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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Induction of the Escherichia coli yijE gene expression by cystine a
b
a
b
Kaneyoshi Yamamoto , Gen Nonaka , Takahiro Ozawa , Kazuhiro Takumi & Akira a
Ishihama a
Department of Frontier Bioscience and Research Center for Micro-Nano Technology, Hosei University, Koganei, Japan b
Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Kawasaki, Japan Published online: 27 Oct 2014.
Click for updates To cite this article: Kaneyoshi Yamamoto, Gen Nonaka, Takahiro Ozawa, Kazuhiro Takumi & Akira Ishihama (2014): Induction of the Escherichia coli yijE gene expression by cystine, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.972328 To link to this article: http://dx.doi.org/10.1080/09168451.2014.972328
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014
Induction of the Escherichia coli yijE gene expression by cystine Kaneyoshi Yamamoto1,*, Gen Nonaka2, Takahiro Ozawa1, Kazuhiro Takumi2 and Akira Ishihama1 1
Department of Frontier Bioscience and Research Center for Micro-Nano Technology, Hosei University, Koganei, Japan; 2Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Kawasaki, Japan Received July 23, 2014; accepted September 15, 2014
Downloaded by [Baskent Universitesi] at 00:28 21 December 2014
http://dx.doi.org/10.1080/09168451.2014.972328
Cystine is formed from two molecules of the cysteine under oxidized conditions, but is reversibly converted to cysteine by reduction. Growth of Escherichia coli is retarded in the presence of excess cystine. Transcriptome analysis showed 11 upregulated and 26 down-regulated genes upon exposure to excess cystine. The reporter assay confirmed regulation by cystine of the expression of one upregulated membrane gene, yijE, and two downregulated membrane genes, yhdT and yihN. In order to identify the as yet unidentified gene encoding cystine efflux transporter, the putative cystine efflux candidate, yijE gene, was over-expressed. Expression of the yijE gene suppressed the slow growth of E. coli in the presence of high concentration of extracellular cystine. In good agreement, the knock-out of yijE gene increased the sensibility to cystine. These observations altogether imply that the yijE gene is involved in response to cystine in E. coli. Key words:
Escherichia coli; cystine; genome expression; yijE; cystine resistance
Cystine, a sulfur-containing amino acid, is formed by oxidation of two molecules of cysteine, and is reversibly converted to cysteine under intracellular reduced conditions. The inter-conversion system between cystine and cysteine plays an important role in defense of Escherichia coli against oxidative stress, such as in the presence of hydrogen peroxide.1) For maintenance of the intracellular concentration of cysteine at an optimum level, E. coli is known to possess multi-transporters including Bcr,2) CydDC,3) EamA (YdeD),4,5) and EamB (YfiK).6) In contrast, the transport system of cystine has never been identified in E. coli. For searching the putative cystine-transport system, we performed transcriptome analysis of the genes affected in the presence of cystine, and then reporter analyses of some candidate membrane genes. Among the genes tested, the hitherto uncharacterized yijE gene was found to confer tolerance to cystine. *Corresponding author: Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Materials and methods E. coli strains and growth conditions. E. coli BW25113 was used as a test strain throughout this study.7) JW4303 is the yijE knock-out strain of BW25113 was provided by NRBP of Japan.8) Cells were grown at 37 °C in LB medium or M9-glucose minimum medium in an elbow glass tube with reciprocal shaking for aerobic condition or without shaking in N2-saturating chamber for anaerobic condition. For the analysis of gene expression, cystine was added to a final concentration of 10 μM, which will not affect cell growth. Construction of plasmids and lysogens. For construction of the yijE expression plasmid, a fragment of the yijE gene was amplified by PCR using the genome of E. coli W3110 type-A strain9) as a template and a pair of primers, pQE-YIJE-LF (5′-CTGGAGGATCCATGTCTGCCGCAGGAAAGA-3′) and pQE-YIJE-LR (5′-GAAGAAAGCTTTCAGATCCTTTTTACACTG-3′). The PCR product was digested with BamH I and Hind III and then ligated into pQE80L (Qiagen) at the corresponding sites. The recombinant plasmid, pYijE, was confirmed by DNA sequencing. A single copy of test promoter-lacZ translational fusion gene on the genome was constructed according to the published procedure.10) In brief, a fragment of the test promoter, −500 and +150 with respect to the initiation codon, including the promoter11,12) and the N-terminal proximal coding sequences, was amplified by PCR using the genome of E. coli W3110 type-A strain as a template and a pair of following primers: YIJE-P-LF (5′-TTTCCGAATTCGAGTCACTGCTGATCGCA-3′) and YIJE-P-LR (5′-TGAATGGATCCAGAGCGCCGAAAATGCAGC-3′) for yijE promoter; YIHN-P-LF (5′-CGCGGGAATTCGTTCCTCGGTCACGGTGC-3′) and YIHN-P-LR (5′-CGAGGGGATCCAACGCCTGGCGCAGCGTCA-3′) for yihN promoter; and YHDT-P-LF (5′-TGCCGGAATTCCGCTGTCGATCAAGCAAGA-3′) and YHDT-P-LR (5′-GCGGCGGATCCATGCAGGCCATCTCAAACC-3′) for yhdT promoter. The PCR product was digested with
