Abstract
We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.
Highlights
Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an endemic disease that continues to cause considerable economic losses in the swine industry worldwide [1,2]
We have previously reported that some strains of A. pleuropneumoniae are capable of natural transformation [19,20]
For sucrose counter-selection, bacteria were plated onto salt-free LB agar consisting of 10 g tryptone, 5 g yeast extract, and 1.5 g agar per L supplemented with 10% filter-sterilised sucrose (LB-S) for E. coli clones, or onto salt-free LB agar supplemented with 10% sucrose, 10% horse serum and 0.01% NAD (LB-SSN) for A. pleuropneumoniae clones
Summary
Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an endemic disease that continues to cause considerable economic losses in the swine industry worldwide [1,2]. After good husbandry practices are taken into account, there are two basic methods used to limit endemic infection: vaccines and antibiotics. Bacterin (killed whole cell) and subunit vaccines have been developed for A. pleuropneumoniae, none has conferred complete protection against infection with all serovars (for a review, see [3]). There is growing interest in development of live attenuated vaccines (LAVs), as they have the potential to protect against homologous and heterologous serovars [4,5,6]. A LAV should not contain antibiotic resistance markers, and ideally should be differentiated from clinical isolates [5,6,7]. An ideal LAV for A. pleuropneumoniae might be used as a vector for heterologous protection against other pig pathogens
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