Characterization of a Novel Riboswitch-Regulated Lysine Transporter inAggregatibacter actinomycetemcomitans

Abstract

Aggregatibacter actinomycetemcomitans is an opportunistic pathogen that resides primarily in the mammalian oral cavity. In this environment, A. actinomycetemcomitans faces numerous host-and microbederived stresses, including intense competition for nutrients and exposure to the host immune system. While it is clear that A. actinomycetemcomitans responds to precise cues that allow it to adapt and proliferate in the presence of these stresses, little is currently known about the regulatory mechanisms that underlie these responses. Many bacteria use noncoding regulatory RNAs (ncRNAs) to rapidly alter gene expression in response to environmental stresses. Although no ncRNAs have been reported in A. actinomycetemcomitans, we propose that they are likely important for colonization and persistence in the oral cavity. Using a bioinformatic and experimental approach, we identified three putative metabolitesensing riboswitches and nine small regulatory RNAs (sRNAs) in A. actinomycetemcomitans during planktonic and biofilm growth. Molecular characterization of one of the riboswitches revealed that it is a lysine riboswitch and that its target gene, lysT, encodes a novel lysine-specific transporter. Finally, we demonstrated that lysT and the lysT lysine riboswitch are conserved in over 40 bacterial species, including the phylogenetically related pathogen Haemophilus influenzae.

Probing Bacterial Metabolism during Infection Using High-Resolution Transcriptomics

“…1). Among these TSS, several previously characterized TSS, including katA, apiA, and lysT were verified by our RNA-seq analyses, thus validating our approach (21,36). Because of the methods used to generate the RNA-seq libraries, it is important to note that the TSS identified represent authentic TSS, as well as the 5= ends of processed RNAs.…”

“…However, most bacterial transcripts are not specifically processed at a defined nucleotide but are processively degraded (37). Moreover, since several of the TSS identified here correspond to TSS identified by other approaches (21,36), many of these TSS likely represent true start sites.…”

“…In total, A. actinomycetemcomitans expressed 210 ncRNAs, including 127 asRNAs, 3 pre-crRNAs, 3 riboswitches, 75 sRNAs, and 1 unidentified tRNA. Among these ncRNAs were housekeeping ncRNAs involved in transcription, translation, and protein secretion (Table 1) and several previously discovered and predicted ncRNAs (21,(42)(43)(44)(45)(46)(47). In addition, many ncRNAs were expressed differently in vivo than during in vitro biofilm growth.…”

“…Differential regulation of ncRNAs also provided insight into the nutritional content of the infection site. For example, the A. actinomycetemcomitans lysine riboswitch and the sRNA GcvB (21,61,62) showed reduced abundance during infection. These ncRNAs regulate the expression of lysine and glycine transport proteins, respectively, and their decreased levels suggest that these amino acids are found at reduced levels in an abscess.…”

“…For RNA-seq, A. actinomycetemcomitans was grown in chemically defined, modified Socransky medium supplemented with 20 mM glucose and 50 mM morpholinepropanesulfonic acid (MOPS), pH 7.2 (CDM) (8,20), while shaking at 150 rpm in a 5% CO 2 atmosphere at 37°C. Prior to biofilm growth, overnight liquid cultures of A. actinomycetemcomitans were diluted 1:2 with fresh CDM, grown for 2 h, centrifuged at Ͼ16,000 ϫ g, resuspended in CDM, and inoculated onto CDM agar plates as colony biofilms (21). Colony biofilms were grown for 4 h on CDM agar, transferred to fresh CDM agar for 4 more h of growth, and collected in RNAlater solution.…”