Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria

Abstract

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellularly) prevented the inhibition by arsenate.In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.An observation made a number of years ago was that cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium, tethered to glass by their flagella, can be made to rotate clockwise (CW) by inclusion of purified CheY in them (16). At that time this observation was intriguing, because CW rotation in intact bacteria was known to require intracellular ATP (1,3,10,17,20); however, these envelopes rotated CW in the apparent absence of ATP. (When envelopes are prepared, the internal content of the bacterial cell is diluted at the lysis step by more than 104-fold; therefore, their ATP levels are presumably negligible.) To verify that the CW-rotating envelopes indeed did not contain residual ATP, we treated them with arsenate, an effective ATP-depleting agent in intact bacteria (10,17). Surprisingly, arsenate immediately stopped not only the CW-rotating envelopes but also the counter-CW (CCW)-rotating, CheY-free envelopes. The purpose of this study is to discriminate between the potential reasons for the inhibition of flagellar rotation by arsenate. (It should be noted that even though the ATP requirement was later found to be for CheY phosphorylation [8,9,23] addition of the respiratory substrate D,L-lactic acid (2 mM) to the flow medium. Exogenous addition of arsenate (on top of the lactic acid) stopped the rotation of both CCW (Fig. 1) and CW (not shown) envelopes within 2 min. Removal of arsenate from the flow medium caused resumption of rotation, provided that the incubation time with arsenate was relatively short: about 67% of the envelopes resumed their rotation when the incubation time was 5 min or less, but only about 17% did so when the incubation time was 8 min or longer. The inhibitory effect of arsenate was much reduced when phosphate was included within the envelopes (Fig. 1), indicating (i) that arsenate acts in the envelopes as a competitive inhibitor of phosphate and (ii) that its site of action is intracellular. (It should be noted in this regard that E. coli has two primary Pi transport systems: the pst system which is ATP dependent, having a higher specificity for phosphate than for arsenate, and the pit system, which is a proton motive force [PMF]-dependent system having similar affinities for phosphate and arsenate [18]. The pit sys...

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