Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static.The animal intestinal tract harbors a vast number of bacteria representing a complex ecosystem in which the microorganisms are present without overgrowing the host but also without being flushed out by the host's intestinal activities, e.g., peristaltic movements and fluid flow. At least 400 to 500 different bacterial species are thought to be present at any time in the healthy human intestinal tract, and up to 10 12 bacteria are found per g of feces (3, 9). One question of interest is whether the intestinal flora grows as a homogeneous population at a low rate or as a heterogeneous population containing fast, slow, and nongrowing organisms.Accurate calculations of the rates of bacterial proliferation in the intestine have so far been represented only by average estimates at the level of populations. For example, the growth rate of Escherichia coli has been estimated in vivo, i.e., in the mouse intestine, by radioisotope techniques (8), by dilution by growth of a nonreplicating genetic marker (17,25), and simply by counting the number of viable cells (12,14). With these techniques, generation times from 30 min to 40 h have been estimated for E. coli (12,14,16,36). Also, continuous-flow cultures have been developed to mimic bacterial interactions in the gut and estimate overall growth rates (13, 27). These systems, however, do not reflect the physiological conditions in the gut, where entrapping of the bacteria in the mucus gel plays an important role. Furthermore, bacterial cell morphology, protein profiles, and growth physiology have been found to be distinct during growth in the intestine when compared with growth in laboratory media (22,33).We have previously found that in test tubes E. coli BJ4 is rod shaped, appears as large cells, and has a great potential for fast growth; however, soon after colonization of mice, E. coli BJ4 differentiates into a coccoid morphology, the so-called small variant, which grow more slowly than the rod-shaped E. coli cells in aerated test tubes (22). Here, we continue our investigation of the growth physiology of E. coli BJ4 present in its natural environment, the large intestine. Bacterial growth rates can be estimated from the cellular RNA and/or DNA contents, since the cellular RNA...

When a parent virus replicates inside its host, it must first use its own genome as the template for replication. However, once progeny genomes are produced, the progeny can in turn act as templates. Depending on whether the progeny genomes become templates, the distribution of mutants produced by an infection varies greatly. While information on the distribution is important for many population genetic models, it is also useful for inferring the replication mode of a virus. We have analyzed the distribution of mutants emerging from single bursts in the RNA bacteriophage 6 and find that the distribution closely matches a Poisson distribution. The match suggests that replication in this bacteriophage is effectively by a stamping machine model in which the parental genome is the main template used for replication. However, because the distribution deviates slightly from a Poisson distribution, the stamping machine is not perfect and some progeny genomes must replicate. By fitting our data to a replication model in which the progeny genomes become replicative at a given rate or probability per round of replication, we estimated the rate to be very low and on the on the order of 10 ؊4 . We discuss whether different replication modes may confer an adaptive advantage to viruses.In recent years, viruses have been increasingly used in the laboratory to study the fitness effects of mutations (1,4,8,10,14,15,16,25). The increase is likely due to two reasons. First, these fitness effects, which are generally quantified as the distribution of the magnitude, the sign (deleterious versus beneficial), and the interaction (additive versus nonadditive) of the mutations, are key to many evolutionary models, and viruses have become a major focus of evolutionary studies. Much of our predictive theories in evolution have come from population genetic models that make assumptions about the fitness effects of mutations. For example, depending on whether the interaction between deleterious mutations is log-additive, the models predict distinctly different advantages for the evolution of recombination (5, 15). Second, many issues addressed by such models have now also been raised in the context of viruses, especially RNA viruses, which have a much higher mutation rate than DNA viruses (11,12). Evolutionary topics as wide ranging as the quasi-species concept, game theory, the origin of life, the constancy of the molecular clock, the divergence and convergence of isolated populations, and the evolution of recombination have all been recently considered in relation to viruses (1-3, 5-7, 15, 17, 18, 22, 24, 29, 31, 32).As the more qualitative aspects of the distribution of mutational effects and interactions have become known, it has become desirable to extract more quantitative information. However, more quantitative analyses require also more exact information on another type of distribution, that of mutations and mutants in a population. The distinction between mutations and mutants is important and is made herein. A mutation is the event...

Single-celled organisms dividing by binary fission were thought not to age [1-4]. A 2005 study by Stewart et al. [5] reversed the dogma by demonstrating that Escherichia coli were susceptible to aging. A follow-up study by Wang et al. [6] countered those results by demonstrating that E. coli cells trapped in microfluidic devices are able to sustain robust growth without aging. The present study reanalyzed these conflicting data by applying a population genetic model for aging in bacteria [7]. Our reanalysis showed that in E. coli, as predicted by the model, (1) aging and rejuvenation occurred simultaneously in a population; (2) lineages receiving sequentially the maternal old pole converged to a stable attractor state; (3) lineages receiving sequentially the maternal new pole converged to an equivalent but separate attractor state; (4) cells at the old pole attractor had a longer doubling time than ones at the new pole attractor; and (5) the robust growth state identified by Wang et al. corresponds to our predicted attractor for lineages harboring the maternal old pole. Thus, the previous data, rather than opposing each other, together provide strong evidence for bacterial aging.

