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1 Graduate School of Information Science, Nara Institute of Science and Technology, Nara 630-0101, Japan
2 Unité des Membranes Bactériennes, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25 rue du Dr Roux, F75724 Paris cedex 15, France
Correspondence
Francis Biville
fbiville{at}pasteur.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Regulators of the LysR family are present in many bacteria and are involved in the control of various processes, including virulence, stress responses and metabolism (Schell, 1993
). LysR family regulators can be easily identified by sequence comparisons because they all contain a characteristic helix–turn–helix DNA-binding motif in the N-terminal part (Schell, 1993). The DNA-binding sites of LysR family members are generally 80–35 bp upstream from the transcription start site (Schell, 1993). No consensus sequence has been identified for the binding sites of LysR family members. More than half of the 21 characterized LysR regulators in E. coli regulate genes involved in catabolic or anabolic processes (Schell, 1993). The structural genes of most of these LysR type regulators are neighbours of their target genes. Other characterized LysR family members, which control various process like transport, stress responses and DNA replication, do not map close to their targets (Schell, 1993). There are about 24 putative LysR regulators in E. coli. One, ygiP, is transcribed divergently from an operon containing two characterized genes (ttdA and ttdB) encoding the two subunits of a tartrate dehydratase (Reaney et al., 1993). Tartrate dehydratase converts tartrate to oxaloacetate. E. coli can use L-, D- and meso-tartrate as reducible substrates for anaerobic growth on glycerol (Reaney et al., 1993). Tartrate dehydratase activity is induced by L- and meso-tartrate during anaerobic growth with glycerol and may be repressed by glucose and oxygen (Reaney et al., 1993). The sequence of the last gene of the operon, ygiE, is consistent with it encoding an integral inner-membrane protein potentially involved in tartrate uptake (Pos et al., 1998). Here, we report the functional identification of ygiP as a positive regulator required for the L-tartrate-dependent induction of the ttdA-ttdB-ygjE operon. Inactivation of ygiP, ttdA or ygjE abolishes L-tartrate-dependent anaerobic growth on glycerol. In a ygiP mutant, the expression of the ttdA-ttdB-ygjE operon is not induced by L-tartrate.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) supplemented with glucose (0.4 %), glycerol (0.4 %) or gluconate (0.4 %), as carbon sources. When specified, tartrate was added to a final concentration of 0.4 %. Anaerobic cultures were grown in Erlenmeyer flasks with rubber stoppers and with shaking. Anoxia was achieved by bubbling with N2 for 2 min. Antibiotics were added to the following final concentrations (µg ml–1): ampicillin (Ap), 100; spectinomycin (Sp), 100; chloramphenicol (Cm), 20; kanamycin (Km), 25. Solid medium contained 1.5 % Difco agar. Deep agar growth tests were performed in 63 B1 minimal medium containing 0.3 % agar supplemented with various carbon sources. Fifteen millilitres of the growth test medium heated at 50 °C were introduced into a screw-topped tube. One hundred microlitres of the cell suspension (OD600=1) was added and gently mixed with a pipette into the test medium. The test tubes were then closed with a screw cap and incubated at 37 °C. After 72 h, the OD600 of the samples in the test tubes was measured in a Spectronic 20D spectrophotometer.
Genetic techniques.
P1 lysates and transductions were performed as described by Miller (1972). Cells were transformed by the calcium chloride method (Maniatis et al., 1982) or electroporation (Dower et al., 1988)
DNA manipulations.
Chromosomal DNA was isolated according to the method of Marmur (1961). Large-scale plasmid DNA preparations were made using the Plasmid Maxi Kit (Qiagen) as recommended by the manufacturer. Small-scale plasmid DNA preparations were done as described by Birnboim & Doly (1979). Restriction, modification and ligation were carried out according to the manufacturer's recommendations. DNA fragments of interest were separated by gel electrophoresis and isolated by using the QIAquick Gel Extraction Kit (Qiagen). DNA fragments were amplified in a Hybaid PCR thermocycler, using the Expand High Fidelity PCR System (Boerhinger).
