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Originally published In Press as doi:10.1074/jbc.M710108200 on December 21, 2007

J. Biol. Chem., Vol. 283, Issue 9, 5738-5747, February 29, 2008
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Involvement of RNA-binding Protein Hfq in the Post-transcriptional Regulation of invE Gene Expression in Shigella sonnei*Formula

Jiro Mitobe{ddagger}, Tomoko Morita-Ishihara{ddagger}, Akira Ishihama§, and Haruo Watanabe{ddagger}1

From the {ddagger}Department of Bacteriology, National Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640 and the §Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, Japan

Received for publication, December 11, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The temperature-dependent regulation of Shigella virulence genes is believed to be accomplished at the transcriptional stage by the regulators VirF and InvE. Several lines of evidence herein described indicate that post-transcriptional regulation of InvE expression plays a key role in the temperature-dependent regulation of virulence gene expression: (i) a considerable amount of invE mRNA continues to be transcribed under low temperature conditions, where the production of InvE protein is tightly repressed; (ii) the stability of invE mRNA markedly decreases, because its decay rate is significantly increased under the repressing conditions. Strikingly, in the hfq mutant of Shigella sonnei, a considerable amount of InvE protein was produced even at low temperature. This increase in the InvE level was found to be associated with the improved stability of invE mRNA, in agreement with the finding that the RNA chaperon Hfq influences post-transcriptional regulations of various genes. Consistently, overexpression of the Hfq protein decreased the production of InvE protein even under the expressing condition at 37 °C. The binding in vitro of purified Hfq protein to invE RNA was shown to be stronger at 30 °C than at 37 °C in two experiments, gel shift analysis and surface plasmon resonance (Biacore) analysis. These results altogether suggest that Hfq plays an important role in the temperature-dependent regulation of invE expression at the post-transcriptional step.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Type III secretion system (TTSS)2 plays a key role in the expression of virulence by pathogenic Shigella. The expression of TTSS is tightly regulated by temperature in such a way that it is high at 37 °C but not at 30 °C (1, 2). This regulation apparently fits the life cycle of Shigella, because the expression of virulence genes is needed for its invasion and propagation in host animals, but the expression of virulence genes might be a potential burden for its survival in natural environment.

The TTSS-associated genes of Shigella are encoded on the virulence plasmid and controlled by two regulator proteins VirF and InvE (VirB) (3, 4). VirF, an AraC type transcriptional regulator, activates transcription of the invE (virB) gene (3, 5-7). The second regulatory protein, InvE, a homolog of a plasmid-partitioning factor, ParB (6), with DNA binding activity (8), activates transcription of the mxi-spa and ipa genes encoding TTSS components by replacement of a global repressor H-NS, a histone-like DNA-binding protein (9).

Various mechanisms have been proposed to explain the temperature-dependent regulation of TTSS (10). For instance, a mutation of the hns gene in Shigella flexneri was reported to result in increased TTSS expression at the repressing temperature 30 °C (11). The H-NS protein is now accepted as one of the key factors for temperature-dependent gene expression in various Gram-negative bacterias through the structural change of its oligomer formation at high temperature (12). In fact, the hns mutation increases the expression of virF promoter-lacZ fusion, and the addition of H-NS protein into the in vitro transcription system results in decreased transcription from the virF promoter in a temperature-dependent manner (13).

On the contrary, detailed analysis for transcription in vivo of virF and invE genes indicated that significant amounts of virF and invE mRNA are transcribed, albeit at a reduced level, at 30 °C in both wild-type S. flexneri and hns mutant at various growth phases (14). Previously, we isolated a cpxA mutant, in which the expression of TTSS genes was significantly decreased. Characterization of the cpxA mutant revealed that the expression of InvE protein is controlled at the post-transcriptional level. Analysis with both transcriptional and translational fusion of invE-lacZ reporter plasmids showed that a sufficient amount of invE mRNA is transcribed, but the protein expression is repressed in the cpxA mutant (15).

Finding of the post-transcriptional regulation in InvE expression allowed us to reexamine the transcription of the invE gene under the repressing condition at 30 °C. Using the invE-lacZ transcriptional fusion plasmid, a considerable level of β-galactosidase activity was detected at 30 °C, but with use of the invE translational fusion plasmid, a similar level of the β-galactosidase activity was not detected at 30 °C. Taking the results together, we propose the concept of post-transcriptional regulation for InvE expression. Supporting this model, an RNA chaperone, Hfq, was found to exert marked influence on the expression of the invE gene.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sequencing—The hfq gene of Shigella sonnei HW383 was sequenced by ABI-PRISMTM 310 genetic analyzer (PerkinElmer Life Sciences), using oligonucleotide primers (see supplemental Table S1) hfq1 and hfq2. The sequenced hfq gene in S. sonnei HW383 was identical to that of Escherichia coli K-12 strain, submitted to the DNA sequence data base DDBJTM with the accession number AB293541. The upstream region of virF gene in S. sonnei HW383 was sequenced using oligonucleotide primers virF1 and virF2 (GenBankTM/EBI Data Bank accession number AB300612). The sequence containing a putative transposase was fully identical to that of S. sonnei strain Ss046 (nucleotides 38934-37955 of GenBankTM/EBI Data Bank accession number CP000039) and Shigella boydii strain Sb227 (nucleotides 37617-38596 of GenBankTM/EBI Data Bank accession number CP000037).

Expression Plasmids—The hfq gene (nucleotides 8002-8368 of GenbankTM/EBI Data Bank Accession number AE000489) was amplified by PCR from genomic DNA of E. coli K-12 strain BW25113 (16) using the primers hfq3 and hfq4. The amplified DNA product was digested with the restriction enzymes NcoI and XbaI, ligated into the plasmid pTrc99A (17) (GenbankTM/EBI Data Bank accession number U13872) to produce pTrc-hfq. The same DNA sequence was amplified with primers hfq5 and hfq6. The amplified DNA product was digested with the restriction enzymes NdeI and XhoI, ligated into the plasmid pET22b (Novagen, Madison, WI) without the addition of the histidine-tag sequence to produce pET-hfq. For construction of pBAD-invE, the invE sequence (nucleotides 209-1190 of GenbankTM/EBI Data Bank accession number M33790) was amplified using oligonucleotide primers invE1 and invE2. The amplified DNA product was digested with the restriction enzymes NheI and HindIII, ligated into the plasmid pBAD24 (18) (GenbankTM/EBI Data Bank Accession number X81837) to produce pBAD-invE. The integrity of the sequences in the above plasmids were checked by a PerkinElmer Life Sciences 310 DNA sequencer. All primers were synthesized by Grainer Co. (Tokyo, Japan).

