Properties of K⁺/H⁺ Antiporter in Alkaliphilic Bacillus sp. No.66 (JCM 9763)

　

Makio KITADAa, Shinya MOROTOMIa,b*, Koki HORIKOSHIb,and Toshiaki KUDOa

　

a Microbiology Laboratory, The Institute of Physical and Chemical Research(Riken), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan

b Bioengineering Laboratory, Department of Applied Chemistry, Toyo University, Kawagoe, Saitama 350, Japan

　

Investigations of how pH homeostasis is achieved in various bacterial species have focused on the role of Na⁺/H⁺ antiporter or K⁺/H⁺ antiporter in the cell membrane. It has been suggested that a Na⁺/H⁺ antiporter is involved in regulation of intracellular pH in alkaline environments. In our previous reports (1), we reported that a ΔΨ-dependent Na⁺/H⁺ antiporter played a crucial role for pH homeostasis in an alkaline range in alkaliphilic Bacillus species. During studies on ΔΨ-dependent /H⁺ antiporter in Bacillus sp. No.66 (JCM9763), however, we detected uphill H⁺ influx into the right-side-out vesicles produced by an outwardly directed K⁺ gradient without imposition of ΔΨ (2). This was the K⁺/H⁺ antiport system which was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp. The present work was conducted to characterize the K⁺/H⁺ antiporter in membrane vesicles of this strain. We also studied its kinetic properties and the reverse reaction.

An outwardly directed K⁺ gradient stimulated uphill H⁺ influx into right-side-out vesicles and created the inside acidic ΔpH. This H⁺ influx was pH dependent and increased as pH increased from 6.8 to 8.4. Addition of 100 mM quinine inhibited the H⁺ influx by 75%. This exchange process was electroneutral, and the H⁺ influx was not stimulated by the imposition of the membrane potential (interior negative). Addition of K⁺ at the point of maximum ΔpH caused a rapid K⁺-dependent H⁺ efflux consistent with the inward exchange of external K⁺ for internal H⁺ by a K⁺/H⁺ antiporter. Rb⁺ and Cs⁺ could replace K⁺ but neither Na⁺ nor Li⁺ could. The H⁺ effiux rate was a hyperbolic function of K⁺ and increased with increasing extravesicular pH (pH_out) from 7.5 to 8.5. Furthermore, this K⁺/H⁺ antiporter in this strain could contribute to acidification of the vesicle interior at an alkaline region. These findings were consistent with the presence of K⁺/H⁺ antiport activity in these membrane vesicles.

　

1. Kitada, M., Hashimoto, M., Kudo T., and Horikoshi, K. (1994) J. Bacteriol., 176, 6464-6469.

2. Kitada, M., Morotomi, S., Horikoshi, K., and Kudo, T. (1997) Extremophiles, 1,135-141.

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