Environmental Microbiology (2003)

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(1), 3–12

© 2003 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology1462-2912Blackwell Science, 20035Original Article

Bacterial adap-

tation to nickel in neocaledonian soilsM. Hery et al.

Received 26 July, 2002; revised 30 September, 2002; accepted30 September, 2002. *For correspondence. E-mail navarro@univ-lyon1.fr; Tel. (

+

33) 4 72 43 29 86; Fax (

+

33) 4 72 43 12 23

Adaptation to nickel spiking of bacterial communities in neocaledonian soils

Marina Héry,

1,2

Sylvie Nazaret,

1

Tanguy Jaffré,

3

Philippe Normand

1

and Elisabeth Navarro

1,2

*

1

Ecologie Microbienne, UMR CNRS 5557 and

2

UR IRD 83, Université Lyon I, Batiment Mendel, 43 bd du 11 Novembre 1918, F-69622 Villeurbanne cedex, France.

3

Laboratoire de Microbiologie et de Botanique, IRD Nouméa, New Caledonia.

Summary

Adaptation to nickel of bacterial communities of twoextreme neocaledonian soils (an ultramafic soil andan acidic soil) was investigated by nickel spiking andcompared with adaptation in a non-neocaledoniansoil used as reference. Soil microcosms wereamended with nickel chloride (NiCl

2

), and bacterialcommunity structure was analysed with the riboso-mal intergenic spacer analysis (RISA) technique.Then, bacterial populations that respond to nickelstress were identified by cloning and sequencing. Inthe ultramafic soil, a shift occurred on day zero on theassay profiles and consisted of the emergence of abacterial group closely related to the

Ralstonia/Oxalo-bacter/Burkholderia

group. It is hypothesized thatNiCl

2

had a physico-chemical impact on soil struc-ture. Fourteen days after nickel spiking, another shiftoccurred in the two soils that concerned a bacterialgroup belonging to the Actinomycete group. Only afew changes occurred in the bacterial communitystructure of the neocaledonian soils compared withthose of the reference soil, which is more affected bynickel spiking. These results suggest that neocale-donian soil bacteria are particularly well adapted tonickel.

Introduction

New Caledonia is characterized by the presence of ultra-mafic rocks, including peridotites and serpentines. Theserocks make up one-third of the main island area, whereasthey represent only 1% of the Earth’s surface-emergedlands (Broock, 1984). Ultramafic soils (serpentine soils)derived from these rocks present extreme edaphic condi-

tions. These extreme soils are characterized by deficien-cies in elementary elements (such as nitrogen, carbonand phosphorus) and by a very high level of heavy metals,particularly nickel (250-fold the average level of nickel inother earth soils). Neocaledonian soils are very interest-ing models for the study of bacterial adaptation to nickelbecause it is not a classical type of pollution, as bacteriahave been in contact with nickel for 40 million years.Intensive nickel mining and extraction of nickel ore fromopen-cut mines has provoked destruction of the vegeta-tion and, therefore, these represent a major environmentalproblem in New Caledonia.

Some work has been done on neocaledonian soil bac-teria: it has been shown that nickel levels had an impacton

Frankia

diversity (Navarro

et al

., 1999). Furthermore,some nickel-resistant bacteria have been isolated fromthese soils (Stoppel and Schlegel, 1995). In this study, theapproach is more global, and the whole community isconsidered. There are only a few papers on whole micro-bial communities of these extreme soils. Recently,Mengoni

et al

. (2001) found a high genetic diversity ofbacterial isolates in Italian serpentine soils in spite of theselective environment.

