Metal-tolerant bacterial populations from natural and metal-polluted soils

003x-07 I7 ‘x3.030?43-0xs03.0~~0 1983 Pergamon Press Ltd

CopyrIght (

METAL-TOLERANT BACTERIAL POPULATIONS FROM NATURAL AND METAL-POLLUTED SOILS T. DUXRURY and B. BICKNELL* Dcpartmcnt

of Microbiology.

University

of Sydney,

Sydney, New South Wales 2006. Australia

Summary-The

toxicity of several metals towards bacterial populations from natural and metal-polluted soils could bc described either partially by a single exponential equation or completely by the sum of two exponential functions. Bacterial populations from both soils contained two subgroups. one of which could tolcratc metals over a grcatcr r;mgc ofconccntrntions than the other. Most bacteria comprising the more metal-tolerant subgroup were Gram-ncgativc and were multiple drug resistant. Exceptions wcrc organisms. tcntativcly idcntilicd as coryncforms. isolated on nickel-supplemcntcd medium. It is suggcstcd that. in gcncral. Gram-nceativc bacteria arc more metal-tolerant than Gram-positive organisms and. in soils containing comparatively low Icvcls of metal pollution. may be able to function without the need for plasmid-mediated metal-tolerance.

Gadd and Griffiths (1978) termed such resistance mechanisms “gratuitous” in that they were components or functions of cells which were not a specific response towards metal-tolerance. Duxhury (1981) showed that whole populations of soil bacteria exhibit characteristic responses to different heavy metals. In this study we have investigated whether metals exert a less toxic effect on whole populations of bacteria from metal-affected soil compared to those from unpolluted soil. In addition, in order to determine whether metaltolerance was associated with a few restricted groups of bacteria, or with individuals from several diverse groups, we have partially characterized some metaltolerant bacteria isolated from the polluted site.

INTRODUCTION contains as diverse an assortment of microorganisms as can be found in any natural ecosystem (Gray and Williams, 1971). When some stress factor such as high temperature, extremes of pH, or chemical pollution, is imposed on a natural environment it often results in a decrease in species diversity and the populations which assume dominance possess fitness traits which allow them to endure these stresses (Alexander. 1976). Microbial populations in metal-polluted environments contain organisms often described as metaltolerant. Jordan and Lechevalier (1975) reported that microbial populations in soils close to a zinc-smelter contained greater proportions of bacteria able to grow on media containing high concentrations of zinc. Similarly, Timoney ct ul. (1978) showed that mercury-resistant bacteria were only isolated in suhstantial numbers from mercury-polluted sediments. However, it was not known whether these were individuals which had acquired tolerance to those particular metals, as with plasmid-mediated metaltolcrancc (Rcanncy. 1976; Chakraharty. 197X) and chromosomal mutation (Drake, 1974), or whether they were members of a group of organisms which were inherently metal-tolerant. Tyler rf ul. (1974) suggested that the nitrifying bacteria might he such a group in that they were possibly more resistant to cadmium than the “general soil microflora”. This was their explanation for the apparent stimulation of nitrifying rates they observed when this metal was added to soil. Such groups of bacteria may he metal-tolerant because of interactions between microbial cell wall components and metal ions which may result in the their detoxitication as may precipitation of metal sulphides by hydrogen sulphide producers. Soil

MATERIALS AND METHODS

The metal-affected soil was taken from Port Kembla, New South Wales, an area which had been exposed to contamination from an industrial complex since 1908 (Beavington. 1973). As far as could be ascertained the soil had remained undisturbed since before the complex was built. Unfortunately, extensive urbanization around the complex precluded the possibility of selecting, close by, an undisturbed site to represent an equivalent control soil. Consequently, the control soil was located in natural bushland 180 km south of Sydney. Duplicate samples of each soil were taken at depths of 4-6 cm and 14-l 6 cm in polycarhonate vials and kept cold until used (O--4 C. overnight). The two samples from each horizon were mixed and duplicate subsamples used in the investigation. The 14-16 cm samples were used only for the metal analysis. Soil pH was measured in soil:water mixtures (I :5 w,Iv). Determinations of soil metal concentrations, soil moisture content, and loss on ignition were performed as described by Duxbury (1981). A tryptone-yeast extract-glucose-salts medium, pH 6.776.8 (T-medium, Duxhury. 1981). with or