Construction of Mutant Strains—For the construction of deletion mutants for hfq, hns, and invE genes, a PCR-based gene disruption technique was applied for wild-type S. sonnei strain MS390 as described previously (15, 16). A kanamycin-resistant gene cassette in pKD13 (16) was amplified with the following primers: for MS4831, hfq7 and hfq8; for MS4841, hns1 and hns2; and for MS1632, invE3 and invE4. The kanamycin-resistant gene cassette was removed as described (16). Integrity of the nonpolar deletion sequences was confirmed by DNA sequencing.

For construction of the hfq::aphA deletion mutants of S. sonnei MS4835 and S. flexneri MF4835, a three-step PCR-based gene disruption technique (19) was applied for both wild-type strains of S. sonnei MS390 and S. flexneri 2457T (20), respectively, using primers hfqU1, hfqU2, hfqD1, and hfqD2. A kanamycin-resistant gene cassette aphA was amplified from pTH18ks5 (21) using Km1 and Km2 primers.

For construction of the S. sonnei pinvE::paraBAD mutant MS5512, a modification of the three-step PCR-based gene replacement technique (22) was applied for MS390 using invEU1, invEU2, invED1, and invED2 primers. The gene cassette, composed of the chloramphenicol acetyltransferase gene, the araC repressor, and the promoter region for the araBAD operon (nucleotides 70188-71339 of GenBankTM/EBI Data Bank accession number AP009048 [GenBank] .1), was amplified from an E. coli strain BW25113 chloramphenicol acetyltransferase-araC (22), using the araC1 and araC2 primers (see Fig. 2A). After construction of all mutants, the presence of the virulence plasmid that encodes the form I antigen was confirmed by agglutination with diagnostic antiserum for S. sonnei (Denka Seiken, Tokyo, Japan) or PCR of the invE gene for S. flexneri 2457T using primers invE1 and invE2. At least two independent deletion mutants were constructed for each deletion, which showed the same results for all experiments. Bacterial strains and plasmids used in this study are listed in Table 1.


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  		TABLE 1
Bacterial strains and plasmids used in this study

 
Bacterial Culture Condition—Luria-Bertani (LB) medium (LB Lenox; Difco) was used for bacterial growth. Concentrations of antibiotics were as follows: ampicillin, 50 µg/ml; chloramphenicol, 12.5 µg/ml; rifampicin, 200 µg/ml (Sigma). Usage of antibiotics is specified by the figure legends for each experiment. For all experiments, each strain was inoculated into 2 ml of LB medium and grown overnight at 30 °C with shaking (150 rpm) in a water bath. The precultures were diluted 50-fold for the 30 °C culture and 100-fold for the 37 °C culture to give similar incubation periods (2.5-3 h) in a pair of 5-ml fresh LB medium samples. The samples were incubated at both 30 and 37 °C with shaking at 150 rpm, monitored for turbidity at 600 nm by a spectrophotometer (SpectronicTM 20+, Shimadzu, Kyoto, Japan), and harvested at A600 = 0.8. Each 50 µl of the whole culture was used for measurement of β-galactosidase activities as previously described (23), or 10 µl of the whole culture was subjected to Western blotting with 10% SDS-PAGE as previously described (24). IpaB and InvE proteins were detected by anti-IpaB monoclonal antibody (25) or anti-InvE polyclonal antibody (15). Hfq and H-NS proteins were detected as previously described (26, 27).

RNA Preparation and Detection—Each 2 ml of the whole culture was quickly mixed with 150 µl of 5% (v/v) water-saturated phenol in ethanol (28). The bacterial cells were immediately collected by centrifugation at 12,000 x g for 1 min, and total RNA was purified by 1 ml of a phenol containing the RNA extraction reagent ISOGENTM (Nippon Gene, Tokyo, Japan). For measurement of invE mRNA stability, S. sonnei strains were inoculated into 35 ml of LB medium without antibiotics and allowed to grow until A600 reached 0.8. After rifampicin (Sigma) was added to give a final concentration of 200 µg/ml, the culture continued to incubate at the initial temperature. An aliquot of the cultures (2 ml) was harvested every 2 min for preparation of RNA samples. The total RNA pellets were dissolved in 40 µl of nuclease-free water (Invitrogen) and measured for the concentration by spectrophotometer ND-1000TM (Nano-Drop Technologies, Wilmington, DE). The concentration of the RNA samples was adjusted to 50 ng/µl, and then the residual DNA was digested by TURBO DNA-freeTM kit (Ambion, Austin, TX) for 30 min at 37 °C. For reverse transcription (RT)-PCR, each 100 ng of total RNA was amplified by TitanTM one-tube RT-PCR kit (Roche Applied Science). Conditions for RT-PCR were as follows: 60 °C for 30 min; 94 °C for 2 min; and 26 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 90 s. For detection of virF mRNA and 6 S RNA, two additional cycles (for a total of 28 cycles) of PCR were employed. Primers used for RT-PCR for virF mRNA were virF-RT1 and virF-RT2. Primers used for invE transcript were invE-RT1 and invE-RT2. Primers used for RT-PCR for 6 S RNA transcript were ssrS-RT1 and ssrS-RT2. Each 10 µl of the PCR product was subjected to electrophoresis in a 1% agarose gel containing 50 µg/ml ethidium bromide. For real time PCR analysis, cDNA was synthesized from 100 ng of total RNA in 10 µl of TranscriptorTM first strand cDNA synthesis kit (Roche Applied Science) for 30 min at 60 °C. Each cDNA was analyzed in a triplicated manner by ABI Prism 2000 thirmal cycler with a Perfect real timeTM PCR kit (Takara Japan) in a 25-µl reaction containing 2 µl of cDNA, 120 nM concentration each of invE84F, invE156R, ssrS36F, and ssrS72R primers, and a 32 nM concentration each of invE100T and ssrS65T TaqmanTM probes. Samples were amplified by 40 cycles of 95 °C for 5 s and 60 °C for 31 s. Primers and TaqmanTM probes were designed by ABI PRISM primer design software and synthesized by ABI Japan. The amounts of invE mRNA and 6 S RNA were determined by ABI prism evaluation software using a standard curve provided by five serial dilutions of cDNA from the sample of time 0 at 37 °C. The amount of invE-mRNA was normalized by the amount of 6 S RNA transcript as an internal control. Relative values against the sample of time 0 at 37 °C were blotted on the semilog plot. The RNA preparation and real time PCR analysis were repeated three times, and similar trends of results were obtained.

