Sensory transduction in Escherichia coli: two complementary pathways of information processing that involve methylated proteins.

Abstract

The properties of two classes of behavioral mutants of Escherichia coli (called tsr and tar) The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.grade. Nickel and cobalt were used as the sulfate salts; aspartate, acetate, and benzoate, as the sodium salts. Bacteria. Chemotactic mutants AW518 and AW539 were isolated from the chemotactically wild-type Escherschia coli K-12 strain AW405 as previously described (5, 6). The double mutant AW569 was constructed by transferring the chemotaxis defect of AW518 to AW539 via cotransduction with thr from a thr+ derivative of AW518. Behavioral Assays. Bacteria used in capillary assays (7) were grown on minimal medium and washed as described previously (7). Responses were quantitated by integrating the areas under concentration-response curves. Bacteria used in temporal assays (2,8,9) were grown on tryptone broth as described previously (10). Five milliliters of cells were washed three times in 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA (chemotaxis medium) and resuspended in this medium. Temporal assays were quantitated by measuring the duration of the response to stimuli; the response to attractants is a suppression pf tumbling (8), while the response to repellents is an enhancement of tumbling (2).Methylation. Cells were grown and washed as described previously (4). All subsequent incubations were carried out at 300 with rotary shaking. Washed cells were resuspended (to 3 X 108 cells per ml) in chemotaxis medium containing chloramphenicol (200 tg/ml) to inhibit protein synthesis, sodium D,L-lactate (10 mM), and L-threonine, L-leucine, and L-histidine (0.1 mM each). After 5 min of incubation [3H]methionine was added (4 ,tM) and 5-ml aliquots were distributed to various flasks. Samples to be stimulated with attractants or repellents received these compounds 30 or 58 min later, respectively. All reactions were terminated after 60 min of labeling by the addition of 0.25% formalin (final concentration). These times of incubation (30 min for attractants and 2 min for repellents) allow the new equilibrium levels of methylation to be established before termination of the experiment. § Samples were centrifuged to sediment the cells and the pellets were resuspended in 100 ,l of Laemmli sample buffer (11)

Aspartate and maltose-binding protein interact with adjacent sites in the Tar chemotactic signal transducer of Escherichia coli

“…The Tar protein of E. coli mediates attractant responses to both aspartate and maltose (43). Aspartate binds directly to the periplasmic domain of Tar (18,37), but maltose binds to the periplasmic maltose-binding protein (MBP) (10).…”

Relationships between C4 dicarboxylic acid transport and chemotaxis in Rhizobium meliloti

“…E. coli cells treated with cycloleucine behave the same as methioninestarved cells (3). Methionine starvation results in loss of the adaptation response in E. coli and Bacillus subtilis, causing abnormally long periods of smooth swimming and loss of chemotactic responsiveness, as measured by capillary assays (28,41,42). Surprisingly, cycloleucine was shown to be a potent attractant for R. meliloti ( Samples were subjected to SDS-PAGE, and a fluorogram of the gel was prepared.…”

Motility and Energy Taxis of Salmonella spp.

“…(Lee & Imae, 1990;Jeffrey & Koshland, 1993;Oosawa & Imae, 1984;Springer et al, 1977). Tsr functions also as thermoreceptor.…”