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PubMed1978

Defeat of colicin tolerance in Escherichia coli ompA mutants: evidence for interaction between colicin L-JF246 and the cytoplasmic membrane.

Escherichia coli ompA mutants are tolerant to colicin L-JF246. This tolerance can be overcome by a variety of treatments that have as their target the outer membrane or the peptidoglycan layers of the cell envelope. Thus, increasing the concentration of colicin L, releasing lipopolysaccharide from the outer membrane by treatment of intact cells with ethylenediaminetetracetic acid (EDTA), converting cells to spheroplasts by treatment with lysozyme-EDTA or penicillin, or trypsin, treatment of intact cells will result in an increased colicin sensitivity. These treatments alter the outer membrane of ompA mutants and suggest that the altered outer membrane may allow the penetration of at least a portion of the colicin L molecule to a site of action located within this barrier. To substantiate this, we have demonstrated that membrane vesicles prepared from ompA mutants are sensitive to colicin L and that 14C-labeled colicin L binds rapidly to both the outer and inner membrane fractions of the cell.

Cell Fractionation↗
PubMed1978

Cell envelope associations of Aquaspirillum serpens flagella.

Specific regions of the cell envelope associated with the flagellar basal complex of the gram-negative bacterium Aquaspirillum (Spirillum) serpens were identified by studying each of the envelope layers: outer membrane, mucopeptide, and plasma membrane. The outer membrane around the flagella insertion site was differentiated by concentric membrane rings and central perforations surrounded by a closely set collar. The perforations in both the outer membrane and the isolated mucopeptide layer were of a size accomodating the central rod of the basal complex but smaller than either the L or the P disks. The P disk of the complex may lie between the mucopeptide and the outer membrane. Electron microscopy of intact, spheroplasted, or autolyzed preparations did not adequately resolve the location of the inner pair of disks of the basal complex. Freeze-etching, however, revealed differentiation within the plasma membrane that appeared to be related to the basal complex. The convex fracture face showed depressions which are interpreted as impressions of a disk surrounded by a set of evenly spaced macromolecular studs and containing a central "plug" interpreted as the central rod. In thin sections, blebs, which appear to be associated with the flagellar apparatus, were seen on the cytoplasmic side of the plasma membrane. Superimposing the dimensions of the flagellar basal complex and the spacings of the cell envelope layers and using the position of the L disk within the outer membrane for reference, showed that the S disk might be within and the M disk beneath the plasma membrane. A tentative model was developed for comparison with that based on the structure of the Escherichia coli basal complex.

Bacteriolysis↗
PubMed1979

Isolation and characterization of protoplasts from Saccharomyces rouxii.

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.

Organoids↗
PubMed1979

Sensitivity of Escherichia coli to cephaloridine at different growth rates.

Steady-state populations of Escherichia coli B/r were treated with cephaloridine at minimal inhibitory concentrations. The antibiotic sensitivity of the cells and the localization of spheroplast emergence along the cell surface were examined as a function of cell length and growth rate. In fast-growing populations (greater than 1 division per h) the sites of cephaloridine interaction occurred preferentially at the cell pole in the smaller cells and at the cell center in dividing cells. At decreasing growth rates the cells became more resistant to cephaloridine, and a gradual shift from the cell pole toward the cell center was observed for the sphere position. A similar growth rate-dependent change in localization was found for sucrose-induced plasmolysis vacuoles.

Cell Division↗
PubMed1980

Localization of enterobacterial common antigen: Proteus mirabilis and its various L-forms.

An investigation of Proteus mirabilis wild-type strains and their various derived L-forms shows that the enterobacterial common antigen (ECA) is localized in the outer membrane of the cell envelope of these strains. In strains where the outer membrane is lacking (stable protoplast L-forms) or where its amount is reduced (spheroplast UL19) no ECA or only reduced amounts of it are detected by serological tests or by ferritin-labeling techniques.

Antigens, Bacterial↗
PubMed1981

Purification of Streptococcus group C bacteriophage lysin.

