Experimental proof that the fast dividing, hypervirulent bacteria are young bacterial population

In the last blog, I assumed that the fast dividing, hypervirulent bacteria isolated after repeated growth in early exponential phase are young bacterial population. This can be confirmed by doing the same experiment in Saccharomyces cerevisiae.

When yeast cells undergo replication by budding, a bud scar is left behind on mother cell’s surface. Bud scars remain permanently deposited on the surface and get accumulated as mother cells undergo more divisions. Thus, an old mother cell will have more number of bud scars whereas the number will be less in relatively young mother cells. Thus, the bud scars are used as a marker for the number of divisions a cell had undergone or the budding index. The budding index can be calculated by analyzing the cell wall for bud scars using confocal microscopy.

For the experiment, when a normally dividing yeast culture reaches an O.D. of 0.3-0.5, 100 ul of the culture should be withdrawn and added to 3 ml of fresh culture medium and incubated for growth. This process should be repeated 3-4 times. At the end of the fourth cycle, when yeast cells remain in early exponential phase itself, the budding index should be calculated. This can be compared with a yeast culture in one-time exponential phase and also with a culture in stationary phase. If the cells obtained after repeated culturing in early exponential phase are indeed young yeast cells, the number of bud scars will be lower than that in other two.

If they are found to be young yeast cells, the same can be true for bacteria also.

Next- Isolation of slow dividing, small colony forming, hypovirulent, senescent bacteria

Isolation of fast dividing, hypervirulent, young bacteria- a simple experiment that can reveal many interesting properties

In one of my experiments aimed at eliminating persister bacteria (a small subset of slow growing bacterial population) from a normally dividing population, a culture of bacteria was repeatedly grown in early exponential phase (Jacob 2007). This experiment is simple and inexpensive (only an incubator and a spectrophotometer are needed) and quick (can be finished in a few days), but give some interesting results regarding bacterial growth kinetics.

In this experiment, when a culture of bacteria reached an O.D. of 0.3- 0.5 ( i.e. at a stage of light turbidity of the medium), 100 ul of the culture was transferred to 3 ml of fresh medium and incubated again. The same procedure was repeated 4 times. Repeated selection of bacteria at early exponential phase eliminated all slow growing bacteria. At the end of 4^th cycle, when the bacterial culture was allowed to continue its growth and reach a stationary phase, some interesting properties were noticed.

1. The growth rate of the selected bacteria was found to be much higher than a normally dividing culture

2. At stationary phase, the number of bacteria per ml of medium was higher

3. Persisters that were absent initially after repeatedly grown in early exponential phase reappeared at stationary phase

4. GFP expression of selected bacteria were higher

5. Activity of ampicillin was much reduced against the selected bacteria especially at higher initial inoculum size (in fact, bacteria grew as if antibiotic was not added; however, this lack of activity was not due to antibiotic resistance)

In the above article, only the third property was reported since the article was focused on the phenotypic shift of persisters. Based on the increased GFP expression and lack of activity of ampicillin (which I attribute to fast growth and increased production of enzymes that destroy the antibiotic, especially at higher inoculum size), I assumed that those bacteria were hypervirulent. Indeed, the hypervirulence of selected bacteria was later reported by Chapuis et al. (2011) after injection of Xenorhabdus nematophila into insects. My interpretation to the above result is that these fast dividing, hypervirulent bacteria are very young bacteria that are found in low in numbers in a normally dividing population (see the earlier blogpost).

Just because the selected bacteria are fast dividing and hypervirulent does not prove that they are young bacteria. However, it is possible to prove it with the help of another experiment.