*Present address: School of MIcrobiology. University of New South Wales. Kensington. New South Wales 2033. Australia. 243

T. DJXII~JKY

7&I

and

without added metals or antibiotics, was used. When soil material was to be plated, cycloheximide (50,flgml ‘, Actidione, Upjohn, Michigan) was included as an antifungal agent. The metals were supplied as the chlorides, except zinc sulphate, and were used individually in agar plates. All chemicals were of analytical reagent grade. The toxicity of the metals towards the soil bacterial populations was determined by surface-plating dilutions of soil suspension onto T-medium supplemented with dilferent concentrations of each metal (Duxbury. 19gl). Colonies were counted after 5 days at 30 C and a further 9 days at 25°C. Bacteria were isolated from metal-supplemented media which had been surface-inoculated with 0. I ml aliquots of IO-fold dilutions of Port Kembla soil suspension. The metals and concentrations (mM) used were: cadmium (OS), cobalt (O.-S),copper (I .O), mercury (0.1). nickel (1.0), and zinc (1.5). Following incubation for I4 days as above morphologicallydilfercnt colonies were subcultured onto metalsupplemented media and purified by repeated streaking. It was decided to use morphological dissimilarity as a criterion for isolate selection instead of the accepted random approach as it had been shown that. when soil populations were plated onto metalcontaining media, population diversity decreased and colonies of the same morphology were represented several times on the same plate. If a random selection technique had been employed this would have probably resulted in the same organism being reisolated several times thereby introducing bias into the later population analysis. Our approach was to isolate as many different organisms as possible with no intention of indicating the relative abundance of each isolate within the total soil population. Isolates were coded according to the metal contained in their isolation medium and maintained as working stocks on metal-supplemented media at 4°C and as freezedried cultures. To test for multiple metal tolerance a replicaplating technique was used. Each isolate was grown at 30 C to visible turbidity in T-broth. The cultures were diluted in the same medium to a faint opalescence and randomly assigned to welb in a sterile, 32-place antibiotic replicator (H. J. Clemens, North Ryde, New South Wales). Duplicate replica plates were prepared on T-medium supplemented with metals at concentrations of 0.1, 0.5, 1.0, 1.5, 2.0 and 3.0 mM, plus an unsupplemented control. Additional

-Table I.

B.

BECKNELL

concentrations of particular metals were included as required. Presence or absence of growth at 30 C was assessed daily for 7 days. To test for antibiotic resistance, duplicate plates of T-medium were swabbed with a faintly opalescent culture, prepared as above, and allowed to dry for I h. The antibiotics (applied as Oxoid Multodisks) used, together with the disc concentrations and the zone widths which indicated sensitivity, were: chloramphenicol (IO pg, 3 mm), erythromycin (IO /~g, 2 mm), sulphafurazole (IOO~g, 3 mm), penicillin (I.5 units, 2 mm), ampicillin (2 /tg, 3 mm). streptomycin (IO pg. 2 mm). tetracycline (5Opg, 7 mm), cephaloridine (25 pg. 2 mm), nitrofurantoin (200 11.g. 3 mm), kanamycin (30 ,ug, 2 mm), nalidixic acid (30 c(g, 4 mm). The widths of the zones around each disc were measured after a suitable period at 30 C. Motility was ascertained by phase contrast microscopy of wet mounts of young cultures grown on T-medium. The Gram stain was Hucker’s modification.

RESULTS

The two soils used in the investigation were different from one another in several respects (Table I). That from Port Kembla contained higher metal concentrations as well as having much more organic matter and a less acid pH. Although the metal concentrations in the bushland soil were almost identical in each horizon the upper horizon at Port Kembla contained much more metal than the lower one. Soil bacterial populations from each site were tested for metal toxicity and, with the exception of those for mercury, the results are displayed in Fig. I. As Duxbury (1981) had found most of the curves could be described by the equation J: =aexp(-h-r)

where Jl is the number of bacteria able to grow on T-medium in the presence of a metal at a concentration x (mM), and a is the number of bacteria able to grow on unsupplemented T-medium. The values of u for each metal and soil were derived during curvefitting analysis and the variation shown in Table 2 resulted from the differences in the goodness-of-fit for each data set. The actual bacterial counts on unsupplemented T-medium for each soil were for the

Soil characteristics Metal

and metal contents content*

Hushland 5cm

15cm

11.2 0.06 48.2 13.5

10.7 ND 31.4 II.3

4.4 1.5

ND ND

(/tg g

’ dry

soil) Port Kembla 5cm 15cm

Cadmium Cobalt Copper Mercury Nickel _ Zinc PH Loss on ignition (“,,) *Mean

of two detcrmin~tions.

