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.

Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen found exclusively in the mammalian oral cavity and is a proposed causative agent of localized aggressive periodontitis (22). Within the oral cavity, A. actinomycetemcomitans resides in the gingival crevice, the area around the tooth bounded by the tooth surface on one side and the gingival epithelium on the other. The gingival crevice is colonized by numerous bacterial species residing in surface-associated biofilm communities and is bathed by a serum exudate called gingival crevicular fluid (29, 32, 39). Microbes growing in the gingival crevice face an array of host- and microbe-derived stresses, including host immune factors such as complement and immunoglobulins (29), potentially toxic metabolites such as H₂O₂ and lactic acid (14, 43), intense competition for nutrients, and steep oxygen gradients that may fluctuate continuously (32). While it is clear that A. actinomycetemcomitans senses and responds to precise cues in the gingival crevice 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.

Bacteria utilize noncoding regulatory RNAs (ncRNAs) to rapidly respond to environmental stresses and alter gene expression (44). In contrast to transcription factors, ncRNAs provide a rapid means of fine-tuning gene expression in response to environmental signals (30). There are two major types of ncRNAs utilized in bacterial gene regulation. trans-acting small regulatory RNAs (sRNAs) regulate gene expression by binding target mRNAs, which typically reduces target gene mRNA levels due to degradation of the mRNA-sRNA duplex or reduces translation of a target gene by sequestration of the ribosomal binding site (18). cis-acting RNAs, or riboswitches, are cotranscribed with the downstream gene(s) they regulate and affect gene expression by binding small molecules, such as amino acids and nucleotides (20, 28, 59). Riboswitches have two functional components: the aptamer domain, which mediates binding to a small molecule, and the expression platform, which controls the downstream gene (59). Riboswitch expression platforms consist of an RNA stem-loop structure that functions in one of two ways to control the downstream gene. They can affect translation by occluding the ribosomal binding site of the target gene, or they can cause premature transcriptional termination of the downstream gene in a rho-independent manner (59).

Based on their importance in numerous other bacteria, we hypothesized that ncRNAs would play important roles in A. actinomycetemcomitans response to external stresses such as nutrient limitation. A. actinomycetemcomitans provides an excellent platform for such studies since it seemingly inhabits a single niche, the mammalian oral cavity, where it must respond quickly to changing environmental conditions. Since nutrient availability in the gingival crevice is limited due to competition with host cells and other microbes, ncRNAs involved in nutrient uptake likely play important roles in A. actinomycetemcomitans physiology in vivo. Using a bioinformatic and experimental approach, we identified three metabolite-sensing riboswitches and nine sRNAs in A. actinomycetemcomitans during planktonic and biofilm growth. We also characterized the first riboswitch in A. actinomycetemcomitans, a lysine riboswitch, and its target, the novel lysine transport gene, lysT. Finally, we demonstrated that this system is conserved in many bacterial species, including the phylogenetically related pathogen Haemophilus influenzae.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The bacterial strains used in this study are listed in Table 1. Escherichia coli was grown aerobically in Luria-Bertani (LB) broth at 37°C, unless otherwise noted. A. actinomycetemcomitans was grown at 37°C in tryptic soy broth supplemented with 0.5% yeast extract (TSBYE) or a chemically defined medium (CDM) containing 20 mM glucose (11, 51) in a 5% CO₂ atmosphere with shaking at 165 rpm. H. influenzae was grown in brain heart infusion broth or CDM supplemented with 10 μg/ml hemin and 10 μg/ml NAD (sBHI and sCDM, respectively) at 37°C in a 5% CO₂ atmosphere with shaking at 165 rpm. Bacterial biofilms were grown as colony biofilms using 0.4-μm-pore-size nylon filters on top of either BHI or TSBYE agar plates (57). Antibiotic concentrations were 100 μg/ml ampicillin and 20 μg/ml kanamycin for E. coli, 2.5 μg/ml rifampin for A. actinomycetemcomitans, and 20 μg/ml kanamycin for H. influenzae.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Genotype or morphologya	Source
Strains
A. actinomycetemcomtians VT1169	Smooth strain	6
A. actinomycetemcomtians Y4	Smooth strain	37
E. coli K-12	Wild type	21
E. coli DH5α	F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK−mK+) (Nalr)
E. coli K-12 lysP	rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 lysP mutant (Kmr)	5
E. coli K-12 lysP pGEM-T Easy	rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 lysP mutant (Kmr Apr)	This study
E. coli K-12 lysP pPJ005	rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 lysP (Kmr Apr) pPJ005 lysTAa	This study
E. coli K-12 lysP pPJ018	rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 lysP (Kmr Apr) pPJ018 lysTH. influenzae	This study
H. influenzae 86-028NP	Nontypeable	7
H. influenzae 86-028NP ΔlysT	Nontypeable, lysT (Kmr)	This study
Plasmids
pGEM-T Easy	TA cloning vector	Promega
pPJ005	A. actinomycetemcomitans lysT in pGEM-T Easy	This study
pPJ018	H. influenzae lysT in pGEM-T Easy	This study

^alysT_Aa, lyst from A. actinomycetemcomitans; lysT_{H. influenzae}, lyst from H. influenzae.