Invasion Assay—Bacterial invasion into HeLa cells was tested using the gentamicin protection assay. Overnight precultures (2 ml of LB-ampicillin) were inoculated in 5 ml of brain-heart infusion broth (Difco). After incubation at 37 °C for 40 min, IPTG was added to a final concentration of 0.1 or 1 mM, and the incubation was continued for an additional 80 min at 37 °C. HeLa cells were grown on glass coverslips to 60% confluence in antibiotic-free Dulbecco's modified eagle medium (Invitrogen) containing 10% fetal calf serum (Invitrogen). Cells were then infected with bacteria grown in BHI at 37 °C at a multiplicity of infection of 100 per cell and centrifuged at 700 x g for 10 min. After cells were incubated at 37 °C in a CO2 incubator for 20 min, gentamicin (Wako) was added to the culture medium at a final concentration of 200 µg/ml, and cells were incubated further at 37 °C in a CO2 incubator for 15 min. After incubation, cells were washed twice with phosphate-buffered saline and lysed in phosphate-buffered saline containing 0.5 ml of 0.5% Triton X-100. A 100-µl aliquot of the lysates was plated onto LB plates and incubated at 37 °C overnight. Colonies grown on LB plates were counted. For the reliability of results, each sample determination was performed in triplicate. The result of a representative assay is shown in Table 2.


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  		TABLE 2
Efficiency of invasion

 
Purification of Hfq Protein—An E. coli strain, BL21(DE3), carrying pET-hfq was grown in 1 liter of LB medium 37 °C for 4 h as described (26). Hfq protein was purified with a 10-ml bed volume of Buthyl-ToyopealTM-650 hydrophobic interaction medium (Tosoh, Tokyo, Japan) in a 35-ml Econo ColumnTM (Bio-Rad) using an Econo SystemTM gradient maker (Bio-Rad) as described (29).

Gel Shift Analysis—Two RNA probes encoding 75- and 140-nucleotide invE sequences from the transcription start site were prepared by in vitro transcription of T7 RNA polymerase (Roche Applied Science). The DNA templates for the T7 transcription were prepared by PCR amplification of the invE sequence in MS390 with primer pairs of invE-T7F and invE75R (for the 75-nucleotide RNA) or invE-T7F and invE140R (for the 140-nucleotide RNA). The T7 transcription mixtures were treated by RNase-free DNase I (Roche Applied Science) for digestion of the DNA template at 37 °C 15 min and purified by phenol/chloroform and a Nuc-awayTM spin column (Ambion). The 5'-end of purified RNA was dephosphorylated by alkaline phosphatase in the Kinase MaxTM labeling kit (Ambion), and 2 pmol of the RNA was labeled by [{gamma}-32P]ATP (catalog number AA0018; GE Healthcare) with T4 polynucleotide kinase in the kit. The labeled RNA was purified by phenol/chloroform and a Nuc-awayTM spin column (Ambion) equilibrated with an RNA binding buffer: 10 mM Tris-HCl, pH 7.5, 100 mM NH4Cl, 5 mM magnesium acetate, 0.1 mM dithiothreitol (30). Each 20 fmol of the labeled RNA was used for probes of gel shift analysis. Hfq protein (0, 1, 2, 4, 8, and 16 nM calculated for the Hfq hexamer) was mixed with the probe in 10 µl of the RNA binding buffer at 30 or 37 °C for 10 min. Then the samples were mixed with 2 µl of 5x hi-Density TBE Sample BufferTM (catalog number LC6678; Invitrogen), subjected to electrophoresis with 6% NovexTM DNA retardation gel (catalog number EC63652; Invitrogen) in 0.5x TBE at 30 °C for 80 min or 37 °C for 70 min. Electrophoresis was performed in an XCell SureLockTM mini-cell electrophoresis tank (catalog number EI0001) with a glass circulation pipe from Nihon Eido Co. (Tokyo, Japan), which was placed in a water bath with a circulation pump (Haake, Germany), heated at the indicated temperature. The gel was fixed by 10% methanol, 10% acetate and dried on filter paper. The signals on the gel were visualized on x-ray film RX-U (Fuji Film, Tokyo, Japan) with an intensifying screen at -80 °C for 4-16 h.

Surface Plasmon Resonance (Biacore) Analysis—The Biacore 2000 system and sensor chip SA (research grade; Biacore International AB) were used for the binding assay. For preparation of the RNA-immobilized sensor tip, 2 pmol of the in vitro transcribed invE RNA used for gel shift analysis (140 nucleotides) were labeled with 0.2 mM biotin-11-ATP (catalog number NEL544; PerkinElmer Life Sciences) by 1 unit of E. coli poly(A) polymerase (Ambion) for 10 min at 37 °C as described (31). The labeled invE RNA was extracted by phenol/chloroform, chloroform and purified by the Nuc-awayTM spin column (Ambion) two times. Before binding of the RNA, the sensor tip was treated with 50 mM NaOH, 1 M NaCl at a flow rate of 20 µl/min for 1 min three times. The invE RNA was diluted in a high salt buffer (10 mM Tris-HCl, pH 7.4, 1 M NaCl) and captured on flow cell 2 of the streptavidin-coated sensor chip at a flow rate of 10 µl/min until a change of at least 100-150 resonance units was detectable. The base line of resonance intensity was then allowed to stabilize for at least 15 min.

