Influence of infected cell growth state on bacteriophage reactivation levels

Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation. Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold. This enhanced reactivation capacity was correlated with the ca. 30-fold-greater UV-C resistance of P. aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation. The dark repair capacity of P. aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined. For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection. For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1. 5 years, respectively. Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential.     

Chemical composition of Enterococcus faecalis in biofilm cells initiated from different physiologic states

Enterococcus faecalis is a ubiquitous bacterium of the gut that is observed in persistent periradicular infections. Its pathogenicity is associated with biofilm formation and the ability to survive under nutrient-poor (starvation) conditions. However, characteristics of chemical composition of biofilm cells developed by starved E. faecalis cells remain poorly understood. In this study, E. faecalis cells in exponential, stationary, and starvation phases were prepared and separately cultured to form biofilms. Confocal laser scanning microscopy was performed to verify biofilm formation. Raman microscopy was used to investigate the chemical composition of cells within the biofilms. Compared to cells in exponential or stationary phase, starved cells developed biofilms with fewer culturable cells (P?E. faecalis.     

Caffeine and artificial insemination

We studied the reactivation of cells and the repair of photomutagenic damage induced by xanthotoxin and visnagin plus NUV in arg-1 cells of Chlamydomonas reinhardii. Maintenance of liquid cultures in the dark resulted only in a slight reactivation of cells, even after 24 h. Repair of photomutagenic damage was more efficient: within 24 h the number of Arg⁺ revertants was reduced by 50% in cells cultured in the dark at 20°C. The repair was more efficient at 30°C. At the beginning of dark cultivation an after-effect could be observed. Cultivation in standard white light instead of dark after treatment resulted in a very strong after-effect. Therefore it was not possible to detect any photoreactivation.After treatment with xanthotoxin plus standard white light (24 h) neither reactivation of cells nor repair of photomutagenic damage was found. The after-effect was higher than after xanthotoxin plus NUV. It is possible that a small amount of repair could be masked by the after-effect.Treatment with visnagin yielded similar results. The photomutagenic effect of visnagin is described for the first time in this paper. The drug is a much less effective photomutagen than xanthotoxin. The photomutagenesis of visnagin may be attributable to photoproducts similar to those formed after treatment with furocoumarins.No definite conclusion can be drawn from the present results regarding the basis for the observed lack of repair (or reduced repair) after standard white light treatment; a possible cause might be a preferential formation of bi-adducts under these conditions.     

Marine Bacterial Isolates Display Diverse Responses to UV-B Radiation

The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except Sphingomonas sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for Vibrio natriegens when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.     

The control of dark repair mechanisms in meiotic cells

Summary The responses to UV irradiation of pre-meiotic and meiotic cells of the unicellular green alga Chlamydomonas reinhardi have been examined in a wild type and a UV-sensitive mutant (UVS1) strain. During late interphase and early prophase the cells become highly sensitive to UV under dark conditions, but show much less change in sensitivity following photoreactivation (PR). At these times the effect of PR is not dose-modifying, but very large PR factors are obtained — at the 50% survival level in some stages values up to 23 being obtained. The enhancement of dark sensitivity is attributed to a diminution in dark repair activity, and it is suggested that this is associated with the process of genetic recombination that occurs in these meiotic cells. In addition at those times when the dark repair capacity is at a low level, a phenomenon akin to UV reactivation occurs, in that within a certain dose range, survival increases with increasing dose. The possible basis of this phenomenon is discussed.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

Indicators for photoreactivation and dark repair studies following ultraviolet disinfection

Repair of DNA in bacteria following ultraviolet (UV) disinfection can cause reactivation of inactivated bacteria and negatively impact the efficiency of the UV disinfection process. In this study, various strains of E. coli (wild-type, UV-resistant and antibiotic-resistant strains) were investigated for their ability to perform dark repair and photoreactivation, and compared based on final repair levels after 4 h of incubation, as well as repair rates. Analysis of the results revealed that the repair abilities of different E. coli strains can differ quite significantly. In photoreactivation, the log repair ranged from 10 to 85%, with slightly lower log repair percentages when medium-pressure (MP) UV disinfection was employed. In dark repair, log repair ranged from 13 to 28% following low-pressure (LP) UV disinfection. E. coli strains ATCC 15597 and ATCC 11229 were found to repair the fastest and to the highest levels for photoreactivation and dark repair, respectively. These strains were also confirmed to repair to higher levels when compared to a pathogenic E. coli O157:H7 strain. Hence, these strains could possibly serve as conservative indicators for future repair studies following UV disinfection. In addition, dimer repair by photoreactivation and dark repair was also confirmed on a molecular level using the endonuclease sensitive site (ESS) assay.     