The physiological asymmetry between daughters of a mother bacterium is produced by the inheritance of either old poles, carrying non-genetic damage, or newly synthesized poles. However, as bacteria display long-term growth stability leading to physiological immortality, there is controversy on whether asymmetry corresponds to aging. Here we show that deterministic age structure landscapes emerge from physiologically immortal bacterial lineages. Through single-cell microscopy and microfluidic techniques, we demonstrate that aging and rejuvenating bacterial lineages reach two distinct states of growth equilibria. These equilibria display stabilizing properties, which we quantified according to the compensatory trajectories of continuous lineages throughout generations. Finally, we show that the physiological asymmetry between aging and rejuvenating lineages produces complex age structure landscapes, resulting in a deterministic phenotypic heterogeneity that is neither an artifact of starvation nor a product of extrinsic damage. These findings indicate that physiological immortality and cellular aging can both be manifested in single celled organisms.

Bacteriophages are the most numerous entities in the biosphere. Despite this numerical dominance, the genetic structure of bacteriophage populations is poorly understood. Here, we present a biogeography study involving 25 previously undescribed bacteriophages from the Cystoviridae clade, a group characterized by a dsRNA genome divided into three segments. Previous laboratory manipulation has shown that, when multiple Cystoviruses infect a single host cell, they undergo (i) rare intrasegment recombination events and (ii) frequent genetic reassortment between segments. Analyzing linkage disequilibrium (LD) within segments, we find no significant evidence of intrasegment recombination in wild populations, consistent with (i). An extensive analysis of LD between segments supports frequent reassortment, on a time scale similar to the genomic mutation rate. The absence of LD within this group of phages is consistent with expectations for a completely sexual population, despite the fact that some segments have >50% nucleotide divergence at 4-fold degenerate sites. This extraordinary rate of genetic exchange between highly unrelated individuals is unprecedented in any taxa. We discuss our results in light of the biological species concept applied to viruses.sex ͉ virus ͉ linkage disequilibrium ͉ Cystoviridae ͉ biogeography

Evidence for ageing in symmetrically dividing bacteria such as Escherichia coli has historically been conflicting. Early work found weak or no evidence. More recent studies found convincing evidence, but negative results are still encountered. Because bacterial ageing is believed to result from non-genetic (e.g. oxidative) damage, we tested the possibility that the negative outcomes resulted from the lack of an extrinsic damage agent. We found that streptomycin, which produces mistranslated proteins that are more vulnerable to oxidation, was able to induce both damage and ageing in bacterial populations. A dosage effect relating the level of damage to the concentration of streptomycin was observed. Our results explain the previous inconsistencies, because all studies that failed to find evidence for bacterial ageing did not use a damage agent. However, all studies that succeeded in finding evidence utilized fluorescent proteins as a visual marker. We suggest that ageing in those studies was induced by the harmful effects of an extrinsic factor, such as the proteins themselves or the excitation light. Thus, all of the earlier studies can be reconciled and bacterial ageing is a real phenomenon. However, the study and observation of bacterial ageing require the addition of an extrinsic damage agent.

Cellular aging, a progressive functional decline driven by damage accumulation, often culminates in the mortality of a cell lineage. Certain lineages, however, are able to sustain longlasting immortality, as prominently exemplified by stem cells. Here, we show that Escherichia coli cell lineages exhibit comparable patterns of mortality and immortality. Through single-cell microscopy and microfluidic techniques, we find that these patterns are explained by the dynamics of damage accumulation and asymmetric partitioning between daughter cells. At low damage accumulation rates, both aging and rejuvenating lineages retain immortality by reaching their respective states of physiological equilibrium. We show that both asymmetry and equilibrium are present in repair mutants lacking certain repair chaperones, suggesting that intact repair capacity is not essential for immortal proliferation. We show that this growth equilibrium, however, is displaced by extrinsic damage in a dosagedependent response. Moreover, we demonstrate that aging lineages become mortal when damage accumulation rates surpass a threshold, whereas rejuvenating lineages within the same population remain immortal. Thus, the processes of damage accumulation and partitioning through asymmetric cell division are essential in the determination of proliferative mortality and immortality in bacterial populations. This study provides further evidence for the characterization of cellular aging as a general process, affecting prokaryotes and eukaryotes alike and according to similar evolutionary constraints.

Non-genetic phenotypic variation is common in biological organisms. The variation is potentially beneficial if the environment is changing. If the benefit is large, selection can favor the evolution of genetic assimilation, the process by which the expression of a trait is transferred from environmental to genetic control. Genetic assimilation is an important evolutionary transition, but it is poorly understood because the fitness costs and benefits of variation are often unknown. Here we show that the partitioning of damage by a mother bacterium to its two daughters can evolve through genetic assimilation. Bacterial phenotypes are also highly variable. Because gene-regulating elements can have low copy numbers, the variation is attributed to stochastic sampling. Extant Escherichia coli partition asymmetrically and deterministically more damage to the old daughter, the one receiving the mother’s old pole. By modeling in silico damage partitioning in a population, we show that deterministic asymmetry is advantageous because it increases fitness variance and hence the efficiency of natural selection. However, we find that symmetrical but stochastic partitioning can be similarly beneficial. To examine why bacteria evolved deterministic asymmetry, we modeled the effect of damage anchored to the mother’s old pole. While anchored damage strengthens selection for asymmetry by creating additional fitness variance, it has the opposite effect on symmetry. The difference results because anchored damage reinforces the polarization of partitioning in asymmetric bacteria. In symmetric bacteria, it dilutes the polarization. Thus, stochasticity alone may have protected early bacteria from damage, but deterministic asymmetry has evolved to be equally important in extant bacteria. We estimate that 47% of damage partitioning is deterministic in E. coli. We suggest that the evolution of deterministic asymmetry from stochasticity offers an example of Waddington’s genetic assimilation. Our model is able to quantify the evolution of the assimilation because it characterizes the fitness consequences of variation.