the physiological and ecological roles of urease activity...

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


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


lactic acid

pHex

pHin

LacS

lactose

lactose

galactose

galactose

glucose

b-galactosidase

glycolysis


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


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


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 onlyA16 only




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


FSC

-A


FSC

-A

Cou

nt


T0

T1

T1 + NH3

T1 + NH3

T1T0

pHex: 6.68pHin: 7.04

pHex: 9.60pHin: >7.80

19


FSC

-A


FSC

-A


T0 T1 + NH3

T1 + NH3T1T0

FSC

-A


Cou

nt

T1

… and on L. delbrueckii bulgaricus pHin



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

Are you sure it is convenient to select urease negative strains?

Thank you for the attention

29

the physiological and ecological roles of urease activity...

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