Julius Adler

The Effect of Environmental Conditions on the Motility of Escherichia coli

SUMMARY: A simple chemically defined medium for examining the motility of Escherichia coli K12 was designed. The essential components were: (1) a chelating agent to protect the motility against inhibition by traces of heavy metal ions; (2) a buffer to keep the pH value at the optimum between pH 6·0 and 7·5; (3) an energy source to stimulate the motility above that allowed by an endogenous energy source. Oxygen was required unless an energy source was provided which yielded energy anaerobically. A temperature optimum was determined. A chemically defined growth medium capable of producing motile bacteria was devised. It was found that the presence of glucose or growth above 37° prevented synthesis of flagella.

Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels

We have modified the procedure of Criado and Keller (1987) to study ion channels of Escherichia coli reconstituted in liposomes. The modifications include (a) excluding the use of any detergent and (b) inducing blisters from liposomes with Mg2+. These blisters, which appear to be unilamellar, are stable for hours. They could be repeatedly sampled with different patch-clamp pipettes each achieving seal resistance greater than 10 GOhms. Activities of three types of ion channels are often observed by use of this method, including two voltage-sensitive cation channels of different conductances. Even the mechanosensitive channel, previously recorded from live E. coli cells (Martinac et al., 1987), was also detected in these blisters. Apparently the channel protein and any accessory structures, postulated to be needed for mechanotransduction, can be reconstituted together by this method.

A Method for Measuring the Motility of Bacteria and for Comparing Random and Non-random Motility

SUMMARY: The motility of Escherichia coli was measured in capillary tubes by determining the distribution of bacteria throughout the tube (the complete assay) or simply by locating the point of furthest advance of the bacteria—the frontier of the migration (the frontier assay). The diffusion of 14C-glucose was similarly measured in capillary tubes by determining the distribution of radioactivity throughout the tube. The diffusion of glucose under the conditions used was correctly described by the known diffusion equation. The method gives a measure of the net forward velocity of the bacteria. Interpretations, advantages and disadvantages of these assays are given. The method also gives a measure of the degree of randomness of the motility. When chemotaxis is taking place, a very high proportion of the bacteria leave the origin and migrate as a band. When bands are not allowed to form, by omitting methionine, the motility of the bacteria qualitatively resembled a random process, such as the diffusion of glucose.

Evolutionary Conservation of Methyl-Accepting Chemotaxis Protein Location in Bacteria and Archaea

The methyl-accepting chemotaxis proteins (MCPs) are concentrated at the cell poles in an evolutionarily diverse panel of bacteria and an archeon. In elongated cells, the MCPs are located both at the poles and at regions along the length of the cells. Together, these results suggest that MCP location is evolutionarily conserved.

Decision-Making in Bacteria: Chemotactic Response of Escherichia coli to Conflicting Stimuli

Motile bacteria presented simultaneously with both attractant and repellent respond to whichever one is present in the more effective concentration. Apparently bacteria have a processing mechanism that compares opposing signals from the chemoreceptors for positive and negative taxis, sums these signals up, and then communicates the sum to the flagella.

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca2+ and glutamate−. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.

Fusion of bacterial spheroplasts by electric fields

Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size.

Motility and Chemotaxis of Filamentous Cells of Escherichia coli

Filamentous cells of Escherichia coli can be produced by treatment with the antibiotic cephalexin, which blocks cell division but allows cell growth. To explore the effect of cell size on chemotactic activity, we studied the motility and chemotaxis of filamentous cells. The filaments, up to 50 times the length of normal E. coli organisms, were motile and had flagella along their entire lengths. Despite their increased size, the motility and chemotaxis of filaments were very similar to those properties of normal-sized cells. Unstimulated filaments of chemotactically normal bacteria ran and stopped repeatedly (while normal-sized bacteria run and tumble repeatedly). Filaments responded to attractants by prolonged running (like normal-sized bacteria) and to repellents by prolonged stopping (unlike normal-sized bacteria, which tumble), until adaptation restored unstimulated behavior (as occurs with normal-sized cells). Chemotaxis mutants that always ran when they were normal sized always ran when they were filament sized, and those mutants that always tumbled when they were normal sized always stopped when they were filament sized. Chemoreceptors in filaments were localized to regions both at the poles and at intervals along the filament. We suggest that the location of the chemoreceptors enables the chemotactic responses observed in filaments. The implications of this work with regard to the cytoplasmic diffusion of chemotaxis components in normal-sized and filamentous E. coli are discussed.

Ion channel activities in the Escherichia coli outer membrane

The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.

Voltage-sensitive ion channel of Escherichia coli.

SummaryA voltage-sensitive, cation-selective ion channel ofEscherichia coli has been reconstituted into liposomes and studied with the patch-clamp method. The single channel conductance was 91 pS in symmetric solutions of 150mm KCl. Many channels were open most of the time, with frequent brief transitions to closed levels. Multiple conducting units could close and reopen simultaneously, and this apparent cooperativity in gating was increases with depolarizing voltages. Above a voltage threshold, the channels closed irreversibly, often in groups.