Pyruvate-Associated Acid Resistance in Bacteria Jianting Wu, Yannan Li, Zhiming Cai and Ye Jin Appl. Environ. Microbiol. 2014, 80(14):4108. DOI: 10.1128/AEM.01001-14. Published Ahead of Print 2 May 2014.
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Pyruvate-Associated Acid Resistance in Bacteria Jianting Wu,a Yannan Li,c Zhiming Cai,a,b Ye Jind Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Shenzhen, Chinaa; Shenzhen Key Laboratory of Genitourinary Tumor, Shenzhen Second People’s Hospital, the First Affiliated Hospital of Shenzhen University, Shenzhen, Chinab; Center for Synthetic Biology Engineering Research, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Shenzhen University Town, Shenzhen, Chinac; Department of Medicine and Therapeutics and State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Shatin, NT, Hong Kongd
G
astric acid (pH 2) in the stomach of the host efficiently kills or inhibits the growth of bacterial pathogens. After being swallowed, enteric organisms have to overcome the low pH of gastric acid. Acidic stresses also come from bacterial growth per se. It is well established that bacteria such as Escherichia coli utilize the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway to generate ATP from acetate production in glucose-containing environments even in the presence of ample oxygen (1). The phenomenon of overflow metabolism has been attributed to an imbalance between the fluxes of glucose uptake and those for energy production and biosynthesis (2), probably caused by improperly controlled glucose uptake and/or limited activity of the tricarboxylic acid (TCA) cycle (3). As a weak acid, acetate is toxic to bacteria and has been found to uncouple the transmembrane pH gradient (4, 5), acidify the cytoplasm, and interfere with methionine biosynthesis (6–8). Therefore, acid resistance (AR) is an important ability that E. coli possesses to survive low pH and flourish. Indeed, E. coli organisms can be so resistant to low pH that they survive gastric acid, colonize the gut, and cause diseases even though small numbers of them (10 to 100) are ingested (9, 10). The cyclic AMP (cAMP) receptor protein (CRP) is a crucial regulator of AR in E. coli. It has been demonstrated that CRP negatively regulates AR by repressing a set of AR genes (11, 12). CRP has to form a complex with the signal metabolite cAMP to be functional (13, 14), and cAMP synthesis requires adenylate cyclase (CyaA) that is activated by the phosphorylated EIIA component of the glucose-specific phosphotransferase system (phosphoEIIAglc) (15, 16). Glucose dephosphorylates the EIIA component, thereby inhibiting cAMP synthesis and consequently derepressing the cAMP-CRP-dependent AR genes (17). Thus, glucose enhances the ability of exponentially growing bacteria to survive low pH (18). CRP-dependent AR functions in the presence of glucose. When glucose is exhausted, the formation of the cAMP-CRP com-
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plex is restored and the CRP-repressed AR genes are inactivated. A question, therefore, arises as to how bacteria resist acidic stresses in the presence of substantial cAMP-CRP complex. Here we show that pyruvate and/or its downstream metabolites induce AR by a mechanism that is independent of cAMP-CRP. We reveal that pyruvate and/or its downstream metabolites enhance AR by activating the small noncoding RNA (sncRNA) Spot42 and that the pyruvate activation of Spot42 does not require the cAMP-CRP complex. Spot42, in turn, confers AR by elevating expression of RpoS, a master regulator of stress resistance (19, 20). Interestingly, Spot42 is repressed by the cAMP-CRP complex (21). Thus, Spot42 is involved both in CRP-independent AR and in CRPdependent AR. Spot42 is widely present in medically important gammaproteobacterial genera such as Escherichia, Pantoea, Xenorhabdus, Salmonella, Citrobacter, Yersinia, Serratia, Edwardsiella, Dickeya, Photorhabdus, Enterobacter, Klebsiella, Rahnella, and Shigella (22). In addition, pyruvate is a metabolic product of many sugars. Therefore, the AR mechanisms revealed here are not limited to E. coli grown on glucose but are applicable to diverse bacteria living on various carbon sources.
Applied and Environmental Microbiology
Received 24 March 2014 Accepted 24 April 2014 Published ahead of print 2 May 2014 Editor: M. Kivisaar Address correspondence to Ye Jin, [email protected], or Zhiming Cai, [email protected]. J.W. and Y.L. contributed equally to this article. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.01001-14
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Glucose confers acid resistance on exponentially growing bacteria by repressing formation of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and consequently activating acid resistance genes. Therefore, in a glucose-rich growth environment, bacteria are capable of resisting acidic stresses due to low levels of cAMP-CRP. Here we reveal a second mechanism for glucose-conferred acid resistance. We show that glucose induces acid resistance in exponentially growing bacteria through pyruvate, the glycolysis product. Pyruvate and/or the downstream metabolites induce expression of the small noncoding RNA (sncRNA) Spot42, and the sncRNA, in turn, activates expression of the master regulator of acid resistance, RpoS. In contrast to glucose, pyruvate has little effect on levels of the cAMP-CRP complex and does not require the complex for its effects on acid resistance. Another important difference between glucose and pyruvate is that pyruvate can be produced by bacteria. This means that bacteria have the potential to protect themselves from acidic stresses by controlling glucose-derived generation of pyruvate, pyruvate-acetate efflux, or reversion from acetate to pyruvate. We tested this possibility by shutting down pyruvate-acetate efflux and found that the resulting accumulation of pyruvate elevated acid resistance. Many sugars can be broken into glucose, and the subsequent glycolysis generates pyruvate. Therefore, pyruvate-associated acid resistance is not confined to glucose-grown bacteria but is functional in bacteria grown on various sugars.
Acid Resistance in Bacteria
MATERIALS AND METHODS
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counting the CFU of acid-treated cells. As untreated controls, cells before the acid treatment were also quantified by counting the CFU as described above. Acid survival (expressed as percent) was calculated with the following formula: 100 ⫻ (CFU of treated cells/CFU of untreated cells). All assays were carried out in quadruplicate on two different occasions. Growth curve. For simultaneous measurement of acid resistance and corresponding cell growth, 100-l quantities of overnight cultures were diluted in 50 ml of LB medium in 250-ml flasks, followed by incubation with shaking (220 rpm) for 12 h at 37°C. Every 2 h, an aliquot was sampled from the culture and optical density at 600 nm was measured after dilution when necessary. Determination of extracellular cAMP, pyruvate, and acetate. To quantify extracellular cAMP, overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h. Cell suspensions were then centrifuged at 12,000 ⫻ g for 5 min at 4°C. The resulting supernatants were then subjected to cAMP determination using a cAMP direct immunoassay kit (BioVision, Mountain View, CA) as directed by the manufacturer. To quantify extracellular pyruvate, overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h before the quantification assay. For time course measurement of extracellular pyruvate, cells were incubated or 6 h and pyruvate was determined at 1-h intervals. Pyruvate in the supernatants was assayed using an EnzyChrom pyruvate assay kit (BioAssay Systems, Hayward, CA). Acetate in the supernatants was quantified using an acetate detection kit (Megazyme, Bray, Ireland) according to the manufacturer’s instructions. Western blotting. Overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h. The bacterial cells were then centrifuged at 12,000 ⫻ g for 5 min at 4°C, and the resulting cell pellets were mixed with sample loading buffer (300 mM Tris-HCl, 6% SDS, 30% glycerol, 0.6% bromphenol blue, 1.2 M betamercaptoethanol), boiled for 10 min, and subjected to 12% SDS-PAGE in duplicate. Proteins of one set of gels were then electrotransferred onto an Immobilon-P membrane (Millipore, MA) and detected with an E. coli CRP monoclonal antibody (NeoClone Biotechnology, Madison, WI) followed by an anti-mouse horseradish peroxidase (HRP)-conjugated antibody (Invitrogen). Proteins were visualized using the SuperSignal West Pico chemiluminescent substrate from Pierce (Rockford, IL). The other set of SDS-PAGE gels was stained with Coomassie brilliant blue to demonstrate equal loading. Statistical analysis. Independent t tests were used to compare means obtained from pyruvate, cAMP, beta-galactosidase activity, and acid resistance assays. P values of ⬍0.05 were considered statistically significant.