Disruption of ygiP and ttdA.
Non-polar mutations that delete the entire ygiP or ttdA gene were created by allelic exchange with the non-polar cat gene cassette from a derivative of pHP45
(Fellay et al., 1987). A three-step PCR procedure was used to produce a PCR product in which the Cm gene cassette is flanked by arms of about 900 bp corresponding to the sequences upstream from the start codon and downstream from the stop codon of the gene of interest.
The cat gene cassette (0.9 kb) was amplified from E. coli FB85 (see Table 1
) using the primers cat3 and cat5 (Table 2). The primers used for ygiP were ygiPav1AV and casav1am for the left 900 bp homology arm, and casamtav and Amtam for the right 900 bp homology arm. (Table 2). For ttdA, the primers used were ygiPamamt and ygiPamtavl for the left 900 bp homology arm, and ttdABavlamt and ttdABavl1 for the right 900 bp homology arm (Table 2).
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). Correct chromosomal insertion was checked by PCR amplification using cat primers cat3 and cat 5 in combination with ygiP external primers ygiPav1av and Amtam, or in combination with ttdA external primers ygiPamamt and ttdABavl1 (Table 2). Mutations were then introduced into MG1655 and POP3 att
: : ttdA-lacZ by P1 transduction.
Disruption of fnr.
The fnr-disrupted strain was constructed using PCR products and a standard one-step gene inactivation protocol for disruption of chromosomal genes that is based on the high efficiency of the phage Red recombinase using BW25113 (Datsenko & Wanner, 2000). The PCR product is amplified from pKD13 as a template with primers PO309 and PO310 (Table 2). To check the mutation, PCR was performed with an upstream-specific primer for the fnr gene, PO312, and a Km-cassette-specific primer, K1 (Datsenko & Wanner, 2000) (Table 2). The fnr mutation was then introduced into MG1655 and POP3 att : : ttdA-lacZ by P1 transduction.
Plasmid constructions
pGPttdA-lacZ.
A DNA fragment corresponding to 390 bp upstream from the E. coli MG1655 ttdA gene was amplified with primers fttdAAMT and fttdAAVL (Table 2). The PCR products were digested with EcoRI and SmaI and ligated to pGPlacZtt (Rossi et al., 2003) digested with EcoRI and SmaI to create pGPttdA-lacZ.
pAM239 : : ygiP.
ygiP was amplified from chromosomal MG1655 DNA using primers ygiPamt and ygiPavl (Table 2). After filling-in by T4 polymerase, the PCR fragment was inserted into the HincII site of pAM239 giving pAM239 : : ygiP.
pAM239 : : ttdA-B.
A ttdA-B DNA fragment was amplified from chromosomal MG1655 DNA with primers ttdA-Bamt and ttdA-Bavl (Table 2). After filling-in by T4 polymerase, the PCR fragment was inserted into the HincII site of pAM239 giving pAM239 : : ttdAB.
Insertion of ttdA-lacZ into the E. coli chromosome.
The EcoRI–HindIII fragment from pGPttdA-lacZ, containing the ttdA-lacZ transcriptional fusion, was inserted between the EcoRI and HindIII sites of pBR322 to give pBR322ttdA-lacZ. The fusion was transferred by homologous recombination into InCh (http://beck2.med.harvard.edu/resources/InCh/lambda_InCh_frames.htm) (Boyd et al., 2000). The recombinant phage was used to lysogenize strain POP3. After integration and stabilization of the recombinant lysogens, integration of the transcriptional fusions at the att site in the E. coli chromosome was confirmed by PCR.
Preparation of labelled cDNA, array hybridization and capture of data
RNA isolation.
RNA was isolated from bacteria cultured anaerobically in 50 ml M63B1 supplemented with gluconate as carbon source in the presence and absence of tartrate. A sample of each culture was taken after 6 h incubation at 37 °C (OD600=0.2–0.3). RNA was isolated using the Qiagen RNAprotect, RNeasy Mini Kit and RNase-free DNase sets, according to the manufacturer's instructions.
