The GASP phenotype in Acinetobacter baylyi A Senior Thesis submitted to the Department of Biology, The Colorado College by Leland Krych Date ____________________________________ Approved by: _________________________________________ Primary Thesis Advisor ________________________________________ Secondary Thesis Advisor Introduction Microbiologist Steven Finkel once referenced Thomas Hobbes’s Leviathan to describe the realities of a bacterium’s life; it is: “solitary, poor, nasty, brutish and short (Finkel 2006).” All theatrics aside, Dr. Finkel makes the point that bacteria have it rough, and that this fact is often overlooked in modern bacterial experimentation. Therefore, common laboratory models for bacterial growth might not reveal how bacteria survive in nature, where resources are scarce and competition is fierce. Dr. Finkel goes on to suggest that a more accurate way of studying bacteria in the lab would be to focus on an overlooked aspect of the bacterial life cycle: long-term stationary phase. In order to address why this concept is significant we need to review the five stages in the typical life cycle of a bacterial population raised in the laboratory. These phases are known as lag phase, exponential phase, stationary phase, death phase, and long- term stationary phase. After inoculation of bacteria into liquid medium there is an initial phase in population growth in the media known as lag phase which is then followed by a phase of exponential growth of the culture. This burgeoning population growth is known as the exponential growth phase. Eventually, the population growth levels off into stationary phase as the amount of space and nutrients start to dwindle. After this period approximately 99% of the cells perish. Survivors from death phase undergo high rates of mutation and begin great rates of cell division in the next stage of the bacterial life cycle: long term stationary phase (LTSP). Long term stationary phase is a highly dynamic phase in which the birth rates and death rates of the bacteria are approximately equal (figure 1). This small population of cells can survive long periods without addition of nutrients. A batch of E. coli in LTSP survived for five years with only the addition of sterile distilled water to maintain the volume and osmolarity (Finkel et al., 2000). Finkel (2006) suggests that the study of LTSP is important because in a natural environment most bacteria probably live under threat of starvation where the activation of stress-response genes and the use of alternative metabolic pathways is essential for life. One phenotype that is associated with cells found in LTSP is the Growth Advantage in Stationary Phase (GASP) phenotype (Zambrano 1996, Finkel 2001, Farrell 2003, Finkel 2006,). The GASP phenotype was first described in 1993 by Zambrano who noticed that 10 day old E coli could out-compete 1 day old E coli when co-incubated under starvation conditions. To test for the GASP phenotype, cells are grown until they reach immediate stationary phase, and then these cells are competed with cells that have already reached LTSP. The older cells are introduced into the competition experiment in a ratio of 1:1000 old:young. Eventually the aged cells increase in population and drive the younger cells to extinction. This phenomenon is known as a class 1 GASP phenotype. The GASP phenotype is determined by observing the population densities of the two separately labeled strains. Three other classes of GASP phenotypes have been reviewed by Dr. Finkel (figure 2). There is the class 2 GASP phenotype whereby the aged cell population increases but this increase does not lead to the demise of the younger cells. The class 3 phenotype where aged cells initially exhibit the GASP phenotype but then die out; leaving the younger cells at stable population levels. And finally, there is the class 4 phenotype in which aged cells die without any serious population growth. The competitive advantage of the older cells is a result of mutations formed in LTSP (Zambrano 1996). Currently, all known gene mutations that cause GASP phenotypes increase the ability of the bacteria to catabolize various substrates as a source of energy (Finkel 2006). An example is DNA, which E. coli cells use as a source of food during LTSP (Finkel 2001). Drake (1998) found that in E. coli, the rate of mutation in LTSP was one point mutation for every 300-400 cells. In contrast, cells in exponential growth phase show a mutation frequency of one in every 10,000 cells. While high mutation frequency is typically not a favorable trait for a cell to possess, it is thought that when environmental pressures are extreme enough, a high mutation frequency might create new alleles that provide a competitive advantage. A possible mechanism for the formation of mutations in the GASP phenotype in E. coli is mutations in the SOS-induced DNA polymerases. SOS DNA polymerases are enzymes that repair DNA lesions that DNA polymerase III cannot copy through. DNA polymerase III is the polymerase that synthesizes the leading and