Physiologic Roles of Soluble Pyridine Nucleotide Transhydrogenase in <Emphasis Type="Italic">Escherichia Coli &L

Annals of Microbiology, 58 (2) 275-280 (2008)

Physiologic roles of soluble pyridine nucleotide transhydrogenase in Escherichia coli as determined by homologous recombination

Hanjun ZHAO, Peng WANG, Enqi HUANG, Yadong GE, Guoping ZHU*

The Key Laboratory of Molecular Evolution and the Institute of Molecular Biology and Biotechnology, Anhui Normal University, 1 Beijing Road, Wuhu, Anhui 241000, P.R. China

Received 28 January 2008 / Accepted 15 April 2008

Abstract - The soluble transhydrogenase is an energy-independent flavoprotein and important in cofactor regenerating system. In order to understand its physiologic roles, the recombinant strain with the deletion of soluble transhydrogenase gene (ΔudhA)in Escherichia coli was constructed using homologous recombination. Then the different genetic backgrounds containing either icdNADP or icdNAD, which encodes NADP-dependent isocitrate dehydrogenase (IDH) or engineered NAD-dependent IDH, were transduced into ΔudhA, creating two strains (icdNADP/ΔudhA, icdNAD/ΔudhA). During growth on acetate, icdNADP/ΔudhA grew poorly and its growth rate was remarkably reduced by 75% as compared with the wild type. However, icdNAD/ΔudhA showed significantly better growth than icdNADP/ΔudhA. Its growth rate was about 3.7 fold of icdNADP/ΔudhA, which was equivalent to the wild type. These results indicated that UdhA is an essential NADH resource for acetate-grown E. coli and is a dominant factor for bacteria to adapt to the stress environment. Furthermore, when UdhA was absence, icdNAD/ΔudhA displayed about 1.5 fold increase in the IDH activity after switching the carbon source from glucose to acetate. And RT-PCR showed that the expression of NADH dehydrogenase II (NDH-2) in icdNAD/ΔudhA was remarkably up-regulated by about 2.8 fold as compared with icdNADP/ΔudhA. The increase of IDH activity and NDH-2 expression can be explained by the reducing excess NADPH production and restoring higher levels of NADH generation in cells.

Key words: soluble transhydrogenase; homologous recombination; growth rate; isocitrate dehydrogenase; NADH dehydrogenase II; real-time PCR.

INTRODUCTION the enzyme activity was strongly activated by NADPH and 2’-AMP and inhibited by NADP+.The More and more attention is paid to the utilisations of inhibition of STH by NADP+ implies that its physio- the soluble pyridine nucleotide transhydrogenase logical role is the conversion of NADPH to NADH that (STH) in the metabolic engineering and industrial can enter the respiratory chain for energy genera- biocatalysis today. STH is found in the cytoplasm of tion (Voordouw et al., 1983). some heterotrophic bacteria. It is a kind of redox Although the functions have not been determined enzyme which catalyses the reversible transfer of definitely, the soluble transhydrogenase has been reducing equivalents between two major cofactors, extensively applied in the metabolic engineering nicotinamide adenine dinucleotide (NAD+)and and industrial biocatalysis today. STH of P. fluo- nicotinamide adenine dinucleotide phosphate rescens was used to a cell-free system for efficient (NADP+). STH specifically transfers the 4B hydrogen cofactor cycling, resulting in high yields of the from the nicotinamide ring of both NADH and important semisynthetic opiate drug hydromor- NADPH to NADP+ and NAD+, respectively (Boonstra phone. In addition, an enzymatic system for the et al., 2000). The enzyme has been studied in some regeneration of redox cofactors NADH and NADPH detail from Pseudomonas fluorescens, Pseudomonas was established in nanostructural reverse micelles aeruginosa and Azotobacter vinelandii. It is a soluble using the soluble transhydrogenase of E. coli cou- flavoprotein containing flavin adenine dinucleotide pled with the expression of glycerol dehydrogenase (FAD+) and is remarkable for its formation of large (Ichinose et al., 2005). polymers. The kinetic behavior of STH showed that In E. coli, STH is encoded by an udhA gene. The protein, UdhA, is composed of 466 amino acids and was found to be 59% identical and 77% similar to * Corresponding author. Phone/Fax: +86-553-3883592; E-mail: [email protected] P. fluorescens STH (Boonstra et al., 1999). 276 H. ZHAO et al.

