The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5′-UTR Variant

Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5′-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5′-untranslated regions (5′-UTR) of 28 and 234 nucleotides. The transcript containing the long 5′-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5′-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5′-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5′-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5′-UTR. These findings emphasize how 5′-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5′-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.


Introduction
Listeria monocytogenes is a facultative intracellular food-borne bacterium responsible for serious clinical manifestations including febrile gastroenteritis, meningitis, encephalitis and maternofetal infections in humans and livestock, with an estimated fatality rate of 20-30% of infected individuals [1][2][3]. Following ingestion, L. monocytogenes is able to cross the intestinal, blood-brain and placental barriers. The bacterium expresses a number of virulence factors that promote entry into phagocytic and non-phagocytic eukaryotic cells, intracellular survival and proliferation, and spreading to adjacent cells [4].
Genome studies show that all Listeria species sequenced to date have more than 40 genes that encode predicted surface proteins bearing an LPXTG sorting motif [5]. This motif is recognized by sortase enzymes, which anchor these proteins covalently to the cell wall. In pathogenic Listeria, some of these LPXTG proteins direct essential steps throughout the infection process, including bacterial adhesion and uptake by the host cell [6][7][8][9]. Proteomic analyses indicated that levels of many of these LPXTG surface proteins change on adaptation to different environments. The Listeria cell wall subproteome thus changes substantially in actively growing and resting bacteria. Mutants that lack sortase SrtA and SrtB activity show impaired LPXTG protein anchoring to the peptidoglycan [10] as well as differences in the relative levels of certain LPXTG proteins [11]. Recent studies also showed major changes in the cell wall proteome when L. monocytogenes proliferate inside epithelial cells [12]. Upregulation of defined LPXTG proteins has been observed in intracellular bacteria, including Internalin-A and Lmo0514 [12]. The mechanisms that regulate the coordinated production of such a large number of LPXTG proteins nonetheless remain largely unknown.
Bacterial small RNAs (sRNA) are a class of bacterial gene expression regulators important in many physiological processes, including virulence and cell envelope homeostasis [13,14]. sRNA coordinate target gene expression in response to environmental changes and have regulatory functions that affect protein activity and mRNA stability/translation in many microorganisms, including bacterial pathogens [15,16]. More than 100 sRNA have been identified for L. monocytogenes by the use of tiling arrays, global RNA sequencing (RNA-Seq) and bioinformatics methods [14,17,18]; more than 30 of these have been validated by northern blot, but their biological function and mechanisms of action are so far unknown [19]. There is little information on the regulation of sRNA expression in L. monocytogenes. Some reports implicate the alternative sigma factor SigB in regulating expression of the sRNA SbrA (Rli11) and SbrE (Rli47) [17,20,21]. In addition, 22 sRNA genes are preceded by putative sigma A boxes in the L. monocytogenes genome [14]. Recent studies also show that the sRNAs Rli31, Rli33-1, Rli38 and Rli50 modulate virulence in L. monocytogenes [14,17]. Despite these studies, there is no model that describes how sRNA expression in L. monocytogenes responds to infection of eukaryotic cells. With the exception of LhrA, which controls expression of the chitinase ChiA post-transcriptionally [22], and of the multicopy sRNA LhrC, which modulates LapB adhesin expression [23], the identity of the functions targeted by L. monocytogenes sRNA inside or outside eukaryotic cells, remains unknown.
Here we studied the regulatory mechanism responsible for the increase in the LPXTG protein Lmo0514 in the cell wall of intracellular bacteria [12]. Our data demonstrate an sRNA that is a key regulatory element in modulating levels of this cell wall surface protein during intracellular infection. This response to the eukaryotic niche is directed by the activity of two promoters in the target gene that generate transcripts with 59-untranslated regions (59-UTR) of distinct length. The relative abundance of these two transcripts differs in extra-and intracellular bacteria. Only the 'long' version, enriched in intracellular bacteria, bears the sRNA binding site. This mechanism confines the regulation of lmo0514 by this sRNA to the intracellular eukaryotic niche.

