Role of the pneumococcal autolysin (murein hydrolase) in the release of progeny bacteriophage and in the bacteriophage-induced lysis of the host cells.

The pneumococcal bacteriophage Dp-1 seems to require the activity of the N-acetylmuramic acid-L-alanine amidase of the host bacterium for the liberation of phage progeny into the medium. This conclusion is based on a series of observations indicating that the exit of progeny phage particles is prevented by conditions that specifically inhibit the activity of the pneumococcal autolysin. These inhibitory conditions are as follows: (i) growth of the bacteria on ethanolamine-containing medium; (ii) growth of the cells at pH values that inhibit penicillin-induced lysis of pneumococcal cultures and lysis in the stationary phase of growth; (iii) addition of trypsin or the autolysin-inhibitory pneumococcal Forssman antigen (lipoteichoric acid) to the growth medium before lysis; (iv) infection of an autolysin-defective pneumococcal mutant at a multiplicity of infection less than 10 (treatment of such infected mutant bacteria with wild-type autolysin from without can liberate the entrapped progeny phage particles); (v) release of phage particles and culture lysis can also be inhibited by the addition of chloramphenicol to infected cultures just before the time at which lysis would normally occur. Bacteria infected with Dp-1 under conditions nonpermissive for culture lysis and phage release secrete into the growth medium a substantial portion of their cellular Forssman antigen in the form of a macromolecular complex that has autolysin-inhibitory activity. We suggest that a phage product may trigger the bacterial autolysin by a mechanism similar to that operating during treatment of pneumococci with penicillin (Tomasz and Waks, 1975).     

Phage Typing Reactions on Brucella Species

The nature of the phage typing reactions on Brucella species was determined by rates of adsorption and infection, one-step growth experiments, and susceptibility to lysis from without. The highest rates of adsorption and infection were obtained on smooth B. abortus cultures, and large clear plaques were produced. One or a few phage particles per B. neotomae cell killed about one-half of the cells, but some went through an infective cycle and released mature phage that resulted in production of small clear plaques. With B. suis, more phage particles per cell were required to kill, replication did not occur, and plaques were not observed. Still greater numbers of phage particles were required to cause some inhibition of growth of B. melitensis lawns. Rough Brucella cultures and species, such as B. ovis and B. canis, were not affected by the highest concentrations of phage. B. abortus cultures of intermediate colonial morphology adsorbed phage, but only a few infected cells (after a delayed latent period) released mature phage. An infected culture or colony appeared normal until spontaneous phage mutants appeared which could penetrate the cell wall more effectively than the parent phage. The mutant phage multiplied more rapidly, and the colony changed to a sticky white form.     

Biological properties of the phage-tail-like particles from Myxococcus coralloides D

Abstract Electron microscopy of preparations of the Myxocococcus coralloides D autolytic supernatants revealed the presence of phage-tail-like particles. The particles consisted of a core, a contractile sheath and a baseplate with fibers. The induction of the defective prophage occurred when the cultures reached the stationary phase, but the events occurring during induction did not lead to cell lysis. Particles nerver appeared during exponential growth. They were not observed when M. coralloides D grew on solid media, either. Purified samples of phage tails were able to inhibit most of the myxobacterial strains which were tested when the particles were in the extended state, but they were unable to multiply on the sensitive strains.     

Variability of Bacillus thuringiensis under various growth conditions

When a lysogenic culture of Bacillus thuringiensis subsp. galleriae 69-6 was grown under the batch conditions, 93-99% of cells in the population produced R-form colonies and ca. 1% yielded S-form colonies. The amount of spore-forming cells was 99% in R-variants and 8% in S-variants. The quantity of S-variants rose abruptly to 99% when the culture was grown under the chemostat conditions. The number of S-variants increased with the rate and the duration of growth. The process was influenced by growth-limiting factors. Temperate phage variants capable of host culture lysis on solid media (i.e. h-mutants) were not found under the conditions of batch cultivation. However, such phage particles (h-mutants) appeared under the conditions of chemostat. The titre of these phage particles reached 10(8), 10(7) and 10(4) particles per 1 ml at limitation with yeast extract, glucose and phosphorus, respectively. Under the conditions of chemostat, the particles behaved as temperate ones and their growth was not found. Irrespective of the limitation, the phage titre did not correlate with the ratio of R and S-forms in the population. When the growth was limited with phosphorus, the quantity of S-forms increased abruptly while the spontaneous induction of the phage was inhibited. The quantity of cells capable of spore formation decreased in the cultures isolated from the chemostat and grown on MPA: 69-80% of the cells in R-forms and merely 8% in S-forms.     

