Journal ong>ofong> Cell and Molecular Biology 5: 87-93, 2006.
Haliç University, Printed in Turkey.
ong>Theong> ong>effectong> ong>ofong> ong>exogenousong> ong>superoxideong> ong>generatorong> ong>chemicalsong> on sodA and flpA
promoters expression in Lactococcus lactis
Kahramanmarafl Sütçü Imam University, Faculty ong>ofong> A g r i c u l t u re, Biometry and Genetics, 46060
Received 12 April 2006; Accepted 15 June 2006
Oxidative stress is caused by ong>superoxideong> anion or hydrogen peroxide under aerobic growth condition. Some bacteria
could eliminate toxic species ong>ofong> oxygen using S o d A enzyme. ong>Theong> ong>effectong> ong>ofong> ong>superoxideong> generating ong>chemicalsong> and
hydrogen peroxide on sodA and f l p Apromoter activities were investigated in this study. ong>Theong> observation indicated
that the f l p A promoter activity increased with menadione addition in the f l p B mutant but not f l p A mutant
backgrounds. Also s o d A activity increased 4-, 7-, 50, and 27-fold in shaking (250 rpm), memadione, paraquat and
plumbagin addition respectfully. Catalase enzyme supplementation had positive ong>effectong> on bacterial growth during
exponential phase ong>ofong> f l p Amutants than f l p B mutants and wild type. Exogenously, hydrogen peroxide addition was
unable to increase either s o d A promoter or S o d Aenzyme activity.
Key Words: Superoxide dismutase, s o d A gene, f l p Agene, paraquat, plumbagin, menadione, hydrogen peroxide
Lactococcus lactis’te d›fl kaynakl› süperoksit üreten kimyasallar›n s o d A ve f l p A
promoter aktivitesine etkisi
Oksijen stresine aerobik büyüme flartlar›nda süperoksit anyonu veya hidrojen peroksit sebep olmaktad›r. Baz›
bakteriler toksik oksijen türlerini S o d A enzimini kullanarak elimine edebilmektedirler. Bu çal›flmada süperoksit
üreten kimyasallar›n ve hidrojen peroksidin s o d A ve f l p A promoter aktivitesine etkisi incelenmifltir. Bulgular f l p A
promotor aktivitesinin menadione eklenmesi ile f l p B mutant bakterisinde yükseldi¤i fakat f l p Amutant bakterisinde
yükselmedi¤i bulunmufltur. Ayr›ca s o d A aktivitesi kültürün sallanmas›, memadione, paraquat ve plumbagin
kimyasallar›n›n eklenmesi ile s›ras›yla 4-, 7-, 50, ve 27-kat yükseltilmifltir. Katalaz enzim eklenmesi f l p A mutant
bakterilere büyüme safhas›nda f l p B mutant› ve mutant olmayan bakterilerden daha fazla pozitif etki sa¤lam›flt›r.
D›flar›dan hidrojen peroksit eklenmesi ne s o d A promotor aktivitesini ne de S o d Aenzim aktivitesini artt›rm›flt›r.
Anahtar Sözcükler: Superoksit dismutaz, s o d A geni, f l p Ageni, parakuat, plumbagin, menadion, hidrojen peroksit
Molecular oxygen is toxic for anaerobic
microorganisms but it is less obvious that oxygen is
poisonous to aerobic microorganisms (Okado-
Matsumoto et al. 2004). Molecular oxygen is a triplet
radical in its ground-stage (.O-O.) and has two
unpaired electrons. ong>Theong> toxic species ong>ofong> oxygen are
created by one electron reduction caused by a
chemical compound and the enzymatic reactions occur
88 Ismail Akyol
in vivo. Although molecular oxygen (O 2) is not
damaging to DNA, other more reactive forms ong>ofong>
oxygen have more electrons than molecular oxygen
including ong>superoxideong> radicals, hydrogen peroxide and
hydroxyl radicals. Aerobe microorganisms have
evolved antioxidant defences to protect themselves
against reactive oxygen species. ong>Theong>se defences
include enzymes (such as ong>superoxideong> dismutase,
catalase and glutathione peroxidase), low molecular
agents and proteins that bind metal ions in forms that
can not catalyse radical reactions (Halliwell, et al.