RESULTS AND DISCUSSION
Pyruvate and/or its downstream metabolites induce AR. It is well known that pyruvate is a product of glycolysis. Our data showed that added glucose (20 mM), lactose (1%), or glycerol (5%) increased extracellular levels of pyruvate (all P ⬍ 0.05) (Fig. 1A), indicating that many sugars are converted to pyruvate. Under normal conditions without exogenous pyruvate, intracellular pyruvate levels are 45-fold higher than extracellular pyruvate levels (28). The increased extracellular pyruvate observed with cells grown on various sugars (Fig. 1A), therefore, indicates a more significant increase in intracellular pyruvate levels. Our time course assays revealed that E. coli quickly consumed pyruvate added to the growth medium so that extracellular pyruvate decreased to low levels after 5 h of incubation with shaking in LB at 37°C (Fig. 1B), suggesting that any effects of pyruvate on biological processes occur primarily in the log phase. Next, we examined if pyruvate affected the ability of bacteria to resist acid. For AR assays, bacterial cells in the early log phase were treated with acidified LB medium at pH 2.0 for 2 h and then the survival rate was determined. As a control, glucose at 20 mM dra-
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E. coli strains and growth conditions. The E. coli K-12 strain MG1655 (from our laboratory stock) and its isogenic mutants (constructed previously or in this study) were used for phenotypic examination in this study. MG1655 is a widely investigated laboratory strain with annotated sequence and biochemical information. It has been completely sequenced so that genetic engineering can be easily designed and performed. For these reasons, we chose MG1655 for this study and our data could be compared with and integrated with previous findings. The MG1655 strains were grown at 37°C in Luria-Bertani (LB) medium (Affymetrix, Cleveland, OH) or on LB agar (Affymetrix) (23, 24). The antibiotics ampicillin (50 g/ml; Sigma, St. Louis, MO) and chloramphenicol (12.5 g/ml; Sigma) were added to growth medium or agar when appropriate. Gene deletion. Gene deletion was performed using the recombineering system as described previously (25, 26). Briefly, the E. coli K-12 strain MG1655 was transformed with plasmid pSim6 (a gift from Donald Court), from which the expression of the recombination proteins was induced for 15 min at 42°C with shaking (220 rpm). The MG1655 strain carrying pSim6 was incubated at 32°C with shaking at 220 rpm until the optical density at 600 nm (OD600) of the bacterial culture reached 0.4 to 0.6. Then, PCR fragments encompassing a loxP-cat-loxP cassette with homology (45 nucleotides [nt]) to the regions immediately flanking the deletion target were transformed via electroporation into the MG1655 cells harboring pSim6. After induction of red functions, recombinants were selected for chloramphenicol resistance (encoded by the cat gene) and were further verified by colony PCR and sequencing. spf cloning. The selectable loxP-cat-loxP cassette was first inserted immediately after the stop codon of the spf gene (encoding Spot42) on the chromosome by recombineering (as described above). We then inserted spf preceded by its native promoter and the loxP-cat-loxP cassette in a pET32a expression vector by recombineering. Specifically, we PCR amplified the spf gene (including its native promoter) and adjacent “floxed” cat cassette using primers that contained homology (45 nt) to the plasmid insertion site. The PCR product and expression vector were cotransformed into MG1655 carrying pSim6 after induction of red. Recombinants were selected for chloramphenicol resistance and verified by PCR and sequencing. In the recombinant plasmid, named pSpot42, the native promoter of the spf gene drove Spot42 overproduction without induction. To construct an empty plasmid, the loxP-cat-loxP cassette was inserted into pET32a (Novagen, Inc., Madison, WI) and selected for chloramphenicol resistance. The resulting plasmid was named pCm. As pCm did not express Spot42 but was otherwise similar to pSpot42, it served as a negative control. Construction of a chromosomal spf-lacZ transcriptional fusion. The loxP-cat-loxP selectable cassette was inserted immediately after the stop codon of the lacZ gene on the MG1655 chromosome using recombineering (as described above). Next, the lacZ-loxP-cat-loxP cassette was PCR amplified and inserted immediately prior to the poly-T string of the spf gene on the chromosome. The inserted lacZ gene together with its native ribosome binding site (15 bp upstream of the start codon) was cotranscribed with spf. Beta-galactosidase assay. Overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h. Expression of lacZ fusions was quantified using a commercially available beta-galactosidase assay kit (Pierce Biotechnology, Rockford, IL) as described previously (23, 27). Levels of beta-galactosidase, expressed as Miller units (MU), were calculated using the following formula: OD420 ⫻ 1,000/(OD600 ⫻ hydrolysis time ⫻ volume of lysate). Acid resistance assay. Overnight cultures were diluted 1:500 in fresh LB medium (pH 6.8) and incubated at 37°C with shaking at 220 rpm for 4 h. Cells were then challenged to pH 2.0 by adjusting the LB medium with HCl. Cells were treated statically with acid for 2 h and then washed with phosphate-buffered saline (PBS) at pH 7.2 to remove acid. Cells were serially diluted in PBS, and 20 l of each diluted suspension was plated on LB agar. After overnight culture at 37°C, viable cells were determined by
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matically increased AR by a factor of more than 1,700 (P ⫽ 0.016) (Fig. 1C). This was expected, as glucose represses the formation of the cAMP-CRP complex, a transcriptional regulator known to negatively regulate AR by repressing AR genes (11, 12). The requirement of the glucose activation of AR for the cAMP-CRP complex was confirmed by our AR assays, in which the survival rate of a crp null mutant was 2.59% (⫾1.5%) after acid challenge (Fig. 1C, lower portion), 2,500 times higher than that of the wildtype strain (Fig. 1C, upper portion) (P ⫽ 0.0096). Moreover, added glucose at 20 mM did not increase AR with the crp null mutant (P ⫽ 0.933) (Fig. 1C, lower portion). Interestingly, added pyruvate at 20 mM significantly increased AR, as observed with glucose (P ⫽ 0.0246) (Fig. 1C, upper portion). In contrast to glucose, however, pyruvate did not lose the AR-inducing ability with the crp null mutant (P ⫽ 0.0054) (Fig. 1C, lower portion). cAMP assays and Western blot assays revealed that added pyruvate had no effects on either cAMP (P ⫽ 0.818) or CRP levels (Fig. 1D). Collectively, these results indicate that pyruvate induces AR by a mechanism independent of the cAMP-CRP complex. It is noteworthy that 20 mM was the concentration of pyruvate added to the growth medium. Due to the consumption of pyruvate, the concentration of pyruvate dramatically decreased after 4 h of incubation when the bacteria were subjected to the AR assays. As revealed by the time course measurement of extracellular pyruvate (Fig. 1B), the extracellular pyruvate concentration was less than 47.5 (⫾3.3) M 4 h after the concentration dropped to 20 mM. It is intracellular pyruvate that mediates AR. However, exogenous pyruvate at high concentrations just mildly increased intracellular levels of pyruvate (28), since pyruvate uptake is an en-
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FIG 2 Elevation of acid resistance as a result of shutting down acetate production. (A) Extracellular levels of acetate in wild-type MG1655 (wt) and the ackA-pta null mutant (⌬ackA-pta) as a function of time. (B) Extracellular levels of pyruvate in the wt and ⌬ackA-pta mutant grown in LB medium for 4 h. (C) Acid survival at pH 2.0 of the wt and ⌬ackA-pta mutant grown in LB medium for 4 h before 2 h of acid treatment. P values of ⬍0.05 (*) were considered statistically significant.
ergy-dependent active transport process (29). For these reasons, the concentration of added pyruvate has to be high to cause remarkable effects on AR and regulation of target genes in this study. In contrast to added pyruvate, added glucose and other carbon sources result in endogenous generation of pyruvate through glycolysis. After growth in LB medium plus 20 mM glucose for 4 h, bacteria displayed extracellular pyruvate concentrations of 40.7 (⫾7.9) M (Fig. 1A), close to the extracellular pyruvate concentrations of the bacteria grown in LB medium supplemented with 20 mM pyruvate. This indicates that pyruvate generated from glucose is sufficient to have significant effects on AR. Increased AR as a result of the addition of pyruvate can result from pyruvate or downstream metabolites such as acetyl coenzyme A (Ac-CoA), acetyl phosphate, and acetate, which are reversibly interconvertible (1). To see if these metabolites are responsible for elevated AR, we tested the effects of Ac-CoA and acetate on log-phase AR. Addition of neither of them had any effect on log-phase AR (both P ⬎ 0.05) (Fig. 1C), excluding roles for Ac-CoA and acetate. Conversion from Ac-CoA to acetate is mainly achieved by the AckA-Pta (acetate kinase-phosphotransacetylase) pathway (1, 30, 31). As the acetate outflow is an outlet for pyruvate discharge, we reasoned that shutting off this pathway could elevate levels of pyruvate, thereby increasing AR. To test this possibility, we deleted the entire ackA-pta operon. We then monitored extracellular acetate over time for the resulting null mutant (⌬ackA-pta) and the wild-type strain to confirm the role of this pathway in acetate outflow. With the wild-type strain, extracellular acetate levels increased during the log phase and dropped after 4 h of incubation (Fig. 2A). This phenomenon has previously been termed “acetate switch” and occurs when glucose is exhausted and bacteria transit from acetate excretion to acetate uptake (1). In contrast, the ⌬ackA-pta mutant produced little acetate throughout the incubation (Fig. 2A), confirming that deleting the ackApta operon effectively shuts off acetate excretion. We then examined if shutting down the acetate outflow caused pyruvate accumulation and had any effect on AR. As shown in Fig. 2B, deleting the ackA-pta operon elevated extracellular levels of pyru-
Applied and Environmental Microbiology
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FIG 1 Pyruvate induces acid resistance independently of the cAMP-CRP complex. (A) Levels of extracellular pyruvate of the E. coli MG1655 strain grown in LB medium alone, LB medium plus 20 mM glucose (Glc), LB medium plus 1% lactose (Lac), and LB medium plus 5% glycerol (Gly) for 4 h. (B) Time course of extracellular pyruvate levels of MG1655 grown in LB medium plus 40 mM pyruvate. (C) Acid survival at pH 2.0 of the wild-type MG1655 (wt) grown in LB medium alone, LB medium plus 20 mM glucose (Glc), LB medium plus 20 mM pyruvate (Pyr), LB plus 10 mM acetyl coenzyme A (coA), and LB medium plus 20 mM acetate (Ac) and of a crp null mutant (⌬crp) grown in LB medium alone, LB medium plus 20 mM glucose (Glc), and LB medium plus 20 mM pyruvate (Pyr). Cells were grown for 4 h and then treated with acidified LB medium (pH 2.0) for 2 h. The survival rate was then determined by counting CFU. (D) Extracellular cAMP and corresponding CRP levels of MG1655 grown in LB medium without pyruvate supplementation and LB medium plus 20 mM pyruvate. Cells were grown for 4 h before quantification of cAMP and CRP. P values of ⬍0.05 (*) were considered statistically significant.