DNA and plasmid manipulations.

DNA and plasmid isolations were performed using standard methods (4). Restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs. Chromosomal DNA was isolated using DNeasy tissue kits (Qiagen), and plasmid isolations were performed using QIAprep spin miniprep kits (Qiagen). DNA fragments were purified using QIAquick mini-elute PCR purification kits (Qiagen), and PCR was performed using the Expand Long Template PCR system (Roche). DNA sequencing was performed by automated sequencing technology using the University of Texas Institute for Cell and Molecular Biology sequencing core facility.

RNA preparation.

Bacterial cultures were mixed 1:1 (vol/vol) with RNAlater (Ambion) and stored up to 2 weeks at 4°C. Total RNA was purified using RNAbee as described by the manufacturer (Tel-Test). RNA samples were treated with DNase to remove contaminating chromosomal DNA as described previously (11).

Northern blot ncRNA screen.

Total RNA was prepared from exponential phase planktonic A. actinomycetemcomitans VT1169, stationary-phase planktonic A. actinomycetemcomitans VT1169, and A. actinomycetemcomitans VT1169, the last growing as a colony biofilm (43, 57). Gel load dye II (Ambion) was mixed with 10 μg total RNA and separated on 8% polyacrylamide-8 M urea gels. An RNA ladder (Ambion) was transcribed with [α-³²P]UTP (Perkin Elmer) from the Century Plus marker template (Ambion) using the T7 MaxiScript in vitro transcription kit (Ambion) and served as the size standard. Gels were stained with ethidium bromide for imaging using a G:BOX imaging apparatus (Syngene) and transferred to nitrocellulose. RNA was UV cross-linked to the nitrocellulose, and blots were prehybridized with ExpressHyb hybridization solution (ClonTech) for 1 h at 55°C in an HL-2000 HybriLinker hybridization oven (UVP). DNA oligonucleotide probes (Table 2) were end labeled with [γ-³²P]ATP (Perkin Elmer) using the Kinase Max system (Ambion). Probes were added to the prehybridization buffer and incubated 17 h at 38°C. Blots were washed in 20 ml 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS for 20 min at 38°C, and the wash was repeated four times. Blots were sealed and exposed to film (Kodak) for 2 to 17 h and developed with an X-Omat 2000 processor (Kodak).

TABLE 2.

Primers and Northern blot probes used in this study

Primer or probe	Sequence
Primers
Aa-lysT-F	GTC AGT GGT TCT CCC TTA ATA TTA G
Aa-lysT-R	GTG CTG GAT CAC GAT TTA ACG
NTHi-lysT-F	GGC TTA AGG GTG CTA CGG TTG TTC TGG TC
NTHi-lysT-R	GGC TTA AGG GCA AGT TTT TGA ATG AGA TCA CAC
NTHi1465KO-P1-F	CCT TCA ACC GCA CTT ATT GG
NTHi1465KO-P1-R	CAG ATA GCC CAG TAG CTG ACA TGT AGT GAT TGG GGA GAT TAC CG
NTHi1465KO-P2-F	GCG GGA CTC TGG GGT TCG AAA TGT GTG ATC TCA TTC AAA AAC TTG
NTHi1465KO-P2-R	GAA GTG CGG TCA AAT TCC AAA C
NTHi1465KO-verify	GTA ATG CGC GAT TCG ATA AAC
Kan-F	ATG TCA GCT GGG CTA TCT G
Kan-R	ATT TCG AAC CCC AGA GTC CCG C
lysRS-PE	CTT TTA ACT GAT TTC GGC AAA CG
Aa-lysT-RT-F	GTT ATT TGC GTT GTT ACT CGC CAT C
Aa-lysRS-lysT-RT-F	GTT GGT GAC GTC TTC GAA AGA ACC G
Aa-lysT-RT-R	AAG GCG CGC TTA CTG CCG GAC ATA C
Probes
Glycine-Probea	AAA GTA CCT TGG ATC TCT CCA AGG G
FMN-Probea	TTT CAC CCT GCC CTG AGA ATG CG
aa35-Probea	GCA ATA CTT AAC AGA TGT GGC TTA TG
aa84-Probea	CTT GGA CGG GGC TTG AAT GAG GTC A
aa89-Probea	TTA CCG CCT TAT CTT CAC AAA GCG C
aa337-Probea	CTC AAG CCT AAA TCC TGA ATT TGC
aa575-Probea	GGT TGT GCT TTT TTA AGC ACT GGG
aa673-Probea	TCC ACC TTT CCA GCC TTC CCA ATT A
aa734-Probea	CCG CAC TTT AGT TTT TAG TTT CCT CCG
Alpha_RBS-Probea	CAG GAT ACT CAA ACA GAG TGT CAA GG
GcvB-Probea	GGA AAC TCT TTA ACC AGT AAG
Aa-lysRS-Probea	CAC CGT TAT CCA CTC GGA AAA TAT AC
Aa-lysRS-Probe-F	GGA ATT CCT AAT ACG ACT CAC TAT AGG GAA CAG TAG CGC TCC ATA GTT G
Aa-lysRS-Probe-R	CCC AAG CTT GGG CAA AAA ACG CGT CGG GGT TTA GCC CGA CGA CTT GTG TAG AGG TGC AAA
NTHi-lysRS-Probe-F	TAA TAC GAC TCA CTA TAG GGC AAC CGT AGC ACT CCA TAG
NTHi-lysRS-Probe-R	GTA GAG GCG CAA TTA TTA TAA G
NTHi-lysT-Probe-F	TAA TAC GAC TCA CTA TAG GGT CGG TGC TGC CGT TGA ATC
NTHi-lysT-Probe-R	TCG TGC CTT TGC TGA ATG G

