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The influence of yggG gene deficiency on the production and assimilation of acetate in Escherichia coli

The influence of yggG gene deficiency on the production and assimilation of acetate in... Ann Microbiol (2011) 61:979–983 DOI 10.1007/s13213-011-0203-z SHORT COMMUNICATION The influence of yggG gene deficiency on the production and assimilation of acetate in Escherichia coli Yoshihiro Ojima & Nobuya Shirasaka & Yuto Fukuda & Mizuho Komaki & Masahito Taya Received: 25 August 2010 /Accepted: 10 January 2011 /Published online: 27 January 2011 Springer-Verlag and the University of Milan 2011 Abstract During the culture of Escherichia coli BW25113 under sub-lethal oxidative stress generated by photo-excited grown on glucose as a carbon source, approximately 1.0 g titanium dioxide (Kim et al. 2005). A gene homology acetate/l was produced after 10 h and, thereafter, acetate search suggested that the yggG gene encodes a 25-kDa was completely consumed as an alternative carbon source protein putatively assigned as a metalloprotease. A recent after glucose starvation. In contrast, during the culture of a report demonstrated that the yggG gene responded to yggG-deficient E. coli mutant JW2903, acetate also accu- stimulation by ultraviolet irradiation and heat shock, and mulated but was not subsequently consumed, even after confirmed the interaction between the yggG gene product glucose starvation during the culture for 24 h. The analysis and the cell membrane (Huang et al. 2007). In addition, the of gene expression revealed that the accumulation of acetate yggG gene was also up-regulated in the presence of zinc was not due to the malfunction of the acetate-assimilating ions in the medium (Hagiwara et al. 2003). Although these pathway in response to glucose starvation but to the lowering responses to external stimuli were reported, the relationship of metabolic flow into the TCA cycle. This was explained by between the yggG gene and central metabolism has not been the lower levels of gltA and acnA gene expressions, and the studied. In our previous study, it was reported that the higher level of arcA gene in JW2903 cells. These results introduction of the yggG gene in L-phenylalanine-producing suggest that the yggG gene contributes to the normalization E. coli strain enhanced the yield of L-phenylalanine, of metabolic pathways including the TCA cycle. associated with the repressed production of acetate (Ojima et al. 2009), which leads to the hypothesis that yggG plays an . . Keywords Acetate metabolism yggG gene TCA cycle important role in the central metabolism of E. coli cells. The present work examined the effect of yggG deficiency on the central metabolic pathway in E. coli, especially its Introduction effect on acetate metabolism. Acetate production profiles were discussed in terms of the expressions of genes involved The functionally-undefined gene, yggG, was previously in the production and in the assimilation of acetate while found to be a stress-response gene when superoxide comparing the culture properties of the wild type E. coli dismutase (SOD)-deficient Escherichia coli was cultured strain and the yggG-deficient mutant on glucose medium. Electronic supplementary material The online version of this article Materials and methods (doi:10.1007/s13213-011-0203-z) contains supplementary material, which is available to authorized users. : : : : Bacterial strains and culture conditions Y. Ojima N. Shirasaka Y. Fukuda M. Komaki M. Taya (*) Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Escherichia coli BW25113 and its yggG-deficient mutant, Osaka University, JW2903, were obtained from the National BioResource 1-3 Machikaneyama-cho, Project (NIG, Japan) (Baba et al. 2006). For the cultures of Toyonaka, Osaka 560-8531, Japan BW25113 and JW2903 strains, the cells from Luria-Bertani e-mail: taya@cheng.es.osaka-u.ac.jp 980 Ann Microbiol (2011) 61:979–983 (LB) agar plates were pre-cultured in 10 ml of liquid LB medium for 14 h at 37°C, and then 1 ml of culture broth was transferred into 100 ml of test culture medium. The test culture medium, denoted as MS medium (Matsui et al. 2001), contained 10 g glucose, 2 g yeast extract, 16 g (NH ) SO , 4 2 4 1g KH PO ,1g MgSO ·7H O, 8 mg MnSO ·5H O, and 2 4 4 2 4 2 10 mg FeSO ·7H O per liter of deionized water. The