The methodology used to study microbial communities

in situ

has been based mostly on cultivation and isolationtechniques (Torsvik

et al

., 1996; Head

et al

., 1998). How-ever, the biases linked to such methods are known, asonly 1–10% of soil bacteria are culturable (Richaume

et al

., 1993; Torsvik

et al

., 1996). Molecular methods per-mit bypass of the limitations and biases associated withcultivation techniques. The aim of this work was to evalu-ate whether long-term nickel-exposed bacterial communi-ties would be more adapted to nickel stress than ‘newly’perturbed soil. We have focused on two types of soilrepresentative of the range of soil encountered in NewCaledonia. The first is an ultramafic alluvial soil, locallycalled mine soil, because of its very high level of nickel(5 mg g

-

1

soil). The second is an acidic alluvial soil derivedfrom volcano-sedimentary acidic rocks and characterizedby an acidic pH and a lower concentration of nickel thanthe ultramafic soil (0.8 mg g

-

1

). A third soil was used asreference: it is a French silt–loam soil with a normal levelof nickel (0.02 mg g

-

1

). To study bacterial adaptation tonickel, soils were amended with nickel chloride, and twoapproaches were realized in parallel during 1 month:counting of culturable bacteria on plate agar (viable het-erotrophic and nickel resistant) and monitoring of the

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structure of the bacterial communities with the ribosomalintergenic spacer analysis (RISA) technique. RISA is aDNA fingerprint method permitting analysis of the struc-ture of bacterial communities without the need for isolationand cultivation steps (Borneman and Triplett, 1997). RISAinvolves DNA extraction of soil bacteria and polymerasechain reaction (PCR) amplification of the ribosomal inter-genic spacer region with primers targeting the 16S and23S genes. This intergenic spacer displays a significantheterogeneity in both length and nucleotide sequences(Gürtler and Stanisich, 1996; Gürtler, 1999), which isexploited in RISA. Therefore, this fingerprint method pro-vides band profiles that are representative of the geneticstructure of the bacterial community. RISA has been usedsuccessfully for the analysis of bacterial communities fromsoils (Borneman and Triplett, 1997; Ranjard

et al

., 2000),from fresh water (Fisher and Triplett, 1999), from seawater (Acinas

et al

., 1999) or from the rhizosphere treatedwith different antibiotics (Robleto

et al

., 1998).

Results

Bacterial counts (Figs 1 and 2)

Before the addition of NiCl

2

, nickel-resistant bacteria (Ni

R

)were present in the acidic soil and represented an aver-age of 1.24% (

±

0.17) of the total culturable community.NiCl

2

treatment had the same negative effect on the viableheterotrophic (VH) (Fig. 1) and on the culturable Ni

R

bac-teria (Fig. 2). The NiCl

2

treatment did not induce a signif-icant change in the percentage of Ni

R

cfu. In unspiked soil,the number of bacteria remained constant over the 30 daytime course.

In the ultramafic soil, nickel-resistant bacteria initially

represented 19.73% (

±

2.33) of the total culturable com-munity. The addition of NiCl

2

led to a decrease in thenumber of cfu in both VH and Ni

R

bacteria (Figs 1 and 2).Results for the ultramafic soil are similar to thoseobserved for the acidic soil: amendment with NiCl

2

had anegative effect on bacterial growth. Concerning the nickel-resistant cfus, a decrease was observed over the first10 days (3.58%

±

0.09 on day 10) and then an increaseoccurred (8.73%

±

2.01 on day 30), but the initial level wasnever reached (data not shown). In the CSA soil, the effectof NiCl

2

on the number of cfu was stronger than in neo-caledonian soils: for the VH, the decrease in the numberof cfu was about 10-fold immediately after NiCl

2

wasadded (day 0) (Fig. 1). The number of Ni

R

was initially verylow and represented an insignificant percentage of thetotal culturable bacteria. After the addition of NiCl

2

, thisnumber fell under the detection threshold (Fig. 2).

In pH controls, the number of bacteria remained con-stant over the 30 day time course, irrespective of the soilor populations considered (data not shown).

RISA

With the usual extraction method, no DNA could be recov-ered from the NiCl

2

-spiked soils. The use of a KCl washingstep before DNA extraction allowed the recovery of DNA.Extracted and purified DNA was then amplifiable, andPCR reproducibility was high.

