Addition Of Chloramphenicol Research Articles

Inclusion bodies as reversible protein reservoirs

Inclusion bodies (IBs) are major protein aggregates that arise owing to a deficiency of the cell's protein folding machinery. This often occurs in recombinant bacteria where plasmid-encoded genes are being highly expressed. In this context, research into the prevention of IB formation or the re-forming of correctly folded protein from IBs is of great importance to the biotechnology industry. Understanding IBs is equally important for research into amyloid diseases (such as Alzheimer's disease), which are caused by misfolding of proteins, resulting in their deposition in the cells as fibrils. Surprisingly, given the significance of IBs, they have generally been poorly investigated. However, a recent paper by Carrio et al. 1xCarrio, M.M et al. : 7–11See all References1 is an exception. They have shown that IB production in vivo is, in fact, caused by an imbalance between protein aggregation and solubility.The authors used two plasmid-encoded proteins expressed in Escherichia coli to study IB production. One was a β-galactosidase fused to a viral capsid protein, and the other was phage 22 tail-spike protein (TSP). In both systems, they calculated the number of IBs formed and their volume, the amount of soluble versus insoluble protein, and the activity of the protein as an indicator of proper folding. These measurements were taken at hourly intervals before and after the addition of chloramphenicol, which arrests protein synthesis. In the β-galactosidase system, using SDS-PAGE and densitometry against known standards, they showed that the amount of insoluble material in the cell cultures sharply decreased upon addition of chloramphenicol, with a concomitant increase in the amount of soluble material. A combination of microscopy and digital imaging also showed a reduction in the number and volume of IBs in the cells. Correct re-folding of the protein was confirmed by an increase in the β-galactosidase activity in the cultures following termination of protein synthesis. The same trend was found with the expression of TSP. Carrio et al. also demonstrated in vitro re-folding of IB proteins by incubating purified IBs from the two strains with cell extracts from plasmid-free BL26 cells, with the result being a recovery of enzymatic activity and soluble peptide in both systems.The authors concluded that IBs are the result of an imbalance in the equilibrium between protein precipitation and re-folding and solubility in the cell. By switching off protein production, the protein folding machinery can catch up, thus redressing the balance. The authors therefore see IBs not as a dead-end product caused by inefficiency of the bacterial protein folding machinery, but rather as a reversible reservoir of protein awaiting correct folding when conditions permit. The significance of the result is already apparent: IB solubility in vitro could provide the answer to the problem of IB formation in biotechnology and, from a clinical viewpoint, it raises possibilities for new therapeutic approaches to amyloid diseases.

Circadian clock-protein expression in cyanobacteria: rhythms and phase setting.

The cyanobacterial gene cluster kaiABC encodes three essential circadian clock proteins: KaiA, KaiB and KaiC. The KaiB and KaiC protein levels are robustly rhythmical, whereas the KaiA protein abundance undergoes little if any circadian oscillation in constant light. The level of the KaiC protein is crucial for correct functioning of the clock because induction of the protein at phases when the protein level is normally low elicits phase resetting. Titration of the effects of the inducer upon phase resetting versus KaiC level shows a direct correlation between induction of the KaiC protein within the physiological range and significant phase shifting. The protein synthesis inhibitor chloramphenicol prevents the induction of KaiC and blocks phase shifting. When the metabolism is repressed by either translational inhibition or constant darkness, the rhythm of KaiC abundance persists; therefore, clock protein expression has a preferred status under a variety of conditions. These data indicate that rhythmic expression of KaiC appears to be a crucial component of clock precession in cyanobacteria.

A cold acclimation protein with refolding activity on frozen denatured enzymes.

We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100 degrees C, cryopreservation at -20 degrees C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12 degrees C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82x10(-10), 4.35x10(-9), 8.98x10(-12), and 3.05x10(-11), respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.

Nonthermal death of Escherichia coli

The destruction of Escherichia coli M23 OR.H– using lethal water activity levels and nonlethal temperatures was investigated. Death rates were measured for a combination of four growth-permissible temperatures (15°C, 25°C, 40°C and 45°C) and four distinctly lethal water activities (0.75, 0.83, 0.88 and 0.90). In addition, death rates were measured at two temperatures (4°C and 50°C) outside the growth range of E. coli. Death rate increased both at higher temperature or lower water activity. Inactivation curves resulting from exposure of E. coli to osmotic stress were biphasic. The initial rate of death was followed by a slower second phase decline, or “tailing” effect. Addition of chloramphenicol to the suspending medium reduced the tailing effect and suggested that tailing was caused by de novo protein synthesis.