initial 4 2 pH of the medium was set to 7.0 and CaCO (20 g/l) was added to the medium to avoid a pH drop during the culture. The cultures were incubated in conical flasks under constant agitation on a rotary shaker at 200 revolutions per minute (NR-20; Taitec) kept at 37°C for the prescribed culture time. Cell growth was recorded by measuring the optical density of culture broth at 660 nm (OD ) after dissolving solid CaCO suspensions with dilute HCl. Dry cell weight (DCW) was calculated using a predetermined relation of DCW= 0.36×OD . Analyses Glucose and acetate concentrations were determined with an analyzer, 300 Plus BioProfile (Nova Biomedical). For studying gene expression, the cells harvested at the described time were subjected to total RNA extraction, according to a previously reported procedure (Ojima et al. 2009), followed Fig. 1 Cell growth, and glucose and acetate concentrations in E. coli by reverse transcription to cDNA with a PrimeScript™ RT BW25113 (closed circle wild-type) and JW2903 (open circle yggG- reagent kit (Takara Bio) using random hexamer primers (see deficient mutant) cultures Supplementary Table S1). The rrsA (16s rRNA) gene was employed as a reference for the real-time PCR assay. Real- cell concentration in the JW2903 culture was approximately time PCR was performed with a Chromo4 and MJ Opticon 60% of that in the BW25113 culture. Acetate production in Monitor™ (Bio-Rad Laboratories) using SYBR Premix Ex the JW2903 culture accumulated to a higher level than that of Taq™ (Takara Bio) under the conditions of 10 s at 95°C, the BW25113 culture and remained in the culture broth until followed by 40 cycles of 5 s at 95°C and afterward 20 s at the end of the culture. Meanwhile, for BW25113, the cells 60°C. For each cDNA sample, a cycle threshold value (C ) started to consume acetate as a carbon source due to glucose of the reference gene was subtracted from that of target gene exhaustion in the broth. This alternative metabolic pathway, to obtain ΔC , and the expression of each gene was named the “acetate switch”, has been well documented for E. -ΔCt quantified by 2 . coli culture (Wolfe 2005). JW2903 strain seemed to fail the switching of carbon source utilization after glucose starvation. Table 1 summarizes the results from triplicate cultures of Results and discussion BW25113 and JW2903 strains for 24 h. The average DCW of BW25113 cells was 2.3 g/l, approximately 1.4 times Effect of yggG deletion on the culture properties of E. coli greater than for JW2903 cells. Furthermore, the cell yields of BW25113 and JW2903 were Y =0.25 and 0.18 g DCW/Glu Figure 1 shows the time courses of cell growth, and glucose and acetate concentrations in typical culture runs of Table 1 Cell growth, cell yield on glucose and acetate concentration BW25113 and JW2903 strains in MS medium. In preliminary in cultures of E. coli BW25113 and JW2903 strains experiments, acetate was confirmed to be a dominant Strain DCW (g/l) Y / (g DCW/g glucose) Acetate (g/l) DCW glu metabolite accumulated in the cultures of both strains. The growth profiles of these strains were similar in the early BW25113 2.3±0.1 0.25±0.02 N.D. culture phase, but after 8 h of culture time, JW2903 cells JW2903 1.7±0.2 0.18±0.03 2.4±0.3 showed a limited growth curve, compared to BW25113 cells. This phase was followed by decreased glucose consumption The data were obtained from triplicate cultures for 24 h. N.D. Not detectable that coincided with the repression of cell growth. The final Ann Microbiol (2011) 61:979–983 981 DCW/g glucose, respectively, which also indicated that synthetase (acs) contributes to the irreversible incorporation JW2903 cells had a lower efficiency of carbon source of acetate into acetyl-CoA (Brown et al. 1977; Kumari et al. utilization. During cell cultures, drastic pH changes were not 1995). The expressions of citrate synthase (gltA), a key observed and the final pH values were 7.6 and 7.5 on average enzyme for entering into the TCA cycle, and two aconitases in the BW25113 and JW2903 cultures, respectively. From (acnA and acnB) can be indicators of the activity of the TCA these results, it is suggested that the yggG deletion causes a cycle. It has been reported that these three genes were notable alteration in central metabolism with an increased negatively regulated by the two-component Arc (aerobic