My model of bacterial senescence

The current model of bacterial senescence proposed by Stewart et al. (2005) suggests that as bacteria undergo aging, 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. In this case, mother cell retains the damaged proteins to itself whereas daughter cells are spared from accumulating damaged proteins.

In my model of bacterial senescence, different subpopulations of bacteria are considered. A bacterial colony comprises many subpopulations which may exhibit different growth rates. These subpopulations can be broadly divided into three - a small subpopulation of fast dividing young bacteria, a major population with intermediate growth rate and a small subpopulation of slow dividing bacteria that are towards the terminal stages of senescence.

The young bacterial population divides fast and is hypervirulent and may not contain any damaged proteins. After many divisions, the mother cell may gradually accumulate some damaged proteins whereas the daughter cells are rejuvenated offsprings. Thus a small population of young virgin bacteria is always maintained.

However, the majority of the population in a colony exhibit intermediate growth rate. Since they comprise the major population, the growth rate of the colony will be similar to the growth rate exhibited by this population. They may carry some amount of damaged proteins which are not segregated to mother cell alone, but are also transferred to daughter cells. However, with increasing amount of damaged proteins in mother cells, the daughter cells will also accumulate more damaged proteins.

Towards the terminal stages of senescence, mother cells may lose their ability to retain the damaged proteins and hence may be shared almost equally with daughter cells. At this stage, mother cell will also give rise to senescent daughter cells. Since both mother and daughter cells are old cells and divide slowly, a single senescent cell gives rise to small colony.

In a population, majority of the cells have only limited carbonylated proteins. This, along with a subpopulation of virgin cells without any damaged proteins, help to maintain a pool of young cells that are large enough to prevent the extinction of the population.

Thus, my model of bacterial aging is close to that of Schizosaccharomyces pombe, the fission yeast (discussed in the blogpost on Sep.6). The similarity may not be surprising given that both bacteria and S. pombe divide by binary fission.

Next- Senescent bacteria and persisters

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

Shifting bacterial population distribution curve to the right or left

As per my model, a bacterial population in a colony is heterogeneous with respect to growth rate and age. There are small subpopulations of both young and senescent bacterial population in addition to the major population which have an intermediate growth rate. The growth rate of the whole population will be close to the intermediate population as they comprise the major population.

However, the bacterial population distribution curve can be shifted either towards the left or the right. It can be shifted towards the left by growing the bacterial culture in early exponential phase. In this case, the growth rate of the culture gradually increases as more young bacteria are selected (as described in the previous blog on Sep.19). However, this increased growth rate of the culture may not be a permanent feature since the bacteria undergo senescence. Thus, if the culture of young bacteria is allowed to grow and reach stationary phase, the growth rate may gradually decrease due to the gradual increase in senescent population. If 50 ul of this stationary phase culture is transferred to 3 ml of fresh medium and allowed to grow, the growth rate may further reduce. If this process is repeated, the growth rate may reach the initial rate.

On the other hand, the bacterial distribution curve can be shifted to right by incubating the bacterial culture with aminoglycosides which results in the selection of slow dividing senescent bacteria (see the blogpost on Oct.5). Whether they can be shifted back to normal depends upon the stage of senescence. Bacteria towards the terminal stage of senescence can not be reverted back and in this case it may not be possible to shift the growth rate to normal.

Next- My model of bacterial senescence.

Why both slow and fast dividing subpopulations of bacteria are missed during routine culture?

During routine culture, the number of bacteria on solid agar medium that can form distinct colonies, separated from each other are usually around 100. Among 100 colonies, we may not notice slow or fast dividing subpopulations.

It is easy to explain why we miss slow dividing bacteria during routine culture- their number is very small and they divide slowly. By the time these subpopulation of bacteria start to grow and form colonies, the normal bacterial population would have already grown and covered the agar. Hence, to isolate those subpopulations of small colony variats (SCV), the normally dividing population needs to be eliminated, which can be done with the help of aminoglycosides (see the blogpost on Oct.5).

On the other hand, one would expect to notice fast dividing subpopulation more frequently. Even if their initial number is low, one would expect them to gradually dominate the population since their growth rate is higher than the normal. However, this may not happen if this fast dividing subpopulation undergoes senescence. As they divide, the growth rate of mother cell gradually reduces and becomes comparable to the normal population whereas the daughter cell may be a rejuvenated offspring. It may be due to the mother cell undergoing senescence that the fast dividing subpopulation does not dominate the culture. However, the fast dividing subpopulation can be selected by removing the normal population by repeatedly growing the culture in early exponential phase (as described in the previous blog on Sep.19).

Thus, bacterial senescence can explain why the fast dividing, hypervirulent subpopulation of bacteria that can be isolated by repeatedly growing the culture in early exponential phase does not dominate the whole population even if they have the growth advantage over the normal population.