Results
The L. monocytogenes gene that encodes the LPXTG surface protein Lmo0514 is expressed as two variants with distinct 59-UTR Lmo0514, a L. monocytogenes LPXTG surface protein of unknown function, is encoded by a gene upregulated by bacteria located within macrophages [24]. Lmo0514 is also more abundant in the cell wall of bacteria that proliferate inside epithelial cells than in bacteria growing in laboratory media [12]. To study the basis of this regulation, we compared lmo0514 expression in extraand intracellular bacteria. Real-time quantitative PCR (qPCR) assays showed enhanced lmo0514 mRNA expression (,6-fold) in intracellular bacteria after infection of JEG-3 human epithelial cells (Fig. 1A). Consistent with our previous work [12], the Lmo0514 protein was detected mainly in the cell wall of intracellular bacteria, with very low levels in extracellular bacteria (Fig. 1B). Changes in relative levels of Lmo0514 protein were estimated to be.200-fold (Fig. 1B), much higher than those for lmo0514 mRNA (,6-fold). This lack of correlation between induction of lmo0514 transcript and protein levels in intracellular bacteria led us to hypothesize that post-transcriptional regulatory mechanisms act on this gene.
To evaluate this possibility, we sought lmo0514 gene expression control mechanisms that operate specifically in intracellular bacteria. Previous in silico predictions by Loh et al. [25] indicated that lmo0514 could be expressed from three promoters at Figure 1. Regulation of the L. monocytogenes gene that encodes the LPXTG surface protein Lmo0514 in intracellular bacteria located inside eukaryotic cells. Total RNA and cell wall protein extracts were prepared from L. monocytogenes wild-type strain EGD-e grown in BHI medium to stationary phase (extracellular) and from bacteria collected in JEG-3 epithelial cells at 6 h post-infection (intracellular). (A) lmo0514 transcript levels monitored by real-time qPCR using primers Lmo0514-F and Lmo0514-R, which map within the lmo0514 coding region (Table S2). (B) Lmo0514 protein levels detected by Western blot. Levels of another LPXTG surface protein, Internalin-A (Inl-A), are shown for comparison. Densitometry analysis of bands is shown as numbers relative to the band detected in wild-type bacteria. Cell wall extracts of extracellular bacteria are concentrated 6-fold relative to those of intracellular bacteria. In intracellular bacteria, note the marked increase in relative levels of Lmo0514 protein (37.566 = 225), which contrasts with the ,6-fold increase in lmo0514 mRNA. doi:10.1371/journal.pgen.1004765.g001

