A simple method to study colony development in Streptomyces

Abstract When sodium azide was added to cultures of Myxococcus coralloides D a rapid loss in turbidity was observed. The lysis occurred irrespective of the culture age. If the azide was added to cultures which had been division-inhibited with puromycin, lysis was also induced. Other uncoupling agents (2,4-dinitrophenol, methyltriphenylphosphonium bromide and N , N '-dicyclohexylcarbodiimide) were effective to induce lysis, but not the ionophores gramicidin D or valinomycin. Energizing the membrane by the addition of glycerol, glucose or ascorbate to prelytic cultures was a means of preventing the lytic events.     

Acrylonitrile induces autolysis Bacillus subtilis

Acrylonitrile (AN) is an industrial chemical used in the manufacture of plastics and other polymers. AN has been reported to be an acute toxin and is a known carcinogen in rodents. When AN was mixed with suspensions of Bacillus subtilis, the bacteria began autolysis. It was determined that AN is partially converted to cyanide, a strong protonophore in B. subtilis. Autolytic enzymes in B. subtilis become active when the protonmotive force is dissipated. The amount of cyanide produced from AN, however, was not enough to promote autolysis in exponential B. subtilis. This is the first report showing that AN may induce autolytic reactions in bacteria. It is suggested the autolysis of B. subtilis may be useful in the environmental monitoring of AN. In addition, the metabolism of AN by bacilli may be useful in bioremediation.     

Regulation of autolysins in teichuronic acid-containing Bacillus subtilis cells

Bacillus subtilis cells grown under phosphate starvation induce teichuronic acid (TUA) synthesis while simultaneously repressing teichoic acid synthesis (TA). The turnover rates of TA-containing and TUA-containing walls are similar, indicating that autolysin function is similar and suggesting that modulation of autolytic function may be similar. In this study, it is demonstrated, utilizing fluorescein isothiocyanate (FITC)-dextran to probe the wall pH, that a low pH exists in the wall matrix. A second probe, cationized ferritin (CF), was used to observe cell surface protonation. Suspensions of B. subtilis cells containing either TA or TUA were aggregated with CF only after the addition of a proton-motive-force-dissipating agent. Respiring B. subtilis TUA-containing cells labelled with FITC-dextran exhibited little fluorescence. Conversely, fluorescence intensities exhibited by cells de-energized with nitrogen gas were significantly greater. The effects of protonmotive force on autolytic activity were studied by adding cell wall protein extract containing concentrated autolysin to exponentially growing TA-containing and TUA-containing B. subtilis cells. Both TUA-containing and TA-containing cells were lysed only after the addition of sodium azide. These data suggest that during normal growth the wall of TUA-containing B. subtilis cells is protonated, and proton-motive force influences autolytic regulation in both TUA-containing and TA-containing B. subtilis cells.     

Induction of autolysis in Bacillus subtilis by ochratoxin A.

Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 microgram/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 microgram/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.     

Composition of the regulators of bacterial autolysis

This work was aimed at studying the composition of agents regulating bacterial autolysis and isolated from the lysate of Bacillus subtilis 402, B. subtilis R2 and Micrococcus lysodeikticus biomass by extraction with 5% TCA followed by precipitation from the extract with 5 volumes of isopropanol. Fractions activating bacterial autolysis and fractions inhibiting it were found in all of the preparations after separation on Acrylex P-60. Fractions with a molecular mass below 12,600 D activated the autolysis whereas fractions with a molecular mass above 18,400 D inhibited it. The activity of fractions inhibiting the autolysis decreased while that of fractions activating the autolysis increased in the regulating agents isolated from B. subtilis cultures with the aging of the latter. The capability of the fractions to activate the autolysis correlated with the content of amino groups and phosphate in them whereas the capacity to inhibit the autolysis correlated with the content of reducing sugars in the fractions. The preparation of the fraction which activated the autolysis from B. subtilis R2 contained 18 amino acids with the predominance of alanine, glutamic acid, lysine and phenylalanine. Apparently, the regulating properties of the preparations are created with the aid of teichoic acids as well as peptidoglycan and protein fragments associated with the acids.     

Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization

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Chloroplast membranes and coupling factor conformations.

The demonstrated role of proton translocation and resulting electrochemical activity gradients (protonmotive force) in ATP synthesis by chloroplasts is noted. Evidence for the participation of conformational changes in the terminal ATPase (coupling factor, or CF1) is reviewed. Hydrogen exchange into ordinarily cyptic groups of the molecule occurs only when the subtending membranes are put under the stress of a protonmotive force. Since up to 100 hydrogen atoms per mole are involved in the energy-dependent exchange the conformational change permitting tham access to the medium must be a major one. Chemical reagents are beginning to be used to attack groups on CF1 that are exposed only when the membranes are energized. N-ethylmaleimide binds covalently, sulfate causes as yet unspecified damage, and permanganate leads to oxidative damage to CF1 under energized conditions. The last two reagents are analogues of phosphate, and ADP must be added for them to inhibit. On the basis of this and other differences between the conditions needed for inhibition by permanganate or sulfate, and that by N-ethylmaleimide or the hydrogen exchange, a somewhat complex scheme involving several successive or alternative conformations of CF1 can be postulated. Questions are raised as to the way in which a conformational change in a bound protein could be caused by a proton activity gradient across its supporting membrane, and as to whether the altered conformations might constitute a part of the energy transformations leading to ATP synthesis.     

Energized membrane may regulate nucleoid conformation in Bacillus subtilis

Abstract It was shown in a previous study, using a temperature-sensitive initiation mutant of Bacillus subtilis that DNA synthesis at the origin and terminus of replication occurred at cell poles. In the current study, it is demonstrated that cells specifically labelled at the origin or near the terminus, show a re-distribution of radioactivity when treated with the uncoupling agent, sodium azide. Since it is most likely that the DNA-cell wall attachment is mediated through membrane interactions, we now propose that the maintenance of this attachment is dependent on the energized state of the membrane.     

The effect of aeration and metabolic inhibitors on resistance to amphotericin in starved cultures of Candida albicans.

The development of resistance to amphotericin methyl ester, measured in terms of the amount of drug required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida albicans in starved cultures under controlled conditions of aeration, stirring and temperature. Resistance develops at a rate which increases with the rate of aeration, limited by the onset of damage due to turbulence. Resistance decreases rapidly if gassing with N2 is substituted for aeration, but sensitivity does not reach that of exponentially growing cells. Resumption of aeration is followed by a slow recovery of resistance. The addition of inhibitors of protein synthesis (trichodermin, verrucarin) or uncoupling agents (2,4-dinitrophenol, sodium azide) at the beginning of starvation results in an increased rate of development of resistance. Adding inhibitors at a later stage, when resistance has developed after 72 h aeration, does not affect the decrease in resistance produced by gassing with N2 but the presence of trichodermin or verrucarin delays the recovery of resistance o     

Relationship between the Latent Form and the Active Form of the Autolytic Enzyme of Streptococcus faecalis

A 10-hr starvation of Streptococcus faecalis ATCC 9790 for the amino acids methionine and threonine results in cells which are resistant to autolysis and which contain greatly reduced quantities of both active and latent (proteinase activable) forms of the autolytic enzyme (an N-acetyl-muramide glycanhydrolase). Cell walls were isolated from cells harvested at various times during the recovery from such starvation and were assayed for active and latent forms of the autolysin. Within 10 min of recovery the latent enzyme began to increase. Only after 30 to 60 min did the active enzyme begin to increase; after a similar lag, the cells' proneness to lysis markedly increased. The intracellular localization of both forms of the autolysin was examined, using as an experimental tool the ability of added cell wall to bind autolysin. (14)C-lysine-labeled, inactivated cell walls were added to exponential-phase cells, which were then disrupted, and the mixed wall population was isolated. Measurement of the (14)C release during wall autolysis indicated that the active enzyme in the cells was not available for binding to the added (14)C-labeled walls and was therefore wall-bound in vivo. In contrast, up to 85% of latent autolysin activity was found to have been efficiently bound to the added (14)C walls. The results obtained suggest (i) cellular autolysis is a reflection of the level of active enzyme and not of latent enzyme, and (ii) autolysin is synthesized and mainly located in the cytoplasm as an inactive latent precursor (proenzyme) which is transported to sites on the cell wall associated with wall biosynthesis, where it becomes activated.     

Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids.

The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids.     

Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli.

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.     

The role of a protonmotive force in genetic transformation of Bacillus subtilis]

The hypothesis on the role of protonmotive force in the transport of DNA through the membrane of Bac. subtilis cell during initial stages of genetic transformation was tested. A genetic transformation of arsenate-treated cells was observed. Treatment of cells by the protonophorous uncoupler of oxidative phosphorylation-carbonylcyanide dichlorophenyl--hydrazone-led to the inhibition of initial stages of genetic transformation having no significant effect on the level of intracellular ATP concentration and on the viability of cells. The dissipation of protonmotive force by means of K+ and H+ fluxes catalyzed by valinomycin and nigericin also caused the inhibition of initial stages of genetic transformation. The inhibitory effect of cationic penetrant tetraphenyl phosphonium was observed, the effect being potentiated by low concentrations of anionic penetrant phenyldicarbaundecaborate. The value of the membrane potential in the energized valinomycin-treated cells calculated from the distribution of K+ was within the range of 70--100 mV (inside minus). These results support the conception that a protonmotive force drives DNA transport through the membrane of Bac. subtilis cells.     

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.     

Export requirements of pneumolysin in Streptococcus pneumoniae

Streptococcus pneumoniae is a major causative agent of otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolysins (CDCs), is produced by virtually all clinical isolates of S. pneumoniae, and ply mutant strains are severely attenuated in mouse models of colonization and infection. In contrast to all other known members of the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains undefined. We show that an exogenously added signal sequence is not sufficient for Sec-dependent Ply secretion in S. pneumoniae but is sufficient in the surrogate host Bacillus subtilis. Previously, we showed that Ply is localized primarily to the cell wall compartment in the absence of detectable cell lysis. Here we show that Ply released by autolysis cannot reassociate with intact cells, suggesting that there is a Ply export mechanism that is coupled to cell wall localization of the protein. This putative export mechanism is capable of secreting a related CDC without its signal sequence. We show that B. subtilis can export Ply, suggesting that the export pathway is conserved. Finally, through truncation and domain swapping analyses, we show that export is dependent on domain 2 of Ply.     

Inhibition of vacuolar H+-ATPases by fusidic acid and suramin

The vacuolar system of eukaryotic cells is energized by a few ATP-driven ion pumps. One of these, the H+-ATPase, plays a major role in providing the protonmotive force for several organelles, as well as maintaining the proper pH inside the organelles. Formation of the protonmotive force in organelles isolated from the vacuolar system was inhibited by fusidic acid. The inhibition results from a combination of uncoupling the proton pumping and inhibition of the H+-ATPase activity. Suramin is also a potent inhibitor of the H+-ATPase from chromaffin granules. A possible connection between these activities and inhibition of HIV infection is pointed out.     

Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.     

Proton-linked D-xylose transport in Escherichia coli.

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.     

Entry of methotrexate into Streptococcus pneumoniae: a study on a wild-type strain and a methotrexate resistant mutant

Entry of methotrexate (MTX) into the folate prototrophic bacterium Streptococcus pneumoniae was poorly inhibited by folate or its natural derivative folinic acid, suggesting that if MTX is transported via a folate transporter, the affinity of that transporter for MTX is higher than for folate. In the range of concentrations tested, MTX uptake was non-concentrative and decreased in ATP-depleted bacteria. When the external concentration of MTX was increased from 1 X 10(-7) M to 1 X 10(-6) M, uptake became saturated and was insensitive to ionophores. However when external MTX concentrations were increased to 1 X 10(-5) M, uptake increased linearly, and was inhibited by the ionophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin, suggesting that the process was energized by the protonmotive force (delta p) at this concentration. A model for MTX entry in S. pneumoniae is proposed with respect to these results. The high level of resistance to MTX of the nonsense mutant amiA9 cannot be entirely explained by a decrease in MTX uptake.