Superoxide dismutase enzymes (SODs) are
metalloenzymes which catalyse dismutation ong>ofong> O 2·into
H 2O 2 and O 2, and play an important role in
oxidative defence systems (McCord, 2002). ong>Theong>re are
three types ong>ofong> SODs: MnSOD, FeSOD, and
Cu/ZnSOD that contain either manganese, iron or a
combination ong>ofong> copper and zinc as their catalytic metal
cong>ofong>actors at the active sites. MnSODs are found in
prokaryotes and in the mitochondria ong>ofong> eukaryotes,
whereas FeSODs are found in prokaryotes and in the
chloroplast ong>ofong> eukaryotes. Although Cu/ZnSODs are
generally present in eukaryotes, they are also present
in a small number ong>ofong> gram-negative bacteria.
MnSODs and FeSODs are structurally closely related
to each other whereas Cu/ZnSODs appear to have
evolved independently (Zelko et al., 2002).
Lactic Acid Bacteria (LAB) are ong>ofong> major economic
importance to the food industry. ong>Theong> subspecies
L a c t o c o c c u s l a c t i s subsp. c re m o r i s MG1363 (Kok et
al. 2001) and Lactococcus lactis subsp. l a c t i s IL1403
(Bolotin et al. 2001) are well studied plasmid-free
lactococcal strains in terms ong>ofong> genetics and molecular
biology. Lactococcus lactis SodA protein (Sanders et
al. 1995) is similar to the N-terminal amino acid
sequences ong>ofong> Bacillus stearo t h e r m o p h i l u s (Bowler et
al., 1990), Escherichia coli (Takeda and Avila, 1986),
and human MnSODs (Zelko et al., 2002). T h e
homology ong>ofong> lactococcal SodA is with the group ong>ofong>
MnSODs rather than the group ong>ofong> FeSODs (Sanders et
al. 1995; Woo-Suk and So, 1999). SOD is induced by
oxygen in Escherichia coli and Lactococcus lactis
(Woo-Suk and So, 1999). SODs are induced in a
number ong>ofong> bacteria during the stationary phase
(Schnell and Steinman, 1995; Leclere et al. 2004). ong>Theong>
relative SOD activities were found to increase upon
entry into the stationary phase in the cultures grown
without shaking. However, SOD activity increased
until the death phase in shaking a culture (Woo-Suk
and So, 1999).
ong>Theong> transcription activator factor fumurate nitrate
reductase (FNR) proteins plays a major role in altering
gene expression (Kiley and Beinert, 1999) and FNR
proteins recognise TTGAT-N 4-ATCA DNA sequence
by Spiro and Guest (1990). Two FNR like proteins
(Flps), FlpA and FlpB, were identified in L a c t o c o c c u s
l a c t i s subsp. c re m o r i s and f l p A and f l p B genes have
FNR binding site (Gostick et al. 1999). FlpB protein
play an activator role in transcriptional regulation
anaerobically but the role ong>ofong> FlpAprotein has not been
clarified clearly so far. Upstream ong>ofong> s o d A g e n e
contains potential FNR protein binding site and
induced by aeration in Lactococcus lactis (Sanders et
al. 1995). Lactococcal s o d A gene regulation by the
FNR like proteins (Flp) in response to oxidative stress
is not known. In this study, the ong>effectong> ong>ofong> ong>superoxideong>
generating ong>chemicalsong> and hydrogen peroxide on s o d A
and f l p A gene expression were studied to determine
which signals ong>ofong> oxidative stress regulate expression
ong>ofong> s o d Aand f l p A genes.
Materials and methods
Chemicals, bacterial strains and growth conditions
Superoxide ong>generatorong> ong>chemicalsong>, paraquat (15 µg/ml),
plumbagin (5 µg/ml) and menadione (10 µg/ml) were
used in this study, purchased from SIGMA.
Lactococcus lactis strains were grown on M17 media
supplemented with 0.5% (w/v) glucose at 30 o C.
Escherichia coli strains were grown on Luria Bertani
(LB) broth shaking or LB agar at 37 o C. Agar plate
media contained 1.5% (w/v) Bacto agar.
Construction ong>ofong> recombinant bacterial strains
ong>Theong> s o d A promoter β-galactosidase reporter gene
fusion plasmid pSOD4 was provided as a kind gift
from J. Sanders (University ong>ofong> Groningen). A 515 bp
region ong>ofong> DNAupstream ong>ofong> the f l p Agene including the
FNR binding site was amplified with B glII and P s tI
site containing primers and cloned into pAK80 by H.
Rawsthorne and plasmid numbered as a pFI2197.
Plasmid DNA was isolated from overnight stationary
phase ong>ofong> Escherichia coli strains cultures using the
QIAGEN plasmid DNA purification kit according to
the supplier’s recommendations (QIAGEN Ltd.
