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RNA. Jul 2006; 12(7): 1383–1396.
PMCID: PMC1484441

Identification of small Hfq-binding RNAs in Listeria monocytogenes

Abstract

The RNA-binding protein Hfq plays important roles in bacterial physiology and is required for the activity of many small regulatory RNAs in prokaryotes. We have previously shown that Hfq contributes to stress tolerance and virulence in the Gram-positive human pathogen Listeria monocytogenes. In the present study, we performed coimmunoprecipitations followed by enzymatic RNA sequencing to identify Hfq-binding RNA molecules in L. monocytogenes. The approach resulted in the discovery of three small RNAs (sRNAs). The sRNAs are conserved between Listeria species, but were not identified in other bacterial species. The initial characterization revealed a number of unique features displayed by each individual sRNA. The first sRNA is encoded from within an annotated gene in the L. monocytogenes EGD-e genome. Analogous to most regulatory sRNAs in Escherichia coli, the stability of this sRNA is highly dependent on the presence of Hfq. The second sRNA appears to be produced by a transcription attenuation mechanism, and the third sRNA is present in five copies at two different locations within the L. monocytogenes EGD-e genome. The cellular levels of the sRNAs are growth phase dependent and vary in response to growth medium. All three sRNAs are expressed when L. monocytogenes multiplies within mammalian cells. This study represents the first attempt to identify sRNAs in L. monocytogenes.

Keywords: Listeria monocytogenes, sRNA, Hfq protein, RNA-binding protein

INTRODUCTION

Small regulatory RNAs (sRNAs) have recently been identified in various organisms, including the pathogenic bacteria Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Vibrio cholerae, and Chlamydia trachomatis (Shimizu et al. 2002; Lenz et al. 2004; Mangold et al. 2004; Pichon and Felden 2005; Grieshaber et al. 2006). A number of these sRNAs have been implicated in the regulation of genes important for bacterial pathogenesis, for example, RNAIII in S. aureus, which controls the expression of virulence genes encoding exoproteins and cell-wall-associated proteins (Novick et al. 1993), and several redundant sRNAs in V. cholerae that regulate genes required for quorum sensing and virulence (Lenz et al. 2004). However, in most cases, the biological functions and mechanisms of action of bacterial sRNAs remain to be elucidated.

Most of our present knowledge on regulatory sRNAs derives from detailed studies of some of the ~70 sRNAs identified in Escherichia coli (for review, see Gottesman 2004; Storz et al. 2004). The majority of these act by controlling gene expression at the post-transcriptional level by base-pairing with complementary sequences in target mRNAs. Alternatively, the sRNAs interact with a regulatory protein, thereby modifying the activity of the protein. In E. coli, the RNA-binding protein Hfq modulates the activity of several sRNAs that act by base-pairing with target mRNAs (for review, see Valentin-Hansen et al. 2004). The Hfq protein binds directly to both sRNAs and target mRNAs and acts as an RNA chaperone to promote sRNA–mRNA duplex formation (Møller et al. 2002; Zhang et al. 2002, 2003; Geissmann and Touati 2004; Rasmussen et al. 2005). The binding of Hfq affects the structure of some, but not all, sRNAs and target mRNAs (Møller et al. 2002; Zhang et al. 2002; Geissmann and Touati 2004). Furthermore, Hfq protects many sRNAs from degradation, most likely by binding to RNase E cleavage sites within these sRNAs (Moll et al. 2003; Zhang et al. 2003). Hfq has been identified as an important virulence factor in several Gram-negative bacteria, including Yersinia enterocolitica, Pseudomonas aeruginosa, Brucella abortus, Legionella pneumophila, and Vibrio cholerae (Nakao et al. 1995; Robertson and Roop 1999; Sonnleitner et al. 2003; Ding et al. 2004; McNealy et al. 2005). In a recent study, we sought to clarify the role of Hfq in stress tolerance and virulence in the Gram-positive pathogen Listeria monocytogenes, which is the cause for listeriosis, a life-threatening disease with clinical symptoms such as febrile gastroenteritis, abortion, septicemia, and meningitis (Vazquez-Boland et al. 2001; Christiansen et al. 2004). L. monocytogenes is a facultative intracellular organism capable of invading a variety of host cells. Following invasion, the bacterium escapes from the phagocytic vacuole, multiplies within the host cell cytosol, and finally spreads to neighboring cells. Using a mutant that carries an in-frame deletion in hfq, we showed that Hfq is important for the tolerance to osmotic and ethanol stress and long-term survival under amino acid limitation (Christiansen et al. 2004). We also found that Hfq contributes to the pathogenicity of L. monocytogenes in mice. We have previously speculated that Hfq in L. monocytogenes could interact with sRNA and that the phenotype caused by lack of Hfq is caused by dysregulation of these sRNAs. The goal of the work presented here was to verify the existence of Hfq-binding sRNA molecules in L. monocytogenes.

In recent years, genome-wide screens for novel small non-coding RNAs have been performed in various bacteria using both computational and experimental approaches (for review, see Vogel and Sharma 2005). Small non-coding RNAs lack open reading frames, and computational approaches rely mainly on the genomic features of known sRNAs combined with comparative genomics (Argaman et al. 2001; Carter et al. 2001; Rivas et al. 2001; Wassarman et al. 2001; Pichon and Felden 2005). In contrast, experimental approaches usually involve the generation of cDNA libraries enriched for small RNA species (Tang et al. 2002, 2005; Vogel et al. 2003; Kawano et al. 2005), transcriptome analysis using microarrays (Wassarman et al. 2001; Tjaden et al. 2002), or coimmunopecipitations with RNA-binding proteins (Zhang et al. 2003; Tang et al. 2005). In the present study, we sought to identify sRNA molecules in L. monocytogenes by coimmunoprecipitation with Hfq followed by sequencing of the Hfq-binding RNA molecules. The identification and initial characterization of three novel sRNAs are presented.