^aDNA oligonucleotide probe.

Primer extension analysis.

Primer extension analysis was performed as previously described (31, 38) using a 6-carboxyfluorescein (FAM)-labeled primer, lysRS-PE (Table 2), to synthesize cDNA from 20 μg A. actinomycetemcomitans VT1169 colony biofilm RNA. Prior to primer extension, RNA integrity was confirmed by agarose gel electrophoresis. A 1-μl portion of a 0.4 μM 5′-FAM-labeled primer was added to the RNA (20 μl final volume), and the mixture was incubated for 10 min at 70°C. The mixture was chilled in an ice water bath, incubated 20 min at 65°C, and held at 42°C. cDNA synthesis reagents (8 μl 5× first-strand buffer, 4 μl 0.1 M dithiothreitol [DTT], 4 μl 10 mM deoxynucleoside triphosphates [dNTPs], and 4 μl SuperScript II [Invitrogen]) were added to the mixture, and the reaction mixture was incubated 2 h at 42°C before ethanol precipitation. cDNA sizing was performed at the University of Oklahoma Health Sciences Center Laboratory for Genomics and Bioinformatics. Fragment analysis was conducted using Peak Scanner software (Applied Biosciences).

RT-PCR for AA02294, lysT, and the lysine riboswitch.

A 1-μg portion of RNA from lysine-starved A. actinomycetemcomitans Y4 was treated with RQ1 DNase (Promega) and used as a template for random primed cDNA synthesis with SuperScript II reverse transcriptase (RT). A negative-control reaction lacking reverse transcriptase was also performed (−RT). Reverse transcriptase reactions were purified with QIAquick spin columns (Qiagen). Three different products were amplified from 10 ng cDNA template that correspond to different regions of the lysT mRNA. A portion of the middle of the coding region of A. actinomycetemcomitans lysT was amplified using Aa-lysT-RT-F and Aa-lysT-RT-R (Table 2). A region spanning the lysine riboswitch and the coding region of lysT was PCR amplified using Aa-lysRS-lysT-RT-F and Aa-lysT-RT-R (Table 2). A region corresponding to the 3′ end of the lysT mRNA was PCR amplified using Aa-lysT-3′-F and Aa-lysT-3′-R (Table 2). A. actinomycetemcomitans chromosomal DNA served as a positive-control template, and the −RT products served as a negative-control template. PCR products were separated on an agarose gel, stained with ethidium bromide, and visualized with a G:BOX imaging apparatus.

Agarose gel Northern blots.

A. actinomycetemcomitans, H. influenzae, or an H. influenzae ΔlysT strain was grown overnight in TSBYE or sBHI, pelleted by centrifugation, washed three times in CDM or sCDM free of l-lysine, resuspended in the same lysine-free medium, and starved for l-lysine for 2 h. A fraction of the cells was removed from the culture for RNA extraction, and 10 mM l-lysine was added to the growth medium. Cells were removed from the culture for RNA extraction at 15, 30, 60, and 120 min after the addition of l-lysine.