Author Summary
Listeria monocytogenes has evolved to adapt to numerous environments, including the intracellular niche of eukaryotic cells. Small RNAs (sRNA) play important regulatory roles in changing environments, and are thus predicted to modulate L. monocytogenes adaption to the intracellular lifestyle. This study shows how the regulatory activity of an sRNA on a defined target is restricted to bacteria in the intracellular infection phase. This regulation relies on a long (234-nucleotide) 59-UTR that bears the sRNA-binding site present in a transcript variant that is upregulated by intracellular L. monocytogenes. The concomitant increase in both the target transcript containing the long 59-UTR and the sRNA, which is postulated to facilitate opening of the Shine-Dalgarno site, culminates in markedly higher protein levels in intracellular bacteria. The limited amounts of both the target and the regulator in extracellular bacteria ensure that production of this bacterial protein is confined mainly to the host rather than the non-host environment.
positions 226, 2104 and 2163. Two of these, 226 and 2163, were assigned as tentatively regulated by sigma A (s A ) and the third, at position 2104, as controlled by sigma B (s B ) [25] ( Fig. 2A). The activity of these putative promoters and the presence of the different transcripts were analyzed by RT-PCR on RNA isolated from L. monocytogenes grown extracellularly and from intracellular bacteria that colonized JEG-3 epithelial cells. lmo0514 transcripts with a long 59-UTR were detected specifically in intracellular bacteria ( Fig. 2A). To confirm these findings, rapid amplification of 59-cDNA ends (59-RACE) assays were used to map transcriptional start sites (TSS) of lmo0514 in bacteria grown extracellularly and in bacteria isolated from eukaryotic cells. These 59-RACE assays revealed two distinct TSS at positions 228 and 2 234 (Fig. 2B, C), and also confirmed expression of the long lmo0514 transcript by intracellular bacteria (Fig. 2C). Putative promoters for these TSS, which we termed P1 and P2, both bear bona fide 210 TATA boxes (Fig. 2B, C). The existence of two lmo0514 transcripts of different length was verified by northern blot (Fig. 3A), with sizes compatible with cotranscription of lmo0514 with the downstream gene lmo0515, which encodes a universal stress protein [26]. lmo0514-lm0515 cotranscription was verified by RT-PCR (Fig. S1). qRT-PCR assays confirmed that expression of the lmo0514 transcript variant with the long 234nucleotide (nt) 59-UTR was upregulated by ,12-fold in intracellular bacteria (Fig. 3B). These findings suggested that the specific induction of this mRNA variant with a longer 59-UTR in intracellular bacteria accounts for or contributes to the 6-fold increase in total lmo0514 mRNA (Fig. 1A). These data supported a model in which intracellular bacteria specifically upregulate expression from the P2 promoter, resulting in an lmo0514 transcript with a long 59-UTR. This assumption takes into account the different ratios between the two lmo0514 transcripts when L. monocytogenes colonizes the eukaryotic cell.
The lmo0514 long 59-UTR variant has a binding site for Rli27, an sRNA induced in intracellular bacteria The increased length of the lmo0514 transcript variant that is upregulated in intracellular bacteria prompted us to test whether the distinctive 234-nt 59-UTR is a target region for sRNAmediated post-transcriptional regulation. We used in silico analysis to search for putative non-coding RNAs in the L. monocytogenes reference strain EGDe [27] that could bind to this lmo0514 long 59-UTR. The targetRNA program (http://cs.wellesley.edu/ ,btjaden/TargetRNA2/) [28] gave a high score to a pairing between defined stretches of the lmo0514 234-nt 59-UTR and a sequence in the lmo0411-lmo0412 intergenic region. A gene in this region encodes an sRNA termed Rli27 that is upregulated by L. monocytogenes in the intestine of infected mice and in human blood, as shown by transcriptomics [17]; RNA-seq corroborated the expression of this sRNA [18]. Although Rli27 was identified as an sRNA induced in infection conditions [17], no further characterization of its function or targets was reported.
Genomic comparisons of pathogenic and non-pathogenic species are usually carried out to identify virulence genes,  [25]. Primers used to amplify the lmo0514 coding sequence are indicated (ORF, primers Lmo0514-F, Lmo0514-R, see Table S2), as well as two fragments of the 59-UTR of different lengths, amplicon ''a'' (254 nt), obtained with primers UTR-B and UTR-1R (Table S2) and amplicon ''b'' (134 nt), obtained with primers UTR-A and UTR-1R (Table S2). Reverse transcriptase-PCR assays showing upregulation in intracellular bacteria of an lmo0514 transcript isoform with a long 59-UTR. 16S rRNA was monitored as loading control. RNA was obtained from extracellular bacteria grown in BHI medium to exponential logarithmic phase (log), stationary phase (stat), and from intracellular bacteria. (B) 59-RACE assay showing a TSS at position 228 relative to the ATG site in extracellular bacteria. Colored bar indicates the position of the primer lmo0514-PE-1rv (Table S2) used for this reaction. (C) 59-RACE assay showing the production by intracellular bacteria of an lmo0514 transcript with a long 59-UTR derived from a TSS at position 2234 relative to the ATG site. Colored bar indicates the position of the primer lmo0514-PE-6rv (Table S2)  including sRNAs [18,29]. We analyzed the genomic region of L. monocytogenes containing rli27 and those of the non-pathogenic species L. innocua and L. welshimeri. In L. monocytogenes, rli27 is flanked by lmo0411 and lmo0412, two genes that map in the opposite DNA strand (Fig. S2), whereas in the L. welshimeri genome, the same intergenic region has a small ORF (lwe0373) that codes for a predicted protein of unknown function (Fig. S2). We nonetheless found that Rli27 is highly conserved in L. innocua (82% identity, Fig. 4A), in contrast with a previous report [17]. The extremely variable rli27 genomic region might thus have been shaped by gain and/or loss of genes during Listeria speciation. Apart from Listeria species, BLAST searches did not identify rli27 orthologs in other bacterial species. Rli27, identified as a 131-nt sRNA [14,18], is not predicted to encode any protein using the Small Open Reading Frame (ORF) tool in the ORF finder program (http://www.bioinformatics.org/sms2/orf_find. html).
Although the existence of Rli27 sRNA was inferred based on its detection by genomic and transcriptomic approaches, it has not yet been formally demonstrated. The presence of rli27 and its flanking genes in different strands ruled out the possibility that its detection by tiling arrays and RNA-seq analyses was due to untranslated regions of neighbor genes. rli27 has its own predicted transcription start site and Rho-independent terminator sequence ( Fig. 4A), and the respective promoter regions in L. monocytogenes and L. innocua showed no significant divergence (Fig. 4A). Northern blot assays using total RNA isolated from L. monocytogenes wild-type EGD-e and an isogenic Drli27 mutant strain demonstrated a small transcript consistent with the ascertained size of Rli27 (,130 nt) (Fig. 4B). Real-time qPCR showed that Rli27 expression is induced (,20-fold) in intracellular bacteria when compared with extracellular bacteria grown in rich medium to logarithmic or stationary phases (Fig. 4C). These findings indicate that Rli27 is a bona fide sRNA that is upregulated by L. monocytogenes inside eukaryotic cells.
Rli27 interacts physically with the 59-UTR specific to the lmo0514 long transcript Rli27 interaction with the lmo0514 59-UTR extends to several regions, although it shows a major predicted pairing region involving Rli27 nucleotides 1 to 21 (Fig. 5A, Fig. S3). We used electrophoretic mobility shift assays (EMSA) to assess the validity of this prediction. We generated in vitro wild-type versions of Rli27 and 59-UTR-lmo0514, together with variants of both RNA  and 210 predicted sites for the rli27 promoter and the rli27 itself (grey background) are highlighted. Nucleotide sequence in orange corresponds to the predicted terminator shared by rli27 and lmo0412. Note that rli27 is absent in L. welshimeri. (B) Northern blot assay performed with total RNAs isolated from bacteria grown in BHI medium to stationary phase. L. monocytogenes strains used included EGDe (WT) and the Drli27 mutant. 5S rRNA was used as loading control. (C) Realtime qPCR showing upregulation of Rli27 expression in intracellular bacteria. Bacteria were grown in BHI medium to exponential (log) or stationary phase, or collected from epithelial cells. Data derive from a minimum of three independent experiments. ***, P#0.001, Student's ttest. doi:10.1371/journal.pgen.1004765.g004 molecules bearing mutations in 3 nt (mut-1) or 14 nt (mut-3) important for pairing (Fig. 5B). Incubation of Rli27 and 59-UTR-lmo0514 wild-type molecules resulted in a duplex with low electrophoretic mobility (Fig. 5C). Conversely, combination of wild-type 59-UTR-lmo0514 with mutated Rli27 (either mut-1 or mut-3 variants), reduced duplex formation (Fig. 5C). Duplex formation was partially restored by combining mutations in Rli27 with compensatory mutations in 59-UTR-lmo0514 (Fig. 5C). Specificity of the Rli27-59-UTR-lmo0514 interaction was confirmed by lack of duplex formation after incubation of the 59-UTR-lmo0514 wild-type molecule with SbrA, an unrelated sRNA (Fig. 5D).