Microbial community analysis by manual RISA providesinformation on the genetic structure of these communities.This method permits the underscoring of changes in thestructure of the bacterial community after environmentalperturbations. In acidic soil spiked with NiCl

2

, a shift

Fig. 1.

Viable heterotrophic (VH) bacterial counts after the addition of NiCl

2

(30 mg of Ni g

-

1

soil).A and B. Controls and assays in CSA soil respectively (dashed lines).C and D. Controls and assays in acidic soil respectively (solid lines).E and F. Controls and assays in ultramafic soil respectively (dotted lines).Bar is standard error.

Time (days)

302010010

5

106

107

108

A

C

D

F

B

E

CFU

/g d

ry s

oil

Fig. 2.

Nickel-resistant (Ni

R

) bacterial counts after the addition of NiCl

2

(30 mg of Ni g

-

1

soil).A. Controls in CSA soil.B and C. Controls and assays in acidic soil respectively.D and E. Controls and assays in ultramafic soil respectively.Bar is standard error.

3020100

103

104

105

106

Time (days)

CFU

/g d

ry s

oil

D

E

C

B

A

Bacterial adaptation to nickel in neocaledonian soils

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appeared in the genetic profile at day 14. The majorchange is the emergence of a band at 350 bp in theassays fingerprints (Fig. 3), a band that was barely detect-able at day 10 (data not shown). For the ultramafic soil,an important shift in the microbial community structure ofthe spiked soils appeared immediately after NiCl

2

wasadded on day 0. This emergence of a 700 bp band in theassay profiles was very sudden: this band was present inthe control profiles, but its intensity was very low. On day14, a second change occurred with the emergence of aband at about 350 bp (Fig. 4). For the two neocaledoniansoils, no other change was noted after day 14. In CSA soilspiked with NiCl

2

, the fingerprints of day 0 and day 30 arevery different (Fig. 5). In soil controls and pH controls, theprofiles did not change over the course of the experiment.The reproducibility of the method is confirmed by thetriplicate profiles that were identical for all soil communi-ties studied.

Visual observations on the gel were confirmed by auto-mated RISA (A-RISA) electrophoregram analysis. Forexample, for the ultramafic soil, the 700 bp band observedin the gel was represented on the electrophoregram by ahigher peak for the assay on day 0 (Fig. 6). Electrophore-grams show complex profiles corresponding to the com-plexity of the soil bacterial communities. The assay and

control profiles were superimposed, showing that the onlyimportant change in the community of the ultramafic soilon day 0 was the initial shift that occurred just after nickelwas added.

Identification

For the 350 bp band from both acidic and ultramafic soil,13 clones were sequenced and analysed by the

BLASTN

program. It is interesting to note that, in the two soils,bacterial groups that respond to nickel stress are related,and many are in the Actinomycetal group (Table 1).

In contrast to the 350 bp band, phylogeny for the 700 bpband from ultramafic soil was done from intergenic spacersequences (tRNA isoleucine and tRNA alanine).Sequences of IGS from

b

-Proteobacteria are availablefrom the databases, and the presence of tRNA permittedinference of the phylogeny, in contrast to IGS from theActinomycetal group. The eight uncultured clonessequenced formed a cluster, which corresponds to a bac-terial group closely related to the

Burkholderia

/

Oxalo-bacter

/

Ralstonia

group in the

b

-Proteobacteria (Fig. 7).Some clones were sequenced on only one strand, but

a phylogenetic tree has been constructed using only thosewith the double strand sequenced.

Fig. 3.

RISA from acid soil spiked with NiCl

2

. The intergenic spacer region was PCR amplified and resolved on a non-denaturating 5% polyacrylamide gel. Lane 1, control day 0; lanes 2 and 3, assays day 0; lanes 4 and 5, assays day 14; lanes 6 and 7, assays day 30; lane 8, control day 30. M, molecular weight marker (100 bp).

