Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

FEMS Microbiology Letters 218 (2003) 305^309

www.fems-microbiology.org

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum Daniel Nottebrock, Ute Meyer, Reinhard Kra«mer, Susanne Morbach



Institut fu«r Biochemie, Universita«t Ko«ln, Zu«lpicher Str. 47, D-50674 Ko«ln, Germany Received 31 October 2002; received in revised form 18 November 2002; accepted 18 November 2002 First published online 3 December 2002

Abstract Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant. 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Mechanosensitive channel; Hypoosmotic stress; Corynebacterium glutamicum

1. Introduction The cytoplasmic membrane of bacteria is permeable to water but forms an e¡ective barrier for most solutes present in the medium or in the cytoplasm. When the external osmolarity increases a rapid e¥ux of water occurs and the cells may plasmolyse. The core physiological response of bacteria to the initial loss of water is the controlled accumulation of so-called compatible solutes in the cytoplasm. The consequent in£ow of water restores the outwardly directed turgor pressure, which is essential for the cell proliferation [1]. If the cell is exposed to a sudden reduction of the external osmolarity, water in£ow causes an increase in the turgor pressure, which could disrupt the cell. As a response cytoplasmic solutes are released via mechanosensitive channels to achieve a rapid reduction of the turgor pressure. The activity of mechanosensitive channels of different size and conductivity was demonstrated in Gramnegative and Gram-positive bacteria [2,3]. At least three

* Corresponding author. Tel. : +49 (221) 470 6464 ; Fax : +49 (221) 5091. E-mail address : [email protected] (S. Morbach).

subfamilies of channels, MscL, MscS and MscM, were identi¢ed in Escherichia coli [4]. Deletion of the mscL gene in E. coli led to a loss in MscL channel activity, but a signi¢cant di¡erence in growth or survival was not observed [5]. Recently, the yggB gene family related to MscS function was identi¢ed including large proteins ( s 700 amino acid residues) only found in Gram-negative bacteria, as well as smaller proteins (V300^500 amino acid residues), which are almost ubiquitous in bacterial genomes [6]. The MscS activity of E. coli protoplasts was eliminated by null mutations in two loci, yggB and kefA. KefA is a large multidomain membrane protein (1120 amino acid residues), while YggB is much smaller (286 amino acid residues) and highly similar to the C-terminal part of KefA. Levina and coworkers [6] demonstrated that mutants lacking YggB and MscL are severely a¡ected in their survival of an osmotic downshock, whereas KefA is not essential. This was the ¢rst demonstration of a physiologically important phenotype provoked by the loss of mechanosensitive channels. In Corynebacterium glutamicum, a Gram-positive soil bacterium, the e¥ux of compatible solutes during the transition from high to low osmolarity was characterized biochemically [7]. E¥ux is characterized by a fast rate of at least 6000 Wmol min31 g31 dry weight and a high speci¢c-

0378-1097 / 02 / $22.00 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(02)01170-9

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ity of the transported solutes. In contrast to other bacteria proline and betaine are signi¢cantly preferred to other amino acids and ATP is not excreted. Patch-clamp analysis of membrane fusion vesicles prepared from C. glutamicum revealed at least two di¡erent types of stretch-activated channels with conductances of 600^700 nS and 1200^1400 nS [8]. These are very similar to values representing MscL and YggB activity in E. coli [4]. Here we present the molecular and biochemical analysis of two open reading frames of C. glutamicum ATCC13032 with similarities to mscL and yggB of E. coli.

