A negative feedback is enough

to tightly regulate a pulse of

gene expression in bacterial
conjugation
Saúl Ares
Grupo Interdisciplinar de Sistemas Complejos (GISC)
and Systems Biology Programme,
Centro Nacional de Biotecnología (CNB-CSIC)
@ omeuxeito

VIII Complexitat.cat, May 21st 2019

Horizontal gene transfer

Doolittle, 1997
 Horizontal gene transfer
in bacteria
Bacterial conjugation is an
important mechanism of
antibiotic resistance spreading
Bacillus pumilus

Gram-positive bacterium

Commonly found in soil

We will study the regulation

of establishment genes of
plasmid p576
Establishment genes
Experimental
results
Promoters P20c, P23c, PardC576 and P27c
control putative establishment genes
Promoters P20c, P23c, PardC576 and P27c
show strong expression

The promoter of gene 27c is

around three times weaker
than the others.
 Gene 27c represses promoters
P20c, P23c, PardC576 and P27c

We denominated gene 27c

reg576
(repressor of establishment genes)
Operator site of Reg576 constituted
by a dual 5’-TTATCCC-3’ motif

Mutations on one of the

heptamer motifs is
enough to inhibit Reg576
binding.
Operator site of Reg576 constituted
by a dual 5’-TTATCCC-3’ motif
Reg576 is a dimer in solution
A for a schematic view of the DNA
Two Reg dimers bind to
absorbance plots for the DNA frag-
Reg576 (OD260 nM) are shown in
576
one operator
, the different DNA fragments in
howed the same s-value of 5.0 S.
did not increase the s-value of frag-
as mutated all the four motifs, but
ncrement (from 5.0 S to 6.3 S) for
rators intact) and moderate (from
nts for fragments F-mut1A (muta-
and F-mut1D (mutations in only
w that Reg576 did not bind to frag-
bound to the other three DNA frag-
ease in s-value it seemed that frag-
mut1D bound the same or similar
that fragment F-P20c bound more
1A and mut1D. However, care has
erpretation since the s-value is not
molecular weight of the complex
hape. To fully extract the maximum
the SV data, besides the hydrody-
complexes, we took advantage of
bance data acquisition at 230 and
0.1 !M was titrated with increasing Reg576
(from 0.1 to 3 !M). Figure 7D shows the bin
built from the experimental buoyant mass
tained at low speed and 260 nm, through an
parameters Hill plot (Equation 6):
axb
y= b
Kd + xb
Where y stands for the number of proteins b
P20c: number of protein
a denotes the maximum
uration, x is the total concentration
b =7.8 of pr
concentration of half-maximal±binding and
Kd = 0.8 0.1μM
cal cooperativity parameter.
Taking into account the complex stoich
mentally determinedmut1A:
for Reg576 –F-P20c , an
erative model (b= 7.8) can explain the exper
b=4.1
isotherm, with a macroscopic ±K
Kd = 0.7 0.1μMd of 0.8 ±
ogously, for Reg576 –F-Mut1A and Reg576 –
plexes, an apparent cooperative model (b
mut1D:
respectively) can account for the binding i
macroscopic Kd of 0.7 ± b=3.2
0.1 !M. These r
with those obtainedKby= MSSV
0.7 ± technique a
0.1μM
d
account that Reg576 is a dimer in solution, d
 Crystal
structure of
Reg576: it is a
dimeric
protein.
Model of the
tetramer
bound to a
double DNA
heptamer.
Experiments: long story short

The repressor Reg576 binds its own promoter

through a double operator that needs 4 molecules
(2 dimers).

Reg576 binds establishment gene promoters

through two operators: 8 molecules at play
(4 dimers).

The repressor’s promoter is 3 times weaker

than establishment gene promoters.
Mathematical
theory
mr pr me
Mathematical model of
gene 20c regulation by Reg576
!!! (!) !
= − !! !! ! mRNA
repressor !" !! !
!!
1+
!!r
Reg576
!!! (!) protein
= ! !! ! − !! !! (!)
!"
!!! (!) 3!
= − !! !! (!) mRNA
!" !
!! ! !
establishment 1+
!!e
20c !!! (!)
= ! !! ! − !! !! (!) protein
!"
where mr is the concentration of repressor mRNA, pr is the c
hr = 4, he = 8, Kr = 0.7, Ke = 0.8
repressor protein, me is the concentration of establishment gene
a = 1, dm of
concentration = 0.1, b = 0.3, dgene
establishment p = 0.03
protein, a is the repres
mr pr me
Mathematical model of
gene 20c regulation by Reg576
!!! (!) !
= − !! !! !
!" !! !
!!
1+ !
!r

