Expression of the Escherichia coli ompW colicin S4 receptor gene is regulated by temperature and modulated by the H-NS and StpA nucleoid-associated proteins

Authors

  • Luciano Brambilla,

    1. Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Rosario, Argentina
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  • Jorgelina Morán-Barrio,

    1. Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Rosario, Argentina
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  • Alejandro M. Viale

    Corresponding author
    1. Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Rosario, Argentina
    • Correspondence: Alejandro M. Viale, IBR, Suipacha 590, 2000 Rosario, Argentina. Tel.: 54 341 4350661; fax: 54 341 4390465; e-mail: viale@ibr-conicet.gov.ar

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Abstract

The OmpW family consists of a ubiquitous group of small outer membrane (OM) β-barrel proteins of Gram-negative bacteria with proposed roles in environmental adaptation but poorly understood mechanisms of expression. We report here that Escherichia coli K-12 OmpW contents are drastically modified by temperature changes compatible with the leap from the environment to warm-blooded hosts and/or vice versa. Thus, while OmpW is present in the OM of bacteria grown at 37 °C, it sharply disappears at 23 °C with the concomitant acquisition of colicin S4 resistance by the cells. ompW::lacZY fusions indicated that temperature regulation operates at the level of transcription, being ompW expression almost abolished at 23 °C as compared to 37 °C. Moreover, E. coli Δhns mutants lacking H-NS showed reductions in ompW transcription and OmpW contents at 37 °C, indicating positive modulatory roles for this nucleoid-structuring protein in ompW expression. Also, ΔhnsΔstpA double mutants simultaneously lacking H-NS and its paralog StpA showed more severe reductions in ompW expression at 37 °C, resulting in the complete loss of OmpW. The overall results indicate that OmpW contents in E. coli are regulated by both temperature and H-NS and reinforce OmpW functions in bacterial adaptation to warm-blooded hosts.

Introduction

The regulation of the production of outer membrane (OM) proteins of Gram-negative bacteria in response to different environmental cues plays a pivotal role in the adaptation of these organisms to changing environments (Lin et al., 2002; Nikaido, 2003; De la Cruz & Calva, 2010; McClean, 2012). OmpW, an OM protein that forms part of the colicin S4 receptor in Escherichia coli (Pilsl et al., 1999; Arnold et al., 2009), is a member of a widespread family of so-called small β-barrel proteins composed by 210–230 amino acid residues structured in 8 transmembrane β-strands and extended loops exposed to the environment (Nikaido, 2003; De la Cruz & Calva, 2010; McClean, 2012). The contents of OmpW have been found to vary significantly in response to different environmental cues both in E. coli and in many other Gram-negative bacterial species, thus suggesting functions in environmental adaptation (Bina et al., 2003; Hu et al., 2005; Nandi et al., 2005; Xu et al., 2006; Gil et al., 2007; Lin et al., 2008, 2010; Wu et al., 2009; McClean, 2012). Most notably, some environmental stimuli increase OmpW contents while others reduce it (Bina et al., 2003; Hu et al., 2005; Nandi et al., 2005; Xu et al., 2006; Gil et al., 2007; Lin et al., 2008, 2010; Wu et al., 2009), thus suggesting the involvement of a complex regulatory network in this process whose details are largely unknown.

Temperature change is a well-known cue used by both commensalistic and pathogenic bacteria signaling the transfer from the external environment into warm-blooded hosts and the necessity to adjust gene expression accordingly (De la Cruz & Calva, 2010; Shapiro & Cowen, 2012). In this work, we demonstrate that E. coli OmpW contents are regulated by temperature changes compatible with the leap from environmental sources to potential hosts and vice versa. We also found that ompW expression was significantly reduced in E. coli Δhns mutant cells bearing deletions in the hns gene encoding the histone-like nucleoid-structuring H-NS protein (Fang & Rimsky, 2008; Stoebel et al., 2008; Dillon & Dorman, 2010; Shapiro & Cowen, 2012). Moreover, ompW expression impairments were exacerbated in ΔhnsΔstpA double mutants additionally lacking StpA, an H-NS paralog and proposed molecular backup. As these nucleoid-binding proteins impose both local and large scale-constraints on DNA topology and their binding activities are sensitive to temperature variations (Fang & Rimsky, 2008; Stoebel et al., 2008; Dillon & Dorman, 2010; Shapiro & Cowen, 2012), the overall results provide evidence for complementary roles of H-NS and StpA in favoring a particular DNA architecture enhancing productive ompW expression at temperatures compatible with potential warm-blooded hosts.

