Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.     

Ultrastructure of Microthrix parvicella from activated sludge

H.M. STRATTON, R. WEBB, E.M. SEVIOUR, L.L. BLACKALL AND R.J. SEVIOUR. 1996. Filaments of Microthrix parvicella grow very poorly in pure culture and have a characteristic uneven appearance containing large numbers of distinctive spherical swollen cells. This feature was only rarely seen with filaments of this organism in activated sludge biomass samples. Ultrastructurally, these spherical swollen cells do not appear to be bacterial endospores or cysts, but do show features that are consistent with them being some form of resting structure. Their production could be a response of M. parvicella to environmental stress, an explanation proposed because of their relative predominance in pure cultures of M. parvicella compared to their infrequent occurrence in filaments of this bacterium observed in activated sludge biomass.     

Phytohemagglutinin isolectin stimulation of glucose utilization by lymphocytes

Normal human lymphocytes were incubated with purified $\text{Phaseolus vulgaris}$ phytohemagglutinin isolectins L4 and E4 and the resulting tritiated thymidine incorporation and glucose utilization were measured. Both isolectins stimulated tritiated thymidine incorporation and glucose utilization, but L4 was at least 30 fold more potent. The dose response of glucose utilization parallelled the dose response of tritiated thymidine incorporation produced by the isolectins. Glucose supplementation of the stimulated cultures did not affect the kinetics or time course of tritiated thymidine incorporation.     

Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus

Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus sequencing, which effectively conceals most intragenomic variation, but cloned sequences containing intragenomic variation are becoming prevalent in DNA databases. To understand effects of using cloned rDNA sequences in phylogenetic analyses we amplified and cloned the ITS region from pure cultures of six Laetiporus species and one Wolfiporia species (Basidiomycota, Polyporales). An average of 66 clones were selected randomly and sequenced from 21 cultures, producing a total of 1399 interpretable sequences. Significant variation (≥ 5% variation in sequence similarity) was observed among ITS copies within six cultures from three species clades (L. cincinnatus, L. sp. clade J, and Wolfiporia dilatohypha) and phylogenetic analyses with the cloned sequences produced different trees relative to analyses with consensus sequences. Cloned sequences from L. cincinnatus fell into more than one species clade and numerous cloned L. cincinnatus sequences fell into entirely new clades, which if analyzed on their own most likely would be recognized as "undescribed" or "novel" taxa. The use of a 95% cut off for defining operational taxonomic units (OTUs) produced seven Laetiporus OTUs with consensus ITS sequences and 20 OTUs with cloned ITS sequences. The use of cloned rDNA sequences might be problematic in fungal phylogenetic analyses, as well as in fungal bar-coding initiatives and efforts to detect fungal pathogens in environmental samples.     

Brevenal Is a Natural Inhibitor of Brevetoxin Action in Sodium Channel Receptor Binding Assays

1. Florida red tides produce profound neurotoxicity that is evidenced by massive fish kills, neurotoxic shellfish poisoning, and respiratory distress. Red tides vary in potency, potency that is not totally governed by toxin concentration. The purpose of the study was to understand the variable potency of red tides by evaluating the potential for other natural pharmacological agents which could modulate or otherwise reduce the potency of these lethal environmental events. 2. A synaptosome binding preparation with 3-fold higher specific brevetoxin binding was developed to detect small changes in toxin binding in the presence of potential antagonists. Rodent brain labeled in vitro with tritiated brevetoxin shows high specific binding in the cerebellum as evidenced by autoradiography. Synaptosome binding assays employing cerebellum-derived synaptosomes illustrate 3-fold increased specific binding. 3. A new polyether natural product from Florida's red tide dinoflagellate Karenia brevis, has been isolated and characterized. Brevenal, as the nontoxic natural product is known, competes with tritiated brevetoxin for site 5 associated with the voltage-sensitive sodium channel (VSSC). Brevenal displacement of specific brevetoxin binding is purely competitive in nature. 4. Brevenal, obtained from either laboratory cultures or field collections during a red tide, protects fish from the neurotoxic effects of brevetoxin exposure. 5. Brevenal may serve as a model compound for the development of therapeutics to prevent or reverse intoxication in red tide exposures.     

