Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Oligonucleotide primers used in this study.
Fig 1.
Schematic presentation of the msm and mal loci encoding relevant carbohydrate ABC transporters in selected streptococci.
The genes within the loci encoding components of the S. suis MsmEFG (A), S. mutans MsmEFGK (B), S. pneuminiae MsmEFG (C), S. suis MalXCD (D), S. mutans MalXFGK (E) and S. pneumoniae MalXCD (F) are represented. Arrows indicate the direction of transcription. Gray arrows, genes encoding the ATPase of ABC transporters; spotted arrows, genes encoding solute binding proteins; black arrows, genes encoding permeases of ABC transporters; blank arrows, other genes adjacent.
Table 3.
Fold changes in transcription of transporter genes compared to glucose-grown bacteria by qRT-PCR.
Fig 2.
(A) Genetic map of the loci encoding predicated carbohydrate ATPase, MsmK, in SC84. Large arrows represent open reading frames and their direction of transcription. Small arrow indicates the promoter of the gene msmK and its transcription direction. (B) SDS-PAGE of purified recombinant protein MsmK. Before electrophoresis, the proteins were exposed to no DTT (left lane,-) or 2 mM DTT (right lane, +DTT). Purified MsmK was formed by a certain amount of monomers (42 KDa) and dimmers (~80 KDa). (C) ATPase activity of MsmK. Protein was incubated with 1 mM ATP for 60 min, and the generation of ADP was quantified. The mixture with BSA protein was used as a negative control. Data represent the average ± SD of at least three independent repeats.
Table 4.
MsmK affects fermentation of different carbohydrates in S. suis.
Fig 3.
MsmK is required for growth in raffinose, melibiose, glycogen and maltotetraose.
The SC-19, ΔmsmK and CΔmsmK strains were grown in CDM containing 1% raffinose (A), 1% melibiose (B), 1% glycogen (C) or 1% maltotetraose (D) as sole carbon source. Growth was measured by the optical density at 600 nm. Data are presented as means from at least three independent experiments.
Fig 4.
Deletion of msmK impacts bacterial growth on maltotriose.
The SC-19, ΔmsmK and CΔmsmK strains were grown on the CDM containing 1% glucose (A), 1% maltose (B), 1% maltotriose (C) or no sugar (D) as sole carbon source. CDM supplemented with glucose/no sugar served as a positive/negative control respectively in growth assays. Growth was measured by the optical density at 600 nm. Data are means from at least three independent experiments.
Fig 5.
Western blot analysis of MsmK expression in SC-19 cultured in different carbohydrates.
(A) Strain SC-19 was cultured in CDM supplemented with 1% (g/v) diverse carbohydrates to log phase. The supernatant of cell lysate was disposed for immunoblot analysis with MsmK or Enolase polyclonal antibodies. Enolase was used as an internal reference. (B) The blots were scanned, and the resultant graphics were quantified with ImageJ2x. Fold change is represented as a comparison to cells grown in glucose, and the expression levels of Enolase as internal references. Data are shown as means ± SEM from at least three independent experiments. Statistical significance was tested by a Two-way ANOVA test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
Fig 6.
MsmK contributes to streptococcal survival in brain.
Mice were received intraperitoneal injection with 3 × 107 CFU of a 1: 1 mix of SC-19 and ΔmsmK strains. At each defined time point, brains of 5 mice were collected and the viable bacteria were counted. The SC-19 and ΔmsmK strains were distinguished by erythromycin added in TSA plates. Solid lines, the means of data of strain SC-19; dotted lines, the means of data of strain ΔmsmK. The means and standard errors of the means are depicted. Statistical significance was tested by a two-tailed Student t test (*, P ≤ 0.05; ***, P ≤ 0.001).
Fig 7.
Schematic summary of carbohydrates utilization by ABC transporters in S. mutans (A), S. pneumoniae (B) and S. suis (C).
Above each ABC complexes is a list of known or putative carbohydrates transported by each ABC transporter. Bidirectional arrow means the MsmK and MalK ATPases can energize permeases interactively. Unidirectional arrows mean the degradation of pullulan and glycogen by pullulanases SpuA or ApuA.