Abstract
A bacterial transcriptome of the primary etiological agent of human dental caries, Streptococcus mutans, is described here using deep RNA sequencing. Differential expression profiles of the transcriptome in the context of carbohydrate source, and of the presence or absence of the catabolite control protein CcpA, revealed good agreement with previously-published DNA microarrays. In addition, RNA-seq considerably expanded the repertoire of DNA sequences that showed statistically-significant changes in expression as a function of the presence of CcpA and growth carbohydrate. Novel mRNAs and small RNAs were identified, some of which were differentially expressed in conditions tested in this study, suggesting that the function of the S. mutans CcpA protein and the influence of carbohydrate sources has a more substantial impact on gene regulation than previously appreciated. Likewise, the data reveal that the mechanisms underlying prioritization of carbohydrate utilization are more diverse than what is currently understood. Collectively, this study demonstrates the validity of RNA-seq as a potentially more-powerful alternative to DNA microarrays in studying gene regulation in S. mutans because of the capacity of this approach to yield a more precise landscape of transcriptomic changes in response to specific mutations and growth conditions.
Highlights
About one fifth of children between the ages of 2 and 19 were reported to have untreated dental caries in the United States (National Center for Health Statistics, 2010)
Bacterial strains and growth conditions Streptococcus mutans strains UA159 and TW1 [11] were maintained on BHI (Difco Laboratories, Detroit, MI) agar plates, and bacterial cultures used for extraction of RNA were prepared with Tryptone-vitamin [17] base medium supplemented with 0.5% of glucose or galactose
All replicates of the same bacterial strain growing in the same carbohydrate conditions clearly clustered together, indicating that the transcriptomic shifts were the results of both sugar specificity and CcpA
Summary
About one fifth of children between the ages of 2 and 19 were reported to have untreated dental caries in the United States (National Center for Health Statistics, 2010). Carbohydrates introduced into the oral cavity provide the preferred energy sources for the majority of the most abundant members of the oral microbiome This is especially true for those organisms that are regarded as significant contributors to the caries process, including aciduric streptococci, certain Actinomyces spp., and various lactobacilli, bifidobacteria and Scardovia spp. Cariogenic bacteria, including the primary etiological agent of human dental caries, Streptococcus mutans, are usually equipped with multiple pathways for the internalization and catabolism of carbohydrates [4]. The development of these repertoires of carbohydrate catabolic pathways likely reflects adaptation to the complex combination of carbohydrates that are secreted by the host in the glycoproteins and other glycoconjugates produced in saliva and gingival exudates, as well as to the variety of simple and complex carbohydrates that became more significant components of the human diet a few thousand years ago. These characteristics include enrichment for the aforementioned aciduric organisms, lower biofilm pH and other changes in microenvironments, e.g. reduced redox, which can alter bacterial gene expression and physiology [5,6,7,8]
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