Effects of Bacteriophages on the Population Dynamics of Four Strains of Pelagic Marine Bacteria

Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.     

Two-stage fermentation with bacteriophage lambda as an expression vector in Escherachia coli

The potential of bacteriophage lambda as an expression vector for a large scale production of cloned-gene proteins was evaluated in batch and continuous bioreactors using a temperature-sensitive mutant in the cl gene, which allows a simple manipulation of temperature as a means to control the phage in the lysogenic or lytic state. A temperature switch from 32 degrees C (or below) to 38 degrees C (or above) forces the phage to go from the lysogenic state to the lytic state. Temperature cycling and a two-reactor system were used for continuous cultures. For the latter the first reactor is maintained in the lysogenic state at a lower temperature to stably maintain the foreign DNA in the host cell, while the second reactor is maintained in the lytic state to force replication of the cloned-gene and overproduction of its products. The results are promising but suggest a greater potential for a mutant which lacks the Q gene which is responsible for host cell lysis and packaging of phage particles.     

Growth and phage production of lysogenic B. megatherium

Cell multiplication and phage formation of lysogenic B. megatherium cultures have been determined under various conditions and in various culture media. 1. In general, the more rapid the growth of the culture, the more phage is produced. No conditions or culture media could be found which resulted in phage production without cell growth. 2. Cultures which produce phage grow normally, provided they are shaken. If they are allowed to stand, those which are producing phage undergo lysis. Less phage is produced by these cultures than by the ones which continue to grow. 3. Cells plated from such phage-producing cultures in liquid yeast extract medium grow normally on veal infusion broth agar or tryptose phosphate broth agar, which does not support phage formation, but will not grow on yeast extract agar. 4. Any amino acid except glycine, tyrosine, valine, leucine, and lysine can serve as a nitrogen source. Aspartic acid gives the most rapid cell growth. 5. The ribose nucleic acid content is higher in those cells which produce phage. 6. The organism requires higher concentrations of Mg, Ca, Sr, or Mn to produce phage than for growth. 7. The lysogenic culture can be grown indefinitely in media containing high phosphate concentrations. No phage is produced under these conditions, but the cells produce phage again in a short time after the addition of Mg. The potential ability to produce phage, therefore, is transmitted through cell division. 8. Colonies developed from spores which have been heated to 100°C. for 5 minutes produce phage and hence, infected cells must divide. 9. No phage can be detected after lysis of the cells by lysozyme.     

Effects of stirring and sparging on cultured hybridoma cells

The response of hybridoma cells to fluid shear caused by stirring and sparging has been investigated in a 2-L turbine-agitated bioreactor. Viable cell count, lactate dehydrogenase (LDH) release, and antibody secretion were measured over the course of batch culture experiments under varied conditions of stirring and gas sparging. The effectiveness of Pluronic F68 as a protective agent in sparged cultures was also studied. Growth was found to be unaffected by stirring of the culture under surface aerated conditions, but gas sparging had a significant detrimental effect on growth and antibody production. The effect of sparging was reduced when cultures were supplemented with Pluronic at a level of 0.4% (w/v). Experimental data were analyzed through formulation of models for LDH release and antibody production. Rates of cell lysis could be estimated by correlating extracellular LDH levels through the model for LDH release. The lysis rate estimated for sparged conditions was sufficiently large to approximately account for the observed decrease in the specific growth rate of the culture. The presence of Pluronic apparently interfered with the LDH release mechanism, so precise estimation of lysis rates under these conditions was not possible. Sparging was found not to have a detrimental effect on antibody production in cultures without Pluronic added. Specific antibody production rates in cultures supplemented with Pluronic were about 25% higher than in sparged cultures without Pluronic added.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