A binding assay was performed at 30 and 37 °C. Purified Hfq protein was diluted in the binding buffer (see above) to give a final concentration of 0, 1, 2, 4, or 8 nM, calculated for the Hfq hexamer, and subsequently injected for 3 min onto two flow cells (flow cell 1, blank as a control; flow cell 2, invE RNA) at a flow rate of 20 µl/min. The nonbinding protein was washed out from the specific RNA-protein by the flow of binding buffer for an additional 700 s. Bound Hfq protein was efficiently removed from the complex by a solution of 2 M NaCl for 2 min at a flow rate of 20 µl/min. The response from the reference cell (flow cell 1, blank) was subtracted from the response from flow cell 2 (biotin-invE RNA) to correct a nonspecific binding. We attempted to determine the Kd value using BIAevaluationTM version 3.1 software (Biacore International AB), but the interaction did not fit the 1:1 Langmuir fitting model because of a multimeric binding between the protein and RNA as observed in the gel shift analysis.

The measurement of CD spectroscopy (32) was performed by Toray Research Center (Kamakura, Japan). The 140-nucleotide invE RNA was diluted to 4 µg/ml in the same buffer used for the gel shift analysis, and 80 µl of the sample was subjected to the CD analyzer, J-820 (Nippon Bunko Inc.), in a 10-mm microcell at 30 and 37 °C; values were integrated from 16 measurements.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transcription of virF and invE in S. sonnei MS390
Transcription of the virF gene encoding the upstream regulator of the invE gene was first examined in the wild-type strain of S. sonnei MS390. For the accurate measurement of intact mRNA, the whole culture was directly mixed with an acidic phenol/ethanol solution for quantitative isolation of mRNA (28), and RT-PCR rather than Northern analysis was employed for the detection of full-length intact mRNA. The level of virF mRNA in the wild-type S. sonnei strain grown in LB medium was almost the same between 30 and 37 °C (Fig. 1A, virF-mRNA), and the level of β-galactosidase activity in wild-type S. sonnei strain MS390 encoded by a virFTL-lacZ translational fusion plasmid (7) was also similar between 30 and 37 °C (Fig. 1B, graph 1). Previously, however, Porter and Dorman (14) reported that virF transcription in S. flexneri strain 2457T at 30 °C was 15-25% of the level at 37 °C (14). In order to identify the molecular basis of this difference, we sequenced the upstream region of the virF promoter of S. sonnei MS390 and found an insertion of a transposon sequence (GenBankTM accession number AB300612). The difference in temperature-dependent transcription of the virF gene between the two wild-type strains might be related to the presence or absence of this insertion sequence. In addition to the intact virF mRNA, we detected an additional band of shorter length by Northern analysis (see "Discussion") (15).

Temperature-dependent regulation of the invE gene, which is under the control of VirF, was next examined by Western blotting and RT-PCR. The production of InvE protein was almost completely repressed at 30 °C (Fig. 1A, lane 1), but under the same culture conditions, a significant amount of invE mRNA was detected by RT-PCR (Fig. 1A, invE-mRNA). The amount of invE transcript at 30 °C was then measured by real time PCR analysis and found to be 10 ± 5% of the level at 37 °C.

The expression of the invE gene was also measured by a β-galactosidase assay using both transcriptional and translational invE-lacZ fusions (15). The transcriptional fusion plasmid was constructed after insertion of the invE promoter to the lacZ gene at 18 bp upstream from the transcriptional start site. Under the repressing conditions at 30 °C, the β-galactosidase activity of wild-type S. sonnei strain MS390 encoded by transcriptional fusion plasmid (invETx-lacZ) was 92% of the level at 37 °C (Fig. 1B, graph 2). In contrast, the β-galactosidase activity encoded by translational fusion plasmid (invETL-lacZ) at 30 °C in LB medium was 11% of the level at 37 °C (Fig. 1B, graph 3). These experiments altogether indicate that the level of transcription and translation does not correlate at a low temperature of 30 °C. Thus, we predicted that the synthesis of InvE is controlled at the stage of post-transcription.


Figure 1
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  		FIGURE 1.
A, RT-PCR for invE and virF mRNA and Western blotting for InvE and IpaB proteins in the wild-type strain of S. sonnei MS390. Overnight culture of MS390 at 30 °C was inoculated into two tubes of LB medium as described under "Experimental Procedures," further cultured at 30 and 37 °C, and harvested at log phase (A600 = 0.8). Each 100 ng of total RNA and 10 µl of the whole culture were subjected to the assays, respectively. The 6 S RNA ssrS gene and a cross-reacted unknown protein by InvE antiserum (loading control) are used as controls for RT-PCR and Western blotting by InvE antiserum, respectively. Primers and antibodies used in the experiments are indicated on the right. Lane 1, culture at 30 °C; lane 2, culture at 37 °C. B, the β-galactosidase activities of the transcriptional fusion (invETx-lacZ) and translational fusion (virFTL-lacZ) (invETL-lacZ) plasmids. Wild-type S. sonnei strain MS390 carrying the plasmids was grown in LB-chloramphenicol medium, and each 50 µl of the whole culture was subjected to the assay. Graph 1, β-galactosidase activities from the virFTL-lacZ plasmid pHW848. Graph 2, β-galactosidase activities from the invETx-lacZ plasmid pJM4320. Graph 3, β-galactosidase activities from the invETL-lacZ plasmid pJM4321. The culture conditions were indicated at the bottom of the graphs as follows: culture at 30 °C (white bars) and at 37 °C (gray bars). Black bar (NC, negative control), β-galactosidase activities of {Delta}cpxR strain MS2830 (graph 1) or virulence plasmid cured strain HW506 (graphs 2 and 3) carrying the respective vectors at 37 °C. Error bars, S.D. of two independent experiments.

