Susceptibility of halobacteria to heavy metals

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1987, p. 1199-1202 0099-2240/87/051199-04$02.00/0 Copy igh © 1987, Ame ican Socie y o Mic obiology Vol. 53, No. 5 Suscep ibili y o Halobac e ia o Hea y Me als J. J. NIETO,* A. VENTOSA, AND F. RUIZ-BERRAQUERO Depa men o Mic obiology, Facul y o Pha macy, Uni e si y o Se ille, Se ille, Spain Recei ed 8 Decembe 1986/Accep ed 18 Feb ua y 1987 Six y-eigh halobac e ia, including bo h cul u e collec ion s ains and esh isola es om widely di e ing geog aphical a eas, we e es ed o suscep ibili y o a sena e, cadmium, ch omium, cobal , coppe , lead, me cu y, nickel, sil e , and zinc ions by an aga dilu ion echnique. The cul u e collec ion s ains showed di e en suscep ibili ies, clus e ing in o i e g oups. Halobac e ium medi e anei and Halobac e ium olcanii we e he mos me al ole an , whe eas Haloa cula cali o niae and Haloa cula sinaiiensis had he highes suscep ibili ies o he cul u e collec ion s ains. Di e en pa e ns o me al suscep ibili y we e ound o all he halobac e ia es ed, and he e was a uni o m suscep ibili y o me cu y and sil e . All s ains es ed we e mul iply me al ole an . Halobac e ia a e ae obic mic oo ganisms which equi e high NaCI concen a ions in a medium o g ow and su i e (9) and which a e included, oge he wi h me hanogens and he moacidophiles, in he a chaebac e ia (22). This mic obial g oup has been ex ensi ely s udied wi h ega d o hei physiology, biochemis y, ecology and, ecen ly, gene ics (2, 9, 10). Al hough he suscep ibili y o an ibio ics and, in some ins ances, he mechanisms o ac ion o some an imi- c obial agen s ha e been s udied in halobac e ia (3, 4, 12), o da e he e a e no epo s conce ning hei na u al suscep i- bili y o hea y me als and o ganome allic compounds, le alone hei abili y o de elop hea y me al esis ances. The la e subjec migh p o ide use ul in o ma ion abou ways o de eloping an ibio ic esis ance, since in nonhalophilic bac- e ia genes de e mining esis ance o an ibio ics and ce ain me als a e o en ca ied on he same plasmids (20). I was ound ecen ly ha megaplasmids o unknown unc ion a e ha bo ed by he majo i y o halobac e ia (6). A knowledge o me al ole ance may e eal some unc ions o some o hese plasmids and, in addi ion, he possible hea y me al esis- ances could be used in halobac e ia as gene ic ma ke s. On he o he hand, me al ole ance could be ele an o he ecology and physiology o halobac e ia, since hey usually g ow in habi a s such as sola sal e ns o hype saline lakes con aining some wa e s which ha e ecen ly been ound o be pollu ed wi h hea y me als (unpublished da a). The aim o he p esen s udy was o examine he suscep- ibili y o a la ge numbe o halobac e ia, including bo h cul u e collec ion s ains and eshly isola ed s ains, o se e al hea y me als. A o al o 68 halobac e ial s ains we e selec ed o his s udy. The 13 cul u e collec ion s ains a e lis ed in Table 1, and he o he 55 s ains ha e been cha ac- e ized in a p e ious axonomic s udy and iden i ied as membe s o he genus Halobac e ium (19). These la e s ains ep esen a e y he e ogeneous g oup o halobac e ia ha can be commonly isola ed om hype saline habi a s in Spain. The cul u es we e g own in a medium ( e e ed o as SWYE) con aining a inal o al sal s concen a ion o ca. 25% (w / ol) and 0.5% (w / ol) yeas ex ac (Di co Labo a o ies, * Co esponding au ho . De oi , Mich.). The composi ion o he sal s solu ion was as ollows (pe cen [weigh / olume]): NaCl, 19.4; MgC92, 1.6; MgSO4, 2.4; CaCl2, 0.1; KCl, 0.5; NaHCO3, 0.02; and NaB , 0.05 (6). The pH was adjus ed o 7.2. Incuba ion was done a 37°C in an o bi al shake (Lab-Line Ins umen s, Inc., Mel ose Pa k, Ill.) a 200 s okes pe min. The 10 hea y me als es ed we e p o ided om s anda d comme cial sou ces as Na2HAsO4, AgNO3, CdCI2, CoC12, CuSO4, K2C O4, HgCl2, NiSO4, Pb(NO3)2, and ZnSO4. S ock solu ions we e made in dis illed wa e , s e ilized by il a ion h ough 0.22-,um-po e memb ane il e s (Millipo e Co p., Bed o d, Mass.), and kep a 4°C o no longe han 1 day. Suscep ibili y was de e mined by an aga dilu ion me hod (21) wi h a S ee s eplica o (17). Pla es con aining 20 ml o SWYE medium solidi ied wi h 2% (w / ol) Bac o-Aga (Di co) and se e al di e en concen a ions o he me al inhibi o s we e p epa ed on he days o he expe imen s. The concen a ions o all he me als we e as ollows (millimola ): 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 20, 40, and 80. This ange co e ed hose concen a ions usually used in s udies o me al esis ance in eubac e ia (7, 20). Be o e use, he pla es we e d ied a 37°C o 30 min; hey we e hen inocula ed wi h 104 o 105 mic oo ganisms om exponen- ially g owing cul u es pe spo . Twen y-one spo s (each om a di e en bac e ial s ain) could be con enien ly es ed pe pla e. The pla es we e hen incuba ed a 37°C o 10 days. Aga pla es wi hou he an imic obial agen s and inocula ed wi h he co esponding es mic oo ganisms we e used as con ols. Simila expe imen s, in which he sal concen a ion in he es medium was 15, 20, o 30% (w / ol), we e also ca ied ou . A e incuba ion, he MIC was de e mined as he lowes concen a ion o me al which p e en ed g ow h. The MICs o all he s ains we e he same when he s ains we e es ed in a leas h ee di e en expe imen s. Fo he pu pose o de ining me al ole ance, hose s ains which we e no inhibi ed by 10 mM As; 1 mM Ag, Cd, Co, C , Cu, Ni, Pb, and Zn; and 0.1 mM Hg we e ega ded as ole an . These concen a ions ha e been used o his pu pose in he majo i y o s udies ca ied ou wi h eubac e ia (11, 14, 20). I is impo an o poin ou ha he a ailabili y o he espec i e me als can be in luenced by chlo ides o o he kinds o sal s con ained in he cul u e 1199 APPL. ENVIRON. MICROBIOL. TABLE 1. MICs o 10 me al ions es ed agains 13 halobac e ial cul u e collec ion s ains MIC (mM) o ': As Ag Ni Co Pb Cd C Hg Zn Cu H. medi e anei ATCC 33500 20 0.5 2.5 1 20 2.5 5 0.01 0.5 1 H. olcanii DS2 20 0.5 2.5 1 20 0.5 5 0.01 0.5 1 H. hispanicum ATCC 33960 10 0.5 2.5 1 10 0.5 5 0.01 0.5 1 H. allismo is ATCC 29715 10 0.5 2.5 1 10 0.5 5 0.01 0.5 1 H. gibbonsii ATCC 33959 10 0.5 2.5 1 10 0.5 5 0.01 0.5 1 H. saccha o o um ATCC 2952 10 0.05 1 0.5 20 0.5 2.5 0.01 0.5 2.5 H. halobium CCM 2090 10 0.05 1 0.5 20 0.5 2.5 0.01 0.5 2.5 H. apanicum NCMB 767 10 0.05 1 0.5 5 0.5 2.5 0.01 0.5 2.5 H. salina ium CCM 2084 20 0.05 1 0.5 5 0.1 2.5 0.01 0.05 1 Haloa cula sp. s ain WS-1 10 0.05 0.1 1 5 0.05 2.5 0.05 0.05 2.5 Haloa cula sp. s ain GN-1 10 0.05 0.1 1 5 0.05 2.5 0.05 0.05 2.5 H. cali o niae ATCC 33799 10 0.05 1 0.5 10 0.1 1 0.05 0.5 2.5 H. sinaiiensis ATCC 33800 10 0.05 1 0.5 5 0.05 2.5 0.05 0.05 1 a Bold ace ype indica es concen a ions in ol ing ole ance o he co esponding me al ion. medium used in he p esen s udy. In addi ion, he o ma ion o me al complexes in his cul u e medium may de e mine he ue soluble me al concen a ions, and, indeed, he oxici y o some me als could be a ibu