TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment

Veterinary Microbiology 193 (2016) 42–48

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TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment Jen-Jie Leea , Ying-Chen Wua , Chih-Jung Kuob , Shih-Ling Hsuana,* , Ter-Hsin Chena,c,* a b c

Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taiwan Department of Veterinary Medicine, National Chung Hsing University, Taiwan Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taiwan

A R T I C L E I N F O

Article history: Received 13 June 2016 Received in revised form 2 August 2016 Accepted 4 August 2016 Keywords: Acid tolerance Oxidative stress Salmonella enterica serovar Choleraesuis TolC

A B S T R A C T

The outer membrane protein TolC, which is one of the key components of several multidrug efflux pumps, is thought to be involved in various independent systems in Enterobacteriaceae. Since the acidic environment of the stomach is an important protection barrier against foodborne pathogen infections in hosts, we evaluated whether TolC played a role in the acid tolerance of Salmonella enterica serovar Choleraesuis. Comparison of the acid tolerance of the tolC mutant and the parental wild-type strain showed that the absence of TolC limits the ability of Salmonella to sustain life under extreme acidic conditions. Additionally, the mutant exhibited morphological changes during growth in an acidic medium, leading to the conflicting results of cell viability measured by spectrophotometry and colonyforming unit counting. Reverse-transcriptional-PCR analysis indicated that acid-related molecules, apparatus, or enzymes and oxidation-induced factors were significantly affected by the acidic environment in the null-tolC mutant. The elongated cellular morphology was restored by adding antioxidants to the culture medium. Furthermore, we found that increased cellular antioxidative activity provides an overlapping protection against acid killing, demonstrating the complexity of the bacterial acid stress response. Our findings reinforce the multifunctional characteristics of TolC in acid tolerance or oxidative stress resistance and support the correlative protection mechanism between oxygen- and acidmediated stress responses in Salmonella enterica serovar Choleraesuis. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction During the course of infection, foodborne pathogens encounter a lethal acidic challenge in the stomach after the ingestion of contaminated food or water. Gastric fluid is extremely acidic characteristic (pH < 2) and is an important defense against infections (Smith, 2003). As an acid-sensitive bacterium, Vibrio cholera requires a higher infective dose to successfully infect a host, as compared to bacteria such as Escherichia coli, Shigella and Salmonella enterica, which can cause diseases when fewer than 100 cells are ingested (Schmid-Hempel and Frank, 2007). Moreover, the elderly, children, and people being administered acid suppression medicine are particularly susceptible to contamination and illness (Howell et al., 2010).

* Corresponding authors at: Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, 145 Xingda Road, Taichung 402, Taiwan. E-mail addresses: [email protected] (S.-L. Hsuan), [email protected] (T.-H. Chen). http://dx.doi.org/10.1016/j.vetmic.2016.08.006 0378-1135/ã 2016 Elsevier B.V. All rights reserved.

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) is a nontyphoid serotype with a highly invasive character and is associated with bacteremia and systemic infection (Chiu et al., 2004). As a foodborne and waterborne organism, Salmonella uses various systems to survive in a wide range of pH values in both, natural and host environments (Riesenberg-Wilmes et al., 1996). Previous reports have suggested that Salmonella survives in a lethal acidic environment by inducing the adaptive acid tolerance response (ATR), which can be characterized by the capability to tolerate extreme acidity and enhance the survival rate after treatment with moderately acidic pH (Foster and Hall, 1990; Alvarez-Ordonez et al., 2011). Further studies provided evidence that there are at least two different types of ATRs used by Salmonella (Lee et al., 1994). The Log-phase ATR is induced in logarithmically growing cells and involves more than 40 acidshock proteins that are distinct from stationary-phase acid-shock proteins (Lee et al., 1994). Interestingly, in addition to pH-inducible systems, rpoS also acts as a non-acid-inducible acid-shock protein during the stationary phase and has been studied in enteric bacteria (Fang et al., 1992).