Rli27 interaction with the lmo0514 long 59-UTR is necessary to increase Lmo0514 protein levels in intracellular bacteria
To determine the biological relevance of the 59-UTR-lmo0514-Rli27 interaction in vivo, we analyzed the specific contribution of Rli27 binding to Lmo0514 protein upregulation in bacteria that infect eukaryotic cells. We generated a Drli27 strain and a second isogenic mutant, Drli27C2T, which bears an artificial strong terminator between the remaining rli27 sequences. This mutant was intended to avoid polar effects on the flanking genes lmo0411 and lmo0412 (Fig. S4); we also included mutants in these flanking genes, Dlmo0411 and Dlmo0412 [30]. In addition, we designed a qPCR assay specific for the lmo0514 long 59-UTR for comparison to the lmo0514 coding region. There were no notable differences among strains in the relative levels of the long 59-UTR region or the lmo0514 ORF (Fig. 6A). In contrast, Lmo0514 protein levels were ,2.5-to 3-fold lower in the cell wall of the two Rli27-lacking mutant strains isolated from the eukaryotic cell (Fig. 6B). This phenotype was complemented by overproduction of wild-type or mut1 versions of Rli27 from a plasmid (Fig. 6C, D). In contrast, when we tested mut3, the Rli27 mutant bearing 14 nt changes in the major region predicted to interact with the lmo0514 59-UTR (Fig. 5A), it did not restore Lmo0514 protein levels in intracellular  (Fig. 6D). Wild-type, mut1 and mut3 Rli27 versions were all produced by the plasmid at similar levels (Fig. 6C). These data showed that Rli27 interaction with the lmo0514 long 59-UTR was essential for induction of the protein in intracellular bacteria, and that elimination of the Rli27-lmo0514 59-UTR interaction interfered with the Lmo0514 protein increase while levels for the long transcript isoform remained unchanged. Our findings thus supported the need for Rli27 binding for efficient Lmo0514 translation. Control qPCR experiments in extracellular bacteria showed similar lmo0514 transcript levels in this mutant series (Fig. 6E), whereas there were no marked changes in Lmo0514 protein levels (Fig. 6F).
These in vivo experiments based on complementation assays with Rli27 variants supported a mechanism that involves Rli27 binding to the 59-UTR of the long lmo0514 transcript variant that is upregulated by L. monocytogenes inside eukaryotic cells. Such an interaction could promote translation, which would lead to increased Lmo0514 protein levels (Fig. 7).