Next- The experiment that can prove that the fast dividing, hypervirulent bacteria are young bacteria

Jacob, J (2007). Persisters show heritable phenotype and generate bacterial heterogeneity and noise in protein expression . Available from Nature Precedings <http://hdl.handle.net/10101/npre.2007.1411.1>

Chapuis et al. (2011). Virulence and pathogen multiplication: A serial passage experiment in the hypervirulent bacterial insect-pathogen Xenorhabdus nematophila. PLoS ONE 6(1): e15872. doi:10.1371/journal.pone.0015872

Population distribution of an E. coli colony- comparison with Stewart et al. (2005) model

As per Stewart et al. (2005) (discussed in the last blog), the daughter cell formed from a mother cell is a rejuvenated offspring with full reproductive potential. So, if we designate x for virgin cells, x+1 for cells that have undergone 1 division, x+2 for those undergone 2 divisions and so on, then approximately 50% of a normally dividing  population in a colony of bacteria will be virgin cells (x), 25% will be x+1, 12.5% will be x+2 and so on. Hence if we plot the percentage of different populations on a graph, a half-bell curve distribution will be obtained. However, it may not be as perfect as one below as the growth rate of all cells are not the same (mother cells have reduced growth rate as it undergoes senescence).

However, my model of E. coli senescence is closer to that of S. pombe senescence (discussed on September 6 blogpost). In this model, the old cells do not give rise to rejuvenated offspring, but generate old cells itself. However, majority of the cells are relatively young cells with limited damaged or carbonylated proteins and this pool of young cells are large enough to prevent the extinction of the population. Another important feature in my model is the presence of a small population of virgin cells with no damaged or carbonylated proteins. Thus a major difference between Stewart et al. (2005) model and my model is that in the former, majority of the population are virgin cells (rejuvenated offspring) whereas in the latter, majority of the population are young bacteria with limited carbonylated proteins, along with a small population of virgin cells.

Thus, the population distribution in my model follows a normal distribution curve. However, it may not be a perfect bell shaped curve but can be skewed in favor of young cells. A bell-shaped curve indicates that there are small populations of fast-dividing young bacterial cells as well as slow-dividing senescent cells in any bacterial colony.

Is it possible to isolate any of these small populations? The answer is yes – both young cells and old cells can be isolated. In the next few sections, I will discus how to isolate these two populations separately.

Next- Isolation of fast dividing, hypervirulent, young bacteria 

Stewart et al. (2005) model of replicative senescence in E.coli

Perhaps, the earliest evidence of replicative senescence in bacteria was provided by Liu (1999). By tracking the bacterial growth in liquid media with high viscosity, Liu (1999) observed the unidirectional growth and reproduction of E. coli. He proposed that the bacterium has an intrinsic cell polarity with one end behaving as a mother compartment and the other end as the daughter compartment resulting in the formation of two bacteria of succeeding generations. His model defined bacterial age by its experienced chronological time. Based on this model, he predicted that, on bacterial division, the old strand of DNA remain with the mother bacterium whereas the new strand goes to the daughter bacterium and that this distribution of old and new strands of DNA between the mother and daughter cells is responsible for the intrinsic differences between the two.

Later, Stewart et al. (2005) studied the senescence in E. coli using automated time lapse microscopy by following repeated cycles of reproduction. They followed individual exponentially growing cells up to nine generations of growth and reproduction. Their findings were comparable to those reported previously by Liu (1999). The bacterium exhibits cell polarities which give rise to an old pole with a reduced growth rate and a new pole with higher growth rate. They found that the average growth rate of old pole cells was 2.2% slower than that of new pole cells and that the new pole cells were larger and divided sooner than the old pole cells. In addition, the old pole cells were also more likely to die than the new pole cells. They concluded that the two apparently identical cells are functionally asymmetrical, with the old pole cell behaving as the aging mother cell and the new pole cell as the rejuvenated offspring.

Thus, according to these models, bacteria undergo aging and that the growth rate of mother cell decreases with age whereas the daughter cell produced from the mother cell is a rejuvenated offspring with high growth rate which helps to maintain the bacterial lineage.