However, its physiological role remains obscure and enzymes were purchased from New England little is known about its three dimensional structure. Biolabs. QIAquick gel extraction kit (Qiagen) was Recently, it was reported that a mutant E. coli strain used to purify DNA from agarose gels. Plasmids and with the deletion of pgi gene (encoding phosphoglu- genomic DNA were extracted using Wizard® purifi- cose isomerase) and udhA gene did not grow on cation kits (Promega). HerculaseTM enhanced DNA glucose, but the growth could be recovered upon polymerase (Stratagene) was used in all PCR tests. plasmid-based expression of UdhA, seeming that Total RNA was extracted using SV Total RNA UdhA catalyses the reoxidation of NADPH. Isolation System (Promega). The reverse transcrip- Additionally, the UdhA mutant did not grow on tion was performed with Superscript II reverse tran- acetate at all, presumably because extensive catab- scriptase (Invitrogen). LightCycler-FastStart DNA olism of acetate through the tricarboxylic acid (TCA) Master SYBR Green I Kit (Roche Applied Science) cycle generated more NADPH than was required for was used for RT-PCR tests. biosynthesis (Sauer et al., 2004). During growth on a rich carbon source, such as glu- Plasmid cloning. A 2.4-kb XhoI-KpnI fragment, cose, E. coli can proliferate from a tiny population to spanning 0.4 kb of upstream sequence (flanking a high density speedily. However, on acetate, E. coli sequence a, Fs-a) and 0.6 kb of downstream has to force the flux of isocitrate through the gly- sequence (flanking sequence b, Fs-b) of udhA gene, oxylate bypass for preventing the quantitative loss was generated by PCR from the genomic DNA of E. of the acetate as CO2 in the TCA cycle (Miller et al., coli MG1655 with the forward primer 5’-TTGTCTC- 2000). When transferred from glucose to acetate, GAGTTCCAGATAGTCCGCAAGTTCCGC-3’ and the the redistribution of NADH/NADPH ratio in E. coli reverse primer 5’-TGTAGGTACCACATTGGTTTGATTC- was required for cell growth because NADH and CCACAGTTG-3’. The product was then digested and NADPH play as major cofactors in energy production ligated to pSP72 (Promega) XhoIand KpnIback- and biosynthesis. UdhA is one of two transhydroge- bone fragment, creating pUW (~ 4.8 kb) (Fig. 1). nases for maintaining the balance between NAD(H) Next, the MfeI-XmaI fragment carrying the kan pool and NADP(H) pool in cells. gene was amplified from pKD13 (Datsenko et al., In this work, we were interested in elucidating the 2000) using the forward primer 5’-TAGTTAGT- important function of UdhA for acetate-grown E. CAATTGTGAGTTGAGCGATTGTGTAGGCTGGAG-3’ coli. We used homologous recombination to con- and the reverse primer 5’-ATAGTATTCCCGGGT- struct recombinant strains with the deletion of udhA TATAGTGATAAGCTGTCAAACATGAG-3’. The resulting gene in different genetic background. The growth product was inserted into MfeIand AgeIsites of rates, activity assay and RT-PCR analysis were pUW, creating pUK (~ 5.1 kb) (Fig.1). employed to investigate the physiologic roles of UdhA in E. coli during growth on acetate. The pres- ent paper is, to our knowledge, the first report Strain construction by chromosomal homolo- about the effect of the absence of UdhA on the isoc- gous recombination. The udhA gene was disrupt- itrate dehydrogenase (IDH) activity and NADH ed using the phage λ recombination (Red) system dehydrogenase II (NDH-2) expression in E. coli. (Datsenko and Wanner, 2000). Linear PCR product containing kan gene and flanking sequence on either side of udhA gene from pUK was introduced MATERIALS AND METHODS into competent E. coli MG1655 carrying pKD46 by CaCl2 transformation (Johnsonans Brooker, 1999). Bacterial strains. Escherichia coli MG1655 provid- Cells were shocked at 42 °C and recovered at 37 °C ed the wild type background for the experiments. in SOC (it is identical to SOB medium, except that it Strain MM294 (glnV44(AS), λ¯, rfbD1?, endA¯, contains 20 mM glucose.), and then spread on LB spoT1?, thi¯, hsdR17, recA¯) was used as host for plates to select kanamycin-resistant transformants. routine cloning. Escherichia coli strains containing The disruption of udhA gene was confirmed by natural NADP-dependent IDH or engineered NAD- sequencing the PCR products whose regions dependent IDH (Zhu et al., 2005) were a gift from spanned 1 kb of flanking DNA on either side of kan Antony M. Dean’s laboratory (BioTechnology gene. The kanr cassette replacing udhA gene was Institute, University of Minnesota, MN 55108, USA). transduced using P1 (cml, clr100) into a fresh E. coli MG1655 genetic background, resulting in strain Media and reagents. LB, SOB, SOC and MOPS- ΔudhA. Then the different genetic backgrounds con- based minimal medium were prepared as described taining either icdNADP or icdNAD, which encodes NADP- elsewhere (Neidhardt et al., 1974; Stueland et al., dependent IDH or engineered NAD-dependent IDH, 1988; Miller, 1992; Zhu et al., 2005). All media were introduced into ΔudhA by cotransduction with were supplemented with 100 µg/ml ampicillin, 15 the tetr cassettes, respectively (Zhu et al., 2005). µg/ml tetracycline, 30 µg/ml kanamycin, and 20 The resulting strains were denoted icdNADP/ΔudhA NAD µg/ml chloramphenicol as required. Restriction and icd /ΔudhA (Fig. 1). The chromosome-inte- Ann. Microbiol., 58 (2) 275-280 (2008) 277

