The physiological and ecological roles of urease activity in
Streptococcus thermophilus
Stefania ARIOLI
pH decrease
Homo-lactic fermentation
Phenotypic properties affecting the acidification rate:
§ Lactose metabolism
§ Proteolytic activity
§ CO2 metabolism
Streptococcus thermophilusOne of the main component of starter cultures
for dairy industry ($40 billion)
0.2 mm
2
0.2 mm
By analyzing the bacterial genome…(Bolotin et al., 2004)
glutamate
NH3
glutamate
glutamine
urea
CO2
CO2
CO2
PEP
oxalacetate
aspartate2-oxoglutarate
ureIABCDEFG
ppc
aspA
glnA
GluT
CO2
CO2
carbamoylphosphate
argininepyrimidine
carB
2ATP
2ADP
glutamate
NH3
glutamate
glutamine
urea
CO2
CO2
CO2
PEP
oxalacetate
aspartate2-oxoglutarate
ureIABCDEFG
ppc
aspA
glnA
GluT
CO2
CO2
carbamoylphosphate
argininepyrimidine
carB
2ATP
2ADP
In the S. thermophilus Core Genome, detected in all strains of a species… necessary for overall viability (Rasmussen et al., AEM 2008)
Aspartate biosynthesis is essential for the growth of S. thermophilus in milk, and aspartate availability modulates the level of urease activity (Arioli et al., 2007)
Carbamoylphosphate synthetase activity is essential for an optimal growth of S. thermophilus in milk (Arioli et al., 2008)
3
4
Urease of S. thermophilus
ü Complex enzyme (11-gene operon)
ü0.9% of the core genome
ü Loss-of-function mutations/acquisition of relevant traits
ü In all previously characterized S. thermophilus strains
ü Urease-negative not common in nature
ü Physiological role: involved in nitrogen metabolism by a mechanism sensitive to
aspartate, gluatamate, glutamine and NH3 concentration
pH 5.8-6
Milk acidification
4,5
5
5,5
6
6,5
7
0 5 10 15 20
Time (h)pH
4,5
5
5,5
6
6,5
7
0 5 10 15 20
Time (h)pHü Urease as a stress response to counteract
acidic challenge?
ü Contribution to the environmental fitness of
the microrganism?
pHex
pHin
urea
The physiological role…
ATP synthesis associated to urea hydrolysis
ATP synthesis urea-dependent was
measured also in cells treated with
UNCOUPLERS (gramicidine, CCCD) or
ATPase inhibitor (DCCD), and in
membrane-free cell extract
In S. thermophilus
ATP synthesis urea-dependent is not a
chemiosmotic process
• Helicobacter pylori• Ureaplasma urealyticum • Bacillus pasteurii• Howardella ureilytica(Meyer-Rosberg et al., 1996; Smith et al., 1993; Janhs, 1996; Cook et al., 2007)
ATP has been reported to be coupled to urea hydrolysis via a chemiosmotic mechanism
urea 2NH3 + CO2urease
5
ATP
ATP
lactate dehydrogenasepyruvate kinase
G - 6P
F6P
F1-6P
ATP
ADP
DHA-P GA-3P
1-3 PGA
3 PGA
2 PGA
PEP
NAD+
NADH +H +
ADPATP
PYR
ADPATP
lactic acid
NADH +H +NAD+
Pi
Lactose
Galactose
Lact
ose
H*
Lactose
Glucose
Galactose
LacS
LacS
LacS
LacS
b-galactosidase
NH4+
CO(NH2)2
2H2O
CO32-
pHin
EMPpathway
… the hypothesis
… looking at S. thermophilus energetic metabolism…
6
… the influence of urea hydrolysis by urease on extracellular pH and ATP production…
energetically discharged cellswere washed and used in a
second experimental step, fed with:
0
5
10
15
20
25
30
35
40
25 27 29 31 33 355
6
7
8
9
10
Time (min)
pHex
urea
ATP
(mM
)
5
6
7
8
9
10
0
5
10
15
20
25
30
35
40
