Table 1.
Strains and plasmids used in the study.
Figure 1.
Mannitol uptake pathway in S. aureus.
The mtlABFD operon encodes the Mtl-specific PTS (MtlAB) and the operon transcriptional repressor (MtlF); Mtl-1-P 5-dehydrogenase, encoded by mtlD, catalyses the conversion of Mtl-1-P to fructose-6-P which enters into the Embden-Meyerhoff and hexosemonophosphate glycolytic pathways.
Figure 2.
Comparative survival of S. aureus strains.
Growth of dilutions from overnight cultures on BHI agar in the presence and absence of 1 mM linoleic acid. SuvB24 (SH1000 mtlD::Tn917) and Liv1023 (SH1000 mtlD::tet) displayed >500-fold reduced survival on linoleic acid relative to wild type (SH1000), Liv1024 (SH1000 mtlABFD::tet) and the complemented mutant strain Liv1098 (SH1000 mtlD::tet pMJH71).
Figure 3.
Schematic representation of the mtlABFD locus.
Position of the transposon insertion and allelic replacements created during this study.
Figure 4.
Mtl fermentation capability of S. aureus strains.
Mtl fermentation is revealed by acid formation and colour change of the pH indicator to yellow. Liv1023 (SH1000 mtlD::tet) and Liv1024 (SH1000 mtlABFD::tet) do not ferment Mtl and this capability was restored by complementation with the entire locus in strains Liv1098 (SH1000 mtlD::tet pMJH71) and Liv1097 (SH1000 mtlABFD::tet pMJH71). Weak growth of Liv1023 was observed.
Figure 5.
Survival on linoleic acid agar.
Comparative survival of strains on BHI agar supplemented with 1 mM linoleic acid. Strains SH1000 (open circles), Liv1023 (SH1000 mtlD::tet) (filled squares), Liv1024 (SH1000 mtlABFD::tet) (open triangles) and Liv1098 (SH1000 mtlD::tet pMJH71) were diluted in PBS and equivalent volumes were plated onto the agar. SE from triplicate experiments is shown with error bars inside symbols.
Figure 6.
Growth phenotype of mtlD inactivated S. aureus.
(A) Culture of strains in broth containing Mtl at 37°C. Liv1023 (SH1000 mtlD::tet) (•) had a significantly reduced growth rate (P<0.01, Student’s t-test) compared to wild-type (SH1000) (▪), Liv1024 (SH1000 mtlABFD::tet) (▴), strains Liv1098 (SH1000 mtlD::tet pMJH71) (□) and Liv1097 (SH1000 mtlABFD::tet pMJH71) (○). Calculated doubling times between 2 h and 3 h of growth: SH1000 = 0.33, Liv1023 (SH1000 mtlD::tet) = 1.7. Error bars indicate 1 SEM (n = 3). (B) Culture of strains in BHI broth at 25°C. Liv1023 (SH1000 mtlD::tet) (•) has a significantly reduced growth rate (P<0.001, Student’s t-test) compared to wild-type (SH1000) (▪), Liv1024 (SH1000 mtlABFD::tet) (▴), strains Liv1098 (SH1000 mtlD::tet pMJH71) (□) and Liv1097 (SH1000 mtlABFD::tet pMJH71) (○). Calculated doubling times between 5 h and 13 h of growth: SH1000 = 2.46, Liv1023 = 3.13. Representative dataset from triplicate assay.
Figure 7.
Growth of S. aureus in the presence of mannitol and linoleic acid.
Bacteria were cultured on BHI agar containing either no or added mannitol (0.1 M, 0.5 M) in the presence (black bars) or absence (white bars) of 1 mM linoleic acid. Differences in viable cells recovered in the presence of mannitol were significantly reduced compared to the absence of mannitol (P = 0.03 and P = 0.001 for 0.1 M and 0.5 M, respectively).
Table 2.
Sugar alcohols present in S. aureus strains.
Figure 8.
Virulence of mtlD in a murine infection.
(A) Effect of WT SH1000 or Liv1023 (SH1000 mtlD::tet) on percentage change in weight of infected mice. There were no significant differences using Dunn’s test. (B) Effect of mutations of mtlD on cfu of S. aureus SH1000 in kidneys of infected mice. There were no significant differences using the Mann Whitney Test.