J.-J. Lee et al. / Veterinary Microbiology 193 (2016) 42–48

TolC is an important protein channel located on the bacterial outer membrane and participates in drug resistance. In E. coli and Salmonella Typhimurium (S. Typhimurium), tolC deficiency leads to increased sensitivity to a wide range of molecules, including bile salts, detergents, antibiotics, dyes, and organic solvents (Horiyama et al., 2010). The well-known AcrAB-TolC tripartite efflux pump, which belongs to the resistance-nodulation-division family of drug efflux systems, is composed of periplasmic lipoprotein AcrA, integral membrane protein AcrB, and outer membrane protein TolC (Poole, 2005). Additionally, TolC associates with a variety of inner membrane efflux proteins and recognizes diverse substrates and molecules (Horiyama et al., 2010). The role of TolC in E. coli physiology has been previously examined; the pleiotropic phenotypes of tolC, which are involved in drug resistance, outer membrane composition, virulence regulation, and acid tolerance, may be linked to the complex network of tolC and other regulatory factors (Zgurskaya et al., 2011). Compared to E. coli, no studies have determined the relationship between TolC and acid tolerance in Salmonella, infection with which is a potential public threat. Our study showed that the TolC membrane protein is required for S. Choleraesuis survival, expression of adequate acid-mediated genes, and maintenance of its normal morphology in acidic environments. We further found that morphological changes in tolC-deficient S. Choleraesuis result from the presence of toxic reactive oxygen species (ROS), which are alleviated by antioxidants. Furthermore, we used antioxidants or cells exposed to hydrogen peroxide to demonstrate that an increased antioxidative capacity can upregulate acid resistance in S. Choleraesuis. 2. Material and methods 2.1. Strains, plasmids, and growth conditions All S. Choleraesuis strains used in this study were derived from ATCC13312 (SCWT). The mutant was constructed using the l red recombination system (Datsenko and Wanner, 2000) and confirmed by gene sequencing. The strains and plasmids used in this study are listed in Table 1. Cells were grown at 37  C either in tryptic soy broth (TSB) or tryptic soy agar (TSA) at specific pH levels. The antibiotics ampicillin (100 mg/mL) and kanamycin (50 mg/mL) were also used.

43

180 rpm for 30 min. Cultures designed for adaptation were cultivated at pH 5 for 16–20 h before challenge with pH 3 followed by incubation as mentioned above. After acid challenge, the samples were immediately diluted with saline (0.85% NaCl) and plated onto TSA. Surviving bacteria were investigated by determining the number of colony forming units (CFU) before acid challenge and after 24 h of incubation. Percent survival was calculated by comparing the CFU before and after challenge and the results were presented as percentages. 2.3. Quantitative reverse-transcriptional (RT)-PCR Total RNA was extracted from S. Choleraesuis strains after acidic treatment. RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Next, 1 mg of RNA was reverse-transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) and suspended in 100 mL nuclease-free water. Quantitative RT-PCR was performed using the oligonucleotides described in the Supplementary material (Fig. S1 in the online version at DOI: http://dx.doi.org/10.1016/j.vetmic.2016.08.006% 20), and primers specific for the mdh gene were used as an internal control. 2.4. Effects of antioxidant and hydrogen peroxide on S. Choleraesuis in acidic environment To investigate whether ROS induced by acid was involved in morphological changes, a 1/100 dilution of the overnight culture was inoculated into TSB containing 1 mM N-acetyl-L-cysteine (NAC), pH 7 or 5, and incubated at 37  C for 4 h. The morphology and viable counts were determined by microscopy and agar plate counting. For the survival assay, Salmonella strains were examined to determine whether an increased antioxidative capability would protect cells from acid killing effects. Cells were challenged with TSB containing 1 mM NAC following overnight growth at pH 3 or 7 or cells cultured in the presence of 1 mM hydrogen peroxide overnight were challenged with TSB, pH 3. Surviving colonies were counted and the data are presented as percent survival. 3. Results 3.1. TolC plays a role in S. Choleraesuis acidic tolerance

2.2. Acid resistance assay The acid resistance assay was induced as described previously (Lee et al., 1994) with some modifications. Briefly, overnight cultures grown at pH 7 were adjusted to McFarland 0.5, inoculated into fresh TSB at pH 3, and then incubated at 37  C with shaking at