 


Figure 2
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  		FIGURE 2.
A, construction of the arabinose-inducible invE promoter. The native invE promoter in S. sonnei MS390 was replaced by the chloramphenicol acetyltransferase-araC-paraBAD gene cassette (not to scale) to generate {Delta}pinvE::paraBAD strain MS5512. The arrowheads indicate the directions of transcription on the gene cassette. +1, a transcriptional start site of native invE mRNA, where the transcription start site of the araBAD promoter was correctly inserted. B, InvE expression in the {Delta}pinvE::paraBAD strain MS5512. Western blotting for IpaB and InvE proteins and RT-PCR for invE mRNA were performed for the {Delta}pinvE::paraBAD strain MS5512 and wild-type strain MS390. Strain MS5512 was grown overnight in LB-chloramphenicol medium containing 50 µM arabinose, washed twice by fresh LB medium, inoculated into LB medium containing increasing concentrations of arabinose, and cultured at 30 and 37 °C, as indicated. The strains (MS5512 ({Delta}pinvE::paraBAD) and wild-type strain MS390 (Wt)), temperatures (30 and 37 °C), and arabinose concentration (0, 0.2, 0.5, and 1 mM) used in the experiments are indicated at the top. Primers and antibodies used in the experiments are indicated on the right. C, stability of InvE protein. The {Delta}invE strain MS1632 carrying pBAD-invE plasmid was grown in LB-ampicillin medium containing 100 µM arabinose at 30 and 37 °C. After the rifampicin treatment at A600 = 0.8, cells were successively harvested at 10-min intervals for 40 min. Each 10 µl of whole culture was subjected to Western blotting for InvE proteins.

 
Promoter-independent Post-transcriptional Regulation of invE
Transcription from the invE promoter is under the control of VirF (3, 5-7). Next, the native invE promoter was replaced by a promoter cassette containing the E. coli araC repressor and araBAD promoter region (22) so as to examine VirF-independent expression of the invE open reading frame under the control of the AraC-dependent araBAD promoter (Fig. 2A). The resulting pinvE::paraBAD strain MS5512 was cultured in a series of LB media containing increasing concentrations of arabinose. An essentially similar amount of invE mRNA was detected between 0.2 and 1.0 mM arabinose at both 30 and 37 °C (Fig. 2B, invE-mRNA). However, the amount of InvE protein was significantly lower at 30 °C than the level at 37 °C (Fig. 2B, {alpha}-InvE). Consequently, the synthesis of TTSS effector IpaB protein, whose expression is activated by InvE, was also repressed at 30 °C (Fig. 2B, {alpha}-IpaB).

In order to examine the metabolic stability of InvE protein at 30 °C, the invE open reading frame was cloned into an expression vector, pBAD24 (18). The resulting plasmid pBAD-invE expressed the InvE protein in an invE deletion mutant MS1632 at both 30 and 37 °C (Fig. 2C, {alpha}-InvE), indicating that the metabolic stability of InvE protein was the same between 30 and 37 °C. These results further confirmed that the InvE production at 30 °C is regulated at a post-transcriptional stage(s).


Figure 3
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  		FIGURE 3.
A, construction of the two nonpolar hfq deletion mutants. For the hfq deletion mutant MS4831, the hfq gene was replaced by a kanamycin-resistant gene cassette from pKD13, and then the cassette was removed by a site-specific recombination. A residual 82-bp nonpolar sequence (16) is indicated by a black box. For construction of the hfq::aphA mutants (MS4835 and MF4835), the hfq gene was replaced by a nonpolar kanamycin-resistant gene cassette aphA (19). The positions of the hfq promoters are indicated (not to scale) as follows: {sigma}32 promoter, P1hfq;{sigma}70 promoters, P2hfq and P3hfq (53). B, InvE and IpaB expression in the hfq deletion mutant. Wild-type strain MS390 and the hfq mutant MS4831 were cultured in LB medium at 30 and 37 °C and subjected to Western blotting as described. Strains (MS390 (Wt) and MS4831 ({Delta}hfq)) and temperature (30 and 37 °C) are indicated above the panels. Antibodies used in the experiment are indicated at the right. C, InvE and IpaB expression in the hfq deletion mutant with anhfq-encodingplasmid. Lysates of the hfq deletion mutant MS4831 carrying hfq expression plasmid (pTrc-hfq) or vector (pTrc99A) were subjected to Western blotting. Both strains were grown in LB-ampicillin medium containing 0.1 mM IPTG at 30 °C or 1 mM IPTG at 37 °C and harvested as described. Strains (MS390 (Wt) and MS4831 ({Delta}hfq)), temperature (30 and 37 °C), and plasmids (pTrc99A (minus) and pTrc-hfq (plus)) are indicated at the top. Antibodies used in the experiment are indicated on the right. Lane 1, {Delta}hfq (pTrc99A) at 30 °C with 0.1 mM IPTG; lane 2, {Delta}hfq (pTrc-hfq) at 30 °C with 0.1 mM IPTG; lane 3, {Delta}hfq (pTrc99A) at 37 °C with 1 mM IPTG; lane 4, {Delta}hfq (pTrc-hfq) at 37 °C with 1 mM IPTG; lane 5, wild-type strain MS390 grown at 37 °C in the LB medium.

 
Involvement of Hfq in the Post-transcriptional Regulation of InvE Synthesis
Since an RNA-binding protein Hfq (33, 34) is known to be involved in post-transcriptional regulation of gene expression, we next constructed a deletion mutant of the hfq gene in S. sonnei (Fig. 3A) for comparison of InvE expression at low and high temperatures. The growth characteristics of the S. sonnei hfq mutant MS4831 were similar to those of E. coli and Salmonella typhimurium; the hfq mutant showed delayed recovery from the stationary phase, but the growth rate was the same as that of wild-type strain in the logarithmic phase (35).