Next- Population distribution of an E. coli colony- a comparison with Stewart et al. (2005) model

Liu, S. V. (1999). Tracking bacterial growth in liquid media and a new bacterial life model. Science in China 42, 644-654.

Stewart et al. (2005). Aging and death in an organism that reproduces by morphologically symmetric division. PLoS Biol 3(2), e45.

Replicative senescence in Caulobacter crescentus

Replicative senescence has been reported in Caulobacter crescentus, a bacterium in which cytokinesis is intrinsically asymmetrical. The bacterium can freely swim in water and this free swimming cell represents the swarmer cell. The swarmer cell is non-reproductive, but after a period of free swimming, it gets differentiated to a sessile reproductive stalked cell. The stalked cell remains attached to the substrate but produces progeny swarmer cells which then separates from the stalked cells and begin free swimming.

Ackerman et al. (2003) studied the replicative senescence in Caulobacter crescentus by using microscopy flow chambers in which the stalked cells were attached to the chamber while the swarmer cells produced from the stalked cells were removed by the medium flowing through the chamber. They noticed that, as the stalked cells gave rise to more progenies, their rate of division slowed and finally stopped completely. However, the progenies produced, i.e. the swarmer cells, were rejuvenated offspring. Moreover, those progenies produced towards the end were indistinguishable from any of the young cells produced earlier in the experiment in terms of cell division time. These experiments proved that the stalked cells undergo senescence and that their reproductive output decreases with increasing aging. However, the major difference was that, unlike S. cerevisiae, S. pombe, or D. melanogaster, the progenies from both the older and younger mother cells were rejuvenated offspring and indistinguishable from each other. i.e. older bacterium produces only young bacterium.

Next- Stewart et al. (2005) model of replicative senescence in E.coli

Ackermann et al. (2003). Senescence in a bacterium with asymmetric division. Science 300(5627), 1920.

Replicative senescence in Schizosaccharomyces pombe

Unlike Saccharomyces cerevisiae which divides by budding, Schizosaccharomyces pombe (S. pombe) divides by binary fission and hence named as fission yeast. They grow by elongation at their ends and divide by medial fission to produce two daughter cells and in this sense, it undergo morphologically symmetric division (however, the division is not symmetric in all aspects).

Minois et al. (2006) studied the replicative senescence of S. pombe by measuring the occurrence and intensity of protein carbonylation (a marker of senescence) in single and symmetrically dividing cells of S. pombe. In S. cerevisiae, cabonylated proteins are segregated unevenly between mother and daughter cells during cytokinesis, with most of the carbonylated proteins retained by the mother cell. However, in S. pombe, proteins are not segregated to one half of the cell but are shared between the two cells. They found that carbonylated proteins are never segregated to mother cell alone but are shared between the two. However, with increase in carbonylation, they are shared less equally. This pattern of sharing is opposite to that of S. cerevisiae. This indicates that in S. pombe, damaged cells give rise to damaged cells itself. In a population, most of the cells have only low levels of carbonylation whereas a few of the cells show high levels of carbonylation which is consistent with senescence model of S. cerevisiae in which a gradual accumulation of carbonylated proteins occurs as the cells undergo aging. Cells with high levels of carbonylation exhibit lower fitness and are less likely to divide. However, since majority of the cells have only limited carbonylated proteins, this indicate that the pool of young cells are large enough to prevent the extinction of the population.

Thus, Minois et al. (2006) have shown that cells of S. pombe also undergo aging. However, unlike S. cerevisiae, the carbonylated proteins are shared between the mother and the daughter cells and hence the old cells of S. pombe generate old cells itself.

My model of E.coli senescence is almost similar to that of S. pombe. This will be discussed later.

Next- Replicative senescence in Caulobacter crescentus

Minois et al. (2006). Symmetrically dividing cells of the fission yeast Schizosaccharomyces pombe do age. Biogerontology 7:261-267.