!!! (!)
= ! !! ! − !! !! (!)
!"
!!! (!) 3!
= − !! !! (!)
!" !! !
! !
1+ !
!e

!!! (!)
= ! !! ! − !! !! (!)
!"
where mr is the concentration of repres
repressor protein, me is the concentration
hr = 4, he = 8, Kr =concentration
0.7, Ke = 0.8of establishment gene pro

a = 1, dm = 0.1, b =transcription rate, dm its mRNA degradatio

0.3, dp = 0.03
 mr pr me
High cooperativity necessary
for tight repression
Supplemental, Val et al. Regulation of p576 establishment genes

!!! (!) !
= − !! !! !
!" !! !
!!
1+ !
!r

!!! (!)
= ! !! ! − !! !! (!)
!"
!!! (!) 3!
= − !! !! (!)
!" !! !
! !
1+ !
!e

!!! (!)
= ! !! ! − !! !! (!)
!"
where mr is the concentration of repres
repressor protein, me is the concentration
h = 1, h = 2, K = 0.7, K = 0.8
r e r concentration
Figure S5. Model predicts that low cooperativity results in poor repression of
establishment genes. e of establishment gene pro
Time evolution of the mRNA and protein concentrations given by our model for the

a = 1, d = 0.1, b = 0.3, d = 0.03 transcription

repressor gene reg576 and the establishment gene 20c, for low values of the Hill rate, dm its mRNA degradatio
m parameters as described in
exponents controlling the repression. he=2 and hr=1, other p
Materials and Methods. Units are arbitrary.
 mr pr me
Differential repression
necessary for tight repression
Supplemental, Val et al. Regulation of p576 establishment genes

!!! (!) !
= − !! !! !
!" !! !
!!
1+ !
!r

!!! (!)
= ! !! ! − !! !! (!)
!"
!!! (!) 3!
= − !! !! (!)
!" !! !
! !
1+ !
!e

!!! (!)
= ! !! ! − !! !! (!)
!"
where mr is the concentration of repres
repressor protein, me is the concentration
h = 8, h = 8, K = 0.7, K = 0.8
Figure S6. Model predicts that equal repression of the repressor and the
r
establishment genes results in sustained establishment e expression. r concentration
gene e of establishment gene pro
Time evolution of the mRNA and protein concentrations given by our model for the

a = 1, d = 0.1, b = 0.3, d = 0.03

epressor gene reg576 and the establishment gene 20c, for equal values of thetranscription
Hill rate, dm its mRNA degradatio
exponents controlling the repression of the repressor mand the establishment gene. p
he=8 and hr=8 (using he=4 and hr=4 produces a result qualitatively similar), other
 Conclusions

We have described a novel regulatory mechanism

of establishment genes of conjugative plasmids.

mr pr me

The high cooperativity and differential repression

found in experiments are necessary for an
efficient pulse regulation.
 Conclusions
A two-gene system based on a negative
feedback loop is sufficient to produce a mr pr me
tightly regulated pulse of expression.

This is simpler than feed-forward loops (Alon 2007)

and other more complicated regulations of pulses
described so far.
 Outlook
The model has been rudimentary characterized.
A more complete study is possible.
Noise effects MUST be included. Conjugation of Gram +
Plasmid Regulated by rap-phr

The study of other

conjugative plasmids, like
Bacillus subtilis’ pLS20,
promises even more fun…

Figure 8. Model of regulatory circuitry of pLS20 conjugation genes. A. Repressed state due to RcoLS20. Gene rcoLS20 (red arrow, rco) encoding
11910–11926 Nucleic Acids Research, 2018, Vol. 46, No. 22 Published online 31 October 2018
doi: 10.1093/nar/gky996

Novel regulatory mechanism of establishment genes

of conjugative plasmids

Downloaded from https://a

Jorge Val-Calvo 1 , Juan R. Luque-Ortega2 , Isidro Crespo 3 , Andrés Miguel-Arribas1 ,
David Abia4 , Dione L. Sánchez-Hevia5 , Ester Serrano1 , César Gago-Córdoba1 , Saúl Ares5,6 ,
Carlos Alfonso2 , Fernando Rojo 5 , Ling J. Wu 7 , D. Roeland Boer 3,* and Wilfried
J.J. Meijer 1,*
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Complex Networks in the Life
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