RESULTS

Identification of sRNAs in L. monocytogenes that coimmunoprecipitate with Hfq

One of the successful methods used to identify novel small non-coding RNAs in E. coli relies on the RNA-binding property of the Hfq protein (Zhang et al. 2003). Hfq immunoprecipitation experiments have resulted in the identification of 20 novel Hfq-binding sRNAs in E. coli. To identify Hfq-binding sRNAs in L. monocytogenes, we used a similar approach, involving RNA sequencing of sRNA molecules that coimmunoprecipitate with Hfq.

For the coimmunoprecipitation experiments, wild-type and Δhfq mutant cells were grown in rich medium (BHI) or an improved minimal medium (IMM) under various stress conditions (see Materials and Methods). Extracts from these cells were prepared and subjected to immunoprecipitations using either Hfq antiserum or control preimmune serum. Next, total immunoprecipitated RNA was 5′-end-labeled and separated by PAGE (Fig. (Fig.1A).1A). On the basis of the autoradiogram, sRNA candidates were selected for purification using the following criteria: the sRNAs were absent in the immunoprecipitations from the Δhfq strain, in immunoprecipitations performed with preimmune serum, or in controls without serum. The purified sRNAs were subjected to RNA sequencing using the enzymes RNase T1 (specific for G residues), RNase U2 (specific for A residues), and RNase Bc (specific for C and U residues) (data not shown). Finally, BLASTN searches against the L. monocytogenes EGD-e genome sequence revealed the genomic location of the regions encoding the sRNAs. The candidates selected for further characterization were encoded primarily from intergenic regions (IGR) between annotated open reading frames (ORFs) and were expected to terminate at a Rho-independent terminator (a G-C-rich stem–loop followed by a U-run). The RNA sequencing step as well as the incorporation of the different criteria narrowed down the list of candidates and resulted in the prediction of three putative sRNAs (Table (Table1).1). One candidate, LhrA (Listeria Hfq-binding RNA), was isolated from samples prepared from stationary-phase cells grown in BHI medium and from cells subjected to iron or glucose starvation in IMM medium. Two candidates, LhrB and LhrC, were identified in immunoprecipitated samples prepared from cells grown in BHI medium at low temperature. To confirm the presence of these three Hfq-binding sRNAs in L. monocytogenes, Northern analysis was performed on total immunoprecipitated RNA from wild-type cells (Fig. (Fig.1B).1B). The Northern analysis was carried out using single-stranded RNA probes of 100–125 nt covering the sequence identified for each of the three sRNAs by RNA sequencing (Table (Table1).1). All three sRNAs were detected in samples treated with Hfq-specific serum (Fig. (Fig.1B).1B). The sRNAs were absent in control samples treated with the preimmune serum (Fig. (Fig.1B).1B). These results confirm the presence of the sRNAs in wild-type cells and their interaction with Hfq. From the Northern blot experiments, the sizes of the sRNAs were estimated to be ~250 bp (LhrA), 150 bp (LhrB), and 100 bp (LhrC), respectively.

FIGURE 1.
Identification of three novel Hfq-binding sRNAs in L. monocytogenes. (A) A representative picture of the gel fractionation of 5′-end-labeled RNAs isolated from coimmunoprecipitation experiments using wild-type or Δhfq mutant cells grown ...
TABLE 1
Summary of the identified small RNAs

Abundance and stability of the Hfq-binding sRNAs

To examine the expression pattern of the sRNA species in L. monocytogenes, we performed Northern blotting using RNA sampled under various growth conditions. In BHI medium, all three sRNAs were present in wild-type cells in a growth-phase-dependent manner (Fig. (Fig.2A).2A). LhrA was present throughout the growth phase, reaching a maximum level in the stationary phase. LhrB was abundant in the exponential growth phase, and low levels could also be detected in stationary-phase cells. LhrC was present in the exponential growth phase only. In the Northern analysis of LhrB, we also detected a larger transcript. The expression pattern of the larger transcript correlates with the expression pattern of LhrB. This finding suggested to us that LhrB may be produced from the processing of this larger transcript, or as a byproduct of a regulatory mechanism such as transcription attenuation. This issue is discussed in more detail below.

FIGURE 2.
sRNA levels during growth phase and under various stress conditions. Total RNA was isolated from wild-type (wt) and hfq mutant (Δhfq or Δ) strains grown at 37°C in (A) BHI to OD600 = 0.4, 0.8, 1.5, and 2.6; (B) IMM or IMM under ...

In IMM medium, the expression pattern of all three sRNAs in wild-type cells peaked during exponential growth (Fig. (Fig.2B).2B). A reduction in the glucose content of the IMM medium strongly diminished the LhrC level in wild-type cells in the exponential growth phase. Moreover, LhrC was induced at low temperature (Fig. (Fig.2B).2B). The levels of all three sRNAs were unaffected by the addition of 4% NaCl or 2% EtOH to exponential phase wild-type cells (data not shown). Thus, the expression pattern of the sRNAs clearly varies under different growth conditions, indicating that the sRNA levels are adjusted in response to specific signals from the environment.

In E. coli, a wide range of half-lives has been reported for the sRNAs (<2 min to >32 min), and the presence of Hfq is known to stabilize at least some of the sRNAs (Masse et al. 2003). To determine if Hfq had a similar effect on the Hfq-binding sRNAs in L. monocytogenes, we performed Northern blot experiments on RNA purified from the Δhfq mutant strain grown under various conditions (Fig. (Fig.2A2A,,B).B). The level of LhrA was strongly diminished in the Δhfq strain in comparison to the wild type, whereas the levels of LhrB and LhrC appeared to be unaffected by the absence of Hfq. To test whether the reduced expression of LhrA in the Δhfq mutant was due to increased sRNA instability, we performed Northern blotting using RNA sampled at different time points after the addition of rifampicin to exponential-phase cells grown in BHI medium (Fig. (Fig.3).3). The half-life of LhrA was estimated to be >32 min in the presence of Hfq and <2 min in the absence of Hfq, clearly demonstrating that Hfq has a stabilizing effect on LhrA. In contrast, LhrB and LhrC were rather unstable in both the wild-type strain and the Δhfq mutant strain (Fig. (Fig.3,3, half-life <2 min).