Strikingly, the hfq mutant MS4831 expressed the InvE and IpaB proteins even under the repressing conditions at 30 °C in LB medium and enhanced their production at 37 °C. The amounts of InvE and IpaB proteins at 30 °C in the hfq mutant were comparable with that of the wild-type strain at 37 °C (Fig. 3B). Consistently, the introduction of an Hfq expression plasmid pTrc-hfq reduced the InvE expression in the hfq deletion mutant MS4831; the hfq mutant carrying the plasmid pTrc-hfq repressed InvE expression at 30 °C in LB medium containing 0.1 mM IPTG (Fig. 3C, lane 2). In addition, overexpression of Hfq protein by 1 mM IPTG repressed the InvE and IpaB expression even under the expressing conditions at 37 °C (Fig. 3C, lane 4).

Stability of invE mRNA
Next we examined the stability of invE mRNA in the hfq mutant by RT-PCR and real time PCR analysis. Under the InvE-expressing conditions of wild-type strain MS390 cultured in LB medium at 37 °C, the invE mRNA was relatively stable at least for 8 min after the rifampicin treatment (t1/2 = 6.19 min), whereas under the repressing conditions at 30 °C, the level of invE mRNA detection was low (10 ± 2% of 37 °C), and moreover, invE mRNA was rapidly degraded, especially in the first 2 min (t1/2 = 2.64 min). Consistent with the increase in InvE expression, the stability of invE mRNA also increased in the hfq deletion mutant MS4831 at 30 °C (t1/2 = 5.82 min) (Fig. 4, A and B).

These results further indicated that the stability of invE mRNA is intimately coupled with the expression of InvE protein. In addition, the recovery of InvE expression in the hfq mutant was also associated with the improvement of invE mRNA stability.

Interaction in Vitro of Hfq with invE RNA
Gel Shift Analysis—In order to examine the interaction between Hfq and invE RNA in vitro, purified Hfq and invE RNA were mixed and subjected to gel shift analysis. The Hfq protein of S. sonnei MS390, which has a sequence identical to that of E. coli, was purified with a hydrophobic interaction column (29). As probes for the gel shift analysis, two types of radioactive invE RNA of 75 and 140 nucleotides in length from the transcription initiation site of the invE gene were prepared after transcription in vitro with T7 RNA polymerase. The Hfq protein bound to both of these invE RNA probes, and the Hfq-invE RNA complex formation was reduced in the presence of excess unlabeled invE RNA (data not shown), indicating that the probe designed can be used for measurement of the Hfq-invE RNA interaction. The Hfq-invE RNA complex formation was examined at both 30 and 37 °C in the presence of increasing concentrations of the Hfq hexamer complex (1-16 nM) and constant concentration (2 nM) of the two RNA probes. At 30 °C, the initial shift of the probes was observed with 4 nM Hfq hexamer (Fig. 5, lane 4), whereas 8 nM was required for its binding at 37 °C (Fig. 5, lane 11). The apparent binding constant determined by disappearance of free probes was 3.2 nM at 30 °C and 6.2 nM at 37 °C. The supershift of the bands was more apparent in longer RNA probe (Fig. 5, top, lanes 4-6 and lanes 11-12), indicating that more than one Hfq hexamer complex bound to the RNA probe.


Figure 4
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  		FIGURE 4.
A, stability of invE mRNA at 30 and 37 °C. Total RNA was isolated from the wild-type strain MS390 and hfq mutant MS4831 grown in LB medium at the indicated temperatures (30 and 37 °C). After the rifampicin treatment at A600 = 0.8, cells were successively harvested at 2-min intervals. Each 100 ng of the purified RNA was used for RT-PCR, and 10 µl of products were subjected to agarose gel electrophoresis. Temperature (30 and 37 °C), strains (wild-type strain MS390 (Wt) and MS4831 ({Delta}hfq)) and the time after the rifampicin treatment (0, 2, 4, 6, 8, 32 min) are indicated at the top. Primers and antibodies used in the experiments are indicated on the right. B, decay curves of the invE mRNA. Each 100 ng of the total RNA were subjected to real time PCR analysis. The amount of RNA was normalized by an internal control using 6 S RNA transcript, and relative values (1.0 at time 0 of wild-type sample at 37 °C) of the corresponding signals were blotted on the y axis. The x axis indicates the time after the rifampicin treatment (0-8 min). Temperature (30 and 37 °C) and strains (wild-type strain MS390 (Wt) and MS4831 ({Delta}hfq)) are indicated in the right. The indicated result is one of three independent experiments that showed similar trends.

 


Figure 5
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  		FIGURE 5.
Gel shift analyses at 30 and 37 °C. Each 2 nM concentration of the 5'-end labeled RNA probe (top, 140 nucleotides; bottom, 75 nucleotides) was mixed with Hfq protein and incubated at 30 or 37 °C for 10 min. The electrophoresis was carried out separately at 30 and 37 °C. Temperature (30 and 37 °C) and the increasing amount of Hfq protein that corresponded to 0-8-fold molar excess are indicated at the top. The final concentration of Hfq hexamer for each electrophoresis was as follows. Lanes 1 and 7, 0; lanes 2 and 8, 1 nM; lanes 3 and 9, 2 nM; lanes 4 and 10, 4 nM; lanes 5 and 11, 8 nM; lanes 6 and 12, 16 nM.

 


Figure 6
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  		FIGURE 6.
Surface plasmon resonance (Biacore) analysis for the interaction between Hfq and invE RNA. The invE RNA (140 nucleotides) was immobilized to a sensor chip SA and placed into a Biacore 2000 optical sensor device. Binding assays were performed at 30 °C (A) and 37 °C (B). Hfq protein was diluted in the RNA binding buffer (0, 1, 2, 4, and 8 nM (right)) and subsequently injected for 180 s onto two flow cells (flow cell 1, blank; flow cell 2, invE RNA) at a flow rate of 20 µl/min. The nonbinding protein was allowed to dissociate from the RNA-protein complex during a wash step (700 s). Bound Hfq protein was subsequently removed with 2 M NaCl. The response value from the reference cell (flow cell 1, blank) was subtracted from the response value from flow cell 2 (invE RNA) to correct the nonspecific binding, the value of which was indicated in difference units (D.U.).