Materials and methods

Bacterial strains and culture conditions

All bacterial strains were grown aerobically in LB liquid or solid medium containing the appropriate antibiotics when corresponding (Km 30 μg mL−1; chloramphenicol 15 μg mL−1) at 23 or 37 °C as indicated in the legends to figures. Solid LB medium included 1.5% (w/v) Bacto agar. The E. coli K-12 strain BW25113 (lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) and the isogenic ΔompW, Δhns, and ΔstpA single mutants (in which the corresponding genes were replaced by a npt cassette conferring kanamycin (Km) resistance) were obtained from the Keio collection (Baba et al., 2006). To construct the ΔhnsΔstpA double mutant of this strain, the npt cassette was first removed from the Δhns::npt mutant by transforming cells with the temperature-sensitive plasmid pCP20 carrying the FLP recombinase (Cherepanov & Wackernagel, 1995). The resulting kanamycin-sensitive single mutants (Δhns*) were then used as hosts for P1 transduction using lysates obtained from ΔstpA::npt bacteria (Miller, 1972) to generate ΔhnsstpA::npt double mutants. A similar procedure was employed to remove the npt cassette from these cells to generate kanamycin-sensitive ΔhnsstpA* double mutants.

The E. coli K-12 strain MC4100 [F, araD139, Δ(argF-lac)169, λ, e14, flhD5301, Δ(fruK-yeiR)725(fruA25), relA1, rpsL150(strR), rbsR22, Δ(fimB-fimE)632(::IS1), deoC1] was from the laboratory stock.

Colicin S4 sensitivity assay

The E. coli 5K strain bearing plasmid pHP189 (Pilsl et al., 1999) was the source of colicin S4. After growing overnight in LB liquid medium at 37 °C, the cells were collected by centrifugation (15 000 g for 10 min), resuspended in 50 mM Tris-HCl (pH 7.5), glycerol 10% (v/v), and subjected to sonic disruption. Crude extracts were then cleared by centrifugation as above, and the supernatant was used for colicin sensitivity assays.

To test colicin S4 sensitivity, the BW25113 (wt) strain was grown overnight in LB liquid medium at 23 or 37 °C. Aliquots of 0.1 mL of this culture were overlaid on LB agar plates previously kept at 23 or 37 °C, and 5 μL of 10-fold serial dilutions of colicin S4 crude extracts prepared as above were then dropped into the agar surface following a clockwise pattern as shown in Fig. 1a. The plates were further incubated for 24–48 h at the corresponding temperature, and the differences in colicin sensitivity were recorded. A BW25113 ΔompW mutant was also included in the assay as a control of colicin S4 resistance (Pilsl et al., 1999; Arnold et al., 2009).

Figure 1.

Temperature regulation of Escherichia coli OmpW contents. (a) Colicin S4 sensitivity assay of BW25113 (wt) grown at 23 or 37 °C. Five μL of 10-fold serial dilutions of colicin S4 crude extracts were dropped into the agar surface following a clockwise pattern as indicated. Isogenic ΔompW mutants (ΔompW) are also included as controls of complete colicin resistance. (b) Left, SDS–polyacrylamide gel electrophoresis analysis of OM fractions isolated from BW25113 cells grown in LB medium at 23 or 37 °C. Right, immunoblot analysis of the corresponding OM fractions using anti-OmpW. The equivalent to 30 μg of total OM proteins was loaded on each well in all cases. (c) Expression of ompW::lacZY transcriptional fusions in BW25113 cells grown at 23 or 37 °C. The schematic representation of the E. coli genomic locus containing the divergently encoded ompW and yciC genes and the corresponding transcription initiation sites (Mendoza-Vargas et al., 2009; Zhou & Rudd, 2013) are shown at the top. β-galactosidase values for a given fusion indicate the percentage with respect to the value measured at 37 °C, and the mean and standard deviation of at least three independent measurements are shown. For details, see 'Materials and methods'.

