Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways

Abstract

The glucose/mannose-phosphotransferase system (PTS) permease EII^Man encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EII^Fru, respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EII^Man Expression of genes for EII^Man and EII^Fru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN Carbohydrate transport by EII^Man had a negative influence on expression of manLMN but not fruRKI In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression.IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries.

Keywords: catabolite repression; dental caries; fructose metabolism; gene regulation; sugar::phosphotransferase.

FIG 1

Diagrams depicting the intergenic regions upstream of manL (A) and fruR (B). Conserved catabolite response elements (cre) are shown in italics and boxed. Putative FruR-binding motifs are shown in bold letters, with engineered mutations (Mfru) listed beneath. Other regulatory elements, including −35, −10, ribosome binding site (RBS), and rho-independent terminator sequences, are labeled. Also indicated are the sites of three nonsense mutations (B, in red) engineered near the beginning of FruR to confirm the correct start codon for FruR, as well as the apparently incorrect start codon (ATG in box) that was originally annotated by the NCBI.

FIG 2

Transcripts of fruI measured by real-time quantitative reverse transcription-PCR (qRT-PCR). (A) Wild-type strain UA159 and two fruR mutants were each grown in TV medium supplemented with 0.5% of fructose or mannose. (B) UA159 and ccpA mutant strain TW1. Asterisks denote statistically significant differences (P < 0.05) relative to the value for the wild-type strain as determined by Student's t test.

FIG 3

Footprints of CcpA mapped onto the promoter regions of manL (A) and fruR (B) by DNA footprint analysis by automated capillary electrophoresis (DFACE). Recombinant His-tagged CcpA protein was used to complex with fluorescently labeled DNA probes containing the promoter regions of manL or fruR, digested with DNase I, and subjected to analysis using capillary electrophoresis. Differential fluorescent labeling (FAM for sense [blue] and VIC for antisense [green]) from both ends of the probes allowed for identification of footprints (bases in red, shown by brackets) on both strands of the DNA.

FIG 4

Growth phenotypes of wild-type UA159 (blue) and the fruR::Sp mutant (red). Cells were grown to mid-exponential phase using BHI and then inoculated into TV medium constituted with 10 mM fructose (A), cellobiose (B), mannose (C), or galactose (D). Error bars represent standard deviations calculated using results from at least three biological repeats. See Table 3 for quantitative information on, and statistical treatment of, the growth curves.

FIG 5

Growth curves of strains UA159 and FruK-13 and levD, fruI, and fruR derivatives of FruK-13. Strains were cultivated in BHI to mid-exponential phase then diluted into TV base medium containing 10 mM glucose (A) or 10 mM fructose (B). Error bars represent standard deviations based on results from at least three biological replicates.

FIG 6

Quantitation of biofilms formed by the wild type (UA159) and mutant strains lacking genes in the fruRKI operon. Exponentially growing cells were inoculated into BM supplemented with 2 mM sucrose plus 18 mM glucose (BMGS) or 18 mM fructose (BMFS) and incubated for 48 h. After a brief washing, the biofilms were stained with crystal violet (CV) and quantified by measuring the optical densities (OD₅₇₀) of the CV solution eluted with a mixture of acetone and ethanol (at 1:4, vol/vol). Error bars indicate standard deviations, and asterisks represent statistical significance relative to values for the wild type (P < 0.05, according to Student's t test).

FIG 7

Biofilms of the wild type (UA159) and mutants with changes in the fruRKI operon observed under confocal laser scanning microscopy. A 24-h biofilm of each strain was formed on a glass surface housed in an 8-well ibidi μ-Slide that was supplied with BM constituted with 18 mM fructose and 2 mM sucrose and then stained with a LIVE/DEAD BacLight bacterial vitality stain kit. Each strain was assayed with three biological repeats, and a representative set of data is shown.

FIG 8

Number of genes in functional categories differentially expressed in the fruK (A) and fruR (B) mutants compared to wild-type UA159. All strains were grown exponentially in BHI. PPNN, purines, pyrimidines, nucleosides, and nucleotides.