Cloning and characterization of the Actinobacillus pleuropneumoniae fur gene and its role in regulation of ApxI and AfuABC expression.

The ferric uptake regulation (fur) gene was cloned and characterized from Actinobacillus pleuropneumoniae and it exhibited 97% amino acid sequence identity to the Haemophilus ducrey fur gene. The flanking regions of the fur gene included an upstream putative flavodoxin (fldA) gene and a downstream possible transmembrane protein gene of unknown function. A single promoter was identified by 5' rapid amplification of cDNA ends (RACE), but there were no sequences homologous to an Escherichia coli Fur box in the 5' upstream sequence. The A. pleuropneumoniae fur clone complemented an E. coli fur deletion mutant. Transcriptional analysis of the divergent promoters of the A. pleuropneumoniae toxin I operon (apxICABD)--and the Actinobacillus ferric uptake operon (afuABC) showed that Fur and calcium together positively regulated the transcription of apxICABD while Fur was a repressor for afuABC. Hemolytic activity was significantly induced by iron and calcium and Fur appeared to act as an activator under high calcium conditions and as a repressor under low calcium conditions. A possible regulator-binding site was suggested by the properties of a point mutation in 33 bp upstream of the apxIC gene. This point mutation affected ApxI and Afu expression in response to iron, calcium, or Fur. These results provide further proof that calcium and the A. pleuropneumoniae Fur protein play a role in the expression of ApxI and Afu.