Analysis of OM proteins by SDS–polyacrylamide gel electrophoresis

Escherichia coli OM fractions were isolated following previously described protocols (Brambilla et al., 2014) and analyzed by SDS-PAGE analysis using 12% (w/v) polyacrylamide gels (Sambrook et al., 1989). The relative levels of OmpW as compared to OmpA in the OM of the different bacterial strains at 23 or 37 °C were estimated by densitometric scanning of the gels with the aid of ImageQuant, version 5.2 (Molecular Dynamics, Inc.). The OmpA protein was selected for this purpose because ompA expression is not subjected to temperature regulation (White-Ziegler & Davis, 2009).

Construction of lacZY transcriptional fusions

The npt cassette was removed from ΔompW::npt or ΔyciC::npt mutants of the Keio collection (Baba et al., 2006) as described earlier. The scar sequence left behind containing the 34 nucleotide FRT site was used to generate the corresponding lacZY transcriptional fusions (Ellermeier et al., 2002). All constructions were verified using PCR using the corresponding forward primers (Baba et al., 2006) and oligonucleotide 5′-GTAACCGACCCAGCGCCC-3′ as the reverse primer annealing 567-bp downstream of the lacZ initiation codon (Ellermeier et al., 2002). To construct Δhns, ΔstpA, or ΔhnsΔstpA mutants containing ompW::lacZY fusions, the npt cassettes were first removed from the corresponding mutants as described above, and the resulting kanamycin-sensitive cells were then used as hosts for P1 transduction using lysates obtained from BW25113 ompW::lacZY bacteria constructed as described above following conventional procedures (Miller, 1972).

β-galactosidase activities were determined at 30 °C in chloroform-permeabilized cells using ortho-nitrophenyl-β-galactoside (ONPG) as substrate following described protocols (Miller, 1972). Aliquots of bacterial cultures grown in LB medium at 23 or 37 °C to an A600 nm of 2.5 were employed for this purpose, thus reflecting ompW expression over long-term growth. The β-galactosidase activities in each case were first calculated in Miller units, and the results expressed as the percentage of the units obtained for each strain as compared to the activity measured for the wt strain at 37 °C.

OmpW purification, antibody production, and immunoblot analysis

The OmpW protein used as antigen was purified from OM fractions of E. coli BL21 (DE3) cells containing plasmid pHP211 (Pilsl et al., 1999) by preparative SDS-PAGE (Sambrook et al., 1989) using 12% polyacrylamide gels. Polyclonal antibodies were elicited in rabbits following conventional procedures (Sambrook et al., 1989). Immunoblot analysis was performed by transferring the proteins from the SDS-PAGE gels to polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia) followed by OmpW detection with rabbit polyclonal antibodies as first antibody and alkaline phosphatase-conjugated anti-rabbit IgG as second antibody as described (Brambilla et al., 2014).

Results and discussion

Escherichia coli OmpW contents are regulated by temperature

In the course of our studies on the biogenesis of E. coli OmpW, we observed that E. coli BW25113 (wt) cells showed markedly increased resistance to colicin S4 when grown at 23 °C as compared to 37 °C (Fig. 1a). As OmpW forms part of the E. coli colicin S4 receptor and bacterial mutants lacking this OM protein (ΔompW mutants) show full resistance to this colicin (Pilsl et al., 1999; Arnold et al., 2009) as also shown in Fig. 1a, the above observations suggested that OmpW contents were strongly reduced or even absent at 23 °C. To analyze this possibility, we first compared the OmpW contents in the OM of E. coli BW25113 cells grown to stationary phase at 23 and 37 °C (Fig. 1b). As seen in the figure, both SDS-PAGE and immunoblot analyses indicated almost nondetectable amounts of OmpW in the OM of cells grown at 23 °C, thus explaining the colicin resistance of cells grown at this temperature. The same results were obtained with the E. coli K-12 strain MC4100 (not shown).

ompW expression is regulated by temperature at the transcription level

The drastic reduction in E. coli OmpW contents observed at 23 °C could reflect temperature regulation of ompW transcription or, alternatively, different post-transcriptional processes at the level of protein secretion or transit to the OM (Lin et al., 2002; Nikaido, 2003; De la Cruz & Calva, 2010; McClean, 2012). We thus constructed ompW::lacZY transcriptional fusions on BW25113 cells to analyze the effects of growth temperature on ompW expression. Expression of the yciC gene located in the same genomic locus than ompW but divergently oriented (Fig. 1c) was also analyzed using yciC::lacZY fusions, to study whether this particular gene clustering bears regulatory relevance. As seen in Fig. 1c, very low ompW transcription was detected at 23 °C as compared to 37 °C, as judged by the levels of β-galactosidase activity obtained in cells bearing ompW::lacZY fusions collected at late exponential phase. Similar differences were also obtained in cells grown at 23 or 37 °C to early exponential phase (A600 nm = 1.0, data not shown).