Application of C Nuclear Magnetic Resonance To Elucidate the Unexpected Biosynthesis of Erythritol by Leuconostoc oenos

Natural-abundance C nuclear magnetic resonance (C-NMR) revealed the production of erythritol and glycerol by nongrowing cells of Leuconostoc oenos metabolizing glucose. The ratio of erythritol to glycerol was strongly influenced by the aeration conditions of the medium. The elucidation of the metabolic pathway responsible for erythritol production was achieved by C-NMR and H-NMR spectroscopy using specifically C-labelled d-glucose. The H-NMR spectrum of the cell supernatant resulting from the metabolism of [2-C]glucose showed that only 75% of the glucose supplied was metabolized heterofermentatively and that the remaining 25% was channelled to the production of erythritol. The synthesis of this polyol resulted from the reduction of the C-4 moiety of the intermediate fructose 6-phosphate. Oxygen has an inhibitory effect on the production of erythritol by L. oenos. Preaeration of a suspension of nongrowing cells of L. oenos resulted in 30% less erythritol and in 70% more glycerol formed during the anaerobic metabolism of glucose. The anaerobic production of erythritol from glucose was also found in growing cultures of L. oenos, although to a smaller extent.     

Accumulation and lethal effect of tritium (tritiated water) inRhodopseudomonas spheroides

Nonsulfur purple photosynthetic bacteria, Rhodopseudomonas spheroides cells were cultured in medium containing tritiated water (THO) under the light-anaerobic and dark-aerobic conditions. The experimental R value defined as specific activity ratio of organic bound 3H to THO in medium was 0.49 and 0.48 for the light-anaerobically grown cells and the dark-aerobically grown cells, respectively. From the relation of R value to number of weight doubling of the cells (n), ratio of experimental R to theoretical R, i.e., (2n-1)/2n derived by assuming no isotope effect, was 0.51 and 0.49 on an average for the light-anaerobically grown cells and the dark-aerobically grown cells, respectively. 3H-incorporation from THO-medium into the light-anaerobic nongrowing cells was affected by the light intensity and suppressed by adding HgCl2, KCN, and 2,4-dinitrophenol as well as 3H-labelling in the dark-aerobic nongrowing cells was affected by oxygen tension and suppressed by adding these metabolic inhibitors. From the fractionation of the lyophilized cells by modified Schneider method, the distribution of exchangeable 3H in cold acid-soluble and ether-ethanol-soluble fractions and nonexchangeable 3H bound to small molecules and macromolecules was 7.4/25.3/67.3 in the growing cells cultured anaerobically in the THO-medium up to late exponential phase in the light. The distribution in the nongrowing cells incubated anaerobically with the THO-medium for 18 h in the light of 300 and 3,000 lux was 82.1/8.4/9.5 and 58.2/19.2/22.6, respectively. These distributions of 3H were changed with growth phase and/or incubation time. On the biological effect of 3H-THO for the cells stocked at -196 degrees C to accumulate 3H-decays, the dark-aerobic nongrowing cells labelled with THO were rather radiosensitive than the dark-aerobically and light-anaerobically grown cells cultured in the THO-medium. The killing efficiencies, i.e., the probability that a single disintegration would be lethal, ranged from 1/200 to 1/275 for the above three kinds of cells labelled with THO. The killing efficiencies for R. spheroides labelled with THO were similar to that for radiosensitive strain CB13 and wild strain Hfr of Escherichia coli labelled with 3H-thymidine and stored at -196 degrees C.     

Improved Enrichment and Isolation Procedures for Obtaining Pure Cultures of Beggiatoa

Scoring agar surfaces with alginate swabs before placing washed filaments of Beggiatoa on the agar has greatly increased the rate at which single filaments move from contaminated areas. Numerous morphological types of pure cultures have been grown in organic media supplemented with either catalase or reducing agents. Aerated sewage was used as the enrichment source.     

Synthesis of interkeratin matrix in differentiating lizard epidermis: an ultrastructural autoradiographic study after injection of tritiated proline and histidine