In order to investigate the role of tolC in the survival of S. Choleraesuis, we initially used the tolC-deficient strain SCDTolC to analyze the importance of tolC in acid resistance in S. Choleraesuis. Significant attenuation of the acid tolerance in the mutant was observed when SCDTolC was exposed to lethal acidic challenge (pH

Table 1 Strains and plasmids used in this study. Strains or plasmids

Characteristic

Reference

Strain SCWT SCDTolC SCDTolC + pKTC

Wild-type tolC null mutant SCDTolC carrying pKTC8

ATCC 13312 This study This study

l Red recombinase expression plasmid

Datsenko and Wanner, 2000 Datsenko and Wanner, 2000 Datsenko and Wanner, 2000 Karimova et al., 1998 This study

Plasmid pKD46 pKD4 pCP20 pKT25 pKTC8

Template for amplifying antibiotic cassette FLP expression plasmid Cloning vector tolC of SCWT cloned into pKT25

44

J.-J. Lee et al. / Veterinary Microbiology 193 (2016) 42–48

1.0E+08 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00

18% 16% 14% 12% 10% 8% 6% 4% 2% 0%

Percent survival CFU

Fig. 1. Acid resistance of S. Choleraesuis. Cells were grown to stationary phase at either pH 7 or pH 5 in TSB and directly challenged by pH 3 medium for 30 min. Cell viability was measured by plate counting. All experiments were performed at least in triplicate.

3) (Fig. 1). The mutant strain exhibited an acid-sensitive phenotype under both unadapted and adapted conditions ( < 0.001% for SCDTolC versus 2.86% for SCWT under unadapted conditions, and <0.002% for SCDTolC versus 11.05% for SCWT in adapted cultures). These results were expected for the role of TolC in general stress resistance (Zgurskaya et al., 2011). The decreased acid resistance of the mutant was recovered by the addition of plasmid containing tolC, and the complemented strain increased its viability by more than that of SCWT. These results indicate that tolC is required for S. Choleraesuis survival in extremely acidic environments, regardless of whether it has been adapted to mild acid conditions. 3.2. Acid-related genes were downregulated in tolC-deficient strain Membrane-bound TolC has been shown to be involved in various mechanisms and influences a wide range of gene expression in E. coli, Salmonella, and Sinorhizobium meliloti (Virlogeux-Payant et al., 2008; Santos et al., 2010; Zgurskaya et al., 2011). However, it remains unknown whether TolC integrity affects acid-tolerance-related genes in Salmonella. RT-PCR was used to analyze and compare the transcription levels of SCDTolC with those of SCWT; the results revealed that disruption of tolC significantly influenced acid-induced genes (Table 2). Activation of tolC decreased the expression of the genes involved in global transcriptional regulation (phoP, ompR, rpoS), iron metabolism (fur), proton efflux pump (cadA, cadC), and cell membrane composition (cfa, ompF) in mild acid (pH 5). Overall, downregulation of these genes was detected after SCDTolC was challenged with pH 3, except for cfa, which was increased. Together, these data suggest that tolC is important for S. Choleraesuis to express proteins essential for coping with excess protons in the bacteria.

Table 2 Expression levels of acid-induced genes. Gene

Function of encoded protein

pH 5 SCWT

SCDTolC

SCWT

SCDTolC

phoP ompR rpoS fur cadA cadC cfa ompF

Two-component system Transcriptional regulator Sigma factor Ferric uptake regulator Lysine decarboxylase enzyme Lysine decarboxylase regulator Cyclic fatty acid Porin

