Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
BackgroundResults
ConclusionsAcknowledgements
The multi-level relationship betweenheterotrophic bacteria and nutrients
Diauxie and co-utilization are not exclusive during growth innutritionally complex environments
Elena Perrin, Michele Giovannini, Francesca Di Patti, Barbara Cardazzo, Lisa Carraro, PaolaTurano, Veronica Ghini, Renato Fani, Marco Fondi
FEMS 2019, Glasgow, July 9, 2019
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
The classic view of microbial growth strategy when multiple carbonsources are available states that they either metabolize them
sequentially (diauxic growth) or simultaneously (co-utilization).
”All you can eat” vs. ”a la cart” strategies result in different growthdynamics (Monod, 1942).
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Diauxic shift is commonly seen as a phase in which the bacteria preparethemselves to use the second sugar. The existence of two stable cell typeswith alternative metabolic strategies emerge and coexist in a culture of the
bacterium has been demonstrated.
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Our knowledge is biased by the fact that this process has been mainlyanalyzed in over-simplified laboratory settings, i.e. using a few modelmicroorganisms and growth media containing only two alternativecompounds.
The marine environment:
• Low average nutrient level (e.g., the concentration of amino acids is inthe range of about 10−9 M)
• Nutrients appear and disappear in a sporadic fashion.
• DOM as a complex medium
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
• Chemotaxis ofPseudoalteromonashaloplanktis TAC125(PhTAC125) toward a pulse ofphytoplankton exudates(Stocker et al. 2010, Science)
• What happens once the patchis colonized?
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
We grew PhTAC125 in a complex medium (peptone, 7 replicates) andanalysed its growth features.
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
• High expression of growth-related genes in the first hours• Stress related genes up between T1 and T2
• Motility-related genes up at the end of the growth
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
A non E. coli-like response to growth lag• 81 TF known to dcontrol central
metabolic enzymes in E. coli
• Homologs for 34 of them inPhTAC125, 8 of them differentiallyexpressed (10%)
• Only RpoS and RpoD aredifferentially expressed among 8selected E. coli global regulators
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
A focus on amino acids degradation. PhTAC125 is know to thrivepreferentially on amino acids (Wilmes et al. 2010).
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
We assembled a defined, nutritionally rich medium including 19 amino acids ([0.2 mM],Cys not included) and analysed growth and metabolic features.
0.2
0.4
0.6
0 1 2 3 4 5 6 7 8 9
O.D
. (60
0 nm
)
C
*
*
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
We assembled a defined, nutritionally rich medium including 19 amino acids ([0.2 mM],Cys not included) and analysed growth and metabolic features.
0.00
0.05
0.10
0.15
0.20
0 1 2 3 4 5 6 7 8 9Time (hours)
Con
cent
ratio
n (m
M)
argasnaspglngluglyhisisoleulysmetpheproserthreotrptyrval
0.2
0.4
0.6
0 1 2 3 4 5 6 7 8 9
O.D
. (60
0 nm
)
A C
*
*
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
We assembled a defined, nutritionally rich medium including 19 amino acids ([0.2 mM],Cys not included) and analysed growth and metabolic features.
0.00
0.05
0.10
0.15
0.20
0 1 2 3 4 5 6 7 8 9Time (hours)
Con
cent
ratio
n (m
M)
argasnaspglngluglyhisisoleulysmetpheproserthreotrptyrval
0.2
0.4
0.6
0 1 2 3 4 5 6 7 8 9
O.D
. (60
0 nm
)
val
ile
leu
thrala
glu
gln
metasn
asp
lysgly
pro
tyr
his
trp
phe
ser
arg
−2
−1
0
1
2
3
−5 0 5
Dim1 (75.5%)
Dim
2 (1
2.7%
)
0.00
0.05
0.10
0.15
0.20
0 1 2 3 4 5 6 7 8 9
Time (hours)
Con
cent
ratio
n (m
M) C1
C2
C3
C4
A C
*
*
B D
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
We assembled a defined, nutritionally rich medium including 19 amino acids ([0.2 mM],Cys not included) and analysed growth and metabolic features.
0.00
0.05
0.10
0.15
0.20
0 1 2 3 4 5 6 7 8 9Time (hours)
Con
cent
ratio
n (m
M)
argasnaspglngluglyhisisoleulysmetpheproserthreotrptyrval
0.2
0.4
0.6
0 1 2 3 4 5 6 7 8 9
O.D
. (60
0 nm
)
0.25
0.50
0.75
1.00
C1 C2 C3 C4
O.D
. (60
0 nm
)
val
ile
leu
thrala
glu
gln
metasn
asp
lysgly
pro
tyr
his
trp
phe
ser
arg
−2
−1
0
1
2
3
−5 0 5
Dim1 (75.5%)
Dim
2 (1
2.7%
)
0.00
0.05
0.10
0.15
0.20
0 1 2 3 4 5 6 7 8 9
Time (hours)
Con
cent
ratio
n (m
M)
0.1
0.2
0.25
0.34
Glu Asp Leu Ala
Gro
wth
rate
(h-1)C1
C2
C3
C4
A C
*
*
E
B D F Order of consumption
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
What is regulating the system?
