1. Sebastian Swanson1
, James Kraemer2
and Michael Laub2,3
1
College of Biological Sciences, University of Minnesota, 2
Department of Biology, Massachusetts Institute of Technology, 3
Howard Hughes Medical Institute
REFERENCES
Katayama, T., Ozaki, S., Keyamura, K., and Fujimitsu, K. (2010). Regulation of the replication cycle:
conserved and diverse regulatory systems for DnaA and oriC. Nature Reviews. Microbiology 8,
163–170.
Messer, W., and Weigel, C. (1997). DnaA initiator—also a transcription factor. Molecular Microbiolo-
gy 24, 1–6.
Mott, M.L., and Berger, J.M. (2007). DNA replication initiation: mechanisms and regulation in bacte-
ria. Nat Rev Micro 5, 343–354.
Conclusions
Experimental Design
Introduction
R
DnaA is well characterized in E. coli as the replication initiator protein, however its role in con-
trolling cellular physiology via transcriptional regulation is only beginning to be understood.
Fig. 1 The four domains of DnaA. Domain I is involved in recruitment of the replisome, domain II
is considered a linker region, domain III has ATPase activity and domain IV is involved in recog-
nizing and binding specific sequences of DNA denoted as “DnaA boxes” (Mott and Berger,
2007).
Fig. 2 As DnaA-ATP is the active form in initiation, the cell has evolved a complex regulatory
system to control nucleotide state and prevent aberrant replication. The proportion of DnaA-ATP
to DnaA-ADP is coupled with replication cycles such that it peaks at the time of initiation (Kata-
yama et. al, 2000).
Fig. 3 DnaA boxes in the promoters of genes thought to be
regulated by DnaA. High affinity sites (white boxes) can be
bound by both DnaA-ATP and DnaA-ADP whereas low affinity
sites (grey boxes) can only be bound by DnaA-ATP (Messer
and Weigel, 1997).
DnaA Repression DnaA Activation
1) Study how the nucleotide state of DnaA alters transcriptional regulation and growth
2) Identify previously unknown gene targets of DnaA
Goals
pDnaAR334A
Cells Accumulate Mass but Lose Viability
Fig. 5 Comparison of growth between
strains in liquid media under experimental
conditions. Following induction during
log-phase growth all strains continued to
accumulate mass at an exponential rate.
Fig. 6 Dilution plates
innoculated from liquid cultures
immediately prior to induction
and 60 minutes afterwards.
pDnaAR334A
cells show an
unhealthy phenotype following
induction while pDnaA and
pDnaAT174P
continue to grow.
0 min
10-1
10-2
10-3
10-4
10-5
10-6
WT
pDnaA
pDnaAR334A
pDnaAT174P
WT
pDnaA
pDnaAR334A
pDnaAT174P
60 min
Fig. 7 Microscopy of cells fixed 90 minutes after induction. pD-
naAR334A
cells have undergone filamentation
WT pDnaA pDnaAR334A
pDnaAT174P
DnaAR334A
Binds to the Origin and Initiates Replication More Frequently Than DnaAT174P
Fig. 8 Relative fold enrichment for binding events at the origin as
determined by qPCR. Samples were immunoprecipitated using
DnaA polyclonal antibodies. Total input of chromatin and relB were
used for normalization. DnaAR334A
showed the strongest enrichment
at the origin when compared to the other mutant and WT.
Fig. 9 Flow cytometry run out experiments for the strains overex-
pressing DnaA 1) prior to induction (black) and 2) 90 minutes after
induction (red). After samples were taken from the cultures, rifampi-
cin and cephalexin were added to inhibit new initations of replication.
The cells were then incubated at 37C for four hours to allow previ-
ously initiated replication forks to complete. 90 minutes after induc-
tion pDnaA and pDnaAR334A
showed overinitiation while pDnaAT174P
showed underinitiation relative to their profiles prior to induction.
DnaAR334A
Binds to DnaA Boxes in Promoters and Differentially Regulates Genes
Fig. 10 Relative fold enrichment for binding events at DnaA boxes within promoters of genes thought to
be regulated by DnaA (Messer and Weigel, 1997) as determined by qPCR. Samples were immunopre-
cipitated using DnaA polyclonal antibodies. Total input of chromatin and relB were used for normaliza-
tion. At all promoters, DnaAR334A
showed the highest number of binding events. This trend was highly
pronounced at PdnaA and PmioC and to a lesser extent PnrdA. Binding at PrpoH did not appear to differ
significantly between the strains.