Northern Max glyoxal load dye (Ambion) was mixed 1:1 (vol/vol) with 20 μg total RNA from each sample, and the mixtures were incubated at 50°C for 30 min. RNA was immediately separated by gel electrophoresis on a 2% agarose gel using the Northern Max buffer system (Ambion). Ethidium bromide-stained RNA gels were imaged using a G:BOX imaging apparatus. Wells were cut away from gels, and RNA was transferred to nitrocellulose by downward transfer for 4 h using a Whatman Turboblotter. RNA was UV cross-linked to the nitrocellulose, and blots were prehybridized with UltraHyb buffer (Ambion) for 1 h at 68°C in an HL-2000 HybriLinker hybridization oven. RNA probe templates were PCR amplified from A. actinomycetemcomitans chromosomal DNA with primers Aa-lysRS-Probe-F and Aa-lysRS-Probe-R (Table 2) or from H. influenzae chromosomal DNA using NTHi-lysRS-Probe-F and NTHi-lysRS-Probe-R (probe 1; see Fig. 6A) or NTHi-lysT-Probe-F and NTHi-lysT-Probe-R (probe 2; see Fig. 6A) (Table 2). RNA probes were in vitro transcribed from PCR templates with [α-³²P]UTP (Perkin Elmer) using the T7 MaxiScript system. Probes were added to the prehybridization buffer, and the mixture was incubated 17 h at 68°C. Blots were washed in 20 ml 2× SSC-0.1% SDS for 10 min at 25°C and washed twice more in 0.1× SSC-0.1% SDS for 15 min each at 68°C. Blots were then sealed and exposed to a phosphor screen (Bio-Rad), imaged using a Personal Molecular Imager (Bio-Rad), and processed with Quantity One software (Bio-Rad).

Lysine riboswitch half-life.

A. actinomycetemcomitans Y4 was grown overnight in TSBYE, washed three times in lysine-free CDM, resuspended in lysine-free CDM, and starved for lysine for 2 h. After starvation, 10 mM l-lysine was added to the growth medium, and 15 min later rifampin was added to the culture to inhibit transcription. At 0, 5, 10, 20, and 40 min after rifampin treatment, cells were removed for RNA extraction. RNA samples were subjected to polyacrylamide urea gel Northern blot analysis (as described above) probing for the A. actinomycetemcomitans lysine riboswitch with Aa-lysRS-Probe (Table 2). Northern blots were exposed to a phosphor screen, imaged on a personal molecular imager, and analyzed by densitometry with Quantity One software. The half-life was calculated using the following equation: H = t{[ln(0.5)]/[ln(S_f/S₀)]}, where H is the half-life, t is time, S_f is the final signal, and S₀ is the original signal.

Expression of A. actinomycetemcomitans lysT and H. influenzae lysT in the E. coli lysP mutant.

A. actinomycetemcomitans lysT was PCR amplified from A. actinomycetemcomitans Y4 chromosomal DNA using the primers Aa-LysT-For and Aa-LysT-Rev (Table 2). The PCR product was ligated into pGEM-T Easy TA cloning vector (Applied Biosystems) to create pPJ005, which was introduced into E. coli DH5α and the lysP mutant obtained from the Keio collection (5). Similarly, lysT was PCR amplified from H. influenzae 86-028NP chromosomal DNA with NTHi-LysT-For and NTHi-LysT-Rev (Table 2). The PCR product was ligated into pGEM-T Easy to construct pPJ018, which was introduced into E. coli DH5α, strain K-12, and the lysP mutant obtained from the Keio collection (5). Plasmid constructs were confirmed by restriction enzyme digest and DNA sequencing.

Constructing an H. influenzae ΔlysT mutant.

Allelic replacement of H. influenzae lysT was performed by natural transformation of H. influenzae strain 86-028NP as previously described (13, 50). The regions flanking lysT on the H. influenzae chromosome were PCR amplified with NTHi1465KO-P1-F, NTHi1465KO-P1-R, NTHi1465KO-P2-F, and NTHi1465KO-P2-R, and a kanamycin resistance gene (aphA) was amplified from pBBR1MCS-2 (27) using Kan-F and Kan-R (Table 2). The kanamycin resistance gene was placed between the two flanking regions by overlap extension PCR (23). The resulting ∼4-kb PCR product was then gel purified with a QIAquick gel purification spin column (Qiagen), and 2 μg product was used to transform H. influenzae 86-028NP by natural transformation (13, 50). H. influenzae ΔlysT mutants were selected on sBHI agar plus 20 μg/ml kanamycin. Chromosomal DNA was extracted from mutants, and allelic replacement was confirmed by PCR amplification with NTHi1465KO-verify and KanR primers (Table 2).

Lysine transport assays.

