Abstract
Carbon catabolite control is required for efficient use of available carbon sources to ensure rapid growth of bacteria. CcpA is a global regulator of carbon metabolism in Gram-positive bacteria like Bacillus subtilis. In this study the genome-wide gene regulation of a CcpA knockout and three specific CcpA mutants were studied by transcriptome analysis, to further elucidate the function of specific binding sites in CcpA. The following three amino acids were mutated to characterize their function: M17(R) which is involved in DNA binding, T62(H) which is important for the allosteric switch in CcpA upon HPr-Ser46-P binding, and R304(W) which is important for binding of the coeffectors HPr-Ser46-P and fructose-1,6-bisphosphate. The results confirm that CcpA was also involved in gene regulation in the absence of glucose. CcpA-M17R showed a small relief of Carbon Catabolite Control; the CcpA-M17R mutant regulates fewer genes than the CcpA-wt and the palindromicity of the cre site is less important for CcpA-M17R. CcpA-T62H was a stronger repressor than CcpA-wt and also acted as a strong repressor in the absence of glucose. CcpA-R304W was shown here to be less dependent on HPr-Ser46-P for its carbon catabolite control activities. The results presented here provide detailed information on alterations in gene regulation for each CcpA-mutant.
Highlights
Gram-negative and Gram-positive bacteria employ a mechanism called carbon catabolite control (CCC) to use carbon sources in a preferential manner (Stülke and Hillen, 2000; Görke and Stülke, 2008; Fujita, 2009)
An unspecific band around 45 kDa was observed for all variants, which was not related to carbon catabolite protein A (CcpA) because it was visible in the knockout strain and not related to the inserted plasmid because it was visible in the wildtype strain
CcpA is a global regulator of carbon catabolite control in B. subtilis and needs HPr or catabolite responsive HPr (Crh) as a coeffector (Moreno et al, 2001; Sonenshein, 2007; Fujita, 2009)
Summary
Gram-negative and Gram-positive bacteria employ a mechanism called carbon catabolite control (CCC) to use carbon sources in a preferential manner (Stülke and Hillen, 2000; Görke and Stülke, 2008; Fujita, 2009). The binding of HPr-Ser46-P to CcpA triggers an allosteric switch in CcpA allowing CcpA to bind its cognate DNA sequences, the catabolite responsive elements (cre) (Stülke and Hillen, 2000; Deutscher, 2008; Görke and Stülke, 2008) These cre sites are semi-palindromic sequences with the following consensus: WTGNNARCGNWWWCAW (R is G or A, W is A or T, and N is any base) (Miwa et al, 2000; Schumacher et al, 2011). The expression of 10% of the genes in B. subtilis are affected by CcpA when glucose is present in the medium (Fujita, 2009), and the expression of 8% of the genes are affected in the absence of glucose (Moreno et al, 2001)
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