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Cra negatively regulates acid survival in Yersinia pseudotuberculosis

Yangbo Hu, Pei Lu, Yong Zhang, Yunlong Li, Lamei Li, Li Huang, Shiyun Chen
DOI: http://dx.doi.org/10.1111/j.1574-6968.2011.02227.x 190-195 First published online: 1 April 2011


Survival in acidic environments is important for successful infection of gastrointestinal pathogens. Many bacteria have evolved elaborate mechanisms by inducing or repressing gene expression, which subsequently provide pH homeostasis and enable acid survival. In this study, we employed comparative proteomic analysis to identify the acid-responsive proteins of a food-borne enteric bacterium, Yersinia pseudotuberculosis. The expression level of eight proteins involved in carbohydrate metabolism was up- or downregulated over twofold at pH 4.5 compared with pH 7.0. The role of a global transcriptional regulator catabolite repressor/activator Cra was further studied in this acid survival process. lacZ-fusion analysis showed that expression of cra was repressed under acidic pH. Deletion of the cra gene increased acid survival by 10-fold, whereas complementation restored the wild-type phenotype. These results lead us to propose that, in response to acidic pH, the expression of cra gene is downregulated to increase acid survival. This is the first study to demonstrate the regulatory role of Cra in acid survival in an enteric bacterium.

  • Cra
  • acid survival
  • Yersinia pseudotuberculosis
  • proteomic analysis
  • carbohydrate metabolism


The acidity of the stomach is a primary barrier through which all food-borne microbial pathogens must pass (Linet al,1995; Foster, 2004). In response to this acid stress, many enteric pathogens have evolved different acid survival systems during long-time host–pathogen interactions. Several such acid survival systems, for example acid resistance (AR) and acid tolerance response, have been defined as helping enteric bacteria to cope with this form of environmental stress (Leeet al,1994; Foster, 1995). In addition to these earlier studies, transcription profiling and proteomic analyses have been applied to globally analyze acid-responsive genes and proteins in enteric pathogens. Expression of genes involved in energy metabolism, stress responses, capsular polysaccharide biosynthesis and gene regulation, have been demonstrated to be acid-induced or -repressed in different bacteria (Stanciket al,2002; Tuckeret al,2002; Chenget al,2007), and provide valuable information to further characterize details of acid survival in enteric bacteria.

Yersinia pseudotuberculosis is transmitted between animals and humans by contaminated food (Naganoet al,1997). Several studies related to acid stress of this bacterium have been reported. An earlier study showed that urease mutant of Y. pseudotuberculosis IP2777.4 loses its ability to survive at pH 3.0 in the presence of urea (Riotet al,1997). The Tat system (tatC), which is essential for virulence, has also been shown to contribute to acid survival of Y. pseudotuberculosis (Lavanderet al,2006). Two-component system regulon assays showed that several regulators, for example PhoP, OmpR and PmrA, control acid survival of Y. pseudotuberculosis (Flamezet al,2008). In our previous work, we have demonstrated that urease is one of the OmpR targets in the acid survival regulation process in Y. pseudotuberculosis (Huet al,2009). We have also characterized the aspartate-dependent acid survival system in Y. pseudotuberculosis and demonstrated the role of aspartase (AspA) in this process (Huet al,2010).

In this study, we first applied two-dimensional (2D) gel analysis to compare the global protein expression changes of Y. pseudotuberculosis cells at pH 4.5 and 7.0. The expression level of eight proteins involved in carbohydrate metabolism was up- or downregulated over twofold at pH 4.5. Genes involved in carbohydrate metabolism are regulated by a transcription factor named Cra for ‘catabolite repressor/activator’ (Saier & Ramseier, 1996); this information led us to speculate on the involvement of Cra in the regulation of this acid survival process. In this report, the role of Cra in acid survival regulation is characterized.