1.289 0.102 0.372 0.377 0.296 0.185 0.488 0.019

0.468 0.099 0.066 0.420 0.208 0.082 0.152 0.006

0.222 0.317 1.478 0.384 0.036 0.249 0.012 0.342

0.068 0.187 0.386 0.217 0.015 0.119 0.173 0.009

pH 3

3.3. Null-tolC mangles S. Choleraesuis growth capability in acidic environment As previously reported, E. coli with truncated tolC showed growth deficiencies in nutrient-limited or mild acidic environments (Dhamdhere and Zgurskaya, 2010; Deininger et al., 2011). We investigated whether the loss of functional TolC in S. Choleraesuis affects bacterial amplification ability. At pH 7, no differences in growth were observed between SCWT and SCDTolC during the 6-h observation (Fig. 2A). Growth at pH 5 exhibited developmental delays in both the parental and mutant strains compared with growth at pH 7, but there was no significant difference in growth between SCWT and SCDTolC in acidic medium; this result differs from that observed in E. coli (Deininger et al., 2011). Therefore, we used a different method rather than spectrophotometry to investigate bacteria capability. A series of dilutions and agar-plating were used for cell counting at each time point. As shown in Fig. 2B, the growth rates of SCWT and SCDTolC at pH 7 were similar during our observation; however, cell growth was apparently arrested in SCDTolC from 4 h to 6 h when the cells were grown at pH 5. These results indicate that tolC plays a role in S. Choleraesuis growth under acidic conditions, although these results were undetectable using a spectrophotometer. 3.4. TolC is important for normal morphology at low pH We also evaluated why different observation methods showed contrasting results when SCWT and SCDTolC were grown at pH 5. One possibility is that the OD values are measured based on the amount of transmitted light, which may be subject to interference by the mass or size of the cell (Merek, 1969). Since the plate counting method involves a lower number of bacteria, we next incubated the strains in acidic medium and identified whether they exhibited morphological alterations. SCWT and SCDTolC showed similar sizes and shapes when the cells were grown at neutral pH (Fig. 3). However, SCDTolC, when grown in an acidic environment, became longer than the SCWT, particularly during the exponential growth phase (4–6 h). Based on our results, TolC is crucial for S. Choleraesuis to maintain its normal phenotype when the cells are grown in acid. 3.5. TolC protects cells from oxidative stress Morphological changes have been reported previously in tolCdeficient E. coli, which was suggested to be caused by starvationinduced oxidative stress (Dhamdhere and Zgurskaya, 2010). This

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45

(A) 0.6

Cell density (OD595)

0.5 0.4 SCWT pH 7

0.3

SCWT pH 5

0.2

SC∆TolC pH 7

0.1

SC∆TolC pH 5

0 0

1

2

3 Time (h)

4

5

6

(B) 3 2.5

CFU (×109)

2 SCWT pH 7

1.5

SCWT pH 5 SCΔTolC pH 7

1

SCΔTolC pH 5 0.5 0 0

1

2

3

4

5

6

Time (h) Fig. 2. TolC is involved in S. Choleraesuis growth in acidic environment. Overnight cultures of SCWT and SCDTolC grown either in pH 7 or pH 5 TSB were diluted into fresh medium. Cell growth was monitored either with (A) spectrophotometer or (B) by plate cell counting. All experiments were repeated at least three times independently.

was also observed in another bacterial species, Campylobacter jejuni, and whether bacteria deficient in sodB, a ROS-detoxification enzyme, exhibited longer shapes was evaluated by microscopy (Oh et al., 2015). Because acid stress is known to involve oxidative stress and other homeostasis systems, we examined whether antioxidative and DNA repair genes were defective in SCDTolC. As shown in Table 3, regardless of whether cells were in a mild or extreme acidic solution, SCDTolC protective or repair genes were inactivated when the cells were in acidic conditions compared to in SCWT, indicating that oxidative stress affects SCDTolC to a greater extent than SCWT. 3.6. Correlation between oxidation- and acid-mediated stresses The results described above suggest that antioxidative capability is affected by tolC deletion when cells are grown in an acidic environment. We evaluated whether the deficiency in oxidative tolerance is a key factor in normal cell division under acid challenge. Overnight cultures were transferred into fresh TSB, pH 5, containing 1 mM NAC, an antioxidant widely used to protect cells from ROS by increasing cellular pools of free radical scavengers, and observed the morphological images. As shown in Fig. 4A, there was no difference between SCWT and SCDTolC regardless of whether the cells were in a neutral or acidic environment in the