During epidermal differentiation in mammals, keratins and keratin-associated matrix proteins rich in histidine are synthesized to produce a corneous layer. Little is known about interkeratin proteins in nonmammalian vertebrates, especially in reptiles. Using ultrastructural autoradiography after injection of tritiated proline or histidine, the cytological process of synthesis of beta-keratin and interkeratin material was studied during differentiation of the epidermis of lizards. Proline is mainly incorporated in newly synthesized beta-keratin in beta-cells, and less in oberhautchen cells. Labeling is mainly seen among ribosomes within 30 min postinjection and appears in beta-keratin packets or long filaments 1-3 h later. Beta-keratin appears as an electron-pale matrix material that completely replaces alpha-keratin filaments in cells of the beta-layer. Tritiated histidine is mainly incorporated into keratohyalin-like granules of the clear layer, in dense keratin bundles of the oberhautchen layer, and also in dense keratin filaments of the alpha and lacunar layer. The detailed ultrastructural study shows that histidine-labeling is localized over a dense amorphous material associated with keratin filaments or in keratohyalin-like granules. Large keratohyalin-like granules take up labeled material at 5-22 h postinjection of tritiated histidine. This suggests that histidine is utilized for the synthesis of keratins and keratin-associated matrix material in alpha-keratinizing cells and in oberhautchen cells. As oberhautchen cells fuse with subjacent beta-cells to form a syncytium, two changes occur : incorporation of tritiated histidine, but uptake of proline increases. The incorporation of tritiated histidine in oberhautchen cells lowers after merging with cells of the beta-layer, whereas instead proline uptake increases. In beta-cells histidine-labeling is lower and randomly distributed over the cytoplasm and beta-keratin filaments. Thus, change in histidine uptake somehow indicates the transition from alpha- to beta-keratogenesis. This study indicates that a functional stratum corneum in the epidermis of amniotes originates only after the association of matrix and corneous cell envelope proteins with the original keratin scaffold of keratinocytes.     

The septin AspB in Aspergillus nidulans forms bars and filaments and plays roles in growth emergence and conidiation

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in ΔaspD and lost in ΔaspA and ΔaspC strains. Interestingly, in the ΔaspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.     

Inactivation and partial degradation of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase in nongrowing cultures of Escherichia coli.

The stability of tryptophan biosynthetic enzyme activities was examined in cultures of repressor-negative (trpR) strains of Escherichia coli K-12 incubated under conditions of nutrient starvation of chloramphenicol inhibition. The results show that four of the five activities examined are stable under most nongrowing conditions, whereas one activity, indoleglycerol phosphate (InGP) synthetase, carried by the trpC protein, is unstable under most conditions tested. Phosphoribosylanthranilate (PRA) isomerase activity, which is also carried by the trpC protein, is unstable during starvation for ammonium, cysteine, or sulfate but is stable under other nongrowing conditions where InGP synthetase is not. InGP synthetase activity but not PRA isomerase activity is also diminished about twofold in cultures using glycerol as a carbon-energy source. These results indicate that one or both activities of the trpC protein is specifically inactivated under several culture conditions. Experiments with antibodies to the trpC protein show that sulfate-starved and ammonium-starved cultures contain 20 to 40% less immunologically reactive trpC protein than unstarved cultures. This indicates that the trpC protein is probably partially degraded under these conditions. During recovery from sulfate starvation or ammonium starvation, cultures slowly regain normal levels of InGP synthetase and PRA isomerase activities, suggesting that inactivation may be reversible.     

The Use of Glucosamine and Actinomycin D in Radioactive Labeling of RNA in Cells in Culture

Pretreatment of confluent cultures of mouse L cells or of well-differentiated nervous system cells in primary cultures with 20–120 mM glucosamine resulted in a stimulation of the uptake of tritiated uridine, but not of adenosine. A marked stimulation of the incorporation of radioactive uridine into acid-precipitable macromolecules was also obtained, while adenosine incorporation was unchanged. Cultures of L cells in log phase of growth were similarly affected by glucosamine pretreatment. Uridine and cytidine uptakes were stimulated by 50%. Tritiated uridine incorporation was stimulated in a biphasic manner, with maximal stimulation (115%) after 15–60 min of labeling and at later times an inhibition of incorporation. The stimulation of cytidine incorporation paralleled the stimulation of its uptake. The data indicate that there is: a) a glucosamine-induced stimulation of pyrimidine nucleoside uptake, b) a marked stimulation of tritiated uridine incorporation into RNA due to depletion of the cellular pools of unlabeled uridine nucleotides during glucosamine pre-treatment, and c) a decrease in the rate of RNA synthesis after several hours of glucosamine treatment, probably related to diminished intracellular supplies of uridine nucleotides. In the presence of glucosamine, high concentrations of actinomycin D could be used to increase nuclear retention of pulse-labeled nascent RNA. Cordycepin treatment did not result in similar retention of RNA. These techniques will be useful in autoradiographic and biochemical studies of nuclear RNA synthesis.     