accumulation of acetate in the culture. respiration control) system, which is encoded by arcA (Iuchi and Weiner 1996;Cunninghamet al. 1997). Investigating metabolic changes of acetate production and Figure 2 shows the detailed expression levels of selected assimilation genes in the cells obtained at 4, 8, 10 and 12 h during the culturing of both strains in MS medium. The tendency of The metabolic pathways involved in the production and gene expression in the PTA-ACKA pathway was not assimilation of acetate in E. coli cells are outlined in significantly changed by the yggG deletion over the Supplemental Figure S1.In E. coli cell culture, acetate is a examined culture period (Fig. 2a, b), indicating that the dominant end-metabolite when grown aerobically on accumulation of acetate in JW2903 culture did not result glucose as a carbon and energy source, especially at a high from the enhancement of the acetate-producing pathway. carbon flux through glycolysis in such a way that it exceeds Furthermore, in the acetate assimilating-pathway, the the turn-over of the TCA cycle flow (Contiero et al. 2000; expression level of acs significantly increased after 10 h Cozzone 1998; Farmer and Liao 1997). The PTA-ACKA in the culture of the BW25113 strain (Fig. 2c), which pathway is a reversible acetate-producing pathway, encoded corresponds to glucose starvation and the switch to the by pta and ackA genes, converting from acetyl-CoA to utilization of acetate as a carbon source (see Fig. 1). The acetate (Brown et al. 1977; Kumari et al. 1995). When the induction of acs was confirmed at 12 h in the culture of TCA cycle activity is not high enough, this pathway may JW2903 in response to a glucose shortage, although the yield redundant acetate. Furthermore, the TCA cycle activity level of expression was about half when compared to the is also important for acetate assimilation after glucose BW25113 strain (Fig. 2c). These results indicate that starvation since acetate as an alternative carbon source is also acetate uptake is not a limiting step for acetate assimilation directed toward the TCA cycle for energy production (Wolfe in JW2903 cells. The expression level of cra gene was also 2005). With respect to acetate assimilation, acetyl-CoA analyzed, which is essential when grown on acetate as a Fig. 2 Gene expressions of selected genes in E. coli BW25113 and JW2903 strains. The closed and open bars show the expression levels in the cells of BW25113 and JW2903 strains, respectively. The data were obtained from three independent culture runs and represented by normalizing on the basis of rrsA (16s rRNA) gene expression. Error bars standard deviation 982 Ann Microbiol (2011) 61:979–983 substrate (Sarkar et al. 2008). Both strains kept certain Conclusion levels of cra expression (Fig. 2d) when acetate was present in the medium, which supports the above discussion. It was concluded that the deletion of the yggG gene in E. coli Next, the expressions of genes relating to the TCA cycle enhanced acetate accumulation in the culture with glucose. were also compared between the two strains. In the case of The accumulation of acetate was not due to the malfunction BW25113 cells, the expression level of gltA kept relatively of the response to the acetate switch but the decreased flow high in an early phase until 8 h, and then decreased by into the TCA cycle. This restriction of the TCA cycle was about half after 10 h (Fig. 2e). Meanwhile, in the case of caused by the low levels of expression of gltA and acnA the JW2903 cells, the expression of gltA until 8 h was about genes regulated by the Arc system. These results suggest that half of that in BW25113 cells and drastically decreased at the yggG gene contributes to the normalization of the aerobic 12 h when the glucose was almost consumed. These results metabolic pathway, including the TCA cycle, through a suggest that the malfunction of acetate uptake can be balance of intracellular phosphorylation. attributable to the decreased flow into the TCA cycle for Acknowledgments We thank Ms Nora Hieke of RWTH Aachen acetate assimilation. Furthermore, the result of acnA University for her assistance with the experiments. We also thank Dr. expression also supports this view (Fig. 2f). In the case of Motomu Nishioka of Osaka Prefectural College of Technology for the BW25113 cells, expression