2. Materials and methods 2.1. Strains, media and growth conditions E. coli strains DH5Kmcr [9] and S17-1 [10] as well as C. glutamicum wild-type strain ATCC13032 [11] and its derivatives (this work) were used. E. coli was grown at 37‡C in Luria^Bertani medium, C. glutamicum at 30‡C in brain heart infusion (BHI, Difco, Detroit, MI, USA) or in MMI medium containing per l: 20 g glucose, 5 g (NH4 )2 SO4 , 5 g urea, 2 g KH2 PO4 , 2 g K2 HPO4 , 0.25 g MgSO4 W7H2 O, 10 mg CaCl2 , 0.2 mg biotin, 1 ml of a solution of trace elements [12]. For the cultivation under salt stress 750 mM NaCl was added, leading to an osmolality of 1.9 osmol kg31 . Due to the chromosomal deletion of yggB the transcription of the ilvBNC operon [12], encoding enzymes for the biosynthesis of branched chain amino acids, was impaired. Therefore, MMI was supplemented with 5 mM L-isoleucine, L-leucine and L-valine. Control experiments in minimal medium (0.3 and 1.9 osmol kg31 ) showed the yggB deletion strains were not impaired in the adaptation to osmotic stress (data not shown). Antibiotics were added in standard concentrations.

pK19mobsacB [13]. The resulting plasmid pKL-BB was transferred to C. glutamicum ATCC13032 by conjugation using E. coli S17-1 as a donor strain. The deletion mutant CgvmscL resulting from a double chromosomal recombination event was checked by PCR (data not shown). For ampli¢cation of yggB and its £anking 5P chromosomal region the sense primer 5P-cagcgaacgcacgaagct and antisense primer 5P-cataggggtggacgtcgg were used. The resulting 2790 bp fragment was phosphorylated and ligated in the SmaI site of pUC18 leading to plasmid pSC. The 380 bp SgrAI/NarI fragment of pSC was removed and the remaining DNA was religated after treatment with Klenow fragment leading to pSC-SN. Subsequently, a 2452 bp HindIII/SacI fragment was isolated and after a ¢ll-in reaction ligated in the SmaI site of pK19mobsacB. The resulting plasmid pKS-SN was transferred to C. glutamicum ATCC13032 by conjugation, the deletion in CgvyggB was veri¢ed by PCR. Double deletion mutants were generated starting either from the parental strain CgvmscL or CgvyggB. 2.3. Construction of complementation plasmids The coding sequence of mscL and yggB was ampli¢ed by PCR using genomic DNA of the wild-type. The following primers were used: mscL sense 5P-ggatccatgcttaaaggtttt, antisense 5P-gaattcctactgaaggcgcttttg, yggB sense 5P-atgattttaggcgtacccattc and antisense 5P-ctaaggggtggacgtcgg. The corresponding 408 and 1189 bp PCR products were phosphorylated and ligated in the SmaI site of pUC18. Subsequently, the mscL gene was isolated after BamHI/EcoRI restriction and ligated in pEKEX2 [14]. The resulting plasmid was designated pEK-mscL. The yggB gene was isolated after a KpnI/BamHI restriction and ligated in pEKEX2 leading to pEK-yggB. 2.4. E¥ux of betaine

2.2. Construction of deletion strains The genes mscL and yggB were deleted by allelic replacement [13]. As parental strain C. glutamicum wildtype ATCC13032 was used. In the ¢rst step the gene of interest together with £anking regions was ampli¢ed by polymerase chain reaction (PCR) using genomic DNA as template. The PCR fragment was sequenced for control. In the case of mscL a 2040 bp fragment containing mscL and the 5P and 3P £anking chromosomal region was ampli¢ed (sense primer 5P-cccccgcaggtgttcgg; antisense primer 5P-ggaagtcttaccgatgttg). The 2040 bp fragment was cloned in the SmaI site of pUC18, leading to plasmid pLC. Subsequently, an internal 235 bp BsmI/BbsI fragment of mscL was removed from pLC. After treatment with Klenow fragment, the remaining plasmid was religated leading to pLC-BB. In the next step, a 1805 bp HindIII/SacI fragment of pLC-BB was isolated, treated with Klenow enzyme and ligated in the SmaI site of

The e¥ux of betaine was measured as recently described by Ru¡ert and coworkers [7]. 2.5. Analysis of cell viability Survival rates of C. glutamicum cells after an osmotic downshock were determined with the LIVE/DEAD BacLight Bacterial Viability Kit of Molecular Probes (Leiden, The Netherlands). Overnight cultures grown in MMI were used for inoculating fresh minimal medium (OD600 = 2) containing 750 mM NaCl (1.9 osmol kg31 ). Cells were incubated for 2 h at 30‡C on a rotary shaker, harvested and washed once either in isoosmotic MMI or in MMI with an NaCl concentration of 500, 250 or 0 mM. Cells were suspended in the identical medium to OD600 = 0.3 and incubated for 15 min in the dark with a mixture of SYTO 9 and propidium iodide. The £uorescence emission spectra of the samples (excitation 470 nm, emission 490^