2
K. Yamamoto et al. 10)
BamH I and EcoR I and then ligated into pRS552 at the corresponding sites. The recombinant plasmids, pTW02 (yijE-lacZ), pHN01 (yihN-lacZ), and pDT04 (yhdT-lacZ), were confirmed by DNA sequencing using Lac30R primer.13) Each lacZ translational fusion plasmid was transformed into MC4100,14) and the transformant was infected with λ RS45 to prepare λ recombinant phage containing the lacZ fusion gene. E. coli BW25113 was infected with the λ lysate, and the lysogen containing the recombinant λ phage was selected by resistance to kanamycin. The isolated lysogens are JE30617 (λyijE-lacZ Kmr), HN81108 (λyihNlacZ Kmr), and DT31108 (λyhdT-lacZ Kmr).
Downloaded by [Baskent Universitesi] at 00:28 21 December 2014
Transcriptome analysis. E. coli BW25113 was grown at 37 °C in M9 medium with and without 10 µM cystine. At the middle of exponential phase, cells were harvested, and total RNAs were prepared
Table 1.
with hot phenol method as previously described.15,16) Preparation of Cy3 or Cy5-labeled cDNA was performed by RNA fluorescence labeling core kit (Takara Bio) using Cy3-dUTP or Cy5-dUTP (GE Healthcare) as a substrate. Hybridization on DNA chip, scanning microarrays, and data analysis were performed as described.17) E. coli CHIPs (Takara Bio) used were Takara Ver. 2 products. All fluorescent intensity data were statically analyzed as described.15–17) The lacZ reporter assay of E. coli. Overnight cultures were diluted 1:1000 into fresh medium, and cells were grown at appropriate time and then subject for β-galactosidase activity. The activity of β-galactosidase was measured according to the standard Miller method.18) The measurement was performed four times to get the average values with standard deviation.
The genes affected by excess of extracellular cystine in E. coli.
Gene name
ORF ID
Description
Ratio (cystine/no-addition) (first Result/second Result)
copA/ybaR cueO/yacK dos/yddU eutB flhA pspA yihG yijE yjiD yjiR (yfjI)
JW0473 JW0119 JW1484 JW2434 JW1868 JW1297 JW3834 JW3915 JW4289 JW4303 JW0267
Copper transporter Multi-copper oxidase cAMP phosphodiesterase Ethanolamine ammonia-lyase Flagellar export pore protein Phage shock protein Predicted endonuclease Predicted permerase DNA repair protein Predicted transcriptional regulator Hypothetical protein
23.27/5.54 15.99/12.51 3.71/4.77 90.01/7.49 3.44/34.01 4.03/5.88 16.31/UP 7.14/3.57 17.88/13.42 UP/5.86 5.00/6.97
citF cusC/ylcB fhiA flgE garD/yhaG malM mcrC mdlB modA nohB pbuX/ygfU tyrP ucpA yaeH ybcV ydiR ydiS yedX ygcE yhaM yhdT yhjA yiaU yihN yihT yqjK
JW0607 JW0561 JW0219 JW1063 JW3097 JW3997 JW4308 JW0439 JW0746 JW0549 JW2856 JW1895 JW2419 JW0159 JW0547 JW1688 JW1689 JW1953 JW2747 JW3079 JW3225 JW3486 JW3557 JW3845 JW3852 JW3071
Citrate lyase Copper/silver efflux system Flagellar biogenesis protein Flagellar hook protein D-galactarate dehydrogenase Periplasmic protein Restriction enzyme Multidrug ATP-binding transporter Molybdate transporter DNA packaging protein Xanthine permerase Tyrosine transporter Oxidoreductase Hypothetical protein Hypothetical protein Putative electrontransport flavoprotein Putative electrontransport flavoprotein Hypothetical protein Predicted kinase Hypothetical protein Inner membrane protein Predicated cytochromeC peroxidase Predicated transcriptional regulator Predicated transporter Predicted aldolase Predicted glycosyl hydrolase
DOWN/0.22 DOWN/DOWN DOWN/0.05 DOWN/0.21 0.29/0.00 DOWN/0.25 0.16/0.13 DOWN/0.16 DOWN/0.08 0.27/0.02 0.17/DOWN DOWN/0.25 DOWN/0.04 0.11/0.27 0.12/0.07 DOWN/DOWN DOWN/0.20 DOWN/DOWN 0.08/0.16 0.14/0.27 0.33/0.31 0.13/0.17 0.21/0.16 DOWN/0.26 0.07/0.28 0.16/DOWN
Notes: Gene name, ID, and function are represented according to the GenoBase database (http://ecoli.naist.jp/GB8/). Experiment was independently performed twice. The ratio is calculated as previously described.15) UP and DOWN indicate the spots where no intensities detected for no- and cystine-addition, respectively.