A simple procedure for the purification of Streptococcus group C phage lysin to apparent homogeneity is described. The electrophoretically pure, enzymatically stable polypeptide of 98,000 molecular weight converted Streptococcus (groups A, F, and H) cells into spheroplasts within 5 min at 0 degrees C or within less than a minute at 37 degrees C.

Bacteriolysis↗
PubMed1983

hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis.

Except for a small fraction of persisters, 10(-6) to 10(-5), Escherichia coli K-12 is killed by prolonged inhibition of murein synthesis. The progeny of persisters are neither more resistant to inhibition of murein synthesis nor more likely to persist than normal cells. Mutants have been isolated in which a larger fraction, 10(-2), persists. The persistent response of the mutants, Hip (high persistence), is to inhibition of murein synthesis at early or late steps by antibiotics (phosphomycin, cycloserine, and ampicillin) or by metabolic block (starvation for diaminopimelic acid). Killing of the parent strain by each of the four inhibitors has two phases: The first is rapid and lasts about 30 min; the second is slower, but still substantial, and lasts 3 to 4 h. The first phase also occurs in the Hip mutants, but then viability of the mutants remains constant after about 30 min. Neither tolerance, resistance, impaired growth, nor reversion of spheroplasts accounts for high-frequency persistence. Two of the mutations map at 33.8 min in a region containing few other recognized functions. This position and the phenotypes define hipA as a newly recognized gene. Transposons Tn5 and Tn10 have been inserted close to hipA making it possible to explore the molecular genetics of persistence, a long recognized but poorly understood phenomenon.

Ampicillin↗
PubMed1984

Induced plasmid-genome rearrangements in Rhizobium japonicum.

The P group resistance plasmids RP1 and RP4 were introduced into Rhizobium japonicum by polyethylene-glycol-induced transformation of spheroplasts. After cell wall regeneration, transformants were recovered by selecting for plasmid determinants. Plant nodulation, nitrogen fixation, serological, and bacterial genetics studies revealed that the transformants were derived from the parental strains and possessed the introduced plasmid genetic markers. Agarose gel electrophoresis, restriction enzyme analysis, and DNA hybridization studies showed that many of the transformant strains had undergone genome rearrangements. In the RP1 transformants, chromosomal DNA was found to have transposed into a large indigenous plasmid of R. japonicum, producing an even larger plasmid, and the introduced R plasmid DNA was found to be chromosomally integrated rather than replicating autonomously or integrated into the endogenous plasmid. Seemingly, a similar section of chromosomal DNA was involved in all the genomic rearrangements observed in the R. japonicum RP1 and RP4 transformant strains.

Chromosomes, Bacterial↗
PubMed1984

Methylammonium transport in Anacystis nidulans R-2.

Methylammonium was taken up rapidly by illuminated cells of Anacystis nidulans R-2, leading to internal concentrations of 1.3 +/- 0.1 mM within 1 min, and a gradient of up to 200 between the cells and medium. Accumulation of 14CH3NH3+ required at least 5 mM NaCl, but the uptake rate was independent of medium pH between 6.5 and 9. The kinetics of uptake could be resolved into an initial fast phase lasting less than 1 min (approximate Km, 7.2 microM; Vmax, 12.5 nmol min-1 mg of protein-1 at 15 degrees C). A second, slower phase associated with product formation was eliminated by preincubation with methionine sulfoximine, a specific inhibitor of glutamine synthetase; the rapid phase was unaffected by this treatment. Ammonium ions competed with 14CH3NH3+ for entry, and addition of 5 microM NH4+ or 100 microM CH3NH3+ released 14CH3NH3+ accumulated during the rapid phase of entry. Small additions of NH4+ made at the same time as additions of 14CH3NH3+ delayed the start of radioactivity uptake by a time which corresponded accurately with the period needed for the complete removal of the added NH4+. The effects of inhibitors on accumulation and carbocyanine dye fluorescence suggest that ATP-dependent membrane potential was needed to drive 14CH3NH3+ transport. Spheroplasts were as active as whole cells in accumulating NH4+ and 14CH3NH3+, indicating that soluble periplasmic components are not involved in the translocation. Some significant differences between the translocation of 14CH3NH3 and that of NH4+ were observed: growth with NH4+ in place of NO3- repressed 14CH3NH3+ accumulation ability without affecting the NH4+ uptake rate Na+ was not required for NH4+ uptake, and concentration of KCl inhibitory with 14C3NH3+ did not reduce NH4+ uptake.

