Evidence for recombination between a sialidase (nanH) of Actinomyces naeslundii and Actinomyces oris, previously named 'Actinomyces naeslundii genospecies 1 and 2'

Abstract

Actinomyces spp., predominant members of human oral biofilms, may use extracellular sialidase to promote adhesion, deglycosylate immunoglobulins and liberation of nutrients. Partial nanH gene sequences (1,077 bp) from Actinomyces oris (n=74), Actinomyces naeslundii (n=30), Actinomyces viscosus (n=1) and Actinomyces johnsonii (n=2) which included the active-site region and the bacterial neuraminidase repeats (BNRs) were compared. The sequences were aligned and each species formed a distinct cluster with five isolates having intermediate positions. These five isolates (two A. oris and three A. naeslundii) exhibited interspecies recombination. The nonsynonymous/synonymous ratio was <1 for both A. oris and A. naeslundii indicating that nanH in both species is under stabilizing selective pressure; nonsynonymous mutations are not selected. However, for A. oris significant negative values in tests for neutral selection suggested the rate of mutation in A. oris was greater than in A. naeslundii but with selection against nonsynonymous mutations. This was supported by the observation that the frequency of polymorphic sites in A. oris, which were monomorphic in A. naeslundii was significantly greater than the frequency of polymorphic sites in A. naeslundii which were monomorphic in A. oris (chi(2)=7.011; P=0.00081). The higher proportions of A. oris in the oral biofilm might be explained by the higher mutation rate facilitating an increased ability to respond successfully to environmental stress.

Fig. 1

Neighbour-joining tree showing relationships between type and reference strains of Actinomyces naeslundii, Actinomyces oris, Actinomyces johnsonii and Actinomyces viscosus and oral and clinical isolates determined by partial nanH gene sequence analysis. Actinomyces naeslundii strains 25, 51 and CCUG 34725 and A. oris strains 60 and 61 (each shown with the nanH sequence accession number) did not cluster with the majority of strains of the same species and were identified as strains with significant evidence of interspecies recombination. Scale bar=0.01 substitutions per site.

Fig. 2

Recombination events found in nanH of Actinomyces oris strains 60 (EU805671) and 61 (EU805672) and Actinomyces naeslundii strains 51 (EU805620), 25 (EU805612) and CUG 34725 (EU805609). Solid line indicates portion of sequence derived from nanH of A. naeslundii and broken line indicates portion of sequence derived from nanH of A. oris. Insertion in strain 25 between 365 and 1038, in strain 51 between 629 and 1038, in strain 60 between 1 and 477, in strain 61 between 432 and 1038 and between 1 and 655 in CUG 34725. Breakpoints determined using RDP suite of programs with significant evidence (P <0.001) for recombination obtained with ≥5 recombination tests in all cases.

Fig. 3

Reverse transcriptase (RT)-PCR assays showing expression of nanH and the housekeeping gene atpA in the type strains of Actinomyces oris (CCUG 34288; EU805602), and Actinomyces johnsonii (CCUG 34287; EU805600), Actinomyces naeslundii (CCUG 2238; EU805601) and Actinomyces viscosus (NCTC 10951; EU805603). Negative controls, omitting the RT in the reaction mix, yielded no amplicons from any strain.