FIGURE 3.
Stability of LhrA, LhrB, and LhrC. Total RNA samples were prepared from wild-type (wt) and hfq mutant (Δhfq) strains before and after rifampicin treatment at the times indicated (in minutes). The RNA samples were analyzed by Northern blot using ...

Mapping the 5′- and 3′-ends of the sRNAs

To identify the 5′-ends of the sRNA molecules, we performed primer extension analysis. For all three sRNAs, 5′-ends were mapped to the regions located just upstream from the sequences identified by RNA sequencing of the immunoprecipitated RNAs (Fig. (Fig.4).4). The primer extension analysis does not differentiate between 5′-ends generated by degradation of an RNA and the primary 5′-ends of a transcript. To investigate this issue, we tested the corresponding regions for promoter activity. DNA fragments containing the putative promoter regions of LhrA, LhrB, or LhrC were fused to lacZ in the transcriptional fusion vector pTCV-lac. The resulting plasmids, plhrA′-lacZ, plhrB′-lacZ and plhrC′-lacZ, were introduced into the wild-type strain, and the level of β-galactosidase activity was determined. We observed that cells containing the srna′-lacZ fusion constructs displayed β-galactosidase activity during growth in BHI medium (Table (Table2).2). These data suggest that the 5′-ends mapped by primer extension represent the bona fide transcription start sites for the three sRNA genes.

FIGURE 4.
Mapping of the 5′-end of the sRNAs. Primer extension analyses were performed to determine the 5′-ends of (A) LhrA, (B) LhrB, and (C) LhrC. Lanes C, T, A, and G are sequencing ladders. The RNA used for these analyses was isolated from a ...
TABLE 2
Transcription analysis of LhrA, LhrB, and LhrC by using lacZ reporter fusions

To identify the 3′-ends of the sRNAs, we performed 3′-RACE. The 3′-ends of the sRNAs identified by this method were consistent with the transcript sizes estimated from the Northern analysis and the position of the 5′-ends (data not shown). At the 3′-end, the sRNAs contain putative stem–loop structures rich in G and C residues followed by a run of U residues, suggesting that all three transcripts terminate at Rho-independent terminators. By using the mfold program (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/), possible structures of the sRNA molecules were made (Fig. (Fig.5).5). The genetic organization and some genomic features of each of the RNAs are discussed in the following.

FIGURE 5.
Genetic organization and putative secondary structures of the sRNAs. (A) lhrA (black arrow) is located between the genes lmo2256 and lmo2258 (white arrows), and overlapping with lmo2257 (dotted arrow). Transcriptional terminators are indicated by lollipop ...

LhrA

LhrA consist of 268 nt and is encoded from a region overlapping with the gene lmo2257 (Fig. (Fig.5A).5A). The putative gene product of lmo2257 has no homology with other proteins in the databases. A coding sequence similar to lmo2257 has not been not annotated in Listeria innocua, although the corresponding region is 97% identical to lmo2257. There is an overlap of 4 nucleotides between lmo2257 and the upstream gene lmo2258, encoding a putative protein belonging to the ribulose-phosphate 3-epimerase family. Furthermore, lmo2257 overlaps with an annotated Rho-independent transcriptional terminator located just downstream from lmo2258. Based on these observations, we question whether lmo2257 encodes a protein product; however, experimental evidence is needed to resolve this issue. We investigated whether LhrA is capable of encoding a small ORF by searching for putative Shine-Delgarno sequences (AGGAGG) upstream from putative start codons (AUG, GUG, or UUG). No putative ORFs could be identified in LhrA.

Transcription of LhrA is initiated 80 nt downstream from the lmo2258 stop codon and terminates in a U-run following an extended stem–loop region corresponding to an annotated Rho-independent terminator in the EGD-e genome. A putative Pribnow box (TAAAAT) and a putative −35 hexamer (TTGCTT) are found upstream from the transcription start site. The start codon for lmo2256, which encodes a putative intracellular protease/amidase, is located 79 bp downstream from this terminator. By BLASTn homology searches, we found that LhrA is conserved among the Listeria species for which genomic sequence information is available (i.e., L. monocytogenes EGD, L. monocytogenes 4b F2365, L. monocytogenes 4b H7858, L. monocytogenes 1/2a F6854, L. innocua Clip 11,262). However, LhrA homologs were not identified in other bacterial species.

LhrB

LhrB is encoded from the IGR between inlC and infC and consists of 140 nt (Fig. (Fig.5B).5B). The inlC gene encodes Internalin C, a known virulence factor in L. monocytogenes. The infC gene encodes the translation initiation factor IF-3. LhrB is conserved between sequenced Listeria species, but was not identified in other bacterial species. Searches for putative ORFs within LhrB were unsuccessful, indicating that LhrB does not encode a protein factor. Transcription of LhrB is initiated 182 nt downstream from the inlC stop codon and is terminated in a U-run following an extended stem–loop region. A putative Pribnow box (TATAGT) and a putative −35 box (TTGACA) are located right upstream from the transcription start site. The terminator structure is located 91 bp upstream from the start codon of infC. This putative Rho-independent transcriptional terminator has not been annotated within the EGD-e genome, and we therefore tested whether this structure functions as a transcription terminator by fusing the entire lhrB gene, including the putative transcription terminator, to the lacZ gene in pTCV-lac, resulting in the plasmid construct plhrB-lacZ. In the plhrB′-lacZ construct, the putative LhrB transcription terminator is absent. The level of specific β-galactosidase activity in wild-type cells containing plhrB′-lacZ was threefold higher than the activity in wild-type cells containing the plhrB-lacZ fusion plasmid (Table (Table2).2). This result indicates that the majority of transcripts initiated at the lhrB promoter are terminated at the stem–loop structure. In the Northern analysis of LhrB, we observed a larger transcript in addition to the 140-bp LhrB transcript (Figs. (Figs.2A,2A, ,3).3). Using a probe directed against infC, we observed a band of the exact same size and expression pattern (data not shown). A primer extension analysis using an infC-specific primer revealed a 5′-end corresponding to the 5′-end of LhrB, suggesting that the large transcript initiates at the LhrB promoter and proceeds through the terminator structure and into infC (data not shown). These observations suggest that LhrB could be the byproduct of a transcription attenuation mechanism controlling the expression of infC. Transcription attenuators are characterized by the presence of mutually exclusive RNA-secondary structures in the leader sequence preceding a gene or operon, one of which could function as a transcription terminator. Depending on the RNA-secondary structure formed in the nascent transcript, the RNA polymerase may either terminate transcription or transcribe the downstream gene(s) (for review, see Winkler and Breaker 2005).