(A)

81.9 0.86 198.8 206.8 6.0 7.4

21.7 ND 54.7 45.7 ND ND

Metal-tolerant

bacterial

populations

from soil

0

0

--\

(bl

QOOI

0.01

I

0.1

0.005

a05

0.5

3 1.5

IO 0.001 5

Cobalt

iB)

0.01

0.1

O.OQ5

I 0.5

0.05

Concentrarton

3 1.5

of metal,

IO O.OOI 5

0.01

0.f

0.005

0.05

I 0.5

3 1.5

IO 5

mM

5

‘i; $ f2

4 0

x -J

3 0.001

0.01 0.005

0.1 0.05

I 0.5

IO O.Ool

3 1.5

Concentration

o.001

0.01 0.005

0.1 0.05

I Q5

of metal,

100.001

3 I.5

O.Of cm_?5

5

5

Concentration

of metal,

0.5

3 t.5

IO 5

mM

0.01 o.cm5

I

0.1 0.05

I

0.1 0.05

0.5

3 I.5

IO 5

mM

Fig. I. Numbers of soil bacteria able 10 grow an T-medium supplemented with individual metals. (-) complete curve; equalicm (A) for Fig. a, b, d and E: equation (B) for Fig. c. f and g. (-----) lower concentration range (equation B, u,/h,curve); (----) extended concentration range (equation B. u:/h, curve). (0 and B) bushl~i~d soil; (0 and PK) Port Kcmbla sod.

T. DUXHUHY and Table 2. Values* for

B. BICKNELL

constants (I, h and r’ (goodness-of-fit), for equations (A) and (B) (a) Lower concentration

Metal

Site

co

B PK

cu

B PK B PK

Hg Ni Zn

B PK B PK

Site

Co Zn

PK B PK

*See text. tB-Bushland;

PK-Port

6.3 9.9 13.1 9.1 5X8.6 432.0 Y.5 5.1 11.7 5.8

2.9 46.8 4.1 51.9 14.3 II.7 3.4 59.2 3.2 60.7

(G-5, 3.1 0.7 20.5

(;;n=MY 0.99 0.98 0.98 0.97 0.80 O.Y3 0.99 0.99 0.99 0.99

2.37 1.79 1.16 I .94 0.03 0.04 1.5Y 3.52 I .28 3.12

h?

rz

(_I‘= I ) (mM)

0.7 0.6 0.x

0.98 0.94 0.98

18.87 19.13 17.72

curve (P2)

Kembla.

bushland soil, 3.1 x 10bg- ’ soil dry wt, and for Port Kembia soil, 6.2 x 10’ g-’ soil dry wt. The factor h is an exponent, also derived during curve-fitting analysis, which is a measure of metal toxicity. This is a combined expression of the inherent toxic qualities of the metal itself and of the metal binding properties of the medium. As Duxbury (1981) had suggested the metals probably fall into broad categories of toxicity i.e. Hg, high toxicity, Cd, intermediate toxicity, and Co, Cu, Ni and Zn, low toxicity. This may be justified because Myhill (1967) reported that even with only 5% error in experimental data two values such as b would only be distinguished if the ratio between them was 4 or greater. However, some data points at the higher metal concentrations deviated from the curve predicted by equation (A). For cobalt (Port Kembla) and zinc (both sites) there was sufficient data available for further analysis. In these cases all the data could be described by the sum of two exponential functions y =cc,exp(--b,x)+a,exp(-b,x)

(PI)

bib,

(b) Extended concentration

MCkll

curve

(B)

