High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis

Abstract

Background: In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity.

Results: The transcriptomes of B. subtilis cultures with three different CcpA expression levels were compared. The higher the amount of CcpA in the cells, the more operons possessing cre sites were differentially regulated. The cre boxes that mediated regulation at low CcpA levels were designated as strong (high affinity) and those which responded only to high amounts of CcpA, as weak (low affinity). Differences in the sequence and position in relation to the transcription start site between strong and weak cre boxes were revealed.

Conclusions: Certain residues at specific positions in the cre box as well as, to a certain extent, a more palindromic nature of cre sequences and the location of cre in close vicinity to the transcription start site contribute to the strength of CcpA-dependent regulation. The main factors contributing to cre regulatory efficiencies, enabling subtle differential control of various subregulons of the CcpA regulon, are identified.

Figure 1

Tight regulation of the CcpA expression level in B. subtilis strain MP902 (Ptet-ccpA, Pxyl-tetR). Lane 1, wild type strain 168; lanes 2 – 11, MP902 grown in presence of 0.2 % xylose and increasing concentration of anhydrotetracycline (ATc): 0.1, 0.2, 04, 0.7, 1, 2, 4, 8, 10 and 20 nM, respectively; lane 12, 200 ng of purified CcpA. The representative graph of three reproducible experiments is shown.

Figure 2

CcpA expression levels in B. subtilis cultures used for DNA microarray experiments. (A) upper panel, CcpA detection using anti-CcpA antibody; lower panel, signal quantification with ImageJ. Four CcpA expression levels were achieved by growing B. subtilis strain MP902 (Ptet-ccpA, Pxyl-tetR) in absence (lanes 1, 3 and 5) and in presence of 0.1, 2 and 20 nM ATc (lanes 2, 4 and 6), respectively. All cultures were grown in presence of 0.2 % xylose and 1 % glucose. Shadows in the background of the picture indicate culture pairs used in microarray experiments (B) Ponceau S control membrane staining for protein load verification. Lane numbers correspond to lane numbers in panel A. The representative graphs of three reproducible experiments are shown.

Figure 3

Analysis of high- (A) and low-affinity (B) cre boxes responsible for gene repression. Weight Matrix (upper panels) and cre box consensus with Position Frequency Matrix (PFM) (lower panels). In the consensus sequence: R is A or G, Y is T or C.

Figure 4

Correlation between the cre to TSS distance to corresponding gene expression level (fold change). (A) High-affinity cre boxes. (B) Low-affinity cre boxes. Black circles - cre boxes of the genes for which TSSs were detected experimentally; grey circles - cre boxes of the genes for which TSSs were predicted in this study, underlined gene names – genes with cre sites known from literature. “0” on the X ax represents the TSS position, negative numbers – cre boxes upstream TSS, positive numbers – cre boxes downstream TSS. For clarity, the outliers were removed (for the full list of cre-TSS distance, see Additional file 5).