• Uptake kinetics (no regulation, dynamics explained by differentuptake kinetics).
• Active regulation The uptake of the different groups is somehowregulated.
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
What is regulating the system?
• Uptake kinetics (no regulation, dynamics explained by differentuptake kinetics).
P + AβA ·φAkA+φA−−−−→ 2 P
P + BβB ·φBkB+φB−−−−→ 2 P
P + CβC ·φCkC+φC−−−−→ 2 P
P + DβD ·φDkD+φD−−−−→ 2 P 0.00
0.05
0.10
0.15
0.20
0 1 2 3 4 5 6 7 8 9
Time (hours)
Con
cent
ratio
n (m
M)
AB
C
D
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
P + AβA ·φAkA+φA−−−−→ 2 P
P + BβB ·φBkB+φB−−−−→ 2 P
P + CβC ·φCkC+φC−−−−→ 2 P
P + DβD ·φDkD+φD−−−−→ 2 P
0 100 200 300 400 500 600
Time (minutes)
0
0.05
0.1
0.15
0.2
Con
cent
ratio
n (m
M)
A
Con
cent
ratio
n (m
M)
0 100 200 300 400 500 600
Time (minutes)
-0.1
0
0.1
0.2
B
0 100 200 300 400 500 600
Time (minutes)
0
0.1
0.2
0.3
Con
cent
ratio
n (m
M)
C
0 100 200 300 400 500 6000.05
0.1
0.15
0.2
Time (minutes)
Con
cent
ratio
n (m
M)
D
0 100 200 300 400 500 600
Time (minutes)
0
0.2
0.4
0.6
0.8
OD
(660
nm
) P
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
What is regulating the system?
• Active regulation The uptake of the different groups issomehow regulated. A cybernetic model (Kompala et al. 1994)is implemented.
P + A E1, ρ1−−−→ 2 PP + B E2, ρ2−−−→ 2 PP + C E3, ρ3−−−→ 2 PP + C E4, ρ4−−−→ 2 P
C A, λ1−−−→ C + E1
C B, λ2−−−→ C + E2
C C, λ3−−−→ C + E3
C D, λ4−−−→ C + E4
dφEi
dt= λi∗u1−(ρ1∗v1+ρ2∗v2+ρ3∗v3+ρ4∗v4)∗φEi−βφEi
dφP
dt= (ρ1 ∗ v1 + ρ2 ∗ v2 + ρ3 ∗ v3 + ρ4 ∗ v4) ∗ φP − αφP
dφSi
dt= ρi ∗ vi ∗ φP
With i = 1,2,3,4 and S = {A, B, C, D}
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
Growth on complex mediumAA metabolismGrowth on a 19 AA mediumA growth mathematica model
0 100 200 300 400 500 600
Time (minutes)
0.2
0.4
0.6
0.8
OD
(60
0 nm
)
0 100 200 300 400 500 600
Time (minutes)
0
0.1
0.2
Con
cent
ratio
n (m
M)
0 100 200 300 400 500 600
Time (minutes)
0
0.1
0.2
Con
cent
ratio
n (m
M)
0 100 200 300 400 500 600
Time (minutes)
0
0.1
0.2
Con
cent
ratio
n (m
M)
0 100 200 300 400 500 600
Time (minutes)
0.1
0.15
0.2
Con
cent
ratio
n (m
M)
MM CyberneticBiomass 0.997 0.994Cluster A 0.9893 0.9983Cluster B 0.9967 0.9977Cluster C 0.9947 0.9904Cluster D 0.9723 0.9908
R2 calculation of the fit
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
• PhTAC125 growth on complex media results in a triauxic growthwith a regulation response to nutrient starvation that does notresemble the model one.
• Growth lag phases are due to nutrient switching.• Sequential and co-utilization are not exclusive. Most efficient C
sources are consumed first.• A simple mathematical model based on Michaelis-Menten
kinetics uptake does not result in a perfect fit with the data.Regulatory mechanisms must be included to explain this growthdynamic at the single cell level
• Emergent sub-populations?
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients
BackgroundResults
ConclusionsAcknowledgements
DBIO, Unifi
• Elena Perrin
• Prof. Renato Fani
• Prof. Alessio Mengoni
• Camilla Fagorzi
• Michele Giovannini
CERM, Unifi
• Veronica Ghini
• Prof. Paola Turano
CSDC, Unifi
• Francesca Di Patti
Unipd
• Barbara Cardazzo
• Lisa Carraro
Marco Fondi The multi-level relationship between heterotrophic bacteria and nutrients