Assessing the role of strain-specific DNA in the growth and biofilm formation of Aggregatibacter actinomycetemcomitans

Strain-specific genes on genomic islands are often acquired by bacteria via horizontalgene transfer (HGT), which may enhance the fitness of the bacteria. Our laboratory has completed a project to compare the genome content of 14 A. actinomycetemcomitans strains recovered from periodontal health and disease. Numerous genomic islands of unknown functions were identified. Some may be nonfunctional vestiges of HGTacquired genes that have undergone further mutations, while others may function to enhance the virulence of the strains. This study developed an approach to identify strainspecific genes that are involved in growth and biofilm formation of A. actinomycetemcomitans . Experimental variables that may affect the assay outcomes wereevaluated. Different approaches for data analysis were also compared. Novel gene genetic protocols were used to construct mutants for A. actinomycetemcomitans strain D7S-1. These mutants were tested for their doubling time and biofilm formation. Mutants with a deletion of a ~5 Kb genomic island (designated as "285-island") exhibited faster doubling time than the wildtype D7S-1 or other mutants. Deletion mutants restored with a copy of the 285-island exhibited doubling time similar to that of wildtype strain D7S-1. Growth of strain-specific genes in pH 4.0 was also tested and two groups of the mutants were distinguished based on their ability to grow at pH 4.0. This is the first study that demonstrated a function of any strain-specific genomic island (except serotype-specific gene cluster) of A. actinomycetemcomitans , and validated our approach to screen for functionally important genomic islands among A. actinomycetemcomitans strains.