pUW (~ 4.8 kb) amp

Fs-a udhA Fs-b

XhoI MfeI AgeI KpnI Digestion of MfeI+AgeI MfeI XmaI Ligation kan cassette pUK (~ 5.1 kb) amp

Fs-a kan cassette Fs-b

Chromosome of wild type Fs-a udhA Fs-b

Double crossover Chromosome of udhA kan cassette

P1 Transduction of icdNADP and icdNAD Chromosome of icdNADP/udhA kan cassette icdNADP Chromosome of icdNAD/udhA

kan cassette icdNAD

FIG. 1 - Schematic illustration of the disruption of udhA gene. The disruption of udhA gene was constructed in wild type Escherichia coli strain MG1655 (Datsenkoand Wanner, 2000). Then the kan cassette was transduced using P1 (cml, clr100) into a fresh E. coli strain MG1655 background and the deletion of udhA gene were confirmed by sequencing chromosomal PCR amplicons. Finally, the icd alleles (icdNADP and icdNAD) were cotransduced with tet cassette (Zhu et al., 2005) into ΔudhA background, respectively. Abbreviations: amp, coding sequence of ampicillin; kan, coding sequence of kanamycin.

grated regions were sequenced to ensure that no Protein extraction and IDH activity assay. Each other mutations had been inadvertently introduced strain (30 ml culture) at exponential phase was col- during strain construction. lected by centrifugation at 3000 x g for 15 min. The equal number of pelleted cells for each strain were Determination of growth curves. Strains were washed twice in 1 ml of extraction buffer, containing cultured in 25 ml of MOPS-based minimal medium of 100 mM KH2PO4 (pH 7.0), 100 mM NaCl, 2 mM containing 2% of either glucose or acetate as the MgCl2, 1 mM EDTA, 2 mM dithiothreitol (DTT), and sole carbon source in a 250 ml flask in an orbital 20% glycerol. After sonication for 10 min in an ice shaker (200 rpm) at 37 °C. Samples were taken bath, the cell debris was removed by centrifugation every 45 min during growth on glucose and every at 12000 x g for 20 min at 4 °C. Protein concentra- two hours during growth on acetate, respectively. tions were determined with the Bio-Rad Bradford The optical density was measured spectrophotomet- Assay using IgG as a standard. rically (UV-2102 spectrophotometer, UNICO Co. Ltd) Total IDH activity was determined spectrophotomet- at a wavelength of 600 nm in a cuvette with a 1 cm rically (Cary300 Bio spectrophotometer, Varian Co. light path. The growth rates were calculated using Ltd) by measuring the reduction of NADP+ or NAD+ the linear regression from logarithmic plots of the at 340 nm in a reaction mixture at 37 °C, contain- O.D. values at 600 nm versus time. ing of 25 mM MOPS (pH 7.5), 2.5 mM NAD(P)+, 0.50 mM D,L-isocitrate, and 5 mM MgCl2 (Chen et al., 278 H. ZHAO et al.