 
Surface Plasmon Resonance Analysis—The interaction between Hfq protein and invE mRNA was also examined by surface plasmon resonance (Biacore) analysis. A fixed amount of the 140-nucleotide-long invE RNA as used above was immobilized onto a sensor tip, and then serially diluted panels of Hfq protein were added. The interaction was stable enough to sustain the binding ability after the flow washing with the buffer containing 100 mM NH4Cl. The binding of Hfq hexamer to invE RNA reached a plateau at a concentration of nearly 8 nM at both 30 and 37 °C (Fig. 6, A and B), the apparent affinity being higher than that (16 nM) observed with gel shift analysis. The distinct difference of RNA binding properties with Hfq protein was, however, observed between the two temperatures of 30 and 37 °C (Fig. 6). The minimum concentration for the initial binding of Hfq protein was 1 nM at 30 °C and 4 nM at 37 °C, respectively. At 30 °C, the sequential binding of several Hfq complexes was observed in a concentration-dependent manner, whereas at 37 °C, a sudden increase of Hfq binding was observed at 4 nM Hfq. The result of Biacore analysis confirmed that the binding of Hfq to invE RNA is influenced by temperature. The slight difference in the minimum concentration of Hfq needed to form the maximum amount of invE RNA-Hfq complex between gel shift and Biacore assays might be due to a lower level of temperature control during the electrophoresis procedures.

Effect of hfq Mutation on the TTSS Expression and Invasion
Expression of IpaB effector molecule in TTSS appeared to increase further in the hfq mutant (see Fig. 3C, lane 3). Consistently, a contact hemolytic assay by sheep erythrocyte, which reflects the secretion activity of the effector molecules, showed the increased activity in the hfq mutant (data not shown). To confirm this finding, an invasion assay was also performed using HeLa cells. Results indicated that the efficiency of invasion into HeLa cells was increased for the hfq mutant, but the increased invasion efficiency was recovered to the wild-type level when Hfq was expressed after introducing the Hfq expression vector, pTrc-hfq, in the presence of increasing concentrations of IPTG (Table 2). These results altogether indicated the intimate involvement of Hfq in virulence gene expression in S. sonnei.

The nucleoid protein H-NS is known to influence the expression of TTSS (13). Finally, in order to examine possible influence in the expression of Hfq and H-NS proteins, both H-NS expression in the hfq mutant and Hfq expression in an hns mutant (MS4841) were examined by Western blotting. The immunoblot analysis indicated that no significant influence on the expression of both proteins was observed in the mutual combinations (data not shown). The immunoblot analysis also indicated that the growth temperature did not affect the expression of Hfq and H-NS proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The precise mechanism for temperature-dependent regulation of virulence genes in Shigella has been left unsolved. Here we propose a post-transcriptional regulation model for the temperature-dependent control of invE expression, because the synthesis of InvE protein is repressed at the low temperature of 30 °C even where transcription of the invE gene is still active. Several lines of evidence support this model in which the temperature-dependent regulation is accomplished in the post-transcriptional stage(s) of the InvE protein expression associating with the stability of invE mRNA and involvement of RNA binding protein Hfq: (i) a significant level of invE mRNA exists in the absence of InvE protein synthesis at 30 °C (34); (ii) the synthesis of β-galactosidase encoded by the invETx-lacZ transcriptional fusion was not repressed under the repressing conditions at 30 °C, but that from the invETL-lacZ translational fusion was repressed under the same conditions (see Fig. 1B, graph 3); (iii) translation of invE mRNA, which was transcribed from an arabinose-inducible promoter, was also repressed at 30 °C even in the presence of a sufficient amount of invE mRNA (see Fig. 2B); (iv) in a deletion mutant of RNA-binding protein Hfq, whose binding to invE RNA is affected by temperature (see Figs. 5 and 6), InvE and IpaB proteins were synthesized even under the repressing conditions at 30 °C (see Fig. 3B); (v) the overexpression of Hfq protein resulted in repression of InvE and IpaB synthesis not only at 30 °C but also at 37 °C (see Fig. 3C); and (vi) the stability of invE mRNA is recovered in the absence of Hfq protein. At the low temperature 30 °C, the decay rate of invE mRNA was significantly higher than that at 37 °C (see Fig. 4). As a result, the level of InvE protein synthesis was markedly reduced at the low temperature. In contrast, in the absence of Hfq, the synthesis of InvE protein was high even at the low temperature because of the improvement of invE mRNA stability. The above features were similar to other post-transcriptionally regulated genes (36, 37) and enforce the our previous result for post-transcriptional regulation of InvE at the expressing conditions (15).

Two previous papers reported the results of different levels of invE (virB) mRNA in Shigella at 30 °C (14, 38). The decrease in invE mRNA had been regarded as a result of transcriptional regulation of regulator cascade, virF and invE genes. The nucleoid protein H-NS was reported to be involved, at least in part, in the transcriptional regulation of virF gene in S. flexneri (11). In the hns mutant, the β-galactosidase activity encoded by virF-lacZ transcriptional fusion plasmid with a limited length of virF promoter increases (13). However, the transcription of native virF mRNA was only partially affected by temperature conditions in the S. flexneri strain (14). Moreover, transcription of the virF gene was virtually unaffected by temperature conditions in our S. sonnei strain, which possesses an insertion sequence-like element in a just upstream region of the virF promoter, whereas the invE expression was affected by temperature. The insertion sequence and the virF promoter sequence were completely identical to those of S. sonnei Ss046 and S. boydii Sb227 strains (see "Experimental Procedures"). The presence of the insertion sequence-like element might be involved in the induction of additional small sized virF transcript, as observed in the Northern analysis (15), and the RT-PCR assay employed in this study could detect the sum of both transcripts that resulted in a similar amount of virF transcription at both 30 and 37 °C. Thus, the contribution of virF transcription for the temperature-dependent regulation is less relevant.

