Bacterial strains, plasmids and culture conditions

To construct plasmids pBADEilA, pBADYgeH₀₄₂, pBADHilA and pBADYgeH_O157, eilA and ygeH genes of strain E. coli 042, hilA gene of S. Typhimurium SL1344 strain and ygeH gene from E. coli O157H7 were amplified using oligonucleotides EILAKPNI5-EILAHINDIII3, YGEHKPNI5-YGEHHINDIII3, HILABADFW-HILABADRV and YGEHO157XbaFW-YGEHO157HindRV, respectively (see Additional file 1). The oligonucleotides add Kpn I and Hind III sites, to the eilA and ygeH genes from E. coli 042, Eco RI and Xba I sites to the hilA gene and Xba I - Hind III sites to ygeH gene from E. coli O157:H7. The corresponding Kpn I-Hind III, Eco RI-Xba I and Xba I-Hind III PCR fragments were cloned into pBAD18 digested with the same enzymes, resulting in plasmids pBADEilA, pBADYgeH₀₄₂, pBADHilA and pBADYgeH_O157, respectively.

Genetic manipulations

Standard molecular and genetic procedures were performed as described by [28]. Enzymes were used according to the manufacturer’s recommendations. Introduction of plasmids into Escherichia coli and S. Typhimurium strains was performed by electroporation of 10% glycerol-washed cells using an Eppendorf gene pulser (Electroporator 2510). Plasmids isolated from E. coli were first passaged through the restriction-deficient S. Typhimurium strain LB5000 [21] before transformation of S. Typhimurium SL1344 competent cells.

Chromosomal deletions of hilA from S. Typhimurium, hns, ihfA and ihfB from E. coli AAG1 and ygeH and hns from E. coli 042 were obtained by the λ Red recombinant method as previously described [23]. The antibiotic-resistance determinant of plasmid pKD4 was amplified using oligonucleotides HNSP1/HNSP2, YGEHP1/YGEHP2 for hns and ygeH genes, and, in the case of hilA, ihfA and ihfB deletions, the chloramphenicol resistance was amplified using as template the pKD3 plasmid and the oligonucleotides HIlAP1/HILAP2 and IHFAP1/IHFAP2, IHFBP1/IHFBP2 (see Additional file 1). Mutants were selected on LB plates containing the appropriate selection marker, and the successful deletion of the gene was confirmed by PCR using the primers CAT-C1 and CAT-C2 (chloramphenicol resistance; Cm^r) or KT and K2 (kanamycin resistance; Km^r) in combination with specific primers located in the remaining gene sequence nearby (Additional file 1, P1UP/P2DOWN series oligonucleotides). When necessary, the antibiotic resistance was then eliminated by transforming the mutant strain with plasmid pCP20 and subsequent incubation at 43°C for two passages according to [23]. The double deletions were obtained by combining one previously deletion with other deletion associated with an antibiotic resistance.

Chromosomal insertions of 3XFLAG sequences to the sipA and ygeH genes were obtained by a modification of the λ Red recombinant method, as described by [25]. The antibiotic-resistance determinant of plasmid pSUB11 was amplified using oligonucleotides SIPA3XP1/SIPA3XP2 and YGEH3XP1/YGEH3XP2 for sipA and ygeH, respectively (Additional file 1). Mutants were selected on LB plates containing kanamycin, and successful 3XFLAG insertion was confirmed by PCR using the oligonucleotides KT and K2 (kanamycin resistance; Km^r) in combination with specific oligonucleotides located in the remaining gene sequence nearby (Additional file 1, 3XP1UP/3XP2DOWN series oligonucleotides). The SV5015SipA strain was used as a donor strain to transfer the SipA::3xFLAG fusion to strain SV5015HilA using phage P22 HT [29], generating strain SV5015SH. The chromosomal fusion YgeH::3xFLAG was constructed in the parental strain E. coli 042 and the isogenic mutant Δhns, generating 042YgeH3X and 042YgeH3XHNS strains, respectively.