of acnA increased in a later helpful suggestions. This study was supported in part by a Grant-in-Aid phase, which coincided with the result in a previous report for Young Scientists (Start-up) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 21860057). (Cunningham et al. 1997). However, in the case of the JW2903 cells, the expression level was relatively low throughout the period examined, compared to the BW25113 cells, and drastically decreased at 12 h, which References was about 3% of the level in BW25113 cells at 12 h. The down-regulation of genes involved in the entrance to the Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko TCA cycle was thought to be a dominant factor for the KA, Tomita M, Wanner BL, Mori H (2006) Construction of accumulation of acetate. However, there was no significant Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:E1–E11 difference in the expression of acnB between the two Brown TDK, Jones-Mortimer MC, Kornberg HL (1977) The enzymic strains (Fig. 2g). As for the regulation of gltA and acnA interconversion of acetate and acetyl-coenzyme A in Escherichia genes, the drastic up-regulation of arcA was recognized at coli. J Gen Microbiol 102:327–336 12 h in the culture of the JW2903 strain, although this up- Contiero J, Beatty C, Kumari S, DeSanti CL, Strohl WR, Wolfe AJ (2000) Effects of mutants in acetate metabolism on high-cell- regulation was not seen in the BW25113 culture (Fig. 2h). density growth of Escherichia coli. J Ind Microbiol 24:421–430 This result can explain the decreased expressions of gltA and Cozzone AJ (1998) Regulation of acetate metabolism by protein acnA at 12 h in the JW2903 culture since arcA is known to phosphorylation in enteric bacteria. Annu Rev Microbiol 52:127– be a negative regulator for these genes (Iuchi and Weiner 164 Cunningham L, Gruer MJ, Guest JR (1997) Transcriptional regulation 1996; Cunningham et al. 1997). As a result, the down- of the aconitase genes (acnA and acnB)of Escherichia coli. regulation of gltA and acnA in JW2903 cells is thought to be Microbiology 143:3795–3805 caused by the increase of arcA expression in a glucose- Farmer WR, Liao JC (1997) Reduction of aerobic acetate production exhausted phase. by Escherichia coli. Appl Environ Microbiol 63:3205–3210 Hagiwara D, Sugiura M, Oshima T, Mori H, Aiba H, Yamashino T, Recently, studies using immunocytochemistry and subcel- Mizuno T (2003) Genome-wide analyses revealing a signaling lular fraction analyses suggested that yggG encodes a network of the RcsC-YojN-RcsB phosphorelay system in membrane-associated protein capable of binding with Era Escherichia coli. J Bacteriol 185:5735–5746 protein directly (Huang et al. 2007, 2008). Era is a Huang Y, Zhang B, Dong K, Zhang XN, Hou L, Wang T, Chen NC, Chen SM (2007) Up-regulation of yggG promotes the survival of membrane-associated GTPase essential for bacterial growth Escherichia coli cells containing Era-1 mutant protein. FEMS (March et al. 1988), but the overproduction of dominant- Microbiol Lett 275:8–15 negative Era has been shown to inhibit cell growth by Huang Y, Dong K, Zhang XN, Zhang B, Hou L, Chen NC, Chen SM decreasing the TCA cycle activity (Pillutla et al. 1996). At (2008) Expression and regulation of the yggG gene of Escherichia coli. Curr Microbiol 56:14–20 the same time, signal transduction of the two-component Arc Iuchi S, Weiner L (1996) Cellular and molecular physiology of system was also reported to be achieved with phosphoryla- Escherichia coli in the adaptation to aerobic environments. J tion of ArcA protein (Iuchi and Weiner 1996). Thus, these Biochem 120:1055–1063 reports lead us to a hypothesis that the deletion of yggG gene Kim SY, Nishioka M, Hayashi S, Honda H, Kobayashi T, Taya M (2005) The gene yggE functions in restoring physiological affects the regulation of the Arc system through disturbing defects of Escherichia coli under oxidative stress conditions. the intracellular balance of the phosphorylation mechanism. Appl Environ Microbiol 71:2762–2765 Further examination will be conducted to elucidate a detailed Kumari SR, Tishel R, Eisenbach M, Wolfe AJ (1995) Cloning, function of yggG. characterization, and functional expression of acs, which encodes Ann Microbiol (2011) 61:979–983 983 acetyl-CoA synthetase in Escherichia coli. J Bacteriol 177:2878– yield of L-phenylalanine with