presence of NAC. These results strongly indicate that cell division failure was correlated with antioxidative disability in SCDTolC, which was confirmed by cell counting on agar plates (Fig. 4B). After confirming the effect of antioxidants on S. Choleraesuis, we next investigated whether strains could be protected from acid killing by increasing bacterial oxidative stress resistance. Phenotypically, the addition of NAC or pre-culture of the cell with hydrogen peroxide enhanced the survival capability of all strains (Fig. 4C), and the acid resistance was more apparent in cells pretreated with hydrogen peroxide than in those grown in antioxidant-containing medium. Taken together, these results show that the antioxidative ability plays an important role in cell division and protecting bacteria from acid killing. 4. Discussion Food-borne pathogens have evolved sophisticated protection systems to enable their infection and survival in susceptible hosts. The ability of microorganisms to sustain in acidic environments occurs mainly through three strategies: pH homeostatic systems, induction of repair-protection cellular proteins, and membrane composition remodeling (Alvarez-Ordonez et al., 2008). Salmonella and E. coli, but not Shigella flexneri, possess an ATR, which protects cells against lethal acidic environments after exposure to mild or

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Fig. 3. TolC is required for cell division under acidic conditions. A 1/100 dilution of the overnight SCWT and SCDTolC were re-inoculated in either in pH 7 or in pH 5 TSB and morphological changes were investigated at 4, 6, and 24 h by optical microscopy.

moderate acid conditions (Foster and Hall, 1990; Lin et al., 1995). In this study, we demonstrated that ATR conferred better survival of S. Choleraesuis strains but could not effectively rescue acid killing in the tolC-deficient strain SCDTolC. TolC acts as an outer-membrane channel that extrudes metabolites or antibiotics by cooperating with several inner-membrane or periplasmic proteins (Koronakis et al., 2004; Li and Nikaido, 2009; Rosner and Martin, 2009). Several reports have suggested a correlation between TolC and pHresistance in E. coli (Foster, 2004; Martins et al., 2009) and Deininger et al. demonstrated that TolC is important for acid adaptation through the regulation of GadAB (Deininger et al., 2011). However, Salmonella does not contain the glutamatedependent acid resistance (Gad) system, indicating that another unknown mechanism involved in TolC-dependent acid tolerance is present, which requires further analysis. In this study, under controlled pH environmental conditions, we determined the expression of acid-regulated genes. We found out that the integrity of TolC affected the function of various systems used for survival under acid conditions, indicating that TolC has multifunctional characteristics and is not just a simple protein channel anchored on the bacterial outer membrane (Benz

Table 3 RT-PCR analysis of oxidative stress response genes. Gene

Function of encoded protein

pH 5 SCWT

SCDTolC SCWT

SCDTolC

nfo recA recB dps katG

Endonuclease Repair and maintenance of DNA DNA repair DNA protection during starvation Catalase peroxidase

0.202 1.823 0.671 2.630 0.880

0.037 1.630 0.137 1.765 0.549

0.092 3.482 0.185 0.613 3.260

pH 3

0.104 4.595 0.475 0.930 3.918

et al., 1993). Inactivation of tolC in Enterobacteriaceae clearly increased drug resistance, attenuated virulence, and influenced porin expression (Zgurskaya et al., 2011). The tolC deletion mutant showed defective expression of molecules required for protecting S. Choleraesuis from acid killing, providing evidence of the correlation between TolC and acid tolerance. Previous reports indicated that culturing cells in an acidic environment leads to bacterial growth defects (Sun et al., 1998), particularly in the tolC deletion mutant (Deininger et al., 2011). However, when grown at pH 5, SCDTolC did not exhibit slower growth than SCWT except for the CFU on agar plates, indicating conflicting results between spectrophotometry and agar counting analysis. Using microscopy, we observed impaired cell division of SCDTolC grown in acidic medium, and morphological changes were greater during the exponential phase than during the stationary phase or in nutrient-limited M9 medium (Dhamdhere and Zgurskaya, 2010). When grown in minimal medium, bacteria prepared for a general stressful environment and initiated various response systems to cope with starvation-induced pressures, including oxidative stress (Navarro Llorens et al., 2010). In Salmonella Typhimurium, deficiency of MacAB, a component of MacAB-TolC multidrug efflux pump, led to ROS sensitization (Bogomolnaya et al., 2013). The outer membrane protein AbuO, a homolog of TolC, protects Acinetobacter baumannii from oxidative stress (Srinivasan et al., 2015). According to above information, we wondered if there is any correlation between TolC and oxidative stress. It is well known that during exposure to acid, microbes suffer from various stresses, such as the denaturation of essential molecules, disruption of enzymatic systems, DNA damage, and oxidative stress (Maurer et al., 2005; Jeong et al., 2008). We hypothesized that acid-induced oxidation pressure would be one

