| Summary: | The aim of the study was to determine whether sub-clusters identified within A. oris and A. naeslundii by Multi locus sequence typing (MLST) of the concatenated sequences of 7 housekeeping genes should be classified as subspecies or distinct species. Determination of whole genome sequences of selected strains of A. oris and A. naeslundii, using Illumina Genome Analyzer IIxe and Roche 454 methods was initiated using paired-end sequencing and single reads respectively. The sequences obtained were aligned using CLC software and annotated using RAST. In preliminary gene-by-gene, analysis a putative Lacto-N-Biose [LNB] gene operon was identified which may mediate growth of A. oris on LNB generated in vivo from the degradation of salivary mucins. The operon were found in all A. oris strains (n=19) but in 18 A. naeslundii strains the operon was dysfunctional with extensive, varied gene loss apparent. Mutants of Actinomyces oris-MG1 were produced with the LNB phosphorylase gene inactivated. All A. oris strains grew on LNB (increase in OD= 0.213 + 0.07 and final pH= 4.99) while none of the A. naeslundii strains (increase in OD=0.09 + 0.05 and final pH=6.3; both p<0.01) or the A. oris mutant did not grow significantly in the presence of LNB. This finding indicated that A. oris strains possess a functional LNB operon and so are better able to utilize LNB generated by the degradation of salivary O-linked glycans. This is the first demonstration of LNB utilization by a member of the oral microbiome and may explain the greater prevalence of A. oris compared to A. naeslundii in oral biofilms. Further the Reverse Transcriptase-PCR (RT-PCR) was carried out to demonstrate the up-regulation of LNBP gene when A. oris-MG1 was grown in LNB. Apart from bio-chemical characterization, comparative genomic analysis was carried out on whole genomic sequences. Digital DNA-DNA homology (DDH) values were obtained using in-silico genome-to-genome comparison and this analysis delivered results consistent with previous traditional classification. Evolutionary analysis using ClonalFrame was also observed. The mutation and recombination events were compared using chi-square test among A. oris and A. naeslundii isolates. A. oris mutation and recombination event were significantly higher than A. naeslundii showing the diversity of A. oris strains in the oral cavity. Seven house keeping genes of publically available strains were included in previous MLST analysis and it was investigated that k20, MG1, c505, OT175 clustered in A. oris group of isolates while OT171, OT170 and A. johnsonii appeared as separate branches in MLST analysis and same was observed in core genome phylogenetic tree. These findings suggest that A. oris forms six distinct groups based on Neighbour-Joining tree analysis, core genome alignment and DDH analysis while A. naeslundii forms a single compact cluster.
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