25 27 29 31 33 35
Time (min)pH
ex
lactoseAT
P (m
M)
0
5
10
15
20
25
30
35
40
25 27 29 31 33 355
6
7
8
9
10
Time (min)
pHex
lactose + urea
ATP
(mM
)
0
5
10
15
20
25
30
35
40
4 9 14 19 245
6
7
8
9
10
ATP
(mM
)
pHex
Time (min)
urea
Urea hydrolysis strongly induces glycolysis and homofermentative metabolism, increasing the
yield of ATP synthesis and lactic acid production7
Mutant MIM945 was obtained by the transformation of the wild-type with pCSS945 vector harboring the Jamaican click beatle
luciferase gene lucGR. In presence of D-Luciferine light emission was dependent on intracelluar ATP concentration
lactose
lactose + urea
lactose + NH3
lactose + urea + oxamate6
6,4
6,8
7,2
7,6
0 2 4 6 8 10 12 14
Time (min)
pHin
intracellular pH
Ligh
t em
issi
on (r
lu/s
)
Time (min)
0
4000
8000
12000
16000
20000
0 5 10 15 20
intracellular ATP
Sodium oxamate
lactate dehydrogenasepyruvate kinase
DHA-P GA-3P
1-3 PGA
3 PGA
2 PGA
PEP
NAD+
NADH +H +
ADPATP
ADPATP
lactic acid
NADH +HNAD+
Pi
+
H
The urea-dependent ATP
synthesis is generated by an
accelerated glycolytic flux due to
an intracellular alkalization
8
In vivo 13C NMR analysis of lactose consumption and lactic acid production in de-energized S. thermophilus cells
13C-Lactose
urea/NH3
[1-13C] Lactose
0
2
4
6
8
10
12
14
16
0 10 20 30 400
2
4
6
8
10
0 10 20 30 40
□ lactose, urea, and sodium oxamate
● lactose and urea 10 mM
○ lactoselactose and urea 0.5 mM
lactose and NH3 1 mM
[1-13C] Glucose
0
2
4
6
8
10
12
14
16
0 10 20 30 400
2
4
6
8
10
0 10 20 30 40
The limitant step in the bioenergetic metabolism of
S. thermophilus is the glycolytic rate rather than
the sugar transport
Increasing glucose concentration in cells fed just with lactose
[1-13C] Lactic acid
0
2
4
6
8
10
0 10 20 30 400,0
0,5
1,0
1,5
0 10 20 30 40
Only lactose
Lactose and urea 10 mM
9
10
Lactose
Lactose/NH3
Lactose/Urea
Raw isothermal titration calorimetry data…
ü The specific changing in enthalpy in S. thermophilus increased 70% and 15% in
the presence of lactose/urea and lactose/NH3
Glu
cose
, lact
icac
id (m
M)
pH
0
2
4
6
8
10
12
14
16
18
20
22
24
4 5 6 7 8 9 10 11 120
0,5
1
1,5
2
2,5
3
3,5
4 5 6 7 8 9 10
pH
Lact
ate
dehy
drog
enas
e(U
/mg)
Effect of pH on lactate dehydrogenase activity
Glu
cose
, lact
icac
id (m
M)
pH
0
2
4
6
8
10
12
14
16
18
20
22
24
4 5 6 7 8 9 10 11 120
0,5
1
1,5
2
2,5
3
3,5
4 5 6 7 8 9 10
pH
Lact
ate
dehy
drog
enas
e(U
/mg)
pH dependency of S. thermophilus
glycolysis activity
§ Effect of the pH on glycolitic enzyme activities
ON
PG
hyd
roly
sis
(OD
415n
m)
pHex
ON
PG
hyd
roly
sis
(OD
415n
m)
A B Effect of NH3 concentration on whole cells b-galactosidase activity
Effect of urea hydrolysis on whole cellsb-galactosidase activity
ON
PG
hyd
roly
sis
(OD
415n
m)
pHex
ON
PG
hyd
roly
sis
(OD
415n
m)
A B
11
time (h)
UFC
/ m
l
1LacS
lactose
lactose
galactose
galactose
glucose
b-galactosidase
glycolysis
lactate dehydrogenase
lactic acid
LacS
lactose
lactose
galactose
galactose
glucose
b-galactosidase
glycolysis
lactate dehydrogenase
lactic acid
1
pH 5.8-6
Supposed model describing the role of urease activity in the bioenergetic metabolism of S. thermophilus...