The symbols are as in equation (A), but at/b, refer to the curve covering the lower metal concentrations (Pl-curve) and az/bz relate to the extended concentration range (PZ-curve). For each metal the b/b, values (Table 2) were very similar for both soils, as were the &-values in the case of zinc. However, there was a 7 to ZO-fold difference between corresponding h, and b, values. Because of the exponential nature of equations (A) and (B), it was not possibie to obtain a value of zero for y which could be used as a criterion of metal tolerance. Extrapolation of the data for each curve to a value of y = I yielded a metal concentration at which only 1 bacterium g-’ of soil should grow and this was regarded as the limit of tolerance for the most metal-tolerant individual in

each subgroup. For each metal similar concentrations were predicted by equation (A) and for the Pl-curve equation (B) (Table 2), irrespective of the soil from which the bacteria came. Likewise, for the P2-curve the cut-off concentrations for both soils were very similar but in this case were 5--15 times higher than for the PI -subgroups. For population analysis metal-tolerant bacteria had been isolated from plates, containing fixed concentrations of metais, prepared from a Port Kembla soil dilution series. Because more bacteria had been able to grow on those plates containing zinc or cobalt, they had been isolated from a soil dilution which represented a detection limit of 1.1 x IO5 bacteria gg’ soil dry wt. The organisms from plates containing cadmium, copper, mercury or nickel were less numerous and their isolation dilution had a detection limit of 1.1 x IO3 bacteria g-’ soil dry wt. Figure 2 shows a diagrammatic representation of the PI- and P2-curves. It can be seen that the concentration range described by the two curves changes depending upon the proportion of the total population which is sampled. In order to calculate the minimum metal concentration which would distinguish PZ-organisms from PI-organisms the Pl-curve parameters (Table 2) and the above detection limits were substituted into equation (A). Isolates were classed as tolerant to a particular metal if they were abie to grow at the metal concentration so calculated. The concentrations (m.Mf derived for the Zn/Co isolates were: cadmium (0.20~. cobalt (0.62), copper (0.68), mercury (0.01) nickel (I .24), zinc( I. 10); and for the Cd/Cu/Hg/Ni isolates were: cadmium (0.36). cobalt (1.09), copper (I. 18) mercury (0.02) nickel (2.14), zinc (I .90). Because most of these values were different from the metal concentrations contained in the isolation plates the individual or-

Metal-tolerant

\ i PI -tolercnce

h

bacterial populations

llmlt 11.1x 10’)

Detection - - llmlt fI.lxI05)

Deteclton limit (l.lx1031 Concenttution

of metal,

mM

Fig. 3. Diagrammatic rcprcscntalion or the PI- and PZ-population response curves. (---) complctc curve. cqua(lon (B); (-----) lower concentration range (cc,/h, curve): (----) cxtendcd conccntraGon range (uZ/h2 curve).

ganisms were retested against the derived concentrations to ensure that they were sufficiently metaltolerant to be included in the P2-population analysis. Such isolates were also tested for multiple metal tolerance, using the same criteria. as well as for multiple drug resistance. Within the whole group of isolates many organisms showed common resistance patterns and could be arranged into similarity groups (Table 3). Most of the organisms were tolerant to a variety of the metals tested except those picked from mercuryor nickel-supplemented media (predominantly groups G and H respectively) which were tolerant to few metals other than mercury and nickel respectively. Amongst all the isolates zinc-tolerance was most frequently encountered followed by tolerance to copper, cadmium. cobalt. mercury and nickel. In the 34 zinc-tolerant isolates cadmium, cobalt and copper tolerances also occurred in 27, 25 and 29 of the organisms respectively. The relative toxicities of the metals towards the P2-organisms was ascertained by determining the number of isolates able to grow at dilrerent concentrations of metals other than the one contained in their initial isolation medium. These results arc shown in Fig. 3a for the Cd/Cu/Hg/Ni isolation group and in Fig. 3b for the Zn/Co isolation group. Within each group, based upon the decrease in numbers of tolerant individuals with increasing metal concentration. it was found that cobalt was more toxic than zinc and that the relative toxicities of the remaining metals was Hg > Cd > Cu > Ni. Many of the individuals in Fig. 3a showed tolerance to 3 mM cobalt and zinc whereas in Fig. 3b no bacteria were tolerant to cobalt in excess of I.5 mM, although four were capable of growth in the presence of 3 mM zinc. These values were in agreement with the limits of tolerance predicted by the P2-parameters for the two groups of isolates. These metal concentrations were, for the I.1 x IO’ detection limit, 8.5 mM cobalt and 9.2 mM zinc. and for the I .I x IO” dctcction limit, I .6 mM cobalt and 3.6 mM zinc. The antibiotic sensitivity spectra ranged from two organisms in group E, which were resistant to all the compounds tested, to one in group I which was

from soil

T. DUXMJKY and B. BICKNELL

24X

(b)