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Fig. 4. Impact of oxidative stress on S. Choleraesuis growth under acidic environment. SCWT and SCDTolC were grown overnight to stationary phase in pH 7 TSB, diluted into either pH 7 or pH 5 medium containing 1 mM NAC, and investigated for (A) morphology 4 h after re-incubation and (B) growth capability. (C) The effect of adding NAC or pre-treating cells with hydrogen peroxide (H2O2) at a tolerable concentration on cell viability was measured by CFU counting 30 min after challenge with pH 3 TSB. Each experiment was conducted independently at least three times.

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of the reasons cause cell-division failure of SCDTolC even under nutrient-rich conditions. Based on our RT-PCR results, acidexposed tolC mutant cells showed less protection against oxidative stress than did SCWT cells, indicating that TolC is involved in coping with excess protons. Furthermore, the acid-induced morphological changes were rescued after the addition of the antioxidant NAC to the culture medium, demonstrating the important role of TolC in protecting cells against oxidative stress. Interestingly, increasing the antioxidative ability protected S. Choleraesuis against acid stress. Numerous studies have reported that acid tolerance provides cross-protection from many environmental stresses such as heat, osmotic change, and hydrogen peroxide (Foster and Hall, 1990; Leyer and Johnson, 1993), but the reverse protective mechanism remained unclear. According to our data, SCDTolC became more resistant to extreme acidic environments when the cells were pretreated with 1 mM hydrogen peroxide than when they were incubated cells with 1 mM NAC, although both treatments significantly increased SCDTolC survival compared to untreated cells. Notably, pretreatment with hydrogen peroxide caused SCDTolC to perform the same as SCWT without treatment. In conclusion, this work shows that the outer membrane protein TolC affects acid tolerance, morphology, and gene expression during acid stress, and further highlights the crucial role of oxidative stress in the acid killing on of S. Choleraesuis. The direct mechanism by which TolC influences these pathways remains to be elucidated. References Alvarez-Ordonez, A., Fernandez, A., Lopez, M., Arenas, R., Bernardo, A., 2008. Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance. Int. J. Food Microbiol. 123, 212–219. Alvarez-Ordonez, A., Begley, M., Prieto, M., Messens, W., Lopez, M., Bernardo, A., Hill, C., 2011. Salmonella spp. survival strategies within the host gastrointestinal tract. Microbiology 157, 3268–3281. Benz, R., Maier, E., Gentschev, I., 1993. TolC of Escherichia coli functions as an outer membrane channel. Zentralbl Bakteriol 278, 187–196. Bogomolnaya, L.M., Andrews, K.D., Talamantes, M., Maple, A., Ragoza, Y., VazquezTorres, A., Andrews-Polymenis, H., 2013. The ABC-type efflux pump MacAB protects Salmonella enterica serovar typhimurium from oxidative stress. MBio 4, e00630–00613. Chiu, C.H., Su, L.H., Chu, C., 2004. Salmonella enterica serotype Choleraesuis: epidemiology, pathogenesis, clinical disease, and treatment. Clin. Microbiol. Rev. 17, 311–322. Datsenko, K.A., Wanner, B.L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97, 6640– 6645. Deininger, K.N., Horikawa, A., Kitko, R.D., Tatsumi, R., Rosner, J.L., Wachi, M., Slonczewski, J.L., 2011. A requirement of TolC and MDR efflux pumps for acid adaptation and GadAB induction in Escherichia coli. PLoS One 6, e18960. Dhamdhere, G., Zgurskaya, H.I., 2010. Metabolic shutdown in Escherichia coli cells lacking the outer membrane channel TolC. Mol. Microbiol. 77, 743–754. Fang, F.C., Libby, S.J., Buchmeier, N.A., Loewen, P.C., Switala, J., Harwood, J., Guiney, D. G., 1992. The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc. Natl. Acad. Sci. U. S. A. 89, 11978–11982. Foster, J.W., Hall, H.K., 1990. Adaptive acidification tolerance response of Salmonella typhimurium. J. Bacteriol. 172, 771–778. Foster, J.W., 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2, 898–907.

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