For the molecular mechanism of post-transcriptional regulation, control by translational efficiency has been proposed. Various factors, such as regulatory small RNAs (37), polyamines (39, 40), and regulatory proteins (41), have been shown to be involved. These translational regulation factors are associated with target mRNAs and induce their conformational changes, leading to activate or repress translation. As a well characterized example for translational regulation by temperature, a novel concept has been presented in a heat-shock operon of a plant bacterium, Bradyrhizobium japonicum. The 5'-untranslated region, named the ROSE element, of heat-shock mRNA, permits translation of heat shock gene mRNA at high temperature and represses the translation initiation at low temperature by forming an intrastrand complementary pair. The ROSE element is considered to be an "RNA thermometer" (32, 42, 43). In order to examine how thermodynamic change affects the structure of invE mRNA like ROSE regulation, the 140-nucleotide invE RNA was subjected to CD spectroscopy (32) at various temperatures. The change of the signal was actually detected between 30 and 37 °C (data not shown). Therefore, the self-limited repression by mRNA structure could not be ruled out as a one of the possible mechanisms for translational repression.

In several examples of post-transcriptional regulation by small RNA, the stability of mRNA is markedly decreased in the absence of active translation under the repressing conditions (37). In our case, the expression of InvE was significantly correlated to the mRNA stability, and the recovery of InvE expression in the hfq mutant at 30 °C was also accompanied by the increase in invE mRNA stability. The two kinds of in vitro binding assays between invE RNA and Hfq protein exhibited a significant change in the RNA binding property at the two different temperatures of 30 and 37 °C. As for the temperature-coupled regulation of invE translation in vivo, however, the involvement of another factor, such as small regulatory RNA, cannot be ruled out, because the thermodynamic change should be more sensitive for small regulatory RNA-target RNA interaction than protein-RNA interaction, as in the case of translational control of rpoS mRNA by DsrA RNA (44). The decay of invE RNA is similar to that of iron-regulated gene sodB mRNA by binding of small RNA RyhB (45, 46). In this case, the specific decay of sodB mRNA is recovered by hfq mutation.

Generally, a complementary pair between a regulatory RNA and the mRNA has a limited length and includes an unmatched pairing, both together making it difficult to find such a homology in the genome data base. In addition, if the interaction between the two RNA molecules is highly sensitive to a slight thermodynamic change in the formation of the complementary pair, the sequence for pairing may be relatively short and the interaction may be weak. Therefore, such a regulatory RNA for the stability control of invE mRNA remains to be identified. Nevertheless, the thermodynamic interaction(s) of invE mRNA with either Hfq or the putative regulatory RNA may contribute to the tight and sensitive repression of InvE expression.

In addition to the expression of TTSS genes, the hfq mutant showed increased efficiency for bacterial cell invasion into HeLa cells. Since the expression of an invasion protein VirG (47) was also increased in the hfq mutant (data not shown), the increased efficiency would represent the sum of effect(s) of the hfq mutation on many factors, including TTSS. Although the enhancement of invasion was observed in the cultured cell line, the hfq mutation may reduce the overall virulence in the natural infection cycle of Shigella. Because the Hfq is involved in the control of expression of a number of genes, including the stress response genes, which are crucial for surviving in the infection cycle, all hfq mutants in various Gram-negative bacteria actually lose the virulence in animal infection models (35, 37, 48-51). However, in these animal infection experiments, the versatile effects of hfq mutation make it difficult to identify specific role(s) of Hfq on virulence gene expression. The specific role of Hfq in virulence has been addressed in the SPI-1 TTSS gene of S. typhimurium. Although the interaction between Hfq and mRNA of regulator protein HilA remains to be resolved, the hfq mutation reduced the SPI-1 expression through the post-transcriptional repression of HilA expression (35).

The result of our invasion assay by use of an HeLa cell was contradictory to the result in a recent study in which an hfq::aphA deletion mutant of S. flexneri lost the ability for invasion into human intestinal cell line Henle 407 (52). This study, however, lacks the complementation experiment with a hfq expression plasmid. In our result, the efficiency of invasion instead increased in the hfq mutant (see Table 2). In order to confirm whether the discrepancy results from the different genetic background or a difference in the two hfq deletion mutants, we constructed the same hfq::aphA deletion of S. flexneri 2457T in addition to our S. sonnei strain MS390 (see Fig. 3A). Both of the two hfq::aphA mutants, MF4835 and MS4835, also expressed InvE and IpaB proteins at 30 °C, and the InvE expression potentially increased at 37 °C as well as the complete hfq deletion mutant MS4831 (data not shown). We noticed that all of the hfq mutants were prone to lose the virulence plasmid. Considerable numbers of too large colonies with rough appearance were isolated whenever single colony isolation was done on selective plates. The large colonies lost the virulence plasmid, which was confirmed by PCR analysis for the invE gene. Therefore, the result in the aforementioned report may be caused by unexpected loss of the virulence or difference of the two human cell lines used. Considering the differentiation to intestinal epithelium in Henle cells, a putative barrier that prevents invasion from potentially weak hfq mutant could be an explanation.

Post-transcriptional control of TTSS expression in S. sonnei can be seen as wasteful that produces the untranslated invE mRNA under repressing conditions. However, considering the energetic consumption for the expression of a huge number of TTSS genes that becomes a potential burden for survival in the environment, the complete repression of TTSS by the post-transcriptional regulation of an upstream regulator may have a biological significance for this organism.


   FOOTNOTES
 
* This work was supported by Ministry of Health, Labor, and Welfare Grant-in-aid H19· kokusai-igaku and by the Ministry of Education, Science and Technology of the Japanese Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 To whom correspondence should be addressed. Tel.: 81-3-5285-1111 (ext. 2201); Fax: 81-3-5285-1171; E-mail: haruwata{at}nih.go.jp.

2 The abbreviations used are: TTSS, Type III secretion system; IPTG, isopropyl-1-thio-β-D-galactoside; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
Cloning vectors, pBAD24 and pTH18ks5 were kindly provided by the cloning vector collection of the National Institute of Genetics (Japan).



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