The role of the yijE in tolerance to cystine in E. coli.
Influence of excess cystine on expression of the putative cystine-transport gene(s) To examine possible influence of excess cystine on expression of three putative cystine-transport genes, yijE, yhdT, and yihN, we performed a reporter assay using the promoter-lacZ fusions. Transformants of E. coli carrying the respective reporter assay vectors, JE30617 (λyijE-lacZ), HN81108 (λyihN-lacZ), and DT31108 (λyhdT-lacZ), were grown under aeration in M9-glucose minimum medium in the presence or absence of cystine until logarithmic phase and then subjected to measure the activity of β-galactosidase. As expected, the promoter for yijE was significantly induced by cystine (Fig. 1(a)), whereas the promoters for yhdT and yihN were repressed (Fig. 1(b) and (c)).
Interestingly, the changes in cystine-dependent expression of these three promoters were not observed when E. coli were grown under anaerobic condition (data not shown). One possible is that cystine is converted to cysteine under anaerobic condition. Previous reports suggest that anaerobic conditions would shift this equilibrium toward cysteine.19,20) These results indicated that the yijE promoter is activated by cystine, while the yhdT and yihN promoters are repressed. Specific response to cystine rather than cysteine was supported by a microarray experiment looking at cysteineresponsive genes where none of these genes was significantly altered in expression upon cysteine shock (fold change of all the three genes in duplicated data ranged within 1.5 at 5 min after addition of 100 μM cysteine; data not shown). Influence of excess cystine on the sensitivity of E. coli lacking the putative cystine-transporter gene Based on the prediction that knock-out of the gene involved in cystine transport should influence the sensitivity to excess cystine, the inducible expression system was constructed for each of the yijE, yhdT, and yihN genes under the control of the IPTGinducible lacZ promoter. These strains were grown in M9-glucose medium with and without excess cystine, and the cell growth was compared in the presence and absence of IPTG induction. The growth of wildtype E. coli BW25113 was significantly reduced in the presence of 80 μM cystine, but after prolonged culture, reached to the same level as measured by optical density at 600 nm (Fig. 2(a)). Among the three candidates of cystine transporter examined, the
(a)
10 1
OD 600 nm
Search for the genes affected in the presence of excess cystine For search of the whole set of cystine-responsive genes, we performed the DNA microarray analysis of E. coli cells growing in M9-glucose medium in the presence or absence of 10 μM cystine. In the middle of exponential growth phase, total RNAs were prepared and subjected to the microarray analysis using an E. coli DNA chip. Among a total of 4000 genes on the DNA chip, transcription of a total of 37 genes exhibited more than three fold change in the presence of cystine (Table 1). A set of 11 genes including 5 hitherto uncharacterized genes were up-regulated, while another set of 26 genes including 13 uncharacterized genes were down-regulated. The set of cystine-responded genes are related to transport and utilization of metals such as copper and flagella formation. As an approach for identification of the putative gene involved in cystine transport, we focused the hitherto uncharacterized membrane genes. We selected three targets: one up-regulated gene, yijE encoding a drug/ metabolite exporter (DME) family17), and two downregulated genes, yhdT encoding the major facilitator superfamily of transporter18) and yihN encoding a short peptide consisting of 80 amino acid residues with two transmembrane domains.