Acid Resistance in Bacteria
and acid survival at pH 2.0 of wild-type E. coli MG1655 (wt) and an spf null mutant (⌬spf) as a function of time. (B) Cell growth (OD600) and acid survival at pH 2.0 of the ⌬spf mutant carrying a control vector (pCm) or pSpot42 that overproduces Spot42 as a function of time. Cells were grown for 4 h before the acid resistance assay. OD600 was measured as follows. Overnight cultures (100 l) were diluted in 50 ml of LB medium in 250-ml flasks and then incubated at 37°C. Every 2 h, an aliquot was taken for determination of OD600 after dilution when necessary.
vate by a factor of 126 (P ⫽ 0.0079). Similar observations have also been reported previously (32, 33). As predicted, deleting the ackApta operon increased AR by a factor of 577 (P ⫽ 0.0236) (Fig. 2C), confirming the role of pyruvate in the elevated log-phase AR. Pyruvate and/or the downstream metabolites enhance acid resistance by inducing Spot42. We next asked how pyruvate and/or the downstream metabolites conferred on bacteria the ability to resist low pH. The following findings linked pyruvateinduced AR with a small noncoding RNA (sncRNA) named Spot42. Our previous work on a knockout library of sncRNAs has revealed that some sncRNAs upregulated AR. One unpublished AR-regulating sncRNA is Spot42, which is encoded by the spf gene. We found that deleting the spf gene reduced AR during the first 8 h of incubation (all P ⬍ 0.05) (Fig. 3A). We then cloned the spf gene into a multicopy pET32a vector, generating pSpot42, which overexpresses Spot42. Consistent with the knockout data, overproduction of Spot42 increased AR through the culture (all P ⬍ 0.05) (Fig. 3B). Specifically, bacterial cells were grown in LB medium and aliquots were measured for both AR and OD600 at different time points. By this means, AR as a function of cell growth phase could be evaluated. Cells overproducing Spot42 were 4,000- to 15,000-fold more acid resistant than those lacking the sncRNA after 2 to 6 h of incubation. This figure dropped to 7-fold after 8 h of incubation and was further reduced to 1.5-fold after 16 h (Fig. 3B). Cell growth curves revealed that 2 to 6 h corresponded to the log phase (Fig. 3A and B). These results indicate that AR regulation by Spot42 is most evident in the early log phase. Spot42 has been identified as a downstream target of the cAMP-CRP complex and is activated by glucose (21). This together with the Spot42 regulation of log-phase AR encouraged us to ask if Spot42 has a role in the pyruvate-conferred log-phase AR. To facilitate evaluation of the possible role of Spot42, we constructed a chromosomally located spf-lacZ transcriptional fusion in E. coli MG1655 so that Spot42 transcription could be easily quantified by measuring beta-galactosidase activity. Subsequent beta-galactosidase assays showed that pyruvate increased Spot42
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FIG 4 Induction of Spot42 transcription by pyruvate. (A) Beta-galactosidase activity of mutants carrying a chromosomal spf-lacZ transcriptional fusion with the crp gene (crp⫹) or without crp (crp⫺). Cells were grown in LB medium supplemented with increasing concentrations of glucose (Glc) or pyruvate (Pyr) for 4 h before quantification of beta-galactosidase activity. (B) Pyruvate dramatically increased acid resistance of wild-type MG1655 (wt) but affected acid resistance of the spf null mutant (⌬spf) to a much lower extent. However, deletion of spf did not abolish pyruvate-conferred acid resistance. P values of ⬍0.05 (*) were considered statistically significant.
expression (P ⫽ 0.00015) (Fig. 4A). Activation of Spot42 by pyruvate was maintained in cells in which crp had been deleted (⌬crp) (P ⬍ 0.05 at all pyruvate concentrations) (Fig. 4A). Thus, in contrast to glucose, pyruvate induces Spot42 expression by a CRPindependent mechanism. The induction of Spot42 expression by pyruvate and upregulation of log-phase AR by Spot42 suggest that pyruvate and/or the downstream metabolites confer log-phase AR at least in part through Spot42. It is noteworthy that deletion of the spf gene, encoding Spot42, did not abolish pyruvate-conferred AR but the effects of pyruvate on AR were not statistically significant (P ⫽ 0.08) (Fig. 4B), confirming the critical role of Spot42 for this AR system. RpoS participates in pyruvate- and Spot42-conferred acid resistance. The phase specificity of Spot42-mediated AR prompted us to link it to growth phase-specific biological processes. One such process is rpoS expression that is not induced until bacteria enter the stationary phase. RpoS is a master regulator of stress resistance (19, 20). This led us to speculate that RpoS may have a role in Spot42-mediated AR. To quantify RpoS expression, we employed a previously constructed MG1655 isogenic mutant carrying an rpoS-lacZ translational fusion on the chromosome (23). In support of the above speculation, deleting the spf gene reduced rpoS-lacZ fusion expression regardless of the pyruvate addition (both P ⬍ 0.05) (Fig. 5A), and overexpressing sncRNA increased the expression (P ⫽ 0.0018) (Fig. 5B). Given the activating effects of pyruvate on Spot42, we predicted that pyruvate enhanced RpoS expression. As expected, pyruvate increased rpoS-lacZ expression (P ⫽ 0.0001) (Fig. 5A). Thus, pyruvate enhances Spot42, which, in
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FIG 3 Effects of Spot42 on bacterial acid resistance. (A) Cell growth (OD600)
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FIG 5 Role of RpoS in pyruvate- and Spot42-conferred acid resistance. (A) Beta-galactosidase activity of mutants carrying a chromosomal rpoS-lacZ translational fusion with the small noncoding RNA Spot42 (Spot42⫹) or without Spot42 (Spot42⫺). Cells were grown in LB medium or LB medium plus 20 mM pyruvate (Pyr) for 4 h before measurement of beta-galactosidase activity. (B) Effects of Spot42 overproduction from pSpot42 on expression of the rpoS-lacZ translational fusion. The fusion strain carrying an empty vector, pCm, served as a negative control. (C) Acid survival of various strains carrying pCm or pSpot42. These include wild-type E. coli MG1655 (wt), an isogenic rpoS null mutant (⌬rpoS), and an isogenic mutant, d567–1342, in which the native regulation of rpoS was removed. All cells were grown in LB medium for 4 h before the acid resistance assay. P values of ⬍0.05 (*) were considered statistically significant.
turn, activates RpoS. However, deleting the spf gene did not abolish the effects of pyruvate on RpoS (P ⫽ 0.00036) (Fig. 5A), indicating the existence of a Spot42-indepenent mechanism. To provide more evidence for the role of RpoS in pyruvate and Spot42 regulation of AR, we tested if removing RpoS abolished the regulation. An rpoS null mutant (⌬rpoS) showed an extremely low survival rate after 2 h of acid treatment, and Spot42 overproduction failed to increase it (Fig. 5C). We then employed a previously constructed rpoS mutant, d567–1342, which loses the natural regulation of rpoS and constitutively expresses rpoS (23). Removing the natural regulation of rpoS significantly diminished the effects of Spot42 on AR, as Spot42 overproduction increased AR ⬃500fold in the wild-type strain but only ⬃27-fold in d567–1342 (P ⫽ 0.012) (Fig. 5C). sncRNAs regulate gene expression either directly by binding to their mRNA targets or indirectly by acting on transcriptional or translational factors that, in turn, regulate the expression (34). If Spot42 regulates rpoS by a direct mechanism, there should be extensive complementarities between the two RNAs. However, complementarity analysis using the program TargetRNA (http://snowwhite.wellesley.edu/targetRNA) (35) did not reveal any extensive complementarities between rpoS and Spot42, suggesting that Spot42 regulates RpoS through an indirect mechanism. The AR system reported in this study is summarized in Fig. 6. It
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appears to be a new AR system different from the currently known systems that are primarily restricted to the stationary phase (24, 36, 37). Acid resistance system 1 (AR1) requires the induction of RpoS but is repressed by glucose, which is contrast to our system. AR2 is glutamate dependent, which requires the presence of glutamate decarboxylase and a putative glutamate:GABA antiporter. Our AR system is not part of AR2, since AR2 is absent from logphase cells grown in LB medium plus 4% glucose (38). AR3 is arginine dependent, requires the presence of arginine decarboxylase (AdiA), and provides a modest level of protection. AR3 is induced by low pH under anaerobic conditions, which is different from our AR system. In contrast to these thoroughly investigated stationary-phase AR systems, much less is known about how logphase cells resist acid, since efforts to study this may have been discouraged by the fact that log-phase cells are acid sensitive. This study reveals for the first time how exponentially growing bacteria such as E. coli resist low pH through pyruvate and/or its downstream metabolites. Bacteria use pyruvate and/or its downstream metabolites as key effector molecules to induce AR by activating expression of the sncRNA Spot42, which, in turn, upregulates RpoS. Any biological processes that affect pyruvate levels would, therefore, have an impact on AR. This is confirmed by our observation that increasing pyruvate accumulation by shutting down pyruvate discharge remarkably elevates AR. Another source of pyruvate is sugar metabolism. Bacteria grown on sugars such as glucose produce pyruvate inside the cells through glycolysis without a need for active uptake of extracellular pyruvate. We show that addition of glucose at 20 mM resulted in a 240-fold increase in extracellular levels of pyruvate and enhanced AR accordingly. The concentration of glucose in foods and drinks easily reaches 20 mM. For instance, the glucose concentrations of orange juices range from 22 to 218 mM (39). Thus, this study reveals that bacteria in sugar-rich foods or drinks are capable of acquiring the ability to resist acid at least partially through the pyruvate- and Spot42-dependent AR system. The resulting acid-resistant bacteria could survive gastric acid and colonize the intestine to become commensal habitants or cause diseases, depending on the virulence of the bacteria. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant C010301), Basic Research Program of Shenzhen (grant
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FIG 6 Pathways of the acid resistance system mediated by pyruvate and Spot42. P-EIIAglc, the phosphorylated EIIA component of the glucose-specific phosphotransferase system; EIIAglc, dephosphorylated EIIA component of the glucose-specific phosphotransferase system; Glc, glucose; Pyr, pyruvate; AR, acid resistance.
