Zinc uptake, oxidative stress and the FNR-like proteins ofLactococcus lactis

Colin Scott a, Helen Rawsthorne b, Manisha Upadhyay a, Claire A. Shearman b,Michael J. Gasson b, John R. Guest a, Je¡rey Green a;*

a Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of She¤eld, Western Bank,She¤eld S10 2TN, UK

b Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

Received 7 July 2000; received in revised form 18 August 2000; accepted 1 September 2000

Abstract

Lactococcus lactis ssp. cremoris MG1363 contains two FNR homologues, FlpA and FlpB, encoded by the distal genes of two paralogousoperons (orfXA=B-orfYA=B-flpA/B). An flpA flpB double mutant strain is hypersensitive to hydrogen peroxide and has a depleted intracellularZn(II) pool. The phenotypes of the flp mutant strains suggest that FlpA and FlpB control the expression of high and low affinity ATP-dependent Zn(II) uptake systems, respectively. Plate tests revealed that expression from a orfXB : :lac reporter was activated by Cd(II),consistent with other Zn(II)-regulated systems. The link between a failure to acquire Zn(II) and hypersensitivity to oxidative stress suggeststhat Zn(II) may be required to protect vulnerable protein thiols from oxidation. ß 2000 Federation of European MicrobiologicalSocieties. Published by Elsevier Science B.V. All rights reserved.

Keywords: Lactic acid bacterium; Zinc; Oxidative stress ; FNR-like protein; Cadmium; Thiol protection

1. Introduction

Recently, two homologues of the oxygen-responsive Es-cherichia coli transcriptional regulator FNR were discov-ered in the lactic acid bacterium Lactococcus lactis [1]. TheFNR-like proteins (FlpA and FlpB) of L. lactis are en-coded by the distal genes of two paralogous operons, thatconsist of three genes orfXA=B, orfYA=B and £pA/B. TheorfXA=B gene products are predicted to belong to the MerPfamily of small metallochaperones [2], the orfYA=B geneproducts are predicted to have homology with the ironstorage protein, ferritin, and the non-speci¢c DNA-bind-ing and protection protein, Dps [3,4]. The £pA/B genesencode proteins that belong to the CRP family of tran-scriptional regulators with greatest similarity to the redox-sensitive FLP of Lactobacillus casei [5]. Both £p operonsare preceded by paralogous open reading frames, eachencoding a potential metal ion transporting P-type ATP-ase (orfWA=B), most similar to the zntA Zn(II) exportingP-type ATPase of E. coli [6,7]. It has also been shown that

both FlpA and FlpB can bind at, and regulate transcrip-tion from, an FNR-dependent promoter in the heterolo-gous host E. coli, and that FlpA binds an FNR-bindingsite in vitro [8]. Furthermore, the £pA and £pB promoterregions contain potential FNR- (Flp-) binding sites at po-sitions pertinent for transcriptional regulation (centredaround 342.5 bp from the transcriptional start of the£pB operon and overlapping the start of the £pA tran-script, centred at +4.5 bp) [1]. Therefore, it is likely thatthe £p operons are regulated by the FlpA and FlpB pro-teins.

Although there was no substantial di¡erence in growthrate between L. lactis wild-type and £p mutant strainseither aerobically or anaerobically [1], preliminary pheno-typic studies revealed that the £pA £pB strain was hyper-sensitive to hydrogen peroxide relative to the parent [1].Furthermore, the intracellular Zn(II) pool of the £pA £pBdouble mutant strain was depleted approximately 8-foldwhen compared with the parental strain [1]. In combina-tion with the predicted functions of the orfW, orfX andorfY gene products, it has been suggested that FlpA andFlpB are regulators of metal ion homeostasis.

Here, further characterisation of L. lactis ssp. cremorisMG1363 strains carrying lesions in the £p genes supports

0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 1 3 - 4

* Corresponding author. Tel. : +44 (114) 2224403;Fax: +44 (114) 2728697; E-mail : je¡.green@she¤eld.ac.uk

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the hypothesis that FlpA and FlpB regulate the expressionof Zn(II) uptake systems, which may confer resistance tooxidative stress.