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700 nm) were measured in a £uorimeter (SLM Aminco, Rochester, USA). The ratio of the integrated intensity of the spectrum between 510 and 540 nm and that between 620 and 650 nm was determined for each sample and the percentage of live cells was calculated by comparing this ratio with a standard curve of live/dead C. glutamicum cells. 2.6. Accession numbers The open reading frame (ORF) numbers for mscL and yggB in the C. glutamicum genome database (http://gib. genes.nig.ac.jp) are Cgl0879 and Cgl1270, respectively.

3. Results and discussion When mechanosensitive channels in C. glutamicum were characterized electrophysiologically [7,8] conductances of 600^700 and 1200^1400 pS were detected, similar to those measured in E. coli, representing MscS and MscL activities encoded by the yggB and mscL genes [4^6]. The C. glutamicum genome project enabled us to identify the corresponding genes by homology searches. These revealed two open reading frames with similarities to mscL and yggB of E. coli, respectively. The MscL homolog (135 amino acid residues) shared 57% identical amino acid residues with MscL of Mycobacterium tuberculosis and 38% with MscL of E. coli and possesses the typical structural characteristics of the MscL family. The Ygg homolog has 533 amino acids and shows a higher identity to the corresponding homologs of di¡erent mycolic acid-containing actinomycetes (up to 36% identity) than to YggB of E. coli, where 29% identical amino acids were found only within the inner part of the two proteins. Using the C. glutamicum wild-type as parental strain, the genes homologous to mscL and yggB were deleted alone (CgvmscL and CgvyggB) and in combination (CgvmscLvyggB or CgvyggBvmscL). By applying hypoosmotic shocks of di¡erent extent, the e¥ux of betaine from C. glutamicum wild-type and deletion mutants was studied. Cells were preloaded with labeled betaine under hyperosmotic conditions (1.92 osmol kg31 ) and subsequently subjected to a sudden reduction of external osmolarity. The amount of excreted betaine was measured after 15 and 60 s, since it had been shown that e¥ux is complete within 60 s [7]. Under isoosmotic conditions only 5% of the labeled betaine was excreted by the wild-type. When the cells were subjected to a hypoosmotic shock we found a linear relationship between the extent of the applied downshock and the amount of released betaine (Fig. 1A). Strain CgvmscL showed an e¥ux behavior more or less similar to the wild-type (Fig. 1B), indicating that the remaining e¥ux system(s) could replace the function of MscL. A similar phenotype was found in an mscL deletion strain of E. coli [15].

