Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zulkifli, L. Articles by Uozumi, N. Search for Related Content PubMed PubMed Citation Articles by Zulkifli, L. Articles by Uozumi, N.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2006, p. 7985-7987, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00886-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mutation of His-157 in the Second Pore Loop Drastically Reduces the Activity of the Synechocystis Ktr-Type Transporter

Lalu Zulkifli and Nobuyuki Uozumi^*

Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan

Received 20 June 2006/ Accepted 4 September 2006

Top Abstract Text References

 
Mutation of a conserved His-157 in the second pore loop of KtrB drastically reduced the activity of the K⁺ transporter from Synechocystis sp. strain PCC 6803. This result suggests that His-157 plays an essential role in the K⁺ transport activity of the transporter system.

Top Abstract Text References

 
The Ktr/Trk/HKT superfamily of K⁺ transporters, which is likely to have evolved from two membrane-spanning K⁺ channels, has a fourfold-repeated membrane-pore-membrane motif (Fig. 1A) (2, 6, 8). KtrB is the K⁺-translocating subunit of the Ktr system from Synechocystis sp. strain PCC 6803 (9) and Vibrio alginolyticus (10). The Ktr system mediates K⁺ uptake in Synechocystis sp. strain PCC 6803. It consists of three kinds of subunits, the transmembrane KtrB subunit and the peripheral KtrA and KtrE subunits (9). Previous studies have shown that the Ktr system is essential for the adaptation of Synechocystis sp. strain PCC 6803 to salinity stress and high osmolality, and in its transport activity is dependent on the proton motive force (9). Synechocystis KtrB possesses two histidines, one located at the extracellular and the other in the intracellular space. The extracellular His-157 in the second pore loop (P_B) is well conserved among Ktr-related proteins of prokaryotic origin (Fig. 1B). In plant K⁺ channels from Solanum tuberosum (KST1) and Arabidopsis thaliana (AKT3), the histidines located at the extracellular space were identified as a determinant for pH dependency (4, 5). The presence of the conserved extracellular His prompted us to investigate the role of His-157 in the activity of Synechocystis Ktr.

View larger version (40K): [in this window] [in a new window]

FIG. 1. Alignment and position of His-157 in the PB of the KtrB subunit of the Synechocystis KtrABE system. (A) Predicted topology of Synechocystis KtrB. His-157 and Arg-149 are located in PB. (B) Amino acid sequence alignment of PB of KtrB and KtrB-related proteins. SynKtrB, Synechocystis sp. strain PCC 6803 KtrB (10); VibKtrB, Vibrio alginolyticus KtrB (11); EcoTrkH, Escherichia coli TrkH (12); EcoTrkG, Escherichia coli TrkG (12). Other KtrB-related proteins found in the databases (by use of NCBI BLAST) that have not been functionally characterized yet are the following: tll0303, Thermosynechococcus elongatus BP-1; Ava_4805, Anabaena variabilis ATCC 29413; BC_1310, Bacillus cereus ATCC 14579; Dace_2354, Desulfuromonas acetoxidans DSM 684; CHY_1575, Carboxydothermus hydrogenoformans Z-129; TTE0196, Thermoanaerobacter tengcongensis MB4; STH1096, Symbiobacterium thermophilum IAM14863; DR_1668, Deinococcus radiodurans R1; FN1725, Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

First, we generated three His variants (H157A, H157E, and H157K) by the overlap extension PCR technique (11). In addition, since another positive residue, Arg-149, is present in P_B, an R149E variant was constructed to compare the transport activities of the His variants. The PCR products were inserted into pPAB404, and mutations were confirmed by DNA sequencing. Native and variant forms were expressed in Escherichia coli LB2003 harboring ktrA and ktrE. E. coli LB2003, which lacks all three K⁺ uptake systems, was used to verify the K⁺ transport activity of KtrB (14).

