The effect of exogenous superoxide generator chemicals on sodA ...

The effect of exogenous superoxide generator chemicals on sodA ...


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

Ismail Akyol

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

hydrogen peroxide.

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

described previously.

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

oxidative damage.

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

without FlpB.


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