LhrC

LhrC is encoded from the 895-bp IGR between cysK and sul and consists of 114 bp (Fig. (Fig.5C;5C; see below). CysK is highly similar to cysteine synthases involved in cysteine metabolism, and Sul is highly similar to the dihydropteroate synthases of the folate biosynthesis pathway. LhrC is conserved between sequenced Listeria species, but was not identified in other bacterial species. No potential ORFs were identified in LhrC. The transcription start site for lhrC is located 725 bp downstream from the cysK stop codon and appears to end at a putative Rho-independent terminator located just 57 bp upstream from the sul start codon. As for LhrB, we tested whether transcription is terminated at this structure by fusing lhrC gene fragments to lacZ in pTCV-lac. For the plhrC-lacZ construct, a DNA fragment encoding the entire LhrC, including the putative transcription terminator, was fused to lacZ, whereas for the plhrC′-lacZ construct, the putative LhrC terminator structure is lacking. The expression from plhrC-lacZ is fivefold lower than the expression from the plhrC′-lacZ fusion (Table (Table2),2), indicating that the LhrC transcript terminates at this structure.

Since the terminator structure is located in very close proximity to the sul gene, it is tempting to speculate that LhrC is a byproduct of an attenuation mechanism controlling the expression of sul and downstream genes. However, an extended Pribnow box (TG-N-TATAAT) is present in the 57-bp region between lhrC and sul, indicating that sul could be transcribed independently of LhrC (data not shown). Furthermore, several observations suggest a far more complex role for LhrC in L. monocytogenes. By inspection of the cysK-sul IGR, three repeats nearly identical to lhrC were found upstream from lhrC, and a fifth copy was identified in the IGR between lmo0946 and lmo0947 (Fig. (Fig.5C).5C). Notably, the fifth LhrC homolog is encoded in the opposite direction of its downstream gene lmo0947. A sequence alignment of LhrC and the four LhrC homologs, including their putative promoter regions, revealed a very high sequence similarity between the five copies (Fig. (Fig.5D).5D). How can we be sure that the sRNA identified in the coimmunoprecipitation experiment is encoded from lhrC and not from the four other copies? When sequencing the sRNA molecule isolated by coimmunoprecipitation with Hfq, an A residue was found at the position corresponding to the A at +16 in LhrC. The four other sequences contain G residues at the corresponding position (Fig. (Fig.5D).5D). This observation shows that the identified sRNA is encoded from lhrC. LhrC was isolated by coimmunoprecipitation using RNA purified from cells grown at 4°C, suggesting that the sRNA encoded from lhrC is most prominent under these conditions. The probes and primers used in the Northern blot and primer extension analyses recognize all five LhrC homologs, and we expect that the bands observed in the expression studies represent a pool of the LhrC homologs. We note that the multiple bands observed in the primer extension analysis of LhrC, indeed, could reflect that this analysis was performed on a mixed population of LhrC homologs (Fig. (Fig.44).

L. monocytogenes Hfq binds to the sRNAs in vitro

The coimmunoprecipitation experiment showed that L. monocytogenes Hfq interacts with LhrA, LhrB, and LhrC in vivo. In E. coli, Hfq binds preferentially to unstructured A/U-rich sequences located in close proximity to more structured regions of the RNA (Møller et al. 2002; Zhang et al. 2002; Geissmann and Touati 2004). According to the proposed secondary structures of the sRNAs (Fig. (Fig.5),5), all three sRNAs identified in L. monocytogenes contain unstructured A/U-rich sequences near stem–loop structures, suggesting that Hfq binds directly to the sRNA molecules. To learn more about the interaction between L. monocytogenes Hfq and the sRNAs, we performed gel mobility shift assays using purified L. monocytogenes Hfq protein (Fig. (Fig.6).6). The binding of Hfq to the sRNAs produces several major shifts, which are believed to reflect the binding of first one and then additional equivalents of Hfq hexamers (Fig. (Fig.6).6). The gel shift experiments revealed that Hfq binds to LhrB with a higher affinity than to LhrA and LhrC, confirming that Hfq binds directly, but with different affinity, to the three sRNAs (Fig. (Fig.66).

FIGURE 6.
Binding of Hfq to the sRNAs tested by in vitro gel mobility shift analysis. 32P-labeled in vitro transcripts of (A) LhrA, (B) LhrB, or (C) LhrC was incubated with increasing amounts of purified Hfq protein to allow complex formation. The binding reactions ...

sRNA expression by L. monocytogenes during infection of mammalian cells

During infection of a host organism, L. monocytogenes enters and multiplies within the intracellular environment of a variety of mammalian cells. To investigate the expression of sRNAs in L. monocytogenes within mammalian cells, we used RT-PCR, a technique that has been used previously to study the expression of virulence genes and stress tolerance genes during infection (Bubert et al. 1999; Gahan et al. 2001). Wild-type L. monocytogenes was used for infection of the mammalian cell line HepG2. Following entry of L. monocytogenes into HepG2 cells, the bacteria escape from the vacuole and start to multiply within the host cytosol, resulting in an ~100-fold increase in the number of intracellular bacteria (Fig. (Fig.7A).7A). At various time points during the infection, cells were lysed and total RNA was purified for RT-PCR analysis (Fig. (Fig.7B).7B). The RT-PCR experiment illustrated that the sRNAs were present at all time points during the exponential growth of L. monocytogenes within the host cytosol. Thus, all three sRNAs are produced by L. monocytogenes when the pathogen multiplies within the intracellular environment of HepG2.

FIGURE 7.
In vivo detection of sRNAs. A cell monolayer of the epithelial cell line HepG2 was infected with wild-type L. monocytogenes EGD. After 1 h of infection, cells were incubated for one-half hour in the presence of gentamycin to kill extracellular bacteria ...