Newly isolated Nodularia phage influences cyanobacterial community dynamics

Cyanophages, that is, viruses infecting cyanobacteria, are a key component driving cyanobacterial community dynamics both ecologically and evolutionarily. In addition to reducing biomass and influencing the genetic diversity of their host populations, they can also have a wider community‐level impact due to the release of nutrients by phage‐induced cell lysis. In this study, we isolated and characterized a new cyanophage, a siphophage designated as vB_NpeS‐2AV2, capable of infecting the filamentous nitrogen fixing cyanobacterium Nodularia sp. AV2 with a lytic cycle between 12 and 18 hours. The role of the phage in the ecology of its host Nodularia and competitor Synechococcus was investigated in a set of microcosm experiments. Initially, phage‐induced cell lysis decreased the number of Nodularia cells in the cultures. However, around 18%–27% of the population was resistant against the phage infection. Nitrogen was released from the Nodularia cells as a consequence of phage activity, resulting in a seven‐fold increase in Synechococcus cell density. In conclusion, the presence of the cyanophage vB_NpeS‐2AV2 altered the ecological dynamics in the cyanobacterial community and induced evolutionary changes in the Nodularia population, causing the evolution from a population dominated by susceptible cells to a population dominated by resistant ones.     

High growth rate and substrate exhaustion results in rapid cell death and lysis in the thermophilic bacterium Geobacillus thermoleovorans

Batch cultures of the thermophilic bacterium Geobacillus thermoleovorans T80 attained extremely high-specific glucose utilization rates leading to high specific growth rates, followed by extensive cell death and lysis with the onset of substrate exhaustion. The dramatic decrease in live cell numbers, as determined by flow cytometry, was accompanied by the release of soluble protein. Once the growth phase reached the point of commitment to lysis created by the impending exhaustion of substrate, the addition of extra carbon substrate did not halt the rapid death rate and lysis, although, towards the end of the exponential growth phase, the substrate was utilized producing only a small additional biomass concentration as a result of the net effect of cell growth and death. This lytic phenomenon was observed when a range of different carbon substrates (glucose, pyruvate, acetate, n-hexadecane, nutrient broth), as well as ammonium (the nitrogen source) in the presence of excess carbon source, reached near exhaustion. The rate and extent of cell death and the ensuing lysis depend on the culture growth rate. Cultures batch grown with a lower initial substrate concentration, or at a lower temperature, or at lower dilution rates for continuous-flow cultures, exhibited a lower rate and extent of cell death and lysis. Batch re-culture of the persister cells resulted in a behavior identical to that of the original culture indicating that these cells were not genetically modified. The glucose utilization, cell growth and death rates were mathematically described based on Monod kinetics and estimated values of pertinent biokinetic constants are reported.     

Effect of light on the attachment of cyanophage AS-1 to Anacystis nidulans.

The effect of illumination on the extent and kinetics of the adsorption of cyanophage AS-1 to the blue-green alga (cyanobacterium) Anacystis nidulans was studied by using 32P-labeled phage. The initial rate of adsorption was not significantly affected by light. However, at Na+ levels used ordinarily to culture the alga ([Na+] = 11.7 mM), the total amount of phage adsorbed was doubled in the illuminated cultures, as compared with the dark-grown ones, over a wide range of multiplicities of infection (0.05 to 20). Upon a 10-fold increase in Na+ concentration in the medium ([Na+] = 0.11 M), the dark adsorption of the phage increased to the level of light adsorption found in low Na+ medium. The effects on phage adsorption of high Na+ concentration and light were not additive.     

Lysis of lysis-inhibited bacteriophage T4-infected cells.

