This doctoral research used single-molecule imaging techniques to study DNA replication in live E. coli cells. Endogenous replication proteins like β2-clamps were labeled with fluorescent proteins using genome engineering. Observations found β2-clamps accumulate on DNA after initiation and remain bound for minutes. Experiments also examined how replisomes encounter natural Tus-Ter roadblocks and determined replication speed decreases but is not stalled. Further work aimed to study primase dynamics and localization of Tus proteins during the cell cycle but faced challenges in strain engineering. The research demonstrated applications of in vivo single-molecule imaging while also highlighting ongoing difficulties in the field.

1. Summary of Doctoral Research
In vivo investigations of E. coli chromosomal replication using single-
molecule imaging
All living organisms pass on their genetic information to their offspring in the form of DNA or
RNA molecules by duplicating them across generations (Meselson & Stahl, 1958). In the
bacteria, their genes are packed in long chains of DNA molecules or chromosomes. One of the
widely studied model organisms, Escherichia coli, replicates its circular chromosome in two
directions starting from an origin region of chromosome with independent replication complexes
or replisomes simultaneously synthesizing the daughter chromosomes (Cooper & Helmstetter,
1968). DNA replication is an important process of the E. coli life cycle because the occurrence of
small errors in its mechanism will affect the cell’s normal state larger (Kogoma, 1997). Much of
our current knowledge about the dynamics of replisome complex has been obtained from in vitro
experiments. However, the natural environment of the cell is considerably different from that of
in vitro solutions (Reyes-Lamothe et al., 2012).
Current advances in genetic engineering and fluorescence imaging enables us to image
proteins in vivo. Single-molecule techniques provide one with added insights into the events that
take place in the cell during replication, and they usually are not apparent while ensemble
averaging used in bulk experiments (Reyes-Lamothe et al., 2010). It is thus essential that
quantitative single-molecule research is conducted before we can fully understand the details of
DNA replication as it occurs in a living cell. Single-molecule imaging in vivo is aided by
employing microfluidics in order to have spatial control over growing cells and provide precisely
regulated growth conditions like temperature, media flow, etc. Similarly, the development of the
accurate and automated image analysis of detected fluorescence signal has become close to
reality now in spite of the heterogeneity and crowding effects in dividing cells (Moolman et al.,
2015). At the same time, the correct labeling of endogenous proteins with fluorescent proteins in
vivo can be achieved through latest genome editing tools (Reyes-Lamothe et al., 2008). Here, I
discuss the combination of these techniques to achieve single molecule imaging of endogenous
proteins to gain insights on the DNA replication mechanism.
Chromosome engineering methods currently use homologous recombination-based
techniques to modify the chromosomal DNA in E. coli, which have application in both industrial
and fundamental research. These methods are employed in this study to currently label the
endogenous proteins by fusing the fluorescent protein coding sequence with the native gene of
interest. However, proper validation of the created strain before using it for the research is
necessary. Modification of chromosomal DNA is currently performed using the recombineering
technique and the fusion of modified sequences in single E. coli strain is done by employing the
P1-phage transduction. Beyond the standard PCR and DNA sequence analysis methods for