Discussion
Given the unique architecture of the cell envelope in Grampositive bacterial pathogens, cell wall-associated proteins have essential functions in the interplay of these microorganisms with the host [31]. Despite the recognized importance of these proteins in infection, relatively few studies address the spatio-temporal regulation of the production of these proteins following host colonization. Obtaining this information is particularly challenging for Gram-positive pathogens such as L. monocytogenes or Staphylococcus aureus, which produce a large arsenal of surface proteins with distinct modes of association to the cell wall [31][32][33].
In this study of the Gram-positive bacterium L. monocytogenes, we identify sRNA-mediated regulation that acts on a cell wallassociated protein, Lmo0514, during the infection process. During the review process of this work, another report showed regulation of L. monocytogenes adhesin LapB by the multicopy sRNA LhrC, although this regulation was not studied in the context of infection [23]. Our data for lmo0514 also distinguish two transcript isoforms with 59-UTR of distinct length that are expressed differentially when the pathogen transits between non-host and host environments. These findings are consistent with a regulatory role for the sRNA Rli27, based on its exclusive binding to the lmo0514 long 59-UTR variant. This long 59-UTR is generated from a promoter, here termed P2, which must respond to environmental cues of the eukaryotic intracellular niche. The regulator itself, Rli27, is also upregulated by L. monocytogenes following entry into host cells. Transcriptional regulators of L. monocytogenes that operate in intracellular bacteria include the alternative sigma factor SigB and the Listeria-specific virulence regulator PrfA. Transcriptomic analyses in sigB and prfA mutants grown in laboratory media did not indicate lmo0514 as a gene regulated by these factors [34]; our results in intracellular bacteria were also negative (Fig. S5A). A yet undetermined regulator might thus be involved in enhancing transcription from the lmo0514 P2 promoter. Neither SigB nor PrfA appear to upregulate Rli27 in intracellular bacteria, as determined by real-time qPCR in sigB and prfA mutants isolated from infected epithelial cells (Fig. S5B).
Comparative transcriptomic studies of L. monocytogenes and L. innocua show that ,87% of the genes are transcribed with 59-UTR shorter than 100 nt, whereas there is a subgroup of approximately 100 genes with long 59-UTR (.100 nt) [18]; this subgroup includes virulence-related genes and genes with riboswitches [17]. Similar distribution of 59-UTR length was also described in the related model organism Bacillus subtilis [35]. About 80 genes shared by L. monocytogenes and L. innocua are produced with different-length 59-UTR [18], which might indicate differences in post-transcriptional regulation of these transcripts. Our data imply a third group of genes based on distinct transcript isoforms that differ in 59-UTR length. lmo0514 is a representative example, as it is expressed as two isoforms with 28-and 234-nt 59-UTR in extra-and intracellular bacteria, respectively. A close parallel is found in a recent work that analyzed sRNA RydC regulation of the Salmonella enterica cfa gene, which encodes a cyclopropane fatty acid synthase [36]. RydC selectively stabilizes the longer of two cfa transcript isoforms, which is associated to the activity of a distal promoter controlled by s A and a proximal promoter modulated by s B [36]. Unlike lmo0514, both cfa isoforms are expressed by S. enterica growing extracellularly in laboratory media. These observations indicate that transcript isoforms with distinct 59-UTR target platforms for sRNAmediated post-transcriptional regulation could profoundly influence protein production. It is noteworthy that long 59-UTRs are frequently associated with genes involved in pathogenesis [18,37].
An interesting feature predicted by the Mfold program is that Rli27 binding to the lmo0514 long 59-UTR could expose the Shine-Dalgarno site, in contrast to the occluded configuration predicted when this 59-UTR folds as single molecule (Fig. S6, S7). This led us to propose that Rli27 positively regulates Lmo0514 protein levels by altering the long 59-UTR conformation. This mechanism resembles that of the translational regulation of the rpoS transcript in Escherichia coli [38]. The Shine-Dalgarno site is blocked by a stem-loop in the rpoS 59-UTR, which is released by base pairing of three distinct Hfq-binding sRNA to the same region. Other paradigmatic cases in L. monocytogenes include the virulence regulator prfA, actA, and the hemolysin (hly) genes [25,[39][40][41]. Our hypothesis for lmo0514 implies that its 234-nt 59-UTR has considerable secondary structural complexity in the absence of Rli27. This assumption is consistent with the study by Wurtzel et al. [18], in which RNA-seq did not define the lmo0514 transcriptional start site, although 2018 such sites were mapped in the L. monocytogenes genome, which account for 88% of all annotated transcriptional units. Our tentative model (Fig. 7) also considers the lmo0514 transcript as 'low-efficiency' in terms of translation; there are marked differences in Lmo0514 protein levels in bacteria isolated from epithelial cells (.200-fold increase) that are not reflected at the transcript level. The secondary structure prediction for the short (28-nt) 59-UTR of the extracellular lmo0514 isoform also suggests probable occlusion of the Shine-Dalgarno site (Fig. S8). Further work is needed to clarify the extent to which such potential structural changes in the 59-UTR might explain Rli27-mediated regulation.
Our EMSA data infer direct Rli27-59-UTR-lmo0514 interaction, which was also relevant in vivo, based on data obtained with the Rli27-mut3 variant. This variant did not restore the Lmo0514 protein levels produced by intracellular bacteria (Fig. 6D). We did not obtain perfect complementation with compensatory mutations in the predicted interacting regions, which allows other interpretations. For example, the targetRNA program might have predicted an incorrect pairing site, pairing between the two molecules might require additional factors with a precise stoichiometry, or the lmo0514 transcript could undergo alternative post-transcriptional regulation; future work will address these possibilities.
We designed in vivo experiments to assess the lmo0514 long 59-UTR requirement in Lmo0514 protein production in the cell wall of bacteria located inside eukaryotic cells. We tested strains that bear chromosomal mutations in the lmo0514 long 59-UTR predicted interaction site or that lack most of the 59-UTR upstream of the P1 promoter 210 and 235 sites (Fig. S9A, S9B). Lmo0514 protein levels dropped markedly inside the eukaryotic cells for some these mutants, especially in that lacking the lmo0514 59-UTR (Fig. S9C, S9D). Nonetheless, lmo0514 transcript levels were affected in these mutants in both extra-and intracellular conditions (Fig. S9C, S9D). Due to the clear side effect of the mutations on transcription, these findings remained inconclusive.
In summary, our results demonstrate that Rli27 is a regulatory sRNA in L. monocytogenes, with an essential role as a positive regulator of the Lmo0514 surface protein during the intracellular infection cycle. We also provide evidence that the Rli27 regulatory role is directed to a transcript isoform that bears the binding site for this sRNA; in addition, we show that this isoform is specifically upregulated by intracellular bacteria. Further research will be necessary to determine how Rli27 might modify the secondary structure of the 59-UTR after binding, and whether such a role requires additional factors also probably upregulated in intracellular bacteria. Another challenge will be to identify the hostderived signal that triggers transcription from the P2 promoter in intracellular L. monocytogenes and the bacterial transcriptional factor responsible.

