Supplementary Method for
Proteomic analysis of spontaneous mutants of Lactococcus lactis: involvement of
GAPDH and arginine deiminase pathway in H2O2 resistance.
Tatiana Rochat(+)*, Samira Boudebbouze, Jean-Jacques Gratadoux, Sébastien Blugeon,
Philippe Gaudu, Philippe Langella and Emmanuelle Maguin(+)
INRA, Institut Micalis, UMR1319/INRA-AgroParisTech, Jouy-en-Josas F78350, France.
* present address: Institut de Génétique et Microbiologie, CNRS, UMR 8621, IFR115, Centre
Scientifique d'Orsay, Université Paris Sud 11, Orsay, France.
+ Corresponding authors:
T. Rochat, Institut de Génétique et Microbiologie, Université Paris-Sud Bât 400, 91405 Orsay
Cedex (FR); tatiana.rochat@u-psud.fr; Phone: +33(0)169154655; Fax: +33(0)169156678.
E. Maguin, INRA, Institut Micalis, Bât 440, Domaine de Vilvert, 78352 Jouy-en-Josas cedex
(FR); emmanuelle.maguin@jouy.inra.fr; Phone: +33(0)134652518; Fax: +33(0)134652727.
Inactivation of gapA, arcA and arcB in L. lactis.
To disrupt genes in L. lactis strains, we used pRV300 vector which is composed of a pBluescript SKreplicon for propagation in E. coli and an erythromycin resistance marker. An internal fragment of the
gene to disrupt was inserted into pRV300 and the resulting plasmids were able to integrate into the
chromosome by homologous recombination as single copies and were maintained stably [1]. After
chromosomal integration of pRV300, the targeted gene was disrupted while the production of a Cterminal truncated form of the protein could remain.
Two pRV300:gapA plasmids were constructed for gapA disruption: the chromosomal integration led
to a protein truncated after the 219th amino-acid for the first one (pRV300:gapA*) and after the 148th
amino-acid for the second one (pRV300:gapA**). Consequently, the gapA mutant resulting from
pRV300:gapA* integration could produce a truncated form of GapA which contains Cys152 residue
contrary the one resulting from pRV300:gapA**.
Internal fragments of gapA were amplified by PCR using primers designed from the database
sequence
of
L.
lactis
MG1363
[2]:
1)
for
gapA*
fragment
with
gapA5'
(5'-
ggtcaaagaagatggttttgatgtcaacgg) and gapA3' (5'-gctggaacaatattttcagctgcagcacgc); 2) for gapA**
fragment with gapAs5' (5'-GATCCTGCAGGGTAGTTAAAGTTGGTATTAACGG) and gapAs3' (5'GATCAAGCTTATTAAGAAATAACTGTTTCACTTCC). The PCR products (named gapA* and
gapA**) were inserted in the cloning vector pCRII-TOPO (Invitrogen) and sequenced. The gapA* and
gapA** fragments were then inserted into pRV300 after digestion, resulting in pRV300:gapA* and
pRV300:gapA** which were transformed and subsequently purified from E. coli Top10 strain
(Invitrogen). pRV300:gapA* and pRV300:gapA** were then electroporated in J60011, SpOx2 and
SpOx3 mutants.
To disrupt arcA and arcB genes in SpOx1 mutant, internal fragments of arcA or arcB genes of 577 or
525
bp
respectively,
were
amplified
by
PCR
using
primers:
arcA5’
(5’-
gcgaattcggacgtacttatgatggattgactg) and arcA3’ (5’-ttgcggccgcccataattcctgggaaaactgtaaattg) or arcB5’
(5’-gcgaattcgaccttggggctcatcctga) and arcB3’ (5’-ttgcggccgccccagttagattctcccattgataccc). The PCR
products (named arc*) were inserted into pRV300 after digestion using EcoRI and NotI, resulting in
two pRV300:arc* plasmids which were transformed and purified from E. coli. After sequencing of the
cloned arc* fragments, pRV300:arc* plasmids were electroporated into SpOx1 strain, resulting in two
SpOx1 arcA and arcB mutants.
Bacterial strains and plasmids used in this study.
Strains
Characteristics
References
MG1363
plasmid-cured L. lactis ssp cremoris used as wild type strain
[3]
J60011
parental strain of SpOx mutants; lac+/prt+ MG1363 derivative
generated by conjugation with NCDO 712, containing the pLP712
conjugative plasmid
[4]
SpOx1
spontaneous mutant of J60011, H2O2 resistant
[4]
SpOx2
spontaneous mutant of J60011, H2O2 resistant
[4]
SpOx3
spontaneous mutant of J60011, H2O2 resistant
[4]
VEL11998
gapA mutant of J60011 (Emr) , pRV300:gapA* integration
This work
VEL12074
gapA mutant of L. lactis SpOx2 (Emr), pRV300:gapA* integration
This work
VEL11999
gapA mutant of L. lactis SpOx3 (Emr), pRV300:gapA* integration
This work
VEL12393
gapA mutant of L. lactis J60011 (Emr), pRV300:gapA** integration
This work
VEL12394
gapA mutant of L. lactis SpOx2 (Emr), pRV300:gapA** integration
This work
VEL12395
gapA mutant of L. lactis SpOx3 (Emr), pRV300:gapA** integration
This work
VEL12278
arcA mutant of L. lactis SpOx1 (Emr)
This work
VEL12280
arcB mutant of L. lactis SpOx1 (Emr)
This work
NZ9000
Plasmids
MG1363 carrying nisRK genes on the chromosome
Characteristics
[5]
References
pRV300 Apr/Emr carrying 453 bp internal fragment of gapA
This work
pRV300:gapA** pRV300 Apr/Emr carrying 444 bp internal fragment of gapA
This work
pRV300:arcA*
pRV300 Apr/Emr carrying 577 bp internal fragment of arcA
This work
pRV300:arcB*
pRV300 Apr/Emr carrying 525 bp internal fragment of arcB
This work
pRV300:gapA*
pRV300
pAM1, Emr
[1]
pVE3655
pWV01, Cmr carrying PnisA, a nisin-inducible promoter
[6]
pNZ:gapA
pWV01, Cmr carrying gapA under control of PnisA
[2]
Apr, Emr and Cmr: resistance to ampicillin, erythromycin and chloramphenicol,
respectively.
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Microbiol 1997, 63, 2117-2123.
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strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3phosphate dehydrogenase. Proteomics 2002, 2, 1041-1046.
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expression in lactic acid bacteria. J.Biotechnol. 1998, 64, 15-21.
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