2. Materials and methods

2.1. Bacterial strains and plasmids

The bacterial strains used were L. lactis ssp. cremorisMG1363 and isogenic single £pA (FI8559), £pB(POP50), and double, £pA £pB (FI8828) mutants [1].The orfXA : :lac reporter plasmid was constructed by clon-ing the £pA operon promoter, as a 515-bp BglII^PstI frag-ment (which extends 154 bp upstream of the transcriptionstart) PCR-ampli¢ed from MG1363 genomic DNA, intothe corresponding restriction sites of pAK80 [9] to createpFI2197. The orfXB : :lac reporter plasmid was constructedby cloning the £pB operon promoter, as a 1055-bp Hin-dIII^BglII fragment (which extends 418 bp upstream ofthe transcription start) isolated from pFI959 [1], into thecorresponding restriction sites of pAK80 to createpFI2184. To con¢rm the ¢delity of the PCR, the fragmentwas sequenced. The parental strain L. lactis ssp. cremorisMG1363 was transformed with pFI2184 and pFI2197,creating the strains FI9377 (JRG4197) and FI9442(JRG4056), which were used to screen for conditionsunder which the £p promoters were active.

2.2. Microbiological methods and DNA manipulation

Unless otherwise stated, bacteria were cultured at 30³Cin GM17 (Difco M17 supplemented with 0.2% (w/v) glu-cose). Media were also supplemented with erythromycin(5 Wg ml31), tetracycline (5 Wg ml31), sodium orthovana-date (Sigma), N,N,NP,NP-tetrakis (2-pyridyl methyl) ethyl-ene diamine (TPEN; Sigma) and X-Gal (40 Wg ml31) whenappropriate. Liquid cultures were routinely incubated in25 ml of medium without shaking to maintain microaero-bic conditions. Zinc-depleted GM17 (ZGM17) was pre-pared by ¢rst stripping GM17 of metal ions using a gravity£ow column packed with Chelex-100 resin (Bio-Rad; 5 gof resin per 100 ml of medium), then supplementing themetal-depleted GM17 with a trace element solution lack-

ing Zn(II) (0.2%, v/v; modi¢ed from Vishniac and Santer[10]), containing EDTA (50 g l31), CaCl2W6H2O (5.54 gl31), MnCl2W7H2O (5.06 g l31), FeSO4W6H2O (4.99 g l31),(NH4)Mo7O24W4H2O (1.1 g l31), CuSO4W5H2O (1.57 g l31)and CoCl2W6H2O (1.61 g l31).

2.3. Phenotypic tests

Phenotypic screening was conducted using GM17 agarplates containing the appropriate antibiotics with an over-lay of GM17 agar seeded with the relevant lactococcalstrain. Antibiotic assay discs were placed in the centre ofeach plate and loaded with 50 Wl of the various test solu-tions at the indicated concentrations. A similar procedurewas used for screening for promoter activity from the £pAand £pB operon reporter strains (JRG4056 and 4197), ex-cept that the GM17 agar also contained X-Gal (40 Wgml31).

3. Results and discussion

3.1. Screening for £p phenotypes

Antibiotic assay discs loaded with a stressor placed inthe centre of seeded agar plates were used to compare theresponses of the parent and £p mutant strains. This meth-od allowed a wide range of compounds to be rapidlytested. Initially, the hypersensitivity of the £pA £pB doublemutant was con¢rmed by comparison of the zones ofgrowth inhibition surrounding discs loaded with hydrogenperoxide (Table 1). However, the parent, £pA and £pBsingle mutants displayed a similar degree of sensitivitytoward hydrogen peroxide, demonstrating that both £pgenes need to be inactivated for hydrogen peroxide resis-tance to be compromised (Table 1).