Protein folding in Escherichia coli: role of 23S ribosomal RNA

Post-translational control of Escherichia coli ribosome on newly synthesised polypeptide leading to its active conformation (protein folding) has been shown in the case of the enzyme β-galactosidase. As expected, antibiotics chloramphenicol and lincomycin, which bind to 23S rRNA/50S subunit and kasugamycin and streptomycin which interact with the 30S subunit instantaneously inhibited protein synthesis when they were added to the growing cells. The increase in β-galactosidase activity, though stopped immediately after the addition of chloramphenicol and lincomycin, went on considerably in the presence of streptomycin and kasugamycin even after the stoppage of protein synthesis.

Short-term Toxicity of Various Pharmacological Agents on the In Vitro Nitrification Process in a Simple Closed Aquatic System.

During the treatment of fish diseases, drugs which inhibit the nitrification process can cause acute ammonia toxicity. The same phenomenon can occur when fish are put into a tank without active cultures of nitrifying bacteria. The purpose of this study was to quantify the inhibitory effects of 15 pharmacological agents, which are often used as therapeutic agents in ichthyopathology, on ammonia removal and nitrate production in a simple closed aquatic system. The experiments were conducted in polyethylene bags containing activated biofilters and synthetic water solutions, held in a water bath. Ammonia was added to initiate the nitrification process, and graded concentrations of various pharmacological agents were added. The effects of the pharmacological agents on in vitro nitrification were assessed by monitoring ammonia and nitrate concentrations compared to controls with no added agents, for 24 hours. Graded concentrations of ampicillin (Albipen®), chloramine T, enrofloxacin (Baytril®), erythromycin, levamisole, methylene blue and polymyxin B induced dose-dependent inhibitions of ammonia removal and nitrate production. The corresponding linear regression curves showed high correlation coefficients and were highly significant (p < 0.05). The addition of chloramphenicol, copper (II ) sulphate, kanamycin disulphate, malachite green, neomycin sulphate, potassium penicillin G, tetracycline and a mixture of trimethoprim and sulphadoxin (Duoprim™) had no significant effects on the nitrification process. A significant dose-related inhibition of nitrate production, but not of ammonia oxidation, was observed with enrofloxacin. The significant correlation (r = 0.940; p < 0.001) between the degrees of inhibition of ammonia oxidation and nitrate production for the various inhibitory pharmacological agents has also been calculated, with a view to validating this method. The data presented suggest that separate tank facilities for hospitalisation or quarantine are necessary when treating diseased fish with ampicillin, enrofloxacin, chloramine T, erythromycin, levamisole, methylene blue or polymyxin B, in order to avoid ammonia poisoning.

Invasion of aortic and heart endothelial cells by Porphyromonas gingivalis.

Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system. The connective tissues of the periodontium are extremely well vascularized, which allows invading microorganisms, such as the periodontal pathogen Porphyromonas gingivalis, to readily enter the bloodstream. However, the ability of P. gingivalis to actively invade endothelial cells has not been previously examined. In this study, we demonstrate that P. gingivalis can invade bovine and human endothelial cells as assessed by an antibiotic protection assay and by transmission and scanning electron microscopy. P. gingivalis A7436 was demonstrated to adhere to and to invade fetal bovine heart endothelial cells (FBHEC), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC). Invasion efficiencies of 0.1, 0.2, and 0. 3% were obtained with BAEC, HUVEC, and FBHEC, respectively. Invasion of FBHEC and BAEC by P. gingivalis A7436 assessed by electron microscopy revealed the formation of microvillus-like extensions around adherent bacteria followed by the engulfment of the pathogen within vacuoles. Invasion of BAEC by P. gingivalis A7436 was inhibited by cytochalasin D, nocodazole, staurosporine, protease inhibitors, and sodium azide, indicating that cytoskeletal rearrangements, protein phosphorylation, energy metabolism, and P. gingivalis proteases are essential for invasion. In contrast, addition of rifampin, nalidixic acid, and chloramphenicol had little effect on invasion, indicating that bacterial RNA, DNA, and de novo protein synthesis are not required for P. gingivalis invasion of endothelial cells. Likewise de novo protein synthesis by endothelial cells was not required for invasion by P. gingivalis. P. gingivalis 381 was demonstrated to adhere to and to invade BAEC (0.11 and 0.1% efficiency, respectively). However, adherence and invasion of the corresponding fimA mutant DPG3, which lacks the major fimbriae, was not detected. These results indicate that P. gingivalis can actively invade endothelial cells and that fimbriae are required for this process. P. gingivalis invasion of endothelial cells may represent another strategy utilized by this pathogen to thwart the host immune response.