0.1 0.01
0.001
0
5
10
15
20
25
20
25
Time (hour)
(b)
yhdT-lacZ
(c)
10
10
10
8
8
8
6
6
6
4
4
4
2
2
2
yihN-lacZ
(b) 10 OD 600 nm
(a) yijE-lacZ -Galactosidase activity (Miller unit)
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Results and discussion
3
1 0.1
0.01 0
5
10
15
Time (hour) 0
0 - +
- +
0
- +
Cystine
Fig. 1. Induction of yijE promoter and repression of yhdT and yihN promoters by addition of cystine. Notes: The reporter strains containing yijE-lacZ (a), yhdT-lacZ (b), and yihN-lacZ (c) (see Materials and methods) were grown in M9glucose with (black bars) and without (white bars) cystine (10 μM) until log phase, and then subjected to β-galactosidase assay.
Fig. 2. Resistance to cystine of E. coli by expression of yijE gene. Notes: BW25113 harboring pQE80L (a) and pYijE (b) were grown in M9-glucose with (triangle) and without (circle) cystine (80 μM). The growth of cultures with reciprocal shaking of an elbow glass tube was automatically measured by TVS062CA (Advantec). This measurement of growth curve was performed three times for each experiment with independent cultures. The fluctuation level among three determinations was less than 20%.
4
K. Yamamoto et al.
OD 600 nm
(a)
Acknowledgments
1
We thank Takahiro Katayama, Takashi Wada, Ayano Tomiyama, and Eri Ishii (Hosei University) for technical assistance. We also thank the National BioResource Project (NBRP) of Japan for providing E. coli JW4303.
0.1
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0.001 0
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Time (hour)
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OD 600 nm
(b)
Funding This work was supported by Grant from Ajinomoto Co. Ltd of Japan and MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2008–2012 (S0801037).
1
0.1
References 0.01
0.001 0
10
20
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Time (hour) Fig. 3. Sensibility to cystine of the yijE-deficient mutant. Notes: BW25113 (a) and JW4303 (b) were grown in M9-glucose with (triangle) and without (circle) cystine (20 μM). The growth of cultures with reciprocal shaking of a glass tube was measured by its turbidity at OD 600 nm. This measurement of growth curve was performed three times for each experiment with independent cultures. The fluctuation level among three determinations was less than 15%. The p-values from Student’s t-distribution were 0.29, 0.33, 021, 0.04,
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Induction of the Escherichia coli yijE gene expression by cystine a
b
a
b
Kaneyoshi Yamamoto , Gen Nonaka , Takahiro Ozawa , Kazuhiro Takumi & Akira a
Ishihama a
Department of Frontier Bioscience and Research Center for Micro-Nano Technology, Hosei University, Koganei, Japan b
Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Kawasaki, Japan Published online: 27 Oct 2014.
Click for updates To cite this article: Kaneyoshi Yamamoto, Gen Nonaka, Takahiro Ozawa, Kazuhiro Takumi & Akira Ishihama (2014): Induction of the Escherichia coli yijE gene expression by cystine, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.972328 To link to this article: http://dx.doi.org/10.1080/09168451.2014.972328
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014
Induction of the Escherichia coli yijE gene expression by cystine Kaneyoshi Yamamoto1,*, Gen Nonaka2, Takahiro Ozawa1, Kazuhiro Takumi2 and Akira Ishihama1 1
Department of Frontier Bioscience and Research Center for Micro-Nano Technology, Hosei University, Koganei, Japan; 2Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Kawasaki, Japan Received July 23, 2014; accepted September 15, 2014
Downloaded by [Baskent Universitesi] at 00:28 21 December 2014
http://dx.doi.org/10.1080/09168451.2014.972328
