Loss of cytochrome oxidase in Saccharomyces cerevisiae during inhibition of mitochondrial protein synthesis by erythromycin and chloramphenicol.

There is a major reduction in respiratory competence, and inhibitionof growth, several hours after the addition of erythromycin or chloramphenicol to Saccharomyces cerevisiae growing in medium containing a non-fermentable carbon source. Spectrographic evidence is presented for a loss of cytochrome oxidase as a consequence of the antibiotic treatment. This loss is prevented by cyanide or oligomycin. When glucose is added, however, the loss occurs irrespective of the presence of the respiratory inhibitors. Cycloheximide does not affect respiratory competence or cause loss of cytochrome oxidase, and it prevents the loss elicited by erythromycin if both compounds are added together. However, if cycloheximide is added some time after the addition of erythromycin, it fails to block the response to the latter drug. The results cannot be accounted for on the basis of the segregation of a finite number of mitochondria into an increasing number of progeny cells but, rather, suggest that the mitochondria are modified during growth in chloramphenicol or erythromycin.     

Flagellum-independent surface migration of Vibrio cholerae and Escherichia coli

Surface translocation has been described in a large variety of microorganisms, including some gram-negative enteric bacteria. Here, we describe the novel observation of the flagellum-independent migration of Vibrio cholerae and Escherichia coli on semisolid surfaces with remarkable speeds. Important aspects of this motility are the form of inoculation, the medium composition, and the use of agarose rather than agar. Mutations in several known regulatory or surface structure proteins, such as ToxR, ToxT, TCP, and PilA, did not affect migration, whereas a defect in lipopolysaccharide biosynthesis prevented translocation. We propose that the observed surface migration is an active process, since heat, protease, or chloramphenicol treatments of the cells have strong negative effects on this phenotype. Furthermore, several V. cholerae strains strongly expressing the hemagglutinin/protease but not their isogenic hap-negative mutants, lacked the ability of surface motility, and the treatment of migrating strains with culture supernatants from hap strains but not hap-null strains prevented surface translocation.     

The possible involvement of protein synthesis in the injection of PL-1 phage genome into its host, Lactobacillus casei.

The process of genome DNA injection, after adsorption, by phage PL-1 into host cells of Lactobacillus casei was monitored by using the electron microscope. Injection of DNA was inhibited by the protein-synthesis inhibitors chloramphenicol and erythromycin at concentrations where the colony-forming ability of cells not infected by phage was unaffected. The results suggest that protein synthesis may be involved in some way in the process of genome injection.     

Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.

Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.     

Effect of Resistance to Chloramphenicol on Bacteriophage Sensitivity of Group A Streptococci

Several phage hosts of group A streptococci became resistant to lysis by bacteriophage as a consequence of having acquired the ability to grow in the presence of chloramphenicol. The phage was adsorbed to the streptococcal cell, and P(32)-labeling of the phage showed that the phage genome penetrated the chloramphenicol (CM)- resistant cells as it did the parent cells. However, artificial lysis of the infected CM-resistant cells with chloroform or enzymes revealed no intracellular mature phage particles. Lysates of infected CM-resistant cells contained no phage-related antigenic materials which possessed serum-blocking power, although they were readily detected in lysates of infected parent cells. The CM-resistant cells were not lysogenized by the phage. Only cells resistant to more than 10 mug/ml of chloramphenicol were resistant to phage, and this threshold effect was taken as an indication of at least two different loci of chloramphenicol resistance on the streptococcal genome. Strains resistant to high levels of other antibiotics, such as streptomycin and erythromycin, showed no resistance to lysis by phage. Evidence indicated that the mutant cells were deficient in an essential function associated with the phage genome.     

Factors influencing the formation and stability of D-glucoside 3-dehydrogenase activity in cultures of Agrobacterium tumefaciens.

