1. Cost of Unneeded Protein in E.coli is Reduced after Several Generations in Exponential Growth Irit Shachrai,Alon Zaslaver,Uri Alon, and Erez Dekel Murugappan Nachiappan April 16, 2015

2. Roadmap Introduction Protein Cost and Impact Experiments ppGpp Systems Results Future Research

3. Introduction

4. E. coli serves as host for protein synthesis Protein cost: E. coli express unneeded proteins during process of synthesis Rate of protein cost varies during exponential growth phase After several generations cells enter a phase with much reduced cost ppGpp system and ribosomal activity are studied with respect to protein cost

5. Protein Cost and Impact

6. Protein cost and Impact What is Protein Cost or Burden? Expression of unneeded protein in E. coli leads to reduced cell growth rate. “Fractional reduction in growth rate due to limited cell resources such as ribosomal capacity” This diverts the resource from producing other beneficial proteins

7. Study of Protein Cost Understanding protein cost is important for improving the use of cells to produce desired proteins. Dong H, Nilsson L and Kurland (1996) experimental measurements showed cost as a function of the levels of the unneeded protein is not a linear function but rather grows faster than linear. Stoebel et al.(2008) studied sources of protein cost in E. coli suggested that cost originates in the production process and not in the products.

8. Background

9. Experiment

10. System for Measuring Protein Cost Unneeded Protein : GFPmut3 (Green Fluorescent Protein) Inducer: Anhydrotetracycline – aTc C 22 H 22 N 2 O 7 HCl System with several plasmids identical except for point mutations in the ribosomal binding sites of gfp gene. Bacteria were grown overnight in a defined medium, M9 + 0.2% glycerol Cell density (OD) and GFP fluorescence were measured every 8 min in a robotic system in 96-well plates. Induced strain was compared to a strain with the same plasmid but without aTc in alternating wells on the 96-well plate. Growth rate was evaluated at each time point by the logarithmic temporal derivative of the OD, g = d(log(OD))/dt.

11. Initial Observations Growth rate of E. coli cells producing Green Fluorescent Protein GFP, an unneeded protein is measured High protein cost observed in early exponential growth After a few generations still in exponential growth, cells enter a phase in which cost is reduced. Though the cost is low, GFP accumulation continues to increase.

12. Further Experiments Experiment was repeated in E. coli cells transformed with 5 variants of the plasmid that had mutated RBSs and thus produced GFP at different rates ranging from about 5% to 15% of total cell protein. Variants followed the same pattern: Phase 1: exponential growth show a high cost of GFP production Phase 2: Cost reduces to near zero Amount of GFP produced has a slight effect on the duration of phase 1: Phase 1 lasts for 2 generations in the variant with lowest expression and three generations in the variant with highest expression.

13. Experiment Observations Measured GFP production rate of the induced and un-induced bacteria. GFP production is high during the first growth phase and further increases as the bacteria enters into the second growth phase. For 3 generations, cells continued their exponential growth and increased their GFP production Beyond this, cost of GFP production reduced to a very low level.

14. Experimental Results Circles are data from plasmids with variant ribosomal binding sites (RBSs) in the GFP gene along with standard errors from 48 parallel measurements in 96-well plates.

15. Growth in Richer and Poorer Medium Rich Medium: M9C  M9 medium + Amino acids Same results observed only difference is it takes more generations (6) to reach phase 2. Poor Medium: Diluted M9 medium Similar results as M9 observed Observation: Time to reach Phase 2 depends on concentration. Higher concentration leads to more growth rate.

16. Background

17. Ribosomal Gene Cost of protein production thought to be dependent on ribosomal genes. Activities of 4 ribosomal RNA promoters – rrnA, rrnB, rrnC, rrnH were measured. Ribosomal promoters are highly active to supply more ribosomes in Phase 1 Ribosome production reduced to for balanced growth in Phase 2

18. ppGpp Systems ppGpp: Guanosine pentaphosphate is an alarmone which is involved in the stringent response in bacteria, causing the inhibition of RNA synthesis when there is a shortage of amino acids present. Alarmone: molecule synthesized due to harsh environmental factors in bacteria. Stringent Response: causes cells to divert resources from growth of cells to secretion amino acid. Ribosomal gene regulation: reduced cost occurs when ribosomes are not only limiting factors for cell growth. ppGpp system is required for growth with reduced cost.

19. Experiment based on ppGpp systems Bacteria starts with low level of ribosome and ppGpp Ribosome production is maximal and limits the growth After 3.5 h, ribosomes increases to a level that they are not only limiting factor Growth Limitation shifts to non ribosomal proteins. ppGpp increases as there are excess of ribosomes leading to stalled ribosomes Hence ribsomal protein concentration reduces and ppGpp level increases till then After 7 h, ribosomal protein count is so low, it starts limiting growth. Hence ppGpp again reduces.

20. Experiments based on ppGpp Regulation (A)Ribosome concentration (B) ppGpp concentration (C) Limiting factors: +ve: ribosomal limit -ve: nonribosomal limit Growth begins with ribosomal limitations leads to balanced growth (D) Bacteria cell count: increases in steady state

21. Experiments based on ppGpp Regulation Growth limitation in balanced growth rapidly switches between ribosomes and non- ribosomal proteins. Protein cost depends on ribosomes, it is high in the first phase when ribosomes are limiting and lower in later phase where ribosomes are limiting only part of the time.

22. Result

23. Ribosomes are not the only limiting factor: When cells reach balanced growth non-ribosomal protein is limiting along with ribosome. Unneeded proteins has a smaller effect on cost in balanced phase as ribosomes are not only limiting.

24. Future Research

25. Alternatives theories for reduced cost Limiting factor in first phase is not due to ribosomes but chaperones or other cellular machinery regulated by ppGpp. It’s possible for proteins or mRNAs compete as limiting factors to add up cost. Source: (Tagkopoulos et al., 2008; Mitchell et al., 2009).

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