We first examined the effect of the histidine mutation on the activity of KtrB by performing a complementation test and a K⁺ uptake assay to determine the kinetic parameters. The K⁺ uptake assay experiment was conducted by the silicone oil filtration technique, and the K⁺ content of the cell pellets was determined by flame photometry (13). In medium supplemented with 7.5 mM KCl, the R149E variant showed better growth than the three His variants did (Fig. 2A). In control experiments, the growth test was carried out using the medium containing K₂SO₄ instead of KCl (Fig. 2A). The growth profile was consistent with that for growth in the medium containing KCl. The control experiments indicate that the depressed growth of the His variants at 7.5 mM KCl was due to K⁺ deprivation but not due to Cl^–. The initial net K⁺ uptake of the His variants was significantly lower, i.e., more than 10-fold and more than 5-fold lower than that for the wild type (WT) and that for the R149E variant, respectively (Table 1) . The V_max value data indicated that replacement of His-157 by Ala, Glu, or Lys resulted in a significant reduction in the K⁺ uptake activity of the variants (Table 2). The apparent K_m values for K⁺ for the three His variants at pH 7.5 exhibited an increase by a factor of at least 24-fold compared to the wild type. The kinetic parameters among the His variants at pH 7.5 were not significantly different (Table 2). These results show that His-157 had a role more crucial than that of Arg-149 and was irreplaceable at that position by the other residues to restore the optimum activity seen in the wild type.

View larger version (45K): [in this window] [in a new window]

FIG. 2. Growth test of E. coli LB 2003. (A) Growth test of E. coli LB 2003 harboring WT KtrB or H157A, H157E, H157K, or R149E variants on synthetic solid medium containing 7.5 mM or 30 mM KCl, 10 mM HEPES-NaOH (pH 7.5). Control experiments using K2SO4 (3.75 and 15 mM) are shown. (B) Growth test of E. coli LB 2003 harboring WT or His variants at various pH values on synthetic solid medium containing 10 mM 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH (pH 5.5 and 6.5), 10 mM HEPES-NaOH (pH 7.0, 7.5, and 8.0), or 10 mM Tricine-NaOH (pH 8.5) at different KCl concentrations. Photographs were taken after overnight incubation at 30°C.

View this table: [in this window] [in a new window]

TABLE 1. Initial velocities of K+ uptake

View this table: [in this window] [in a new window]

TABLE 2. Kinetic parameters for the K+ uptake by E. coli LB2003 containing WT or variant KtrB

Next, we determined the effect of the histidine mutation on K⁺ uptake activity at various pH values through complementation testing and determination of kinetic parameters. The functional complementation test using the E. coli LB2003 mutant showed that the wild type grew well at all pH values of the medium, indicating that the Ktr system from Synechocystis sp. strain PCC 6803 could operate in a wide range of pH values when expressed in E. coli. Growth test data for liquid culture also supported this result (data not shown). His variants could not rescue the mutation of the E. coli mutant LB2003 on acidic medium (Fig. 2B). The lack of complementation by His variants might be due to its low K⁺ uptake activity, which can lead to severe inhibition of growth. Escherichia coli cannot maintain an internal pH of more than 2 units higher than the external pH (3). Under acidic growth conditions, cytoplasmic accumulation of K⁺ and proton extrusion are involved in cytoplasmic pH regulation (1, 7). This result showed the physiological role of the K⁺ uptake system in the acidic pH condition. Thus, in this respect, the accumulation of K⁺ through the high activity of wild-type KtrB supported the hypothesis for the regulation of internal pH homeostasis for normal growth. The lower activity of His variants, which did not compensate for this, led to the cessation of metabolic pathways and cell growth in medium with a lower pH (Fig. 2B).

Changes in the kinetic parameters of the wild type at various pH values were observed. Decreases in affinity to K⁺ and V_max were observed when the external pH of the buffer used for the uptake assay was decreased or increased from the optimum pH (pH 7.5) (Table 2). A marked decrease in V_max was observed at pH 5.5 (Table 2). The kinetic parameter results for WT at various pH values showed that the activity of the transporter is pH dependent. To test whether His-157 is involved in the pH response, we determined the kinetic parameters of the His variants at different pH values. The apparent K_m and V_max values could be obtained only at pH 7.0 and 8.0 from the three His variants, and the values did not show significant difference from those obtained at pH 7.5 (Table 2). The involvement of His-157 in the pH response of transporter KtrB was not found.

