Stabilizing Effect of Colicin E2 on the “Spheroplast Membrane”

Cells of Escherichia coli W3110 and its thymineless mutant, both of which are colicin E2 sensitive, were treated with colicin E2, and then converted to spheroplasts. These spheroplasts seemed to be more stable than those from untreated cells; suspensions of spheroplasts of untreated cells were lysed spontaneously and the turbidity was reduced by approximately 45% on incubation with ethylenediaminetetraacetic acid-lysozyme, whereas suspensions of spheroplasts of colicin E2-treated cells showed 25% reduction in turbidity. This change was irreversible and 5 min of treatment with colicin E2 at 37 C was necessary for stabilization. This process was inhibited by 2,4-dinitrophenol or streptomycin. Cells harboring the colicin E2 factor were not affected by treatment in this way with colicin E2. Alteration of composition of phospholipids was not observed.     

Action of penicillin onSerratia marcescens

The action of penicillin onSerratia marcescens was studied. In culture media containing sucrose (0.33m) and in the presence of magnesium ions, cell wall lesions occurred giving rise to osmotically fragile spheroplasts. However, in the absence of sucrose and magnesium ions it was still possible to induce some spheroplast formation. Quantitative aspects of the conversion of rods into spheroplasts were studied as well as physical properties of the spheroplasts.     

Identification of the plasmid-encoded immunity protein for colicin El in the inner membrane of Escherichia coli

A set of plasmids containing portions of the Col El plasmid were transformed into recA⁻ cells. These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins. UV-irradiated cells label a 14.5 kDa band if they are phenotypically immune to colicin E1, and do not contain this band if they are sensitive to colicin E1. We conclude that the 14.5 kDa protein is the colicin E1 immunity protein. When the inner and outer membranes of these cells are fractionated, the labelled band appears in the inner membrane. The immunity protein must be an intrinsic inner membrane protein, confirming the predictions made by hydrophobicity calculations from primary sequence data.MaxicellCol El plasmidImmunity proteinHydrophobicity calculation     

Spheroplast of Acetic Acid Bacteria

A procedure, preparing spheroplast of acetic acid bacteria, was established to elucidate the membrane structure of the organisms. Of the acetic acid bacteria, only Acetobacter aceti cells were converted into spheroplasts by the sucrose-EDTA-lysozyme system. To Gluconobacter suboxydans, a method exchanging sucrose in the system for NaCl was indispensable. This NaCl-EDTA-lysozyme system was adequate for almost all acetic acid bacteria, which were converted efficiently into spheroplasts. The existence of EDTA was not essential to the genus Gluconobacter.     

Interaction of 125I-Labeled Colicin E1 with Escherichia coli

Colicin E1 protein was labeled with ¹²⁵I to specific activities of up to 2 × 10⁸ cpm/mg of protein and with no loss of the colicin biological activity. The labeled colicin bound to colicin E1-sensitive, tolerant, and immune E1-colicinogenic Escherichia coli. An E. coli mutant resistant to colicin E1 exhibited a much lower colicin-binding capacity. The average number of bound colicin molecules per sensitive cell increased as a function of the colicin concentration in the colicin cell interaction mixture and continued to increase even after loss of viability of the entire culture. Up to 2,400 colicin E1 molecules bound per cell, but saturation was not reached. Binding kinetics showed that maximum binding occurred within 2 to 5 min of colicin addition. Survival and binding assays indicated that one colicin killing unit corresponded to an average of about 100 colicin molecules bound per bacterial cell. This number, however, decreased to about 8 in more extensively washed cells. Trypsin digestion of the colicin-treated cells removed the majority of the cell-bound colicin, but in general provided little rescue from colicin killing. At low colicin concentrations, a linear relationship existed between survival and the number of trypsin-inaccessible colicin molecules. Under these circumstances and in agreement with single-hit kinetics, the relationship between the number of colicin killing units and the number of trypsin-inaccessible colicin molecules was close to 1. After trypsin digestion, cells that were nearly saturated with colicin retained about 200 trypsin-inaccessible colicin molecules per cell. The trypsin-inaccessible colicin might represent those colicin molecules that bound to the specific E colicin receptors of E. coli cells.     