Oligonucleotide microarray construction and transcriptomic experiments.
E. coli Oligo Library (Sigma-Genosys) was printed by a Lucidea microarray spotter (Amersham Biosciences) on Corning UltraGAPS slides (Corning) and treated with a DNA cross-linking reagent (Amersham Biosciences) diluted 1 : 2. Microarray experiments were performed as described by Kershaw et al. (2005). For each experiment, 20 µg total RNA was used to produce Cy3- and Cy5-labelled cDNA using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE Healthcare), according to the manufacturer's instructions. The array slides were prehybridized for 4 h in filtered prehybridization solution (25 % formamide, 5x SSC, 10 mg BSA l–1, 0.1 % SDS) at 42 °C, and then briefly washed in ethanol and dried by centrifugation at 1000 g for 5 min. The probe in hybridization solution [25 % formamide, 5x SSC, 0.1 % SDS, 0.1 µg poly(A) ml–1, 1x Denhardt's solution and 80 pmol Cy3 and Cy5 combined probe] was heated to 95 °C for 3 min and hybridized with an ArrayBooster (Adveritix), according to the manufacturer's instructions. After hybridization, slides were washed in wash buffer I (2x SSC/0.1 % SDS) for 2 min at 42 °C, wash buffer II (0.2x SSC) for 2 min at room temperature and then twice in wash buffer III (0.05x SSC) for 2 min at room temperature. All washes included vigorous shaking of the microarray slides. The slides were dried by centrifugation at 1000 g for 5 min and then analysed using an FLA8000 scanner (Fuji film).
Microarray data analysis.
The 4289 individual genes on the microarrays were classified into three groups. To compare the wild-type strains and their derivatives, we measured Cy3 signal intensities for the reference strain and Cy5 signal intensities for the mutant. To analyse the effect of the addition of tartrate, we measured Cy3 signal intensities for the strain grown in the absence of tartrate and Cy5 signal intensities for the strain grown in the presence of tartrate. Spots were classified into three groups. In group 1, both Cy3 and Cy5 signal intensities were higher than 0.5 (the value of 0.5 is low, but nevertheless significant in this experiment: the negative control oligo spot, an O157-specific oligo, gave intensities below 0). In group 2, either the Cy3 or Cy5 signal intensity was more than 1 SD higher than the negative intensity value. In group 3, both signals were lower than the negative intensity value. Initially, all spots of group 1 were normalized by defining the mean of the ratios (Cy5/Cy3) of all spots as 1.0. The ratios of group 2 spots could not be determined by this method due to absence of significant Cy3 or Cy5 fluorescence. Group 2 spots with high Cy3 or Cy5 intensity values (>1000) were selected. Spots in group 3 were not considered further, being undetectable. Genes that were significantly differently expressed in different conditions were then selected according to the following criteria, provided that similar values were seen in two independent experiments. From group 1, we selected genes that showed relative ratios less than 0.33 or greater than 3.0 as being down- or upregulated, respectively. For genes classified into group 1 in one experiment and group 2 in another identical experiment, those that showed a relative ratio less than 0.33 or higher than 3.0 in one hybridization and high intensity values (>1000) in the other were scored as being down- or upregulated, respectively. Genes classified into group 2 in both hybridizations were recognized as genes with altered regulation. Complete microarray datasets, in compliance with MIAME guidelines (http://www.mged.org/miame), are available at http://genome.naist.jp/array/ (raw and processed data).
-Galactosidase assay.