Fig. 4.

RISA from ultramafic soil spiked with NiCl

2

. The intergenic spacer region was PCR amplified and resolved on a non-denaturating 5% polyacrylamide gel. Lane 1, control day 0; lane 2, control day 30; lanes 3 and 4, assays day 0; lanes 5 and 6, assays day 14; lanes 7 and 8, assays day 30. M, molecular weight marker (100 bp).

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Cloning and sequencing of the IGS of the isolatedstrains were successful for only three and two strains fromultramafic and acidic soils respectively. Positioning of theisolates from ultramafic and acidic soils permitted us toplace these cultivable bacteria in the Actinomycetal group.

Discussion

Heavy metals are known to be toxic at high concentrationsfor all microorganisms. Heavy metals in natural environ-ments cause disturbances in microbial communities withnegative effects on total microbial number and biomass(Chander and Brookes, 1991), on enzyme activity(Ohtonen

et al

., 1994) and on microbial diversity (Del Val

et al

., 1999; Sandaa

et al

., 1999; Speir

et al

., 1999), andcould result in changes in the microbial community struc-ture (Pennanen, 2001). The case of the neocaledoniansoils is of interest because of the presence of heavymetals, and particularly nickel, for 40 million years(Eocene period), and so it can be expected that the impactof nickel on the bacterial communities will be different fromthat of recent pollution treated in most studies. Bacteriahave been in contact with nickel for several million yearsand so are probably well adapted. In contrast, bacterialcommunities in CSA soil, which have never been exposedto high concentrations of nickel, might be more perturbedby nickel addition.

With the usual extraction method, DNA could not beextracted from the NiCl

2

-spiked soil. Some divalent metalcations, such as Ni

2

+

, can interact with DNA molecules toform a complex, the structure of which is likely to be amodified B-type helix. Ethidium bromide cannot bind to

Fig. 5.

RISA from CSA soil spiked with NiCl

2

. The intergenic spacer region was PCR amplified and resolved on a non-denaturating 5% polyacrylamide gel. Lanes 1 and 2, controls day 0; lanes 3 and 4, assays day 0; lanes 5 and 6, assays day 30; lanes 7 and 8, controls day 30. M, molecular weight marker (20 bp).

M 1 2 3 4 5 6 7 8

Fig. 6.

Electrophoregram of A-RISA profiles of the bacterial communities in the ultramafic soil on day 0.

Bacterial adaptation to nickel in neocaledonian soils

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Table 1.

Blast results of clones derived from the 350 bp band.

CloneAccession no. Closest uncultured relative %

Accessionno. Closest cultured relative %

Accessionno.