Tolerant

to

Cd Co Cu Hg Ni Zn

II11119

Concentration of Fig. 3. Frequency

and degree of unselected

metal,

mM

metal tolerances. (a) Cd/Cu/Hg/Ni

resistant to none, but the majority were generally resistant to a range of antibiotics. However, as with the metal tolerances, exceptions to this were those organisms in groups G and H which showed considerably more sensitivity towards the various compounds. None of these organisms was resistant to tetracycline or kanamycin, and only one or two were resistant to chloramphenicol, erythromycin, streptomycin or cephaloridine. Table 3 also shows the Gram-morphology and motility results. The majority of the bacteria were Gram-negative rods, more than half of which were motile. All but one of the remainder were Gramvariable rods in young cultures which changed to Gram-positive cocci during a 7-day incubation. All but one of these bacteria were isolated on nickelsupplemented medium. The isolate in group I was a Gram-positive, spore-forming rod. No true cocci were represented. DISCUSSION

The microflora in the upper parts of the Port Kembla soil had been exposed to greater amounts of metals than were originally present as a result of aerial contamination from a nearby industrial complex (Beavington, 1977). This contamination had occurred slowly over a long period (Ellis and Kanamori, 1977) and it was considered that the soil population would represent one which had gradually changed in its metal-tolerance characteristics in response to rising soil metal concentrations. It was thought that whatever mechanisms of population development had been operating, either selection of phenotypic groups or of individuals carrying plasmids or chromosomal mutations, these should be well represented in a more or less stable condition. By analysing such a population, and comparing it to one which had not experienced changes in soil metal content, it was thought that some insight may be obtained as to the ways in which natural soil bacterial populations respond to environmental changes. In a study of bushland soil Duxbury (1981) de-

isolates. (b) Zn/Co

isolates.

scribed the toxicity of various metals towards soil bacterial populations by a single exponential equation. A second exponential function was not detected because of insufficient data for higher metal concentrations. In our study, although only data for cobalt (Port Kembla) and zinc (both sites) could be analyzed sufficiently, the position of the data points in the upper concentration range for the other metals strongly suggested that the bacterial populations from both soils contained two subgroups, one of which could tolerate metals over a greater range of concentrations than the other. Examination of the bacteria isolated from Port Kembla soil revealed that the majority of the more metal-tolerant organisms were Gram-negative. Terrestrial and aquatic environments contain extremely diverse populations of both Gram-positive and Gram-negative bacteria and selection can take place, in the presence of heavy metals, for resistant organisms. The predominance of Gramnegative bacteria in polluted sediments and water has been reported previously. In the polluted water and sediments of Chesapeake Bay the genus Pseurfomona.s accounted for 66’;/ of all HgC&-resistant bacteria (Nelson and Colwell, 1975). Later, in a taxonomic study of metal-tolerant bacteria from the same location, Austin et al. (1977) reported that the majority of metal-tolerant bacteria were mainly Pseudomonas spp and a large group of Gram-negative organisms tentatively identified as Flauobacterium spp, together with significant numbers of certain Gram-positive genera, in particular Bacillus and some coryneforms. Pseudomonas was also the predominant genus to which Houba and Remacle (1980) assigned metalresistant strains of bacteria in a study of cadmium pollution of three aquatic ecosystems in Belgium. Contrary to this Timoney et al. (1978) found that Bacillus was by far the most common genus observed in all sediments, polluted and natural. However, they suggested that the apparent lack of Gram-negative bacteria in these environments was due to a reduction in the viability of these organisms as a result of exposure to elevated temperatures during pour plate preparation.