The parental and £p lesion strains were equally sensitiveto stresses induced by acid (HCl and acetic acid), sodiumchloride, ethanol and Fe(II) as indicated by the very sim-ilar zones of inhibition for all four strains (not shown).However, enhanced resistance to Zn(II), Cd(II) andMn(II) was observed for all three £p strains, and the£pA £pB strain was more resistant to Co(II) (Table 1).The increased Zn(II) tolerance of the £p strains was of

Table 1The e¡ect of potential stressors on the growth of £p mutants

Stress factor (concentration) MG1363 (parent) FI8559 (£pA) POP50 (£pB) FI8828 (£pA £pB)

H2O2 (1 M) 1010 1070 1070 1520ZnCl2 (1 M) 910 380 490 380CdCl2 (1 mM) 1385 616 531 616MnCl2 (1 M) 800 490 570 450CoCl2 (1 M) 750 710 750 570

Areas (mm2) of zones of clearance around antibiotic assay discs loaded with stress factors (50 Wl of each stressor, concentrations in parentheses) placedin the centre of overlay plates seeded with the indicated strain. The areas shown are the averages from three independent experiments that varied by6 10%.

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particular interest, as previous work had shown that theintracellular Zn(II) pool of the £pA £pB mutant (36 Wg perg dry weight of bacterial cells) was 8-fold lower than thatof the parent (300 Wg per g dry weight of bacterial cells) [1]whereas the levels of Mn(II) (7.7 Wg per g dry weight ofbacterial cells for the parent compared to 8.3^11.2 for themutants), Cd (II) (0.11 Wg per g dry weight of bacterialcells for the parent compared to 0.11^0.12 for the mu-tants) and Co(II) (0.01 Wg per g dry weight of bacterialcells for both the parent and mutant strains) were unaf-fected. Thus these data suggest that the Flp proteins reg-ulate Zn(II) uptake in L. lactis. The enhanced resistance toMn(II) and Co(II) suggests that these ions may also betransported by Flp-dependent systems. Alternatively, atthe high concentrations used in these experiments,Mn(II) and Co(II) may be substrates for the Zn(II) trans-porter. The endowment of multiple resistance to heavymetal ions following mutations that a¡ect a single uptakesystem has been observed in Enterococcus hirae where in-activation of the copper P-type ATPase (CopA) confersmild resistance to both Cu(II) and Ag(II) ions [11].Thus, the phenotypic di¡erences revealed by the screeningreported here suggest that FlpA and FlpB are involved inZn(II) ion homeostasis and oxidative stress survival.

3.2. Attempts to activate the £pB and £pA promoters invivo

Identifying environmental cues that modulate expres-sion of the £p operons could provide insight into theirfunction. Therefore, two lac reporter plasmids were con-structed carrying the promoter regions of the £p operons,and these were used to screen for £p operon expression ina wild-type background (lac3) using the method describedabove. However, none of the stress reagents used in thephenotypic tests (Table 1) altered the expression from ei-ther operon promoter.

However, the pattern of heavy metal resistance (Table1) suggests that FlpA and FlpB are required for the reg-ulation of metal (Zn(II)) uptake. The recent report thatthe Zn(II)-responsive regulator of E. coli (ZntR) responds

to much lower concentrations of Cd(II) than Zn(II) [12]prompted an investigation of the ability of Cd(II) to alterexpression of the £p operons. Expression of the £pB oper-on was clearly induced by just sub-lethal levels of Cd(II)ions, as evidenced by a distinct blue halo indicative ofL-galactosidase activity at the border of the zone of inhi-bition in seeded X-Gal plates (not shown). Attempts toestimate Cd(II)-mediated £pB operon expression havethus far been unsuccessful for reasons that are not yetclear, but may be related to the observation that upregu-lation occurs at just sub-lethal Cd(II) concentrations. Incombination with the £p-dependent depletion of theintracellular Zn(II) pool, this is consistent with Zn(II) asa natural substrate for an Flp-dependent metal ion im-porter.

In contrast to the £pB operon, enhancement of £pAoperon expression by Cd(II) ions was not detected bythis method. This may be a re£ection of their di¡erentpromoter architectures, in which the £pA operon wouldbe derepressed (Flp box at +4.5) whereas the £pB operonwould be activated (Flp box at 342.5) by the Flp regula-tors [1]. Thus, the repression/derepression transition of the£pA operon may be undetectable using the screening pro-cedure employed.