Bacterial strains and growth conditions
The L. monocytogenes strains of serotype 1/2a used here are isogenic to wild-type strain EGD-e [27] (listed in Table S1). For sRNA overexpression analyses, the rli27 wild-type allele was cloned in the pP1 plasmid [42] using Lmorli27-pP1-F and Lmorli27-pP1-R primers (Table S2). Relative expression of cloned sRNA was monitored by semi-quantitative RT-PCR using Lmorli27-F and Lmorli27-R primers (Table S2). L. monocytogenes strains were grown at 37uC in brain heart infusion (BHI) broth. For cloning, E. coli strains were grown in Luria Bertani (LB) broth at 37uC. When appropriate, media were supplemented with erythromycin (1.5 mg/ml) or ampicillin (100 mg/ml).

Construction of Rli27-defective L. monocytogenes mutants
To generate the Drli27 mutant strain, fragments of ,500-bp DNA flanking rli27 were amplified by PCR using chromosomal DNA of L. monocytogenes strain EGD-e and cloned into the thermo-sensitive suicide integrative vector pMAD [43] with primers Lmorli27-A, Lmorli27B, Lmorli27-C and Lmorli27-D (Table S2). Genes were deleted by double recombination as described [43], and deletion was verified by PCR. To generate the Drli27 mutant, we left 9 nt in the 59 end and 50 nt in the 39 end of the rli27 gene, to avoid interference with the lmo0412 terminator (shared with rli27) and the lmo0411 predicted promoter sequence (Fig. S4). This Drli27 mutation affected lmo0411 transcript levels slightly. A new deletion mutant was generated (Drli27C2T), which retains a 59 extended region of the predicted lmo0411 promoter, thus maintaining 21 nt in the 59 end and 50 nt in the 39 end of rli27 (Fig. S4). In addition, a strong artificial terminator sequence between the remaining rli27 sequences was introduced in the Drli27C2T mutant (Fig. S4). All deletions were confirmed by PCR and sequencing, using primers listed in Table S2.
Construction of L. monocytogenes mutants with chromosomal mutations in the lmo0514 59-UTR Three types of mutants were constructed with the following chromosomal mutations: i) changes in 3 nt of the long 59-UTR-lmo0514 to compensate the mutation in Rli27-mut1 (see Fig. S3, S9), ii) changes in 14 nt of the long 59-UTR-lmo0514 to compensate the mutation in Rli27-mut3 (see Fig. S3, S9), and iii) a 174-nt deletion upstream of the 210 and 235 sites of the P1 lmo0514 promoter (Fig. S9). These changes were generated by double recombination as described [43] and when required, using overlapping SOEing PCR. The oligonucleotide primers for these procedures included D0514_P2_A, D0514_P2_B, D0514_P2_C, D0514_P2_D, Mut0514pXG_1-overlap, Mut0514pXG_2-over-lap, Mut0514pXG_5-overlap and Mut0514pXG_6-overlap (Table S2).

Isolation of intracellular bacteria for RNA expression and proteomic analyses
Intracellular bacteria were collected from the human epithelial cell line JEG-3 at 6 h post-infection, as described [12]. For total RNA isolation, epithelial cells cultured in BioDish-XL plates (351040, BD Biosciences) at ,80% confluence (,5.6610 7 cells) were infected (30 min) with L. monocytogenes grown in BHI medium (37uC, overnight) in static non-shaking conditions. RNA was purified using the TRIzol reagent method [17]. For cell wall protein analysis, intracellular bacteria were obtained from JEG-3 cells cultured on four BioDish-XL plates and infected for 6 h [12]. Subcellular fractions containing protoplasts and peptidoglycanassociated proteins were obtained by mutanolysin treatment of intact bacteria as described [10,12], except that bacterial pellets were incubated for 5 h in lysis buffer (10 mM Tris HCl pH 6.9, 10 mM MgCl 2 , 0.5 M sucrose, 60 mg/ml mutanolysin, 250 mg/ml RNAse-A, 16 protease inhibitor).

Bacterial fractionation and western blot analysis
Subcellular fractions containing protoplasts and cell wallassociated proteins of L. monocytogenes grown at 37uC in BHI media were obtained as described [10]. A volume of protoplasts and the cell wall fraction was analyzed by SDS-PAGE followed by Western blot using B. subtilis RecA-specific rabbit polyclonal antibody (a gift of Dr. JC Alonso, Centro Nacional de Biotecnología-CSIC) and rabbit poyclonal sera to the L. monocytogenes LPXTG surface proteins Lmo0263 (InlH), Lmo0433 (InlA) and Lmo0514 [12]. RecA (for the protoplast fraction) and LPXTG proteins InlA and InlH (for the cell wall fraction) were used as loading controls. Goat anti-rabbit antibodies conjugated to horseradish peroxidase (Bio-Rad) were used as secondary antibodies. Proteins were visualized by chemoluminescence using luciferin-luminol reagents.