CIU350-2 AJ417004 Actinobacterium yo11 87 AF268444

Frankia

sp. 84 AJ404870CIU350-4 AJ417005 Actinobacterium yo11 88 AF268444

Frankia

sp. 86 AJ404870CIU350-8 AJ417006

Actinomadura spadix

92 AF163142CIU350-9 AJ417002 Actinobacterium yo11 88 AF268444

Methylobacterium organophilumStreptosporangium longisporum

8584

AF338181AF192143

CIU350-10 AJ417003 Actinobacterium yo11 90 AF268444

Frankia

sp. 87 AJ404870CIU350-59 AJ417011 Actinobacterium yo9 90 AF268442

Streptosporangium longisporum

88 AF192143CIU350-60 AJ416969 Actinobacterium yo11 91 AF268444

Thermobispora bispora

92 U83912CIU350-65 AJ417001 Actinobacterium yo11 92 AF268444

Streptosporangium longisporum

86 AF192143CIU350-67 AJ417000 Actinobacterium yo11 89 AF268444

Streptosporangium fragile

87 AF116233CIU350-70 AJ417007 Actinobacterium MB11C06 85 AY033302

Frankia

sp. 83 AJ404870UM350-3 AJ417008

Bradyrhizobium japonicumFrankia

sp.9188

AF338850AJ404869

UM350-10 AJ417012 Fibrobacter/Acidobacteria MB13C05 86 AY033318

Bradyrhizobium japonicumFrankia

sp.8584

AF293382AJ404869

UM350-12 AJ417009

Frankia

sp. 87 AJ404870UM350-16 AJ417010 Actinobacterium yo11 87 AF268444

Methylobacterium organophilum

85 AF338181CIU-isolate 1

Streptomyces coelicolor

92 AL109848CIU-isolate 14

Streptomyces ambofaciens

97 M27245CIU-isolate 18

Streptomyces ambofaciens

98 M27245CIU-isolate 3

Streptomyces coelicolor

92 AL109848UM-isolate 2

Streptomyces rimosus

96 X62884UM-isolate 8

Streptomyces rimosus

95 X62884UM-isolate 21

Streptomyces rimosus

98 X62884UM-isolate 23

Streptomyces rimosus

98 X62884

Pstutzeri-AJ390590

Tcuprinus-U51430

Neuropaea-AJ005551

Nmeningitidis-L31411

Xampelinus-U76357

Nmultiformis-AJ005558

Rpickettii-L28163

Rsolanacearum-AJ277856

Rsolanacearum-D87086

Rpaucula-AF237657

Reutropha-AJ416498

Bmallei-L28158

Bcepacia-L28152

Bcepacia-D87086

UM700-48-AJ416490

UM700-15-AJ416491

UM700-18-AJ416493

UM700-21-AJ416496

UM700-37-AJ416494

UM700-43-AJ416495

UM700-25-AJ416492

UM700-24 -AJ4164970.05

100

100

100

100

100

100

100

89

98

92

98

95

Uncultured clones

Burkholderia Oxalobacter Ralstonia group

Ammonia-oxidizing bacteria group

Comamodaceae

Ammonia-oxidizing bacteria group

Pseudomonas

Neisseriaceae

Comamodaceae

Fig. 7.

Phylogenetic tree of uncultured clones with related bacteria based on the IGS sequences. UM are uncultured clones from RISA profiles of ultramafic soil spiked with nickel on day 0.

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such a complex (Lee

et al

., 1993; Aich

et al

., 1999) andso, even if DNA was extracted, it might not be detectableon a gel. To overcome this problem, NiCl

2

had to beremoved from the soil before DNA extraction. KCl is aneffective extractant for divalent metal cations at soil pH(Becquer

et al

., 1995). Washing of the soil with KCl beforeDNA extraction permitted the removal of the NiCl

2

fromthe soil and allowed the recovery of amplifiable DNA, andsubsequent PCR reproducibility was high.

Considering the culturable community, surprisingresults were obtained; in fact, NiCl

2

had a negative effecton bacterial growth, irrespective of the soil or populationsconsidered. These decreases were not expected, partic-ularly for the ultramafic soil, which is very rich in nickel, ifwe consider the average level of nickel in normal soils(20 mg of nickel kg-1 soil; McGrath, 1995). A chemicalstress could explain these results. Mobility, availabilityand, therefore, toxicity of a metal ion in soil are determinedby its chemical form and also by the soil properties (Allo-way, 1995; Davison et al., 1995). The addition of a highconcentration of NiCl2 in only one go could lead to a highconcentration of available nickel at the beginning of theexperiment and could explain the bacterial deathobserved. Moreover, addition of NiCl2 in soil microcosmsled to a pH decrease of about 2 units. Acidification con-secutive to amendment might be responsible for theeffects observed on the number of culturable bacteria(Knight et al., 1997; Pennanen et al., 1998; Kelly et al.,1999; Speir et al., 1999). In order to verify the impact ofthis acidification on culturable bacterial communities, achloride acid spiking (2 units pH decrease) was per-formed. No significant impact was observed on bacterialcounts, confirming that the decreases observed afternickel spiking appear to result from nickel toxicity.

