ArrumD MICROBIOLOGY, Feb. 1975, p. 275-286Copyright 0 1975 American Society for Microbiology

Vol. 29, No. 2Printed in U.SA.

Effects of Mercuric Chloride on Growth and Morphology ofSelected Strains of Mercury-Resistant Bacteria

Z. VAITUZIS, J. D. NELSON, JR.,' L. W. WAN,2 Am R. R COLWELL*Department of Microbiology, University of Maryland, College Park, Maryland 20742

Received for publication 6 September 1974

A survey of the comparative cytological effects of growth in the presence ofmercury by a group of mercury-resistant bacterial cultures and a characteriza-tion of the process of bacterial adaptation to Hg2+ ion was accomplished.Mercury resistance was found to be dependent upon the ability to volatilizemercury from the medium and upon the amount of mercury accumulated by thecells. The results indicate that most cultures which adapt to growth in thepresence of HgCl2 exhibit extensive morphological abnormalities. Significanteffects are delay in the onset of growth and cell division and numerous structuralirregularities associated with cell wall and cytoplasmic membrane synthesis andfunction. A detailed analysis of the adaptation process and the resulting effectson morphology was performed on an Enterobacter sp. During the periodpreceding active multiplication, a selection for mercury-resistant mutantsoccurred. It was also demonstrated that growth commenced only at aspecific threshold concentration of Hg2+.

Phytoplankton (1) and various bacterial spe-cies (9, 12, 20) are capable of adapting to growthin the presence of mercury. Investigations ofmercury resistance in microorganisms have re-vealed several possible mechanisms by whichthis process takes place. In some bacteria,resistance is attained through the acquisition ofa plasmid, an extrachromosomal element whichcan be transferred intra- and intergenerically (9,10, 13, 17, 19). Generally, microorganisms havebeen shown to detoxify mercurial compoundsmetabolically by the formation of volatile mer-cury (1, 4, 9, 12, 20) or mercury mercaptides (16,18).Comparatively little information is available

concerning the morphological effects of mercuryupon microorganisms. Tingle et al. (21) re-ported mitochondrial damage and swelling, pel-licular membrane disruption, and loss of motil-ity in Tetrahymena pyriformis incubated in amedium containing 0.5 ug of HgCl2 per ml.Exposure of Pseudomonas aeruginosa cells toHgCl2 was shown to cause swelling which couldbe reversed by addition of sulfhydryl com-pounds (2). Also, extremely low levels of phen-ylmercuric acetate (0.9 to 3 ng/ml) have beenreported to cause gross changes in the cellularform of growing cultures of Phaeodactylum

X Present address: Biology Department, University ofBridgeport, Bridgeport, Conn. 06602.

'Present address: Department of Civil Engineering, How-ard University, Washington, D.C. 20059.

tricornutum and Chlorella (14). Thin sectionsof a fungus (15) and a yeast (3) exposed toHgCl2 were shown to have electron-dense in-clusions associated with the nucleus and cyto-plasm, respectively, indicating possible mer-cury accumulation.Quantitative evidence linking mercury-resist-

ant bacteria in a mercury-contaminated habitatwith in situ generation of Hg0 has been obtained(J. D. Nelson and R. R. Colwell, J. MicrobialEcol., in press). The positive correlation ob-served between numbers of aerobic, hetero-trophic bacteria resistant to HgCl2 and ambientmercury concentration found in ChesapeakeBay sediments suggests that environmentalmercury contamination can exert an effect uponthe population structure of the estuarine micro-flora. To elucidate possible mechanisms bywhich mercury manifests selective pressures, weexamined the effects of inorganic mercury upongrowth and morphology of representativestrains of bacteria isolated from the ChesapeakeBay. (Preliminary results of this investigationwere presented at the 73rd Annual Meeting ofthe American Society for Microbiology, MiamiBeach, Fla., 1973.)