Acid Resistance in Bacteria
JCYJ20120614155650545), and National Program on Key Basic Research Project (grant 2014CB745200).
21.
REFERENCES
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22.
23. 24. 25.
26.
27.
28. 29. 30.
31.
32.
33.
34. 35. 36. 37. 38. 39.
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1. Wolfe AJ. 2005. The acetate switch. Microbiol. Mol. Biol. Rev. 69:12–50. http://dx.doi.org/10.1128/MMBR.69.1.12-50.2005. 2. Farmer WR, Liao JC. 1997. Reduction of aerobic acetate production by Escherichia coli. Appl. Environ. Microbiol. 63:3205–3210. 3. Veit A, Polen T, Wendisch VF. 2007. Global gene expression analysis of glucose overflow metabolism in Escherichia coli and reduction of aerobic acetate formation. Appl. Microbiol. Biotechnol. 74:406 – 421. http://dx .doi.org/10.1007/s00253-006-0680-3. 4. Repaske DR, Adler J. 1981. Change in intracellular pH of Escherichia coli mediates the chemotactic response to certain attractants and repellents. J. Bacteriol. 145:1196 –1208. 5. Kihara M, Macnab RM. 1981. Cytoplasmic pH mediates pH taxis and weak-acid repellent taxis of bacteria. J. Bacteriol. 145:1209 –1221. 6. Roe AJ, O’Byrne C, McLaggan D, Booth IR. 2002. Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity. Microbiology 148:2215–2222. 7. Roe AJ, McLaggan D, Davidson I, O’Byrne C, Booth IR. 1998. Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J. Bacteriol. 180:767–772. 8. Russell JB, Diez-Gonzalez F. 1998. The effects of fermentation acids on bacterial growth. Adv. Microb. Physiol. 39:205–234. 9. Schmid-Hempel P, Frank SA. 2007. Pathogenesis, virulence, and infective dose. PLoS Pathog. 3(10):e147. http://dx.doi.org/10.1371/journal .ppat.0030147. 10. Griffin PM, Tauxe RV. 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13:60 –98. 11. Castanie-Cornet MP, Foster JW. 2001. Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes. Microbiology 147:709 –715. 12. Gosset G, Zhang Z, Nayyar S, Cuevas WA, Saier MH, Jr. 2004. Transcriptome analysis of Crp-dependent catabolite control of gene expression in Escherichia coli. J. Bacteriol. 186:3516 –3524. http://dx.doi.org/10.1128 /JB.186.11.3516-3524.2004. 13. Tagami H, Aiba H. 1998. A common role of CRP in transcription activation: CRP acts transiently to stimulate events leading to open complex formation at a diverse set of promoters. EMBO J. 17:1759 –1767. http://dx .doi.org/10.1093/emboj/17.6.1759. 14. Busby S, Ebright RH. 1999. Transcription activation by catabolite activator protein (CAP). J. Mol. Biol. 293:199 –213. http://dx.doi.org/10.1006 /jmbi.1999.3161. 15. Park YH, Lee BR, Seok YJ, Peterkofsky A. 2006. In vitro reconstitution of catabolite repression in Escherichia coli. J. Biol. Chem. 281:6448 – 6454. http://dx.doi.org/10.1074/jbc.M512672200. 16. Bettenbrock K, Sauter T, Jahreis K, Kremling A, Lengeler JW, Gilles ED. 2007. Correlation between growth rates, EIIACrr phosphorylation, and intracellular cyclic AMP levels in Escherichia coli K-12. J. Bacteriol. 189:6891– 6900. http://dx.doi.org/10.1128/JB.00819-07. 17. Hogema BM, Arents JC, Bader R, Eijkemans K, Yoshida H, Takahashi H, Aiba H, Postma PW. 1998. Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc. Mol. Microbiol. 30:487– 498. http://dx.doi.org/10.1046/j.1365-2958.1998.01053.x. 18. Rowbury RJ, Goodson M. 1998. Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP. J. Appl. Microbiol. 85:615– 620. http://dx.doi.org/10.1046/j.1365 -2672.1998.853569.x. 19. Battesti A, Majdalani N, Gottesman S. 2011. The RpoS-mediated general stress response in Escherichia coli. Annu. Rev. Microbiol. 65:189 –213. http://dx.doi.org/10.1146/annurev-micro-090110-102946. 20. Lacour S, Landini P. 2004. SigmaS-dependent gene expression at the onset of stationary phase in Escherichia coli: function of sigmaSdependent genes and identification of their promoter sequences. J.
Bacteriol. 186:7186 –7195. http://dx.doi.org/10.1128/JB.186.21.7186 -7195.2004. Polayes DA, Rice PW, Garner MM, Dahlberg JE. 1988. Cyclic AMPcyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli. J. Bacteriol. 170:3110 –3114. Hansen GA, Ahmad R, Hjerde E, Fenton CG, Willassen NP, Haugen P. 2012. Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida. BMC Genomics 13:37. http://dx.doi.org/10.1186/1471-2164-13-37. Jin Y, Watt RM, Danchin A, Huang JD. 2009. Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli. BMC Genomics 10:165. http://dx.doi.org/10.1186/1471-2164-10-165. Lin J, Smith MP, Chapin KC, Baik HS, Bennett GN, Foster JW. 1996. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl. Environ. Microbiol. 62:3094 –3100. Sharan SK, Thomason LC, Kuznetsov SG, Court DL. 2009. Recombineering: a homologous recombination-based method of genetic engineering. Nat. Protoc. 4:206 –223. http://dx.doi.org/10.1038/nprot.2008 .227. Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 97:5978 –5983. http://dx.doi.org /10.1073/pnas.100127597. Jin Y, Watt RM, Danchin A, Huang JD. 2009. Use of a riboswitchcontrolled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation. J. Biol. Chem. 284: 28738 –28745. http://dx.doi.org/10.1074/jbc.M109.028076. Yang YT, Bennett GN, San KY. 2001. The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli. Metab. Eng. 3:115–123. http://dx.doi.org/10.1006/mben.2000.0166. Lang VJ, Leystra-Lantz C, Cook RA. 1987. Characterization of the specific pyruvate transport system in Escherichia coli K-12. J. Bacteriol. 169: 380 –385. Hesslinger C, Fairhurst SA, Sawers G. 1998. Novel keto acid formatelyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate. Mol. Microbiol. 27:477– 492. http://dx.doi.org/10.1046/j.1365-2958 .1998.00696.x. Klein AH, Shulla A, Reimann SA, Keating DH, Wolfe AJ. 2007. The intracellular concentration of acetyl phosphate in Escherichia coli is sufficient for direct phosphorylation of two-component response regulators. J. Bacteriol. 189:5574 –5581. http://dx.doi.org/10.1128/JB.00564-07. Dittrich CR, Vadali RV, Bennett GN, San KY. 2005. Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate. Biotechnol. Prog. 21:627– 631. http://dx.doi.org/10 .1021/bp049730r. Sadykov MR, Thomas VC, Marshall DD, Wenstrom CJ, Moormeier DE, Widhelm TJ, Nuxoll AS, Powers R, Bayles KW. 2013. Inactivation of the Pta-AckA pathway causes cell death in Staphylococcus aureus. J. Bacteriol. 195:3035–3044. http://dx.doi.org/10.1128/JB.00042-13. Repoila F, Darfeuille F. 2009. Small regulatory non-coding RNAs in bacteria: physiology and mechanistic aspects. Biol. Cell 101:117–131. http: //dx.doi.org/10.1042/BC20070137. Tjaden B. 2008. TargetRNA: a tool for predicting targets of small RNA action in bacteria. Nucleic Acids Res. 36:W109 –W113. http://dx.doi.org /10.1093/nar/gkn264. Foster JW. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2:898 –907. http://dx.doi.org/10.1038 /nrmicro1021. Lin J, Lee IS, Frey J, Slonczewski JL, Foster JW. 1995. Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J. Bacteriol. 177:4097– 4104. Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW. 1999. Control of acid resistance in Escherichia coli. J. Bacteriol. 181:3525– 3535. Li WJ, Goovaerts P, Meurens M. 1996. Quantitative analysis of individual sugars and acids in orange juices by near-infrared spectroscopy of dry extract. J. Agric. Food Chem. 44:2252–2259. http://dx.doi.org/10.1021 /jf9500750.
