Susceptibility of halobacteria to heavy metals

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1987, p. 1199-1202 0099-2240/87/051199-04$02.00/0 Copyright © 1987, American Society for Microbiology Vol. 53, No. 5 Susceptibility of Halobacteria to Heavy Metals J. J. NIETO,* A. VENTOSA, AND F. RUIZ-BERRAQUERO Department of Microbiology, Faculty of Pharmacy, University of Seville, Seville, Spain Received 8 December 1986/Accepted 18 February 1987 Sixty-eight halobacteria, including both culture collection strains and fresh isolates from widely differing geographical areas, were tested for susceptibility to arsenate, cadmium, chromium, cobalt, copper, lead, mercury, nickel, silver, and zinc ions by an agar dilution technique. The culture collection strains showed different susceptibilities, clustering into five groups. Halobacterium mediterranei and Halobacterium volcanii were the most metal tolerant, whereas Haloarcula californiae and Haloarcula sinaiiensis had the highest susceptibilities of the culture collection strains. Different patterns of metal susceptibility were found for all the halobacteria tested, and there was a uniform susceptibility to mercury and silver. All strains tested were multiply metal tolerant. Halobacteria are aerobic microorganisms which require high NaCI concentrations in a medium to grow and survive (9) and which are included, together with methanogens and thermoacidophiles, in the archaebacteria (22). This microbial group has been extensively studied with regard to their physiology, biochemistry, ecology and, recently, genetics (2, 9, 10). Although the susceptibility to antibiotics and, in some instances, the mechanisms of action of some antimicrobial agents have been studied in halobacteria (3, 4, 12), to date there are no reports concerning their natural susceptibility to heavy metals and organometallic compounds, let alone their ability to develop heavy metal resistances. The latter subject might provide useful information about ways of developing antibiotic resistance, since in nonhalophilic bacteria genes determining resistance to antibiotics and certain metals are often carried on the same plasmids (20). It was found recently that megaplasmids of unknown function are harbored by the majority of halobacteria (6). A knowledge of metal tolerance may reveal some functions for some of these plasmids and, in addition, the possible heavy metal resistances could be used in halobacteria as genetic markers. On the other hand, metal tolerance could be relevant to the ecology and physiology of halobacteria, since they usually grow in habitats such as solar salterns or hypersaline lakes containing some waters which have recently been found to be polluted with heavy metals (unpublished data). The aim of the present study was to examine the susceptibility of a large number of halobacteria, including both culture collection strains and freshly isolated strains, to several heavy metals. A total of 68 halobacterial strains were selected for this study. The 13 culture collection strains are listed in Table 1, and the other 55 strains have been characterized in a previous taxonomic study and identified as members of the genus Halobacterium (19). These latter strains represent a very heterogeneous group of halobacteria that can be commonly isolated from hypersaline habitats in Spain. The cultures were grown in a medium (referred to as SWYE) containing a final total salts concentration of ca. 25% (wt/vol) and 0.5% (wt/vol) yeast extract (Difco Laboratories, * Corresponding author. Detroit, Mich.). The composition of the salts solution was as follows (percent [weight/volume]): NaCl, 19.4; MgC92, 1.6; MgSO4, 2.4; CaCl2, 0.1; KCl, 0.5; NaHCO3, 0.02; and NaBr, 0.05 (6). The pH was adjusted to 7.2. Incubation was done at 37°C in an orbital shaker (Lab-Line Instruments, Inc., Melrose Park, Ill.) at 200 strokes per min. The 10 heavy metals tested were provided from standard commercial sources as Na2HAsO4, AgNO3, CdCI2, CoC12, CuSO4, K2CrO4, HgCl2, NiSO4, Pb(NO3)2, and ZnSO4. Stock solutions were made in distilled water, sterilized by filtration through 0.22-,um-pore membrane filters (Millipore Corp., Bedford, Mass.), and kept at 4°C for no longer than 1 day. Susceptibility was determined by an agar dilution method (21) with a Steers replicator (17). Plates containing 20 ml of SWYE medium solidified with 2% (wt/vol) Bacto-Agar (Difco) and several different concentrations of the metal inhibitors were prepared on the days of the experiments. The concentrations of all the metals were as follows (millimolar): 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 20, 40, and 80. This range covered those concentrations usually used in studies of metal resistance in eubacteria (7, 20). Before use, the plates were dried at 37°C for 30 min; they were then inoculated with 104 to 105 microorganisms from exponentially growing cultures per spot. Twenty-one spots (each from