1995; Singh et al., 2002). All compounds of the TABLE 1 - Growth rates of Escherichia coli mutants in vari- reaction mixture were pipetted into a cuvette with 1 ous genetic backgrounds cm light path and the reaction initiated by adding the cell extract to give a final volume of 1 ml, and a unit (U) of activity was defined described in Dean and Golding (1997).

RT-PCR. Total RNA of culture from each strain at exponential phase was extracted according to the manufacturer’s instructions. RNA quantification and purity were determined by measuring A260 and the ratio of A260/A280, respectively. Total RNA was then reverse transcripted into cDNA that was used for RT-PCR with specific primers of ndh gene encoding NDH-2 (the forward primer 5’-CAGCGGCTTAC- Strains were grown in 25 ml of MOPS-based minimal medi- CGTTCATCT-3’ and the reverse primer 5’-GTAGTC- um containing 2% of either glucose or acetate as a sole CACGCCGTAAACGA-3’). RT-PCR reactions were car- carbon source, in a 250 ml side-arm flask shaking at 200 rpm at 37 °C. Cell densities were determined every 45 min ried out by the initiation step of 10 min at 95 °C, fol- or 2 h and the growth rate determined by regression of the lowed by 40 cycles of 15 s at 95 °C, 5 s at 58 °C, OD600 against time. Standard errors were calculated from 15 s at 72 °C. At the end of each cycle, the fluores- at least three replicate experiments. cence data was collected at 76 °C using LightCycler Software Version 3.5 (Roche Diagnostics). Reaction containing no reverse transcripted total RNA sam- Lag phase is an important period for bacteria ples was processed to demonstrate absence of adaptation to a new niche, which depends not only genomic DNA contamination. Quantification of the on the environment conditions (temperature, pH, ndh gene to the control gene (ratio) was performed oxygen and nutrient) but also on the cell density using a mathematical model as that described in and cell viability. When switching to growth on Pfaffl (2001). acetate after the depletion of glucose, bacteria dif- ferentiate into two ecotypes that differ in their growth profiles, ‘fastswitcher’ and ‘slowswitcher’ RESULTS AND DISCUSSION (Spencer et al., 2007). We observed that icdNADP/ΔudhA, the ‘slowswitcher’, exhibited a very Effect on bacterial growth long switching lag and was strongly dependent on Table 1 presents the exponential growth of strains in the initial cell density (OD ≈0.06) as compared various genetic backgrounds. There was no differ- 600 with those of icdNAD/ΔudhA and wild type strain. ence of the maximum growth rate between icdNADP Therefore, UdhA is a dominant factor for E. coli to /ΔudhA and icdNAD/ΔudhA, which are consistent with adapt to the stress environment. the report that UdhA does not play obviously physiological role during growth on glucose (Sauer et al., Effect on IDH activity 2004). When using acetate as a sole carbon source, IDH plays essential roles in energy metabolism, icdNADP/ΔudhA grew poorly and its growth rate was ammonia fixation, amino acid biosynthesis and vita- remarkably reduced by 75% as compared with the min production from bacterial to human, using NAD+ wild type. However, icdNAD/ΔudhA showed signifi- or/and NADP+ as a cofactor. In E. coli,NADP- cantly better growth than icdNADP/ΔudhA. Its growth dependent IDH (EC 1.1.1.42) catalyses the oxida- rate was about 3.7-fold of icdNADP/ΔudhA, which was tion of isocitrate to αketoglutarate in the TCA cycle equivalent to the wild type. These results indicated and is required for glutamate biosynthesis (Golding that icdNADP/ΔudhA was incapable to reduce excess and Dean, 1998). The engineered IDH changed the NADPH production, which may cause an imbalance coenzyme specificity from NADP+ to NAD+ by seven in NAD(H)/NADP(H) ratio and result in its poor fit- replacements using protein engineering. Kinetic ness on acetate. Additionally, icdNAD/ΔudhA exhibited studies showed that specificity was inverted from a great adaptability to acetate, which may be 7000-fold preference for NADP+ to a 200-fold pref- explained by the reduction of NADPH and restoring erence for NAD+ (Chen et al., 1995; Dean and higher levels of NADH production. Therefore, the Golding, 1997). physiological role of UdhA is to convert NADPH into Table 2 showed that during growth on acetate NADH for energy generation during growth on after cultured in glucose. The IDH activity in the acetate. wild type was decreased by about 22%, which may be caused by the regulation of IDH phosphorylation (Stueland et al., 1988). But no major effect of car- Ann. Microbiol., 58 (2) 275-280 (2008) 279