Amplification of ColE1 related plasmids in recombinant cultures of Escherichia coli after IPTG induction

ColE1-derived plasmids containing different recombinant genes which are controlled by the tac promoter were amplified following induction with IPTG, but no amplification occurred if product formation was not induced. The plasmid copy number of recombinant E. coli increased three- to sixfold within a period of about 6 h in shake flask experiments, batch cultures, and glucose-limited fed-batch cultivations. Plasmid amplification occurred in E. coli B strains as well as in K-12 strains with different plasmids ( rop + and rop −) coding for various heterologous proteins. The amplification was not caused by a toxic effect of IPTG, but was related to a strong inhibition of translation and chromosomal replication after the induction of heterologous gene expression. Similar to the amplification after chloramphenicol addition, plasmid replication proceeded even if oriC replication and translation were inhibited following strong induction of a recombinant gene. In accordance with the effect of chloramphenicol, the level of ppGpp, which is a negative regulator of ColE1 derived plasmid replication, decreased after induction.

Randomly amplified polymorphic DNA analysis of starved and viable but nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is an estuarine bacterium capable of causing a rapidly fatal infection in humans. Because of the low nutrient levels and temperature fluctuations found in the organism's natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of V. vulnificus strains grown in rich media and has been applied to starved and VBNC cells of V. vulnificus in the present study. As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period. Most noticeable was a uniform loss of RAPD amplification products. By 4 h of starvation, the cells were undetectable by the RAPD method. Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm. The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state. VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method. The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states. This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify V. vulnificus DNA in these states.

Evidence for chloramphenicol/H+ antiport in Cmr (MdfA) system of Escherichia coli and properties of the antiporter.

We detected chloramphenicol/H+ antiport activity in membrane vesicles of Escherichia coli and cloned a gene for the antiporter from chromosomal DNA of E. coli. Introduction of the gene into E. coli cells conferred resistance to chloramphenicol and ethidium. A slight increase in resistance to acridine orange was also observed. Elevated chloramphenicol efflux and ethidium efflux were observed in cells harboring a plasmid carrying the gene. Addition of chloramphenicol to the assay mixture reduced the efflux of ethidium. Elevated chloramphenicol/H+ antiport activity was observed in membrane vesicles prepared from cells harboring the plasmid. The pH optimum for the activity was 6.5. We sequenced the gene and deduced the amino acid sequence of its product. A sequence homology search revealed that it was same as that of Cmr (or MdfA). Thus, it became clear that Cmr (MdfA) is the chloramphenicol(and ethidium)/H+ antiporter.

Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002.

Transcription regulation and transcript stability of a light-repressed transcript, lrtA, from the cyanobacterium Synechococcus sp. PCC 7002 were studied using ribonuclease protection assays. The transcript for lrtA was not detected in continuously illuminated cells, yet transcript levels increased when cells were placed in the dark. A lag of 20 to 30 min was seen in the accumulation of this transcript after the cells were placed in the dark. Transcript synthesis continued in the dark for 3 h and the transcript levels remained elevated for at least 7 h. The addition of 10 microM rifampicin to illuminated cells before dark adaptation inhibited the transcription of lrtA in the dark. Upon the addition of rifampicin to 3-h dark-adapted cells, lrtA transcript levels remained constant for 30 min and persisted for 3 h. A 3-h half-life was estimated in the dark, whereas a 4-min half-life was observed in the light. Extensive secondary structure was predicted for this transcript within the 5' untranslated region, which is also present in the 5' untranslated region of lrtA from a different cyanobacterium, Synechocystis sp. PCC 6803. Evidence suggests that lrtA transcript stability is not the result of differences in ribonuclease activity from dark to light. Small amounts of lrtA transcript were detected in illuminated cells upon the addition of 25 microg mL-1 chloramphenicol. The addition of chloramphenicol to dark-adapted cells before illumination allowed detection of the lrtA transcript for longer times in the light relative to controls without chloramphenicol. These results suggest that lrtA mRNA processing in the light is different from that in the dark and that protein synthesis is required for light repression of the lrtA transcript.

Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of Nocardia corallina B276

The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete Nocardia corallina B276 was investigated. Suspensions of N. corallina cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of N. corallina was stimulated by the addition of CO2 and inhibited by the depletion of CO2. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO2, NAD+, and a reductant (NADPH or dithiothreitol). In the absence of CO2, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in N. corallina occurs by a carboxylation reaction forming beta-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium Xanthobacter strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121-32128, 1997). The addition of epoxide carboxylase component I from Xanthobacter strain Py2 to methylepoxypropane-inactivated N. corallina extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II from Xanthobacter Py2 to active N. corallina extracts stimulated epoxide isomerase rates to the same levels observed with the purified Xanthobacter system. Antibodies raised against Xanthobacter strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grown N. corallina extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO2 fixation and the activity of a multicomponent epoxide carboxylase enzyme system.