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In contrast, the yggB deletion caused a more pronounced e¡ect upon lowering external osmolarity. This strain had a reduced ability to excrete betaine compared to the wild-type (Fig. 1C), i.e. the other e¥ux systems were not able to fully substitute the loss of YggB. As a consequence it took longer in CgvyggB (up to 5 min) to counterbalance an osmotic change (data not shown). This fact was detectable even at a moderate downshock from 1.92 to 1.32 osmol kg31 . Furthermore, if the cells where exposed to a more severe hypoosmotic shock only 50% of betaine (after 15 s) were excreted independent of the extent of the applied downshock. In contrast to the wild-type where we found a linear dependence of the amount of excreted betaine and the extent of the hypoosmotic shock, the e¥ux activity of the yggB deletion strain showed a sigmoidal dependence (Fig. 1C). From this observation we conclude that YggB of C. glutamicum seems to be activated by smaller turgor pressure di¡erences as MscL. This is similar to E. coli [4], where a correlation between the conductances of MscM, MscS and MscL and the activation thresholds was found, whereby the channel with the lowest conductance became activated ¢rst. Furthermore, one might assume that the activation threshold of the corynebacterial MscL (and/or of the remaining channels) seems to be high, because small osmotic downshifts were not su⁄cient for activation, only lowering the external osmolality from 1.9 to 1.0 osmol kg31 led to a signi¢cant activation (Fig. 1C). Surprisingly, the e¥ux capacity of strain CgvmscLvyggB was in-between that of the wild-type and of CgvyggB (Fig. 1D). This result was unexpected, since both open reading frames being homologs to mechanosensitive channels have been deleted in this strain which could lead to a reduced or at least similar e¥ux as shown for CgvyggB. As a control, an independently made clone was analyzed, with the deletions introduced in the reverse order using CgvyggB instead of CgvmscL as parental strain. The e¥ux characteristics of the di¡erently constructed double deletion strains were identical (data not shown), leading to the hypothesis that a third mechanosensitive channel is present in C. glutamicum, which is either regulated in its activity or in its synthesis. For a complementation assay CgvmscLvyggB was transformed either with pEK-mscL or pEK-yggB. Compared to the parental strain the e¥ux of betaine of CgvmscLvyggB/pEK-mscL was only enhanced when the strain was exposed to a severe downshock. The plasmid encoding yggB led to noticeable increased e¥ux capacities, in particular when the cells were exposed to moderate downshocks (data not shown). The physiological role of the mscL and yggB homologs was determined in C. glutamicum by investigating the survival rate after a sudden reduction of the external osmolarity. For this purpose cells adapted to MMI medium (0.3 osmol kg31 ) were exposed to a hyperosmotic upshift in MMI containing 750 mM NaCl (1.9 osmol kg31 ), which

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A

90

90

80

80

70

70

Efflux [%]

Efflux [%]

100

60 50 40

60 50 40

30

30

20

20

10

10

0 2.0

1.8

1.6

1.4

1.2

1.0

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C

0 2.0

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Osmolality of the downshock buffer [osmol/kg]

100

100

B

90

80

80

70

70

Efflux [%]

Efflux [%]

90

60 50 40

20

10

10 1.4

1.2

1.0

0.8

0.6

0.4

1.0

0.8

0.6

0.4

0.2

0.0

0.2

D

40 30

1.6

1.2

50

20

1.8

1.4

60

30

0 2.0

1.6

Osmolality of the downshock buffer [osmol/kg]

0 2.0

0.0

Osmolality of the downshock buffer [osmol/kg]

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Osmolality of the downshock buffer [osmol/kg]

Fig. 1. Dependence of the amount of the excreted betaine on the extent of the downshock after 15 s (closed symbols, solid line) and 60 s (open symbols, dotted line). Cells were preloaded with radiolabeled betaine before they were suspended in bu¡er of the indicated osmolality. A: Wild-type; B: CgvmscL ; C: CgvyggB; and D: CgvmscLvyggB. The dashed line represents the e¥ux rates of the wild-type after 15 s (linear ¢t of the graph in A).

led to the biosynthesis of proline within the next 2 h (from 10 Wmol to 600 Wmol g31 cell dry weight). After 2 h the cells were either suspended in isoosmotic medium or exposed to a downshock by dilution in MMI containing 500 mM, 250 mM or no NaCl. The survival rates (% of living cells) were determined by using £uorescent dyes, which either label damaged cells only or both damaged and intact cells (Table 1). Although the di¡erent deletion strains showed changes in their e¥ux behavior and/or kinetics (cf.

Fig. 1), almost no di¡erence in cell viability was detected under downshocks of di¡erent extents. The slightly higher mortality rates of the double deletion strain in the case of a severe downshock were signi¢cant but by far not as pronounced as in the case of an E. coli double deletion mutant where mortality rates of almost 100% were detected [6]. Taken together the properties of the double deletion mutant indicated that besides MscL and YggB at least

Table 1 Survival rates of C. glutamicum wild-type and mscL and yggB deletion mutants Strain

13032 (wild-type) CgvyggB CgvmscL CgvmscLvyggB

NaCl concentration of the downshock medium 750 mM NaCl

500 mM NaCl

250 mM NaCl

Without salt

100% 100% 100% 100%

88 U 13% 93 U 13% 92 U 10% 89 U 10%

51 U 8% 58 U 9% 44 U 10% 32 U 5%

25 U 1% 26 U 5% 24 U 3% 17 U 3%

The survival rate of cells after dilution in isoosmotic medium (MMI with 750 mM NaCl) was set to 100%.