Novel Differentially Regulated Genes Identified by RNA-seq
Fig. 11 Relative expression levels for previously identified transcriptional targets (Messer and
Weigel, 1997) of DnaA at 0, 15 and 30 minutes after induction as quantified by RNA-seq.
RNA was purified and converted to cDNA which was sequenced using Illumina technology.
Singly mapped reads were then aligned to coding regions in the genome. Following induction
nrdA was expressed more highly in both pDnaA and pDnaAR334A
than pDnaAT174P
. MioC was
highly repressed in pDnaAR334A
between 0 and 15 minutes.
Fig. 12 Relative expression levels for an initial subset of genes found to be differentially ex-
pressed between the different conditions 0, 15 and 30 minutes after induction as quantified by
RNA-seq in the same manner as (fig. 11). NrdG and nrdG were expressed more highly in
pDnaA and pDnaAR334A
15 minutes after induction when compared to pDnaAT174P
. Both recA
and lexA were more highly expressed in DnaAR334A
than the other conditions after 30 minutes.
MutH was downregulated 15 minutes after induction in DnaAR334A
. FliY was strongly re-
pressed in pDnaAT174P
between 0 and 15 minutes.
Overexpression of DnaAR334A
Increases Initiation Frequency and
Activates the SOS Response
DnaAR334A
overexpression renders the majority of cells in liquid culture non-viable (fig. 6). Mi-
croscopy of these cells reveals filamentation, a phenotype of the SOS response (fig. 7).
Indeed, RNA-seq confirms the upregulation of recA and lexA over the 30 minutes following in-
duction. In addition, flow cytometry run-outs reveal that 1) this DNA damage is due to overiniti-
ation and 2) this strain is resistant to rifampicin and continues initiating new replication events
after addition of the antibiotic (fig. 9). This could suggest that the abnormaly high numbers of
DnaAR334A
at the origin are able to overcome the inhibitory effects of rifampicin binding on
RNAP or that DnaAR334A
can interact directly with RNAP and prevent rifampicin binding.
DnaAR334A
Binding at DnaA Boxes in Promoters Results in Differen-
tial Gene Expression
Genes identified previously as transcriptional targets of DnaA (Messer and Weigel, 1997) were
analyzed for differential expression following overexpression of the two mutants (fig. 4). NrdA
and mioC showed the highest fold change when comparing DnaA-ATP (DnaAR334A
) and
DnaA-ADP (DnaAT174P
) (fig. 11). While the model for mioC repression by ATP-DnaA binding to
the promoter is well established, the mechanism for nrdA activation by ATP-DnaA is still un-
clear. The promoters of both mioC and nrdA showed higher fold enrichment for DnaA-ATP than
the other forms (fig. 10). Previously unidentified genes were shown to be both activated and re-
pressed by DnA-ATP including lexA, recA and mutH (fig. 12). However, without ChIP data it is
impossible to determine if these are direct transcriptional effects or merely side effects of unre-
lated pathways responding to accumulated DNA damage.
pDnaA
pDnaAR334A
pDnaAT174P
MG1655
Cells are transformedSite directed mutagenesis
to introduce mutations
Cells grown in M9 + .4%
glycerol + .2% CAA at 37C
Addition of arabinose
at 0.2 OD. to in-
duced plasmid ex-
pression
Fig. 4 The experimental procedure used to generate samples. A plasmid containing WT dnaA
behind an arabinose-inducible promoter was amplified using mutagenic primers to yield pD-
naAR334A
and pDnaAT174P
.
DnaAR334A
had reduced intrinsic ATPase activity, thus it functioned as DnaA-ATP.
DnaAT174P
had increased intrinsic ATPase activity, thus it functioned as DnaA-ADP.
After these transformed cells were grown up and induced by arabinose, samples were
taken at different time points for downstream analysis.
pBAD33
ACKNOWLEDGEMENTS
James Kraemer, Allen Sanderlin, Peter Culviner, Monica Guo
The E. coli Replication Initiator Regulates Expression as a
Transcription Factor
Results