Cystine is formed from two molecules of the cysteine under oxidized conditions, but is reversibly converted to cysteine by reduction. Growth of Escherichia coli is retarded in the presence of excess cystine. Transcriptome analysis showed 11 upregulated and 26 down-regulated genes upon exposure to excess cystine. The reporter assay confirmed regulation by cystine of the expression of one upregulated membrane gene, yijE, and two downregulated membrane genes, yhdT and yihN. In order to identify the as yet unidentified gene encoding cystine efflux transporter, the putative cystine efflux candidate, yijE gene, was over-expressed. Expression of the yijE gene suppressed the slow growth of E. coli in the presence of high concentration of extracellular cystine. In good agreement, the knock-out of yijE gene increased the sensibility to cystine. These observations altogether imply that the yijE gene is involved in response to cystine in E. coli. Key words:
Escherichia coli; cystine; genome expression; yijE; cystine resistance
Cystine, a sulfur-containing amino acid, is formed by oxidation of two molecules of cysteine, and is reversibly converted to cysteine under intracellular reduced conditions. The inter-conversion system between cystine and cysteine plays an important role in defense of Escherichia coli against oxidative stress, such as in the presence of hydrogen peroxide.1) For maintenance of the intracellular concentration of cysteine at an optimum level, E. coli is known to possess multi-transporters including Bcr,2) CydDC,3) EamA (YdeD),4,5) and EamB (YfiK).6) In contrast, the transport system of cystine has never been identified in E. coli. For searching the putative cystine-transport system, we performed transcriptome analysis of the genes affected in the presence of cystine, and then reporter analyses of some candidate membrane genes. Among the genes tested, the hitherto uncharacterized yijE gene was found to confer tolerance to cystine. *Corresponding author: Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Materials and methods E. coli strains and growth conditions. E. coli BW25113 was used as a test strain throughout this study.7) JW4303 is the yijE knock-out strain of BW25113 was provided by NRBP of Japan.8) Cells were grown at 37 °C in LB medium or M9-glucose minimum medium in an elbow glass tube with reciprocal shaking for aerobic condition or without shaking in N2-saturating chamber for anaerobic condition. For the analysis of gene expression, cystine was added to a final concentration of 10 μM, which will not affect cell growth. Construction of plasmids and lysogens. For construction of the yijE expression plasmid, a fragment of the yijE gene was amplified by PCR using the genome of E. coli W3110 type-A strain9) as a template and a pair of primers, pQE-YIJE-LF (5′-CTGGAGGATCCATGTCTGCCGCAGGAAAGA-3′) and pQE-YIJE-LR (5′-GAAGAAAGCTTTCAGATCCTTTTTACACTG-3′). The PCR product was digested with BamH I and Hind III and then ligated into pQE80L (Qiagen) at the corresponding sites. The recombinant plasmid, pYijE, was confirmed by DNA sequencing. A single copy of test promoter-lacZ translational fusion gene on the genome was constructed according to the published procedure.10) In brief, a fragment of the test promoter, −500 and +150 with respect to the initiation codon, including the promoter11,12) and the N-terminal proximal coding sequences, was amplified by PCR using the genome of E. coli W3110 type-A strain as a template and a pair of following primers: YIJE-P-LF (5′-TTTCCGAATTCGAGTCACTGCTGATCGCA-3′) and YIJE-P-LR (5′-TGAATGGATCCAGAGCGCCGAAAATGCAGC-3′) for yijE promoter; YIHN-P-LF (5′-CGCGGGAATTCGTTCCTCGGTCACGGTGC-3′) and YIHN-P-LR (5′-CGAGGGGATCCAACGCCTGGCGCAGCGTCA-3′) for yihN promoter; and YHDT-P-LF (5′-TGCCGGAATTCCGCTGTCGATCAAGCAAGA-3′) and YHDT-P-LR (5′-GCGGCGGATCCATGCAGGCCATCTCAAACC-3′) for yhdT promoter. The PCR product was digested with
2
K. Yamamoto et al. 10)
BamH I and EcoR I and then ligated into pRS552 at the corresponding sites. The recombinant plasmids, pTW02 (yijE-lacZ), pHN01 (yihN-lacZ), and pDT04 (yhdT-lacZ), were confirmed by DNA sequencing using Lac30R primer.13) Each lacZ translational fusion plasmid was transformed into MC4100,14) and the transformant was infected with λ RS45 to prepare λ recombinant phage containing the lacZ fusion gene. E. coli BW25113 was infected with the λ lysate, and the lysogen containing the recombinant λ phage was selected by resistance to kanamycin. The isolated lysogens are JE30617 (λyijE-lacZ Kmr), HN81108 (λyihNlacZ Kmr), and DT31108 (λyhdT-lacZ Kmr).