Crawley, UK). Constructed plasmids were introduced
into Lactococcus lactis by electroporation according to
Holo and Nes, 1989 method. Constructed and used
Table 1. Lactococcus lactis strains used in this study.
bacterial strains in this study are described in Table 1.
Promoter activity ong>ofong> f l p Aand pSOD4 were carried out
according to the procedure ong>ofong> Akyol (2002).
P rotein extraction and non-denaturing Polyacrylamide
Gel Electro p h o resis (PA G E )
Lactococcal cells were harvested by centrifugation at
3000 g for 15 min and washed twice in ice cold 50 mM
Tris HCl (pH 7.0). ong>Theong> cell pellets were resuspended
in 1 ml 50 mM Tris HCl and transferred to 5 ml Bijoux
bottles. ong>Theong> cells were broken using a Micro-
Dismembrator (Raun UK Ltd) at 1600 rpm (4 times
for 30s) using 1 ml sterile 0.1 mm-diameter glass
beads. Unbroken cells and debris were removed by
centrifugation at 12000 rpm 4 o C for 30 min. Clear
supernatant was transferred to a clean tube and protein
concentration were determined by the method ong>ofong>
Bradford (1976) with bovine serum albumin as a
Ready made 12.5% non-denaturing PAGE gels
(NOVEX Electrophoresis GmbH, Frankfurt,
Germany) were run at 200 V. ong>Theong> same protein sample
was run on two gels and one ong>ofong> them was used for
SOD activity detection and the other gel developed
with Cooumassie brilliant blue for total protein
S u p e roxide dismutase activity detection
ong>Theong> SOD activity on 12.5% non-denaturing PAGE
gels was done according to Steinman (1978) with
some modifications. One nitro blue tetrazolium tablet
(DNT) was dissolved in 30 ml water and the nondenaturing
(12.5%) acrylamide gel was soaked for 30
minutes while shaking. ong>Theong> gel was shaken in 40 ml
SOD solution (0.028 M tetramethylethylenediamine
(TEMED), 2.8x10 - 5 M ribong>ofong>lavin, and 0.036 M
potassium phosphate at pH 7.8) for 15 min. ong>Theong> gel
was placed on a clean acetate sheet and illuminated for
5 to 15 min. ong>Theong> gel became purple except at the
position containing SOD. ong>Theong> gel was scanned when
the maximum contrast between the band and
background had been achieved.
Superoxide ong>generatorong>s ong>effectong> 89
Bacterial Strains Resistance Properties References
MG1363 - Plasmid free strain Gasson, 1983
FI9077 Rif, Str flpA - strain Rawsthorne, 2000
FI9124 - flpB - strain Rawsthorne, 2000
FI9241 - flpAB - strain Rawsthorne, 2000
FI8841 Tet flpA - and nisA - strain Rawsthorne, 2000
FI9627 Rif, Str flpB - and nisA - strain This study
FI9391 Ery, Cap LL108 containing pSOD4 Sanders et al., 1995
FI9641 Rif, Str, Cap, Ery FI9627 containing pFI2116 and pFI2197 This study
FI9693 Rif, Str, Ery FI9627 containing pFI2197 This study
FI9678 Tet, Str, Ery FI8841containing pFI2197 This study
FI9694 Tet, Ery, Cap FI8841containing pFI2116 and pFI2197 This study
pSOD4 pORI13 containing 0.8kb sodA fragment Sanders et al., 1995
pFI2197 pAK80 containing 515 bp flpAfragment Rawsthorne, 2000
pAK80 Promoterless‚ β galactosidase Israelsen et al., 1995
Effect ong>ofong> ong>exogenousong> oxidative stress on sodA g e n e
e x p re s s i o n
ong>Theong> s o d Apromoter activity levels were detected under
aerated, external ong>superoxideong> generating ong>chemicalsong> or
hydrogen peroxide supplemented conditions. T h e
s o d Apromoter fusion activity increased when the cells
were incubated in the presence ong>ofong> paraquat, plumbagin
and menadione. ong>Theong>se values were found as a
maximum sub-lethal concentration for each ong>chemicalsong>
after experimental observation. ong>Theong> pSOD4 promoter
90 Ismail Akyol
Figure 1. Non-denaturing-12.5% PAGE analysis ong>ofong> cell extracts ong>ofong> oxygen-stressed Lactococcus lactis. A. Cooumassie brilliant
blue stained gel and molecular masses are indicated on the gel. B. Non-denaturing (12.5%) PAGE gel assayed for only SOD
activity. Antibiotic supplemented culture conditions are static (st), shaken at 250 rpm (s), menadione added 10 µg/µl(m).