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Pyruvate-Associated Acid Resistance in Bacteria Jianting Wu,a Yannan Li,c Zhiming Cai,a,b Ye Jind Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Shenzhen, Chinaa; Shenzhen Key Laboratory of Genitourinary Tumor, Shenzhen Second People’s Hospital, the First Affiliated Hospital of Shenzhen University, Shenzhen, Chinab; Center for Synthetic Biology Engineering Research, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Shenzhen University Town, Shenzhen, Chinac; Department of Medicine and Therapeutics and State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Shatin, NT, Hong Kongd
G
astric acid (pH 2) in the stomach of the host efficiently kills or inhibits the growth of bacterial pathogens. After being swallowed, enteric organisms have to overcome the low pH of gastric acid. Acidic stresses also come from bacterial growth per se. It is well established that bacteria such as Escherichia coli utilize the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway to generate ATP from acetate production in glucose-containing environments even in the presence of ample oxygen (1). The phenomenon of overflow metabolism has been attributed to an imbalance between the fluxes of glucose uptake and those for energy production and biosynthesis (2), probably caused by improperly controlled glucose uptake and/or limited activity of the tricarboxylic acid (TCA) cycle (3). As a weak acid, acetate is toxic to bacteria and has been found to uncouple the transmembrane pH gradient (4, 5), acidify the cytoplasm, and interfere with methionine biosynthesis (6–8). Therefore, acid resistance (AR) is an important ability that E. coli possesses to survive low pH and flourish. Indeed, E. coli organisms can be so resistant to low pH that they survive gastric acid, colonize the gut, and cause diseases even though small numbers of them (10 to 100) are ingested (9, 10). The cyclic AMP (cAMP) receptor protein (CRP) is a crucial regulator of AR in E. coli. It has been demonstrated that CRP negatively regulates AR by repressing a set of AR genes (11, 12). CRP has to form a complex with the signal metabolite cAMP to be functional (13, 14), and cAMP synthesis requires adenylate cyclase (CyaA) that is activated by the phosphorylated EIIA component of the glucose-specific phosphotransferase system (phosphoEIIAglc) (15, 16). Glucose dephosphorylates the EIIA component, thereby inhibiting cAMP synthesis and consequently derepressing the cAMP-CRP-dependent AR genes (17). Thus, glucose enhances the ability of exponentially growing bacteria to survive low pH (18). CRP-dependent AR functions in the presence of glucose. When glucose is exhausted, the formation of the cAMP-CRP com-
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plex is restored and the CRP-repressed AR genes are inactivated. A question, therefore, arises as to how bacteria resist acidic stresses in the presence of substantial cAMP-CRP complex. Here we show that pyruvate and/or its downstream metabolites induce AR by a mechanism that is independent of cAMP-CRP. We reveal that pyruvate and/or its downstream metabolites enhance AR by activating the small noncoding RNA (sncRNA) Spot42 and that the pyruvate activation of Spot42 does not require the cAMP-CRP complex. Spot42, in turn, confers AR by elevating expression of RpoS, a master regulator of stress resistance (19, 20). Interestingly, Spot42 is repressed by the cAMP-CRP complex (21). Thus, Spot42 is involved both in CRP-independent AR and in CRPdependent AR. Spot42 is widely present in medically important gammaproteobacterial genera such as Escherichia, Pantoea, Xenorhabdus, Salmonella, Citrobacter, Yersinia, Serratia, Edwardsiella, Dickeya, Photorhabdus, Enterobacter, Klebsiella, Rahnella, and Shigella (22). In addition, pyruvate is a metabolic product of many sugars. Therefore, the AR mechanisms revealed here are not limited to E. coli grown on glucose but are applicable to diverse bacteria living on various carbon sources.
Applied and Environmental Microbiology
Received 24 March 2014 Accepted 24 April 2014 Published ahead of print 2 May 2014 Editor: M. Kivisaar Address correspondence to Ye Jin, [email protected], or Zhiming Cai, [email protected]. J.W. and Y.L. contributed equally to this article. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.01001-14
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Glucose confers acid resistance on exponentially growing bacteria by repressing formation of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and consequently activating acid resistance genes. Therefore, in a glucose-rich growth environment, bacteria are capable of resisting acidic stresses due to low levels of cAMP-CRP. Here we reveal a second mechanism for glucose-conferred acid resistance. We show that glucose induces acid resistance in exponentially growing bacteria through pyruvate, the glycolysis product. Pyruvate and/or the downstream metabolites induce expression of the small noncoding RNA (sncRNA) Spot42, and the sncRNA, in turn, activates expression of the master regulator of acid resistance, RpoS. In contrast to glucose, pyruvate has little effect on levels of the cAMP-CRP complex and does not require the complex for its effects on acid resistance. Another important difference between glucose and pyruvate is that pyruvate can be produced by bacteria. This means that bacteria have the potential to protect themselves from acidic stresses by controlling glucose-derived generation of pyruvate, pyruvate-acetate efflux, or reversion from acetate to pyruvate. We tested this possibility by shutting down pyruvate-acetate efflux and found that the resulting accumulation of pyruvate elevated acid resistance. Many sugars can be broken into glucose, and the subsequent glycolysis generates pyruvate. Therefore, pyruvate-associated acid resistance is not confined to glucose-grown bacteria but is functional in bacteria grown on various sugars.
Acid Resistance in Bacteria
MATERIALS AND METHODS
July 2014 Volume 80 Number 14
counting the CFU of acid-treated cells. As untreated controls, cells before the acid treatment were also quantified by counting the CFU as described above. Acid survival (expressed as percent) was calculated with the following formula: 100 ⫻ (CFU of treated cells/CFU of untreated cells). All assays were carried out in quadruplicate on two different occasions. Growth curve. For simultaneous measurement of acid resistance and corresponding cell growth, 100-l quantities of overnight cultures were diluted in 50 ml of LB medium in 250-ml flasks, followed by incubation with shaking (220 rpm) for 12 h at 37°C. Every 2 h, an aliquot was sampled from the culture and optical density at 600 nm was measured after dilution when necessary. Determination of extracellular cAMP, pyruvate, and acetate. To quantify extracellular cAMP, overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h. Cell suspensions were then centrifuged at 12,000 ⫻ g for 5 min at 4°C. The resulting supernatants were then subjected to cAMP determination using a cAMP direct immunoassay kit (BioVision, Mountain View, CA) as directed by the manufacturer. To quantify extracellular pyruvate, overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h before the quantification assay. For time course measurement of extracellular pyruvate, cells were incubated or 6 h and pyruvate was determined at 1-h intervals. Pyruvate in the supernatants was assayed using an EnzyChrom pyruvate assay kit (BioAssay Systems, Hayward, CA). Acetate in the supernatants was quantified using an acetate detection kit (Megazyme, Bray, Ireland) according to the manufacturer’s instructions. Western blotting. Overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h. The bacterial cells were then centrifuged at 12,000 ⫻ g for 5 min at 4°C, and the resulting cell pellets were mixed with sample loading buffer (300 mM Tris-HCl, 6% SDS, 30% glycerol, 0.6% bromphenol blue, 1.2 M betamercaptoethanol), boiled for 10 min, and subjected to 12% SDS-PAGE in duplicate. Proteins of one set of gels were then electrotransferred onto an Immobilon-P membrane (Millipore, MA) and detected with an E. coli CRP monoclonal antibody (NeoClone Biotechnology, Madison, WI) followed by an anti-mouse horseradish peroxidase (HRP)-conjugated antibody (Invitrogen). Proteins were visualized using the SuperSignal West Pico chemiluminescent substrate from Pierce (Rockford, IL). The other set of SDS-PAGE gels was stained with Coomassie brilliant blue to demonstrate equal loading. Statistical analysis. Independent t tests were used to compare means obtained from pyruvate, cAMP, beta-galactosidase activity, and acid resistance assays. P values of ⬍0.05 were considered statistically significant.
