536
Bacterial Growth and Division
At other growth rates, the patterns of
synthesis appear more complicated, but
this is due to the possibility of initiation
occurring before completion of previous
rounds of replication and the possibility
of initiation of replication occurring in
one division cycle and replication of
the chromosome being completed in a
subsequent division cycle.
3.2.2
Regulation of Initiation of DNA
Replication
Why
is
replication
initiated
at
these
particular times during the division cycle?
How is the initiation process regulated?
Rounds of replication are initiated in a
bacterial cell when the amount of cell
mass or something proportional to the
cell mass (the ‘‘initiator’’) is present at a
certain amount per origin. Initiation of
new rounds of replication occurs once
per division cycle when the mass (or
‘‘initiator’’) per origin reaches a Fxed value.
In the 60 min cell, initiation occurs at
one origin with a cell of unit size (size
1.0). In the 30 min cells, there are two
origins in the newborn cell and the size
of the newborn cell is twice that of a
60 min cell. And the 20 min cell has four
origins initiated in the newborn cell and
the cell size is four times the newborn
60 min cell. Thus, not only does the
DNA per cell increase with growth rate
but the average cell size increases with
growth rate as well. In the cells shown in
±ig. 3, the relative sizes of the newborn
cells, as well as the average cell in the
culture, are in the ratio of1:2:4. This
increase in cell size with growth rate is
consistent with the exponential increase
in cell size as a function of the inverse of
the doubling time.
The actual nature of the initiator is
not known, but a number of candidate
molecules have been identiFed. Among
these, the DnaA protein appears as a
good candidate for the regulator of DNA
initiation.
Alternative models of initiation have
been
proposed,
such
as
the
sudden
accumulation of an inhibitor of initiation
that is diluted out by cell growth. This
model does not accommodate the constant
synthesis of cell-cytoplasmic components.
In all cases, it is indistinguishable from the
initiator accumulation model, and there is
no evidence for such an inhibitor being
synthesized during the division cycle.
3.2.3
The SpeciFc Origin of Replication
A speciFc sequence of DNA in the bacterial
genome is the origin of replication (oriC).
The required sequence (for
E. coli
)i
s
245 bp. Within this sequence are 5 highly
conserved sequence blocks of 15 to 20 bp.
These conserved regions are interspersed
with sequences that are random. Regions
of random sequence but of Fxed length
separate the conserved regions. Thus,
the length of separation of conserved
sequences is more important than the
speciFc sequence between the conserved
sequences. ±our sequences of 9 bp are
speciFc recognition sites for the binding
of dnaA, the speciFc initiator protein. In
addition, there are three 13 bp regions
that are sites for opening the duplex DNA
for initiation. ±inally, there are 14 GATC
sequences located in the origin (oriC)
region and the neighboring regions. These
14 four bp sequences are expected to occur
at a much lower frequency if they were not
a necessary part of the
E. coli
origin.
The ultimate proof that this sequence is
the origin of replication is the production
of minichromosomes, small replicating
circles of DNA that have the bacterial
origin of replication. An
E. coli
cell can
have many copies of the minichromosome
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