decreased acetic acid production in a 2886 recombinant Escherichia coli.Biotechnol Lett31:525–530 March PE, Lerner GC, Ahnn J, Cui X, Inoue M (1988) The Pillutla RC, Ahnn J, Inoue M (1996) Deletion of the putative effector Escherichia coli Ras-like protein (Era) has GTPase activity and region of Era, and essential GTP-binding protein in Escherichia is essential for cell growth. Oncogene 2:539–544 coli, causes a dominant-negative phenotype. FEMS Microbiol Matsui H, Kawasaki H, Shimaoka M, Kurahashi O (2001) Investiga- Lett 143:47–55 tion of various genotype characteristics for inosine accumulation Sarkar D, Siddiquee KAZ, Arauzo-Bravo MJ, Oba T, Shimizu K in Escherichia coli W3110. Biosci Biotechnol Biochem 65:570– (2008) Effect of cra gene knockout together with edd and iclR 578 genes knockout on the metabolism in Escherichia coli. Arch Ojima Y, Komaki M, Nishioka M, Iwatani S, Tsujimoto N, Taya M Microbiol 190:559–571 (2009) Introduction of a stress-responsive gene, yggG, enhances the Wolfe AJ (2005) The acetate switch. Microbiol Mol Biol Rev 69:12–50 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

The influence of yggG gene deficiency on the production and assimilation of acetate in Escherichia coli

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Springer Journals
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Copyright © 2011 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-011-0203-z
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Abstract

Ann Microbiol (2011) 61:979–983 DOI 10.1007/s13213-011-0203-z SHORT COMMUNICATION The influence of yggG gene deficiency on the production and assimilation of acetate in Escherichia coli Yoshihiro Ojima & Nobuya Shirasaka & Yuto Fukuda & Mizuho Komaki & Masahito Taya Received: 25 August 2010 /Accepted: 10 January 2011 /Published online: 27 January 2011 Springer-Verlag and the University of Milan 2011 Abstract During the culture of Escherichia coli BW25113 under sub-lethal oxidative stress generated by photo-excited grown on glucose as a carbon source, approximately 1.0 g titanium dioxide (Kim et al. 2005). A gene homology acetate/l was produced after 10 h and, thereafter, acetate search suggested that the yggG gene encodes a 25-kDa was completely consumed as an alternative carbon source protein putatively assigned as a metalloprotease. A recent after glucose starvation. In contrast, during the culture of a report demonstrated that the yggG gene responded to yggG-deficient E. coli mutant JW2903, acetate also accu- stimulation by ultraviolet irradiation and heat shock, and mulated but was not subsequently consumed, even after confirmed the interaction between the yggG gene product glucose starvation during the culture for 24 h. The analysis and the cell membrane (Huang et al. 2007). In addition, the of gene expression revealed that the accumulation of acetate yggG gene was also up-regulated in the presence of zinc was not due to the malfunction of the acetate-assimilating ions in the medium (Hagiwara et al. 2003). Although these pathway in response to glucose starvation but to the lowering responses to external stimuli were reported, the relationship of metabolic flow into the TCA cycle. This was explained by between the yggG gene and central metabolism has not been the lower levels of gltA and acnA gene expressions, and the studied. In our previous study, it was reported that the higher level of arcA gene in JW2903 cells. These results introduction of the yggG gene in L-phenylalanine-producing suggest that the yggG gene contributes to the normalization E. coli strain enhanced the yield of L-phenylalanine, of metabolic pathways including the TCA cycle. associated with the repressed production of acetate (Ojima et al. 2009), which leads to the hypothesis that yggG plays an . . Keywords Acetate metabolism yggG gene TCA cycle important role in the central metabolism of E. coli cells. The present work examined the effect of yggG deficiency on the central metabolic pathway in E. coli, especially its Introduction effect on acetate metabolism. Acetate production profiles were discussed in terms of the expressions of genes involved The functionally-undefined gene, yggG, was previously in the production and in the assimilation of acetate while found to be a stress-response gene when superoxide comparing the culture properties of the wild