3.3. Growth in presence of Zn(II)/Cd(II)/TPEN

The depletion of the intracellular Zn(II) pool and theenhanced resistance of the £p strains to heavy metal stresssuggests that FlpA and FlpB mediate the expression ofZn(II) uptake genes. Therefore, it was predicted that the£p strains would also exhibit a greater requirement forZn(II) and the ability of the four strains to grow in thepresence of the heavy metal chelator TPEN was assessed(Table 2). In addition, the ¢nal culture densities of the £pmutant and parental strains in the presence of ZnCl2 (1000WM) and CdCl2 (100 WM) were measured (Table 2). Aspredicted from the observations above, the parent wassensitive to Cd(II) and Zn(II) and all three £p strainswere relatively resistant. Furthermore, the parent wasmore resistant to heavy metal starvation by TPEN, where-

Table 2The e¡ect of the £pA and £pB genes on the response of L. lactis to elevated levels of Zn(II) and Cd(II) and to heavy metal starvation

Medium MG1363 (parent) FI8559 (£pA) POP50 (£pB) FI8828 (£pA £pB)

GM17 1.30 (0.20) 1.26 (0.29) 1.36 (0.13) 1.19 (0.12)GM17 plus 100 WM CdCl2 0.01 (0.01) 1.12 (0.23) 0.54 (0.16) 0.96 (0.24)GM17 plus 1000 WM ZnCl2 0.01 (0.01) 0.72 (0.24) 0.69 (0.18) 0.65 (0.20)GM17 plus 50 WM TPEN 1.13 (0.15) 0.01 (0.01) 0.75 (0.01) 0.1 (0.01)ZGM17 0.57 (0.09) 0.11 (0.02) 0.06 (0.02) 0.01 (0.01)ZGM17 plus 100 WM ZnCl2 0.85 (0.23) 0.56 (0.17) 0.80 (0.21) 0.11 (0.02)ZGM17 plus 500 WM ZnCl2 0.48 (0.24) 0.59 (0.29) 0.86 (0.32) 0.62 (0.25)ZGM17 plus 1000 WM ZnCl2 0.04 (0.01) 0.51 (0.20) 0.50 (0.19) 0.50 (0.26)

The ability of L. lactis ssp. cremoris MG1363, FI8559 (£pA), POP50 (£pB) and FI8828 (£pA £pB) to grow in GM17 and GM17 supplemented withCdCl2 (100 WM), ZnCl2 (1000 WM) or TPEN (50 WM), or in ZGM17 (Zn(II)-depleted medium) and ZGM17 supplemented with ZnCl2 (100, 500 and1000 WM) was assessed. The ¢nal culture densities (after 18 h growth) are expressed as the absorbance of the cultures at 600 nm (OD600). Values aregiven as the average of at least three experiments and standard deviations are indicated in parentheses.

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as all the mutants grew relatively poorly under these con-ditions. Thus, it seems likely that the parent can scavengeessential heavy metals from the medium (possibly evenfrom TPEN). Moreover, the observation that the £pAand £pA £pB strains grew less well than the £pB strainin the presence of 50 WM TPEN (Table 2) suggests that thepresence of a functional £pA gene aids growth at very lowZn(II) concentrations. Thus, FlpA may be required for theexpression of a high a¤nity importer, whilst FlpB may berequired for the expression of a low a¤nity (high capacity)importer.

3.4. Zinc requirement of the £pA, £pB single and £pA £pBdouble mutant strains

Because of the reduced intracellular Zn(II) pool of the£pA £pB mutant, it was predicted that it would requiremore Zn(II) than the parent strain for growth. Incubationwith TPEN showed that the £p strains of L. lactisMG1363 did require a higher concentration of heavy met-als in the medium. However, TPEN does not speci¢callychelate Zn(II) cations, therefore a medium (ZGM17) spe-ci¢cally depleted of Zn(II) was used to determine the ¢nalculture densities of all four strains in the absence andpresence of additional Zn(II) (100^1000 WM).