-Galactosidase was assayed by the method described by Pardee et al. (1959).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Regulation of ttdA expression level
The inability to use tartrate for anaerobic growth could be a consequence of a loss of tartrate dehydratase activity (Reaney et al., 1993), possibly due to insufficient expression of the ttdA-ttdB-ygjE operon. We constructed a ttdA-lacZ transcriptional fusion and inserted it into the chromosome of strain POP3 (POP3 att : : ttdA-lacZ) to study the regulation of the operon. We tested growth conditions to identify those allowing better expression of the ttdA-lacZ fusion (Table 3). Cells grown aerobically with glucose or glycerol as carbon source expressed a low basal level of -galactosidase activity (about 4–7 units). This low level of expression of the ttdA-lacZ transcriptional fusion agrees with the absence of tartrate dehydratase activity in E. coli grown in the presence of oxygen (Reaney et al., 1993). Strain POP3 att : : ttdA-lacZ grown with glucose or glycerol as carbon source expressed ttdA-lacZ fusion similarly in the presence and absence of oxygen. For bacteria grown anaerobically with glycerol as carbon source, the presence of tartrate increased ttdA-lacZ expression about fivefold. In contrast, when glucose was used as carbon source, the addition of tartrate had no effect (Table 3). These results are consistent with those showing that, with glycerol as carbon source, tartrate dehydratase activity is induced by L-tartrate but repressed by glucose and oxygen (Reaney et al., 1993). As ygiP and ttdA mutants are impaired for anaerobic growth with glycerol (see above) we used gluconate, which does not provoke catabolic repression of ttdA-ttdB-ygjE expression. We observed L-tartrate-dependent induction of the ttdA-lacZ fusion with gluconate as carbon source (Table 3).
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: : ttdA-lacZ. Strains POP3 att : : ttdA-lacZ and POP3 ygiP att : : ttdA-lacZ were grown in conditions inducing and non-inducing for ttdA-lacZ expression and then assayed for -galactosidase activity. Disruption of ygiP had no effect on expression of the ttdA-lacZ fusion during aerobic growth on gluconate in either the presence or absence of tartrate (Table 4). In contrast, during anaerobic growth on gluconate, ygiP disruption completely abolished the tartrate-dependent induction of the ttdA-lacZ fusion (Table 4). Thus YgiP may be a positive regulator responsible for the tartrate-dependent induction of the ttdA-ttdB-ygjE operon.
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: : ttdA-lacZ and POP3 ttdA att : : ttdA-lacZ. For each strain, we measured -galactosidase activity under conditions inducing and non-inducing for ttdA-ttdB-ygjE operon expression. Disruption of ygjE and ttdA had no effect on the expression of the ttdA-lacZ fusion in the absence of tartrate (Table 4). Disruption of ygjE abolished the tartrate-dependent induction of the ttdA-lacZ fusion (Table 4). This shows that YgjE is necessary for the entry of tartrate, which is required for induction of the ttdA-ttdB-ygjE operon. In contrast, disruption of ttdA substantially increased tartrate induction of the ttdA-lacZ fusion (Table 4). This latter result suggests that when tartrate is not degraded, in this case due to a ttdA mutant, it accumulates inside the cell resulting in very strong induction of the ttdA-ttdB-ygjE operon. Thus, tartrate is a co-inducer of the ttdA-ttdB-ygjE operon.
Genome-wide transcriptome analysis of target genes for YgiP
Both its position on the chromosome and genetic analysis indicate that YgiP is a positive regulator required for L-tartrate-dependent anaerobic growth on glycerol. The effect of ygiP disruption could be the consequence of YgiP having a direct and specific effect on ttdA-ttdB-ygjE operon expression, or of pleiotropic regulatory activity. We used microarray analysis to investigate the possible pleiotropic activity of YgiP, using bacteria grown anaerobically with gluconate in the presence or absence of tartrate. Three different transcriptome profiles were determined and compared: the transcriptome profiles of strain MG1655 grown in the presence and absence of tartrate; MG1655 wild-type and ttdA mutant; and MG1655 and its ygiP mutant. In the wild-type strain, tartrate strongly induced only the expression of the ttdA-ttdB-ygjE operon. Thus, inducing conditions for tartrate dehydratase activity induce the expression of the ttdA-ttdB-ygjE operon but have little effect on the rest of the transcriptome. The transcription product of the ttdA-ttdB-ygjE operon is absent from the ttdA mutant even when bacteria are grown under inducing conditions. The ttdA disruption did not significantly modify the expression of other genes in either the presence or absence of tartrate. Thus, the absence of tartrate dehydratase activity does not cause significant alteration of the transcriptome. The comparison of MG1655 and its ygiP derivative showed that tartrate-dependent induction of the ttdA-ttdB-ygjE operon is abolished by ygiP disruption. Furthermore, the effect of ygiP disruption on transcriptome expression was mainly restricted to the ttdA-ttdB-ygjE operon, consistent with YgiP being a specific regulator. The physiological effects of ygiP disruption could all be consequences of the downregulation of the expression of only ttdA, ttdB and ygiE. All transcriptome data and the experimental information are available at http://genome.naist.jp/array/, in compliance with the MIAME guidelines (http://www.mged.org/miame).
