mapping reads,logarithmic scale

Federal Department of Economic Affairs, Education and Research EAER Agroscope

16.09.2015

Influence of pH on Gene

Expression in Lactobacillus parabuchneri FAM21731

Claudia Wenzel

Lactic acid bacteria

 cheese making

 Lactobacillus casei, Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus

 Lactobacillus parabuchneri

 formation of histamine

 might lead to:

 headache, stomache cramps, vomitting





Histamine Histidine

Histidine decarboxylase

Lactobacillus parabuchneri

 hdcA gene cluster

 contains four genes

 formation of 1,2-propanediol

 from lactic acid

 synthesis only at low pH

hisS hdcB hdcA hdcC

tRNA ligase unknown function histidine decarboxylase transporter

Experimental Setup

pH 3,8 pH 6,8

Growth conditions:

 Modified MRS

 45mM D-/L-lactate

 2,7g/L glucose

 3d @ 30°C Analyses:

 Transcriptome

 1,2-propanediol

 lactate

Transcriptome Analysis

 419 significantly regulated genes (p-value < 0.05, N=3)

 228 up-regulated and 191 down-regulated

 Histidine decarboxylase gene cluster: only hisS up-regulated

 Reductases possibly involved in formation of 1,2-propanediol:

aldA (lactaldehyde dehydrogenase) and put. OR (putative oxidoreductase)

1 10 100 1000 10000

hisS hdcB hdcA hdcC aldA put. OR mapping reads, logarithmic scale

pH 3.8 pH 6.8

* *

*

hisS hdcB hdcA hdcC

tRNA ligase unknown function histidine decarboxylase transporter

Analyses of 1,2-propanediol and lactate

 No formation of 1,2-propanediol at pH 6.8 (GC/MS)

 Culture supernatant at pH 3.8 contained less L-lactate

(enzymatic method)

0 50 100 150 200 250

pH 3.8 pH 6.8

[mM/OD600]

lactate D-lactate L-lactate 0

100 200 300 400 500 600 700

pH 3.8 pH 6.8

[mg/kg*OD600]

1,2-propanediol

*

0 10 20 30 40 50 60 70

pH 3.8 pH 6.8

[%] of lactate

L-lactate

*

* p-value < 0.05, N=6

Conclusions

 pH does not influence histamine production

 pH influences 1,2-propanediol formation

 two genes encoding reductases up-regulated

 L-lactate might be an intermediate

Proposed Pathway

Lactaldehyde

L-Lactate (+ D-Lactate)

1,2-Propanediol

Pyruvate + Acetate + CO₂

NADH + H⁺ NAD⁺ + H₂O aldA

NADH + H⁺ NAD⁺

put. OR

NADH + H⁺ NAD⁺

3 NAD⁺ 3 NADH + H⁺ Glucose

1 10 100 1000 10000

aldA put. OR mapping reads, logarithmic scale

pH 3.8 pH 6.8

*

*

Future Work

 qPCR systems for all genes of hdcA cluster and the two reductases possibly involved

 Growth without lactate, with L-lactate, with D-lactate

 Experiments on cell free extract with L-lactate and 1,2-propanediol

 Feeding Experiments with ¹³C-glucose and ¹³C-lactate

 Cloning and heterologous expression of the two candidate reductases

Acknowledgments

 Stefan Irmler

 Tharmatha Bavan, Cornelia Bär

 René Badertscher, Charlotte Egger

 Daniel Wüthrich, Rémy Brugmann

 Magali Chollet

 Partially funded by: Commission for Technology and Innovation (CTI)

 Interested in more cheese research?

 take a look at Poster 22 - Detection and Diversity of Lactobacillus helveticus in dairy products

Thank you for your attention

Agroscope good food, healthy environment

Transcriptome Analysis

 419 significantly regulated genes (p-value < 0.05, N=3)

 228 up-regulated

 191 down-regulated

1) anatomical structure development 2) biosynthetic process

3) carbon utilization 4) catabolic process

5) cellular component biogenesis 6) cellular component organization 7) cellular metabolic process 8) establishment of localization 9) macromolecule localization 10) methylation

11) multi-organism cellular process

12) nitrogen compound metabolic process

13) organic substance metabolic process 14) primary metabolic process

15) regulation of biological process 16) regulation of biological quality 17) response to chemical stimulus 18) response to external stimulus 19) response to stress

20) single organism signaling

21) single-organism cellular process

22) single-organism developmental process 23) single-organism metabolic process 1

2

3 45 6 7

8 9 11 10 13 12

14 15

16 17

18 19 20

21 22

23