Sequence analysis and transcription of the apxI operon (hemolysin I) from Actinobacillus pleuropneumoniae

The DNA sequence of the entire apxI operon from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 has been determined. This 8292-bp fragment of the chromosomal DNA contains four open reading frames (ORFs) of the strongly hemolytic ApxI toxin. These ORFs correspond to the genes apxIC, apxIA, apxIB and apxID, encoding the activator, the structural toxin protein and the two secretion proteins, respectively. Each of the four ORFs is preceded by a consensus sequence for a putative ribosome-binding site (RBS). The region upstream from apxIC contains several sites that could act as promoters. The transcription start point (tsp) of the apxI operon in A. pleuropneumoniae has been determined by primer extension analysis and was found to be located 133-bp upstream from the translation start codon. The tsp is preceded by sequences matching the -10 and -35 consensus sequence of promoters from Escherichia coli. This is the first promoter identified in A. pleuropneumoniae. The same tsp was used when the expression of apxI was induced by a high concentration of free Ca2+ in the growth medium, as well as when the expression of apxI was not induced by growing the cells in medium depleted of free Ca2+ ions. However, the signal strength of the primer extension was approximately tenfold stronger in Ca(2+)-grown cells. The leader sequence of the transcript is unusually long and very A+U rich (75% A+U).