LacS
lactose
lactose
galactose
galactose
glucose
b-galactosidase
glycolysis
lactate dehydrogenase
lactic acid
pHex
pHin
LacS
lactose
lactose
galactose
galactose
glucose
b-galactosidase
glycolysis
lactate dehydrogenase
lactic acid
pHex
pHin
3
3
... decrease in intra- and extracellular pH and a reduction
of energetic metabolism
2
2
Milk acidification
LacS
lactose
lactose
galactose
galactose
glucose
b-galactosidase
glycolysis
lactate dehydrogenase
lactic acid
pHex
pHin
NH4+
CO(NH2)2
2H2O
CO32-
urease
NH4+
CO(NH2)2
2H2O
CO32-
ureasepHin
LacS
lactose
lactose
galactose
galactose
glucose
b-galactosidase
glycolysis
lactate dehydrogenase
lactic acid
pHex
pHin
NH4+
CO(NH2)2
2H2O
CO32-
urease
NH4+
CO(NH2)2
2H2O
CO32-
ureasepHin
4,5
5
5,5
6
6,5
7
0 5 10 15 20
Time (h)
pH
4,5
5
5,5
6
6,5
7
0 5 10 15 20
Time (h)
pH
... the urea hydrolysis determines a transient cytoplasmic
alkalinization that positively affect the energetic metabolism
Not acid stress response systems
12
Milk sub-culturesWild-type
Urease-negative
1 2 3 4 5 6 7 8 9 10 M
ΔureC 1669 bp ureC 2362 bp
1° 2° 3° 4° 5° 6° 7° 8° 9° 10° M
plating TotalDNA
extraction
May urease activity represent an advantage for milk colonization?Growth competition experiment…
ureC gene (wild-type)
DureC gene (urease-negative)
DureCureC
…urease negative strain looses competition with the wild-type in milk colonization …
wild-type
urease-negative
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10Time (days)
Num
bero
fclo
nes
N. of urease-negative/positive colonies
A16 + 5% of wild-type
A16 only
A16 + 10% of wild-type
A16 + 20% of wild-type
A16 + 50% of wild-type
A16 + 5% of wild-type
A16 onlyA16 only
A16 + 10% of wild-type
A16 + 20% of wild-type
A16 + 50% of wild-type
0
100000
200000
300000
400000
500000
0 5 10 15 20Time (min)
Ligh
t em
issi
on (r
lu/s
)
Urease-negative intracellular ATP inmixed cell suspension
lactose + urea
wild-type A16(urease-negative)bioluminescent
urea
urea
urea
urea
CO2NH3
CO2NH3
NH3
CO2
CO2
NH3
pH
… which is the effect of medium alkalinization on the energetic metabolism of urease-negative microrganisms sharing the
same environment ?
13
A new role of alkalinizing reactions in bacterial metabolism?
acid stress response systems
systems to prevent/postpone the acid stress
and/or
systems to increase the metabolic fitness in adverse environmental conditions
energetic metabolism
NH4+
CO(NH2)2
2H2O
CO32-
pHin
14
15
Cooperation between species in the yogurt consortium is affected
by modulation of intracellular pH driven by urease of
Streptococcus thermophilus
The ecological role….
aminoacids
peptides,putrescine,purine
CO2,pyruvate,formate,
folate,longchain fatty acids,ornithine
lacticacid
lacticacid
urea
urea
urease
2NH3 ?modulation of pHex pHin
pHin
pHin
2NH3
MUTUALISM (Boucher et al., 1982)
ü an interaction between species/strains that is beneficial to both (+/+ interaction)
ü exchanges of benefits
ü yogurt consortium: Streptococcus thermophilus + Lactobacillus delbrueckii bulgaricus
ü nitrogen sourcesü pyruvic acid, folic acid, formic acidü carbon dioxide
üpurine, amino acids, long-chain fatty acidü iron metabolism
16
Alkalizing reactions streamline cellular metabolism in acidogenic bacteria (Arioli et al., 2010)
ü Urea hydrolysis increases the catabolic efficiency of S. thermophilus
