Compensatory mutations in receptor function: a reevaluation of the role of methylation in bacterial chemotaxis.

Abstract

During bacterial chemotaxis membrane receptor proteins are methylated and demethylated at glutamate residues. The generally accepted view is that these reactions play an essential role in the chemosensing mechanism. Strains may be isolated, however, that exhibit chemotaxis in the complete absence of methylation. These are readily obtained by selecting for chemotactic variants of a mutant that completely lacks the methylating enzyme. Methyltransferase activity is not restored; instead, the sensory-motor apparatus is genetically restructured to compensate for the methylation defect. Genetic and biochemical analyses show that the compensatory mutational locus is the structural gene for the demethylating enzyme. Thus, although mutants lacking either the methylating or demethylating enzymes are nonchemotactic, strains defective in both activities exhibit almost-wild-type chemotactic ability.Escherichia coli and Salmonella typhimurium effect chemotaxis by controlled fluctuations between two modes of motor behavior, smooth swimming and tumbling (1, 2). If a cell swims towards attractants its tendency to tumble is suppressed and movement continues. This response is determined by rates of change in attractant concentration, rather than absolute levels (2, 3). It has been hypothesized that receptor methylation plays a central role in this process. According to this hypothesis, methylated receptors modulate motor behavior in response to imbalances between their levels of modification and their degree of occupancy by stimulatory ligands (4-6). Responses occur whenever the rate of change in receptor occupancy exceeds the rate of change in methylation.Each methylated receptor consists of two domains separated by a transmembrane sequence (7-10). Stimulatory ligands bind to the extracellular domain, and methylation occurs at glutamate residues within the cytosol. Methylation increases with attractants and decreases with repellents. The addition reaction is catalyzed by a specific methyltransferase, the product of the cheR gene (11), and groups are removed by an esterase that is the product of cheB (12 (24) using, as a selectable marker, the tetracycline-resistance gene in a TnlO transposon, zea-2::TnlO (21), located roughly 5 kilobases to the cheZ side of the che operon just beyond flaM and flaC (Fig. 1). For instance, the cheR mutation in ST1038 was transduced into PS1 by first transducing TnlO from a chemotactic (Che') strain, ST314zea-2::TnlO, into ST1038. A cheR-tetracycline-resistant recombinant from this cross was then used to transduce the cheR mutation into PS1. At each stage recombinants were first selected for tetracycline resistance and then screened for chemotactic ability, using semisolid tryptone swarm plates (0.35% Bacto-agar/1.3% tryptone/0.7% NaCl). recA-strains for complementation tests were constructed by P22 transduction using as donor a strain contain-

N-terminal half of CheB is involved in methylesterase response to negative chemotactic stimuli in Escherichia coli

“…This apparently resulted from the ability of these overexpressed catalytically inactive proteins to diminish the extremely tumbly bias of the AcheB host. (The limited ability of cells to swarm in the absence of methylation-mediated adaptation has been discussed by Block et al [3] and by Stock et al [53][54][55]). Effects on swimming behavior were also observed when these mutant proteins were overexpressed in wild-type host cells (that had normal levels of functional, wild-type CheB): the swarming ability of the wild-type host was diminished to 4 / tA b Methylesterase activity and swarming ability (relative to wild-type strain D213) associated with the various forms of CheB were measured by using AcheB host D263 transformed with the indicated plasmids.…”

Purification and characterization of the CheZ protein of bacterial chemotaxis

“…13). Furthermore, complementation and recombination analyses with a set of previously defined deletions of the S. typhimurium che region (46) showed that ST171 both complemented and gave Che+ recombinants with deletions extending through cheZ into che Y but was unable to complement or yield Che+ recombinants with cheB deletions (Table 2). From CheZ appears to be an essential component of the chemotaxis system, although its molecular role is unknown.…”

Methylation-Independent Aerotaxis Mediated by the Escherichia coli Aer Protein

“…Mutants defective in CheR or CheB function readily acquire mutations in other components of the chemotaxis machinery that restore a pseudo-wild swimming pattern and enhance the rate of migration in soft agar (16,44,47). Part of the speed-up effect is due to the phenomenon of pseudotaxis, the ability of cells that both run and tumble to percolate through an agar matrix faster than cells that incessantly run or tumble (5,54).…”

“…Part of the speed-up effect is due to the phenomenon of pseudotaxis, the ability of cells that both run and tumble to percolate through an agar matrix faster than cells that incessantly run or tumble (5,54). However, the faster-spreading pseudorevertant colonies also have a sharp ring at their perimeter suggestive of a chemotactic response (44,(46)(47)(48)54). Stock and colleagues (46,47) have argued that cheR cheB double mutants, which have a wild-type swimming pattern, use a methylation-independent adaptation mechanism to move away from repellent compounds produced by the growing colony.…”

“…However, the faster-spreading pseudorevertant colonies also have a sharp ring at their perimeter suggestive of a chemotactic response (44,(46)(47)(48)54). Stock and colleagues (46,47) have argued that cheR cheB double mutants, which have a wild-type swimming pattern, use a methylation-independent adaptation mechanism to move away from repellent compounds produced by the growing colony. Although controversial, this methylation-independent chemotaxis appears to be a more purposeful behavior than pseudotaxis (cf.…”