Replicative senescence in Saccharomyces cerevisiae

Kennedy et al. (1994) studied the replicative aging of the budding yeast S. cerevisiae by microscopically following the mother cells through a number of cell divisions. They noticed that the mother cell underwent a finite number of divisions and that the size of both the mother cell and the daughter cell increased with age. They also noticed that older mother cells underwent a more symmetrical division in which the daughter cell that budded from the mother cell was almost the same size as the mother cell at division. Similarly, the daughter cells of older mother cells had a shorter lifespan than those of younger mother cells. The daughter cells of older mother cells in the last 10% of their lifespan was found to undergo only 7.9 divisions, whereas those daughter cells budded from the mother cells in the first 70% of the lifespan, on an average, divided 26.5 times. Symmetrical division, therefore, does not give any advantage to the daughter cells as they undergo a smaller number of divisions than those derived from younger mother cell.

Aging is associated with the accumulation of damaged proteins, genetic materials or dysfunctional mitochondria. In S. cerevisiae, the levels of protein oxidation increase with the replicative age of mother cells (Aguilaniu et al. 2003). Oxidized proteins are unevenly distributed between the mother and daughter cells during cytokinesis, with mother cells retaining most of the oxidized proteins. However, with increasing age, mother cells lose their ability to retain the oxidized proteins (Aguilaniu et al. 2003). Thus, the amount of oxidized proteins gradually increases in daughter cells as the mother cell undergoes aging.

What happens to the progeny produced from symmetrical daughters? Even though the lifespan of the daughters arising from symmetric divisions is reduced, daughters formed from the daughters of old mother cells are restored back to normal (Kennedy et al. 1994). In other words, progeny from symmetrical daughters recover full life span potential following asymmetric division, indicating that the decrease in the lifespan in daughters of old mother cells is not heritable. Thus the damages accumulated in older mother cells may be diluted in subsequent generations.

Next- Replicative senescence in Schizosaccharomyces pombe

Aguilaniu et al. (2003). Asymmetric inheritance of oxidatively damaged proteins during cytokinesis. Science 299(5613), 1751-3.

Kennedy et al. (1994). Daughter cells of Saccharomyces cerevisiae from old mothers display a reduced life span. J Cell Biol 127(6 Pt 2), 1985-93.

Aging/senescence- introduction

Despite the obvious disadvantages of aging, it is not opposed by natural selection probably because it can be beneficial to the species by avoiding overcrowding and promoting further evolution, thus increasing the fitness of subsequent generations. There are many theories of aging which can be broadly divided into stochastic and non-stochastic theories of aging. The former views aging as a phenomenon resulting from random events leading to cellular damage whereas the latter considers aging a programmed or a predetermined phenomenon occurring in all organisms within a particular time-frame.

Senescence can be conditional or replicative. When a bacterial culture is exposed to stressful conditions such as starvation for prolonged periods, they gradually accumulate oxidative damages and their ability to recover from the damages may also be lost and the cells may finally undergo death. This conditional senescence (Nystrom 2003) is different from the replicative senescence in that the latter results from the sequential loss of fitness following multiple rounds of replication.

Whereas aging is visible and very evident in higher eukaryotic organisms, it is also observable in simple eukaryotes like the fruitfly Drosophila or yeast Saccharomyces. However, senescence in prokaryotes like bacteria was not known till recently. For more than a century, bacteria were considered to be functionally immortal organisms because of their symmetrical division. It was believed that a parent bacterium split symmetrically into two equal daughter cells and that both the cells receive damaged and new cellular constituents equally. Thus, bacteria were considered to be organisms immune to natural aging and death, even though they can be killed by many external agents like starvation or other stressors.

Before moving into bacterial senescence, I will discuss the aging model in simple eukaryotes.

Next- Replicative senescence in Saccharomyces cerevisiae

Nystrom, T. (2003). Conditional senescence in bacteria: death of the immortals. Mol Microbiol 48(1), 17-23