RESULTS AND DISCUSSION
Pyruvate and/or its downstream metabolites induce AR. It is well known that pyruvate is a product of glycolysis. Our data showed that added glucose (20 mM), lactose (1%), or glycerol (5%) increased extracellular levels of pyruvate (all P ⬍ 0.05) (Fig. 1A), indicating that many sugars are converted to pyruvate. Under normal conditions without exogenous pyruvate, intracellular pyruvate levels are 45-fold higher than extracellular pyruvate levels (28). The increased extracellular pyruvate observed with cells grown on various sugars (Fig. 1A), therefore, indicates a more significant increase in intracellular pyruvate levels. Our time course assays revealed that E. coli quickly consumed pyruvate added to the growth medium so that extracellular pyruvate decreased to low levels after 5 h of incubation with shaking in LB at 37°C (Fig. 1B), suggesting that any effects of pyruvate on biological processes occur primarily in the log phase. Next, we examined if pyruvate affected the ability of bacteria to resist acid. For AR assays, bacterial cells in the early log phase were treated with acidified LB medium at pH 2.0 for 2 h and then the survival rate was determined. As a control, glucose at 20 mM dra-
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E. coli strains and growth conditions. The E. coli K-12 strain MG1655 (from our laboratory stock) and its isogenic mutants (constructed previously or in this study) were used for phenotypic examination in this study. MG1655 is a widely investigated laboratory strain with annotated sequence and biochemical information. It has been completely sequenced so that genetic engineering can be easily designed and performed. For these reasons, we chose MG1655 for this study and our data could be compared with and integrated with previous findings. The MG1655 strains were grown at 37°C in Luria-Bertani (LB) medium (Affymetrix, Cleveland, OH) or on LB agar (Affymetrix) (23, 24). The antibiotics ampicillin (50 g/ml; Sigma, St. Louis, MO) and chloramphenicol (12.5 g/ml; Sigma) were added to growth medium or agar when appropriate. Gene deletion. Gene deletion was performed using the recombineering system as described previously (25, 26). Briefly, the E. coli K-12 strain MG1655 was transformed with plasmid pSim6 (a gift from Donald Court), from which the expression of the recombination proteins was induced for 15 min at 42°C with shaking (220 rpm). The MG1655 strain carrying pSim6 was incubated at 32°C with shaking at 220 rpm until the optical density at 600 nm (OD600) of the bacterial culture reached 0.4 to 0.6. Then, PCR fragments encompassing a loxP-cat-loxP cassette with homology (45 nucleotides [nt]) to the regions immediately flanking the deletion target were transformed via electroporation into the MG1655 cells harboring pSim6. After induction of red functions, recombinants were selected for chloramphenicol resistance (encoded by the cat gene) and were further verified by colony PCR and sequencing. spf cloning. The selectable loxP-cat-loxP cassette was first inserted immediately after the stop codon of the spf gene (encoding Spot42) on the chromosome by recombineering (as described above). We then inserted spf preceded by its native promoter and the loxP-cat-loxP cassette in a pET32a expression vector by recombineering. Specifically, we PCR amplified the spf gene (including its native promoter) and adjacent “floxed” cat cassette using primers that contained homology (45 nt) to the plasmid insertion site. The PCR product and expression vector were cotransformed into MG1655 carrying pSim6 after induction of red. Recombinants were selected for chloramphenicol resistance and verified by PCR and sequencing. In the recombinant plasmid, named pSpot42, the native promoter of the spf gene drove Spot42 overproduction without induction. To construct an empty plasmid, the loxP-cat-loxP cassette was inserted into pET32a (Novagen, Inc., Madison, WI) and selected for chloramphenicol resistance. The resulting plasmid was named pCm. As pCm did not express Spot42 but was otherwise similar to pSpot42, it served as a negative control. Construction of a chromosomal spf-lacZ transcriptional fusion. The loxP-cat-loxP selectable cassette was inserted immediately after the stop codon of the lacZ gene on the MG1655 chromosome using recombineering (as described above). Next, the lacZ-loxP-cat-loxP cassette was PCR amplified and inserted immediately prior to the poly-T string of the spf gene on the chromosome. The inserted lacZ gene together with its native ribosome binding site (15 bp upstream of the start codon) was cotranscribed with spf. Beta-galactosidase assay. Overnight cultures were diluted 1:500 in fresh LB medium and incubated at 37°C with shaking at 220 rpm for 4 h. Expression of lacZ fusions was quantified using a commercially available beta-galactosidase assay kit (Pierce Biotechnology, Rockford, IL) as described previously (23, 27). Levels of beta-galactosidase, expressed as Miller units (MU), were calculated using the following formula: OD420 ⫻ 1,000/(OD600 ⫻ hydrolysis time ⫻ volume of lysate). Acid resistance assay. Overnight cultures were diluted 1:500 in fresh LB medium (pH 6.8) and incubated at 37°C with shaking at 220 rpm for 4 h. Cells were then challenged to pH 2.0 by adjusting the LB medium with HCl. Cells were treated statically with acid for 2 h and then washed with phosphate-buffered saline (PBS) at pH 7.2 to remove acid. Cells were serially diluted in PBS, and 20 l of each diluted suspension was plated on LB agar. After overnight culture at 37°C, viable cells were determined by
Wu et al.
matically increased AR by a factor of more than 1,700 (P ⫽ 0.016) (Fig. 1C). This was expected, as glucose represses the formation of the cAMP-CRP complex, a transcriptional regulator known to negatively regulate AR by repressing AR genes (11, 12). The requirement of the glucose activation of AR for the cAMP-CRP complex was confirmed by our AR assays, in which the survival rate of a crp null mutant was 2.59% (⫾1.5%) after acid challenge (Fig. 1C, lower portion), 2,500 times higher than that of the wildtype strain (Fig. 1C, upper portion) (P ⫽ 0.0096). Moreover, added glucose at 20 mM did not increase AR with the crp null mutant (P ⫽ 0.933) (Fig. 1C, lower portion). Interestingly, added pyruvate at 20 mM significantly increased AR, as observed with glucose (P ⫽ 0.0246) (Fig. 1C, upper portion). In contrast to glucose, however, pyruvate did not lose the AR-inducing ability with the crp null mutant (P ⫽ 0.0054) (Fig. 1C, lower portion). cAMP assays and Western blot assays revealed that added pyruvate had no effects on either cAMP (P ⫽ 0.818) or CRP levels (Fig. 1D). Collectively, these results indicate that pyruvate induces AR by a mechanism independent of the cAMP-CRP complex. It is noteworthy that 20 mM was the concentration of pyruvate added to the growth medium. Due to the consumption of pyruvate, the concentration of pyruvate dramatically decreased after 4 h of incubation when the bacteria were subjected to the AR assays. As revealed by the time course measurement of extracellular pyruvate (Fig. 1B), the extracellular pyruvate concentration was less than 47.5 (⫾3.3) M 4 h after the concentration dropped to 20 mM. It is intracellular pyruvate that mediates AR. However, exogenous pyruvate at high concentrations just mildly increased intracellular levels of pyruvate (28), since pyruvate uptake is an en-
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FIG 2 Elevation of acid resistance as a result of shutting down acetate production. (A) Extracellular levels of acetate in wild-type MG1655 (wt) and the ackA-pta null mutant (⌬ackA-pta) as a function of time. (B) Extracellular levels of pyruvate in the wt and ⌬ackA-pta mutant grown in LB medium for 4 h. (C) Acid survival at pH 2.0 of the wt and ⌬ackA-pta mutant grown in LB medium for 4 h before 2 h of acid treatment. P values of ⬍0.05 (*) were considered statistically significant.
ergy-dependent active transport process (29). For these reasons, the concentration of added pyruvate has to be high to cause remarkable effects on AR and regulation of target genes in this study. In contrast to added pyruvate, added glucose and other carbon sources result in endogenous generation of pyruvate through glycolysis. After growth in LB medium plus 20 mM glucose for 4 h, bacteria displayed extracellular pyruvate concentrations of 40.7 (⫾7.9) M (Fig. 1A), close to the extracellular pyruvate concentrations of the bacteria grown in LB medium supplemented with 20 mM pyruvate. This indicates that pyruvate generated from glucose is sufficient to have significant effects on AR. Increased AR as a result of the addition of pyruvate can result from pyruvate or downstream metabolites such as acetyl coenzyme A (Ac-CoA), acetyl phosphate, and acetate, which are reversibly interconvertible (1). To see if these metabolites are responsible for elevated AR, we tested the effects of Ac-CoA and acetate on log-phase AR. Addition of neither of them had any effect on log-phase AR (both P ⬎ 0.05) (Fig. 1C), excluding roles for Ac-CoA and acetate. Conversion from Ac-CoA to acetate is mainly achieved by the AckA-Pta (acetate kinase-phosphotransacetylase) pathway (1, 30, 31). As the acetate outflow is an outlet for pyruvate discharge, we reasoned that shutting off this pathway could elevate levels of pyruvate, thereby increasing AR. To test this possibility, we deleted the entire ackA-pta operon. We then monitored extracellular acetate over time for the resulting null mutant (⌬ackA-pta) and the wild-type strain to confirm the role of this pathway in acetate outflow. With the wild-type strain, extracellular acetate levels increased during the log phase and dropped after 4 h of incubation (Fig. 2A). This phenomenon has previously been termed “acetate switch” and occurs when glucose is exhausted and bacteria transit from acetate excretion to acetate uptake (1). In contrast, the ⌬ackA-pta mutant produced little acetate throughout the incubation (Fig. 2A), confirming that deleting the ackApta operon effectively shuts off acetate excretion. We then examined if shutting down the acetate outflow caused pyruvate accumulation and had any effect on AR. As shown in Fig. 2B, deleting the ackA-pta operon elevated extracellular levels of pyru-
Applied and Environmental Microbiology
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FIG 1 Pyruvate induces acid resistance independently of the cAMP-CRP complex. (A) Levels of extracellular pyruvate of the E. coli MG1655 strain grown in LB medium alone, LB medium plus 20 mM glucose (Glc), LB medium plus 1% lactose (Lac), and LB medium plus 5% glycerol (Gly) for 4 h. (B) Time course of extracellular pyruvate levels of MG1655 grown in LB medium plus 40 mM pyruvate. (C) Acid survival at pH 2.0 of the wild-type MG1655 (wt) grown in LB medium alone, LB medium plus 20 mM glucose (Glc), LB medium plus 20 mM pyruvate (Pyr), LB plus 10 mM acetyl coenzyme A (coA), and LB medium plus 20 mM acetate (Ac) and of a crp null mutant (⌬crp) grown in LB medium alone, LB medium plus 20 mM glucose (Glc), and LB medium plus 20 mM pyruvate (Pyr). Cells were grown for 4 h and then treated with acidified LB medium (pH 2.0) for 2 h. The survival rate was then determined by counting CFU. (D) Extracellular cAMP and corresponding CRP levels of MG1655 grown in LB medium without pyruvate supplementation and LB medium plus 20 mM pyruvate. Cells were grown for 4 h before quantification of cAMP and CRP. P values of ⬍0.05 (*) were considered statistically significant.