2. validating the created strain, we describe the essential verification by Southern blotting for
detecting the extraneous insertions that may occur in recombineering. These unwanted mutations
can be removed by transducing the gene of interest into wildtype strain using virulent P1 phages.
However, phage stock used can become contaminated with temperate phages in which case they
exist as a large plasmid in growing cells resulting in contaminated strains. We then describe
assays to detect the presence of temperate phages in the created strain using the cross-streak agar
and Evans blue-uranine agar plating. We also explain ways to detect cell growth and cell
morphology defects and a stepwise flow of validating the created strain (Tiruvadi Krishnan et al.,
2015).
In order to utilize the techniques we have developed, we began by studying the β2 sliding
clamp proteins in growing cells for an in-depth understanding of the DNA replication and repair
since β2-clamps play an important role in both the processes. Being one of the main components
of the replisome complex β2-clamps ensure high accuracy of the DNA replication and proper
regulation of the processivity to coordinate the timely replication before cell division depending
on the growth conditions. Using the widefied and photoactivable fluorescence microscopy, we
identified the number of β2-clamps bound to DNA and their effective loading and unloading
rates. We found from our observations that β2-clamps accumulate on DNA after initiation of
replication until the DNA-bound β2-clamps attains a steady state, which is maintained during
most of the replication. This accumulation of β2-clamps is due to the relatively slow unloading of
β2-clamps from DNA in comparison to the other replisome components. Through our single
molecule imaging experiments we observed that each β2-clamp is bound to DNA in the orders of
minutes in vivo. Our results show the versatility of the β2-clamp proteins in recruiting different
proteins to the DNA to complete the replication process with high accuracy as it is involved in
various processes of DNA replication and repair (Moolman et al., 2014).
The replisome components at the fork of DNA replication face various protein barriers,
which are bound to DNA and surpass them to complete the DNA replication. We describe the
outcome of a replisome encountering the endogenous Tus-Ter roadblock of E.coli strain with
ectopic origin in vivo. The replisomes initiated from the ectopic origin progress in such a way
that the clockwise (CW) fork approaches the non-permissive side of the Tus-Ter roadblock
considerably earlier than the counter-clockwise fork. From the results, we found that the
replisomes, which are detected by the labeled β2-clamps, continue binding to DNA even after the
CW fork faces the Tus-TerC barrier. By observing the replication times of the ectopic origin
strains we determine that the speed of replication decreases when the fork meets the Tus-Ter
roadblocks while it is not stalled forever. We found the presence of Tus-Ter barriers influence
the sister chromosome alignment (SCA) patterns. From these results, we demonstrate the
flexibility of the replisomes at the replication fork to the Tus-Ter roadblocks and the sensitivity
of the SCA patterns to the progression of the forks (Moolman, Tiruvadi Krishnan et al., 2016).
Polar arrest mechanism of replication forks by the Tus-Ter roadblocks tries to answer the
important questions of how and where the termination of chromosome replication occurs in E.

3. coli. We attempted to ascertain the localization and stoichiometry of native Tus proteins during
the cell cycle using the quantitative widefield fluorescence microscopy and microfluidics. From
our initial observations, we found that Tus is expressed in low copy numbers in the order of eight
to thirteen molecules per growing cell, of which around five molecules form foci (signal above
the background) through possibly binding to Ter site. We also observe that the intensity of the
foci do not change much during the cycle implicating that the Tus is constantly bound to Ter
sites in the chromosome. Due to the low fluorescence signal of labeled Tus, no observable
binding or unbinding events was detected in the cells. The labeled Tus foci were found to be
distributed from cell poles to mid-cell similar to the expected localization of Ter sites. Our
results depict an overview of the intricate details of the Tus-Ter dynamics in vivo during the cell
cycle and aid in the design of future experiments for further studies on the mechanism of
replication termination.
The replisome components synthesize the chromosome in a semi-conservative fashion
with the forks of replication formed during the initiation phase using the RNA primers
synthesized on the DNA strands by the primase. During the elongation phase, the leading strand
of the replication fork is synthesized continuously while the lagging strand is synthesized
discontinuously with the help of primase. In order to understand the in vivo dynamics of
primases during the cell cycle, we attempted to label the E.coli primase proteins. Using the scar-
less recombineering technique we successfully engineered the native primase gene fused with the
fluorescent protein coding sequence. However, the fusion protein was found to be non-
fluorescent due to unknown post-translational modifications while being expressed from the
macromolecular synthesis operon, since the fusion protein was fluorescent when expressed from
an inducible promoter. Hence, we attempted to express the fluorescent-primase ectopically from
the chromosome in which the native primase gene is then knocked-out. The knockout strategies
resulted only in false positive strains necessitating a highly efficient genome-editing tool to
engineer primase gene in E.coli.
The research work discussed here demonstrates the utility of various methods and
experiments performed to expand our knowledge on the replication of chromosome in live
growing cells. An inter-disciplined approach was employed to understand the intricate details of
the DNA replication in action. The applicability of in vivo single molecule imaging techniques
ranges widely from determining the dynamics of endogenous proteins to their stoichiometry
during chromosome replication. In this research work, we have validated the effectiveness of the
above-said techniques in gaining valuable insights on the replication process at the single-
molecule level. We have also presented the challenges in strain creation techniques and the
importance of them in the implications of in vivo single molecule research. As with any scientific
research, the valuable results obtained from our study raise more innovative questions, which the
researchers will be able to answer with further technical developments in future.

4. References
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