Conclusion
In this report we have predicted, using in silico analysis, and confirmed, using a physiological approach, the function of YgiP. Like other regulators of the LysR family involved in the control of metabolic processes, the structural gene for YgiP is adjacent to its target genes. Moreover, transcriptome analysis clearly showed that the regulatory activity of YgiP, now renamed TtdR, is mainly restricted to the adjacent ttdA-ttdB-ygjE operon. Thus, TtdR is essential for anaerobic growth on glycerol as carbon source in the presence of tartrate through its activation of the ttdA-ttdB-ygjE operon. Induction of ttdA-ttdB-ygjE operon expression, which requires tartrate addition, is strongly enhanced in a ttdA strain unable to convert tartrate to oxaloacetate and thus accumulates tartrate. Tartrate is thus the co-inducer of TtdR for induction of the ttdA-ttdB-ygjE operon. Tartrate dehydratase activity is induced by L- and meso-tartrate during anaerobic growth with glycerol and is repressed by glucose and aerobiosis (Reaney et al., 1993). In E. coli, many transitions from aerobic to anaerobic metabolism are controlled at the transcriptional level by the activity of the dual global regulator FNR (Kang et al., 2005), and by the two-component regulatory system ArcAB (Liu & De Wulf, 2004). The activity of these two regulators is differentially modulated in the presence or in the absence of oxygen. A canonical FNR-binding site had been identified in the promoter of the ttdA-ttdB-ygjE operon (Reaney et al., 1993). We first investigated the effect of fnr disruption on ttdA-lacZ fusion expression in bacteria grown with gluconate and tartrate in the absence or in the presence of oxygen. Disruption of fnr had no effect on ttdA-lacZ expression in both the absence and presence of oxygen (Table 5). Also, disruption of arcA only slightly decreased the tartrate-dependent induction of the ttdA-lacZ fusion when bacteria were grown in the absence of oxygen (Table 5). As a consequence, we hypothesize that most of the regulatory effect of oxygen on the ttdA-ttdB-ygjE operon expression is mediated by TtdR. TtdR, which shares 44 % identity with OxyR (data not shown), contains two cysteines, cysteine288 and cysteine296, which might participate in the constitution of a redox sensor as has been shown for cysteine199 and cysteine208 of OxyR (Zheng et al., 1998). Sequence analysis of TtdR orthologues deposited in the MBGD database (http://mbgd.genome.ad.jp/) showed that these two cysteines are found only in E. coli TtdR. In addition, two highly conserved cysteines, cysteine170 and cysteine193, are found in many TtdR orthologues. There are several well characterized cysteines in many redox-sensing proteins, such as SoxS, OxyR, Fnr, ArcB and BagS (Bauer et al., 1999; Malpica et al., 2004; Swem et al., 2003). Cysteines in these proteins exist under two different states, oxidized and reduced. By changes in these states, proteins can sense changes in the cellular redox environment. In the case of TtdR, since there was no induction of ttdA-lacZ transcriptional fusion in the presence of tartrate and oxygen, we propose that the redox states of cysteine170 and cysteine193, on one hand, and cysteine288 and cysteine296 on the other hand, change during a transition from anaerobic to aerobic conditions. This change would lead to inactivation of TtdR which, in turn, would not be able to activate the transcription of the ttdA-B-ygjE target operon. To test this hypothesis, a deeper analysis of the importance of cysteines for TtdR regulatory function is required.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 9 December 2005;
revised 8 March 2006;
accepted 13 March 2006.
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