lagging strand at the replication fork of DNA. When DNA polymerase III encounter abasic sites, photodimers, or other damaged bases the SOS DNA polymerases are called in to repair the damage (Finkel 2006). Yeiser et al. (2002) demonstrated that in competition with wild type E. coli, mutants lacking one or more SOS-induced DNA polymerases show a dramatic reduction in fitness. Additionally, Yeiser et al (2002) performed GASP tests on wild type cells and the polymerase deficient mutants and found that 10 day old wild type cells experienced a class 1 GASP phenotype when competed against 1 day old wild type cells. In contrast, when 10 day old mutants were competed with 1 day old mutants, the mutants displayed variable GASP phenotypes (Yeiser 2002) indicating that DNA polymerases play some role in generating a class 1 GASP phenotype. It is also prescient to note that the mutants displayed type 1 GASP phenotypes when in competition with other polymerase deficient mutants. It is important to note that A. baylyi does not possess the lexA gene, a regulator of the SOS DNA response (Hare et al. 2006). Recent findings suggest, however, that this process might be mediated by the UmuDAb gene that possesses similar characteristics (Hare et al. 2012). The model in which we chose to study the GASP phenotype is the gram-negative soil bacterium Acinetobacter baylyi. While Finkel has speculated that bacteria in addition to E. coli should exhibit a GASP response, GASP has been demonstrated in only a few species. A. baylyi and E. coli are distant relatives, found in the same Class, Gammaproteobacteria, but different Orders (Pseudomonadales and Enterobacteriales, respectively). One striking physiological trait that is very different between the two bacteria is that A. baylyi is well known for high natural transformation efficiency, while E. coli have never been shown to be naturally competent. Competence for natural transformation is a physiological state that permits the uptake of exogenous DNA from the environment. It requires synthesis of a complex competence machine, encoded by proteins in the Type IV Pilus family. E. coli does have genes homologous to this system, but appears to use them for catabolism of DNA under starvation conditions (Finkel 2001). The evolutionary advantage caused by natural competence is highly debated and as of now there are many competing hypotheses as to why it evolved. Here are two examples: 1) Bacteria exploit natural transformation for nutrient acquisition. Palmen et al. (1994) found that the addition of a five-fold saturation amount of DNA in comparison to normal amounts of DNA present in solution did not result in growth of Acinetobacter calcoaceticus cells under carbon and nitrogen limitation. However, under conditions of limited phosphate, the addition of DNA facilitated further growth. Under these conditions, none of the cells were transformed (as evidenced by lack of radio-labeled DNA in the bacteria). Finkel et al. (2001) found that wild-type strains of E. coli showed 50 times greater growth yield in minimal media with added DNA than mutants lacking specific homologs of competence genes. 2) Bacteria use exogenous DNA as fodder for DNA repair. Redfield et al (1988) found using a computer simulated model that natural transformation could reduce mutational load but that this benefit hinged upon the source of DNA. The reduction of mutation load was reduced and or negligible when the DNA came from cells that were killed by selection against deleterious mutations; which is what one would expect from unfit cells under high stress conditions. Redfield (1997) also found that according to her numerical model that the benefit of mutational reduction deriving from natural transformation is only very slight. Redfield’s models do not consider the possibility that healthy cells might release DNA in response to environmental changes. Discussion of natural transformation would not be complete without an understanding of the competence machinery. The mechanism for DNA uptake through natural transformation is mediated by the competence genes; however the overall structure of the multi-protein competence machine has never been visualized. Efforts are being taken by Kristine Lang and Phoebe Lostroh to realize this goal. Sequence analysis has revealed that competence machinery is related to the type-IV secretion system (Chen 2004). Type IV secretion systems have been adapted to several distinctive physiological purposes. The best understood is use for building type IV pili, which are surface-exposed, retractable fibers that mediate the formation of biofilms, fruiting bodies and motility (Averhoff 2003). Other type IV secretion systems, however, have been adapted for secretion of other protein substrates or for uptake of DNA during natural competence. In all cases, the machines are comprised of homologous protein subunits. Averhoff et al. (2003) proposed a model for DNA translocation through the Acinetobacter baylyi competence system based on homologues to Type IV secretion systems (figure 3).