Alkaline Phosphatase↗
PubMed1987

Parasexual genetics of Torulopsis glabrata.

Prototrophic hybrids were generated in the asexual yeast Torulopsis glabrata by the fusion of spheroplasts derived from parent strains which bore complementing auxotrophic markers. The DNA content (per cell) of two hybrids was essentially that predicted by summing the corresponding parental values. UV irradiation of these two hybrids resulted in the formation of sectored colonies with genetic properties consistent with their origin by either mitotic recombination or chromosomal nondisjunction.

Candida↗
PubMed1987

Glucose uptake by the cellulolytic ruminal anaerobe Bacteroides succinogenes.

Glucose uptake by Bacteroides succinogenes S85 was measured under conditions that maintained anaerobiosis and osmotic stability. Uptake was inhibited by compounds which interfere with electron transport systems, maintenance of proton or metal ion gradients, or ATP synthesis. The most potent inhibitors were proton and metal ionophores. Oxygen strongly inhibited glucose uptake. Na+ and Li+, but not K+, stimulated glucose uptake. A variety of sugars, including alpha-methylglucoside, did not inhibit glucose uptake. Only cellobiose and 2-deoxy-D-glucose were inhibitory, but neither behaved as a competitive inhibitor. Metabolism of both sugars appeared to be responsible for the inhibition. Cells grown in cellobiose medium transported glucose at one-half the rate of glucose-grown cells. Spheroplasts transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose-binding protein. Differences in glucose uptake patterns were detected in cells harvested during the transition from the lag to the log phase of growth compared with cells obtained during the log phase. These differences were not due to different mechanisms for glucose uptake in the cell types. Based on the results of this study, B. succinogenes contains a highly specific, active transport system for glucose. Evidence of a phosphoenolpyruvate-glucose phosphotransferase system was not found.

Anaerobiosis↗
PubMed1988

Role of cell wall in Saccharomyces cerevisiae mutants resistant to Hg2+.

Hg2+-resistant mutants were isolated from Saccharomyces cerevisiae. Although they were very much like the parental strains in terms of colony-forming ability, they grew faster than the parental strains in the presence of sublethal doses of Hg2+. The Hg2+-resistant mutations were dominant. They were centromere linked and were divided into two groups by means of recombination; one of the mutations, designated HGR1-1, was mapped on chromosome IV because of its linkage to the TRP1 locus. The Hg2+-resistant mutants took up Hg2+ as much as, or slightly more than, the parental strains did. The mutants and parental strains retained only about 5 and 15%, respectively, of the cell-associated Hg2+ after removal of the cell wall; therefore, the mutants had less spheroplast-associated Hg2+ than did the parental strains. These results indicate that the cell wall plays an important role in protection against Hg2+ by acting as an adsorption filter and that the mutations described confer Hg2+ resistance by increasing the Hg2+-binding capacity of the cell wall.

Biological Transport↗
PubMed1989

Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.

ATP-Binding Cassette Transporters↗
PubMed1989

Molybdenum accumulation in chlD mutants of Escherichia coli.

The content of molybdenum in wild-type and chlD cells was measured under a variety of growth conditions to determine if cells with a defective chlD gene were able to accumulate molybdenum. The chlD cells accumulated less molybdenum than wild-type cells did but concentrated molybdenum to a level at least 20-fold higher than the concentration in the culture medium. Molybdenum was present within spheroplasts of chlD cells and was not dialyzable. The chlD cells accumulated as much molybdenum as wild-type cells did when grown in medium containing 0.1 mM molybdate; thus, the capability of incorporation of molybdenum into cellular component(s) was equivalent to that of the wild type under these conditions.

