Escherichia coli MUTANTS DEFECTIVE IN CHEMOTAXIS TOWARD SPECIFIC CHEMICALS

Abstract

Abstract.-Mutants of Escherichia coli K12 have been found which fail to carry out chemotaxis toward certain chemicals only. One mutant exhibits greatly reduced chemotaxis toward L-serine but has no detectable defect either in uptake or in oxidative metabolism of that compound. Another mutant is not attracted to D-galactose and certain related sugars. There is a correlation between the galactose chemotaxis defect and a defect in galactose uptake, perhaps indicating a common component for chemotaxis and uptake systems. The results are discussed in terms of a model for chemotaxis in which attractants are detected by specific "chemoreceptors."Escherichia coli are chemotactic, that is, they are attracted to certain chemicals.1, 2 A model for bacterial chemotaxis has been proposed2 in which classes of sterically related compounds are recognized by specific "chemoreceptors." Information from all chemoreceptors is channeled through a common pathway to the flagella where a response is initiated. The model predicts two classes of nonchemotactic mutants. One class is defective in the common pathway, reducing chemotaxis to all chemicals ("generally nonchemotactic mutants"). The other has defects in specific chemoreceptors, reducing chemotaxis to certain chemicals ("specifically nonchemotactic mutants"). Generally nonchemotactic mutants of Escherichia coli K12 have been isolated, characterized,3 and studied genetically4' 5 in our laboratory. In this report we describe two specifically nonchemotactic mutants, one defective in taxis to a group of amino acids and the other to a group of sugars. Materials and Methods.-Bacteria: The serine taxis mutant, AW518, was isolated from AW405 (E. coli K12, wild type for chemotaxis, gal-i, gal-2, thr, leu, his-4, thi, lac, xyl, ara, str-r, ton A-r, tsx-r) by means of a selection procedure described previously.3 In this procedure, cells are placed at the center of a tryptone semisolid plate. Rings of chemotactic bacteria swarm out from the center in response to amino acid gradients created by bacterial growth.1 Cells remaining at the center after swarming has occurred are used to inoculate a fresh plate. After several such transfers, the center is enriched in nonflagellated, abnormally flagellated, paralyzed, and nonchemotactic mutants.W3109 (gal-9), a galactose operon extreme polar mutant of E. coli K12 obtained from Dr. E. Lederberg, was found to be a galactose taxis mutant. AW520, a gal+ revertant of W3109 (also lami-r and ton A-r), was used in the experiments described in this paper. To select for revertants to normal galactose taxis, about 109 cells of AW520 (grown, harvested, and washed as in chemotaxis experiments) in 0.1 ml of chemotaxis medium were spread in a streak on a semisolid galactose plate. On such a plate, bacteria chemotactic toward galactose form a dense ring' easily visible after 12 hr. After 3 days at 350C in a wet incubator, chemotaxis rings were faintly visible along the border of the AW520 streak. A single colony isolate, AW521, from one of the rings showed normal c...

Analysis of protein structure in intact cells: Crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor

“…Cells were inoculated from a frozen glycerol stock into 2 mL tryptone broth containing 50 pg/mL ampicillin; incubated overnight with vigorous agitation at 35 "C; inoculated from that culture to 1.0 X IO8 cells/mL in 4 mLHl salts medium (Hazelbauer et al, 1969) containing required amino acids at 0.5 mM, 0.4% sodium succinate, 50 pg/mL ampicillin, 25 pg/mL chloramphenicol (for cells harboring pAI 12), and 50 p M isopropyl P-D-thiogalactoside; incubated as above until reaching 2.5 X 10' cells/mL; inoculated from that culture to 2.5 X IO' cells/mL in the same minimal medium; incubated as above until reaching 2.5 X 10' cells per mL; and then rapidly cooled in an ice slurry to be kept for use that day. For experiments using Cu-phenanthroline, 0.5 rnL cell suspension was placed in a 15 mL beaker, incubated at 35 "C with vigorous agitation to provide generous aeration and after 5 min an equal volume of water (for control cells) at 35 "C or solution of Cuphenanthroline, prepared as described (Lee et al, 1995b), was added.…”

A supplemented soft agar chemotaxis assay demonstrates the Helicobacter pylori chemotactic response to zinc and nickel

“…A widely used assay for H. pylori and other bacteria in general is the spatial soft agar migration assay. This assay analyses movement of the bacterial cells over time in low-percentage-agar media (Hazelbauer et al, 1969;Miller et al, 2009;Wolfe & Berg, 1989). In this assay, bacteria create gradients by nutrient consumption and waste product excretion, and migrate to fresh regions.…”

Control of transducer methylation levels in Escherichia coli: investigation of components essential for modulation of methylation and demethylation reactions

“…During adaptation to attractants, the relevant transducer protein becomes more methylated (22). In response to a step increase in repellent concentration, the bacteria initially tumble incessantly and then return to random behavior; during this tumbling episode the methylation level of the relevant transducer protein decreases (6,22,28).Methylation and demethylation of the transducer proteins are accomplished by the products of the cheR and cheB genes, which encode the chemotaxis-specific methyltransferase and methylesterase, respectively. There are two general mechanisms by which chemotactic stimuli could affect the rates of transducer methylation and demethylation to * Corresponding author.…”

“…During adaptation to attractants, the relevant transducer protein becomes more methylated (22). In response to a step increase in repellent concentration, the bacteria initially tumble incessantly and then return to random behavior; during this tumbling episode the methylation level of the relevant transducer protein decreases (6,22,28).…”