Bacteria were grown overnight in either LB or sBHI for E. coli and H. influenzae, respectively. Cultures were diluted 1:1 in the same medium and incubated 1 h with shaking at 37°C. E. coli was washed three times in lysine-free MOPS (50 mM MOPS [morpholinepropanesulfonic acid], 43 mM NaCl, 93 mM NH₄Cl, 2 mM KH₂PO₄, pH 7.2, 20 mM glucose, 5 μg/ml Fe₂SO₄, 700 μg/ml MgSO₄, 100 μg/ml CaCl₂), and H. influenzae was washed in lysine-free sCDM and resuspended in 500 μl of the same medium to an optical density at 600 nm (OD₆₀₀) of 0.40. Bacteria were incubated for 5 min at 37°C; 4 μCi (100 nM) l-[¹⁴C]lysine was added, and the mixture was incubated at 37°C. At 10 and 20 min, 250 μl was removed from each transport reaction and quenched with 2.5 ml ice-cold MOPS or sCDM with 10 mM l-lysine. The quenched transport reactions were filtered through a 0.4-μm-pore-size filter, the filters were washed with 3 ml ice-cold MOPS or sCDM with 10 mM l-lysine, and the filters were placed in 4 ml Ecolite scintillation fluid and counted on a Beckman-Coulter LS6500 liquid scintillation counter. Endpoint cold competition transport assays were carried out for 20 min, as described above; however, the medium used for the transport assay contained a 20 mM concentration of the competing amino acid (i.e., l-arginine, l-glycine, or l-lysine).

Lysine riboswitch RNA secondary structure analyses.

The A. actinomycetemcomitans lysine riboswitch nucleotide sequence was analyzed with Mfold to predict the RNA secondary structure (61). The homologous sequence upstream of NTHi1465 was also analyzed with Mfold (28, 61). Both riboswitch structures were compared to lysine riboswitch crystal structures from Thermotoga maritima to identify conserved lysine-binding nucleotides and structural elements (17, 48).

Identifying LysT homologs and putative lysine riboswitches.

BLASTp analysis was used to find proteins homologous to A. actinomycetemcomitans LysT, with an E value cutoff of 10⁻¹⁰⁰ (2). ClustalW (version 1.83) was used to align amino acid sequences of proteins from 64 species and conduct phylogenetic analysis with 1,000 bootstrap replicates (55). Phylogenetic trees were drawn with TreeView software (60). All loci encoding BLASTp LysT homologs were analyzed for putative lysine riboswitches in the 400-bp region upstream of their start codons. The 400-bp region was analyzed with RiboSW software which scanned for any riboswitches in the sequence. When a putative lysine riboswitch was identified by RiboSW, the predicted riboswitch start location relative to the start codon was recorded, along with the free energy associated with the secondary structure and the corresponding HMM E value (12).

Glycine riboswitch RNA secondary structure analyses.

The intergenic region upstream of AA00167 was analyzed with RiboSW to identify the putative glycine riboswitch sequence (12). Mfold software was used to generate a secondary structure, which was then compared to the consensus glycine riboswitch structure determined by Kwon and Strobel (28, 61).

RESULTS

Discovery of A. actinomycetemcomitans regulatory RNAs.

While there has not been an investigation of ncRNAs in A. actinomycetemcomitans, a recent in silico analysis predicted 35 ncRNAs encoded in intergenic regions of the A. actinomycetemcomitans genome (http://www.oralgen.lanl.gov/). To determine if these predicted ncRNAs are produced by A. actinomycetemcomitans, RNA was isolated from logarithmic, stationary-phase, and colony biofilm cells, and Northern blot analysis was used to probe for 23 of the predicted ncRNAs. Twelve ncRNAs were detected, including three putative riboswitches and nine sRNAs ranging in size from ∼110 to 500 nucleotides (nt) (Table 3). Of the 12 ncRNAs identified, 8 were identified under all growth conditions, and 4 were observed only in colony biofilm-grown A. actinomycetemcomitans. Additionally, several ncRNAs (putative glycine riboswitch, putative lysine riboswitch, Aa84, Alpha_RBS, Aa337, GcvB, and Aa575) were expressed at higher levels during biofilm growth. We chose to further characterize the putative lysine riboswitch since it was observed only in colony biofilms, it possessed high homology to characterized lysine riboswitches, and it was positioned upstream of a gene of unknown function that is conserved in a large number of bacteria (see below).

TABLE 3.

Riboswitch and sRNA discovery in A. actinomycetemcomitans

^aA. actinomycetemcomitans VT1169 grown planktonically to exponential phase.

^bA. actinomycetemcomitans VT1169 grown planktonically to stationary phase.

^cA. actinomycetemcomitans VT1169 grown as a colony biofilm.

^dORF predicted upstream of riboswitch or sRNA.