Acid Resistance in Bacteria
and acid survival at pH 2.0 of wild-type E. coli MG1655 (wt) and an spf null mutant (⌬spf) as a function of time. (B) Cell growth (OD600) and acid survival at pH 2.0 of the ⌬spf mutant carrying a control vector (pCm) or pSpot42 that overproduces Spot42 as a function of time. Cells were grown for 4 h before the acid resistance assay. OD600 was measured as follows. Overnight cultures (100 l) were diluted in 50 ml of LB medium in 250-ml flasks and then incubated at 37°C. Every 2 h, an aliquot was taken for determination of OD600 after dilution when necessary.
vate by a factor of 126 (P ⫽ 0.0079). Similar observations have also been reported previously (32, 33). As predicted, deleting the ackApta operon increased AR by a factor of 577 (P ⫽ 0.0236) (Fig. 2C), confirming the role of pyruvate in the elevated log-phase AR. Pyruvate and/or the downstream metabolites enhance acid resistance by inducing Spot42. We next asked how pyruvate and/or the downstream metabolites conferred on bacteria the ability to resist low pH. The following findings linked pyruvateinduced AR with a small noncoding RNA (sncRNA) named Spot42. Our previous work on a knockout library of sncRNAs has revealed that some sncRNAs upregulated AR. One unpublished AR-regulating sncRNA is Spot42, which is encoded by the spf gene. We found that deleting the spf gene reduced AR during the first 8 h of incubation (all P ⬍ 0.05) (Fig. 3A). We then cloned the spf gene into a multicopy pET32a vector, generating pSpot42, which overexpresses Spot42. Consistent with the knockout data, overproduction of Spot42 increased AR through the culture (all P ⬍ 0.05) (Fig. 3B). Specifically, bacterial cells were grown in LB medium and aliquots were measured for both AR and OD600 at different time points. By this means, AR as a function of cell growth phase could be evaluated. Cells overproducing Spot42 were 4,000- to 15,000-fold more acid resistant than those lacking the sncRNA after 2 to 6 h of incubation. This figure dropped to 7-fold after 8 h of incubation and was further reduced to 1.5-fold after 16 h (Fig. 3B). Cell growth curves revealed that 2 to 6 h corresponded to the log phase (Fig. 3A and B). These results indicate that AR regulation by Spot42 is most evident in the early log phase. Spot42 has been identified as a downstream target of the cAMP-CRP complex and is activated by glucose (21). This together with the Spot42 regulation of log-phase AR encouraged us to ask if Spot42 has a role in the pyruvate-conferred log-phase AR. To facilitate evaluation of the possible role of Spot42, we constructed a chromosomally located spf-lacZ transcriptional fusion in E. coli MG1655 so that Spot42 transcription could be easily quantified by measuring beta-galactosidase activity. Subsequent beta-galactosidase assays showed that pyruvate increased Spot42
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FIG 4 Induction of Spot42 transcription by pyruvate. (A) Beta-galactosidase activity of mutants carrying a chromosomal spf-lacZ transcriptional fusion with the crp gene (crp⫹) or without crp (crp⫺). Cells were grown in LB medium supplemented with increasing concentrations of glucose (Glc) or pyruvate (Pyr) for 4 h before quantification of beta-galactosidase activity. (B) Pyruvate dramatically increased acid resistance of wild-type MG1655 (wt) but affected acid resistance of the spf null mutant (⌬spf) to a much lower extent. However, deletion of spf did not abolish pyruvate-conferred acid resistance. P values of ⬍0.05 (*) were considered statistically significant.
expression (P ⫽ 0.00015) (Fig. 4A). Activation of Spot42 by pyruvate was maintained in cells in which crp had been deleted (⌬crp) (P ⬍ 0.05 at all pyruvate concentrations) (Fig. 4A). Thus, in contrast to glucose, pyruvate induces Spot42 expression by a CRPindependent mechanism. The induction of Spot42 expression by pyruvate and upregulation of log-phase AR by Spot42 suggest that pyruvate and/or the downstream metabolites confer log-phase AR at least in part through Spot42. It is noteworthy that deletion of the spf gene, encoding Spot42, did not abolish pyruvate-conferred AR but the effects of pyruvate on AR were not statistically significant (P ⫽ 0.08) (Fig. 4B), confirming the critical role of Spot42 for this AR system. RpoS participates in pyruvate- and Spot42-conferred acid resistance. The phase specificity of Spot42-mediated AR prompted us to link it to growth phase-specific biological processes. One such process is rpoS expression that is not induced until bacteria enter the stationary phase. RpoS is a master regulator of stress resistance (19, 20). This led us to speculate that RpoS may have a role in Spot42-mediated AR. To quantify RpoS expression, we employed a previously constructed MG1655 isogenic mutant carrying an rpoS-lacZ translational fusion on the chromosome (23). In support of the above speculation, deleting the spf gene reduced rpoS-lacZ fusion expression regardless of the pyruvate addition (both P ⬍ 0.05) (Fig. 5A), and overexpressing sncRNA increased the expression (P ⫽ 0.0018) (Fig. 5B). Given the activating effects of pyruvate on Spot42, we predicted that pyruvate enhanced RpoS expression. As expected, pyruvate increased rpoS-lacZ expression (P ⫽ 0.0001) (Fig. 5A). Thus, pyruvate enhances Spot42, which, in
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FIG 3 Effects of Spot42 on bacterial acid resistance. (A) Cell growth (OD600)
Wu et al.
FIG 5 Role of RpoS in pyruvate- and Spot42-conferred acid resistance. (A) Beta-galactosidase activity of mutants carrying a chromosomal rpoS-lacZ translational fusion with the small noncoding RNA Spot42 (Spot42⫹) or without Spot42 (Spot42⫺). Cells were grown in LB medium or LB medium plus 20 mM pyruvate (Pyr) for 4 h before measurement of beta-galactosidase activity. (B) Effects of Spot42 overproduction from pSpot42 on expression of the rpoS-lacZ translational fusion. The fusion strain carrying an empty vector, pCm, served as a negative control. (C) Acid survival of various strains carrying pCm or pSpot42. These include wild-type E. coli MG1655 (wt), an isogenic rpoS null mutant (⌬rpoS), and an isogenic mutant, d567–1342, in which the native regulation of rpoS was removed. All cells were grown in LB medium for 4 h before the acid resistance assay. P values of ⬍0.05 (*) were considered statistically significant.
turn, activates RpoS. However, deleting the spf gene did not abolish the effects of pyruvate on RpoS (P ⫽ 0.00036) (Fig. 5A), indicating the existence of a Spot42-indepenent mechanism. To provide more evidence for the role of RpoS in pyruvate and Spot42 regulation of AR, we tested if removing RpoS abolished the regulation. An rpoS null mutant (⌬rpoS) showed an extremely low survival rate after 2 h of acid treatment, and Spot42 overproduction failed to increase it (Fig. 5C). We then employed a previously constructed rpoS mutant, d567–1342, which loses the natural regulation of rpoS and constitutively expresses rpoS (23). Removing the natural regulation of rpoS significantly diminished the effects of Spot42 on AR, as Spot42 overproduction increased AR ⬃500fold in the wild-type strain but only ⬃27-fold in d567–1342 (P ⫽ 0.012) (Fig. 5C). sncRNAs regulate gene expression either directly by binding to their mRNA targets or indirectly by acting on transcriptional or translational factors that, in turn, regulate the expression (34). If Spot42 regulates rpoS by a direct mechanism, there should be extensive complementarities between the two RNAs. However, complementarity analysis using the program TargetRNA (http://snowwhite.wellesley.edu/targetRNA) (35) did not reveal any extensive complementarities between rpoS and Spot42, suggesting that Spot42 regulates RpoS through an indirect mechanism. The AR system reported in this study is summarized in Fig. 6. It
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appears to be a new AR system different from the currently known systems that are primarily restricted to the stationary phase (24, 36, 37). Acid resistance system 1 (AR1) requires the induction of RpoS but is repressed by glucose, which is contrast to our system. AR2 is glutamate dependent, which requires the presence of glutamate decarboxylase and a putative glutamate:GABA antiporter. Our AR system is not part of AR2, since AR2 is absent from logphase cells grown in LB medium plus 4% glucose (38). AR3 is arginine dependent, requires the presence of arginine decarboxylase (AdiA), and provides a modest level of protection. AR3 is induced by low pH under anaerobic conditions, which is different from our AR system. In contrast to these thoroughly investigated stationary-phase AR systems, much less is known about how logphase cells resist acid, since efforts to study this may have been discouraged by the fact that log-phase cells are acid sensitive. This study reveals for the first time how exponentially growing bacteria such as E. coli resist low pH through pyruvate and/or its downstream metabolites. Bacteria use pyruvate and/or its downstream metabolites as key effector molecules to induce AR by activating expression of the sncRNA Spot42, which, in turn, upregulates RpoS. Any biological processes that affect pyruvate levels would, therefore, have an impact on AR. This is confirmed by our observation that increasing pyruvate accumulation by shutting down pyruvate discharge remarkably elevates AR. Another source of pyruvate is sugar metabolism. Bacteria grown on sugars such as glucose produce pyruvate inside the cells through glycolysis without a need for active uptake of extracellular pyruvate. We show that addition of glucose at 20 mM resulted in a 240-fold increase in extracellular levels of pyruvate and enhanced AR accordingly. The concentration of glucose in foods and drinks easily reaches 20 mM. For instance, the glucose concentrations of orange juices range from 22 to 218 mM (39). Thus, this study reveals that bacteria in sugar-rich foods or drinks are capable of acquiring the ability to resist acid at least partially through the pyruvate- and Spot42-dependent AR system. The resulting acid-resistant bacteria could survive gastric acid and colonize the intestine to become commensal habitants or cause diseases, depending on the virulence of the bacteria. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant C010301), Basic Research Program of Shenzhen (grant
Applied and Environmental Microbiology
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FIG 6 Pathways of the acid resistance system mediated by pyruvate and Spot42. P-EIIAglc, the phosphorylated EIIA component of the glucose-specific phosphotransferase system; EIIAglc, dephosphorylated EIIA component of the glucose-specific phosphotransferase system; Glc, glucose; Pyr, pyruvate; AR, acid resistance.