MATERIALS AND METHODSOrganisms and cultural methods. Microorga-

nisms used in this study were isolated by spreadingsuitable dilutions of water and sediment on an artifi-cial estuarine salts nutrient broth and agar medium

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consisting of 0.2% glucose, 1.0% Casamino Acids(Difco), 0.1% yeast extract (Difco), 1.0% NaCl, 0.23%MgCl2-6H20 and 0.03% KCl (pH 7.2). The mediumwas solidified with 2.0% agar (Difco) and supple-mented with varying concentrations of HgCl2. Theidentity of the isolates was determined to the genuslevel by established cultural and biochemical proce-dures. A listing of the microorganisms, together withtheir strain designations, appears in Table 1.Assay for Hg2+ content. Portions (0.1 ml) of

mid-log phase Enterobacter 85, grown without mer-cury, were inoculated into cotton-stoppered flaskscontaining 5 ml of fresh estuarine salts nutrient broth.One group of flasks, containing the medium alone,was used as uninoculated controls. The assays weremade under two sets of conditions. In the first, bothinoculated and control flasks contained 2 jig of 203Hg-labeled HgCl2 per ml (Amersham/Searle, ArlingtonHeights, Ill.). In the second set, the amount of labeledHgCl2 was increased to 4 ig/ml. All flasks were placedin a shaker-incubator at room temperature, and therate of growth was monitored on a Klett-Summersonphotoelectric colorimeter (Klett Manufacturing Co.,New York, N.Y.) and expressed as optical densityrelative to the uninoculated control. Duplicate 0.1-mlvolumes were periodically removed from all flasks andeither added directly to 10 ml of preblended liquidscintillation mixture 3a70 (Research Products Inter-national, Elk Grove Village, Ill.) or filtered through a0.45-jim membrane filter. The filter membranes werewashed with three 1-ml volumes of buffered threesalts solution (see assay for HgCl2 metabolism below)and were placed in 10 ml of liquid scintillationmixture. An ABAC SL40 liquid scintillation spec-trometer was used for determination of total andnonfilterable radioactivity. Portions of cells grown inmedium containing unlabeled HgCl2 were removedaseptically at varying time intervals (see arrows, Fig.1) for electron microscopy or plating for viable countdetermination.Assay for HgCl2 metabolism. One milliliter of

each culture, previously grown in HgCl2-containingbroth, was inoculated into 5 ml of fresh mediumcontaining a previously determined threshold concen-tration of HgCl2 for each particular culture (Table 2).The flasks were incubated with shaking for 2 h at25 C. The cultures were then centrifuged and sus-pended in 1 ml of 0.01 M potassium phosphate-buff-ered (pH 7.0) "three salts" solution (1.0% NaCl, 0.23%MgCl2.6H2O, and 0.03% KCl). The cells were cen-trifuged again and resuspended in 5 ml of bufferedthree salts with 1 jig of 203Hg-labeled HgCl2 per ml.The suspensions were aerated by bubbling moist airthrough capillary tubes to ensure uniformity of sus-pension and to enhance the evolution of volatilemercury. Portions (0.1 ml) of the suspensions wereremoved at 0, 15, and 30 min and either addeddirectly to 10 ml of liquid scintillation mixture orfiltered through a 0.45-jim membrane filter. The filtermembranes were washed with three 1-ml volumes ofbuffered three salts and were placed in 10 ml of liquidscintillation mixture to determine the percentage ofmercury accumulated by the cells. Mercury metabo-lism was measured by the loss of 203Hg from the

aerated cell suspension. The decrease in radioactivityafter a 30-min incubation was expressed as thepercentage of mercury metabolized relative to theremaining concentration of HgCl, in an uninoculatedcontrol tube containing 1 jg of 203Hg-labeled HgCl2per ml.

Electron microscopy. Cells taken from late log-phase cultures were transferred to fresh broth (0.1 mlof inoculum per 5 ml of broth) in flasks with andwithout sublethal concentrations of HgCl2. Thesewere incubated at 25 C with shaking for three to fourgeneration times or until equal turbidities were ob-tained during early log phase of growth. The cultureswere fixed according to the procedure described byKellenberger and Ryter (7). After a stepwise dehydra-tion through a 25, 50, 75, 85,95, and 100% ethanol andpropylene oxide series, the specimens were embeddedin Epon (11) and were thin sectioned by using an LKB800 Ultrotome III fitted with a diamond knife. Thesections were collected on collodion-coated, 200- or300-mesh copper grids and poststained with saturatedaqueous uranyl acetate (25) and alkaline lead citrate(24). The specimens were viewed and photographed inan RCA EMU 3F or Hitachi HU-11A electron micro-scope operating at an accelerating voltage of 50 kV.