^eBeginning coordinates from intergenic region (IGR) from A. actinomycetemcomitans strain HK1651 genome (http://oralgen.lanl.gov/cgi-bin/coordinate.cgi?dbname=aact).

^fEnding coordinates from intergenic region (IGR) from A. actinomycetemcomitans strain HK1651 genome (http://oralgen.lanl.gov/cgi-bin/coordinate.cgi?dbname=aact).

^gORF predicted downstream of riboswitch or sRNA.

^hApproximate size of riboswitch or sRNA, as determined by Northern blot analysis.

Mapping the transcriptional start site and promoter of the putative lysine riboswitch.

To determine a transcriptional start site and map a putative promoter region for the putative lysine riboswitch, primer extension was used. The major primer extension product was 93 nt in length (Fig. 1A), corresponding to an adenine transcriptional start site (Fig. 1B) positioned 7 bp upstream of the predicted riboswitch aptamer domain and 267 bp upstream of the hypothetical gene AA02294 (Fig. 1D). Examination of the DNA sequence upstream of the transcriptional start site revealed a sequence with some homology to a canonical E. coli σ⁷⁰ promoter and an open reading frame (ORF) encoding the global transcriptional regulator H-NS (52). A canonical rho-independent terminator was identified 4 bp downstream of the aptamer domain (Fig. 1C).

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.

Structural predictions demonstrate features characteristic of lysine riboswitches.

Structures of riboswitch aptamers are critical for their function. Structural studies of lysine riboswitch aptamers show two characteristic elements: an “RNA kissing loop” that binds l-lysine at the base of the two loops and a loop E motif that enables RNA twisting (9, 17, 48). The secondary structure prediction program Mfold was used to generate an RNA secondary structure of the putative A. actinomycetemcomitans lysine riboswitch based on free energies of internal base pairing (61) (Fig. 2A). The predicted structure has the characteristic kissing loops and loop E motif. Conserved nucleotides known to be important for coordinating l-lysine at the base of the kissing loops were also present (48) (Fig. 2A), providing strong in silico support that this ncRNA is a lysine riboswitch.

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.

The riboswitch accumulates in the presence of lysine.

Riboswitches are cotranscribed with the genes they regulate (40). To determine whether this putative riboswitch formed a contiguous transcript with the downstream gene AA02294, RT-PCR was used. Primers were designed to amplify a region spanning the riboswitch and the AA02294 coding region (Fig. 2B). Our results reveal that A. actinomycetemcomitans does produce a contiguous mRNA containing both the putative riboswitch and AA02294. Based on the location of this mRNA, we hypothesize that the putative riboswitch serves as a cis-regulatory element in the 5′ untranslated region of AA02294.

Upon binding lysine, most lysine riboswitch expression platforms prematurely terminate transcription of the downstream gene (9, 10, 20, 40, 48, 54). Because we identified a potential rho-independent terminator downstream of the lysine aptamer domain and upstream of AA02294 (Fig. 1C), we hypothesized that A. actinomycetemcomitans grown in the presence of lysine would produce high levels of the prematurely terminated riboswitch transcript compared to that produced by lysine-starved cells. To test this hypothesis, Northern blot analysis was carried out to probe for the riboswitch in the absence and presence of lysine. As anticipated, the terminated riboswitch transcript is present only after the addition of lysine to the medium (Fig. 2C), consistent with previous studies of lysine riboswitches (20, 40). The full-length mRNA (approximately 2,000 nt) was not observed by Northern blot, although in the absence of lysine, an RNA larger than the riboswitch was detected (0 min, Fig. 2C). Thus, it appears that the full-length transcript is unstable or there is a technical issue in detecting the riboswitch when present in the full-length transcript. Importantly, the full-length transcript could be detected by RT-PCR (Fig. 2B), and the full-length transcript along with the riboswitch were detected in the closely related bacterium H. influenzae (see below).

Riboswitch half-life.

Since the prematurely terminated transcript accumulated to high levels upon exposure to lysine (Fig. 2C), we used Northern blot analysis to calculate the lysine riboswitch half-life. For these experiments, A. actinomycetemcomitans grown in the absence of lysine was exposed to lysine to induce production of the riboswitch. After 15 min, the transcription inhibitor rifampin was added and degradation of the riboswitch over time was monitored using Northern blots and densitometry (Fig. 3). The results indicate that the riboswitch has a half-life of 17 ± 4 min, which is nearly five times the average half-life of an E. coli transcript (3.69 min) (8).

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.