RNA preparation and reverse transcriptase PCR assays
Total RNA from extracellular bacteria grown to exponential (OD 600 ,0.2) and non-shaking stationary phase (OD 600 ,1.0) was prepared as described [11]. Oligonucleotides for RT-PCR assays were designed using Primer Express v3.0 (Applied Biosystems)(listed in Table S2). RNA was treated with DNase I (Turbo DNA-free kit, Ambion/Applied Biosystems) at 37uC for 30 min. RNA integrity was assessed by agarose-TAE electrophoresis. RT-PCR was performed using the one-step RT-PCR kit (Qiagen). Briefly, RT-PCR were carried out with 10 to 70 ng RNA (depending on the gene analyzed) in the following conditions: 50uC for 35 min, 95uC for 15 min, followed by 30 cycles (16 cycles for the 16S rRNA gene) of 94uC for 30 s, 55uC for 30 s, and 72uC for 1 min, and then an additional elongation step at 72uC for 10 min. The gene that encodes 16S rRNA was used as a housekeeping gene for all strains in all experimental conditions [44].

cDNA libraries and real-time quantitative PCR (qPCR)
For cDNA library construction, we used 1 mg of total DNA-free RNA and the High-Capacity cDNA Archive kit (Applied Biosystems) including a random hexamer mix. Reverse transcription was performed at a one-step run of 25uC for 10 min, 37uC for 2 h and 85uC for 5 min. Primers for qPCR were designed using Primer3 [45](listed in Table S2). qPCR was performed in a 10 ml final volume with 1 ng of the cDNA library as template, 500 nM of gene-specific primers and the Power SYBR Green PCR Master Mix (Applied Biosystems). Reactions and data analysis were carried out as described [46].

59-rapid amplification of cDNA ends (59-RACE)
59-RACE was performed as described [47], with minor modifications. To convert 59triphosphates to monophosphates, 15 mg DNA-free RNA, isolated from L. monocytogenes growing extracellularly at 37uC to stationary phase or from intracellular bacteria collected at 6 h post-infection of epithelial cells, was treated with 25 U tobacco acid pyrophosphatase (TAP) (Epicentre Technologies) at 37uC for 60 min in a total reaction volume of 50 ml containing 50 mM sodium acetate (pH 6.0), 1 mM EDTA, 0.1% b-mercaptoethanol, 0.01% (v/v) Triton X-100 and 80 U RNAsin (Promega). TAP-negative (TAP2) control RNA was processed in the same conditions in the absence of TAP. Following TAP treatment, RNA was phenol/chloroform-extracted and precipitated with sodium acetate and ethanol. Pellets were rinsed with 70% ethanol in DEPC-dH 2 O, then resuspended in 65 ml DEPC-dH 2 O; 29 ml of these TAP+ or TAP-treated RNA were combined with 5.5 ml 106 buffer, 120 U RNasin, 10% (v/v) dimethylsulfoxide, 70 U RNA ligase, 150 mM ATP and 150 ng RNA oligonucleotide adapter, in a total reaction volume of 55 ml. Samples were denatured (95uC, 5 min) and then chilled on ice. RNA adapter ligation was performed (17uC, 12 h). Following ligation, RNA was phenol/chloroform-extracted and converted to cDNA with a lmo0514-specific primer (Lmo0514-Pe-3rv) and the Thermoscript RT System (Invitrogen). Reverse transcription was performed in three cycles (55uC, 60uC and 65uC; 20 min each), followed by RNAseH treatment (37uC, 20 min). lmo0514 cDNA (2 ml) was amplified by PCR with oligonucleotides RaceIN and lmo0514-PE-1rv (30 cycles of 95uC for 15 s, 55uC for 30 s, and 72uC for 1 min), or with oligonucleotides RaceIN and lmo0514-PE-6rv in the same cycling conditions. PCR products were resolved on 2% agarose gels and bands of interest were excised and subcloned into pCR 2.1 TOPO-vector (Invitrogen). Plasmids containing inserts were purified using the QIAprep Spin Miniprep Kit (QIAgen) and sequenced.

Northern blot assays
To detect the sRNA Rli27 and the 5S rRNA, 15 mg total RNA were electrophoresed in a 6% polyacrylamide 8 M urea gel (1 h, 200 V in 16 TBE). RNA was transferred to a Hybond membrane (Amersham) for 2.5 h at 40 V in 0.56 TBE at 4uC and RNA was UV-crosslinked to the membrane. Membranes were pre-hybridized with UltraHyb buffer (Ambion; 65uC, 2 h) and hybridized with 10 6 cpm 32 P-labeled specific riboprobes (65uC, overnight). Membranes were washed with 26 SSC, 0.5% SDS and 16 SSC, 0.1% SDS and exposed to X-ray film. 5S rRNA was used as control [22]. A nonradioactive digoxigenin (DIG)-based RNA detection protocol was used for Northern blot analysis of lmo0514 and the 16S rRNA. Total RNA (1 mg for lmo0514 or 200 ng for 16S rRNA) was separated on a 1.5% agarose denaturing gel (2% formaldehyde, 16 MOPS), overnight capillary transferred to a Hybond membrane in 206 SSC, and UV-crosslinked. The membrane was prehybridized (68uC, 1 h) and then hybridized with DIG-labeled lmo0514 and 16S rRNA probes (68uC, overnight). Immunological detection of RNA was performed (DIG Northern starter kit; Roche) and exposed to X-ray film.