RESULTS AND DISCUSSIONSurvey of morphological effects. Electron

microscopic examination of thin sections of thebacterial cultures grown at sublethal or thresh-old concentrations of HgCl, showed a widerange of morphological abnormalities. Growthin the presence of mercury commenced aftervarying lengths of time, with an array of mor-phological effects which did not appear incontrol cultures grown without mercury of thesame physiological age (Table 1). In gram-nega-tive bacteria, the most frequently observeddefect was an apparent loss of regulation in thecell wall synthesizing process. This was sug-gested by the appearance of irregular cell wallcontours, elongated pleomorphic as well asgiant cells and spheroplasts, and the develop-ment of cross-walls and branching in lieu of celldivision by constriction. In gram-positive bacte-ria, the major defect occurred in cross-wallformation. Thin sections showed cross-wallswith irregular contours, and a failure of somecells to divide resulted in elongated pleo-morphic forms with a number of septa withinthem at apparently random irregular locationswithin the bacteria. A number of cultures alsoshowed plasmolysis and changes in mesosomalstructure, indicating possible interference bythe mercury with cytoplasmic membrane syn-thesis and function. These effects may be re-lated to the finding that most of the mercurybound to bacterial cells is associated with eitherthe cell wall or cytoplasmic membrane (unpub-lished data; 22, 23).

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TABLE 1. Survey of morphological changes in bacteria grown in the presence of HgCI2

Culture HgCl2 concn Lag phase (h) Morphological effectsa

Genus Strain(g/l

Arthrobacter 72 5 24 Irregular septum formationBacillus 394 15 41 Formation of irregular and

multiple septaCitrobacter 132 20 70 Plasmolysis, membrane damageEnterobacter 85b 1 0 Pleomorphic cells, outgrowth of

cell wall, plasmolysisFlavobacterium 119 10 40 Convoluted cell wall, plasmoly-

sis, elongated cells, irregularmesosomes

Pseudomonas 244 25 0 NonePseudomonas 94 10 24 Convoluted cell wallPseudomonas B-16c 15 6 days Small spherical cells bounded

by common outer membraneVibrio 639 5 72 Giant, pleomorphic cells,

spheroplasts, elongated cells

a Four generation times after the lag phase as determined by turbidimetric comparison with mercury-freecontrol cultures.

" See Fig. 4 to 9 for detailed morphological changes.c Supplied by R. A. MacLeod, McGill University, Montreal, Que., Canada.

Further examination of Table 1 reveals avarying lag period prior to onset of growth.Furthermore, some organisms are capable ofadapting to growth in the presence of mercurymore rapidly than others. The initial concentra-tion of mercury that any given culture willadapt to was also found to be different. Themost resistant culture, Pseudomonas strain 244,did not exhibit any appreciable lag at all beforethe onset of growth. Thus, both the reasons forresistance to varying amounts of mercury andthe transpiration of events during the character-istically prolonged lag phase were investigatedby monitoring the amount of mercury in themedium and within the cells.Metabolism and accumulation of HgCl.

The comparative metabolism of Hg2+ to volatilemercury is presented in Table 2. Generally, themore resistant cultures metabolized a largeproportion of the Hg2+ (70 to 81%) and rela-tively less Hg2+ was cell associated (16 to 37%).Likewise, electron microscopic examination ofthin sections showed a direct correlation be-tween mercury accumulation by the cells andthe amount of electron-dense clusters of ribo-somes in the cytoplasm. These data suggestthat the ability to convert Hg2+ to volatilemercury is part of the basis for bacterial resist-ance. This trait, which is presumably responsi-ble for the reduction in Hg accumulation by thecells, is observed after the induction of mercury-metabolizing enzyme(s) (5, 8, 12). During our

survey of morphological effects of growth in thepresence of mercury, the cells had not been

TABLE 2. Comparative metabolism andaccumulation of HgCI2 in a buffer solution containing

1 ug of HgCI2 per ml

HgCl, Dry(Alg/ml) wegt HgCI2 Cell-

Culture in wfeight metab- associatedgrowth (mg/ml) olized (%) HgCl, (%)medium(g/)

