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

Muzi Zhu,Jufang Wang,Xiaoning Wang,Yanping Lu,Shuang Li,Zezhang Wen

doi:10.1371/journal.pone.0142121

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 (K D) 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.

Highlights

In nature, microorganisms develop a mechanism to metabolize a variety of carbon sources to acquire energy in order to survive complex environment
To confirm whether carbon catabolite repression (CCR) phenomenon exits in T. aotearoense SCUT27, cells were cultured in anaerobic serum bottles at 55°C containing 50 mL MTC medium using 5 g/L glucose and 5 g/L a different carbohydrate as carbon source, i.e. xylose, mannose, galactose, arabinose and cellobiose
The functional complementation test was performed using the strains of B. subtilis 168, MA-1 and TA-1. 10 μL of overnight bacterial culture was spread on LB plates containing 1.0% (w/v) starch and 2.0% (w/v) glucose as carbon sources, which was supplemented with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG)

Summary

Introduction

Microorganisms develop a mechanism to metabolize a variety of carbon sources to acquire energy in order to survive complex environment. 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 phosphorylation of HPr at different spots catalyzed by HPrK/P controls the CCR-related gene transcription and expression [12].

Results

Conclusion