type E. coli dismutase (SOD)-deficient Escherichia coli was cultured strain and the yggG-deficient mutant on glucose medium. Electronic supplementary material The online version of this article Materials and methods (doi:10.1007/s13213-011-0203-z) contains supplementary material, which is available to authorized users. : : : : Bacterial strains and culture conditions Y. Ojima N. Shirasaka Y. Fukuda M. Komaki M. Taya (*) Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Escherichia coli BW25113 and its yggG-deficient mutant, Osaka University, JW2903, were obtained from the National BioResource 1-3 Machikaneyama-cho, Project (NIG, Japan) (Baba et al. 2006). For the cultures of Toyonaka, Osaka 560-8531, Japan BW25113 and JW2903 strains, the cells from Luria-Bertani e-mail: taya@cheng.es.osaka-u.ac.jp 980 Ann Microbiol (2011) 61:979–983 (LB) agar plates were pre-cultured in 10 ml of liquid LB medium for 14 h at 37°C, and then 1 ml of culture broth was transferred into 100 ml of test culture medium. The test culture medium, denoted as MS medium (Matsui et al. 2001), contained 10 g glucose, 2 g yeast extract, 16 g (NH ) SO , 4 2 4 1g KH PO ,1g MgSO ·7H O, 8 mg MnSO ·5H O, and 2 4 4 2 4 2 10 mg FeSO ·7H O per liter of deionized water. The initial 4 2 pH of the medium was set to 7.0 and CaCO (20 g/l) was added to the medium to avoid a pH drop during the culture. The cultures were incubated in conical flasks under constant agitation on a rotary shaker at 200 revolutions per minute (NR-20; Taitec) kept at 37°C for the prescribed culture time. Cell growth was recorded by measuring the optical density of culture broth at 660 nm (OD ) after dissolving solid CaCO suspensions with dilute HCl. Dry cell weight (DCW) was calculated using a predetermined relation of DCW= 0.36×OD . Analyses Glucose and acetate concentrations were determined with an analyzer, 300 Plus BioProfile (Nova Biomedical). For studying gene expression, the cells harvested at the described time were subjected to total RNA extraction, according to a previously reported procedure (Ojima et al. 2009), followed Fig. 1 Cell growth, and glucose and acetate concentrations in E. coli by reverse transcription to cDNA with a PrimeScript™ RT BW25113 (closed circle wild-type) and JW2903 (open circle yggG- reagent kit (Takara Bio) using random hexamer primers (see deficient mutant) cultures Supplementary Table S1). The rrsA (16s rRNA) gene was employed as a reference for the real-time PCR assay. Real- cell concentration in the JW2903 culture was approximately time PCR was performed with a Chromo4 and MJ Opticon 60% of that in the BW25113 culture. Acetate production in Monitor™ (Bio-Rad Laboratories) using SYBR Premix Ex the JW2903 culture accumulated to a higher level than that of Taq™ (Takara Bio) under the conditions of 10 s at 95°C, the BW25113 culture and remained in the culture broth until followed by 40 cycles of 5 s at 95°C and afterward 20 s at the end of the culture. Meanwhile, for BW25113, the cells 60°C. For each cDNA sample, a cycle threshold value (C ) started to consume acetate as a carbon source due to glucose of the reference gene was subtracted from that of target gene exhaustion in the broth. This alternative metabolic pathway, to obtain ΔC , and the expression of each gene was named the “acetate switch”, has been well documented for E. -ΔCt quantified by 2 . coli culture (Wolfe 2005). JW2903 strain seemed to fail the switching of carbon source utilization after glucose starvation. Table 1 summarizes the results from triplicate cultures of Results and discussion BW25113 and JW2903 strains for 24 h. The average DCW of BW25113 cells was 2.3 g/l, approximately 1.4 times Effect of yggG deletion on the culture properties of E. coli greater than for JW2903 cells. Furthermore, the cell yields of BW25113 and JW2903 were Y =0.25 and 0.18 g DCW/Glu Figure 1 shows the time courses of cell growth, and glucose and acetate concentrations in typical culture runs of Table 1 Cell growth, cell yield on glucose and acetate concentration BW25113 and JW2903 strains in MS medium. In preliminary in cultures of E. coli BW25113 and JW2903 strains experiments, acetate was confirmed to be a dominant Strain DCW (g/l) Y / (g DCW/g glucose) Acetate (g/l) DCW glu metabolite accumulated in the cultures of both strains. The growth profiles of these strains were similar