Arthrobacter 72 1.2 0.24 0.0 36.8Bacillus 394 3.0 0.28 36.6 64.2Citrobacter 132 6.0 0.42 80.4 15.6Enterobacter 85 1.2 0.51 10.5 90.6Pseudomonas 94 3.0 0.42 80.9 23.4Pseudomonas 244 6.0 0.44 70.6 37.1Vibrio 639 1.2 j 0.51 1.5 99.8

previously exposed to mercury and thus dis-played characteristic lag phases of varyinglength. It appears, then, that the onset ofgrowth and cell division in the presence of Hg iscontingent upon the induction of the necessaryenzyme(s) for the detoxification of the growthmedium. The following experiments were de-signed to examine this process of adaptation tomercury, and to correlate it with morphologicalchanges.Growth and morphological changes during

adaptation to HgCl,. A gram-negative specieswas selected for a detailed investigation toclarify events occurring while bacteria wereapparently quiescent in mercury-containingbroth and to relate these events to cytologicalchanges associated with adaptation.Enterobacter strain 85 was serially transferredseveral times in the absence of mercury before

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inoculation into flasks with and without 2 ,ug of203Hg-labeled or unlabeled HgCl2 per ml. Aftera 24-h lag phase, the mercury-containing culturegrew at a rate comparable to that of the controlculture (Fig. 1). Although the turbidity of theHgCl2-containing culture remained constant,an initial 3-log drop in viable count occurred.Viable count and turbidity increased at the endof the lag phase. An "isotope effect" was sug-gested by the slightly shorter lag phase in theunlabeled mercury-containing culture. Duringthe initial phase of incubation, radioactivitywas rapidly lost from both the inoculated brothand from the sterile control. Other investigatorshave shown that reducing agents such as glu-cose (6) and yeast extract (A. D. Murray, and D.K. Kidby, Abstr. Annu. Meet. Am. Soc. Micro-biol. 1972, E98, p. 17) in the growth mediapromote the reduction of Hg2+. The loss ofradioactivity from the control broth representedthe volatilization of mercury and did not arise

I-

I-

m0w1r01

2 4 6 24

INCUBATION TIME (hr.)

26 28 30

FIG. 1. Metabolism and uptake of 2 ug of HgCl2per ml by Enterobacter strain 85. Shaker flaskscontaining broth with and without 203Hg and unla-beled HgCl2 (2 jg/ml) were inoculated with an 18-hculture. Turbidity of the culture without mercury (@)and the culture with labeled mercury (A) were

measured. Total (A) and nonfilterable (U) activity ofthe latter were also measured. An uninoculated,203Hg-labeled control flask (0) was also assayed fortotal radioactivity. Portions were removed from flaskswithout and with unlabeled mercury at intervals forexamination by electron microscopy (arrows). Theopen circles (0) represent total viable counts in theunlabeled mercury culture.

from adsorption to the glass walls of the flask,since only 8.9% of the radioactivity was re-covered in sequential washes of the vessel withwater, 0.5 N HNOa, and 10% Radiac wash(Atomic Products Corp., Center Moriches,N.Y.). An initial, small loss of cell-bound mer-cury coincided with the drop in viable count,indicating that lysis of the cells may haveoccurred. When the mercury concentrationdropped approximately 45.0%, to 1.1 gg/ml,growth was initiated. At this point, the rate ofmercury loss from the broth accelerated in theinoculated flask, and the rate of cellular uptakeper unit volume of broth increased.

After incubation of the spread plates pre-pared for determination of total viable counts,small colonial variants (85-S) of the HgCl,-con-taining culture began to appear (Fig. 2). Thesecolonies contained nonmotile, pleomorphiccells, in contrast to the large "parent"-likecolonies (85-L) comprising the remainder of thepopulation. The comparative properties of thetwo types are discussed in a subsequent section.The growth experiment was repeated by using

an elevated concentration of mercury (4 ,ug/ml)to test the hypothesis that the length of the lagphase demonstrated by this organism was de-pendent upon reduction of mercury concentra-tion to a specific threshold concentration of 1.1gg/ml. The pattern of growth and loss of mer-cury was as observed with 2 gg of HgCl1 per ml,except that the lag phase was found to bealmost 3 days long (Fig. 3). During the lagphase, the mercury concentration dropped by70%, to a level of 1.2 Ag/ml, a concentrationclosely approximating the concentration of 1.1Mg of HgCl2 per ml at which growth commencedin the first experiment. An increased isotopeeffect was observed in the second experiment,where the difference between the lag phase forlabeled and unlabeled cultures was nearly 10 h.Again, a drop in viable count was accompaniedby a drop in cell-bound mercury. As in the firstexperiment, the rate of loss of mercury from theinoculated flask accelerated at the end of lagphase. Numbers of small colonial variants com-prised approximately 1% of the total viablecount, and these small colony variants in-creased in parallel with the total population(Fig. 3).An electron microscopic examination of thin

sections of cells harvested from the unlabeledbroth with and without mercury was made.Control cells examined at the early, mid, andlate logarithmic phases of growth were found tobe normal in all respects, namely, array andmorphology of the deoxyribonucleic acid fibrils,ribosomes, and cell envelope, as well as mode of