ed o a me al complex a he han a me al ca ion. The MICs o he hea y me als es ed agains he halobac e ial collec ion s ains used in he p esen s udy a e shown in Table 1. On he basis o he simila MICs o all he me als es ed, hese s ains can be g ouped in o i e majo g oups. (i) Halobac e ium medi e anei and Halobac e ium olcanii appea ed o be he leas suscep ible. (ii) Halobac- e ium hispanicum, Halobac e ium allismo is, and Halobac e ium gibbonsii had he same esponse o each me al and we e equally a ec ed by h ee me al ions (C , Ni, and Pb). (iii) Halobac e ium halobium, Halobac e ium sac- cha o o um, Halobac e ium apanicum, and Halobac e- ium salina ium had a simila esponse o Ag, C , Co, Hg, and Ni ions. Howe e , he MICs o Cd, Cu, and Zn ions o H. salina ium we e lowe , bu his s ain was he only one in his g oup o which he MIC o As ion was highe . O he - wise, all hese s ains we e ela i ely ole an o C and Pb ions (MICs, 2.5 and 5 mM, espec i ely), and h ee o hem we e also ole an o Cu ion (MIC, 2.5 mM). (i ) Haloa cula sp. s ain WS-1 and Haloa cula sp. s ain GN-1 had he same esponse o each o he me als es ed and we e ole an o C , Cu, and Pb ions. ( ) Haloa cula cali o niae and Haloa cula sinaiiensis we e inhibi ed by he lowes concen- a ions o mos o he me als es ed. TABLE 2. Suscep ibili y o 68 halobac e ial s ains o 10 me al ions Cumula i e % o s ains suscep ible o he ollowing me al Me al ion ion concn (mM): 0.005 0.01 0.05 0.1 0.5 1 2.5 5 10 20 As 0 0 0 0 0 5 12 41 85 100 Ag 0 28 35 71 90 100 100 100 100 100 Cd 0 0 0 28 66 91 100 100 100 100 Co 0 0 0 7 38 75 100 100 100 100 C 0 0 0 0 0 32 41 85 100 100 Cu 0 0 0 0 5 20 98 100 100 100 Hg 0 70 100 100 100 100 100 100 100 100 Ni 0 0 0 7 10 55 86 100 100 100 Pb 0 0 0 0 0 0 6 20 66 100 Zn 0 0 10 43 84 94 100 100 100 100 All he cul u e collec ion s ains we e ela i ely ole an o lead and ch omium (excep o H. cali o niae o he la e me al). Only he s ains belonging o g oup iii (excep o H. salina ium) and he Haloa cula s ains (excep o H. sinaiiensis) we e ole an o coppe . Only h ee s ains (H. medi e anei, H. olcanii, and H. salina ium) we e ole an o a sena e. H. medi e anei showed he highes ole ance o cadmium o all he s ains es ed. The cumula i e pe cen ages o s ains suscep ible o a - ious concen a ions o me al ions a e shown in Table 2. A wide ange o concen a ions was es ed because o he pauci y o in o ma ion on he suscep ibili y o halobac e ia o hea y me als. I was hoped ha an inhibi o y concen a- TABLE 3. Pa e ns o ole ance o 10 hea y me al ions in 68 halobac e ial s ains No. o di e en No. (%) ole ances o s ains 8 As, Cd, Co, C , Cu, Ni, Pb, Zn 1 (1.5) 7 Cd, Co, C , Cu, Ni, Pb, Zn 1 (1.5) 6 C , Co, Cu, Ni, Pb, Zn 1 (1.5) 5 Co, C , Cu, Ni, Pb 7 (10.3) As, C , Cu, Ni, Pb 2 (2.9) As, Cd, C , Ni, Pb 1 (1.5) As, C , Co, Cu, Pb 1 (1.5) Cd, C , Cu, Ni, Pb 1 (1.5) Co, C , Cu, Ni, Pb 1 (1.5) 4 C , Cu, Ni, Pb 6 (8.8) Co, Cu, Ni, Pb 4 (5.9) As, C , Ni, Pb 1 (1.5) Cd, C , Cu, Pb 1 (1.5) C , Cu, Pb, Zn 1 (1.5) Cd, Co, Cu, Pb 1 (1.5) 3 C , Cu, Pb 12 (17.6) C , Ni, Pb 4 (5.9) As, C , Pb 3 (4.5) As, Cu, Pb 1 (1.5) Cu, Ni, Pb 1 (1.5) 2 Cu, Pb 12 (17.6) C , Pb 5 (7.5) 1200 NOTES NOTES 1201 ion would be ob ained which could be used as a guide o u he in es iga ions, despi e he ac ha he oxici ies o some me als could be a ibu ed o me al complex o ma ion. On he o he hand, in expe imen s in which he sal concen- a ion in he es medium was 15, 20, o 30% (w / ol), ins ead o he s anda d 25%, no