Sizes of G1 and G2 populations in starved Tetrahymena

Feulgen cytophotometry and autoradiography were used to study DNA content and DNA synthesis in starved and starved-refed Tetrahymena pyriformis GL-C. It was found that (1) the cell population shows a limited increase in cell number during starvation and this increase is restricted to the first 7 h of starvation; (2) at the end of starvation, there is a portion of the cell population whose DNA content is similar to that for standard G2 cells; (3) a significant portion of the dividing cells at the first division following refeeding in the presence of [³H]TdR are unlabeled; (4) these unlabeled cells are among the first to divide and, upon division, generally enter into a cell cycle either lacking a G1 phase or with a shortened G1 phase.     

DNA repair and survival in UV-irradiated chicken embryo fibroblasts

The role of the pyrimidine dimer in cell killing, DNA synthesis and repair has been studied by utilizing the light-requiring DNA-repair mechanism of photo- reactivation in UV-irradiated chicken-embryo fibroblasts. Survival, as measured by colony-forming ability at 41°C, is increased in cells left in the light. The initial inhibition of DNA synthesis by UV is much less in light-treated cells, and levels reach that of unirradiated controls much faster than when the cells are left in the dark. The number of endonuclease-sensitive sites (dimers)_measured by an assay with a crude extract from M. luteus, rapidly decreases as the cells are allowed to photoreactive. However, in the dark, significant amounts of repair also occur, but at a much lower rate and with a lag phase of several hours. Unscheduled DNA synthesis occurs to a similarly low extent in both dark- and light-treated cells, confirming the finding that some amount of excision repair occurs that is light-independent. When survival is examined as a function of the number of dimers present, the dimers, not the non-dimer products, appear to be responsible for cell killing. In this study, the removal of dimers in vivo by photoreactivation has made it possible to demonstrate directly that dimers are primarily responsible for the deleterious effects of UV on DNA synthesis and survival.     

Clostridium cellulolyticum Viability and Sporulation under Cellobiose Starvation Conditions

Depending on the moment of cellobiose starvation, Clostridium cellulolyticum cells behave in different ways. Cells starved during the exponential phase of growth sporulate at 30%, whereas exhaustion of the carbon substrate at the beginning of growth does not provoke cell sporulation. Growth in the presence of excess cellobiose generates 3% spores. The response of C. cellulolyticum to carbon starvation involves changes in proteolytic activities; higher activities (20% protein degradation) corresponded to a higher level of sporulation; lower proteolysis (5%) was observed in cells starved during the beginning of exponential growth, when sporulation was not observed; with an excess of cellobiose, an intermediate value (10%), accompanied by a low level of sporulation, was observed in cells taken at the end of the exponential growth phase. The basal percentage of the protein breakdown in nonstarved culture was 4%. Cells lacking proteolytic activities failed to induce sporulation. High concentrations of cellobiose repressed proteolytic activities and sporulation. The onset of carbon starvation during the growth phase affected the survival response of C. cellulolyticum via the sporulation process and also via cell-cellulose interaction. Cells from the exponential growth phase were more adhesive to filter paper than cells from the stationary growth phase but less than cells from the late stationary growth phase.     

TOR controls translation initiation and early G1 progression in yeast.

Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early G1 phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (G0). A G1 cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced G1 arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the G1 arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, G1 progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early G1 progression and growth in the absence of nutrients.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.

This study analyzes the effects of polyamine starvation on cell cycle traverse of an arginase-deficient CHO cell variant (CHO-A7). These cells grow well in serum-free medium, provided that it contains ornithine or polyamines or both. In the absence of ornithine or polyamines or both, the CHO-A7 cells develop severe polyamine deficiency and, as a consequence, grow more slowly. When grown to a stationary phase in the presence of ornithine or putrescine or both, the CHO-A7 cells became arrested in G0/early G1. However, when starved for ornithine and polyamines, they accumulated in the S and G2 phases. Ornithine and polyamine starvation of CHO-A7 cells causes an increase in ornithine decarboxylase activity. When this increase was prevented by treatment with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, growth was further suppressed, and a greater fraction of cells were found in the S and G2 phases of the cell cycle.     

Studies of the cell cycle of in vitro cultured skin fibroblasts in goats: work in progress