D-glucoside 3-dehydrogenase specific activity in Agrobacterium tumefaciens was maximal towards the end of the exponential growth phase of batch cultures; over 90% of the activity disappeared within the next 15 h. Manganese ions, although essential for growth of the organism, strongly repressed D-glucoside 3-dehydrogenase synthesis in sucrose medium but had little effect when the carbon source was methyl alpha-D-glucoside. D-Glucoside 3-dehydrogenase activity increased linearly with increasing specific growth rate in chemostat cultures limited by carbon, nitrogen, phosphate or manganese when methyl alpha-D-glucoside was the carbon source. High enzyme activity was found with sucrose as carbon source only when the growth medium was manganese-limited. D-Glucoside 3-dehydrogenase activity disappeared from A. tumefaciens incubated in carbon- and nitrogen-free medium or in nitrogen-free medium containing succinate, but on continued incubation the activity returned and was then stable. The recovery of activity could be prevented by chloramphenicol or erythromycin. Bacteria containing the recovered dehydrogenase activity could not convert sucrose to 3-ketosucrose when oxygen acted as the terminal electron acceptor, but produced 3-ketosucrose at the normal rate in the presence of ferricyanide. D-Glucoside 3-dehydrogenase activity disappeared irreversibly from bacteria incubated in nitrogen-free medium containing sucrose. Loss of activity followed first order kinetics in bacteria taken from nitrogen-, phosphate- or manganese-limited chemostat steady states; an accelerating rate of decay occurred in cells grown under carbon-limitation. 8-Hydroxyquinoline, chloramphenicol, erythromycin, 2,4-dinitrophenol and manganese ions could reduce the rate of decay.     

Reversible inhibition of the motility of human spermatozoa by tetraphenylboron.

The motility of washed suspensions of human spermatozoa was completely inhibited by tetraphenylboron at concentrations that had little effect on sperm energy metabolism. The inhibition of motility was reversed by quaternary ammonium salts, albumin, caffeine, dibutyryl cyclic AMP and potassium ions. The addition of ouabain to cells redndered immotile by tetraphenylboron prevented reinitiation of motility by potassium but not by the other compounds. These observations, together with the effect of tetraphenylboron on the fluorescence of sperm suspensions treated with 1-anilinonaphthalene 8-sulphonic acid, suggest that the binding of tetraphenylboron to sites on the sperm plasma membrane is involved in the inhibition of sperm motility and the cyclic AMP may be involved in the regulation of ion transport across the plasma membrane.     

Cloning and Characterization of the Flavobacterium johnsoniae Gliding Motility Genes gldD and gldE

Cells of Flavobacterium johnsoniae move over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Cells of F. johnsoniae propel latex spheres along their surfaces, which is thought to be a manifestation of the motility machinery. Three of the genes that are required for F. johnsoniae gliding motility, gldA, gldB, and ftsX, have recently been described. Tn4351 mutagenesis was used to identify another gene, gldD, that is needed for gliding. Tn4351-induced gldD mutants formed nonspreading colonies, and cells failed to glide. They also lacked the ability to propel latex spheres and were resistant to bacteriophages that infect wild-type cells. Introduction of wild-type gldD into the mutants restored motility, ability to propel latex spheres, and sensitivity to bacteriophage infection. gldD codes for a cytoplasmic membrane protein that does not exhibit strong sequence similarity to proteins of known function. gldE, which lies immediately upstream of gldD, encodes another cytoplasmic membrane protein that may be involved in gliding motility. Overexpression of gldE partially suppressed the motility defects of a gldB point mutant, suggesting that GldB and GldE may interact. GldE exhibits sequence similarity to Borrelia burgdorferi TlyC and Salmonella enterica serovar Typhimurium CorC.     