activity was increased 4, 7, 50, and 27 fold in shaking
(250 rpm),with paraquat, menadione and plumbagin
supplemented cultures, respectfully. In contrast, no
promoter activity increase was obtained in the
presence ong>ofong> hydrogen peroxide (data not shown). So
the increase in s o d A gene expression in response to
aeration is mediated by ong>superoxideong> rather than
S o d Ap rotein expression in response to oxidative stre s s
ong>Theong> ong>effectong> ong>ofong> 10 µg/ml menadione addition on Sod
protein expression in different strains was analysed on
SOD activity gels (Figure 1.). It was obvious that Sod
protein expression increased with menadione addition.
S o d A activity was increased in f l p A - and f l p B -
background strains (FI9641, FI9693, FI9678, and
Figure 2. Effects ong>ofong> catalase enzyme on bacterial growth
curve. wt : MG1363; f l p A - : FI9077; f l p B - : FI9124; f l p A B - :
FI9694) with menadione addition so mutation in f l p
genes, f l p A and f l p B, had not affected SodA
ong>Theong>re were no significant differences between
static and hydrogen peroxide added treatment in the
following strains LL108, FI9641, FI9693, FI9678, and
Superoxide ong>generatorong>s ong>effectong> 91
FI9694 (data not shown). Sod protein activity was not
induced by H 2O 2 addition.
Effect ong>ofong> hydrogen peroxide and catalase addition on
the growth ong>ofong> wild type and flp mutant strains
D i fferent sub-lethal concentrations ong>ofong> hydrogen
peroxide, 0.5 mM, 0.75 mM, 1mM, 1.25 mM, 1.5 mM
and 2 mM, were added to wild type (MG1363) and f l p
mutant strains, FI9077, FI9124and FI9241, and
growth was monitored using the Bioscreen C
(Taransgalatic Ltd., Finland). Increasing the hydrogen
peroxide concentration decreased the growth (data not
shown). However, these changes were not different
between wild type and f l p mutants. Both MG1363 and
f l p mutants were sensitive to 2.5 mM hydrogen
peroxide due to DNA replication blocking by high
level ong>ofong> hydrogen peroxide.
Catalase enzyme protects the cell from the toxic
e ffects ong>ofong> hydrogen peroxide by catalyzing its
decomposition into molecular oxygen and water
without the production ong>ofong> free radicals. L a c t o c o c c u s
l a c t i s does not have catalase and considering the
importance ong>ofong> catalase in the oxidative stress response
ong>ofong> facultative anaerobes it is not clear how
Figure 3. Growth curve for FI9641 strains under sub-lethal concentrations ong>ofong> menadione (different shaped lines). b-galactosidase
activity ong>ofong> FI9641 strains under sub-lethal concentrations ong>ofong> menadione were measured 100 ml cultures which contains 5 ( ),
7.5 ( ), or 10 ( ) µg/ml menadione were harvested at 24 h ong>ofong> growth (blocks in graph). No b-galactosidase activity was detected
from 2.5 µg/ml menadione contains and control cultures.
92 Ismail Akyol
Lactococcus lactis tolerates oxygen. 2.5 μl catalase
(9240 U) was added to 100 ml growth media to
determine its ong>effectong>. Samples were taken as indicated
and measured at OD 6 0 0
using Bio Photometer
(Eppendorf Ltd. Cambridge, UK). Catalase
supplementation had a more positive ong>effectong> on
bacterial growth during the exponential phase ong>ofong> f l p A
mutants growth than f l p B and wild type strains.
However, when growth reached the stationary phase
the catalase added cultures were quite similar to the
controls (Figure 2.). FlpA proteins could be
controlling the response to the hydrogen peroxide
induced stress that can be mimicked by adding
catalase in the absence ong>ofong> FlpA.
