Characterization of a novel riboswitch-regulated lysine transporter in Aggregatibacter 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 microbe-derived 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 metabolite-sensing 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.

FIG. 1.

Mapping the transcriptional start site of the A. actinomycetemcomitans AA02294 lysine riboswitch with primer extension analysis. (A) Fluorescent intensities of primer extension products synthesized from A. actinomycetemcomitans colony biofilm RNA. Fluorescent peak height corresponds to cDNA levels. (B) Transcriptional start site (+1) and promoter sequence for the A. actinomycetemcomitans AA02294 lysine riboswitch. The sequence complementary to the primer used for primer extension is boxed, the transcriptional start site is boldfaced, and the putative −10 and −35 promoter sequences are underlined. Sequence begins at 1,563,524 and ends at 1,563,723 on the A. actinomycetemcomitans chromosome. (C) The AA02294 lysine riboswitch rho-independent terminator. The terminator begins at +168 and terminates at +202 relative to the transcriptional start site. (D) Schematic of the A. actinomycetemcomitans AA02294 lysine riboswitch genomic context. The arrow indicates the transcriptional start site determined by primer extension analysis, and the rho-independent terminator is shown as a hairpin. Image is drawn to scale.

FIG. 2.

The A. actinomycetemcomitans AA02294 lysine riboswitch. (A) Predicted structure of the A. actinomycetemcomitans lysine riboswitch aptamer. The structure was predicted using Mfold software (61). Structural motifs conserved in all lysine riboswitches, kissing loops and loop E motif, are identified. Boxed nucleotides are predicted to be involved in binding lysine based on sequence and structural homology to the crystal structure of the lysine riboswitch from T. maritima (17, 48). (B) RT-PCR detecting the AA02294 mRNA (products 1 and 3) and the readthrough transcript containing both the lysine riboswitch and AA02294 (product 2). The positive-control reactions (gDNA) show PCR amplification from A. actinomycetemcomitans chromosomal DNA, while the negative control (−RT) represents PCR amplification from cDNA synthesis reactions without reverse transcriptase added, and the final lane (cDNA) depicts PCR amplification from cDNA synthesized from RNA-harvested, lysine-starved A. actinomycetemcomitans. (C) The AA02294 lysine riboswitch accumulates in the presence of lysine. A. actinomycetemcomitans lysine-starved cells (−lys, 0 min) were exposed to lysine (+lys), and samples were removed at 15, 30, 60, and 120 min postaddition. The arrow indicates the predicted size of the full-length lysT transcript containing the riboswitch. RNA from each time point was subjected to Northern blot analysis using a probe for the lysine riboswitch.

FIG. 3.

The A. actinomycetemcomitans lysine riboswitch has a half-life of 17 ± 4 min. A. actinomycetemcomitans lysine-starved cells were exposed to lysine and treated with rifampin (Rif) to stop transcription. Lysine riboswitch levels were monitored by Northern blot analysis, and the half-life was calculated as described in Materials and Methods. The ethidium bromide-stained gel was used to ensure equal RNA loading.

FIG. 4.

A. actinomycetemcomitans lysT restores lysine transport in an E. coli lysine transport mutant. (A) Transport of l-[¹⁴C]lysine by the E. coli lysP mutant with an empty vector (−, black) or a vector expressing A. actinomycetemcomitans lysT from pPJ005 (lysT, white). Transport was measured in counts per minute at 10 and 20 min after the addition of l-[¹⁴C]lysine to the assay. (B) Unlabeled l-lysine inhibits uptake of l-[¹⁴C]lysine by A. actinomycetemcomitans LysT. Lysine transport assays were carried out in the presence of excess unlabeled glycine, l-arginine, or l-lysine. −, negative-control assay where no competing amino acid was added. The data in both graphs represent the means of results of three independent experiments; error bars in all assays represent the standard error, and statistical significance was determined by an unpaired two-tailed Student's t test: *, P < 0.05; **, P < 0.005.

FIG. 5.

The H. influenzae lysT homolog NTHi1465 encodes an l-lysine transporter. (A) H. influenzae lysT restores lysine transport in an E. coli lysine transport mutant. Transport of l-[¹⁴C]lysine by the E. coli lysP mutant with an empty vector (−, black) or a vector expressing H. influenzae lysT from pPJ018 (lysT, white) was measured in counts per minute at 10 and 20 min. (B) NTHi1465 encodes the only lysine transporter in H. influenzae. Transport of l-[¹⁴C]lysine by wild-type H. influenzae (wt, black) and the H. influenzae ΔlysT (ΔlysT, white) mutant was measured in counts per minute at 10 and 20 min after the addition of l-[¹⁴C]lysine to the assay. ND, no lysine uptake was detected. The data are the means of results of three independent experiments, error bars in all assays represent the standard error, and statistical significance was determined by an unpaired two-tailed Student's t test: *, P < 0.05; **, P < 0.005.

FIG. 6.

Lysine riboswitch and lysT homolog in H. influenzae. (A) Genomic context of the H. influenzae lysT homolog (NTHi1465) and its lysine riboswitch. RNA probes for Northern blot analysis are depicted as arrows 1 and 2. (B) Predicted secondary structure of the H. influenzae lysine riboswitch aptamer. Mfold was used to predict RNA structure (61). Structural motifs are noted, and predicted lysine-binding nucleotides are boxed.

FIG. 7.

The lysine riboswitch is conserved in H. influenzae. (A) The NTHi1465 lysine riboswitch accumulates in the presence of lysine. H. influenzae wild-type (NTHi) and ΔlysT lysine-starved cells (−lys, 0 min) were exposed to lysine (+lys), and samples were removed at 15, 30, and 60 min postaddition. RNA from each time point was subjected to Northern blot analysis using a probe for the NTHi1465 l-lysine riboswitch. (B) H. influenzae lysT diminishes upon addition of lysine. The RNA samples from the lysine starvation experiment described above were subjected to Northern blot analysis and probed for lysT. Arrows indicate the full-length lysT transcript in both panels.