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FIG. 2. Colonial variation in Enterobacter strain 85. Samples of culture grown in 2 ug of unlabeled HgCI2 perml containing broth were diluted and plated on basal nutrient medium and incubated at 25 C for 7 days. Small(85-S) and large (85-L) colonial forms were observed.

100 division. At 0 h, the mercury-containing culturewas found to be identical to the O-h control (Fig.

'10.0 4). After 2 h (Fig. 5), the cells appeared normalso|/ in overall morphology. The deoxyribonucleic/0 acid fibrils in some cells appeared condensed,

/ 0 rather than dispersed throughout the cyto-plasm, and electron-dense areas among theribosomes were seen. At 6 h, intact cells similar

f to those at 2 h were accompanied by swollen and0 ° plasmolyzed cells (Fig. 6). Approximately one-= third of all the cells at this stage showed signs of

2.0 *,40 a lysis. At 24 h (Fig. 7), the culture was character-£ . ized by lysed cell debris as well as viable, but

4Z 4.00 pleomorphic, cells with abnormal cross-wallF 7 formation. Electron-dense clusters of ribosomes0 I.20 A s / ^ were more frequently seen in these cells. As

. -- ---- growth progressed through the 26th and 28th h,0.5 .. .to the cells became relatively more rod shaped,j IA although irregular cell walls and electron-dense

1020 30 .00 go io ribosome clusters were still present (Fig. 8, 9).INCUUTION TIME (hr.) The latter clusters are similar to electron-dense

FIG. 3. Metabolism and uptake of 4 gg of HgCI, structures seen in other microorganisms ex-per ml by Enterobacter strain 85. Shaker flasks posed to mercury (3, 15). A high-resolutioncontaining broth with 203Hg-labeled and unlabeled analytical technique, however, such as electronHgCI2 (4 Mg/ml) were inoculated with an 18-h culture. probe microanalysis, would be required to de-Turbidity (A) and total (A) and nonfilterable (U) termine whether the density is due to preferen-radioactivity in the labeled culture were measured.An uninoculated, labeled control flask (0) was also (0) and numbers of small colonial variants (85-S) (a)assayed for total radioactivity. Total viable counts were obtained.

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FIG. 4. Thin section of Enterobacter 85 immediately after addition to broth containing 2 fig of HgCI2 per ml.The cells show dispersed chromatin, normal ribosomes, and cell envelope constituents, as well as typicalgram-negative cell division by constriction. The bar in all electron micrographs represents 1 tim.

tial intracellular binding of Hg.Properties of mercury-selected colonial

variants. After repeated serial transfers inmercury-free medium, the small colonial vari-ants (85-S) lost their pleomorphism and re-gained motility, but retained their colonial

morphology. The variants were examined to seeif they differed from the parent stock, withrespect to mercury resistance and/or the abilityto reduce Hg2+ (Tables 3 and 4). It is evidentthat the small colonies selected by growth in thepresence of HgCl2 represent a stable mutation

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FIG. 5. Thin section of Enterobacter 85 after a 2-h incubation in broth containing 2 ug of HgCl2 per ml. Theoverall morphology is similar to that of cells at zero time, except for condensed chromatin and the appearance ofelectron-dense groups of ribosomes (arrow).

to mercury resistance (Table 3). The basis forincreased resistance is unclear from the resultsof assays of mercury metabolism in the parentand variant cultures (Table 4). Culture 85-Sshowed only a slight increase in metabolism ofHgCl2. The mechanism of resistance might bedue to a lowered mercury accumulation. This,however, does not appear to be an acceptableexplanation, because each of the cultures accu-mulated a similar amount of labeled Hg2 .