Effect ong>ofong> ong>exogenousong> menadione addition on bacterial
g rowth and flpA gene fusion activity
ong>Theong> pFI2197 containing f l p B mutant strain (FI9693)
and f l p A (FI9678) mutant strains were constructed.
f l p Apromoter expression can not be detected in strains
FI9641, FI9693, FI9678 or FI9694 under static or
shaking conditions or in presence ong>ofong> H 2O 2 or the
ong>superoxideong> generating ong>chemicalsong> (plumbagin and
paraquat). However f l p A promoter expression was
observed in the f l p B - mutant strains (FI9641
[21.1±0.68 μmoles/mins x 10 -5 ], FI9693 [20.7± 0.68
μmoles/mins x 10 - 5 ]) when supplemented with
menadione, the most ong>effectong>ive ong>superoxideong> ong>generatorong> as
ong>Theong> f l p A promoter activity was measured at
d i fferent concentrations ong>ofong> menadione. When the
concentration ong>ofong> menadione increased, culture growth
decreased gradually while the f l p A promoter activity
increased (Figure 3.). Cultures containing 5, 7.5, and
10 mg/ml menadione grew up to OD 6 0 0: 0.4. It
appeared that the expression ong>ofong> f l p A increased when
the conditions were harsh.
Oxidative stress can be produced by ong>superoxideong> anion
or hydrogen peroxide. Active oxygen species can be
present in growth media, generated by ong>exogenousong>
ong>chemicalsong> or produced as a result ong>ofong> metabolic activity.
In Escherichia coli there are two key transcriptional
regulators that respond to oxidative stress. OxyR
regulates the adaptive response to hydrogen peroxide
controlling 16 genes and SoxRS regulates 14 genes in
response to the ong>superoxideong> anion (Asad et al. 2004).
ong>Theong> adaptive responses to ong>superoxideong> and hydrogen
peroxide are different but overlapping. Genomic
database searches showed that homologs ong>ofong> OxyR
regulated genes are present in Lactococcus lactis
subsp. l a c t i s IL1403 ( f u r, dps, hemH, sufABC, yaiA)
and in Lactococcus lactis subsp. c re m o r i s MG1363
(hemH, sufABC, yaiA) as are homologs ong>ofong> SoxRS
regulated genes in IL1403 (lyslR, zwf, fur, ribA)and
MG1363 (sodA, zfw).
ong>Theong> SodAprotein ong>ofong> L a c t o c o c c u sl a c t i s is involved
in the oxidative stress response and s o d A mutant cells
have delayed aerobic growth compared to the wild
type. ong>Theong> s o d Agene expression showed that s o d Awas
activated only two fold during aeration (Sanders et al.
1995). Chang and So, (1999) demonstrated that SodA
in Lactococcus Lactis is expressed in a growth phase
dependent manner therefore the expression ong>ofong> SodAin
Lactococcus lactis could be affected by oxidative
stress and other stresses, presumably starvation, toxic
reagents, and acids. Exogenous ong>chemicalsong> can be used
to generate ong>superoxideong> at a higher level than that
produced during aerobic growth. ong>Theong> s o d A promoter
activity is highly induced by paraquat, menadione and
plumbagin. Increased expression ong>ofong> SodA protein by
menadione culture supplementation was confirmed on
non-denaturing PAGE gels assayed for SodA activity.
SodA protects the cell from ong>superoxideong> but not from
hydrogen peroxidase. Exogenously H 2O 2 addition was
unable to increase either sodA promoter or S o d A
Promoter sequence analysis, using the song>ofong>tware
Fuzznuc, showed that lactococcal s o d A p r o m o t e r
contains potential FNR and Flp binding site. ong>Theong>
presence ong>ofong> a FNR binding site suggests that the Flp
proteins may be involved in regulating s o d A
expression in response to oxidative stress. ong>Theong> SodA
activity was monitored in single and double f l p mutant
strains under static and shaking growth conditions.
However the results showed that f l p deletions had no
ong>effectong> on expression ong>ofong> the SodA protein. Thus it
appears that the Flps do not induce the s o d A gene in
response to oxidative stress. It may be that there are a
number ong>ofong> different overlapping transcriptional
regulators that control s o d A so the ong>effectong> ong>ofong> deleting
one or two is not seen. It is not clear if the Flps induce
alternative protection or repair systems to overcome
ong>Theong> f l p Apromoter expression needs both FlpAand
FlpB (Akyol, 2002; Rawsthorne, 2000) but here
induction ong>ofong> f l p Aexpression occurred with menadione
and did not require FlpB. Expression ong>ofong> the f l p A
promoter increased gradually in the f l p B mutant but
not f l p A mutant backgrounds as the level ong>ofong>
menadione (ong>superoxideong> ong>generatorong>) was increased
while the bacterial growth decreased. It appears that
menadione addition either induces an unknown
protein that can substitute for FlpB or perhaps the
chemical can cause a direct change in the FlpA
conformation, which allows it to bind to the promoter
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