T4 bacteriophage (phage)-infected cells show a marked increase in latent-period length, called lysis inhibition, upon adsorption of additional T4 phages (secondary adsorption). Lysis inhibition is a complex phenotype requiring the activity of at least six T4 genes. Two basic mysteries surround our understanding of the expression of lysis inhibition: (i) the mechanism of initiation (i.e., how secondary adsorption leads to the expression of lysis inhibition) and (ii) the mechanism of lysis (i.e., how this signal not to lyse is reversed). This study first covers the basic biology of the expression of lysis inhibition and lysis of T4-infected cells at high culture densities. Then evidence is presented which implies that, as with the initiation of lysis inhibition, sudden, lysis-associated clearing of these cultures is likely caused by T4 secondary adsorption. For example, such clearing is often observed for lysis-inhibited T4-infected cells grown in batch culture during T4 stock preparation. The significance of this secondary adsorption-induced lysis to wild T4 populations is discussed. The study concludes with a logical argument suggesting that the lytic nature of the T4 phage particle evolved as a novel mechanism of phage-induced lysis.     

Flow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters

Flow cytometry has been used to study the contents of macromolecular compounds and light-scatter parameters in batch and continuous cultures of a recombinant Escherichia coli strain that forms protein inclusion bodies. Changes in relative DNA and RNA contents and cell mass as estimated by forward-angle light scatter were detected and tightly correlated in batch culture. In addition, heterogeneity of wide-angle light scatter (WALS), which we related to the presence of cellular inclusion bodies, was observed. In contrast, the relative RNA content and cell mass did not change during continuous culture, and homogeneity of WALS was found. In addition, unexpected changes in relative DNA content were observed after 67 h of culture, indicating a change in bacterial physiology.     

Rifamycin: XXXIII. Isolation of Actinophages Active on Streptomyces mediterranei and Characteristics of Phage-Resistant Strains

Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.     

Rifamycin: XXXIV. Physicochemical Characterization of Actinophages Active on Streptomyces mediterranei

Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.     

Elevated Lytic Phage Production as a Consequence of Particle Colonization by a Marine <Emphasis Type="Italic">Flavobacterium</Emphasis> (<Emphasis Type="Italic">Cellulophaga</Emphasis> sp.)

Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."     

Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.     

Exopolysaccharide production by the cyanobacterium Anabaena sp. ATCC 33047 in batch and continuous culture

The halotolerant, filamentous, heterocystous cyanobacterium Anabaena sp. ATCC 33047 released, during the stationary growth phase in batch culture and, at low dilution rate, in continuous culture, large amounts of an exopolysaccharide (EPS) to the culture medium. Different environmental, nutritional and physical parameters affected production and accumulation of the EPS. The presence of either a combined nitrogen source or NaCl at high concentration led to decreased EPS production, without affecting cell growth. In contrast, generation of the EPS was markedly enhanced in response to an increase in either air flow rate, temperature or irradiance. In continuous culture, accumulation of EPS in the medium increased in response to a decrease in the dilution rate, with maximal EPS productivity being reached at a dilution rate of 0.03 h⁻¹.     

pH-controlled continuous cultivation of mycoplasmas.

The continuous cultivation of mycoplasmas in a pH-controlled metabolistat was investigated with the fermentative strain Mycoplasma mobile 163K and the nonfermentative strain Mycoplasma arthritidis ISR1. The addition of medium and the removal of culture suspension were regulated by acid production from glucose by M. mobile 163K and by ammonium production from arginine by M. arthritidis ISR1, respectively. For both strains the optimal pH for continuous growth was 7.0. The steady state could be maintained for at least 21 days. With CFU of 8.4 X 10(9) ml-1 (M. mobile 163K) and 3.2 X 10(9) ml-1 (M. arthritidis ISR1), the cell concentrations were slightly higher than those obtained in batch cultures. The dependence on the adjusted pH values was measured for several parameters, such as flow rate, CFU, glucose fermentation or production of ammonia, and gliding velocity. Since the long lag phases of batch cultures can be avoided, pH-controlled continuous cultures provide an appropriate system for the production of mycoplasma cells.