DISCUSSION

Here, we present the identification and preliminary characterization of three novel sRNAs in L. monocytogenes. Prokaryotic sRNAs were originally defined as small RNA molecules encoded from independent genes bordered by promoters and terminator structures located in the intergenic regions (Zhang et al. 2003; Vogel et al. 2003; Gottesman 2004). At present, the definition has evolved also to include small RNA molecules derived from 5′- or 3′-untranslated regions (UTRs) of mRNAs or even from inside the protein-coding regions (Vogel et al. 2003; Kawano et al. 2005). The gene encoding the sRNA LhrA in L. monocytogenes overlaps with the open reading frame lmo2257; however, it remains to be determined whether a protein is actually encoded from lmo2257. Owing to the annotation of lmo2257, LhrA would most likely be missed in computational-based searches for small RNAs, since these approaches favor the identification of sRNAs encoded from intergenic regions. Our finding of an sRNA encoded from an annotated open reading frame emphasizes the importance of using experimental-based approaches for the identification of sRNAs.

LhrA displays several features that are characteristic for regulatory sRNAs in E. coli. One of the major groups of E. coli sRNAs controls gene expression via base-pairing with target mRNAs in an Hfq-dependent fashion (Gottesman 2004; Valentin-Hansen et al. 2004). Hfq has been shown to bind to sRNAs and stimulate their pairing to target mRNAs. In our studies, we found that LhrA is highly abundant in the stationary growth phase, like many of the sRNAs identified in E. coli (Argaman et al. 2001; Wassarman et al. 2001; Vogel et al. 2003). Furthermore, Hfq binds to LhrA in vivo and in vitro and clearly increases the stability of the sRNA. We find it likely that LhrA is a regulatory RNA, which controls gene expression at the post-transcriptional level by base-pairing with a target mRNA. Work is currently in progress in our laboratory to further elucidate the function of LhrA in L. monocytogenes.

The LhrB sRNA appears to originate from a transcription attenuation mechanism controlling the expression of the downstream gene infC. In E. coli, sRNAs originating from 5′-UTRs of mRNAs have been identified in several cloning-based screens for sRNAs (Vogel et al. 2003; Kawano et al. 2005). Many of the 5′-UTR-derived transcripts were found to overlap with regions known to be required for transcription attenuation. Genes controlled by transcription attenuation often encode proteins involved in biosynthetic pathways providing vitamins, purines, lysine, or other amino acids. The regulatory strategy of transcription attenuation relies on the binding of small metabolites to the mRNA leader transcripts, which favors the formation of either termination or antitermination structures (for review, see Winkler and Breaker 2005). tRNA-, protein-, and ribosome-mediated transcription attenuation mechanisms have been described in various bacteria as well. Intriguingly, it has been suggested that the 5′-UTRs could have independent functions in the cells as well, such as binding and storage of the molecules interacting with the mRNA (Vogel et al. 2003; Kawano et al. 2005). The infC gene in Gram-positive bacteria, including L. monocytogenes, was recently identified in a computer-based search for all of the genes in 180 fully sequenced bacterial genomes likely to be controlled by attenuation (Merino and Yanofsky 2005). The attenuator-prediction procedure applied by Merino and Yanofsky was based on the identification of two mutually exclusive RNA-secondary structures in the sequences preceding the coding regions present in these genomes, one of which could function as a transcription terminator. Indeed, our experimental observations support the prediction of a transcription attenuator in the sequence preceding infC in L. monocytogenes. In E. coli, the InfC protein has been found to be an essential part of the translation apparatus (Laursen et al. 2005). InfC is known to be involved in the recycling of ribosomal subunits, and it discriminates against the use of unusual start codons; a feature that appears to be an important element in the autoregulation exerted by InfC on its own expression. We note that L. monocytogenes InfC is translated from the unusual start codon AUU, indicating that the autoregulatory mechanism is conserved. Importantly, E. coli infC does not appear to be regulated by a transcriptional attenuation mechanism (Merino and Yanofsky 2005), suggesting that this regulatory strategy could be specific to infC in Gram-positive bacteria. To understand how the balance between the formation of termination and antitermination structures in the 5′-UTR is controlled, an important task now is to identify the nature of the signal(s) that affect the formation of the RNA-secondary structures within the 5′-UTR of the infC mRNA. Interestingly, we identified LhrB as an Hfq-binding sRNA, suggesting a role for this RNA-binding protein in the transcription attenuation mechanism. Hfq is known to affect a variety of physiological processes in bacteria through its RNA-binding activity (for review, see Valentin-Hansen et al. 2004). In E. coli, Hfq's role as an RNA chaperone is well-documented, and it is therefore tempting to speculate that the binding of Hfq to the 5′-UTR of infC mRNA under certain conditions could influence the RNA-secondary structure, and thereby support the formation of either the termination or antitermination structures. Alternatively, the binding of Hfq to infC mRNA could mediate the binding of additional regulatory factors, such as a regulatory sRNA, which may be induced under specific growth conditions. A comparison of the levels of LhrB and infC mRNA in the presence and absence of Hfq did not reveal an effect of Hfq on the switch between the termination/antitermination structures, at least not under the growth conditions tested here (i.e., BHI medium) (see Figs. Figs.2A,2A, ,3).3). In future work, we will focus on the putative role of Hfq in the expression of infC under various growth conditions.

The gene encoding LhrC sRNA is present in five copies at two different locations in the genome of L. monocytogenes EGD-e. Multiple copies of sRNAs have recently been observed in other bacteria as well, including S. aureus, P. aeruginosa, and V. cholerae (Lenz et al. 2004; Wilderman et al. 2004; Pichon and Felden 2005). In P. aeruginosa, two tandem sRNAs are involved in iron homeostasis (Wilderman et al. 2004). The two sRNAs are highly homologous, and deletion of both copies is required to affect the iron-dependent regulation of their target genes in P. aeruginosa (Wilderman et al. 2004). In V. cholerae, functional studies have provided evidence that four sRNA homologs are involved in the regulation of quorum sensing (Lenz et al. 2004). Together with Hfq, these four sRNAs control the destabilization of the luxR mRNA, encoding the quorum-sensing regulatory protein LuxR. The four sRNAs are clearly functionally redundant, since the simultaneous deletion of all gene copies is required in order to observe a mutant phenotype (Lenz et al. 2004). The reason for this functional redundancy is not yet known; however, the presence of multiple sRNAs could allow the fine-tuning of target gene expression in response to multiple signals from the environment. The functional role(s) of the five LhrC homologs in L. monocytogenes is the subject of ongoing investigations in our laboratory.

