Comprehensive Mutational Analysis of Sucrose-Metabolizing Pathways in Streptococcus mutans Reveals Novel Roles for the Sucrose Phosphotransferase System Permease

Abstract

Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO 4 hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIA Lev ) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA. Sucrose is among the most cariogenic carbohydrates (1, 2), and this disaccharide, composed of ␤2,1-linked fructose and glucose, influences the development of caries in multiple ways. First, sucrose can serve as a readily metabolizable carbon and energy source for many members of the oral microbiome and is a particularly effective substrate for generation of organic acids via glycolysis by the abundant oral streptococci and Actinomyces spp. that comprise a large proportion of the oral microbiome. Many oral bacteria possess transport systems for sucrose but can also hydrolyze sucrose outside the cell. Sucrose is a substrate for a variety of glucosyltransferase enzymes (GTFs) that are secreted mainly by oral streptococci. For example, the GTF enzymes of Streptococcus mutans, the primary etiologic agent of human dental caries, release free fructose and form high-molecular-mass ␣1,3-and ␣1,6-linked homopolymers of glucose, commonly called glucans or mutan, which act as an adhesive scaffolding to promote the formation of oral biofilms, particularly on the smooth surfaces of the teeth (3, 4). Many oral streptococci and certain Actinomyces spp. also produc...

Sucrose- and Fructose-Specific Effects on the Transcriptome of Streptococcus mutans, as Determined by RNA Sequencing

“…To construct mutants deficient in extracellular sucrolytic activities, strain MMZ950 (gtfBC::Tc gtfD M9stop ftf M9stop ) (20) was used in a transformation assay as the recipient of the suicide plasmid pBGE, which replaced the gtfA gene with an erythromycin resistance marker, and a PCR product that replaced the fruAB coding sequence with a nonpolar kanamycin marker. The resultant strain, MMZ1009 (gtfABCD ftf fruAB), was validated by allele-specific PCR (28) for point mutations in gtfD and ftf that were established previously through site-directed mutagenesis (20).…”

“…Growth phenotypes of these mutants indicated that gtfBCD, ftf, and the scrAB pathway constitute the majority of the sucrose-catabolizing capacity. Importantly, we also observed sucrose-dependent induction of the fructan hydrolase operon (fruAB) (5) and a fructose-PTS operon (levDEFGX or EII Lev ), with both being mediated by an unusual four-component signal transduction system (LevQRST) (21) and modulated by the sucrose-specific PTS enzyme II (ScrA) (20). Specifically, in a mutant that lacked all known extracellular sucrolytic activities, addition of sucrose resulted in increased expression of a levD::cat promoter fusion.…”

“…We previously reported the construction of a number of otherwise isogenic sucrase-deficient mutants derived from the cariogenic human isolate S. mutans UA159 and the systematic assess-ment of the contributions to sucrose metabolism by various enzymes and pathways (20). Growth phenotypes of these mutants indicated that gtfBCD, ftf, and the scrAB pathway constitute the majority of the sucrose-catabolizing capacity.…”

“…However, sucrose-specific impacts on S. mutans gene regulation can be studied only in a genetic background that is devoid of all extracellular enzymes capable of modifying sucrose; otherwise, the observed results will be due to combined effects of sucrose, liberated hexoses, and hydrolysis products of sucrose-derived exopolysaccharides. To assess the global impact of sucrose metabolism on bacterial gene regulation, we applied next-generation RNA sequencing technology (RNA-Seq) (24) to mRNAs from selected sucrase-deficient mutants of S. mutans (20). The exceptional depth and resolution of RNA-Seq allowed for a comprehensive examination of sucrose and the sucrose-PTS in the transcriptome.…”

Modification of Gene Expression and Virulence Traits in Streptococcus mutans in Response to Carbohydrate Availability

“…Interestingly, it has been demonstrated that CcpA has very little influence on CCR in S. mutans (19)(20)(21), whereas transcriptomic analyses have revealed a prominent role for CcpA in the regulation of global carbon flow, control of the transition between homoand heterofermentative growth, and regeneration of NADH, as failure to maintain adequate NADH levels in S. mutans can have detrimental effects (14,22). While it has been shown that HPr can exert effects on CCR in S. mutans, CCR in this organism is dominantly controlled by the glucose/mannose EIIAB permease, ManL (EIIAB Man ), but also the FruI and FruCD fructose permeases, the EII Lev fructose/mannose permease, and the sucrose PTS permease have been shown to be capable of influencing CCR in this cariogenic pathogen when their cognate sugars are present (23,24). Notably, deletion of manL resulted in a loss of diauxic growth in medium containing a combination of glucose and the fructose homopolymer inulin as the carbohydrate sources, and a general dysregulation of many genes involved in carbohydrate transport, catabolite repression, and energy metabolism was noted in a manL deletion mutant compared with the gene regulation in the parental strain UA159 (25).…”