Construction of an ygeH::lacZ transcriptional fusion

A transcriptional ygeH::lacZ fusion was constructed by cloning the promoter region of the ygeH gene from EAEC 042 into the pRS551 plasmid. A single copy of the fusion was then inserted into the chromosome of the AAG1 strain by using the bacteriophage λRS45 as described by [27]. First of all, the promoter region of the ygeH gene was amplified using the oligonucleotides YGEHBAMHI5/YGEHBAMHI3 (Additional file 1) that add a Bam HI site, and the resulting PCR product was purified using the kit High Pure PCR Product Purification Kit (Roche). Subsequently, the purified PCR product was digested with Bam HI enzyme (Fermentas) and ligated into the Bam HI-digested pRS551, generating the plasmid pYgeH. The transcriptional fusion ygeH::lacZ cloned into the plasmid pYgeH was then transferred to the λRS45 bacteriophage according to [27] and integrated as a single copy into the attB locus of the AAG1 chromosome. To confirm the correct insertion from the selected resulting colonies, a PCR screening was performed according to [30].

Beta-galactosidase assay

β-galactosidase activity measurements were performed as described by [31].

Cell-free supernatant preparation

The supernatants from cultures were obtained utilizing trichloroacetic acid (TCA) as agent for protein precipitation and acetone for removal traces from TCA treatment. The cultures were grown in LB medium until O.D._600nm at 2.0, and then 10 ml were centrifuged at 6.000 rpm for 10 minutes at room temperature. The supernatant was filtered with a 0.22 μm pore size filter (Millex GP, Millipore) and proteins were precipitated by adding TCA at a final concentration of 10% and maintaining samples on ice for 1 hour. The samples were then centrifuged for 30 minutes at 13.400 rpm at 4°C, the supernatants were discarded and 0.5 ml of acetone was added. The samples were centrifuged once more as described above. After removal of acetone the pellet was air-dried for 10 minutes and the samples were reconstituted in sample buffer (Laemmli sample buffer, Bio-Rad).

SDS-PAGE and western blotting

Protein samples were analysed by SDS-PAGE at 10% and 12.5%. Proteins were transferred from the gels to PVDF membranes. Western blot analysis was performed with monoclonal antibodies raised against FLAG-epitope (1:10.000, Sigma) or against GAPDH (1:2000, Thermo Scientific) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2500, Promega). Detection was performed by enhanced chemiluminescence using Quantity One software (Bio-Rad).

RNA isolation

Total RNA was extracted from bacteria using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction. Potential traces of DNA were removed by digestion with DNase I (Turbo DNA-free, Ambion), according to the manufacturer’s protocol. RNA concentration and RNA quality were measured using a Nano- Drop 1000 (Thermo Fisher Scientific).

Real-time qRT-PCR

Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed as previously described [32]. Briefly, 1 μg of total RNA was reverse transcribed to generate cDNA using the High-capacity cDNA Reverse Transcription kit (Applied Biosystems) as recommended by the manufacturer. As a control, parallel samples were run in which reverse transcriptase was omitted from the reaction mixture. Oligonucleotides complementary to the genes of interest were designed using primer3 software. Real-time PCR using SYBR green PCR master mix (Applied Biosystems) was carried out on the ABI Prism 7700 sequence detection system (Applied Biosystems). After analysis of amplification plots with the ABI Prism sds software package, relative quantification of gene transcription was performed using the comparative threshold cycle (CT) method. The relative amount of target cDNA was normalized using the gapA gene as an internal reference standard.

Electrophoretic mobility shift assay (EMSA)

For EMSA, a 228 bp fragment corresponding to the promoter region of the ygeH operon (nucleotides -207 to +21) was amplified using oligonucleotides YGEHBAMHI5/YGEHBAMHI3 (Additional file 1). For each reaction, 50 ng DNA were mixed with increasing concentrations of the purified H-NS-His protein in binding buffer (250 mM HEPES, pH 7.4, 350 mM KCl, 5 mM EDTA, 5 mM DTT, 500 μg BSA ml^-1, 25% glycerol). After incubation for 30 min at room temperature, 20 μl of samples were separated on 5% polyacrylamide/0.5 TBE gel. The bands were stained using ethidium bromide and visualized using Quantity One software (Bio-Rad). For competitive EMSA, the fragment corresponding to the ygeH promoter region DNA (50 ng) was mixed with a 3 fold excess of competitor DNA (hlyR[33]) in a final volume of 20 μl, using the same binding buffer as described above. Increasing concentrations of H-NS His were added as indicated, and the reaction was incubated at room temperature for 30 min. The samples were resolved, stained and visualized as described above. Purified H-NS His protein was obtained as described [34], and the hlyR DNA was obtained as described by [33].