in the early BW25113 2.3±0.1 0.25±0.02 N.D. culture phase, but after 8 h of culture time, JW2903 cells JW2903 1.7±0.2 0.18±0.03 2.4±0.3 showed a limited growth curve, compared to BW25113 cells. This phase was followed by decreased glucose consumption The data were obtained from triplicate cultures for 24 h. N.D. Not detectable that coincided with the repression of cell growth. The final Ann Microbiol (2011) 61:979–983 981 DCW/g glucose, respectively, which also indicated that synthetase (acs) contributes to the irreversible incorporation JW2903 cells had a lower efficiency of carbon source of acetate into acetyl-CoA (Brown et al. 1977; Kumari et al. utilization. During cell cultures, drastic pH changes were not 1995). The expressions of citrate synthase (gltA), a key observed and the final pH values were 7.6 and 7.5 on average enzyme for entering into the TCA cycle, and two aconitases in the BW25113 and JW2903 cultures, respectively. From (acnA and acnB) can be indicators of the activity of the TCA these results, it is suggested that the yggG deletion causes a cycle. It has been reported that these three genes were notable alteration in central metabolism with an increased negatively regulated by the two-component Arc (aerobic accumulation of acetate in the culture. respiration control) system, which is encoded by arcA (Iuchi and Weiner 1996;Cunninghamet al. 1997). Investigating metabolic changes of acetate production and Figure 2 shows the detailed expression levels of selected assimilation genes in the cells obtained at 4, 8, 10 and 12 h during the culturing of both strains in MS medium. The tendency of The metabolic pathways involved in the production and gene expression in the PTA-ACKA pathway was not assimilation of acetate in E. coli cells are outlined in significantly changed by the yggG deletion over the Supplemental Figure S1.In E. coli cell culture, acetate is a examined culture period (Fig. 2a, b), indicating that the dominant end-metabolite when grown aerobically on accumulation of acetate in JW2903 culture did not result glucose as a carbon and energy source, especially at a high from the enhancement of the acetate-producing pathway. carbon flux through glycolysis in such a way that it exceeds Furthermore, in the acetate assimilating-pathway, the the turn-over of the TCA cycle flow (Contiero et al. 2000; expression level of acs significantly increased after 10 h Cozzone 1998; Farmer and Liao 1997). The PTA-ACKA in the culture of the BW25113 strain (Fig. 2c), which pathway is a reversible acetate-producing pathway, encoded corresponds to glucose starvation and the switch to the by pta and ackA genes, converting from acetyl-CoA to utilization of acetate as a carbon source (see Fig. 1). The acetate (Brown et al. 1977; Kumari et al. 1995). When the induction of acs was confirmed at 12 h in the culture of TCA cycle activity is not high enough, this pathway may JW2903 in response to a glucose shortage, although the yield redundant acetate. Furthermore, the TCA cycle activity level of expression was about half when compared to the is also important for acetate assimilation after glucose BW25113 strain (Fig. 2c). These results indicate that starvation since acetate as an alternative carbon source is also acetate uptake is not a limiting step for acetate assimilation directed toward the TCA cycle for energy production (Wolfe in JW2903 cells. The expression level of cra gene was also 2005). With respect to acetate assimilation, acetyl-CoA analyzed, which is essential when grown on acetate as a Fig. 2 Gene expressions of selected genes in E. coli BW25113 and JW2903 strains. The closed and open bars show the expression levels in the cells of BW25113 and JW2903 strains, respectively. The data were obtained from three independent culture runs and represented by normalizing on the basis of rrsA (16s rRNA) gene expression. Error bars standard deviation 982 Ann Microbiol (2011) 61:979–983 substrate (Sarkar et al. 2008). Both strains kept certain Conclusion levels of cra expression (Fig. 2d) when acetate was present in the medium, which supports the above discussion. It was concluded that the deletion of the yggG gene in E. coli Next, the expressions of genes relating to the TCA cycle enhanced acetate accumulation in the culture with glucose. were also compared between the two strains. In the case