The effect of serum starvation and olomoucine treatment on the cell cycle and apoptosis of goat skin fibroblasts cultured in vitro is reported in this paper. The cells were obtained from the ear of a female goat 1.5 years of age. Analysis of cell cycle distribution by fluorescence-activated cell sorting (FACS) showed that 3.4, 60.8 and 15.1% of normally cycling cells were at G1, G0 and S phase, respectively. Serum starvation for 1, 3 and 5 days arrested 70.1, 70.2 and 83.4% cells, respectively, at G0/G1 phase. Seventy-eight percent of confluent cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Of cells not deprived of serum, 73.6% were arrested at G1/G0 when treated with 100 microM olomoucine for 9 h compared to 85.5% of cells that had been starved of serum for 2 days (co-inhibition) (P<0.01). After co-inhibition, 45% of cells entered S phase when re-cultured in normal medium for 5 h, indicating that the inhibition was reversible. Under normal culture conditions, 1.2% of cells underwent apoptosis. Serum starvation for 1, 2, 3, 5 and 10 days caused apoptosis in 1.7, 3.9, 4.5, 11.7 and 90.3% of cells, respectively. Treatment with 100 microM olomoucine for 9h did not increase the number of apoptotic cells significantly (1.9%, P>0.05). When cells were co-inhibited, 4.1% of cells underwent apoptosis. In conclusion, although serum withdrawal for 5 days or more effectively arrested cells at G0/G1 stages, it increased apoptosis of cells significantly. However, co-inhibition by serum withdrawal and olomoucine treatment was found to be an appropriate treatment to obtain more healthy G0/G1 cells based on the low percentage of apoptotic cells after treatment.     

Survival of parental and genetically modified derivatives of a soil isolated Pseudomonas fluorescens under nutrient-limiting conditions

Wild type, a rifampicin-resistant mutant and three genetically modified derivatives of the soil isolate Pseudomonas fluorescens R2f were starved in pure cultures for periods of up to 70 d. Cells were starved after harvesting at a point early in the stationary phase of the growth curve and all five strains demonstrated the ability to survive nutrient deprivation and resuscitate rapidly when growth nutrients became available. No difference in total counts and metabolic activity was detected between the strains. Plate counts were similar for all strains up to day 35. Wild type and the rifampicin-resistant mutant strain showed greater recovery than the genetically modified strains on day 70. During the starvation period there was a significant decrease in cell lengths of all five strains, however, there was no significant difference between the strains. The shape of the starved cells varied with the growth phase at which they had been harvested. Cells taken from early stationary phase and starved produced predominantly rod-shaped cells whereas those taken from early log phase and starved produced small round cells. In experiments when the rifampicin-resistant mutant and the genetically modified strain Art-3 were starved at early log phase the cells were significantly smaller than respective cultures not exposed to the nutrient limiting conditions, and there was no significant difference in the response of the two strains. None of the cultures produced ultramicrobacteria, and none of the cultures entered a non-culturable state. Starvation at different cell densities did not affect the recoverability of the cells. The results of this study demonstrate that responses to starvation conditions by the genetically modified and parental strains are similar.     

The novel gene trs1 encodes an essential protein for the transition from mitotic cell cycle to resting state in Schizosaccharomyces pombe

A novel gene trs1 in the fission yeast Schizosaccharomyces pombe has been genetically defined. The trs1 mutant showed several intriguing phenotypes. Cells were sensitive to starvation and rapidly lost viability in the stationary phase; cells in the stationary phase were sensitive to heat shock. Some heat-shock proteins were not induced and the heat-shock response in log-phase cells was defective. These mutant phenotypes strongly suggest a vital function of the trs1 gene product for transition from the G1 to G0 phase on starvation and for the normal heat-shock response.     

Viability and Endogenous Substrates Used During Starvation Survival of Rhodospirillum rubrum

Cells of Rhodospirillum rubrum were grown photoorganotrophically and chemoorganotrophically and then starved for organic carbon and combined nitrogen under four conditions: anaerobically in the light and dark and aerobically in the light and dark. Illumination prolonged viability and suppressed the net degradation of cell material of phototrophically grown cells, but had no effect on chemotrophically grown cells that did not contain bacteriochlorophyll. The half-life survival times of carbohydrate-rich phototrophically grown cells during starvation anaerobically or aerobically in the light were 17 and 14.5 days, respectively. The values for starvation aerobically and anaerobically in the dark were 3 and 0.5 days, respectively. Chemotrophically grown cells had half-life survival times of 3 and 4 days during starvation aerobically in the light and dark, respectively, and 0.8 day during starvation anaerobically in the light or dark. Of all cell constituents examined, carbohydrate was most extensively degraded during starvation, although the rate of degradation was slowest for phototrophically grown cells starved anaerobically in the light. Phototrophically grown cells containing poly-beta-hydroxybutyrate as carbon reserve were less able to survive starvation anaerobically in the light than were carbohydrate-rich cells starved under comparable conditions. Light intensity had a significant effect on viability of phototrophically grown cells starving anaerobically. At light intensities of 320 to 650 lx, the half-life survival times were 17 to 24 days. At 2,950 to 10,500 lx, the survival times decreased to 1.5 to 5.5 days. The kinetics of cell death correlated well with the rate of loss of cell mass of starving cells. However, the cause of death could not be attributed to degradation of any specific cell component.     

TL,the new bacteriophage of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and its application for the search of halo-producing bacteriophages

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.