The epiphyte vegetation of Annona glabra on Barro Colorado Island, Panama

Aim Information on the community composition, structure, and dynamics of epiphyte vegetation is scarce. A survey of the epiphytes occurring on all individuals of one particular host tree species in a well-studied neotropical research site allowed us a comparison of the epiphyte flora of this tree with the local epiphyte flora, the analysis of spatial distribution patterns and the use of these patterns as indications for changes in time. In the future, our results can be used as a baseline data-set for the direct observation of the long-term dynamics in epiphyte communities. Location The study was conducted on Barro Colorado Island (BCI), Panama. Methods We recorded all individuals of the vascular epiphytes growing on Annona glabra L., a flood-tolerant, multiple-stemmed tree, which is restricted to the shoreline of BCI. Data on tree biometrics, epiphyte species, and epiphyte abundances were collected for more than 1200 trees. Results In total, we encountered almost 15,000 epiphytic individuals in sixty-eight species, corresponding to more than one third of the entire epiphyte flora of Barro Colorado Island. The component species differed strongly in abundance: the four most important species accounted for >75% of all individuals. In most cases, the same four species were also the first to colonize a tree (=phorophyte). Colonization patterns indicated no replacement of early colonizers by late arrivals. Species richness and epiphyte abundances showed a positive correlation with the size and the density of the host trees. All species showed a highly clumped distribution and the physiognomy of epiphyte communities of individual trees was dominated either by one or several of the four most common species or by a set of frequently co-occurring tank bromeliads. Other species were dominant only in exceptional cases. Most species were always rare. A distance effect on community composition was mostly confined to a local scale with an increased similarity in the species assemblage of stems of a tree v. neighbouring trees. Main conclusions The epiphytes on a single small phorophyte species may encompass a surprisingly large proportion of the local epiphyte flora. The observations that most tree crowns are inhabited by a single or only very few species, and that all epiphyte species show highly clumped distributions suggest a predominance of very local dispersal within a tree crown, which is only infrequently interrupted by successful long-distance dispersal between crowns.     

PILINIA EARLEAE N. SP. (CHLOROPHYCEAE,CHROOLEPIDACEAE) FROM THE FLORIDA WEST COAST1

Pilinia earleae n. sp. family Chroolepidaceae, was found in the Tampa Bay area of the Florida west coast in October, 1976, on the tunic of an ascidian, later on dead mollusc shells, a nylon faking line, plastic strips, and as an epiphyte of Gracilaria foliifera, a red alga. It is described on the basis of abundant material from the natural environment and laboratory cultures. The type locality is the southernmost known for the genus in the western North Atlantic.     

低分子量神经丝蛋白（NF—L）的体外组装

Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins: NF-L, NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0.15 mol/L NaCl, 2 mmol/L MgCl2, neutral pH(pH 6.8) and 37 degrees C. In 6 mol/L urea, NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes, some irregular filaments were formed. Further dialyzed for another 40 minutes, the long smooth filaments appeared. Some filaments were unraveled at the end regions, where existed 2-4 subfilaments. Four subfilaments were more often observed. That is to say, the 10 nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0.15 mol/L NaCl, NF-L reconstituted into 45-180 nm-long, 10 nm-width filaments, which were not able to elongate into long filaments.     

Hormonal influence on photocontrol of the protandry in the genus Helianthus

Under natural photoperiodic conditions protandry in hermaphrodite disc flowers of sunflower (Helianthus annuus L.) is determined by the different elongation rates of the style and filaments. The elongation of the filament and style starts simultaneously after the daily dark period, but the style growth rate is slower. When plants close to anthesis are exposed to continuous white light (WL) a loss of protandry occurs: the filaments do not grow far enough to extrude the anthers from the corolla. The histological analyses show that the number of filament epidermal cells remains unaltered after organ elongation and that cells respond to photoperiod only by cell expansion. Emasculation does not substantially inhibit filament cell expansion, whereas isolation of the filament or stamen from the corolla suggests that this organ could be the perception site of the filament growth stimulus. In vitro treatments with auxin (indole-3-acetic acid, IAA or alpha-naphthaleneacetic acid, NAA) reverses the inhibition of cell expansion caused by continuous WL, whereas gibberellic acid (GA(3)) at high concentrations reproduces the effect of continuous WL. Experiments carried out on various Helianthus spp. show that all these plants have evolved the same photo- and hormonal-control of the protandry. In experiments in which the light treatments were continued for 24 h, the auxins drastically reduced the inhibiting effect of red light (R) and dichromatic treatments FR (far red)+R, whereas GA(3) repressed filament extension regardless of light quality. As far as auxins are concerned, the response of sunflower filaments does not appear to be connected with the polar transport of the hormone. Moreover, the promoting effect of darkness is not mediated by an increase of endogenous free IAA in disc flowers. However, sunflower filaments manifested a similar temporal pattern of response to the light/dark cycle and to auxin.     