Metal-tolerant

bacterial

Most of the Port Kembla isolates could be grouped according to their patterns of multiple metal tolerance and drug resistance. Kelch and Lee (1978) noted that particular groups of bacteria had characteristic patterns of drug resistance, even when isolated from different environments. In reasoning the origin of these patterns they discounted similar exposure to antibiotics as well as the possession of similar chromosomal genes, but postulated a sharing of a pool of r-factors. If plasmids were a major factor in determining the metal tolerance characteristics of a population then the development of Gram-negative organisms might be favoured because of the apparent case with which these extrachromosomal elements can be transferred across broad taxonomic groups, although plasmid transfer in natural soil ecosystems has yet to be confirmed. Even so, populations of such bacteria would be expected to show differences from non-tolerant populations in the relative toxicities of the various metals as plasmid-mediated tolerance is usually specific for one or two individual metals. This was not found to be the case for the PZ-population. However, antibiotic resistance in Gram-negative organisms has also been ascribed to intrinsic factors (Richmond and Curtis, 1974) and to barriers in the cell envelope (3oman PI d., 1974). Indeed the Gramnegative cell envelope has long been regarded as a barrier to many substances in addition to antibiotics (Leive, 1974; Nikaido and Nakae, 1979). In addition, it is well known that metal ions interact with cell walls of both Gram-positive (Marquis et ul., 1976) and Gr~lln-negative (Beveridge and Koval, 1981) bacteria. Heavy metals have a multiplicity of potential target sites of toxic action ranging from the cell wall to cytoplasmic constituents. However, the h-values in equations (A) and (B) take into account not only the efrect of the growth medium but also the kinds of toxicity target involved as well as their susceptibility and accessibility to metal ions. The need for a higher range of metal concentrations to effect an inhibitory response from the P2-population, together with the low h-values. may be due to a reduction in the accessibility of the target sites. Although at this stage we have no conclusive evidence we would suggest that the Gram-neg~~tive cell envelope does indeed present a permeability barrier to metal ions thereby impeding their entry into the cell interior. Tentative evidence for this may be found in a report by Suzuki et al. (1976) who observed that a mutant of Escherichia di. with a structural change in a lipoprotein in its outer membrane, was more susceptible to mercuric coInpounds than the wild type. Although we have suggested that Gram-negative organisms may become predominant in metalpolluted environments due to phenotypic selection, this group of organisms would also be favoured if chromosomal mutation was involved. Although mutation rates may possibly be the same in both Grampositive and Gram-negative organisms (Drake, 1974) the cell envelope of the latter group would be a unique feature of the ccl1 in which mutational changes could markedly alter its selectivity or permeLlhility characteristics. For cxamplc, Lutkenhaus (1077) obtained mutants of I:‘. co/i 13/r lacking a major outer membrane protein (protein b) by selecting for copper resistance. These mutants showed a

populations

from soil

249

decrease in their ability to utilize a variety of metabolites when present in low concentrations and Lutkenhaus suggested that this resulted from a decrease in the perm~dbility of the outer cell membrane. Indeed, he observed that these mutants were still sensitive to high concentrations of CL?’ and suggested that this may have been due to its penetration into the cell via alternative membrane pores. The simple subdivision into Gram-positive and Gram-negative subgroups could be the reason why only two subgroup responses were detected among such diverse bacterial populations as can be found in soils. However, it is reasonable to expect some degree of overlap between these two major divisions as particular groups of bacteria may exhibit tolerance towards specific metals. For example, most of the organisms isolated from nickel-supplemented media showed many characteristics of the coryneform group. Indeed the pronounced morphogenic change from rods to cocci during growth of these organisms would suggest that these bacteria belong to the genus Ar/hrohacter. These organisms showed a common tolerance to nickel but were restricted in their tolerance to other metals and were sensitive to a wide range of antibiotics. However, nickel tolerance would appear not to be a general characteristic of Au/hrohacter because A. nzurinus (ATCC 25374) failed to grow in concentrations in excess of 0.4 mM NiCI? (Cobet rt ul., 1970) although the composition of the growth medium would influence the toxicity of the metal (Ramamoorthy and Kushner, 1975). it is also interesting to note that, in the studies of Nelson and Colwell (1975), Austin et uf. (1977) and Timoney et ul. (1978), the genus Bud/us was very prevalent among Hg-resistant organisms. The organism in group I (Table 3) was tentatively identified as a Bacillus sp. and it too was resistant to mercury. Of course, multiple antibiotic resistance and metal tolerance may be plasmid-borne and, because of the involvement of Gram-negative bacteria. should not be discounted. But we suggest that, at comparatively low levels of metal pollution, Gram-negative soil bacteria may be able to endure this stress without the need for plasmids, although these genetic elements may become more important as environments become more heavily contaminated. /tekno~~led~ements-We financial assistance of Scheme.

gratefully acknowledge the the Australian Research Grants

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250

T. DUXBUKY an d B. BICKNELL

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