A. actinomycetemcomitans lysT encodes a novel lysine transporter.

Lysine riboswitches have been shown to regulate lysine biosynthetic genes in Bacillus subtilis and the lysine transport gene lysP in E. coli (20, 45). The gene downstream of the A. actinomycetemcomitans lysine riboswitch, AA02294, is a hypothetical gene of unknown function. A previous study hypothesized that genes downstream of several lysine riboswitches encoded novel lysine transporters, although no empirical evidence was provided to test this hypothesis (45). There are several well-characterized bacterial lysine transporters, including the lysine-specific LysP transporter in E. coli (15, 53), the lysine-arginine-ornithine (LAO) transporter in E. coli (25), and LysI in Corynebacterium glutamicum (47). While LysI and LysP uniquely transport lysine, the LAO transporter preferentially transports arginine and ornithine (46). Since A. actinomycetemcomitans does not possess homologs to known lysine transporters, and bioinformatic analysis with Phyre software predicted that AA02294 encodes an inner membrane transport protein (26) with an NhaC domain (24, 36, 58), we hypothesized that AA02294 encodes a novel lysine transporter.

To determine whether A. actinomycetemcomitans AA02294 encodes a lysine transporter, we tested its ability to heterologously restore lysine transport in an E. coli lysine transport mutant. A plasmid carrying A. actinomycetemcomitans AA02294 (pPJ005) was transformed into an E. coli lysP mutant that is unable to take up l-lysine, and the ability of this strain to transport l-[¹⁴C]lysine was assessed. Expression of AA02294 in trans restored l-lysine transport to the E. coli lysP mutant (Fig. 4A), indicating that AA02294 encodes a novel lysine transporter (referred to as LysT). It was also important to determine the specificity of LysT, since some lysine transporters have been shown to also transport l-arginine (46, 53). To test the specificity of LysT, the ability of unlabeled l-lysine, l-arginine, and l-glycine to inhibit uptake of l-[¹⁴C]lysine by the E. coli lysP mutant expressing A. actinomycetemcomitans lysT was assessed. The addition of unlabeled l-lysine reduced uptake of l-[¹⁴C]lysine to background levels, while addition of unlabeled l-arginine inhibited l-[¹⁴C]lysine uptake approximately 50% (Fig. 4B). The addition of unlabeled glycine had no effect on lysine uptake.

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.

Haemophilus influenzae LysT is a lysine transporter.

Although LysT lacks homology to known lysine transporters such as LysP in E. coli (15, 53), a BLASTp homology search revealed LysT homologs from 64 species of bacteria, including Gammaproteobacteria, Betaproteobacteria, and bacilli (E value < 10⁻¹⁰⁰; see Fig. S1 in the supplemental material) (2, 3). Of the genes encoding the LysT homologs, 48 are predicted to have lysine riboswitches within 400 bp of their start codons (see Table S1 in the supplemental material) (12). Included within this group containing riboswitches is the LysT homolog (ORF NTHi1465) from the human pathogen Haemophilus influenzae, which causes otitis media, bacterial meningitis, and pulmonary infections (16). The H. influenzae LysT is a hypothetical inner membrane protein with 75% identity and 92% similarity to A. actinomycetemcomitans LysT over 249 amino acids in the predicted loop regions. To determine if H. influenzae NTHi1465 encoded a lysine transporter, NTHi1465 was expressed in the E. coli lysP mutant from pPJ108 and tested for its ability to restore lysine transport. As expected, heterologous expression of NTHi1465 restored the ability of the E. coli lysP mutant to transport lysine (Fig. 5A), indicating that H. influenzae NTHi1465 encodes an l-lysine transporter. In addition, NTHi1465 appears to be the only lysine transporter in H. influenzae, as deletion of lysT eliminated the ability of H. influenzae to transport lysine (Fig. 5B).

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.

H. influenzae lysine riboswitch.

As previously discussed, bioinformatics also predicted a putative lysine riboswitch upstream and contiguous with H. influenzae lysT. Similar to A. actinomycetemcomitans, hns was located upstream of lysT and in the opposite orientation (Fig. 6A). Structural predictions of the H. influenzae lysine riboswitch aptamer domain using Mfold revealed conserved structural elements, as well as complete conservation of the nucleotides required for lysine binding (Fig. 6B) (61). Although predicted to be structurally similar and contiguous with their respective lysT genes, the A. actinomycetemcomitans and H. influenzae riboswitches are localized to different positions in regard to the lysT start codons. In A. actinomycetemcomitans, the riboswitch is predicted to terminate approximately 50 bp upstream of the start codon for lysT; however, in H. influenzae the riboswitch is predicted to overlap the start codon of lysT.

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.