of The accumulation of acetate was not due to the malfunction BW25113 cells, the expression level of gltA kept relatively of the response to the acetate switch but the decreased flow high in an early phase until 8 h, and then decreased by into the TCA cycle. This restriction of the TCA cycle was about half after 10 h (Fig. 2e). Meanwhile, in the case of caused by the low levels of expression of gltA and acnA the JW2903 cells, the expression of gltA until 8 h was about genes regulated by the Arc system. These results suggest that half of that in BW25113 cells and drastically decreased at the yggG gene contributes to the normalization of the aerobic 12 h when the glucose was almost consumed. These results metabolic pathway, including the TCA cycle, through a suggest that the malfunction of acetate uptake can be balance of intracellular phosphorylation. attributable to the decreased flow into the TCA cycle for Acknowledgments We thank Ms Nora Hieke of RWTH Aachen acetate assimilation. Furthermore, the result of acnA University for her assistance with the experiments. We also thank Dr. expression also supports this view (Fig. 2f). In the case of Motomu Nishioka of Osaka Prefectural College of Technology for the BW25113 cells, expression of acnA increased in a later helpful suggestions. This study was supported in part by a Grant-in-Aid phase, which coincided with the result in a previous report for Young Scientists (Start-up) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 21860057). (Cunningham et al. 1997). However, in the case of the JW2903 cells, the expression level was relatively low throughout the period examined, compared to the BW25113 cells, and drastically decreased at 12 h, which References was about 3% of the level in BW25113 cells at 12 h. The down-regulation of genes involved in the entrance to the Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko TCA cycle was thought to be a dominant factor for the KA, Tomita M, Wanner BL, Mori H (2006) Construction of accumulation of acetate. However, there was no significant Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:E1–E11 difference in the expression of acnB between the two Brown TDK, Jones-Mortimer MC, Kornberg HL (1977) The enzymic strains (Fig. 2g). As for the regulation of gltA and acnA interconversion of acetate and acetyl-coenzyme A in Escherichia genes, the drastic up-regulation of arcA was recognized at coli. J Gen Microbiol 102:327–336 12 h in the culture of the JW2903 strain, although this up- Contiero J, Beatty C, Kumari S, DeSanti CL, Strohl WR, Wolfe AJ (2000) Effects of mutants in acetate metabolism on high-cell- regulation was not seen in the BW25113 culture (Fig. 2h). density growth of Escherichia coli. J Ind Microbiol 24:421–430 This result can explain the decreased expressions of gltA and Cozzone AJ (1998) Regulation of acetate metabolism by protein acnA at 12 h in the JW2903 culture since arcA is known to phosphorylation in enteric bacteria. Annu Rev Microbiol 52:127– be a negative regulator for these genes (Iuchi and Weiner 164 Cunningham L, Gruer MJ, Guest JR (1997) Transcriptional regulation 1996; Cunningham et al. 1997). As a result, the down- of the aconitase genes (acnA and acnB)of Escherichia coli. regulation of gltA and acnA in JW2903 cells is thought to be Microbiology 143:3795–3805 caused by the increase of arcA expression in a glucose- Farmer WR, Liao JC (1997) Reduction of aerobic acetate production exhausted phase. by Escherichia coli. Appl Environ Microbiol 63:3205–3210 Hagiwara D, Sugiura M, Oshima T, Mori H, Aiba H, Yamashino T, Recently, studies using immunocytochemistry and subcel- Mizuno T (2003) Genome-wide analyses revealing a signaling lular fraction analyses suggested that yggG encodes a network of the RcsC-YojN-RcsB phosphorelay system in membrane-associated protein capable of binding with Era Escherichia coli. J Bacteriol 185:5735–5746 protein directly (Huang et al. 2007, 2008). 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Arch Ojima Y, Komaki M, Nishioka M, Iwatani S, Tsujimoto N, Taya M Microbiol 190:559–571 (2009) Introduction of a stress-responsive gene, yggG, enhances the Wolfe AJ (2005) The acetate switch. Microbiol Mol Biol Rev 69:12–50

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Published: Jan 27, 2011

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