Next- Shifting bacterial population distribution to the right or left

Experiment that could indicate that SCVs are senescent bacteria

One of the markers of senescence is the accumulation of oxidative carbonylated proteins. Cells of an E. coli population show asymmetry not only with respect to growth rate, but also with respect to protein oxidation levels (Desnues et al. 2003; Aguilaniu et al. 2003). An E. coli population consists of relatively low damaged daughter cells (low protein oxidation) that are reproductively competent and damaged mother cells with reduced reproductive ability (Desnues et al. 2003). In exponentially growing E. coli, the amount of protein aggregates increases over time and were found to be more prevalent in dead cells than in culturable cells (Maisonneuve et al. 2008a). Similarly, aggregated proteins accumulate in cells with older poles, which are associated with a decrease in reproductive ability (Lindner et al. 2008).

In the earlier blog, I had hypothesized that small colony variants of E. coli isolated using subinhibitory concentrations of aminoglycosides are senescent bacteria which are hypovirulent, slow dividing and form small colonies on solid medium. Measuring the levels of protein carbonylation can give an indication whether they are senescent bacteria. If they are senescent bacteria, one can expect the protein carbonylation levels to be high. As far as I know, level of protein carbonylation in small colony variants has never been measured (a search of “small colony variants” and protein carbonylation returned only two results in Google scholar).

However, increased protein carbonylation may not be conclusive evidence that SCVs are replicative senescent bacteria. Increased carbonylation can be a feature of both conditional and replicative senescence. Hence further research may be required to differentiate between these two. In fact, there is possibility that mutants that form SCVs (like hemin, menadione or thiamine mutants) may also show increased carbonylation which may be due to conditional senescence and not replicative senescence. Hence, I assume that both mutant SCVs and non-mutant SCVs may show increased protein carbonylation, the former due to conditional senescence and the latter due to replicative senescence.

If the yeast cells isolated by repetitively growing in early exponential phase have low number of bud scars (described earlier) and SCVs isolated using aminoglycosides have increased protein carbonylation levels, I can say with increased confidence that my model of bacterial aging is different from that proposed by Stewart et al. (2005) and that SCVs are senescent bacteria

Next- Why both slow and fast dividing subpopulation of bacteria are missed during routine culture?

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

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

Desnues et al. (2003). Differential oxidative damage and expression of stress defence regulons in culturable and non-culturable Escherichia coli cells. EMBO Rep 4(4), 400-4.

Maisonneuve et al. (2008). Protein aggregates: an aging factor involved in cell death. J Bacteriol 190(18), 6070-5.

Lindner et al. (2008). Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation. Proc Natl Acad Sci U S A 105(8), 3076-81.

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

                   

                   In a normally dividing population, a small subpopulation of slow dividing bacteria is present which can be isolated using aminoglycoside antibiotics. They are termed as small colony variants (SCV). SCVs had already been discussed before (please check the posts in the month of August). They constitute a naturally occurring, slow-growing subpopulation of bacteria that form small colonies (less than one-tenth of the size of parent colonies) on solid media (Proctor et al. 2006). Much has been published on the biochemical aspects and the significance of SCVs. However, there are two areas where I have difference of opinion from those in published articles.

1. SCVs are mutants that revert to normal growth in the presence of auxotrophic agents

2. SCVs are responsible for chronic infections

Whereas a number of mutants form SCVs and can be reverted to normal growth after adding hemin, menadione, thiamine or thymidine, all SCVs isolated in vitro after adding aminoglycosides may not be specific mutants. In fact all SCVs are not similar and may exhibit different protein profiles (Kriegeskorte et al. 2011). Similarly, the role of SCVs in chronic infections is questionable (please read the previous posts).

A pure culture of SCVs of E. coli DH-5alpha cells can be isolated after treating cells with subinhibitory concentration of aminoglycosides like kanamycin as explained in Jacob (2007). In short, 50 ul of stationary phase culture is added to 3 ml of fresh LB medium containing kanamycin at different concentrations and incubated for 2 days. Three factors are important to get a pure culture of SCVs- initial inoculum size, concentration of antibiotic and the total time of incubation. If the inoculum size is very low, SCVs may be missed, but if high, some normally dividing bacteria that have escaped killing may overgrow and mask SCVs. Since they are slow dividing bacteria, SCVs may take longer time to grow. With different concentrations of kanamycin, colonies of different sizes can be obtained.

The slow dividing SCVs have been shown to be hypovirulent also (Sifri et al. 2006). But, how can it be proved that they are senescent bacteria?

Next- Experiment that could indicate that SCVs are senescent bacteria

 

Proctor et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4(4), 295-305.

Kriegeskorte et al. (2011). Small colony variants of Staphylococcus aureus reveal distinct protein profiles. PROTEOMICS, 11: 2476–2490.

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

Sifri et al. (2006). Virulence of Staphylococcus aureus small colony variants in the Caenorhabditidis elegans infection model. Infection and Immunity, 74(2);1091-1096.