The overall results thus reinforce the notion that temperature regulation of ompW expression resides mainly at the transcriptional level. On the contrary, no significant differences in the expression of the yciC gene were observed between 23 and 37 °C (Fig. 1c), thus indicating that temperature regulation is restricted to the ompW gene in this common genomic locus.

Genetic identification of the H-NS and StpA nucleoid-associated proteins as modulators of ompW expression

The nucleoid-structuring H-NS protein is an abundant DNA-binding protein involved in the regulation of a substantial number of temperature-responsive E. coli genes (White-Ziegler & Davis, 2009). We thus decided to analyze whether H-NS could also mediate temperature regulation of ompW expression, and for this purpose, we measured both ompW expression and OM OmpW contents in E. coli Δhns mutants grown at 23 and 37 °C (Fig. 2). As seen in Fig. 2a, ompW transcription was negatively affected at 37 °C by H-NS loss as judged by the almost 50% reduction in the relative levels of ompW::lacZY expression found in Δhns mutants when compared to wt cells grown at this temperature (Fig. 2a). The ompW transcript levels attained at 37 °C in Δhns mutants, however, were still sufficient to induce significant synthesis of OmpW as judged by the relative content of this protein in the OM of these mutants which were around 60% of the wt strain (Fig. 2b and c). At 23 °C, ompW transcription in Δhns mutants was highly impaired similarly to the case of wt cells (Fig. 2a). This reduced ompW transcriptional activity was also negatively affected by H-NS loss as judged by further reductions in β-galactosidase activity observed in Δhns mutants as compared to wt cells at 23 °C (Fig. 2a). Thus, the overall results shown above suggest separable effects of temperature and H-NS on ompW expression.

Figure 2.

Positive modulation of Escherichia coli ompW expression by H-NS. (a) Expression of ompW in BW25113 (wt), Δhns, ΔstpA, and ΔhnsΔstpA cells grown at 23 or 37 °C. β-galactosidase values indicate the percentage with respect to 37 °C, and the mean and standard deviation of at least three independent measurements are shown. (b) SDS-PAGE analysis of OM fractions derived from the above cells grown at 37 °C. The final positions of OmpW as well as the OmpC/OmpF and OmpA porins (Brambilla et al., 2014) are shown on the left margin. The final positions of the molecular mass markers are also indicated. (c) Relative OmpW contents (as compared to OmpA) in the OM fractions of cells shown in (b). For details, see 'Materials and methods'.

Escherichia coli contains an H-NS paralog, StpA, which shares around 58% identity with H-NS and also belongs to the nucleoid-associated proteins group (Sonden & Uhlin, 1996; Zhang et al., 1996; Stoebel et al., 2008; Dillon & Dorman, 2010). Although stpA deletion does not promote a notable phenotype to E. coli as compared to hns deletion, the current evidence suggests both complementary as well as individual roles for these two proteins on gene expression and also in thermoregulation (Sonden & Uhlin, 1996; Zhang et al., 1996; Johansson et al., 1998; Dillon & Dorman, 2010).

We thus analyzed next whether StpA could play complementary roles to H-NS in mediating ompW expression, and for this purpose, we analyzed ompW::lacZY expression and OmpW contents in ΔstpA and ΔhnsΔstpA mutants and compared these results with those of Δhns single mutants discussed earlier (Fig. 2). As seen in Fig. 2, ompW expression at 37 °C was impaired in ΔhnsΔstpA cells to an extent even larger than that observed in Δhns single mutants, as judged by both β-galactosidase activity values (Fig. 2a) and the absence of OmpW in the corresponding OM fractions (Fig. 2b and c). On the contrary, no impairment on ompW::lacZY expression at 37 °C was observed in ΔstpA single mutants, and actually, relative increases of β-galactosidase activity values were obtained in these cells (Fig. 2a). Still, this increased ompW transcriptional activity observed in ΔstpA mutants was not reflected in higher OmpW contents as judged by the similar relative amounts of this OM protein between ΔstpA mutants and wt cells (Fig. 2b and c). At 23 °C, ompW transcription was highly impaired in both ΔstpA and ΔhnsΔstpA mutants, similarly to the case of wt and Δhns mutants shown above (Fig. 2a).

