Localization of proteins controlling motility and chemotaxis in Escherichia coli.

Flagellar proteins controlling motility and chemotaxis in Escherichia coli were selectively labeled in vivo with [35S]methionine. This distribution of these proteins in subcellular fractions was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The motA, motB, cheM, and cheD gene products were found to be confined exclusively to the inner cytoplasmic membrane fraction, whereas the cheY, cheW, and cheA (66,000 daltons) polypeptides appeared only in the soluble cytoplasmic fraction. The cheB, cheX, cheZ, and cheA (76,000 daltons) proteins, however, were distributed in both the cytoplasm and the inner membrane fractions. The hag gene product (flagellin) was the only flagellar protein examined that copurified with the outer lipopolysaccharide membrane. Differences in the intracellular locations of the che and mot gene prodcuts presumably reflect the functional attributes of these components.     

Effect of morphine analogues on chemotaxis in Escherichia coli.

Pretreatment of Escherichia coli w3110 with levorphanol, a morphine analogue, reduced chemotaxis to serine, aspartic acid and galactose. This decreased chemotaxis was not due to decreased viability or motility. Pretreatment with 1.1 mM-levorphanol for 1 h, followed by washing to remove the drug prior to determination of chemotaxis, inhibited chemotaxis to each of the attractants by at least 80%. Pretreatment with dextrorphan, the enantiomorph of levorphanol, or levallorphan, the N-allyl analogue of levorphanol, resulted in a similar inhibition of chemotaxis. Reversal of the inhibition produced by pretreatment with levorphanol required a period of growth of at least one generation time.     

Effect of outer membrane permeability on chemotaxis in Escherichia coli.

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.     

Light-scattering study of the temperature dependence of Escherichia coli motility.

Two light-scattering techniques are used to study the temperature dependence of translational and rotational motility in Escherichia coli. The method of number fluctuation spectroscopy is developed theoretically and experimentally to measure the translational swimming speed of a smooth swimming strain of E. coli. Interference fluctuation techniques are used to study the rotational component of the motion. The results demonstrate that the thrust remains proportional the the torque generated by the flagella throughout the range studied and also show that relative changes in translational swimming speed may be inferred from the dynamics of rotational motion.     

Effect of gamma-ray irradiation on Escherichia coli motility

The effects of ionizing radiation on bacteria are generally evaluated from the dose-dependent survival ratio, which is determined by colony-forming ability and mutation rate. The mutagenic damage to cellular DNA induced by radiation has been extensively investigated; however, the effects of irradiation on the cellular machinery in situ remain unclear. In the present work, we irradiated Escherichia coli cells in liquid media with gamma rays from ⁶⁰Co (in doses up to 8 kGy). The swimming speeds of the cells were measured using a microscope. We found that the swimming speed was unaltered in cells irradiated with a lethal dose of gamma rays. However, the fraction of motile cells decreased in a dose-dependent manner. Similar results were observed when protein synthesis was inhibited by treatment with kanamycin. Evaluation of bacterial swimming speed and the motile fraction after irradiation revealed that some E. coli cells without the potential of cell growth and division remained motile for several hours after irradiation.     

Effect of amino acids and oxygen on chemotaxis in Escherichia coli

Adler, Julius (University of Wisconsin, Madison). Effect of amino acids and oxygen on chemotaxis in Escherichia coli. J. Bacteriol. 92:121-129. 1966.-Motile cells of Escherichia coli placed at one end of a capillary tube containing a mixture of the 20 amino acids commonly found in proteins migrate out into the tube in two bands. The bands are clearly visible to the naked eye, and they can also be demonstrated by microscopy, photography, and densitometry, and by assaying for bacteria throughout the tube. The occurrence of more than one band is not due to heterogeneity among the bacteria, since each band can be used over to give rise to two again. The first band uses all the oxygen to oxidize portions of one or more of the amino acids, including serine, and the second band consumes the residual serine anaerobically. The results are interpreted to mean that E. coli shows chemotaxis toward oxygen and serine. When no energy source is added to the medium, a band of bacteria still appears. It consumes all the oxygen to oxidize an endogenous energy source. The addition of any one of 10 oxidizable amino acids stimulates the rate of travel of this band. Alanine, an example that was studied in detail, supports such a band that consumes all the oxygen to oxidize a portion of the alanine. Serine, the only amino acid that this strain can use either aerobically or anaerobically when grown under the conditions used here, gives rise to two bands.     

Negative chemotaxis in Escherichia coli

Several methods for detecting or measuring negative chemotaxis are described. Using these, we have surveyed a number of chemicals for their ability to repel Escherichia coli. Although most of the repellents are harmful compounds, harmfulness is neither necessary nor sufficient to make a compound a repellent. The repellents can be grouped into at least nine classes according to (i) competition experiments, (ii) mutants lacking certain of the negative taxes, and (iii) their chemical structure. The specificity of each class was studied. It is suggested that each class corresponds to a distinct chemoreceptor. Generally, non-chemotactic mutants lack both positive and negative chemotaxis, and l-methionine is required for both kinds of taxis. Repellents at very low concentrations are not attractants, and attractants at very high concentrations are not repellents.     

Studies on bacterial chemotaxis. II. Effect of cheB and cheZ mutations on the methylation of methyl-accepting chemotaxis protein of Escherichia coli.