ü The local transient increase of pH due to the urease activity positively affects the energetic
metabolism of an urease-negative microrganism17
AIM of the STUDY
Effect of urea hydrolysis by S. thermophilus on the variation of the pHin
of L. delbrueckii subsp. bulgaricus to add new information on the proto-
cooperation existing between the two species during yogurt production
ü Flow cytometry
üStaining: membrane-permeating cFDASE cleaved by intracellular esterases,
the cFSE fluorescence decreases while pHin is decreasing
18
1. Effect of ammonia on S. thermophilus pHin
ü Saline solution
ü Absence of carbon source
FSC
-A
cFSE fluorescence - A
cFSE fluorescence - A
FSC
-A
cFSE fluorescence - A
FSC
-A
Cou
nt
cFSE fluorescence - A
T0
T1
T1 + NH3
T1 + NH3
T1T0
pHex: 6.68pHin: 7.04
pHex: 9.60pHin: >7.80
19
cFSE fluorescence - A
FSC
-A
cFSE fluorescence - A
FSC
-A
cFSE fluorescence - A
T0 T1 + NH3
T1 + NH3T1T0
FSC
-A
cFSE fluorescence - A
Cou
nt
T1
… and on L. delbrueckii bulgaricus pHin
pHex: 5.16pHin: 5.65
pHex: 9.70pHin: 7.72
20
2. The ammonia released by urease activity of S. thermophilus modulates the pHin of
urease-negative microrganismsü Mixed populations
ü Saline solution
pHin 6.8
pHex 6.8
pHin 6.6
pHex 6.5
cFSE
fluorescence-A
FSC - A
cFSE
fluorescence-A
FSC - A
pHin >7.8
pHex 8.6
pHex 6,4
pHin 6.6
FSC - A
FSC - A
T0 T1
T0 T1
+ Urea 2.5 mM
S. thermophilus A16 urease-negative
(stained)
S. thermophilusCNRZ385 (unstained)
21
pHin 6.3
pHex 6.3
pHin >7.8
pHex 8.5
pHin 6.5
pHex 6.3
pHin 6.6
pHex 6,2
FSC - A FSC - AcFSE
fluorescence-A
cFSE
fluorescence-A
FSC - A FSC - A
T0 T1
T0 T1
+ Urea 2.5 mM
L. delbrueckii bulgaricus MIM91 (stained)
S. thermophilusCNRZ385 (unstained)
üThe ammonia released by the urease-positive population diffuses inside urease-negative
strain thus determinig an increase of pHin
3. Effect of urea hydrolysis or ammonia alkalization on pH homeostasis and homolactic
fermentation of S. thermophilus CNRZ385…
ü urea-free skimmed milk
CNRZ385 CNRZ385, urea (2,5 mM) CNRZ385, NH3 (5 mM)
Time(min) Time(min) Time(min)
pHex-pH i
n
pHex-pH i
n
pHex-pH i
n
6
6,5
7
7,5
8
0 5 10 15
pHex pHin
L-lactic acid (mM)8.9 ± 0,2 9,4 ± 0,1 (+6%) 9.7± 0,1 (+8%)
(Arioli et al., 2010)23
… of L. delbrueckii bulgaricus MIM91…
MIM91 MIM91, NH3 (5 mM)pH
ex-pH i
n
pHex-pH i
n
D-lactic acid (mM)7,9 ± 0,2 9,4 ± 0,1 (+16%)
Time(min) Time(min)
(Siegufeldt et al., 2000)
ΔpH=pHin-pHex~ 0
24
25
Monitoring the lactic acid production by 13C-NMR analysis
13C
13C
glycolysis
Homolacticfermentation
Time (min)
13C
-Lac
tic a
cid
rela
tive
amou
nt
Time(min) Time(min) Time(min)
pHex
cFSE
fluorescence-A
… and of yogurt consortium mixed cultures
+ urea (2,5 mM)mixed culture + NH3 (5 mM)
Lactic acid (mM)7.0 ± 0,3 8,8 ± 0,3 (+21%) 8.4± 0,3 (+17%)D+L3.6 ± 0,1 4.0 ± 0,2 (+10%) 4.0± 0,1 (+10%)L3.4 ± 0,2 4.8 ± 0,1 (+30%) 4.4± 0,2 (+23%)D
26
CONCLUSION
ü The urease activity of S. thermophilus modulates the pHin of L. delbrueckii
bulgaricus during milk fermentation
üThe alkalization of the pHin positively affects the energetics of both single
cultures and protocooperation
ü Urease as an altruistic cooperative trait
ü New benefit occuring in yogurt consortium
27
(Gerosa and Sauer, 2011)
METABOLISM and ITS REGULATION
Layers of cellular regulatory mechanisms
Urease activity28