Cell growth in Escherichia coli: Study of fluctuations and asymmetry in cell extension

Abstract

Cell dimensions are defined for each species. Several cellular pathways guarantee faithful duplication of cell during binary fission. However, phenotypic heterogeneity has been observed in genetically homogenous populations. Advanced scientific studies have proved such fluctuations crucial in the development, for they have been associated with phenomena like division of labour and rise of survivors under harsh environmental situations. Thesis provides an insight into possible mechanisms for the origin of variations in cell lengths of clonal E. coli populations. Series of experiments helped assess an elongation in Escherichia coli cells and its effect on the population length distributions. In our studies with E. coli batch cultures, we observed a linear increase in cell length variation above growth rate 1 generation per hour. On the other hand, below this threshold phenotypic heterogeneity was observed to be reduced but it stayed almost constant for all the growth rates. ‘The point of inflection’ in the cell length variation was identified and correspond to the growth rate at which multi- fork replication is triggered in a bacterial cell. Molecular analysis of RecA dynamics showed increased occurrence of replication fork stalling events at high growth rates. We inferred that stochastic arrest in the replication increases multiplicatively at higher growth rates because of multi- fork replication in E. coli cell which in turn, increases the probable halts in cell division through SOS response, producing non- genetic cell length variation in an isogenic population. Deviation from defined cell dimensions has previously been assigned to the fluctuations in gene expression. Ergo, next step was to probe the effect of replication stochsticity on genetic expression and connect it with phenotypic noise in isogenic populations of E. coli MG22. By examining hydroxyurea treated populations at different growth rates, we detected that the total gene expression noise can be modulated by the population growth rate. Growth rate dependent dissection of the noise showed that intrinsic noise dominates in genetic circuitry, at higher growth rates. In recovered cells, intrinsic noise was observed to be linear with increasing stalls in DNA replication till HU concentration hits 13 mM but then it surprisingly drops monotonously for higher HU concentrations, breaching the connection. Extrinsic gene expression noise takes over at slower growth rates. We predict that at sub- lethal HU dosages, replication fluctuations, in highly proliferating populations can cause phenotypic variability through elevated intrinsic gene expression noise. On a slightly different note, analysis of E. coli elongation, revealed the presence of inherent asymmetry in growth of an individual cell. We found that, an asymmetry in growth introduces difference in the cell division time of two sisters born at same time. Inspection of MreB dynamics showed bias in the distribution of MreB molecules along cell length, suggesting possible role of MreB cytoskeleton in growth bias. Though, its connection with phenotypic variation is not clear, we report a new finding as opposed to the classical description of E. coli growth. The work combined with published data, sets a new paradigm for growth in an Escherichia coli cell.