This report represents the first attempt to identify sRNAs in L. monocytogenes. The success of the immunoprecipitation approach relies on the ability to capture and purify sRNAs with a strong interaction to Hfq. In our approach, we identified the Hfq-binding sRNAs by RNA sequencing, which requires relatively large quantities of RNA. It is known that some sRNAs are expressed only under specific growth conditions or at low levels, making the sRNAs difficult or even impossible to detect under standard growth conditions (Wassarman et al. 2001). More sensitive detection methods, such as the microarray approach applied for the identification of Hfq-binding sRNAs in E. coli, would most likely increase the outcome of the coimmunoprecipitation experiment (Zhang et al. 2003). Alternative methods could be successful as well, such as the generation of specialized cDNA libraries (Vogel et al. 2003; Kawano et al. 2005). We expect that use of alternative methods could support the future discovery of additional sRNAs in L. monocytogenes.

MATERIALS AND METHODS

Bacterial strains and growth media

The L. monocytogenes EGD serotype 1/2a strain and the EGDΔhfq mutant strain (Christiansen et al. 2004) were routinely grown at 37°C with shaking in brain heart infusion media (BHI, Oxoid), Luria-Bertani (LB) medium, or improved minimal media (IMM) (Phan-Thanh and Gormon 1997). When required, erythromycin or kanamycin was added to final concentrations of 5 μg/mL and 50 μg/mL, respectively.

Coimmunoprecipitation of sRNA with α-Hfq

For identification of small regulatory RNAs in L. monocytogenes, coimmunoprecipitations were performed with Listeria α-Hfq serum and total cellular extracts of EGD and Δhfq strains. Immunizing rabbits with peptides (Ross-Petersen A/S) created the Listeria α-Hfq serum identical to the N terminus (MKQGGQGLQDYYLNQ) and C terminus (TFSPQKNVALNPDAE) of the Listeria Hfq protein. The specificity of the antibody was confirmed by Western blot analysis on protein extracts from wild-type and hfq mutant strain (data not shown). The coimmunoprecipitations were performed by incubating the α-Hfq serum (0, 20, and 40 μL) or control preimmune serum (40 μL) with protein A Sepharose beads (~20 μL beads; Amersham) in 500 μL of TMK buffer (20 mM Tris-acetate at pH 7.25, 10 mM Mg-acetate, 200 mM K-glutamate, and 1 mM DTT) in 2-mL flat-bottom Eppendorf tubes overnight at 4°C with gentle shaking. After five washes with 1 mL of TMK buffer, the serum-protein A Sepharose complex was incubated with 500 μL of cleared lysate for 2 h at 4°C. The lysate was prepared from 60 mL of stationary-phase or 120 mL of mid-log-phase culture bacteria grown in BHI to exponential or stationary phase and exposed to 4% NaCl, 2% EtOH, or 48°C for 20 min, 4°C for 2 h, or grown in IMM; IMM containing 0.1% glucose (normal concentration in IMM: 2.0%); IMM containing 0.002% Fe-citrat (normal concentration in IMM: 0.88%); or IMM containing 0.002% L-leucine, 0.002% DL-isoleucine, 0.002% DL-valine, 0.002% DL-methionine, 0.002% L-arginine, 0.002% L-tryptophan, 0.002% L-phenylalanine, and 0.002% L-histidine (normal concentration in IMM: 1%). The cells were harvested and resuspended in 3 mL of lysis buffer (20 mM Tris-HCl at pH 8.0, 150 mM KCl, 1 mM MgCl2, and 1 mM DTT) containing 10 units of RNase inhibitor (RNAguard; Promega) and lysed by sonication. Following the incubation, the immunoprecipitated complexes were washed five times with 1.5 mL of TMK buffer. The sRNAs were phenol/chloroform extracted from the protein A Sepharose beads and ethanol-precipitated. Dried sRNAs were resuspended in 8 μL of H2O, 1 μL of dephosphorylation buffer, and 1 μL of calf intestine alkaline phosphatase (Roche) and allowed to incubate for 1 h at 50°C for dephosphorylation to proceed. The sRNAs were again phenol/chloroform-extracted. Following, the dephosphorylated sRNAs were 5′-end-labeled with T4 Polynucleotide kinase as described by the manufacturer (New England Biolabs) using 0.5 μL of γ-32P-ATP (10 mCi/mL; Amersham) at 37°C for 1 h. The reaction was stopped by addition of 2 μL of Stop formamide loading buffer (95% formamide, 20 mM EDTA, 0.05% XC, 0.05% BPB), and the samples were incubated at 95°C for 2 min and placed on ice. Afterward, the samples were run on a 10% urea polyacrylamide gel at 10 W for 2 h. The gel was then exposed to autoradiography at −80°C for 2–4 h or overnight. Following development of the film, sRNA candidates were cut out of the gel using the film as pattern, and sRNAs were extracted from the gel slices with extraction buffer (250 mM NaOAc at pH 6.0, 1 mM EDTA) and phenol overnight. The sRNA candidates were sequenced by enzymatic RNA sequencing (Donis-Keller et al. 1977) with the RNases T1, U2, and Bc. The RNase digests were run together with a basic hydrolytic reaction on a 20% polyacrylamide gel. The gel was wrapped in Vita-wrap and exposed to autoradiography at −80°C. The obtained sequences were BLASTed against the L. monocytogenes EGD-e genome database (http://genolist.pasteur.fr/ListiList/) to localize the genomic position of the genes encoding the sRNAs. Twenty-four bands, corresponding to 24 putative Hfq-binding RNAs of various sizes, were cut out and analyzed by enzymatic RNA sequencing. Eight candidates were successfully sequenced. Two candidates corresponded to fragments of 23S rRNA, which were not further analyzed in the current study. The six remaining candidates were encoded from putative intergenic regions. All six candidates appeared to terminate at putative Rho-independent transcription terminators. Unfortunately, the RNA sequences of 16 RNA samples were illegible. In these samples, the amounts of RNA were either too low or contained multiple RNA species of similar sizes, which made it impossible to identify the individual RNAs by RNA sequencing.