Carbon and nitrogen dynamics in a grassland soil with varying pH: effect of pH on the denitrification potential and dynamics of the reduction enzymes

Abstract Soil pH is known to affect most soil biological processes, including denitrification. There are conflicting results as to the effect of pH on the denitrification process and the ability of denitrifiers to adapt to acid soil conditions. The denitrification potential and dynamics of the reduction enzymes involved were investigated for four soils of different pH values from the Park Grass Experiment at Rothamsted Experimental Station. Soils were incubated aerobically or anaerobically after being amended with 500 mg NO3--N kg-1. Anaerobic treatments were with or without C2H2 or with or without chloramphenicol (known to inhibit protein synthesis). All samples were incubated for 48 h, while CO2 and N2O production and NO3-, NO2- and NH4+ concentrations were monitored. CO2 and N2O production decreased with decreasing pH under both aerobic and anaerobic conditions. The addition of chloramphenicol under anaerobic conditions appeared to have no effect on CO2 production, but decreased N2O production in soils of pHCaCl2 greater than or equal to 5, but not in soils of pHCaCl2 less than or equal to 4, where N2O production was generally slow. Investigations of the enzyme dynamics involved in denitrification using the DETRAN model, suggested that the initial concentrations of N reductase enzymes were small in all soils and the biomass of Park Grass was not adapted to anaerobiosis. Data also suggested that soils of pHCaCl2 greater than or equal to 4.9, NO2- could accumulate to high concentrations (15-30 mg N kg-1) during anaerobic periods of 6-36 h. In the aerobic incubations nitrification was observed in all soils even at pHCaCl2 less than or equal to 3.9. Antecedent water regime of the soil appeared to be important in controlling denitrification activity and the dynamics of the reduction enzymes involved in the process. There were significant effects of pH on the number of microorganisms, the time to de novo enzyme synthesis and on the kinetics of the reduction enzymes involved in denitrification.

Temperature‐regulated mRNA accumulation and stabilization for fatty acid desaturase genes in the cyanobacterium Synechococcus sp. strain PCC 7002

Cyanobacteria acclimate to low-temperature conditions by desaturating their membrane lipids. The desB (omega 3 desaturase) and desC (delta 9 desaturase) genes of Synechococcus sp. strain PCC 7002 were cloned and characterized, and the expression of the desA (delta 12 desaturase), desB and desC genes was studied as a function of temperature. The steady-state mRNA abundance for the desA gene was threefold higher in cells grown at 22 degrees C than in cells grown at 38 degrees C. desB transcripts were not detected at 38 degrees C, but were abundant in cells grown at 22 degrees C. Levels of desC mRNA were similar at both growth temperatures. The mRNA levels of each desaturase gene increased within 15 min of a temperature shift-down to 22 degrees C, and mRNA levels recovered within 15 min after a shift-up to 38 degrees C. The cold-induced accumulation of transcripts from the desA and desB genes was suppressed by the addition of chloramphenicol, but the transient elevation of the desC transcript levels at 22 degrees C was not affected by chloramphenicol. The half-lives of the desA and desB mRNAs were significantly longer in cells grown at 22 degrees C than in cells grown at 38 degrees C, but the desC mRNA had a similar half-life at both temperatures. These studies reveal three patterns of temperature regulation for the desaturase genes, whose expression is tightly controlled by a combination of mRNA synthesis and stabilization. These studies demonstrate that elevation of desaturase mRNA levels is not the rate-limiting event during the low-temperature acclimation of cyanobacteria.

Phosphomannose isomerase of Xanthomonas campestris: a zinc activated enzyme.

Phosphomannose isomerase (pmi, EC 5.3.1.8) was purified to homogeneity from a wild strain of Xanthomonas campestris. The apparent molecular weight as determined by SDS-PAGE and Sephadex G-100 Superfine was found to be 58 kDa. The purified enzyme showed a single band on acrylamide gel electrophocusing with pI = 5.25. The optimum pH was 7.0 and the Km for D-mannose-6-phosphate was 2 mM. Pmi can be activated by bivalent cations with the order of Co2+>Zn2+>Mn2+>Ni2+>Ca2+. Addition of low concentration of ZnCl2 (2 x 10[-7] M) in the growth medium resulted in the enhancement of pmi activity to around 2.5 x fold. The half life of pmi, as it was measured by the addition of chloramphenicol, was 110 min, whereas in the medium supplemented with ZnCl2 was 270 min. Chemical modification experiments implied the existence of one histidyl residue located at or near the active site.