Tryptophanless Death in Bacillus subtilis

A decline in colony-forming ability is observed in actively growing cultures of a tryptophan arginine auxotroph of Bacillus subtilis after removal of tryptophan (tryptophanless death). This phenomenon can be prevented by simultaneous starvation of the other required amino acid or by chloramphenicol administered in bacteriostatic concentration but not by actinomycin. Addition of tryptophan analogues not only prevents the death but also allows recovery of the cells that have lost the ability to form colonies on solid media. The term tryptophanless death is therefore inappropriate. Chloramphenicol but not actinomycin inhibits the recovery brought about by tryptophan analogues.     

Some properties of two erythromycin-dependent strains of Escherichia coli

Strains of Escherichia coli can be isolated that require erythromycin for growth. With one strain, AM, a range of antibiotics, including chloramphenicol, tetracycline, spectinomycin, kasugamycin and rifampicin, will substitute for erythromycin on solid and in liquid media; nalidixic acid supports growth in liquid but not on solid media. With a second strain, 103, chloramphenicol, tetracycline and spectinomycin support growth in liquid media but on solid medium only chloramphenicol substitutes for erythromycin. In media of higher than normal ionic strength, strain AM, but not strain 103, can grow in the absence of antibiotics. Possible reasons for these complex phenotypes are discussed.     

Cytophaga-flavobacterium gliding motility

Flavobacterium johnsoniae, like many other members of the Cytophaga-Flavobacterium-Bacteroides group, displays rapid gliding motility. Cells of F. johnsoniae glide over surfaces at rates of up to 10 microm/s. Latex spheres added to F. johnsoniae bind to and are rapidly propelled along cells, suggesting that adhesive molecules move laterally along the cell surface during gliding. Genetic analyses have identified a number of gld genes that are required for gliding. Three Gld proteins are thought to be components of an ATP-binding-cassette transporter. Five other Gld proteins are lipoproteins that localize to the cytoplasmic membrane or outer membrane. Disruption of gld genes results not only in loss of motility, but also in resistance to bacteriophages that infect wild-type cells, and loss of the ability to digest the insoluble polysaccharide chitin. Two models that attempt to incorporate the available data to explain the mechanism of F. johnsoniae gliding are presented.     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

Sensitivity of Haloquadratum and Salinibacter to antibiotics and other inhibitors: implications for the assessment of the contribution of Archaea and Bacteria to heterotrophic activities in hypersaline environments

Antibiotics and bile salts have been used to differentiate between heterotrophic activity of halophilic Archaea and Bacteria in saltern ponds. In NaCl-saturated brines of crystallizer ponds, most activity was attributed to Archaea. Following the recent isolation of Haloquadratum, the dominant archaeon in the salterns (reported to be sensitive to chloramphenicol and erythromycin), and the discovery of Salinibacter, a representative of the Bacteria, in the same ecosystem, reevaluation of the earlier data is required. The authors measured amino acid incorporation by Haloquadratum and Salinibacter suspended in crystallizer brine to investigate the suitability of antibiotics and bile salts to distinguish between archaeal and bacterial activities. The amino acid uptake rate per cell in Salinibacter was two orders of magnitude lower than that of Haloquadratum under the same conditions. Salinibacter was inhibited by chloramphenicol, erythromycin, and deoxycholate, but not by taurocholate. Erythromycin did not inhibit incorporation by Haloquadratum, but moderate inhibition was found by chloramphenicol at 10-50 microg mL(-1). Deoxycholate was highly inhibitory, but only partial inhibition was obtained in the presence of 25 microg mL(-1) taurocholate. Inhibition by chloramphenicol and taurocholate increased with increasing salt concentration. Erythromycin and taurocholate proved most valuable to differentiate between archaeal and bacterial activities in saltern brines.     