EVIDENCE FOR MECHANOSENSITIVE TRANSMEMBRANE ION CHANNELS OF SMALL CONDUCTANCE IN MAGNETOTACTIC BACTERIA

To determine whether or not mechanosensitive (MS) ion channels are present in the magnetotactic bacterium Magnetospirillum magnetotacticum, techniques for spheroplast preparation in Escherichia coli were adapted for this bacterium. This resulted in the formation of 2–3-μm spheroplasts, which were used for patch clamp analysis. Ion channel activity in M. magnetotacticum was compared with that of the MS of small conductance (MscS) in E. coli. This comparison reveals the presence of MscS-like channels in M. magnetotacticum and, as this bacterium produces intracellular magnetite (Fe₃O₄) particles similar to those found in the human brain, provides a model for investigation of the effects of magnetic fields on MS ion channels in magnetite-bearing cells.     

Molecular characterisation of the colicin E2 operon and identification of its products

Summary The DNA sequence of the entire colicin E2 operon was determined. The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells. A potential LexA binding site is located immediately upstream from ceaB, and a rho-independent terminator structure is located immediately downstream from celB. A comparison of the predicted amino acid sequences of colicin E2 and cloacin DF13 revealed extensive stretches of homology. These colicins have different modes of action and recognise different cell surface receptors; the two major regions of heterology at the carboxy terminus, and in the carboxy-terminal end of the central region probably correspond to the catalytic and receptor-recognition domains, respectively. Sequence homologies between colicins E2, A and E1 were less striking, and the colicin E2 immunity protein was not found to share extensive homology with the colicin E3 or cloacin DF13 immunity proteins. The lysis proteins of the ColE2, ColE1 and CloDF13 plasmids are almost identical except in the aminoterminal regions, which themselves have overall similarity with lipoprotein signal peptides. Processing of the ColE2 prolysis protein to the mature form was prevented by globomycin, a specific inhibitor of the lipoprotein signal peptidase. The mature ColE2 lysis protein was located in the cell envelope. The results are discussed in terms of the functional organisation of the colicin operons and the colicin proteins, and the way in which colicins are released from producing cells.     

A Function of 40 kDa Outer Membrane Protein in Serratia marcescens

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium-binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.     

A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from escherichia coli

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca²⁺ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (>98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cell, and in investigating envelope-related synthesis and assembly processes.     

Lipopolysaccharide Defective Mutant of Escherichia coli with Decreased Sensitivity to Colicin E2

Several colicin-sensitivity mutants were isolated from Escherichia coli K-12. The mutants could not form colonies in the presence of colicin E2, but recovered their colony-forming ability on trypsin treatment even after prolonged incubation with the colicin. They showed increased sensitivity to hydrophobic antibiotics and detergents, as well as resistance against P1 and T4 phages, both of which seemed due to structural changes of lipopolysaccharide (LPS). Quantitative analysis by gas-liquid chromatography revealed that the mutant-LPS contained a different stereoisomer of heptose with decreased amounts of neutral sugars (rhamnose, glucose and galactose). LPS extracted from the parental colicin-sensitive strain could neutralize the killing activity of colicin E2 in vitro, but the mutant-LPS could not. The mutant strains retained functional receptor proteins for colicin E2. These observations suggest that LPS plays an important role in the early stage of the interaction of colicin E2 with E. coli cells.     

Genetics of colicin E susceptibility inCitrobacter freundii

The insensitivity ofCitrobacter freundii to the E colicins is based on tolerance to colicin E1 and resistance to colicins E2 and E3. Spontaneous colicin A resistant mutants ofC. freundii also lost their colicin E1 receptor function. Sensitivity to colicin E1 can be induced by F′gal ⁺ tol ⁺ plasmids, thetol A⁺ gene product of which is responsible for this effect. Receptor function for colicins E2 and E3 is induced by theE. coli F′14bfe ⁺ plasmid, which is also able to enhance notably the receptor capacity for colicin E1. Thebfe ⁺ gene product ofE. coli, which is responsible for these phenomena, also restores the receptor function for colicin A and E1 in colicin A resistant mutants ofC. freundii. All results show that there is a remarkable difference between theE. coli bfe ⁺ gene product and thebfe ⁺ gene product ofC. freundii and also between thetol A⁺ gene products of these strains. The sensitivity to phage BF23 parallels the sensitivity to colicins E2 and E3 and is also induced by the F′14bfe ⁺ plasmid.     

