Table 1.
Strains and plasmids used in this study.
Table 2.
Primers used in this study.
Fig 1.
Effects of 2-DG on the cell growth for T. aotearoense SCUT27 using different sugars as the main carbon source.
0.5 g/L of 2-DG was added to the culture medium containing 5 g/L of glucose (A), xylose (B), mannose (C), cellobiose (D), galactose (E), arabinose (F) or starch (G). Data were plotted as mean ± SD (n = 2).
Fig 2.
The inhibitory effects of 2-DG concentration on xylose-related CCR of T. aotearoense SCUT27.
Overnight cultures were inoculated into fresh medium containing 5 g/L xylose supplemented with increasing concentrations of 2-DG (0, 0.005, 0.05, 0.5 or 5 g/L). After incubation at 55°C for different hours, culture optical densities were measured and expressed as mean ± SD (n = 2). *, significant difference at p<0.05 (T-test); **, very significant difference at p<0.01.
Fig 3.
Glucose inhibition assays on carbohydrates.
T. aotearoense SCUT27 was cultured at 55°C in serum bottle containing 50 mL medium using mixed sugar as carbon sources (5 g/L glucose and 5 g/L different sugar). (A), xylose (B), mannose (C), cellobiose (D), galactose and (E), arabinose. Carbohydrate measurements were performed in duplicates.
Fig 4.
The phylogenetic trees of ccpA (A), ptsH (B) and hprK (C) from T. aotearoense SCUT27.
The phylogenetic trees were constructed by Neighbor-Joining (NJ) method with 1000 replicates of bootstrap test using MEGA4.0. The numbers for gene “locus tag” on the chromosome of some strains in the GenBank were shown.
Fig 5.
Functional verification of ccpA from T. aotearoense SCUT27 in B. subtilis mutant MA-1, which was defective in the ccpA gene (BSU29740).
(A) Starch utilization of B. subtilis 168 (a), MA-1 (b) and TA-1 (c) on agar plates containing 1% (w/v) starch and 2% (w/v) glucose. After the plates incubated at 37°C for 2 h, 5 h and 8 h, the plates were flooded with I2/KI (0.5:5.0%, w/v) solution to examine the starch hydrolysis halos. (B) Comparison of expression levels of the amyE, xylA and xylB genes. The bars represent the mean values of the expression levels of different genes. All the experiments were done in triplicate and statistically tested (T-test).
Fig 6.
Phos-tag™ PAGE assay of HPr phosphorylation by HPrK/P.
27 μg of HPr or HPrM and 5 μl of HPrK/P were mixed in 50 μl 100 mM Tris-Cl buffer (pH 7.0) containing 5 mM MgCl2, 10 mM NaCl and incubated at 37°C for 10 min. 5 mM ATP, 40 mM FBP and 40 mM Glc-6-P were selectively added into the reaction mixture. 0.5 μg protein samples were load on Phos-tag™ SDS-PAGE.
Fig 7.
Interaction analysis of CcpA with HPr.
The affinity purified HPr and HPrM were phosphorylated with HPrK/P as previously described. Heat-treated cell extracts containing CcpA-NH mixed with different protein samples were loaded onto the HiTrap™ Chelating HP column. The eluted fractions were separated by SDS-PAGE using a 12% acrylamide gel and stained with coomassie brilliant blue. M: protein standards, 1: purified HPr, 2: HPrSerP, 3: CcpA-NH mixed with HPrSerP, 4: CcpA-NH mixed with HPrSerP and formaldehyde, 5: CcpA-NH mixed with HPr, 6: CcpA-NH mixed with HPrM, 7: Nitrilase mixed with HPrSerP as negative control.
Table 3.
Association and dissociation rate constants and equilibrium binding constants a.