Acid Resistance in Bacteria
JCYJ20120614155650545), and National Program on Key Basic Research Project (grant 2014CB745200).
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REFERENCES
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23. 24. 25.
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27.
28. 29. 30.
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1. Wolfe AJ. 2005. The acetate switch. Microbiol. Mol. Biol. Rev. 69:12–50. http://dx.doi.org/10.1128/MMBR.69.1.12-50.2005. 2. Farmer WR, Liao JC. 1997. Reduction of aerobic acetate production by Escherichia coli. Appl. Environ. Microbiol. 63:3205–3210. 3. Veit A, Polen T, Wendisch VF. 2007. Global gene expression analysis of glucose overflow metabolism in Escherichia coli and reduction of aerobic acetate formation. Appl. Microbiol. Biotechnol. 74:406 – 421. http://dx .doi.org/10.1007/s00253-006-0680-3. 4. Repaske DR, Adler J. 1981. Change in intracellular pH of Escherichia coli mediates the chemotactic response to certain attractants and repellents. J. Bacteriol. 145:1196 –1208. 5. Kihara M, Macnab RM. 1981. Cytoplasmic pH mediates pH taxis and weak-acid repellent taxis of bacteria. J. Bacteriol. 145:1209 –1221. 6. Roe AJ, O’Byrne C, McLaggan D, Booth IR. 2002. Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity. Microbiology 148:2215–2222. 7. Roe AJ, McLaggan D, Davidson I, O’Byrne C, Booth IR. 1998. Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J. Bacteriol. 180:767–772. 8. Russell JB, Diez-Gonzalez F. 1998. The effects of fermentation acids on bacterial growth. Adv. Microb. Physiol. 39:205–234. 9. Schmid-Hempel P, Frank SA. 2007. Pathogenesis, virulence, and infective dose. PLoS Pathog. 3(10):e147. http://dx.doi.org/10.1371/journal .ppat.0030147. 10. Griffin PM, Tauxe RV. 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13:60 –98. 11. Castanie-Cornet MP, Foster JW. 2001. Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes. Microbiology 147:709 –715. 12. Gosset G, Zhang Z, Nayyar S, Cuevas WA, Saier MH, Jr. 2004. Transcriptome analysis of Crp-dependent catabolite control of gene expression in Escherichia coli. J. Bacteriol. 186:3516 –3524. http://dx.doi.org/10.1128 /JB.186.11.3516-3524.2004. 13. Tagami H, Aiba H. 1998. A common role of CRP in transcription activation: CRP acts transiently to stimulate events leading to open complex formation at a diverse set of promoters. EMBO J. 17:1759 –1767. http://dx .doi.org/10.1093/emboj/17.6.1759. 14. Busby S, Ebright RH. 1999. Transcription activation by catabolite activator protein (CAP). J. Mol. Biol. 293:199 –213. http://dx.doi.org/10.1006 /jmbi.1999.3161. 15. Park YH, Lee BR, Seok YJ, Peterkofsky A. 2006. In vitro reconstitution of catabolite repression in Escherichia coli. J. Biol. Chem. 281:6448 – 6454. http://dx.doi.org/10.1074/jbc.M512672200. 16. Bettenbrock K, Sauter T, Jahreis K, Kremling A, Lengeler JW, Gilles ED. 2007. Correlation between growth rates, EIIACrr phosphorylation, and intracellular cyclic AMP levels in Escherichia coli K-12. J. Bacteriol. 189:6891– 6900. http://dx.doi.org/10.1128/JB.00819-07. 17. Hogema BM, Arents JC, Bader R, Eijkemans K, Yoshida H, Takahashi H, Aiba H, Postma PW. 1998. Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc. Mol. Microbiol. 30:487– 498. http://dx.doi.org/10.1046/j.1365-2958.1998.01053.x. 18. Rowbury RJ, Goodson M. 1998. Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP. J. Appl. Microbiol. 85:615– 620. http://dx.doi.org/10.1046/j.1365 -2672.1998.853569.x. 19. Battesti A, Majdalani N, Gottesman S. 2011. The RpoS-mediated general stress response in Escherichia coli. Annu. Rev. Microbiol. 65:189 –213. http://dx.doi.org/10.1146/annurev-micro-090110-102946. 20. Lacour S, Landini P. 2004. SigmaS-dependent gene expression at the onset of stationary phase in Escherichia coli: function of sigmaSdependent genes and identification of their promoter sequences. J.
Bacteriol. 186:7186 –7195. http://dx.doi.org/10.1128/JB.186.21.7186 -7195.2004. Polayes DA, Rice PW, Garner MM, Dahlberg JE. 1988. Cyclic AMPcyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli. J. Bacteriol. 170:3110 –3114. Hansen GA, Ahmad R, Hjerde E, Fenton CG, Willassen NP, Haugen P. 2012. Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida. BMC Genomics 13:37. http://dx.doi.org/10.1186/1471-2164-13-37. Jin Y, Watt RM, Danchin A, Huang JD. 2009. Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli. BMC Genomics 10:165. http://dx.doi.org/10.1186/1471-2164-10-165. Lin J, Smith MP, Chapin KC, Baik HS, Bennett GN, Foster JW. 1996. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl. Environ. Microbiol. 62:3094 –3100. Sharan SK, Thomason LC, Kuznetsov SG, Court DL. 2009. Recombineering: a homologous recombination-based method of genetic engineering. Nat. Protoc. 4:206 –223. http://dx.doi.org/10.1038/nprot.2008 .227. Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 97:5978 –5983. http://dx.doi.org /10.1073/pnas.100127597. Jin Y, Watt RM, Danchin A, Huang JD. 2009. Use of a riboswitchcontrolled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation. J. Biol. Chem. 284: 28738 –28745. http://dx.doi.org/10.1074/jbc.M109.028076. Yang YT, Bennett GN, San KY. 2001. The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli. Metab. Eng. 3:115–123. http://dx.doi.org/10.1006/mben.2000.0166. Lang VJ, Leystra-Lantz C, Cook RA. 1987. Characterization of the specific pyruvate transport system in Escherichia coli K-12. J. Bacteriol. 169: 380 –385. Hesslinger C, Fairhurst SA, Sawers G. 1998. Novel keto acid formatelyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate. Mol. Microbiol. 27:477– 492. http://dx.doi.org/10.1046/j.1365-2958 .1998.00696.x. Klein AH, Shulla A, Reimann SA, Keating DH, Wolfe AJ. 2007. The intracellular concentration of acetyl phosphate in Escherichia coli is sufficient for direct phosphorylation of two-component response regulators. J. Bacteriol. 189:5574 –5581. http://dx.doi.org/10.1128/JB.00564-07. Dittrich CR, Vadali RV, Bennett GN, San KY. 2005. Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate. Biotechnol. Prog. 21:627– 631. http://dx.doi.org/10 .1021/bp049730r. Sadykov MR, Thomas VC, Marshall DD, Wenstrom CJ, Moormeier DE, Widhelm TJ, Nuxoll AS, Powers R, Bayles KW. 2013. Inactivation of the Pta-AckA pathway causes cell death in Staphylococcus aureus. J. Bacteriol. 195:3035–3044. http://dx.doi.org/10.1128/JB.00042-13. Repoila F, Darfeuille F. 2009. Small regulatory non-coding RNAs in bacteria: physiology and mechanistic aspects. Biol. Cell 101:117–131. http: //dx.doi.org/10.1042/BC20070137. Tjaden B. 2008. TargetRNA: a tool for predicting targets of small RNA action in bacteria. Nucleic Acids Res. 36:W109 –W113. http://dx.doi.org /10.1093/nar/gkn264. Foster JW. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2:898 –907. http://dx.doi.org/10.1038 /nrmicro1021. Lin J, Lee IS, Frey J, Slonczewski JL, Foster JW. 1995. Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J. Bacteriol. 177:4097– 4104. Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW. 1999. Control of acid resistance in Escherichia coli. J. Bacteriol. 181:3525– 3535. Li WJ, Goovaerts P, Meurens M. 1996. Quantitative analysis of individual sugars and acids in orange juices by near-infrared spectroscopy of dry extract. J. Agric. Food Chem. 44:2252–2259. http://dx.doi.org/10.1021 /jf9500750.