Northern blotting

Total RNA from L. monocytogenes was prepared by using the hot acid-phenol procedure (Podbielski et al. 1995). Total nucleic acid concentrations and purity were estimated using absorbance readings (260 nm/280 nm) on an Ultraspec II spectrophotometer (LKB Biochrom). Northern blot analyses were carried out as described elsewhere (Franch et al. 1997). Briefly, RNA samples containing equal amounts of RNA (10 μg) and similar levels of 16S RNA were denatured for 3 min at 96°C in loading buffer containing 96% formamide, separated on 8% urea polyacrylamide gels, and transferred to a Zeta-probe membrane by capillary blotting. The total RNA extracts were prepared from cells growing in BHI medium to stationary phase or from exponential-phase cells treated with 4% NaCl or 2% EtOH for 20 min, incubated at 4°C for 2 h, or from cells grown to OD600 = 0.6 or 2.6 in IMM medium adjusted to contain low amounts of glucose, FeCitrat, or amino acids (described above). For detection of sRNA candidates, the membranes were hybridized with 32P-labeled RNA probes, which were generated by in vitro transcription with T7 RNA polymerase on PCR fragments constructed to contain the T7 promoter, and exposed to autoradiography. The transcription mixtures (20 μL) contained 1μg of PCR-generated DNA fragment; 5× transcription buffer (Promega); 10 mM DTT; 500 μM each ATP, GTP, UTP and 12 μM CTP; 50 μCi of α-[32P]-CTP, 32 units of RNase inhibitor (RNAguard; Promega); and 10 units of T7 RNA polymerase (Promega). The in vitro transcription was carried out at 37°C for 120 min followed by treatment with 1 unit of DNase (Promega) for 15 min. Before use, the probe was heated to 80°C for 2 min. For generating the LhrA probe, the primers lhrA-8 and lhrA-9 were used, for the LhrB probe the primers lhrB-1 and lhrB-2, and for the LhrC probe the primers lhrC-1 and lhrC-2 were used. As RNA markers, in vitro transcripts on PCR fragments created from primers lhrA-8 and lhrA-10 resulted in a 300-bp marker, and PCR fragments created from primers lhrA-8 and lhrA-11 resulted in a 100-bp marker (Table (Table33).

TABLE 3
PCR primers used in this study

Half-life determination

Cultures were grown in BHI medium at 37°C to an OD600 of 0.4 and treated with rifampicin (10 μg/mL). At the indicated time points, 40-mL samples were collected by centrifugation at 0°C. Total RNA purification and Northern blot analysis were performed as described elsewhere.

Primer extension analysis

Primer extensions were performed as previously described (Christiansen et al. 2004). Briefly, the primer extensions were performed using 15 μg of total RNA per reaction. 5′-32P-labeled primers (LhrA: lhrA-22; LhrB: lhrB-3; LhrC: lhrC-1) (Table (Table3)3) were used for detection of sRNA transcription start sites. Total RNA was prepared using the hot acid phenol procedure as described above from wild-type cell culture grown in BHI to OD600 = 0.8. PCR fragments created by the primers lhrA-1 and lhrA-2, lhrB-5 and lhrB-6, and lhrC-3 and lhrC-4 (Table (Table3)3) were used as templates in DNA sequencing reactions.

Constructions of promoter-lacZ fusions and β-galactosidase assays

DNA fragments containing regions of lhrA, lhrB, and lhrC were amplified by PCR. For a truncated lhrA fragment, lhrA′, a 109-bp fragment (ranging from position −100 to position +9 relative to the LhrA transcription start site) was amplified using primers lhrA-12 and lhrA-19 (Table (Table3).3). Additionally, a fragment containing full-length lhrB, including the transcriptional terminator, was amplified using primers lhrB-10 and lhrB-18, giving a 326-bp fragment (ranging from position −158 to position +168 relative to the lhrB transcription start site) (Table (Table3).3). For the truncated lhrB fragment, lhrB′, a 186-bp fragment (ranging from position −158 to position +28 relative to the lhrB transcription start site) was amplified using primers lhrB-10 and lhrB-11 (Table (Table3).3). Furthermore, a fragment containing full-length lhrC, including the transcriptional terminator, was amplified using primers lhrC-3 and lhrC-8, giving a 253-bp fragment (ranging from position −134 to position +119 relative to the lhrC transcription start site) (Table (Table3).3). For the truncated lhrC fragment, lhrC′, a 202-bp fragment (ranging from position −134 to position +68 relative to the lhrC transcription start site) was amplified using primers lhrC-3 and lhrC-7 (Table (Table3).3). To construct plasmids plhrA′-lacZ, plhrB′-lacZ, and plhrC′-lacZ, containing the truncated sRNA gene fragments fused to lacZ, and plasmids plhrB-lacZ and plhrC-lacZ, containing the full-length sRNA gene fragments fused to lacZ, the PCR fragments were digested with EcoRI and BamHI and cloned into EcoRI–BamHI-digested pTCV-lac (Poyart and Trieu-Cuot 1997). Cells grown in BHI medium were harvested at OD600=0.6. β-Galactosidase assays were performed as previously described in Christiansen et al. (2004). The specific activity of β-galactosidase was calculated as follows: [(OD420 of the reaction mixture)−(OD550 of the reaction mixture)]/[(reaction time in minutes)×(OD600 of cells used in the reaction mixture)]. The background activity corresponding to the specific β-galactosidase activity of the empty vector pTCV-lac was subtracted from the specific β-galactosidase activity of the promoter-lacZ constructs. The presented specific β-galactosidase activities are the averages of three independent experiments in which the observed variations did not exceed 10%.

