Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
CLASSROOM S Mahadevan Survival in Stationary Phase Department of Molecular Reproduction, Development and Genetics Indian Institute of Science Bangalore 560 012, India. Email: [email protected] Keywords E. coli, GASP. The challenges faced by microorganisms in their natural environments are much more than what they may experience under controlled growth conditions in the laboratory. While they are provided with abundant nutrients and stable conditions such as temperature and aeration in the laboratory, most microorganisms are subjected to prolonged starvation and other types of stress in their natural habitats. In an attempt to understand microbial physiology under conditions seen in nature, there has been a resurgence of interest in what is generally known as ‘stationary phase’ where laboratory cultures of bacteria apparently cease to grow. What happens if a culture of an organism such as Escherichia coli is left shaking in the flask without the addition of fresh medium for several days? For most students of microbiology, the answer is simple – death. They are taught that the culture will enter what is known as the death phase where the curve plunges sharply and hits zero at some point (Figure 1). Can one verify this experimentally? This is a simple enough experiment to do in any reasonably equipped college microbiology laboratory. All one has to do is to keep measuring the viable count for several days after the onset of stationary phase. If one attempts to do this simple experiment, one will be in for many surprises. Cell number 109 108 Stationary phase 107 Death phase 106 105 104 Exponential phase 103 Lag phase Figure 1. A typical ‘text book’ bacterial growth curve. 92 1 2 3 Time (days) RESONANCE July 2007 CLASSROOM Contrary to expectations, it has been shown that laboratory cultures of E. coli grown in standard Luria-Bertani medium stabilize and show a constant cell count that is about 1% of the original titre [1]. If one started with 108 cells per ml, this will be still a million cells! How do they survive prolonged starvation and osmotic and pH stress? This happens in three phases. At the onset of starvation, several physiological changes take place in the cell. The overall size of the cell reduces, the cytoplasm is more condensed and the peptidoglycan cell wall has a higher degree of cross-linking. These result in higher tolerance to osmotic, pH and temperature stress. Most of these changes are primarily due to the induction of a large number of genes triggered by the product of the rpoS gene that codes for the stationary phase – specific sigma factor S. This form of microbial “differentiation” is reminiscent of endospore formation in Gram-positive organisms such as bacilli at the onset of starvation. But what happens to the population upon prolonged starvation in stationary phase? How do cell numbers stabilize? The answer to this question is more difficult, at the same time more interesting. Cell numbers stabilize after the majority of cells die during the death phase. This happens after 2-3 days of starvation [2]. The survivors of prolonged stationary phase (beyond three days) undergo a phenomenon that has been aptly named GASP, which is an acronym for Growth Advantage in Stationary Phase. The GASP phenomenon is a typical case of Darwinian evolution – “survival of the fittest”. Rather than being dormant, the population of cells in stationary phase is highly dynamic. Mutations occur periodically and those that confer an advantage to the cells by enabling them to grow on the available resources from the dying cells get selected. Those cells that carry such mutations will now be able to grow and multiply at the cost of the dying cells – a case of microbial cannibalism. Early stationary phase cultures may have fewer such mutations and as the cultures age, there is progressive accumulation of more such mutations. During prolonged stationary phase, many successive population turnovers occur (Figure 2). Several subpopulations of GASP mutants may coexist in aged cultures and as a result, they will RESONANCE July 2007 93 CLASSROOM Cell number 109 108 107 106 105 GASP2 GASP3 GASP4 GASP1 104 103 3 4 5 6 7 8 9 10 Time (days) Figure 2. The GASP phenomenon in prolonged stationary phase characterized by successive population takeovers. be genetically heterogeneous compared to the starting cultures. Interestingly, the first GASP mutation seen is in the rpoS locus that results in a S that shows reduced activity. How such a mutation helps the cell survive is still not clear, but it has been shown that it leads to enhanced metabolic capabilities. The fact that the phenomenon has a genetic basis and is not simply a case of microbial adaptation has been demonstrated by the fact that GASP mutations can be propagated even after switching to exponential growth. In addition, when the mutant locus is introduced into the parent strain that has not undergone starvation, the stain can now exhibit the GASP phenotype right away without having to undergo selection. A relatively simple experiment can show that starved populations of bacteria are dynamic. One needs two isogenic strains of bacteria that carry two different selectable markers such as resistance to tetracycline or kanamycin (Strains A and B). These markers must be neutral which can be verified by a simple competition experiment. Grow the two cultures A and B separately to stationary phase in LB medium and mix equal proportions of cells without addition of fresh medium. Allow the cultures to grow for several days and monitor the viable count of each strain by plating on tetracycline and kanamycin-containing plates. Once the markers are shown to be neutral, one can mix culture A and B that have been grown for different days (for example 1 day old culture A with 10 day old 94 RESONANCE July 2007 CLASSROOM culture B) and do similar competition experiments. This can be done reciprocally with different ratios of A and B in different experiments. If the 10-day old culture can out-compete the one day old culture, it is an indication that the former has more GASP mutants than the latter. This can be repeated with cultures that have been in stationary phase for different number of days. As the cultures age, one can also look at colonies of the survivors to record if there are any morphological variations. If the laboratory has a PCR machine, one can also attempt to find differences at the genomic level by using methods such as RAPD (this involves amplification of different segments of the genomic DNA using random pairs of PCR primers). The genetic characterization of loci that confer a GASP phenotype will be the real challenge. So far, only very few such loci conferring GASP phenotype have been characterized at the functional level [2]. Thus studying aged cultures in the laboratory can give several hints about microbial survival in the real world. Suggested Reading [1] M M Zambrano and R Kolter, GASPing for life in stationary phase, Cell, Vol.86, pp.181–184, 1996. [2] S Finkel, Long-term survival during stationary phase: evolution and the GASP phenotype, Nature Micobiol. Rev., Vol.4, pp.113–120, 2006. Our Readers Write ... I am a freelance engineer and take classes for children in the nearby school to motivate them to excel in science. I enjoyed the Resonance article on Louis Pasteur. It was very nice; so were the articles on Einstein in the 2005 Resonance issues. I do not know how the journal will appeal to our school children. I wish they read it. I also hope that you can publish pictorial biographies on such seminal researchers for children. Shankar, Chennai ********* Please accept my sincere wishes. I read the article ‘The Legend of Louis Pasteur’ by S Mahadevan (Resonance, January 2007). Thank you for such an encouraging and inspiring article. I found the article ‘Mechanochemistry: The Amazing Viral DNA Packaging Molecular Motor’ by K L Sebastian (Resonance, May 2007) was worthy reading, interesting and encouraging. Ajay K Runthala, Pilani, Rajasthan RESONANCE July 2007 95