L. lactis MG1363 grew well in ZGM17 without supple-mentation (OD600 = 0.57, Table 2), con¢rming that an ac-tive Zn(II) scavenging system is in operation in MG1363.The ¢nal culture densities for the £pA, £pB single and £pA£pB double mutants were low when grown in the Zn(II)starvation medium (Table 2). The growth defect waslargely alleviated in the £pA and £pB strains by the addi-tion of 100 WM ZnCl2 to the growth medium. However,the £pA £pB strain failed to attain a high OD600 evenwhen Zn(II) was supplied at 100 WM. At ZnCl2 concen-trations of 500 WM and 1000 WM, the growth yields of the£pA, £pB and £pA £pB mutants were greater than that ofthe parent (which failed to grow at all in the presence of1000 WM ZnCl2 ; Table 2), con¢rming that £p lesions con-fer resistance to Zn(II) cations.

3.5. ATP-dependent sensitivity

The increased resistance of the L. lactis £p strains toZn(II) was assumed to be caused by a reduced rate ofZn(II) uptake by speci¢c transport proteins whose genesfall within the FlpA/FlpB regulon. Because of the closeproximity of a gene encoding a potential metal transport-ing P-type ATPase upstream of both £p operons [1], thee¡ects of sodium orthovanadate (a phosphate analoguethat inhibits ATP-dependent processes [13]) on theZn(II) sensitivity of the parental strain were tested(Fig. 1). Zinc toxicity was partially relieved by the pres-ence of orthovanadate. Hence it seems likely that the sen-sitivity to Zn(II) is due to ATP-dependent transmembranetransport.

4. Conclusion

The aim of the work presented here was to elucidatea functional role for the products of the £p genes ofL. lactis, and clear evidence suggesting that FlpA andFlpB regulate metal uptake (particularly Zn(II) uptake)has been uncovered. Growth in the presence of the heavymetal ion chelator, TPEN, suggests that FlpA is requiredfor the expression of a high a¤nity, low capacity Zn(II)transport system, whilst FlpB controls a high capacity, butlow a¤nity, uptake system. Further, it has been demon-strated that these processes are ATP-dependent. This isconsistent with the composition of the £p operons andthe upstream open reading frames, which perhaps formthe basis of two Zn(II) uptake (OrfWA=B), transport(OrfXA=B) and storage (OrfYA=B) mechanisms.

Hypersensitivity to hydrogen peroxide is only observedwith the £pA £pB lesion strain, and is coincident with thestark depletion of the intracellular Zn(II) pool [1]. Thissuggests that Zn(II) uptake is used by L. lactis to counteroxidative stress. Indeed, it has been demonstrated thatZn(II) actively protects the thiol groups of metallothioneinagainst oxidation to disul¢des [14,15]. In addition, the re-dox-mediated activation of the chaperone Hsp33 involvesthe conversion of a dithiol co-ordinated Zn(II) centre to adisul¢de bond [16], and Zn(II) may be intimately involvedin setting the redox poise of this transition so that it re-sponds to oxidative stress rather than normoxia. More-over, FlpA isolated from aerobic cultures of E. coli (whichprobably correspond to oxidative stress conditions forL. lactis proteins) contains 1.32 Zn atoms per monomer

Fig. 1. Increased Zn(II) tolerance of MG1363 in the presence of sodiumorthovanadate. Cultures of L. lactis ssp. cremoris MG1363 (triangles)and the £pA £pB derivative FI8828 (squares) were grown in GM17(closed symbols) or GM17 supplemented with 1 mM ZnCl2 (open sym-bols) at three concentrations of sodium orthovanadate. The ¢nal growthyields were obtained from two independent experiments and plottedagainst sodium orthovanadate concentration.

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[8]. It is a possibility, then, that one role of Zn(II) in the L.lactis cytoplasm is to protect thiol groups (including thoseof FlpA) from oxidative conversion to disul¢de bonds,and that depletion of the intracellular Zn(II) pool leavesvulnerable thiols open to oxidative attack by hydrogenperoxide. Thus, the provision of Zn(II) to the thiol groupsof L. lactis proteins may constitute a general redox stresssurvival mechanism.

Acknowledgements

We thank Neil Wyborn for helpful discussions, theBBSRC for ¢nancial support. J.G. is a BBSRC AdvancedFellow.

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