Downloaded by [Baskent Universitesi] at 00:28 21 December 2014
Transcriptome analysis. E. coli BW25113 was grown at 37 °C in M9 medium with and without 10 µM cystine. At the middle of exponential phase, cells were harvested, and total RNAs were prepared
Table 1.
with hot phenol method as previously described.15,16) Preparation of Cy3 or Cy5-labeled cDNA was performed by RNA fluorescence labeling core kit (Takara Bio) using Cy3-dUTP or Cy5-dUTP (GE Healthcare) as a substrate. Hybridization on DNA chip, scanning microarrays, and data analysis were performed as described.17) E. coli CHIPs (Takara Bio) used were Takara Ver. 2 products. All fluorescent intensity data were statically analyzed as described.15–17) The lacZ reporter assay of E. coli. Overnight cultures were diluted 1:1000 into fresh medium, and cells were grown at appropriate time and then subject for β-galactosidase activity. The activity of β-galactosidase was measured according to the standard Miller method.18) The measurement was performed four times to get the average values with standard deviation.
The genes affected by excess of extracellular cystine in E. coli.
Gene name
ORF ID
Description
Ratio (cystine/no-addition) (first Result/second Result)
copA/ybaR cueO/yacK dos/yddU eutB flhA pspA yihG yijE yjiD yjiR (yfjI)
JW0473 JW0119 JW1484 JW2434 JW1868 JW1297 JW3834 JW3915 JW4289 JW4303 JW0267
Copper transporter Multi-copper oxidase cAMP phosphodiesterase Ethanolamine ammonia-lyase Flagellar export pore protein Phage shock protein Predicted endonuclease Predicted permerase DNA repair protein Predicted transcriptional regulator Hypothetical protein
23.27/5.54 15.99/12.51 3.71/4.77 90.01/7.49 3.44/34.01 4.03/5.88 16.31/UP 7.14/3.57 17.88/13.42 UP/5.86 5.00/6.97
citF cusC/ylcB fhiA flgE garD/yhaG malM mcrC mdlB modA nohB pbuX/ygfU tyrP ucpA yaeH ybcV ydiR ydiS yedX ygcE yhaM yhdT yhjA yiaU yihN yihT yqjK
JW0607 JW0561 JW0219 JW1063 JW3097 JW3997 JW4308 JW0439 JW0746 JW0549 JW2856 JW1895 JW2419 JW0159 JW0547 JW1688 JW1689 JW1953 JW2747 JW3079 JW3225 JW3486 JW3557 JW3845 JW3852 JW3071
Citrate lyase Copper/silver efflux system Flagellar biogenesis protein Flagellar hook protein D-galactarate dehydrogenase Periplasmic protein Restriction enzyme Multidrug ATP-binding transporter Molybdate transporter DNA packaging protein Xanthine permerase Tyrosine transporter Oxidoreductase Hypothetical protein Hypothetical protein Putative electrontransport flavoprotein Putative electrontransport flavoprotein Hypothetical protein Predicted kinase Hypothetical protein Inner membrane protein Predicated cytochromeC peroxidase Predicated transcriptional regulator Predicated transporter Predicted aldolase Predicted glycosyl hydrolase
DOWN/0.22 DOWN/DOWN DOWN/0.05 DOWN/0.21 0.29/0.00 DOWN/0.25 0.16/0.13 DOWN/0.16 DOWN/0.08 0.27/0.02 0.17/DOWN DOWN/0.25 DOWN/0.04 0.11/0.27 0.12/0.07 DOWN/DOWN DOWN/0.20 DOWN/DOWN 0.08/0.16 0.14/0.27 0.33/0.31 0.13/0.17 0.21/0.16 DOWN/0.26 0.07/0.28 0.16/DOWN
Notes: Gene name, ID, and function are represented according to the GenoBase database (http://ecoli.naist.jp/GB8/). Experiment was independently performed twice. The ratio is calculated as previously described.15) UP and DOWN indicate the spots where no intensities detected for no- and cystine-addition, respectively.
The role of the yijE in tolerance to cystine in E. coli.
Influence of excess cystine on expression of the putative cystine-transport gene(s) To examine possible influence of excess cystine on expression of three putative cystine-transport genes, yijE, yhdT, and yihN, we performed a reporter assay using the promoter-lacZ fusions. Transformants of E. coli carrying the respective reporter assay vectors, JE30617 (λyijE-lacZ), HN81108 (λyihN-lacZ), and DT31108 (λyhdT-lacZ), were grown under aeration in M9-glucose minimum medium in the presence or absence of cystine until logarithmic phase and then subjected to measure the activity of β-galactosidase. As expected, the promoter for yijE was significantly induced by cystine (Fig. 1(a)), whereas the promoters for yhdT and yihN were repressed (Fig. 1(b) and (c)).
