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Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense.

Zhu M, Lu Y, Wang J, Li S, Wang X - PLoS ONE (2015)

Bottom Line: By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG).The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization.The equilibrium binding constant (KD) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.

View Article: PubMed Central - PubMed

Affiliation: Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China.

ABSTRACT
The strictly anaerobic, Gram-positive bacterium, Thermoanaerobacterium aotearoense SCUT27, is capable of producing ethanol, hydrogen and lactic acid by directly fermenting glucan, xylan and various lignocellulosically derived sugars. By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG). However, the xylose and mannose consumptions were not markedly affected by 2-DG at the concentration of one-tenth of the metabolizable sugar. Accordingly, T. aotearoense SCUT27 could consume xylose and mannose in the presence of glucose. The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization. The highly purified Histidine-containing Protein (HPr) could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase (HPrK/P) with no need to add fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc-6-P) in the reaction mixture. The specific protein-interaction of catabolite control protein A (CcpA) and phosphorylated HPr was proved via affinity chromatography in the absence of formaldehyde. The equilibrium binding constant (KD) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0142121.g006: 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.

Mentions: Binding of HPr to CcpA requires its phosphorylation by HPrK/P at the Ser-46 [4, 43, 44]. To detect the activity of HPrK/P, the recombinant HPr and HPrK/P were incubated with 5 mM ATP for 10 min. The product analysis showed that HPr was phosphorylated by HPrK/P with the existence of ATP judged by its migration on Phos-tag™ SDS-PAGE (Fig 6A and 6B) [26]. HPrK/P kinase activity was reported to be stimulated by FBP or Glc-6-P in Gram-positive bacteria [4, 45, 46]. However, FBP or Glc-6-P was not essential for HPrK/P kinase activity from T. aotearoense SCUT27 in this study (Fig 6C and 6D). Some researchers made the same observation that FBP or Glc-6-P could be omitted for highly purified recombinant HPrK/P in Enterococcus faecalis [26] and Streptococcus sp. [47]. HPrK/P lost its kinase activity when there was no ATP in the reaction mixture, indicating that it was ATP-dependent (Fig 6E). In order to investigate the specificity of HPrK/P, we introduced the serine 46 to alanine mutation in the HPr. No phosphoryltion of HPrM was observed on the non-denaturing PAGE (Fig 6F–6I), indicating the ATP-dependent protein kinase of HPrK/P specifically phosphorylated the 46 seryl residue in HPr.


Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense.

Zhu M, Lu Y, Wang J, Li S, Wang X - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4634974&req=5

pone.0142121.g006: 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.
Mentions: Binding of HPr to CcpA requires its phosphorylation by HPrK/P at the Ser-46 [4, 43, 44]. To detect the activity of HPrK/P, the recombinant HPr and HPrK/P were incubated with 5 mM ATP for 10 min. The product analysis showed that HPr was phosphorylated by HPrK/P with the existence of ATP judged by its migration on Phos-tag™ SDS-PAGE (Fig 6A and 6B) [26]. HPrK/P kinase activity was reported to be stimulated by FBP or Glc-6-P in Gram-positive bacteria [4, 45, 46]. However, FBP or Glc-6-P was not essential for HPrK/P kinase activity from T. aotearoense SCUT27 in this study (Fig 6C and 6D). Some researchers made the same observation that FBP or Glc-6-P could be omitted for highly purified recombinant HPrK/P in Enterococcus faecalis [26] and Streptococcus sp. [47]. HPrK/P lost its kinase activity when there was no ATP in the reaction mixture, indicating that it was ATP-dependent (Fig 6E). In order to investigate the specificity of HPrK/P, we introduced the serine 46 to alanine mutation in the HPr. No phosphoryltion of HPrM was observed on the non-denaturing PAGE (Fig 6F–6I), indicating the ATP-dependent protein kinase of HPrK/P specifically phosphorylated the 46 seryl residue in HPr.

Bottom Line: By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG).The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization.The equilibrium binding constant (KD) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.

View Article: PubMed Central - PubMed

Affiliation: Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China.

ABSTRACT
The strictly anaerobic, Gram-positive bacterium, Thermoanaerobacterium aotearoense SCUT27, is capable of producing ethanol, hydrogen and lactic acid by directly fermenting glucan, xylan and various lignocellulosically derived sugars. By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG). However, the xylose and mannose consumptions were not markedly affected by 2-DG at the concentration of one-tenth of the metabolizable sugar. Accordingly, T. aotearoense SCUT27 could consume xylose and mannose in the presence of glucose. The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization. The highly purified Histidine-containing Protein (HPr) could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase (HPrK/P) with no need to add fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc-6-P) in the reaction mixture. The specific protein-interaction of catabolite control protein A (CcpA) and phosphorylated HPr was proved via affinity chromatography in the absence of formaldehyde. The equilibrium binding constant (KD) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.

No MeSH data available.


Related in: MedlinePlus