The invasion gene regulator coded by ygeH shares significant homology to EilA and HilA proteins

The complete genome sequence of E. coli 042 strain has led to the identification of two open reading frames encoding two proteins EilA (565 aminoacids) and YgeH (458 aminoacids) that exhibit similarity with the S. Typhimurium HilA protein (37% and 29% of similarity with HilA, respectively). In addition to the already reported EilA regulator [19], the putative invasion gene regulator YgeH shows a significant degree of conservation (Figure 1). Whereas EilA appears to be restricted to E. coli 042 and other enteroaggregative strains, YgeH is also present in other E. coli strains, such as K12, enterohemorrhagic O157:H7, enteroaggregative hemorrhagic O104:H4 and others [18]. In spite of the significant degree of identity at the amino acid level between HilA and YgeH, no information is available about its biological activity, neither regulatory role nor its targets. Thus, we decided to further characterize this latter protein from strain 042.

EilA and YgeH proteins from E. coli strain 042 complement the HilA− phenotype in S. Typhimurim strain SV5015

We addressed first the question whether the YgeH proteins from strain 042 (YgeH₀₄₂) and from strain O157 (YgeH_O157) could be functionally equivalent to HilA. To test this, we first constructed a ΔhilA mutant of S. Typhimurium strain SV5015 (see Methods section). To monitor HilA activity we tested the expression of the sipA gene. The sipA gene is encoded in the sic/sip operon. The SipA effector is secreted by the type III secretion system encoded in the Salmonella pathogenicity island 1 (SPI1). Expression and translocation of SipA requires HilA activation of SPI1 operons [12],[35]. To detect SipA, a 3xFLAG was fused to its C-terminal end. For the complementation studies, eilA and ygeH genes from strain 042 and the ygeH gene from strain O157 were cloned in the expression vector pBAD18 [26]. Wild-type SV5015SipA (SipA::3xFLAG) and its ΔhilA derivative (SV5015 SipA::3xFLAG ΔhilA) were transformed with plasmids pBAD18, pBADHilA, pABDEilA, pBADYgeH₀₄₂ and pBADYgeH_O157. Cultures of the different transformants were grown in LB medium containing carbenicillin. Proteins present in the corresponding supernatants were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining (Figure 2A). Moreover SipA::3xFLAG was immunodetected with anti-flag specific antibodies. As expected, SipA protein could only be detected in wt strain SV5015 SipA::3xFLAG, but not in the corresponding ΔhilA derivative (Figures 2C). Alternatively, cultures of these strains were grown in LB medium containing carbenicillin and 0.2% arabinose. Overexpression of EilA, YgeH₀₄₂, and YgeH_O157 upon arabinose induction was confirmed by qRT-PCR. Upon arabinose inducing conditions, plasmids pBADHilA, pBADEilA, and pBADYgeH₀₄₂ complemented in trans the ΔhilA mutation (Figure 2B and D). Interestingly, plasmid pBADYgeH_O157 does not complement ΔhilA. The amount of expressed SipA::3xFLAG was very similar in strains harbouring either pBADHilA, pBADEilA or pBADYgeH. Other effector proteins could also be detected in cells expressing any of the three HilA-like modulators (Figure 2B). These results provide evidence for E. coli strain 042 EilA or YgeH proteins being able to functionally replace HilA in Salmonella, but not for YgeH_O157. It is noteworthy that, compared to YgeH₀₄₂, YgeH_O157 presents 22 amino acid substitutions, that might result in loss of protein function (see Discussion section).

EilA and YgeH042 activate sipA expression via InvF in Salmonella

The regulatory cascade leading to sipA expression requires HilA-mediated activation of InvF which, in turn, activates SipA [36]. To check if either EilA or YgeH₀₄₂ would influence sipA expression by activating InvF, strain SV5258 (invF::lacZ ) was used. A ΔhilA derivative was constructed by transducing the ΔhilA::Cm allele from strain MH-C1. Strain SV5258 was transformed with plasmid pBAD18, and strain SV5258hilA was transformed with plasmids pABD18, pBADHilA, pBADEilA and pBADYgeH₀₄₂. The corresponding transformants were grown at 37°C to the beginning of the stationary phase (OD₆₀₀ 2.0) either in LB medium or in LB medium containing 0.02% arabinose. As expected, neither arabinose nor the vector pBAD18 affected invF expression in the hilA ⁺ strain (Figure 3). In contrast, in ΔhilA mutant strain, the presence of plasmids pBADHilA, pBADEilA or pBADYgeH₀₄₂ drastically increased invF transcription when cells were grown in LB medium containing arabinose (Figure 3). Hence, the effect of both EilA and YgeH₀₄₂ influencing invF transcription is similar to that of HilA.