Nevertheless, bacterial counts allowed us to study onlya small part of the bacterial community. To evaluate nickelimpact on the whole community, a molecular approachsuch as RISA is more suitable.

On day 0, a 700 bp band emerged in the assay RISAfingerprints from the ultramafic soil. If we consider visualobservations on the gel, the 700 bp band appears to bedominant in the assay profiles, with a very high intensity,suggesting that this bacterial population is a dominant onein the total community.

This change occurs immediately after NiCl2 addition,and no more than 5 min had passed between the addi-tion of NiCl2 and DNA extraction. Bacterial populationsneed more time for growth and to become the majority ina bacterial community. Given the delay involved, a bio-logical explanation was considered less likely than achemical one. The hypothesis that can be proposed isthat NiCl2 has an impact on soil aggregation, changingthe soil microstructure. This change in soil structurecould lead to the liberation of a bacterial population,

which then becomes accessible to DNA extraction. Theimpact of acidification on RISA profiles was monitored inHCl-spiked microcosms; as for bacterial counts, no sig-nificant impact was observed on RISA profiles (data notshown).

The 350 bp band that emerged in both acidic and ultra-mafic soils on day 14 corresponds to adapted bacterialpopulations, the development of which was favoured whennickel levels in the soil were high. In the two soils, theshifts occurred at the same time (day 14) and concernedthe same bacterial group. In 14 days, bacterial popula-tions responded to nickel stress in the two soils andemerged in the total community. These populationsbelong to the Actinomycetal group. Actinomycetes areessential actors in fundamental ecological processes inthe soil, such as biochemical cycles, and their activity iscrucial for soil fertility (Goodfellow and Williams, 1983).The interference of heavy metals in such processes cangreatly affect biosphere quality, and so the developmentof efficient adaptation strategies is primordial for thesebacteria. Adaptation of Actinomycetes to heavy metalshas not been studied much compared with other bacteria,but some research has shown that Actinomycetes canhave a large adaptation capacity in polluted environments(Golab et al., 1990; Margesin and Schinner, 1996; San-daa et al., 1999). Moreover, some nickel-resistant bacteriacharacterized from anthropogenically or naturally nickel-polluted soils have been identified as Arthrobacter orStreptomyces (Stoppel and Schlegel, 1995; Mengoniet al., 2001).

Bacterial community structure of CSA soil is very dis-turbed by nickel addition, with several bands that appearor disappear. In view of these results, we postulate thatneocaledonian soil response to nickel amendment is spe-cific to adapted soil bacterial communities.

In order to identify culturable resistant bacteria growingon plate agar with nickel added on day 14, we have clonedand sequenced their intergenic region. The results sug-gest that culturable resistant bacteria from both acidic andultramafic soils are Actinomycetes closely related to theStreptomyces genus. So, uncultured bacteria thatrespond to nickel stress in the neocaledonian soils are notthe same as the bacteria that grow on plate agar. Thissuggests that the adapted population corresponding tothe shift in assay profiles of day 14 is not culturable withthe conditions used.

For the 700 bp band, phylogeny was based on inter-genic spacer sequences. tRNA present in these IGS isvery conserved and suitable for inference of phylogeny.For the 350 bp band, phylogeny was not possible,because IGS of Actinomycetes do not contain tRNAsequences and so, results are illustrated by BLAST resultsin Table 1. The major limitation of RISA is the smallamount of information available in the database concern-