Atomic-scale models of Ktr transporters proposed by Durell and Guy have shown that His-157, together with Phe-156 and Ser-158, faced the transport pathway (2). The three residues are likely to be placed in juxtaposition to Gly-166, which is a constituent of the selectivity filter in P_B (2, 8). The replacement of His-157 by Ala, Glu, or Lys might affect the proper conformation required for optimal activity.

 
This work was supported by grants-in-aid for scientific research (17380064 and 17078005 to N.U.) and by the 21st Century COE Program from MEXT and JSPS and the Institute for Advanced Research Project Funds from Nagoya University.

 
* Corresponding author. Mailing address: Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. Phone: 81-52-789-5202. Fax: 81-52-789-5206. E-mail: uozumi{at}agr.nagoya-u.ac.jp.

Published ahead of print on 15 September 2006.

Top Abstract Text References

 

1
1. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49:359-378.[Free Full Text]
2
2. Durell, S. R., and H. R. Guy. 1999. Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K⁺. Biophys. J. 77:789-807.[Medline]
3
3. Foster, J. W. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2:898-907.[CrossRef][Medline]
4
4. Geiger, D., D. Becker, B. Lacombe, and R. Hedrich. 2002. Outer pore residues control the H⁺ and K⁺ sensitivity of the Arabidopsis potassium channel AKT3. Plant Cell 14:1859-1868.[Abstract/Free Full Text]
5
5. Hoth, S., P. Dreyer, P. Dietrich, D. Becker, B. Muller-Rober, and R. Hedrich. 1997. Molecular basis of plant-specific acid activation of K⁺ uptake channels. Proc. Natl. Acad. Sci. USA 94:4806-4810.[Abstract/Free Full Text]
6
6. Kato, Y., M. Sakaguchi, Y. Mori, K. Saito, T. Nakamura, E. P. Bakker, Y. Sato, S. Goshima, and N. Uozumi. 2001. Evidence in support of four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na⁺/K⁺ translocating AtHKT1, a member of the superfamily of K⁺ transporters. Proc. Natl. Acad. Sci. USA 98:6488-6493.[Abstract/Free Full Text]
7
7. Kobayashi, H., H. Saito, and T. Kakegawa. 2000. Bacterial strategies to inhibit acidic environments. J. Gen. Appl. Microbiol. 46:235-243.
8
8. Mäser, P., Y. Hosoo, S. Goshima, T. Horie, B. Eckelman, K. Yamada, K. Yoshida, E. P. Bakker, A. Shinmyo, S. Oiki, J. I. Schroeder, and N. Uozumi. 2002. Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants. Proc. Natl. Acad. Sci. USA 99:6428-6433.[Abstract/Free Full Text]
9
9. Matsuda, N., H. Kobayashi, H. Katoh, T. Ogawa, L. Futatsugi, T. Nakamura, E. P. Bakker, and N. Uozumi. 2004. Na⁺-dependent K⁺ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phase of cell adaptation to hyperosmotic shock. J. Biol. Chem. 279:54952-54962.[Abstract/Free Full Text]
10
10. Nakamura, T., R. Yuda, T. Unemoto, and E. P. Bakker. 1998. KtrAB, a new type of bacterial K⁺-uptake system from Vibrio alginolyticus. J. Bacteriol. 180:3491-3494.[Abstract/Free Full Text]
11
11. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
12
12. Schlösser, A., M. Meldorf, S. Stumpe, E. P. Bakker, and W. Epstein. 1995. TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli. J. Bacteriol. 177:1908-1910.[Abstract/Free Full Text]
13
13. Tholema, N., E. P. Bakker, A. Suzuki, and T. Nakamura. 1999. Change to alanine of one out of four selectivity filter glycines in KtrB causes a two magnitude decrease in the affinities for both K⁺ and Na⁺ of the Na⁺ dependent K⁺-uptake system KtrAB from Vibrio alginolyticus. FEBS Lett. 450:217-220.[CrossRef][Medline]
14
14. Uozumi, N. 2001. Escherichia coli as an expression system for K⁺ transport systems from plants. Am. J. Physiol. Cell Physiol. 281:C733-C739.[Abstract/Free Full Text]

---

Journal of Bacteriology, November 2006, p. 7985-7987, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00886-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zulkifli, L. Articles by Uozumi, N. Search for Related Content PubMed PubMed Citation Articles by Zulkifli, L. Articles by Uozumi, N.