Materials and methods

2D gel analysis

Overnight culture (100mL) of Y. pseudotuberculosis YpIII strain grown in Yersinia–Luria–Bertani (YLB) broth (1% tryptone, 0.5% yeast extract and 0.5% NaCl) at pH 7.0 at 28°C was shifted to 37°C for 2h or diluted into YLB at pH 4.5 (adjusted with hydrochloric acid) for acid challenge assay and then incubated at 37°C for 2h. Protein sample preparation and 2D gel running were performed as described previously (Huet al,2009). Gels were stained with colloidal CBB G-250 and then scanned with a PowerLook 1000 (UMAX Technologies). Spot densities were quantified and analyzed with the pd quest software package (version 7.3.0, Bio-Rad). Each sample was prepared and analyzed in triplicate. Proteins with densities which increased or decreased ≥2-fold in all three experiments (P<0.01 in Student's t-test) were excised and digested with trypsin and identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS.

lacZ-fusion construction

To construct plasmids containing the translational genelacZ fusions, two primers were designed for each gene in which the reverse primer was designed at the 3′-end (missing the stop codon), and the forward primer of each gene was designed around 600bp upstream of the stop codon. The primers were listed in Supporting Information, Table S1. Each PCR product was inserted between the SalI and SpeI sites of pDM4-lacZ (Huet al,2009) to generate a series of plasmids named pDM4-2762Z, pDM4-2764Z and pDM4-3529Z, which was transformed into Escherichia coli S17-1. Homologous recombination and subsequent selection were carried out as described (Huet al,2009).

β-Galactosidase assay

YpIII strains carrying the genelacZ fusions were cultured overnight at 28°C in YLB broth and diluted into fresh YLB (pH 4.5) to ∼108CFUmL−1. After incubation at 37°C for 0 and 2h, cells were collected and washed with phosphate-buffered saline (PBS; pH 7.0). β-Galactosidase activity was determined and calculated as described previously (Huet al,2010). Data were analyzed by Student's t-test.

Mutant construction

For Δcra construction, two DNA fragments (493 and 500bp) up- and downstream of the cra gene, which omitted the entire cra gene were amplified using two pairs of primers, P3529-u-F/R and P3529-d-F/R (Table S1). These two PCR products were digested with the appropriate restriction enzymes and inserted into the similarly digested pDM4 to obtain pDM4-cram, which was subsequently transformed into E. coli S17-1. Transconjugation was performed to obtain Δcra strain. For complementation, the cra gene was amplified from YpIII genomic DNA using primer P3529-SF/XR (Table S1) and inserted into the low copy plasmid pKT100 (Huet al,2009) to obtain pKT-cra, which was then transformed into the Δcra strain.

Acid survival assays

Stationary-phase overnight cultures grown in YLB medium at pH 7.0 were diluted to 106CFUmL−1 in PBS at pH 4.5 and incubated at 37°C for 2h. The cultures were serially diluted and plated onto YLB agar plates and colonies were counted after 20h growth at 37°C. Percent survival was calculated as described previously (Huet al,2009). All assays were repeated at least three times and the data were analyzed by Student's t-test.


Increased expression of the fruBKA operon at acidic pH

We applied 2D gel to screen proteins whose expression was induced or repressed at pH 4.5, which is a sublethal pH for YpIII (Huet al,2009); 21 proteins showed more than twofold changes in all three replicate experiments (Fig. 1). These proteins were identified by MALDI-TOF MS and are summarized in Table 1. Among these proteins, eight proteins involved in carbohydrate metabolism were up- or downregulated over twofold at pH 4.5 (Fig. 2a). It is worth noting that the three proteins that were involved in the beginning step of fructose metabolism (FruB-1, FruB-2, FruK) (Owet al,2007) were all upregulated by acid challenge (Fig. 2a and b).

Figure 1

Comparative proteomic analysis of YpIII at pH 7.0 and pH 4.5 using 2D gels. The horizontal axis represents the pH of the isoelectric focusing gradients; the vertical axis represents molecular weights in thousands, based on migration in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numbers indicate proteins that showed more than twofold changes in all three replicate experiments.