Electrophoretic mobility shift assays (EMSA)
Gel mobility shift assays were performed with 1.48 pmol in vitro-transcribed RNA corresponding to the lmo0514 59-UTR (nucleotides 2234 to 214 from the lmo0514 ATG codon) and increasing concentrations of in vitro-transcribed RNAs for Rli27 wild-type, Rli27-mut1 and Rli27-mut3. These in vitro-transcribed molecules included Rli27 nucleotides 1 to 131 plus an additional 60 nt at the 39 end, as designed for optimal amplification. We produced lmo0514 59-UTR variants with compensatory mutations in 3 nt (mut-1) or 14 nt (mut-3) for those generated in Rli27. Oligonucleotide primers used are listed in Table S2. We also generated an amplified molecule corresponding to RNA SbrA (Rli11) encompassed nucleotides 1 to 69 of the total of 71 nucleotides. The reaction was carried out in 10 ml of 16 binding buffer (20 mM Tris-acetate pH 7.6, 100 mM sodium acetate, 5 mM magnesium acetate, 20 mM EDTA) (37uC, 1 h). The binding reactions were mixed with 2 ml loading dye (48% glycerol, 0.01% orange G) and loaded on native 4% polyacrylamide gels, followed by electrophoresis in 0.56TBE buffer (200 V, 4uC). Gels were stained with Gel Red nucleic acid stain (Biotium) and photographed under UV transillumination with the GelDoc 2000 system (Bio-Rad).

Statistical and densitometry analyses
Statistical significance was analyzed with GraphPad Prism v5.0b software (GraphPad Inc.) using Student's t-test. A P value#0.05 was considered significant. For densitometry of bands obtained in western blots, we used ImageJ software (National Institutes of Health of USA [http://imagej.nih.gov/ij/]). Figure S1 lmo0514 is cotranscribed with the downstream gene lmo0515, which codes for a universal stress protein in L. monocytogenes [26]. PCR assays performed on reverse-transcribed RNA (cDNA) and genomic DNA (gDNA). C(-) refers to a control sample that lacks a template. Colors indicate relative position of the Lmo0514-F and 0515-R primers (Table S2). RNA and DNA were isolated from L. monocytogenes wild-type strain EGD-e (WT) grown to stationary phase (OD 600 ,1.0) at 37uC in BHI medium in non-shaking conditions. (TIF) Figure S2 Comparison of the rli27 region of L. monocytogenes EGD-e strain (Lmo) with the respective regions of non-pathogenic species L. innocua (Lin) and L. welshimeri (Lwe). Genomes were compared using the WebACT tool (http://www.webact.org/ WebACT/home). Red indicates similar genomic organization; blue indicates inversions. Orthologous genes are shown in same color. The rli27 gene of L. monocytogenes has no ortholog in L. welshimeri and is flanked by lmo0411 and lmo0412, two genes in the opposite DNA strand predicted to encode a protein similar to phosphoenolpyruvate synthase and a protein of unknown function, respectively. (TIF) Figure S3 Scheme of all predicted interaction sites between Rli27 and the lmo0514 59-UTR. Interactions were predicted using the targetRNA program (http://cs.wellesley.edu/,btjaden/ TargetRNA2/). Scheme shows the exact positions of the predicted interaction regions between the lmo0514 59-UTR and Rli27 as well as the mutant variants Rli27-mut1 and Rli27-mut3, with changes highlighted in yellow. Note that the hybridization energy is lower in the case of the Rli27 variants. Compensatory mutant variants of the 59-UTR-lmo0514 molecule (lmo0514-mut1, lmo0514-mut3) are also shown. (TIF) Figure S4 Genome region of the L. monocytogenes EGD-e strain bearing rli27 and its flanking genes, and the exact location of the deletions generated in the Drli27 and Drli27C2T mutants. Blue boxes represent the predicted 210 and 235 sites of the lmo0411 promoter. Note that a strong artificial terminator was introduced in Drli27C2T to avoid expression of the remaining Rli27-specific sequences. Both rli27 and lmo0412 genes share a Rhoindependent terminator. (TIF) Figure S5 lmo0514 and rli27 expression are not regulated by SigB or PrfA in extra-or intracellular bacteria. (A) qPCR data relative to lmo0514 obtained from total RNA isolated from extracellular bacteria grown in BHI medium to stationary phase (extracellular) or collected from epithelial cells (intracellular). Primers Utr0514_qPCR_F and Utr0514_qPCR_R were used. , 59-UTR-mut1 (3-nt change) and 59-UTR-mut3 (14-nt change). (C) Effect of these chromosomal mutations on Lmo0514 protein and lmo0514 ORF levels in extracellular bacteria grown in BHI medium to stationary phase. (D) Effect of these chromosomal mutations on Lmo0514 protein and lmo0514 transcript levels (differentiating production of the long 59-UTR isoform) in intracellular bacteria collected from epithelial cells. Note the marked decrease in Lmo0514 protein by the D59-UTR-lmo0514 and the 59-UTR-mut3 mutants in the intracellular niche of the eukaryotic cell. These mutants nonetheless have low lmo0514 transcript expression, probably due to side effects linked to loss of the 59-UTR region between the P2 and P1 promoters. (TIF)