Cytolytic and phospholipase C activity in Legionella species

To examine one possible mechanism of damage to leucocytes and tissue cells in legionellosis, seven species of Legionella were examined for cytolytic activity and for elaboration of phospholipase C, an enzyme that can damage mammalian cell membranes. Cytolysis was assessed using erythrocytes in agar. Phospholipase C was assayed by release of p-nitrophenol from p-nitrophenylphosphorylcholine and of tritiated phosphorylcholine from L-alpha-dipalmitoyl-[choline-methyl-3H]phosphatidylcholine. L. pneumophila, L. bozemanii, L. micdadei, L. dumoffii, L. gormanii, L. longbeachae and L. jordanis all lysed dog red blood cells, which have a high ratio of membrane phosphatidylcholine to sphingomyelin. The same strains hydrolysed varying amounts of p-nitrophenylphosphorylcholine; L. bozemanii exhibited the greatest activity. L. pneumophila, L. bozemanii, L. dumoffii, L. longbeachae and L. jordanis, but not L. micdadei, released tritiated phosphorylcholine from labelled substrate. These results indicate that several species of Legionella possess cytolytic capability; exotoxins with activity may play a role.     

Membranes of animal cells. II. The metabolism and turnover of the surface membrane

Turnover studies of the surface membrane and of cell particulate matter of L cells in tissue culture in logarithmic and plateau phase of growth have been made. The rate of incorporation of isotope into these fractions and the rate of fall of specific activities of labeled L-cell fractions have been observed. The following interpretation of the data appears most likely although other interpretations are possible. Growing and nongrowing cells synthesize approximately similar amounts of surface membrane and particulate material. In the growing cell the material is incorporated with net increases in substance. There is relatively little turnover. In the nongrowing cell newly synthesized material is incorporated, but a corresponding amount of material is eliminated so that there is turnover without net increase of substance. Our results suggest that there is no gross differential turnover between the protein, lipid, and carbohydrate of the surface membrane under the conditions of our experiments. Metabolic inhibitors or omission of amino acids in the culture medium lead to a decrease in synthesis of surface membrane and cell particulates and cause an equivalent decrease in the rate of degradation of surface membrane and of particulates; therefore the synthetic and degradative aspects of turnover appear to be coupled. As cultures of nongrowing cells in suspension or on a glass surface age, their synthetic and turnover capacities diminish. Our results suggest that the cell may exist in a nongrowing state with a level of synthesis similar to that of a growing cell. It can exist in this state with a high level of turnover.     

Involvement of the relA gene in the autolysis of Escherichia coli induced by inhibitors of peptidoglycan biosynthesis

It is generally assumed that inhibitors of peptidoglycan biosynthesis do not kill nongrowing bacteria. An exceptional case is reported here. The addition of chloramphenicol to amino acid-deprived cultures of relA+ strains of Escherichia coli which were treated with beta-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis. This phenomenon is termed chloramphenicol-dependent lysis. To be effective, chloramphenicol had to be present at its minimum growth-inhibitory concentration (or higher). Analogs of chloramphenicol which did not bind to ribosomes were completely ineffective. Amino acid deprivation was actually not required to demonstrate chloramphenicol-dependent lysis, and cultures treated with growth-inhibitory levels of chloramphenicol alone were lysed when challenged with inhibitors of peptidoglycan synthesis. Peptidoglycan synthesis has been shown previously to be under stringent (relA+) control, and chloramphenicol is known to be an antagonist of stringent control. Thus, it is proposed that the mechanism of chloramphenicol-dependent lysis is based on the ability of chloramphenicol to relax peptidoglycan synthesis in nongrowing relA+ bacteria. This is also consistent with the observation that treatment of amino acid-deprived relA mutants with inhibitors of peptidoglycan synthesis resulted in lysis, i.e., without the mediation of chloramphenicol.