View this table:
Table 1

Identification of differentially displayed proteins on 2D gels

Log 2 (pH 4.5/pH 7.0)
1GrcAAutonomous glycyl radical cofactor1.700.24
2YPK_2821Hypothetical protein1.290.20
3FruB-1Bifunctional fructose-specific PTS IIA/HPr protein2.350.41
4FruB-2Bifunctional fructose-specific PTS IIA/HPr protein1.510.41
5OsmYOsmotically induced periplasmic protein1.590.16
6UreGUrease accessory protein1.090.11
7RbsB2Ribose ABC transporter, periplasmic ribose-binding protein−1.660.42
8YPK_1579D-isomer-specific 2-hydroxyacid dehydrogenase family protein1.320.07
9YPK_0796Hypothetical protein−1.680.90
10LysSLysyl-tRNA synthetase3.410.04
11SdaAl-Serine dehydratase 1−2.410.93
12CpdB2′,3′-Cyclic-nucleotide 2′-phosphodiesterase−1.460.46
13GpmAPhosphoglycerate mutase 1 family−1.490.27
14AspAAspartate ammonia lyase1.360.26
15LigADNA ligase, NAD dependent−1.610.42
16ClpBProtein disaggregation chaperone1.830.46
17RsdAnti-RNA polymerase σ 70 factor−3.780.25
19AsnAAsparagine synthetase−1.520.32
20UreC-1Urease, α-subunit2.490.32
21UreC-2Urease, α-subunit1.980.32
Figure 2

Up- and downregulation of proteins involved in carbohydrate metabolism at pH 7.0 and pH 4.5. (a) Average changes of acid-regulated protein expression involved in carbohydrate metabolism from 2D gels (P<0.01 in Student's t-test for all these proteins compared with nonregulated protein). SE in triplicate experiments is shown as error bar. (b) Comparison of the expression of FruB-1, FruB-2 and FruK at pH 7.0 and pH 4.5 by 2D gel.

To further confirm the increased expression of fruBKA at acidic pH, we constructed translational lacZ fusions of fruBlacZ and fruAlacZ, which are located at the beginning and end of the fruBKA transcription unit (Fig. 3a). As seen in Fig. 3b, in accordance with our 2D gel results, higher β-galactosidase activities of both fruBlacZ and fruAlacZ fusions were observed at pH 4.5 than at pH 7.0, suggesting that expression of fruBKA is acid induced.

Figure 3

Expression of fruB and fruA at pH 7.0 and pH 4.5 by lacZ-fusion analysis. (a) Gene organization of fruBKA on YpIII genome. (b) β-Galactosidase activities of fruBlacZ and fruAlacZ at pH 7.0 and pH 4.5. **P<0.01.

Acid represses Cra production to induce fruBKA expression

Expression of the fruBKA operon encoding FruB, FruK and FruA was reported to be negatively controlled by a transcription factor Cra at physiological pH in several bacteria (Saier & Ramseier, 1996). This raised the question of whether the acid-induced fruBKA expression is mediated by Cra. To address this question, we constructed translational cralacZ fusion and compared the β-galactosidase activities with or without acid challenge. β-Galactosidase activities of cells challenged with acid were obviously lower than those without challenge, suggesting cra expression is repressed by acid (Fig. 4a). Furthermore, we constructed the cra deletion strain named Δcra and compared fruB and fruA expressions in Δcra and YpIII wild-type strains. Expressions of fruB and fruA were both acid induced in YpIII wild-type strain (Fig. 4b and c). But there was no significant difference of β-galactosidase activities at pH 7.0 and at pH 4.5 in Δcra, although the values in Δcra were obviously higher than in YpIII, which confirmed the Cra regulates fruBKA expression in YpIII. Together, these results suggested that the acid induction of fruBKA expression is mediated by repressed expression of Cra at acidic pH.

Figure 4

Expression of cra at acidic pH and regulation of Cra to fruBKA expression. (a) Comparison of cra expression at natural and acidic pH in YpIII by lacZ-fusion analysis. Acid-induced expression of fruB (b) and fruA (c) in YpIII and in Δcra. **P<0.01.