Characterization of the cea gene of the ColE7 plasmid.

Summary The complete nucleotide sequence (1731 nucleotides) of the gene encoding colicin E7 (cea) of plasmid CoIE7-K317 was determined. This sequence encoded a deduced polypeptide of 576 amino acids of molecular weight 61349 Da. Comparison of the nucleotide and amino acid sequences ofcea E7 with those of other E-group colicins revealed that colicin E7 was closely related to colicin E2, both in gene sequence and in predicted secondary structure of the deduced protein. Judging from the results of cross-immunity tests, we postulated that CoIE7 is probably a proximate ancestor of Co1E2 and Co1E8. Based on results from colicin production tests on cells harboring a 5 end deleted form of thecea E7 gene, we propose, that a previously unknown, non-inducible promoter may be involved in regulation of the constitutive expression of thecea E7 gene.     

Binding domains of colicins E1, E2 and E3 in the receptor protein btub ofEscherichia coli

Eight reagents specifically modifying amino acids were applied to cells of a standardEscherichia coli colicin indicator strain to followin vivo changes of its binding capacity for colicins E1–E3 and hence the binding domains (epitopes) for them in the outer membrane receptor protein BtuB. The effect of these reagents was also investigated in a mutant strain carrying an extensive BtuB deletion. The following differences of the binding epitopes could be ascertained.Colicin E1: Blockage of OH-groups, just as N-substitution of His and modification of Arg and Trp enhance binding of colicin E1. In the deleted receptor, also abolition of carboxylic anion bonds enhances its affinity for colicin E1. It follows that colicin E1 is bound, most of all, to the hydrophobic domain A (loops 1+2) of BtuB.Colicins E2 and E3: both exert rather analogous binding parameters. In contrast to E1, O-substitution of Ser and Thr dramatically decreases the E2 and E3 binding, similarly to modification of Lys. There is also a clear difference in the binding affinity of the domain for E2 and/or E3 and for E1 following modifications of their Arg and His. Colicins E2 and E3 are rather bound to the hydrophilic domain B (loops 5–7) of the receptor. In this respect, interactions of colicins E2 and E3 with deeper parts of A and B domains (Trp, several Arg, Lys and His residues) exhibited subtle differences. Acidic pH (4.5–6.0) shows a positive, while pH 7.0–8.5 a rather negative impact on the receptor-binding function for the colicins. It was clearly demonstrated that there is just a partial difference between the binding behavior of colicins E1, E2 and/or E3.     

Formation and regeneration ofHalobacterium spheroplasts

Spheroplasts ofHalobacterium cutirubrum were formed upon suspension of cell pellets in 0.1M MES buffer, pH 7.0, containing 0.5M sucrose, 0.25M NaCl, and 0.01M MgCl₂. The spheroplasts regenerated into rod-shaped bacteria when plated on a complex medium containing 15% (wt/vol) sucrose, undergoing several divisions as spherical bodies before the rod shape developed. The frequency of regeneration was approximately 5% of the total spheroplasts plated. The yield of regenerants was increased significantly (to approximately 35%) when bovine serum albumin was present in the spheroplasting buffer and dilution media. The conditions for spheroplast formation and regeneration inH. cutirubrum were also found effective forHalobacterium salinarium but not forHalobacterium halobium.NRCC Paper no. 23080.     

SPHEROPLAST FORMATION AND ASSOCIATED ULTRASTRUCTURAL CHANGES IN A SYNCHRONOUS CULTURE OF ANACYSTIS NIDULANS TREATED WITH LYSOZYME1,2

Cells of Anacystis nidnlans were grown in synchronous culture using a light-dark alternation to obtain synchronization. Two synchronous cycles were obtained with, decay of synchrony beginning with the third cycle. Cells of various ages in the growth cycle were treated with lysozyme to form spheroplasts. The percentage of spheroplast formation varied with age of the cells. After extended periods of lysozyme treatment, up to 90% of the cells of all ages showed spheroplast formation. Some cells were resistant to the action of lysozyme regardless of age or length of treatment. An ultrastructure study of the spheroplast was made. The electron-dense inner layer of the cell wall was removed by the action of lysozyme on the glucosamine residues of the cell wall, indicating true spheroplast formation. The photosynthetic apparatus became more pronounced with extended treatment with lysozyme.     