Energetics of gliding motility in Mycoplasma mobile

Mycoplasma mobile glides on surfaces at up to 7 microm/s by an unknown mechanism. We studied the energetics that power gliding by using a novel, growth medium-free system. We found that cells could glide in defined media if the glass substrate is preconditioned by exposure to horse serum. The active component that potentiates gliding is sensitive to proteinase K treatment. We used the defined medium system to test the effect of various inhibitors, ionophores, and poisons on motility of M. mobile. Valinomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), N,N'-dicyclohexylcarbodiimide, phenamil, amiloride, rifampin, and puromycin had no short-term effects on gliding. We also confirmed that we were able to modulate the membrane potential with valinomycin and FCCP by using a potential-sensitive dye. Shifting the pH likewise had no effect on motility. These results rule out the use of conventional ion motive forces to power gliding. Arsenate had a dramatic inhibitory effect on gliding, and both the speed and the fraction of cells moving tracked ATP levels. Sodium orthovanadate had a slight but significant inhibitory effect on gliding. Taken together, these results suggest that the motor system of M. mobile is likely an ATPase or is directly coupled to an ATPase.     

Effects of p-Fluorophenylalanine and Chloramphenicol on Chemotaxis in Escherichia coli

The effects of chloramphenicol and p-fluorophenylalanine (p-FPA) on growth, proportion of motile cells, average rate of motility, and the chemotactic response of a methionine auxotroph of Escherichia coli K-12 were studied. Kinetic studies revealed that the inhibition of chemotaxis by p-FPA can be explained by the effect on growth, proportion of motile cells, and average rate of motility rather than a selective inhibition of chemotaxis per se. The effect of chloramphenicol on chemotaxis could not be explained in terms of these characteristics. It is concluded that low concentrations of chloramphenicol, unlike p-FPA, selectively inhibit chemotaxis.     

Receptor-mediated in vitro gene transformation by a soluble DNA carrier system

We present, here, evidence that foreign DNA can be specifically delivered to cells by a soluble carrier system that takes advantage of receptor-mediated endocytosis. Our experiments were based on the following concepts: hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo-)glycoproteins; DNA can bind to polycations in a strong but noncovalent manner forming soluble complexes; and the gene for chloramphenicol acetyltransferase, a bacterial enzyme that acetylates chloramphenicol, is not present in mammalian cells. We coupled asialoorosomucoid (ASOR) to poly-L-lysine to form an asialoorosomucoid-poly-L-lysine conjugate. The plasmid, pSV2 CAT, was complexed to the conjugate in a molar ratio of 1:2. To test this complex, a model system was used consisting of hepatoma cell lines, Hep G2, asialoglycoprotein receptor (+), and SK-Hep 1, receptor (-). Each cell line was incubated with filtered ASOR X poly-L-lysine X DNA complex, or controls consisting of DNA plus ASOR, DNA plus poly-L-lysine, or DNA alone. Cells were assayed for the presence of chloramphenicol acetyltransferase activity as a measure of gene transformation. SK-Hep 1, receptor (-) cells, produced no detectable acetylated chloramphenicol derivatives under any condition. However, Hep G2, receptor (+) cells, incubated with the ASOR X poly-L-lysine X DNA complex were transformed as indicated by the presence of chloramphenicol acetyltransferase activity (0.028 chloramphenicol acetyltransferase units/10(6) cells). Mixtures of individual components of the complex failed to transform these cells. Competition by a 10-fold excess of ASOR prevented gene transformation by the ASOR X poly-L-lysine X DNA complex.     

Gliding Motility Mutants of Myxococcus xanthus

Two gliding motility mutants of Myxococcus xanthus are described. The semimotile mutant (SM) originated by high-frequency segregation from the motile FB(t) strain. Segregation was enhanced by acridine dye treatment. SM cells glide only when apposed to other cells in a swarm. The nonmotile strain (NM) originated by mutation from SM. NM cells neither glide individually nor cooperatively. FB(t), SM, and NM are indistinguishable with respect to fine structure, vegetative growth rate, glycerol-induced microcyst formation, spheroplasting, bacteriophage sensitivity, and responses to light. The motility mutants are more resistant to penicillin and more sensitive to actinomycin D than is the gliding wild type. The NM mutant is also a morphogenetic mutant; it is unable to form fruiting bodies.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.