T4 cannot produce a burst in stationary phase cells, but little is known about what happens when phage encounters such cells. Traditionally, research of stationary phase bacteria has not been a priority. It has been seen as a confusing stagnant period leading to "death". More recently, however, it has been found that cells can endure long periods deprived of previously-thought essential substances. In fact, up to 10⁷ colony forming units per mL have been observed after one year of incubation (1). Deprivation conditions are probably quite prevalent in the natural environment.

Stationary phase bacteria have their own unique morphology and physiology. At the onset of stationary phase, the composition of the cell undergoes chemical and structural changes. The periplasmic space increases and the entire cell shrinks, becoming both more compact and spherical. Three different forms of RNA polymerase holoenzyme are detected, all with distinctly altered promoter specificity. The unsaturated fatty acids from cell membranes are converted to cyclopropyl derivatives. (2) The ratio between phosphatidylglycerol and phosphatidylethanolamine increases from about 0.3 to 0.9 as the cells approach stationary phase. The peptidoglycan layer also changes, potentially contributing to an autolytic resistance which has been observed to protect stationary phase cells from penicillin, trichloroacetic acid, and cycles of freeze-thaw (3).

T4 infection of exponentially growing E. Coli cells completely disrupt the host genome structure and expression; transcription is terminated, DNA replication disrupted, host DNA degraded, translation cut off, and host mRNA dismantled. Little is known about how these mechanisms are affected when T4 infects stationary cells. It is important to note that infected stationary cells do not lyse, even when chloroform has been added. However, at least 20 minutes after infection, infective centers were observed when cells were diluted and plated on a rich medium. It is unknown what state T4 are in during stationary phase infection, and mechanisms allow the formation of infective centers when the cell is reintroduced to rich medium.

During starvation, protein synthesis drops to approximately 20% of the initial rate (4) observed during exponential growth, then stabilizes for at least the next 47 hours. Transcription of proteins induced during starvation is controlled by alternate sigma factors. Thirteen proteins common to several different forms of nutrient deprivation have been detected. Acetate, which accumulates towards the end of log phase, may act as an activator of these sigma factors (5). Many early-starvation proteins appear to be proteases and peptidases (6); this data supports results claiming that protein turn-over increases 5-fold in starved E. coli cells. Excess dimerized ribosomes, resistant to degradation during stationary phase, separate upon addition of nutrients to the environment; these free ribosomes may then act as an energy source for endogenous metabolism. (7).

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Purpose

The purpose of our experiments is to explore the production of proteins in stationary phase infections and compare these to proteins produced during exponential phase infections. The experiments done were carried out under nutrient exhaustion in minimal media utilizing glucose, acetate, or glycerol separately as energy sources.

We have tested for trends in unadsorbed phage and infective center counts in infections of stationary phase cells. Protein patterns on 1D gels of stationary phase were analyzed and compared to 1D gels of log phase proteins, both infected and noninfected cells. In high MOI infections (10 phage/bacterium), protein patterns reveal the repression of host protein synthesis. In low MOI infections, this pattern has not been confirmed. Unadsorbed phage counts, and the production of infective centers in low MOI stationary infections, decreased as a function of elapsed time when samples were plated on rich media. The majority of phage rapidly adsorbed during stationary phase infections; however, a residual number of phage do not produce infective centers, nor do they appear as unadsorbed phage. This phenomenon may be explained as an example of reversible adsorption.

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Materials and Methods

Media: We supplemented basic M-9 salts with .02% casamino acids, .6% glycerol, .1% of each of the following salts: CaCl2, MgSO4, FeCl3, ZnCl2.

Bacteria: We used W3110 taken from a plate and grown in M9 complete.

See Protocols.

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Goals for Future Research

Experiments will soon be undertaken using phage anti-serum in conjunction with high MOI infections to more precisely determine the trends concerning infective centers and unadsorbed phage. Further research into the rate, stability, and window of viability for phage infection would greatly help illuminate the confounding mechanisms involved, such as "pseudo-lysogeny" which has been observed in Tbilisi, Georgia by Chanishvili. We hope to identify phage proteins in stationary phase and compare them to both bacterial and log phase proteins. This inquiry raises many interesting questions that have profound implications regarding the life cycles of phage and bacterium in the natural world.

We hope, based upon our results on the 1D gels, to identify proteins expressed during T4 infection of stationary phase E. coli. Ultimately, we would like to carry out experiments based on other conditions we believe to be characteristic of E. coli's natural environment. Oxygen starvation, alternate food sources, and bacterial genotype are all variables that we would like to explore. We have been using radioactive amino acids to label our proteins, assuming that those amino acids are incorporated. Unfortunately, however, our amino acids could have been broken down to carbon monomers and utilized via gluconeogenesis. Using mutant strains of E. coli incapable of this conversion could eliminate this possibility.

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Acknowledgments

We would like to extend our gratitude towards Pia Aronsson, Barbara Anderson, and the rest of the T4 staff for their patient help and enduring support in our endeavours.

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References

1. Kolter, Roberto. 1992. Life & Death in Stationary Phase. ASM News. 58:75-79

2. Cronan, John E. 1968. Phospholipid Alterations during growth of E. Coli. Journal of Bact. 95:2054-61.

3. Tuomanen, E., Z Markiewicz, and A. Tomasz. 1988. Autolysis-Resistant Peptidoglycan of Anomalous Composition in Amino-Acid-Starved E. Coli. Journal of Bact. 170:1373-76

4. Reeve, Carole A., Penny S. Amy, and Abdul Matin. 1984. Role of Protein Synthesis in the Survival of Carbon-Starved E. Coli K-12. Journal of Bact. 160:1041-46

5. Kolter, Roberto. 1993. The Stationary Phase of the Bacterial Life Cycle. Annu. Rev. Microbiol. 47:855-74

6. Groat, R., J. E. Schultz, E. Zychlinsky, A. Bockman, and A. Matin. 1986. Starvation Protein in E. Coli: Kinetics of Synthesis and Role in Starvation Survival. Journal of Bact. 168:486-493

7. Siegele, Deborah, and Roberto Kolter. 1992. Life After Log. Journal of Bact. 174:345-48

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Resources on the Web

2D gels of stationary phase E. coli by Andy Link at Harvard Medical School.

The Ecology of T4 bacteriophage.

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