Cra negatively regulates acid survival

It was established that Cra acts as a global regulatory protein (Crasnier-Mednanskyet al,1997) but its role in bacterial acid survival has never been reported. To test whether Cra is involved in acid survival, we compared the percent survival of Δcra and the wild-type strains at acidic condition. The percent survival of Δcra was 10-fold higher compared with the wild type in PBS at pH 4.5 (Fig. 5). Complementation of cra deletion by a low copy plasmid carrying the cra gene restores the percent survival to the level of wild-type strain. No significant difference was observed in the percent survival of Δcra strain and Δcra carrying control plasmid pKT100 (Fig. 5). These results demonstrated that Cra negatively controls acid survival and suggested that depressed cra expression at acidic pH would increase acid survival.

Figure 5

Negative regulation by Cra of acid survival. Percent survivals of YpIII parent strain (YpIII), cra-deleted strain (Δcra), cra-complemented strain (pKT-cracra) and cra-deleted strain carrying the control vector pKT100 (pKT100/Δcra) in PBS at pH 4.5. **P<0.01.


Many regulatory proteins, such as RcsB, H-NS, EvgA and GadEXW, have been characterized to be involved in acid survival process (Foster, 2004; Tramontiet al,2006; Krinet al,2010). Most of these proteins were functionally related to amino acid-dependent AR systems. Carbohydrate metabolism has been demonstrated for many years to be important for overcoming acidic stress (Linet al,1995) but the mechanism remains unclear (Foster, 2004). Cyclic AMP receptor protein (CRP), a regulator that participates in glucose metabolism regulation (Perrenoud & Sauer, 2005), has been demonstrated to be a global regulator in the glucose-repressed AR system in E. coli (Castanie-Cornetet al,1999). It is so far the only regulator linking carbohydrate metabolism and bacterial acid survival, although the regulatory mechanism of CRP in acid survival process is still obscure.

In this study, we demonstrated the participation of another carbohydrate metabolism-related regulator Cra in the acid survival process. The Cra protein was initially characterized as the fructose repressor FruR and was demonstrated to be a global regulatory protein in carbohydrate metabolism (Saier & Ramseier, 1996). Although it has been shown that Cra regulates numerous genes involved in carbohydrate metabolism (Saier & Ramseier, 1996; Sarkaret al,2008), growth rate (Owet al,2007) and bacterial virulence (Allenet al,2000), there is no report showing the role of Cra in acid survival. And here, we also detected the decreased expression of cra at acidic pH (Fig. 4a). Based on these data, we propose that acidic pH downregulates cra expression, which will then increase bacterial acid survival.

After confirming the role of Cra in the acid survival process, it would be interesting to find the targets of Cra in regulating acid survival. Although we have confirmed the regulation of Cra to fruBKA, fruBKA is not directly involved in the acid survival process because deletion of fruBKA did not decrease acid survival (data not shown). The link between Cra and acid survival is not clear. Stationary σ factor RpoS, an important factor responsible for bacterial acid survival (Coldeweyet al,2007), has been shown to be Cra-depressed in E. coli at physiological pH (Sarkaret al,2008). We tried to compare the expression of rpoS gene in wild-type and cra-deleted strains at pH 7.0 and pH 4.5 by lacZ-fusion analysis. However, the β-galactosidase activities of rpoSlacZ in all these conditions were very low even at stationary phase (data not shown). Whether Cra regulates RpoS in the acid survival process is unclear and needs further studies.

In summary, we have demonstrated the regulatory role of Cra in the acid survival process in Y. pseudotuberculosis. This is the first report linking Cra to acid survival regulation, although establishing the targets for Cra in acid survival regulation requires further studies. Our current study provides information to characterize the details of the relationship between carbohydrate metabolism and acid survival in enteric bacteria.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. Primers used in this study.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.


We thank Prof. P. Williams for the YpIII strain. This study was supported by a grant from China National Science and Technology Specific Projects (2009ZX10004-207).


  • Editor:: Stephen Smith


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