The observations above thus indicated positive roles for H-NS in the regulation of E. coli ompW expression. Moreover, they also pointed for complementary roles between H-NS and StpA in mediating ompW expression, thus reinforcing the idea that StpA can function as a molecular backup for the activating role of H-NS (Sonden & Uhlin, 1996; Zhang et al., 1996; Johansson et al., 1998). However, the observation that ompW expression was significantly affected in Δhns but not in ΔstpA single mutants (Fig. 2) also disclosed differences between H-NS and StpA in mediating this regulation. The higher ompW transcription levels found in ΔstpA cells at 37 °C (Fig. 2) could be explained by the loss of the StpA repressive activity on hns expression (Zhang et al., 1996). The higher H-NS contents expected on these cells could result in consequent stimulations of ompW expression as suggested from the results of this work.

H-NS is an abundant bacterial protein whose binding to the chromosome imposes both local and large scale-constraints on DNA topology which may affect gene expression, usually in a negative manner (Fang & Rimsky, 2008; Stoebel et al., 2008; Dillon & Dorman, 2010; Shapiro & Cowen, 2012). Moreover, H-NS binding to DNA seems extremely sensitive to temperature fluctuations, a property that may account for its role in regulating expression of many temperature-responsive genes (Fang & Rimsky, 2008; Stoebel et al., 2008; Dillon & Dorman, 2010; Shapiro & Cowen, 2012). The formation of complex nucleoprotein structures obstructing RNA polymerase movements has generally been accounted for transcriptional arrest (Fang & Rimsky, 2008; Stoebel et al., 2008; Dillon & Dorman, 2010; Shapiro & Cowen, 2012). However, positive roles of H-NS in the expression of a number of E. coli genes have also been reported (Johansson et al., 1998; White-Ziegler et al., 2000; Müller et al., 2006; White-Ziegler & Davis, 2009; Park et al., 2010). In this context, it has been also noted that some configurations may exist in which H-NS could stabilize a particular DNA conformation that favors the correct recognition of promoter sequences by the RNA polymerase and/or a positive transcriptional regulator, or exclude a repressor (Fang & Rimsky, 2008; Stoebel et al., 2008; Singh & Grainger, 2013). ompW thus represents another example of a temperature-regulated gene whose expression is positively mediated by H-NS. It is noteworthy, however, that cells lacking H-NS showed reduced but not complete impairments of ompW transcription at 37 °C and that the additional removal of StpA was required to abolish OmpW synthesis (Fig. 2). This argues for a regulatory mechanism in which these nucleoid-binding proteins act better by stabilizing a particular DNA topology that favors the productive recognition of the ompW promoter by the RNA polymerase, rather than indicating an absolute requirement of these proteins for ompW expression.

The results above also support the notion that OmpW roles are more related to the interaction of the E. coli cell with its warm-blooded hosts (McClean, 2012), and, in fact, the shielding of this colicin receptor outside the host probably increases survival advantage (Govan, 1986). Providing the various causes that can differentially modify OmpW contents in bacteria (Bina et al., 2003; Hu et al., 2005; Nandi et al., 2005; Xu et al., 2006; Gil et al., 2007; Lin et al., 2008, 2010; Wu et al., 2009), it seems likely that both positive and negative factors concertedly act in regulating ompW expression. Further work is being conducted to elucidate these factors and the roles played by OmpW in bacterial physiology.

Acknowlegements

This work was supported by grants from CONICET and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT). A.M.V. and J.M.B. are career investigators of CONICET, and L.B. a former Fellow of this institution. We are indebted to Dr H. Pilsl (Universität Tübingen, Tübingen, Germany) for the generous gift of E. coli strains and plasmids from his collection.

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