Multiple Unnecessary Protein Sources and Cost to Growth Rate in E.coli

The fitness and macromolecular composition of the gram-negative bacterium E.coli are governed by a seemingly insurmountable level of complexity. However, simple phenomenological measures may be found that describe its systems-level response to a variety of inputs. This thesis explores phenomenological approaches providing accurate quantitative descriptions of complex systems in E.coli. Chapter 1 examines the relationship between unnecessary protein production and growth rate in E.coli. It was previously unknown whether the negative effects on growth rate due to multiple unnecessary protein fractions would add linearly or collectively to produce a nonlinear response. Within the regime of this thesis, it appears that the interplay between growth rate and protein is consistent with a non-interacting model. We do not need to account for complex interaction between system components. Appendix A describes a novel technique for real-time measurement of messenger RNA in single living E.coli cells. Using this technique, one may accurately describe the transcriptional response of gene networks in single cells.Physic

Monitoring lineages of growing and dividing bacteria reveals an inducible memory of mar operon expression

In Gram negative bacteria, the multiple antibiotic resistance or mar operon, is known to control the expression of multi-drug efflux genes that protect bacteria from a wide range of drugs. As many different chemical compounds can induce this operon, identifying the parameters that govern the dynamics of its induction is crucial to better characterize the processes of tolerance and resistance. Most experiments have assumed that the properties of the mar transcriptional network can be inferred from population measurements. However, measurements from an asynchronous population of cells can mask underlying phenotypic variations of single cells. We monitored the activity of the mar promoter in single Escherichia coli cells in linear micro-colonies and established that the response to a steady level of inducer was most heterogeneous within individual colonies for an intermediate value of inducer. Specifically, sub-lineages defined by contiguous daughter-cells exhibited similar promoter activity, whereas activity was greatly variable between different sub-lineages. Specific sub-trees of uniform promoter activity persisted over several generations. Statistical analyses of the lineages suggest that the presence of these sub-trees is the signature of an inducible memory of the promoter state that is transmitted from mother to daughter cells. This single-cell study reveals that the degree of epigenetic inheritance changes as a function of inducer concentration, suggesting that phenotypic inheritance may be an inducible phenotype

Minimally invasive determination of mRNA concentration in single living bacteria

Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to ∼40 nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria

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Minimally invasive determination of mRNA concentration in

single living bacteri