In the case of the Enterobacter strain studied,

it appears that during the lag phase most of thecells are lysed and mercury-resistant mutantsare selected, and, when the mercury concentra-tion is reduced to ca. 1 gg/ml via chemical andbiological reduction of Hg2+, the surviving cellsinitiate growth at a normal rate. Whereas theexact mechanism of resistance to HgCl. for thisparticular organism is unclear, we have shownthat resistance in other organisms is related tothe ability to reduce Hg2+ ion to Hg0 (12). Ourresults suggest that in the presence of toxic

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FIG. 6. Thin section of Enterobacter 85 after a 6-h incubation in broth containing 2 Aig of HgCI2 per ml. Thisfigure shows the typical morphology of the intact cells. The major portion of the cells is lysed at this stage.

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FIG. 7. Thin section of Enterobacter 85 after a 24-h incubation in broth containing 2 ,gg of HgCl2 per ml.Viable, pleomorphic cells and frgments of lysed cells are seen. Cellular division occurs by cross-wall formation(arrows) rather than by normal constriction. Numerous electron-dense groups of ribosomes are also seen.

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FIG. 8. Thin section of Enterobacter 85 after a 26-h incubation in broth containing 2 ttg of HgCI2 per ml.Viable, pleomorphic cells predominate. Electron-dense clusters of ribosomes are present in all cells.

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EFFECTS OF MERCURIC CHLORIDE ON BACTERIA

II II

An.- j~~~~~~

FIG. 9. Thin section of Enterobacter 85 after a 28-h incubation in broth containing 2 9g of HgCI2 per ml. Thecells show reversion to rod-shaped morphology, although cell wall contours are still somewhat irregular.Electron-dense clusters of ribosomes are present in fewer numbers.

levels of mercury, reduction of the mercuryconcentration to a definite threshold level,either by metabolic or chemical volatilization,permits growth of the organism. In the early

stages of growth the cells appear pleomorphicand contain dense inclusions. As the concentra-tion of mercury in the medium continues todecrease, the bacteria revert to normal mor-

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286 VAITUZIS ET AL.

TABLE 3. Mercury resistance0 by Enterobacter 85before and after growth in the presence ofHgCI,

(strains 85-L and 85-S)Growth in HgCl, concn (jug/ml)

Culture2 4 6 8 10

85 + + - - -

85-L + + + _ _85-S + + + + +

aThe cultures were serially subcultured in theabsence of mercury. Each tube of broth containing theconcentrations of mercury indicated above was inocu-lated with 3 drops of a log-phase culture and incu-bated, with shaking, at 25 C for 4 days.

TABLE 4. Mercury metabolism by Enterobacter 85before and after growth in the presence of HgCl2

(strains 85-L and 85-S)a

Dryt of HgCl2 Cell-Culture Dry wt of metabolized associatedcells (mg/mi) (%) HgCl, (%)

85 0.41 9.8 92.385-L 0.30 7.1 93.085-S 0.38 11.3 91.7

a See footnote to Table 3.

phology. Experiments are now in progress todetect the involvement of plasmids in the mer-

cury resistance of this particular group of orga-nisms.

ACKNOWLEDGMENT

This work was supported by Environmental ProtectionAgency grant RE-02529-01.

LITERATURE CITED

1. Ben-Bassat, D., G. Shelef, N. Gruner, and H. Shuval.1972. Growth of Chlamydomonas in a medium contain-ing mercury. Nature (London) 240:43-44.

2. Bernheim, F. 1971. The effect of cyanogen iodide andmercuric chloride on the permeability of cells of Pseu-domonas aeruginosa and the antagonistic action ofsulfhydryl compounds. Proc. Soc. Exp. Biol. Med.138:444-447.

3. Brunker, R. L., and T. L. Bott. 1974. Reduction ofmercury to the elemental state by a yeast. Appl.Microbiol. 27:870-873.

4. Furukawa, K., T. Suzuki, and K. Tonomura. 1969.Decomposition of organic mercurial compounds bymercury-resistant bacteria. Agric. Biol. Chem.33:128-130.

5. Furukawa, K., and K. Tonomura. 1972. Induction ofmetallic mercury-releasing enzyme in mercury resist-ant Pseudomonas. Agric. Biol. Chem. 36:2441-2448.

6. Gillespie, D. C. 1972. Mobilization of mercury fromsediments into guppies (Poecilia reticulata). J. Fish.Res. Board. Can. 29:1035-1041.

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