signi ican di e ences in he MICs we e obse ed in he ange o 20 o 30% sal s. Howe e , a he lowes sal concen a ion, an enhanced oxici y o he hea y me als es ed was ound. This esul may ha e been due o a highe a ailabili y o cells o ake up he me al ions in he medium wi h he lowes sal concen a- ion (closely ela ed o physiological cellula changes which ake place a his low salini y) o o a lowe deg ee o in e ac ion be ween he ee me al ions and he sal s p esen in he medium. The equencies o ole ance o each me al ion in all he s ains es ed we e as ollows: As, 15%; Ag, 0%; Cd, 9%; Co, 25%; C , 72%; Cu, 80%; Hg, 0%; Ni, 45%; Pb, 100%; and Zn, 6%. All halobac e ia es ed we e ole an o lead, and a g ea pe cen age o hem we e ole an o e en a 10 mM concen a ion o his me al. Fu he mo e, a g ea ac- ion o he s ains we e also ole an o coppe and ch o- mium. Howe e , i should be poin ed ou ha his high ole ance o lead, coppe , and ch omium could ha e been due o he binding o hese me al ions o some o he componen s o he es medium, as has been epo ed by Ramamoo hy and Kushne (13). These in es iga o s showed ha di e en componen s o he medium (yeas ex ac , pep one, yp one, e c.) exhibi ed high binding o lead, coppe , and cadmium. Besides, lead can be accumu- la ed in he cell wall and memb ane (18). Mo eo e , i may be ha he e was much less lead a ailable in he cul u e medium han ini ially added, since he e was a g ea amoun o sul a e p esen in he medium. As a esul , he soluble lead would be a a lowe concen a ion. Ne e heless, since e y li le esea ch has been conduc ed on he gene ic basis o lead esis ance in bac e ia, he ole ance exhibi ed by halobac e ia appea s o be an in e es ing ield o addi ional esea ch. O he wise, all he s ains demons a ed a high suscep ibil- i y o me cu y and sil e and e y li le ole ance o zinc, cadmium, and a sena e. Me cu y was he agen ha showed he highes ac i i y agains he halobac e ia es ed; all we e inhibi ed by a 0.05 mM concen a ion o his me al ion, and mos o hem we e inhibi ed by 0.01 mM. The suscep ibili y o halobac e ia o me cu y has been p e iously used as a pheno ypic ea u e in some axonomic s udies on halobac- e ia (5, 15). Howe e , he concen a ion o HgCl2 used in hose epo s was highe han 0.1 mM, a concen a ion which inhibi ed all he s ains es ed. I was epo ed (1) ha , in he p esence o high concen a ions o NaCl, he oxici y o zinc was inc eased, and his was he esul no o a syne gis ic in e ac ion be ween Zn2+ and ele a ed osmo ic p essu es bu o he o ma ion o complex anionic ZnCl- species, which exe ed g ea e oxici ies han did ca ionic Zn2+. This ac could be esponsible o he high suscep i- bili y o halobac e ia o zinc. All he s ains es ed we e ole an o mul iple me al ions: 17 we e ole an o wo me al ions (25%), 21 we e ole an o h ee (30.9%), 14 we e ole an o ou (20.7%), 13 we e ole an o i e (19.2%), and 1 (1.5%), 1 (1.5%), and 1 (1.5%) we e ole an o six, se en, and eigh me al ions, espec- i ely. S ains wi h iple ole ance we e ound mos e- quen ly, ollowed by hose wi h double ole ance. Howe e , s ains ole an o i e o ou di e en me al ions we e signi ican ly de ec ed (abou 40% o he o al s ains). The pa e ns o ole ance o he hea y me als es ed in he 68 halobac e ial s ains es ed a e shown in Table 3. I is well documen ed ha many me al esis ances can be go e ned by plasmid-encoded mechanisms in bo h g am- nega i e and g am-posi i e eubac e ia (8, 16, 20). 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