligofermentans

Abstract

bStreptococcus oligofermentans is an oral commensal that inhibits the growth of the caries pathogen Streptococcus mutans by producing copious amounts of H 2 O 2 and that grows faster than S. mutans on galactose. In this study, we identified a novel eightgene galactose (gal) operon in S. oligofermentans that was comprised of lacABCD, lacX, and three genes encoding a galactosespecific transporter. Disruption of lacA caused more growth reduction on galactose than mutation of galK, a gene in the Leloir pathway, indicating that the principal role of this operon is in galactose metabolism. Diauxic growth was observed in cultures containing glucose and galactose, and a luciferase reporter fusion to the putative gal promoter demonstrated 12-fold repression of the operon expression by glucose but was induced by galactose, suggesting a carbon catabolite repression (CCR) control in galactose utilization. Interestingly, none of the single-gene mutations in the well-known CCR regulators ccpA and manL affected diauxic growth, although the operon expression was upregulated in these mutants in glucose. A double mutation of ccpA and manL eliminated glucose repression of galactose utilization, suggesting that these genes have parallel functions in regulating gal operon expression and mediating CCR. Electrophoretic mobility shift assays demonstrated binding of CcpA to the putative catabolite response element motif in the promoter regions of the gal operon and manL, suggesting that CcpA regulates CCR through direct regulation of the transcription of the gal operon and manL. This provides the first example of oral streptococci using two parallel CcpA-dependent CCR pathways in controlling carbohydrate metabolism.T he human oral cavity harbors between 700 and 19,000 operative taxonomic units (16,19,45), among which streptococci are the most abundant microorganisms, contributing about 80% of the total microbial population in the dental biofilm (5, 31). Complex interspecies competitions occur among the oral streptococci due to limited space and shared nutritional requirements (30,37,38,50). The outcome of these interspecies competitions may determine the health status of the oral cavity.Carbohydrates are common carbon and energy sources for all streptococci, and as such, differences in uptake and metabolic efficiency among the species affect each species' competitiveness in the oral biofilm. The carbohydrate metabolism of bacteria is usually under the control of carbon catabolite repression (CCR) (13,34,35,42), in which rapidly metabolizable carbohydrates, usually glucose, repress the utilization of nonpreferred carbohydrates. It is generally believed that catabolite control protein A (CcpA) (8, 15) and the histidine phosphocarrier protein HPr (9) function in the regulation of CCR in most low-GϩC Gram-positive bacteria. HPr phosphorylation (HPr-Ser-P) is triggered by fructose-1,6-bisphosphate (9), a glycolysis intermediate. In the presence of a preferred carbohydrate, i.e., glucose, HPr-Ser-P serves as a cofactor to assist CcpA binding ...

The carbon catabolite repressor CcpA mediates optimal competence development in Streptococcus oligofermentans through post‐transcriptional regulation

“…The capability of efficiently utilizing carbohydrates and coping with environmental stress would determine the interspecies competition (Zheng et al , ; Moye et al , ; Roberts and Kreth, ; Giacaman et al , ), so the oral health. Although it has been shown that CCR control of carbohydrate metabolism in S. mutans is achieved mainly by PTS permeases and HPr (Zeng and Burne, ; Zeng and Burne, ; Tong et al , ), our study demonstrated that CcpA functions as a master regulator of CCR control in S. oligofermentans (Cai et al , ). As a similar gene arrangement of a tRNA Arg gene situated immediately upstream of the comCDE operon (Fig. )…”

“…CcpA functions as a global regulator for optimizing carbon source utilization in Gram‐positive bacteria (Deutscher et al , ; Carvalho et al , ) and also serves as the main CCR control protein in carbohydrate metabolism in S. oligofermentans (Cai et al , ). In addition, CcpA is involved in the regulation of competence development in S. gordonii ; however, its inactivation reduced the transcription of the competence genes comC , comD and comYA (Zheng et al , ), in contrast to controlling TRT as found in this study.…”

“…The amino‐terminal His 6 tagged CcpA protein of S. oligofermentans was overexpressed and purified from BL21(DE3)‐pLysS‐ ccpA strain as described previously (Cai et al , ). Briefly, cells were grown at 37°C to OD 600 0.4–0.6, and added with 1 mM of isopropyl‐β‐ d ‐thiogalactopyranoside (Sigma‐Aldrich, St Louis, MO).…”

“…Previously, we found that inactivation of the ccpA gene in Streptococcus oligofermentans , which is a close relative of S. pneumoniae and also uses ComCDE to control competence (Tong et al , ), caused a reduction in transformation efficiency (Cai et al , ). S. oligofermentans is frequently isolated from the healthy tooth surface of humans (Tong et al , ) and it suppresses the growth of the dental caries pathogen Streptococcus mutans by producing abundant amounts of hydrogen peroxide (H 2 O 2 ) (Tong et al , ; Tong et al , ; Zhang et al , ; Liu et al , ; Zhou et al , ).…”

The roles of RelA/(p)ppGpp in glucose-starvation induced adaptive response in the zoonotic Streptococcus suis

“…Some cre -dependent operons were only up-regulated in SC-19. For example, the operon galKT (SSU05_0360, SSU05_0361) encoding the enzymes for conversion of galactose to Glc-1-p, was more than 38-fold overexpressed in SC-19, suggesting that use of galactose during glucose starvation was very important in S. suis 36. The glgABCD operon (SSU05_1013–1016) encoding the enzymes for glycogen biosynthesis37 was approximately 20-fold up-regulated in SC-19 during glucose starvation.…”