Radioactive proteins from chemotactic mutants of Escherichia coli with continuous tumbling phenotype (cheB and cheZ) and their otherwise isogenic parent were compared by two-dimensional gel electrophoresis. The system was capable of separating non-methylated methyl-accepting chemotaxis protein (MCP) from its methylated equivalent. The analysis of proteins from the envelope fraction of the bacteria showed that the cheB mutants contained a larger portion of methylated MCP than did the parent. However, the change of MCP methylation level was small, if any, in cheZ strains. The results suggest that the product of cheB gene and the product of cheZ gene are not functional complementary. The product of cheB gene functions in controlling the level of methylation at the stationary state of the organisms. In addition to known MCP species, a new MCP of about 43,000 daltons was found. This MCP appears to be involved in transducing signals of some sugars.     

Effect of temperature on the size of Escherichia coli cells.

The distributions of cell volumes of steady-state Escherichia coli ML30G cultures at various temperatures were measured. For cultures in a minimal medium, the distributions were indistinguishable at several temperatures between 15 and 30 C; at higher temperatures the cells were slightly smaller, and at lower temperatures they were slightly larger. For cultures in a complex medium, the cells were slightly larger at both high and low temperatures of growth. An abrupt change of temperature within the middle range led to a transient change in the distribution of cell volume, suggesting that the size of dividing cells is well regulated. No synchrony of division was induced by a change in temperature.     

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.     

Studies on bacterial chemotaxis. III. Effect of methyl esters on the chemotactic response of Escherichia coli.

The methylation-demethylation reaction of methyl-accepting chemotaxis protein (MCP) is tightly coupled to the appearance of the chemotactic response in Escherichia coli. The bacteria might therefore show a unique response upon the addition of a compound containing a methyl group. We selected methyl N-methyl anthranilate (NMMA) and its analogs for examination. When NMMA was added to a suspension of E. coli (wild type), the bacteria tumbled as it does in the presence of a repellent. NMMA caused tumbling of wild-type bacteria for at least 20 min, while a conventional repellent makes the bacteria tumble for at most one min. The effect of NMMA requires functional MCP, cheA gene product, cheB gene product, and possibly cheX gene product. A positive signal of NMMA (i.e. sudden dilution) was detected by cheZ mutants with much higher sensitivity than that of a conventional repellent, indole, while both signals were rather poorly but equally detected by cheB mutants. These results suggest that the drug is related to the function of cheB gene product, a possible demethylating enzyme of MCP.     

Identification of polypeptides necessary for chemotaxis in Escherichia coli.

Molecular cloning techniques were used to construct Escherichia coli-lambda hybrids that contained many of the genes necessary for flagellar rotation and chemotaxis. The properties of specific hybrids that carried the classical "cheA" and "cheB" loci were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the hybrids redefined the "cheA" and "cheB" regions. Six genes were resolved: cheA, cheW, cheX, cheB, cheY, and cheZ. They directed the synthesis of specific polypeptides with the following apparent molecular weights: cheA, 76,000 and 66,000; cheW, 12,000; cheX, 28,000; cheB, 38,000; cheY, 8,000; and cheZ, 24,000. The presence of another gene, cheM, was inferred from the protein synthesis experiments. The cheM gene directed the synthesis of polypeptides with apparent molecular weights of 63,000, 61,000, and 60,000. The synthesis of all of these polypeptides is regulated by the same mechanisms that regulate the synthesis of flagellar-related structural components.     

Characterization of lambda Escherichia coli hybrids carrying chemotaxis genes.

Molecular cloning techniques were used to construct hybrid Escherichia coli lambda phage and isolate Col E1 factors that carried the cheB region of the E. coli genome. The products of these genes were examined by using a series of deletions in the phage to stimulate specific polypeptide synthesis in ultraviolet-irradiated cells and by using Col factor to program protein synthesis in minicells. Seven flagellar related polypeptides were synthesized. Three of these with apparent molecular weights of 38,000, 28,000, and 8,000 were associated with the cheB region; three polypeptides 63,000, 61,000, and 60,000 were associated with the region that maps between cheB and cheA. These bands were referred to as the triplet group. We suggest that these polypeptides are the same as the methyl-accepting chemotaxis protein described by Kort et al. (Proc. Natl. Acad. Sci. U.S.A. 72:3939-3943, 1975). Another polypeptide with a molecular weight of 12,000 is associated with the cheA region which also produces at least three gene products. We conclude that the cheA-cheB region in E. coli is complex. Further genetic and biochemical analyses are required to describe all of these products.     

Unidirectional motility of Escherichia coli in restrictive capillaries.

In a 6-microns capillary filled with buffer and in the absence of any chemotactic stimuli, Escherichia coli K-12 cells swim persistently in only one direction. This behavior of E. coli can be simply explained by means of the length and relative rigidity of their flagella. Single-cell motility parameters--swimming speed, turn angle, and run length time--were measured. Compared with the motility parameters measured in bulk phase, turn angle was influenced because of the effect of the geometrical restriction.     

Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions.

The major cotransduction gap of the Escherichia coli chromosome extends from mini 31 to 34. We have inserted transposons through this gap which, by sequential transduction, link sbcA (min 29.8) with manA (min 35.7) and thus eliminate the gap. These results indicate that the length of DNA in the region, as measured by transduction, is not significantly different from the length obtained by conjugational time of entry. Since this segment of the E. coli chromosome has few known genes, these transposon insertions will be useful for genetic manipulations in the region of the gap. We describe the usefulness of these markers for rapidly mapping mutations which may be isolated in the region from min 27 to 37.     

Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coli. Among other nitroso compounds tested only N-methyl-N′-nitro-N-nitrosoguanidine (MNG) was an active mutagen in the system used.