Interstrain spheroplast fusion of Alternaria alternata

Summary Auxotrophic and drug resistant colonies of Alternaria alternata were selected following UV mutagenesis of spheroplasts and genetic transformation with pDH25. Intrastrain cell fusion of certain A. alternata parental strains induced by polyethylene glycol occurred at an average rate of 0.35%; interstrain fusions occurred at a rate of 0.08%. Mitotic recombination resulted from UV mutagenesis of spheroplasts from several fusants from 6hy1 × 1ar1. Fusants synthesized different levels of the cyclic tetrapeptide, tentoxin; some colonies produced higher levels than either parent. These results demonstrate that spheroplast fusion may have a potential application for genetic analysis of secondary metabolite production and for strain improvement in A. alternata.Mention of a trademark of proprietary product does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.     

The interaction betweenE. coli spheroplasts and plant protoplasts: A proposed procedure to deliver foreign genes into plant cells

Summary E. coli spheroplasts can be used to deliver DNA vectors into plant protoplasts. The use of fluorescent dyes showed that 25–100% of the protoplast population was associated with 1–9 spheroplasts following incubation with several fusogens. Electron microscopy demonstrated spheroplasts attached to protoplasts via a plasma membrane protrusion after high pH/Ca²⁺ treatment, but PEG-high pH/Ca²⁺ promoted endocytosis of spheroplasts into a plasma membrane bounded vesicle. Ultrastructural profiles showed that fusion between spheroplasts and protoplasts did not occur. Immunofluorescence studies detectedE. coli antigens associated with tobacco protoplasts, and after fusogen treatment the antigens were dispersed within the peripheral cytoplasm. The elimination of residual contaminatingE. coli cells from protoplasts was achieved by lysozyme and antibiotic treatment, thus allowing DNA vector assessment in axenic culture.     

The immunity genes of colicins E2 and E8 are closely related

We have determined the nucleotide sequence of the newly characterized colicin E8imm gene which exists in tandem with the colicin E3imm gene in the: ColE3-CA38 plasmid. Comparison of these immunity structures reveals considerable sequence divergence) but the ColE8imm gene is markedly homologous to the colicin E2imm gene from the ColE2-P9 plasmid.Issued as NRCC no. 23586 and as CBRI no. 1480.     

Physical Properties of Escherichia coli Spheroplast Membranes

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.     

The influence of colicinogenic plasmids ColIb-P9, ColIa-CA53 and ColV-K30 on the repair, mutagenesis and induction of colicin E1 synthesis

Summary The presence of colicinogenic plasmids ColIb-P9 and ColIa-CA53 in E. coli K-12 cells, wild-type with respect to repair, enhanced the survival of cells after UV irradiation and increased the frequency of UV-induced argE3 and his-4 reversions, while the presence of ColV-K30 negatively affected repair and mutagenesis. The plasmid ColIb-P9 showed a UV-protective effect in E. coli cells carrying mutations in genes uvrA, uvrB, uvrC, polA, recB, recF, though in none of the mutants did cell survival reach the wild-type level. The effect of ColIb-P9 on mutagenesis did not depend on the uvrA or recB genes. The plasmids' protective effect and the enhancement of mutagenesis depended on the recA ⁺ lexA⁺ genotype. The frequency of 2-aminopurine-induced mutations was not affected by ColIb-P9 or ColV-K30. The presence of ColIb-P9 decreased the ability of ColEl-carrying cells to induce colicin E1 synthesis caused by DNA-damaging agents: UV, MNNG, mitomycin C, whereas ColV-K30 increased the percentage of colicin E1-producing cells. These plasmid effects on the level of induction of colicin E1 synthesis were not observed in the case of induction caused by chloramphenicol which did not depend on the products of recA and lexA genes.Abbreviations AP 2-aminopurine - MNNG N-methyl-N-nitro-N-nitrosoguanidine - ICS induction of colicin synthesis - CM chlorampheniol - MC mitomycin C