To determine if the H. influenzae riboswitch acts at the transcriptional level, H. influenzae was grown with and without l-lysine, and Northern blot analysis was used to probe for both the riboswitch and lysT. As with A. actinomycetemcomitans, two transcripts were present when using the riboswitch-specific probe: a small transcript corresponding to the lysine riboswitch and a larger transcript likely corresponding to the degraded riboswitch-lysT transcript (Fig. 7A). As expected, the lysine riboswitch accumulated upon addition of l-lysine, while the larger transcript disappeared. Since we were not able to definitively detect the full-length transcript with the riboswitch probe, we also utilized a probe specific for lysT. This probe detected a specific mRNA at ∼2,000 nt, corresponding to the full-length lysT mRNA. Also, the probe hybridized to the full-length lysT transcript only in the lysine-starved samples and did not detect any transcripts in cells exposed to lysine. However, as expected from Northern blots with riboswitch-specific probes (Fig. 2C and Fig. 7A), it appears that the full-length transcript is highly unstable, as a number of smaller transcripts were also detected (Fig. 7B). Importantly, no hybridization was observed when RNA from the H. influenzae lysT deletion strain was used (Fig. 7A and B), indicating that the transcripts identified in the wild-type RNA samples are specific for the riboswitch and lysT. Together, these results demonstrate that the lysine riboswitch is conserved in H. influenzae and regulates lysT at the transcriptional level.

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.

DISCUSSION

The study describes the identification of 12 new ncRNAs in A. actinomycetemcomitans. While most of these ncRNAs were produced by planktonic and biofilm bacteria, several appear to be produced at higher levels in biofilm-grown bacteria. Interestingly, three ncRNAs that are induced in biofilm bacteria have predicted functional overlap; specifically, they appear to regulate amino acid transport. Along with the lysine riboswitch characterized in this study, a putative glycine riboswitch was identified upstream of AA00167 (Table 3; see Fig. S2 in the supplemental material). The riboswitch has structural and sequence homology to other glycine riboswitches (28), and AA00167 belongs to the sodium/alanine symporter superfamily (35), which includes a known glycine transporter (34). We also observed high levels of an sRNA homologous to GcvB in A. actinomycetemcomitans biofilms. In Salmonella enterica serovar Typhimurium and E. coli, GcvB regulates more than 10 genes involved in amino acid transport (41, 42, 49, 56). Since all three of these ncRNAs are proposed to downregulate amino acid transport in the presence of high levels of intracellular amino acids, these data suggest that within the colony biofilm, A. actinomycetemcomitans possesses high intracellular amino acid concentrations. In the gingival crevice, A. actinomycetemcomitans and other bacteria are known to release proteases that could increase free amino acid pools in the surrounding environment (1, 19). However, free amino acid levels have not been directly measured in gingival crevicular fluid. Further studies will be needed to determine if these ncRNAs are important for biofilm formation and biofilm-specific phenotypes.

While it is not surprising that a lysine riboswitch controls transcription of a gene encoding a lysine transporter, the characterization of LysT describes a new family of lysine transporters. LysT homologs were indentified in 64 species of bacteria, including Gram-negative and Gram-positive species (see Fig. S1 in the supplemental material), and our studies of H. influenzae LysT suggest (Fig. 5) that many of the LysT homologs will be involved in lysine transport. Interestingly, as in A. actinomycetemcomitans and H. influenzae, riboswitches are predicted to regulate transcription of 75% of the LysT homologs (see Table S1 in the supplemental material). The latter observation suggests that riboswitch regulation is a common mechanism for regulating transcription of lysine transporters in bacteria. This mode of regulation allows transcription to be linked to intracellular lysine concentrations, thus allowing bacteria to fine-tune expression in real time in response to changing lysine levels.

While not examined in this study, lysine riboswitches could also play important regulatory roles aside from regulating genes involved in lysine transport and biosynthesis. Because our Northern blot analyses showed that the lysine riboswitch has a relatively long half-life of approximately 17 min (Fig. 3), the lysine riboswitch could act as a trans-regulatory sRNA in a lysine-dependent signaling process. Interestingly, Phan and Schumann noted a similar result in Bacillus subtilis (40) and also postulated that the prematurely terminated lysine riboswitch might serve additional functions within the cell. This is not unreasonable, as the S-adenosylmethionine riboswitch in Listeria monocytogenes is known to regulate an important virulence regulator in trans (33).

This study used bioinformatic and experimental approaches to identify ncRNAs in A. actinomycetemcomitans, some of which appear to be highly expressed in biofilms and others which have no known homologs (Aa35, Aa84, Aa337, and Aa575). These results provide the first insight into ncRNAs in A. actinomycetemcomitans, providing a basis for future functional studies. There is no doubt that more ncRNAs are produced by A. actinomycetemcomitans, many of which may be specific for other growth environments, such as the oral cavity or polymicrobial communities. Future studies will address the overall expression of ncRNAs in A. actinomycetemcomitans and determine specific regulatory functions that ncRNAs serve, not only within a single bacterium but within complex polymicrobial communities.