TABLE 2 - IDH activity of toward NADP and NAD on vari- terial oxidative and stress protection (Rapisarda et ous carbon sources al., 2002; Rodriguez-Montelongo et al., 2006). In this study, we found that the physiological role of UdhA was correlated with the expression level of NDH-2. On acetate, NDH-2 expression was down-regulated in both mutant strains. NDH-2 in icdNADP/ΔudhA was significantly down-regulated by about 4 fold with respect to the wild type, whereas NDH-2 in icdNAD/ΔudhA was only slightly repressed (Fig. 2). However, the NDH-2 expression in icdNAD/ΔudhA was much higher than that in icdNADP/ΔudhA, which was up-regulated by about 2.8 fold. It implies that NADH was severely deficient in icdNADP/ΔudhA so that the expression of NDH-2 was IDH activity was determined at 340 nm by measuring the reduction of NADP or NAD in 1 ml reaction mixture at 37 intensively decreased to reduce NADH depletion, °C. Standard errors were calculated from at least three whereas NADH catalysed by NAD-IDH in replicate experiments. icdNAD/ΔudhA may remedy the NADH loss caused by the deletion of UdhA. Therefore, UdhA is an important NADH resource for E. coli growth on the highly bon source exchange on the IDH activity was oxidized compound and the bacteria has self-regu- observed in icdNADP/ΔudhA. By contrast, the IDH lation mechanism corresponding to the insufficient activity of icdNAD/ΔudhA was obviously enhanced NADH. about 1.5 fold during growth on acetate (with respect to glucose). The significant increase of NAD- dependent IDH activity was probably induced by the Acknowledgements absence of UdhA. In order to overcome the loss of We are very much indebted to Professor Antony M. UdhA, icdNAD/ΔudhA initiated stress response mech- Dean and his laboratory for their kind advices and anism in bacteria to recruit NADH from alternative technical assistance. This research was financially pathways for energy production. supported by the National Natural Science Foundation of China (No. 30500300), New Century Excellent Talents in University of the Education Effect on NDH-2 expression Ministry of China (NCET-06-0558) and the Two distinct membrane-bound NADH dehydroge- Outstanding Youth Foundation of Science and nases have been identified in E. coli respiratory Technology of Anhui Province (No. 06043089). chain (Matsushita et al., 1987). In contrast to NDH- 1, NDH-2 (encoded by the ndh gene) catalyses the reoxidation of NADH exclusively and transfers elec- REFERENCES trons to ubiquinone-1 (Calhounand Gennis, 1993; Boonstra B., French C.E., Wainwright I., Bruce N.C. Gyan et al., 2006). In the cell, NDH-2 links the (1999). The udhA gene of Escherichia coli encodes a major catabolic and energy-producing pathways soluble pyridine nucleotide transhydrogenase. J. (Jaworowski et al., 1981) that contribute in the bac- Bacteriol., 181: 1030-1034. Boonstra B., Rathbone D.A., French C.E., Walker E.D., Bruce N.C. (2000). Cofactor regeneration by a soluble pyridine nucleotide transhydrogenase for biological production of hydromorphone. Appl. Environ. Microbiol., 66: 5161-5166. Calhoun M.W., Gennis R.B. (1993). Demonstration of sep- arate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli. J. Bacteriol., 175: 3013-3019. Chen R., Greer A., Dean A.M. (1995). A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity. Proc. Natl. Acad. Sci. USA, 92: 11666-11670. Datsenko K.A., Wanner B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using FIG. 2 - The relative mRNA level of NDH-2 from mutant PCR products. Proc. Natl. Acad. Sci. USA, 97: 6640- Escherichia coli strains as compared with the wild 6645. type strain. It was determined by RT-PCR during growth on acetate. The results are expressed as the average values of three independent experiments. 280 H. ZHAO et al.

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