α‐Galactosidase in immobilized cells of Papaver somniferum L.

AbstractA poppy cell suspension culture was permeabilized by Tween 80 and immobilized by glutaraldehyde. The α‐Galactosidase in these cells showed an optimum pH level at 5.2 and an optimum temperature at 70 °C. Enzyme hydrolysis was linear for 3 h, reaching 86% conversion. A very good level of storage stability was achieved when using dry catalyst and immobilized cells in 0.15 M NaCl solution (with the addition of chloramphenicol, [1‐methyldodecy1)‐dimethylamin‐4‐oxide (ATDNO), chlortetracycline hydrochloride (CLCTC)] or by freezing them in 0.15 M NaCl solution.

β‐Galactosidase in immobilized cells of Daucus carota L.

AbstractThe cell suspension culture Daucus carota L. was permeabilized by Tween 80 and immobilized by glutaraldehyde. β‐Galactosidase showed an optimum pH of 4.7 and an optimum temperature of 55 °C. The enzyme hydrolysis was linear for 3 h, reaching a 65% conversion. A very good level of storage stability was achieved when using dry catalyst, or a solution of 0.15 M NaCl with the addition of chloramphenicol, (l‐methyldodecyl)‐dimethylamin‐4‐oxide (ATDNO), chlortetracycline hydrochloride (CLCTC) or by freezing the immobilized cells in 0.15 M NaCl. The cells characterized by high enzyme activity and stability in long‐term storage showed convenient physicomechanical properties.

Enhancement of apparent resistance to ethanol in Lactobacillus hilgardii

The survival of Lactobacillus hilgardii, a highly ethanol-tolerant organism, after an ethanol challenge at 25% (v/v) for 10 min, increased by several log cycles when cells, grown in the absence of ethanol, were pre-treated with 10% (v/v) ethanol, 15% (v/v) methanol or 2% (v/v) butanol for 4 h. A temperature upshift (25 to 40°C) before ethanol challenge demonstrated a similar enhancement of apparent resistance to ethanol. Ethanol shock enhanced apparent resistance to methanol, butanol and heat challenges. The addition of chloramphenicol to cells prior to any pre-treatment did not significantly diminish the increase in ethanol tolerance, suggesting that de novo protein synthesis is not required for induced tolerance in this organism.

Influence of Escherichia coli on motility parameters of human spermatozoa in vitro.

The influence of E. coli on human sperm motility was studied in vitro. Semen samples were prepared by a swim-up technique and adjusted to 22 x 10(6) spermatozoa/ml. Samples were then inoculated with different concentrations of a uropathogenic strain of E. coli, serotype 06, with initial sperm/bacteria ratios varying between 10:1 and 10000:1. Motion parameters were analysed by computer-aided motility analysis directly, and 2, 4 and 6 h after inoculation. In a second series of experiments, bacterial replication was inhibited by addition of chloramphenicol. In a third series, the effect of E. coli culture filtrates on sperm motility was investigated. The direct inhibitory effect of E. coli on progressive motility of spermatozoa was found to depend upon the bacterial concentration. A distinct inhibitory effect was observed only at a sperm/bacteria ratio of approximately 1, achieved by growth of E. coli during the experiments. For modality of motion, no distinct changes were observed. When growth of bacteria was prevented by chloramphenicol, no inhibitory effect on sperm motility was detected. Sperm motility was not inhibited by E. coli culture filtrates. Analysis by electron microscopy revealed multiple adhesions of E. coli to spermatozoa, causing variable ultrastructural damage as probable morphological correlates of immobilization.

Activation of stable DNA replication in rapidly growing Escherichia coli at the time of entry to stationary phase.

The conditions are described in which DNA replication can occur, in the absence of protein synthesis, in wild-type Escherichia coli cells. Chromosome replication, which is normally inhibited by addition of chloramphenicol, becomes resistant to this drug after nutritional shiftup, e.g. from minimal medium to Luria broth. This replication activity appears transiently when nutritionally upshifted cells enter stationary phase. The activity strictly requires recA+, but it is independent of recB+ and dnaA+. It can occur in the absence of concomitant transcription. Activation of the replication does not result from induction of the SOS response. As the characteristics of this DNA replication resemble those of the previously characterized stable DNA replication, it is termed nutritional shiftup-activatable stable DNA replication, nSDR. Possible mechanisms of the activation of nSDR in rapidly growing cells at the time of entry to stationary phase are discussed.