a different bacterial strain) could be conveniently tested per plate. The plates were then incubated at 37°C for 10 days. Agar plates without the antimicrobial agents and inoculated with the corresponding test microorganisms were used as controls. Similar experiments, in which the salt concentration in the test medium was 15, 20, or 30% (wt/vol), were also carried out. After incubation, the MIC was determined as the lowest concentration of metal which prevented growth. The MICs for all the strains were the same when the strains were tested in at least three different experiments. For the purpose of defining metal tolerance, those strains which were not inhibited by 10 mM As; 1 mM Ag, Cd, Co, Cr, Cu, Ni, Pb, and Zn; and 0.1 mM Hg were regarded as tolerant. These concentrations have been used for this purpose in the majority of studies carried out with eubacteria (11, 14, 20). It is important to point out that the availability of the respective metals can be influenced by chlorides or other kinds of salts contained in the culture 1199 APPL. ENVIRON. MICROBIOL. TABLE 1. MICs of 10 metal ions tested against 13 halobacterial culture collection strains MIC (mM) of': As Ag Ni Co Pb Cd Cr Hg Zn Cu H. mediterranei ATCC 33500 20 0.5 2.5 1 20 2.5 5 0.01 0.5 1 H. volcanii 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. vallismortis 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. saccharovorum 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. trapanicum NCMB 767 10 0.05 1 0.5 5 0.5 2.5 0.01 0.5 2.5 H. salinarium CCM 2084 20 0.05 1 0.5 5 0.1 2.5 0.01 0.05 1 Haloarcula sp. strain WS-1 10 0.05 0.1 1 5 0.05 2.5 0.05 0.05 2.5 Haloarcula sp. strain GN-1 10 0.05 0.1 1 5 0.05 2.5 0.05 0.05 2.5 H. californiae 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 Boldface type indicates concentrations involving tolerance of the corresponding metal ion. medium used in the present study. In addition, the formation of metal complexes in this culture medium may determine the true soluble metal concentrations, and, indeed, the toxicity of some metals could be attributed to a metal complex rather than a metal cation. The MICs of the heavy metals tested against the halobacterial collection strains used in the present study are shown in Table 1. On the basis of the similar MICs of all the metals tested, these strains can be grouped into five major groups. (i) Halobacterium mediterranei and Halobacterium volcanii appeared to be the least susceptible. (ii) Halobacterium hispanicum, Halobacterium vallismortis, and Halobacterium gibbonsii had the same response to each metal and were equally affected by three metal ions (Cr, Ni, and Pb). (iii) Halobacterium halobium, Halobacterium saccharovorum, Halobacterium trapanicum, and Halobacterium salinarium had a similar response to Ag, Cr, Co, Hg, and Ni ions. However, the MICs of Cd, Cu, and Zn ions for H. salinarium were lower, but this strain was the only one in this group for which the MIC of As ion was higher. Otherwise, all these strains were relatively tolerant of Cr and Pb ions (MICs, 2.5 and 5 mM, respectively), and three of them were also tolerant of Cu ion (MIC, 2.5 mM). (iv) Haloarcula sp. strain WS-1 and Haloarcula sp. strain GN-1 had the same response to each of the metals tested and were tolerant of Cr, Cu, and Pb ions. (v) Haloarcula californiae and Haloarcula sinaiiensis were inhibited by the lowest concentrations of most of the metals tested. TABLE 2. Susceptibility of 68 halobacterial strains to 10 metal ions Cumulative % of strains susceptible to the following metal Metal 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 Cr 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 the culture collection strains were relatively tolerant of lead and chromium (except for H. californiae for the latter metal). Only the strains belonging to group iii (except for H. salinarium) and the Haloarcula strains (except for H. sinaiiensis) were tolerant of copper. Only three strains (H. mediterranei, H. volcanii, and H. salinarium) were tolerant of arsenate. H. mediterranei showed the highest tolerance of cadmium of all the strains tested. The cumulative percentages of strains susceptible to various concentrations of metal ions are shown in Table 2. A wide range of concentrations was tested because of the paucity of information on the susceptibility of halobacteria to heavy metals. It was hoped that an inhibitory concentra- TABLE 3. Patterns of tolerance of 10 heavy metal ions in 68 halobacterial strains No. of different No. (%) tolerances of strains 8 As, Cd, Co, Cr, Cu, Ni, Pb, Zn 1 (1.5) 7 Cd, Co, Cr, Cu, Ni, Pb, Zn 1 (1.5) 6 Cr, Co, Cu, Ni, Pb, Zn 1 (1.5) 5 Co, Cr, Cu, Ni, Pb 7 (10.3) As, Cr, Cu, Ni, Pb 2 (2.9) As, Cd, Cr, Ni, Pb 1 (1.5) As, Cr, Co, Cu, Pb 1 (1.5) Cd, Cr, Cu, Ni, Pb 1 (1.5) Co, Cr, Cu, Ni, Pb 1 (1.5) 4 Cr, Cu, Ni, Pb 6 (8.8) Co, Cu, Ni, Pb 4 (5.9) As, Cr, Ni, Pb 1 (1.5) Cd, Cr, Cu, Pb 1 (1.5) Cr, Cu, Pb, Zn 1 (1.5) Cd, Co, Cu, Pb 1 (1.5) 3 Cr, Cu, Pb 12 (17.6) Cr, Ni, Pb 4 (5.9) As, Cr, Pb 3 (4.5) As, Cu, Pb 1 (1.5) Cu, Ni, Pb 1 (1.5) 2 Cu, Pb 12 (17.6) Cr, Pb 5 (7.5) 1200 NOTES NOTES 1201 tion would be obtained which could be used as a guide for further investigations, despite the fact that the toxicities of some metals could be attributed to metal complex formation. On the other hand, in experiments in which the salt concentration in the test medium was 15, 20, or 30% (wt/vol), instead of the standard 25%, no significant differences in the MICs were observed in the range of 20 to 30% salts. However, at the lowest salt concentration, an enhanced toxicity of the heavy metals tested was found. This result may have been due to a higher availability of cells to take up the metal ions in the medium with the lowest salt concentration (closely related to physiological cellular changes which take place at this low salinity) or to a lower degree of interaction between the free metal ions and the salts present in the medium. The frequencies of tolerance of each metal ion in all the strains tested were as follows: As, 15%; Ag, 0%; Cd, 9%; Co, 25%; Cr, 72%; Cu, 80%; Hg, 0%; Ni, 45%; Pb, 100%; and Zn, 6%. All halobacteria tested were tolerant of lead, and a great percentage of them were tolerant of even a 10 mM concentration of this metal. Furthermore, a great fraction of the strains were also tolerant of copper and chromium. However, it should be pointed out that this high tolerance of lead, copper, and chromium could have been due to the binding of these metal ions to some of the components of the test medium, as has been reported by Ramamoorthy and Kushner (13). These investigators showed that different components of the medium (yeast extract, peptone, tryptone, etc.) exhibited high binding to lead, copper, and cadmium. Besides, lead can be accumulated in the cell wall and membrane (18). Moreover, it may be that there was much less lead available in the culture medium than initially added, since there was a great amount of sulfate present in the medium. As a result, the soluble lead would be at a lower concentration. Nevertheless, since very little research has been conducted on the genetic basis of lead resistance in bacteria, the tolerance exhibited by halobacteria appears to be an interesting field for additional research. Otherwise, all the strains demonstrated a high susceptibility to mercury and silver and very little tolerance of zinc, cadmium, and arsenate. Mercury was the agent that showed the highest activity against the halobacteria tested; all were inhibited by a 0.05 mM concentration of this metal ion, and most of them were inhibited by 0.01 mM. The susceptibility of halobacteria to mercury has been previously used as a phenotypic feature in some taxonomic studies on halobacteria (5, 15). However, the concentration of HgCl2 used in those reports was higher than 0.1 mM, a concentration which inhibited all the strains tested. It was reported (1) that, in the presence of high concentrations of NaCl, the toxicity of zinc was increased, and this was the result not of a synergistic interaction between Zn2+ and elevated osmotic pressures but of the formation of complex anionic ZnClspecies, which exerted greater toxicities than did cationic Zn2+. This fact could be responsible for the high susceptibility of halobacteria to zinc. All the strains tested were tolerant of multiple metal ions: 17 were tolerant of two metal ions (25%), 21 were tolerant of three (30.9%), 14 were tolerant of four (20.7%), 13 were tolerant of five (19.2%), and 1 (1.5%), 1 (1.5%), and 1 (1.5%) were tolerant of six, seven, and eight metal ions, respectively. Strains with triple tolerance were found most frequently, followed by those with double tolerance. However, strains tolerant of five or four different metal ions were significantly detected (about 40% of the total strains). The patterns of tolerance of the heavy metals tested in the 68 halobacterial strains tested are shown in Table 3. It is well documented that many metal resistances can be governed by plasmid-encoded mechanisms in both gramnegative and gram-positive eubacteria (8, 16, 20). Since we have recently found that 75% of all halobacteria used in this study carry at least one plasmid and that in the majority of these strains three or four megaplasmids can be detected (6), additional experiments to correlate the presence of plasmids and the tolerance of some heavy metal ions in each individual strain are now in progress in our laboratory. This investigation was supported by grants from the Comisi6n Asesora para el Desarrollo de la Investigaci6n Cientifica y Tecnica and from the Junta de Andalucia. LITERATURE CITED 1. Babich, H., and G. Stotzky. 1978. Toxicity of zinc to fungi, bacteria, and coliphages: influence of chloride ions. Appl. Environ. Microbiol. 36:906-914. 2. Bayley, S. T., and R. A. 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