The cyclic-AMP receptor protein (CRP) regulon in Aggregatibacter actinomycetemcomitans includes leukotoxin

Abstract

The cyclic-AMP receptor protein (CRP) acts as a global regulatory protein among bacteria. Here, the CRP regulon has been defined in Aggregatibacter actinomycetemcomitans using microarray analysis of A. actinomycetemcomitans strain JP2 wild type cells compared to an isogenic crp deletion mutant. Genes whose expression levels changed at least 2-fold with p ≤ 0.05 were considered significant. Of the 300 genes identified as being CRP-regulated, 139 were CRP-activated, including leukotoxin, with the remaining being CRP-repressed. The 300 genes represent 14.2% of ORFs probed which is significantly higher than what has been reported for CRP regulons in other bacteria. If the CRP-regulated genes are put into 17 functional classes, all 17 categories had at least 1 CRP-regulated gene. Several functional categories, mainly transport and binding proteins and energy metabolism proteins, were disproportionately represented in the CRP-regulated subset of genes relative to their overall representation in the genome. This is similar to the patterns seen in other bacteria. Finally, quantitative RT-PCR was used to show that the leukotoxin RNA levels were repressed 16-fold in the CRP mutant indicating that CRP activates leukotoxin transcription. However, this regulation appears to be acting through another regulatory protein since the leukotoxin promoter, unlike ~129 other promoters of CRP-regulated genes, does not have a match to the consensus CRP binding site. Several candidate genes for this intermediary transcription factor have been identified in the CRP-regulon.

Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

“…Two transcription factors that regulate leukotoxin synthesis have been identified previously; IHF represses leukotoxin transcription by binding to the leukotoxin promoter (38), and CRP activates ltxA transcription (33). However, the effect of CRP appears to be indirect since there is no apparent CRP binding site in the leukotoxin promoter.…”

“…To construct the Mlc ϩ complementation plasmid pDK941, a DNA segment that contained a 326-bp region immediately upstream of the mlc gene, the entire mlc gene, and 31 bp of DNA from immediately downstream of the mlc gene was cloned into the EcoRI and SalI sites of the shuttle vector pMMB67EH (45). Plasmid pDK941 DNA was electroporated into the ⌬mlc strain AAM155 and into AAM167, a ⌬crp deletion mutant (33), and ampicillin-resistant colonies were selected. One of each of the resulting ampicillin-resistant isolates was used in subsequent experiments.…”

“…Aliquots from each sample were electrophoresed on SDS-polyacrylamide gels, and the proteins were then visualized by staining with Coomassie brilliant blue. Western blot analysis of leukotoxin expression was done with rabbit polyclonal antileukotoxin antibody as described previously (33) except that LumiGLO reagent (Cell Signaling Technologies, Danvers, MA) and autoradiography were sometimes used to detect the horseradish peroxidase on the secondary antibody.…”

“…This led Inoue et al (30) to speculate that the classic carbon catabolite repression system, including the global regulatory protein cAMP receptor protein (CRP) (32), is involved in regulating leukotoxin production in A. actinomycetemcomitans. Indeed, deletion of the A. actinomycetemcomitans crp gene results in a significant reduction in leukotoxin RNA expression, indicating that CRP is a transcriptional activator of leukotoxin (33). However, in silico analysis indicates that CRP must be acting through another protein to modulate leukotoxin transcription; the leukotoxin promoter does not have a good match (30,33) to the consensus DNA sequence for CRP-specific binding, as defined in E. coli (34,35,36), even though 65 other CRP-regulated operons in A. actinomycetemcomitans are predicted to have CRP binding sites (33).…”

“…Indeed, deletion of the A. actinomycetemcomitans crp gene results in a significant reduction in leukotoxin RNA expression, indicating that CRP is a transcriptional activator of leukotoxin (33). However, in silico analysis indicates that CRP must be acting through another protein to modulate leukotoxin transcription; the leukotoxin promoter does not have a good match (30,33) to the consensus DNA sequence for CRP-specific binding, as defined in E. coli (34,35,36), even though 65 other CRP-regulated operons in A. actinomycetemcomitans are predicted to have CRP binding sites (33). Thus, the identity of the intermediary between CRP and the leukotoxin promoter is unknown.…”