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one further e¥ux channel is present in C. glutamicum, because the e¥ux kinetics in the double knockout strain were faster than in the single deletion mutant CgvyggB. The additional channel(s) should have a transport capacity similar to MscS or MscL, since the detected e¥ux rates were comparable to the capacity of MscS and MscL (Fig. 1). This assumption is supported by patch-clamp analyses performed recently with fusion vesicles from E. coli liposomes and C. glutamicum membranes where conductances typical for MscL and MscS but no e¥ux activities similar to MscM of E. coli were detected [8]. Furthermore, di¡erent from the situation in E. coli where the double deletion of mscL and yggB is lethal when cells are exposed to an osmotic downshock [6], the remaining channel(s) in C. glutamicum was (were) su⁄cient to protect the cells in vivo. In the C. glutamicum genome no similarity to a known gene encoding an osmoresponsive channel involved in the downshock adaptation besides the here investigated mscL and yggB genes could be found, neither a second homolog of MscL, YggB, KefA nor a homolog of Fps1 (the e¥ux system identi¢ed in yeast). Therefore, future work must reveal whether a channel of an up to now unknown family of mechanosensitive channels is present in C. glutamicum.

Acknowledgements We thank Brigitte Bathe and Bettina Mo«ckel (Degussa AG) for providing the sequences of mscL and yggB and Andreas Tauch (University of Bielefeld) for help with database searches.

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[3] Zoratti, M. and Petronelli, V. (1988) Ion-conducting channels in a Gram-positive bacterium. FEBS Lett. 240, 105^109. [4] Berrier, C., Besnard, M., Ajouz, B., Coulombe, A. and Ghazi, A. (1996) Multiple mechanosensitive ion channels from Escherichia coli, activated at di¡erent thresholds of applied pressure. J. Membr. Biol. 151, 175^187. [5] Sukharev, S.I., Blount, P., Martinac, B., Blattner, F.R. and Kung, C. (1994) A large mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265^268. [6] Levina, N., To«temeyer, S., Stokes, N.R., Louis, P., Jones, M.A. and Booth, I.R. (1999) Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identi¢cation of genes required for MscS activity. EMBO J. 18, 1730^ 1737. [7] Ru¡ert, S., Lambert, C., Peter, H., Wendisch, V.F. and Kra«mer, R. (1997) E¥ux of compatible solutes in Corynebacterium glutamicum mediated by osmoregulated channel activity. Eur. J. Biochem. 247, 572^580. [8] Ru¡ert, S., Berrier, C., Kra«mer, R. and Ghazi, A. (1999) Identi¢cation of mechanosensitive ion channels in the cytoplasmic membrane of Corynebacterium glutamicum. J. Bacteriol. 181, 1673^1676. [9] Grant, S.G.N., Jessee, J., Bloom, F.R. and Hanahan, D. (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc. Natl. Acad. Sci. USA 87, 4645^4649. [10] Simon, R., Priefer, U. and Pu«hler, A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. BioTechnology 1, 784^791. [11] Abe, S., Takayama, K. and Kinoshita, S. (1967) Taxonomical studies on glutamic acid-producing bacteria. J. Gen. Appl. Microbiol. 13, 279^301. [12] Morbach, S., Junger, C., Sahm, H. and Eggeling, L. (2000) Attenuation control of ilvBNC in Corynebacterium glutamicum : Evidence of leader peptide formation without the presence of a ribosome binding site. J. Biosci. Bioeng. 90, 501^507. [13] Scha«fer, A., Tauch, A., Ja«ger, W., Kalinowski, J., Thierbach, G. and Pu«hler, A. (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of de¢ned deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69^73. [14] Eikmanns, B.J., Kleinertz, E., Liebl, W. and Sahm, H. (1991) A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene 102, 93^98. [15] Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. and Ghazi, A. (1998) Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli. J. Biol. Chem. 273, 26670^26674.

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