Interestingly, the changes in cystine-dependent expression of these three promoters were not observed when E. coli were grown under anaerobic condition (data not shown). One possible is that cystine is converted to cysteine under anaerobic condition. Previous reports suggest that anaerobic conditions would shift this equilibrium toward cysteine.19,20) These results indicated that the yijE promoter is activated by cystine, while the yhdT and yihN promoters are repressed. Specific response to cystine rather than cysteine was supported by a microarray experiment looking at cysteineresponsive genes where none of these genes was significantly altered in expression upon cysteine shock (fold change of all the three genes in duplicated data ranged within 1.5 at 5 min after addition of 100 μM cysteine; data not shown). Influence of excess cystine on the sensitivity of E. coli lacking the putative cystine-transporter gene Based on the prediction that knock-out of the gene involved in cystine transport should influence the sensitivity to excess cystine, the inducible expression system was constructed for each of the yijE, yhdT, and yihN genes under the control of the IPTGinducible lacZ promoter. These strains were grown in M9-glucose medium with and without excess cystine, and the cell growth was compared in the presence and absence of IPTG induction. The growth of wildtype E. coli BW25113 was significantly reduced in the presence of 80 μM cystine, but after prolonged culture, reached to the same level as measured by optical density at 600 nm (Fig. 2(a)). Among the three candidates of cystine transporter examined, the
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Search for the genes affected in the presence of excess cystine For search of the whole set of cystine-responsive genes, we performed the DNA microarray analysis of E. coli cells growing in M9-glucose medium in the presence or absence of 10 μM cystine. In the middle of exponential growth phase, total RNAs were prepared and subjected to the microarray analysis using an E. coli DNA chip. Among a total of 4000 genes on the DNA chip, transcription of a total of 37 genes exhibited more than three fold change in the presence of cystine (Table 1). A set of 11 genes including 5 hitherto uncharacterized genes were up-regulated, while another set of 26 genes including 13 uncharacterized genes were down-regulated. The set of cystine-responded genes are related to transport and utilization of metals such as copper and flagella formation. As an approach for identification of the putative gene involved in cystine transport, we focused the hitherto uncharacterized membrane genes. We selected three targets: one up-regulated gene, yijE encoding a drug/ metabolite exporter (DME) family17), and two downregulated genes, yhdT encoding the major facilitator superfamily of transporter18) and yihN encoding a short peptide consisting of 80 amino acid residues with two transmembrane domains.
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Results and discussion
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Fig. 1. Induction of yijE promoter and repression of yhdT and yihN promoters by addition of cystine. Notes: The reporter strains containing yijE-lacZ (a), yhdT-lacZ (b), and yihN-lacZ (c) (see Materials and methods) were grown in M9glucose with (black bars) and without (white bars) cystine (10 μM) until log phase, and then subjected to β-galactosidase assay.
Fig. 2. Resistance to cystine of E. coli by expression of yijE gene. Notes: BW25113 harboring pQE80L (a) and pYijE (b) were grown in M9-glucose with (triangle) and without (circle) cystine (80 μM). The growth of cultures with reciprocal shaking of an elbow glass tube was automatically measured by TVS062CA (Advantec). This measurement of growth curve was performed three times for each experiment with independent cultures. The fluctuation level among three determinations was less than 20%.
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Acknowledgments
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We thank Takahiro Katayama, Takashi Wada, Ayano Tomiyama, and Eri Ishii (Hosei University) for technical assistance. We also thank the National BioResource Project (NBRP) of Japan for providing E. coli JW4303.
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Funding This work was supported by Grant from Ajinomoto Co. Ltd of Japan and MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2008–2012 (S0801037).
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Time (hour) Fig. 3. Sensibility to cystine of the yijE-deficient mutant. Notes: BW25113 (a) and JW4303 (b) were grown in M9-glucose with (triangle) and without (circle) cystine (20 μM). The growth of cultures with reciprocal shaking of a glass tube was measured by its turbidity at OD 600 nm. This measurement of growth curve was performed three times for each experiment with independent cultures. The fluctuation level among three determinations was less than 15%. The p-values from Student’s t-distribution were 0.29, 0.33, 021, 0.04,