Cytochromes↗
PubMed1992

Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide.

beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.

Ampicillin↗
PubMed1992

Genetics of the white-opaque transition in Candida albicans: demonstration of switching recessivity and mapping of switching genes.

Spheroplast fusion has been used to analyze the genetics of the reversible phenotypic transition, white-opaque, in Candida albicans WO-1. This transition involves changes in cell shape, permeability, and colony morphology. Fusion of switching with nonswitching cells gave nonswitching fusants, suggesting that the white-opaque phenotype is recessive. Chromosome loss induced by heat shock gave segregants of the fusants which were able to undergo the transition, indicating that the repressor function is genetically defined and probably limited to one or two chromosomes. Chromosomes R, 1, 3, 4, and 7 were eliminated as unique sites for the repressor, leaving 2, 5, and 6 as possible locations. When a ura3 (chromosome 3) nonswitching strain was fused with a switching strain, all ura3 segregants induced by heat shock were incapable of the phenotypic transition. Therefore, some or all of the genes (called SWI genes) essential for the transition are located on chromosome 3. UV irradiation-induced recombination did give rise to Ura- switching progeny, showing that the failure to switch was not due to a side effect of the pyrimidine requirement. The failure to isolate normally switching ura3 progeny generated by UV irradiation suggests a close linkage between the two genes.

Candida albicans↗
PubMed1993

Membrane topologies of the TolQ and TolR proteins of Escherichia coli: inactivation of TolQ by a missense mutation in the proposed first transmembrane segment.

The TolQ and TolR proteins of Escherichia coli are required for the uptake of group A colicins and for infection by filamentous phages. Their topology in the cytoplasmic membrane was determined by cleavage with aminopeptidase K, proteinase K, and trypsin in spheroplasts and cell lysates. From the results obtained, it is proposed that the N terminus of TolQ is located in the periplasm and that it contains three transmembrane segments (residues 9 to 36, 127 to 159, and 162 to 191), a small periplasmic loop, and two large portions in the cytoplasm. The N terminus of TolR is located in the cytoplasm and is followed by a transmembrane segment (residues 21 to 40), and the remainder of the protein is located in the periplasm. A tolQ mutant, which rendered cells resistant to group A colicins and sensitive to cholate, had alanine 13 replaced by glycine and was lacking serine 14 in the first transmembrane segment. The membrane topologies of TolQ and TolR are similar to those proposed for ExbB and ExbD, respectively, which is consistent with the partial functional substitution between ExbB and TolQ and between ExbD and TolR. The amino acid sequences of these proteins display the highest homology in the transmembrane segments, which indicates that the membrane-spanning regions play an important role in the activities of the proteins.

Amino Acid Sequence↗
PubMed1993

The Vibrio fischeri luminescence gene activator LuxR is a membrane-associated protein.

The Vibrio fischeri luminescence (lux) genes are activated at sufficiently high culture densities by the transcriptional activator LuxR in combination with a diffusible signal compound termed autoinducer. We have used antibodies directed against LuxR in immunoprecipitation experiments to study the subcellular location of this transcription factor. The LuxR polypeptide was detected in membranes and not in the soluble pool of cytoplasmic proteins from V. fischeri. LuxR was not released from the membranes by 0.6 M KCl or by the nonionic detergents Nonidet P-40, N-octyl-beta-D-glucopyranoside, and Triton X-100. LuxR and a number of other V. fischeri proteins were released from the membranes by EDTA. The autoinducer had no detectable influence on the subcellular location of LuxR. In spheroplasts, neither the abundance nor the molecular mass of the LuxR antigen was influenced by treatment with proteinase K. Together with other information, these results indicate that LuxR is an amphipathic protein that is associated with the cytoplasmic membrane of V. fischeri.

Bacterial Proteins↗