Regulation of ygeH042 expression

We recently shed light on the role of the nucleoid-associated proteins H-NS and Hha modulating hilA expression in Salmonella in a temperature-and growth phase-dependent manner. IHF appeared to antagonize H-NS-mediated repression of hilA when cells reach the stationary phase of growth [20]. We decided to study the growth conditions influencing ygeH ₀₄₂ expression and the hypothetical role of the global modulator H-NS on ygeH ₀₄₂ expression. We tried to construct a transcriptional ygeH::lacZ gene fusion in the chromosome of strain 042. Under our working conditions, it was not possible to obtain this fusion in the 042 chromosome. We therefore decided to construct it in strain AAG1 (a ΔlacZ derivative from strain MG1655). Expression studies were performed in wt cells and in cells lacking the modulators H-NS and/or IHF at varying temperatures and osmolarities, in exponentially growing cells as well as in cells entering the stationary phase. The most relevant regulatory transcriptional data obtained in strain AAG1 were thereafter validated in strain 042 by detecting YgeH::3xFLAG by qRT-PCR and Western Blotting, as well (see below).

The expression profile of ygeH ₀₄₂ in different culture condition shows that stationary growth phase influences its expression, moderately increasing it. No effects of growth temperature or osmolarity are apparent (Figure 4A). With respect to the modulators H-NS and IHF, H-NS significantly represses ygeH ₀₄₂ expression in the different conditions tested, including the onset of the stationary phase. IHF appears to be required for a proper expression of ygeH ₀₄₂. The fact that deletion of either ihfA or ihfB only decreases ygeH ₀₄₂ expression when the H-NS protein is available, suggest that, in the ygeH ₀₄₂ regulatory region, IHF might antagonize H-NS (Figure 4B and C).

To confirm that the observed regulatory effect of H-NS on ygeH ₀₄₂ expression in strain AGG1 also takes place in strain 042, ygeH ₀₄₂ transcription was detected in strains 042 and 042Δhns by both real-time qRT-PCR and YgeH protein immunodetection. qRT-PCR shows an upregulation in the Δhns mutant of the +4.5 ± 0.17 compared to wt strain. To detect YgeH₀₄₂, 3xFLAG was fused to its C-terminal end. The YgeH::3xFLAG was immunodectected by using the monoclonal anti-flag antibody. As expected from the results obtained in strain AAG1, expression of ygeH ₀₄₂ increased in the absence of H-NS (Figure 5).

We studied next if H-NS binds with specificity to ygeH ₀₄₂ regulatory region. A virtual footprinting analysis of this region showed that it includes three putative sequences, predicted to be H-NS binding sites (see Additional file 2). A 228 bp DNA fragment that includes the ygeH ₀₄₂ promoter region was used for EMSA assays. According to the transcriptional data, binding of H-NS could be shown for the selected DNA fragment (Figure 6A). To provide evidence for specificity of binding, a competitive band shift experiment was performed, using both the fragment corresponding to the ygeH ₀₄₂ regulatory region, and a DNA fragment, corresponding to the regulatory region of the hly operon (407 bp). H-NS does not show specific binding to this fragment [33]. The competitive band shift assay showed that H-NS preferentially binds to the ygeH ₀₄₂ regulatory region (Figure 6B).

YgeH upregulates ETT2 determinants

Taking into account that YgeH is encoded in the ETT2 island, we decided to test the effect of YgeH depletion on expression of different ETT2 encoded genes. Genes selected corresponded to most of the ETT2-encoded operons, and were the eivF gene, encoding an InvF-like protein, eivA, etrA, eprH, ygeG, ygeK and yqeI (locus tag: EC042_3070, EC042_3060, EC042_3059, EC042_3049, EC042_3053, EC042_3045, respectively). Expression of these genes was first monitored by qRT-PCR in the wt 042 strain and in the ygeH ₀₄₂ single mutant. As expected from YgeH being a transcriptional activator, depletion of ygeH resulted in downregulation of the different ETT2 genes (Figure 7A). Taking into account that ygeH expression is increased in an hns mutant, we decided to further investigate whether, as a response to reduced H-NS levels, ETT2 genes are also upregulated in a Δhns mutant. As expected, all the ETT2 genes tested are upregulated in strain 042 Δhns (Figure 7B). Hence, expression of ETT2 genetic determinants is subjected to H-NS-mediated repression via YgeH.