3′-RACE

3′ rapid amplification of cDNA ends (RACE) assays were performed according to the manufacturer's (Invitrogen) instructions on 5 μg of total RNA isolated from EGD wild type grown in BHI to stationary phase in the case of LhrA and to exponential phase (OD600=0.8) for LhrB and LhrC. sRNAs were ligated to the RNA adapter, and reverse transcription was performed to create cDNA. After RNase H treatment, cDNA was amplified by PCR with a primer complementary to the adapter and a gene-specific primer. The gene-specific primers used were lhrA-21, lhrB-8, and lhrC-3 in combination with the complementary adapter primer (Table (Table3).3). The amplification products were cloned into the plasmid vector LITMUS 28i (New England Biolabs, Inc.), and the 3′-ends were identified by DNA sequencing.

Purification of Hfq

The intein system (Impact-CN; New England Biolabs) was used for Hfq purification. The L. monocytogenes hfq was amplified by PCR using oligonucleotides HfqN and HfqC as primers (Table (Table3)3) and EGD chromosomal DNA as template. PCR products were digested with SapI and cloned into SapI–SmaI-digested pTyb11. Protein purification was done according to the manufacturer's recommendations but using the strain BL21hfq (T. Møller, unpubl.). The lysis/wash buffer used was 20 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and 1 mM EDTA. Finally, the protein was dialyzed against 50 mM Tris-HCl (pH 7.5), 1 mM EDTA. The amino acid sequence of the Hfq protein was analyzed by mass spectrometry to confirm the protein purification. The concentration was determined by the same procedure to 8.33 μM Hfq monomer.

Gel mobility shift assay

In vitro transcripts of sRNAs for gel mobility shifts were generated by in vitro transcription with T7 RNA polymerase on PCR fragments constructed to contain the T7 promoter. For in vitro transcription, PCR fragments were amplified with primers lhrA-17 and lhrA-21, lhrB-8 and lhrB-9, and lhrC-8 and lhrC-9 (Table (Table3)3) containing the transcription start site and the terminator, giving fragments of 273 bp, 143 bp, and 118 bp, respectively. The transcription mixtures (50 μL) contained 0.2 μg of the PCR-generated DNA fragment; 5× transcription buffer (Promega); 10 mM DTT; 500 μM each ATP, GTP, UTP, and 64 μM of CTP; 20 μCi of α-[32P]CTP; 32 units of RNase inhibitor (RNAguard; Promega); and 10 units of T7 RNA polymerase (Promega). The transcription was carried out at 37°C for 60 min and followed by purification from a 5% urea-polyacrylamide gel as previously described. The binding assays were performed by mixing binding buffer (500 mM KCl, 100 mM HEPES, 5 mM DTT), 1 μg of tRNA (corresponding to a 12.500-fold molar excess of competitor RNA), purified Listeria Hfq, and 0.005 pmol of labeled sRNA; they were incubated for 20 min at 37°C and transferred to ice for 10 min. The binding reactions (10 μL) were mixed with 5 μL of loading dye (15% glycerol, 0.15% bromophenol blue) and analyzed on a 5% nondenaturing polyacrylamide gel in TBE buffer at 200 V at 4°C for 1.5 h. Subsequently, the gel was dried and subjected to autoradiography.

Intracellular detection

The human liver epithelial cell line HepG2 (ATCC no. HB-8065) was propagated in Dulbecco's modified Eagle's medium (D-MEM with 4500 mg/L glucose, L-glutamine, and pyruvate; GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen). Cells were incubated in the presence of 5% CO2 at 37°C. For the infection assays, the concentration of cells were adjusted to 5 × 105 cells/mL and grown in tissue culture flasks to monolayer (20 h at 37°C). Bacteria were grown overnight, washed, and adjusted to ~1 × 108 colony-forming units (CFU) per milliliter of cell culture medium. Bacteria were added to each flask, resulting in a multiplicity of infection of ~200 bacteria per cell for infection of HepG2, and were incubated at 37°C. After 1 h of incubation, the infected monolayer was washed twice with phosphate-buffered saline (PBS) and overlaid with cell culture medium containing 25 μg/mL gentamycin to kill extracellular bacteria. After one-half hour, the monolayer was washed twice with PBS and lysed with 0.1% Triton X-100. The number of bacteria released was expressed in colony forming units per milliliter (CFU/mL) by plating appropriate dilutions on BHI agar plates. For RNA purifications, the bacteria were collected by centrifugation, and total RNA was prepared by using the hot acid-phenol procedure as previously described (Christiansen et al. 2004). For detection of sRNAs in vivo, semiquantitative reverse transcription (RT)-PCR was performed. One microgram of the purified RNA was treated with RNase-free DNase I (Amersham) according to the manufacturer's recommendations. For cDNA synthesis, 0.1 pmol of primer lhrA-3, lhrB-3, and lhrC-1 (Table (Table3)3) was allowed to anneal to 2 μg of RNA in AMW buffer (Finnzymes) and 1.1 pmol of dNTP in a total volume of 10 μL. To initiate cDNA synthesis, 2 units of AMW Reverse Transcriptase (Finnzymes) was added, and the reaction was allowed to proceed for 30 min at 42°C. One microliter of the cDNA reaction was used as template in 25 μL of PCR amplification reactions using forward primers lhrA-21, lhrB-8, and lhrC-2; reverse primers lhrA-3, lhrB-3, and lhrC-1 (Table (Table3);3); and Taq DNA polymerase (Promega) as described by the supplier, giving fragments of 261 bp, 96 bp, and 102 bp, respectively, when analyzed on a 10% acrylamide gel stained with ethidium bromide. PCR performed on total DNA from bacteria grown in BHI was used as a positive control, and RT-PCR performed without reverse transcriptase was used as a negative control.

ACKNOWLEDGMENTS

We thank Christina Kirkegaard for excellent technical assistance. This work was supported by grants from the FREJA Program, the Danish Natural Science Research Council, and the Danish National Research Foundation.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.49706.

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