Bacterial adaptation to nickel in neocaledonian soils 9

© 2003 Blackwell Publishing Ltd, Environmental Microbiology, 5, 3–12

ing intergenic sequences. Phylogeny based on 16S couldbe more informative but, in this study, we just wanted toposition uncultured clones in relation to principal bacterialgroups rather than infer precise phylogeny. However,fingerprint methods such as RISA are valuable tools forcharacterizing complex bacterial communities anddetecting variations after environmental stress. They areless time-consuming and labour intensive than strategiessuch as small-subunit rRNA gene clone library construc-tion (Ranjard et al., 2000) or techniques based on 16S,which have also their limitations [e.g. the lack of selectiv-ity of the denaturing gradient gel electrophoresis (DGGE)as a result of the GC clamp]. Shifts observed in thegenetic profiles correspond to bacterial populations thatrespond to the stress, and these populations can beidentified by excising, cloning and sequencing the shiftedRISA bands. The RISA technique permits us to bring tothe fore populations adapted to nickel in the two neocale-donian soils studied and to identify them as Actino-mycetes. No other change could be observed, and itseems that there is no important change in microbialstructure, contrary to what has been observed by Smitet al. (1997), Del Val et al. (1999) and in this study for theCSA soil. Microbial communities in polluted soils havebeen studied via the application of various molecularmethods (for a review, see Kozdroj and Van Elsas, 2001).Pollution by heavy metals or organic compounds andtheir consequences on microbial communities generallystudied is mostly caused by industrial practices. The con-tamination by nickel in neocaledonian soils is an atypicalkind of pollution that is natural, existing for millions ofyears and resulting from the geological properties of theultramafic rocks, and so it may explain the fact that micro-bial communities are not disturbed in the same way asmicrobial communities in normal soils. New Caledonia isthus an original model for studying bacterial adaptation tonickel.

We can compare the amendment with nickel with whatoccurs in the open-cut mine where nickel ore is extracted.Mine soils are richer in nickel than ultramafic soils andpoorer in elementary elements. In such soils, only a fewplants other than the endemic Gymnostoma can grow. Abetter understanding of the interactions between microor-ganisms and soil and the identification of adapted bacteriawill permit us to improve the revegetation conditions. Thepresence of a plant in a soil influences the microbialdiversity and soil fertility, and so it is interesting to knowwhat is the plant impact on the microbial communities ofthe soil where it is planted. After the interactions betweenthe microorganisms and the soil, our future works willfocus on the interactions between plants and microorgan-isms. Moreover, neocaledonian soils could provide newstrains and new genetic determinants for nickel resis-tance; further characterization of isolated resistant strains

will be of interest and could be exploited in revegetationor bioremediation practices.

Experimental procedures

Soils and microcosm set-up

Ultramafic soil. The alluvial soil used, which derived fromultramafic rocks, is characterized by a very high level of nickel(5 mg g-1 soil). Only the A horizon (0–10 cm) was used. Itwas taken under Gymnostoma leucodon, Rivière desPirogues, New Caledonia.

Acidic soil. The acidic alluvial soil used derived from acidicrocks and is characterized by an acidic pH (6.1) and a nickelconcentration of 0.8 mg g-1. Only the A horizon (0–10 cm)was used. It was taken under Gymnostoma nodiflorum, Cas-cade de Ciu, New Caledonia.

Reference soil. The soil used was collected from a culti-vated silt–loam soil planted with corn at La Côte Saint André(CSA, France). It was chosen as reference because it had anormal level of nickel. Soil samples were collected from theupper layer (0–20 cm).

Soil characteristics are listed in Table 2. The soils weresieved (2 mm) and stored at room temperature.

Microcosms were set up by placing 10 g (dry weight) ofsoil into 125 ml plasma flasks (Verre Equipements) sealedwith a plastic stopper. In the assays, 2 ml of a NiCl2 solutionwas added to obtain a final concentration of 30 mg (0.5 mmol)of Ni g-1 soil (i.e. 120 mg of NiCl2 g-1). In controls, 2 ml ofultrapure water was added in place of the NiCl2 solution. Inorder to test the effect of pH on bacterial community structure(pH controls), concentrated HCl was added to decrease soilpH to the same level as that obtained by adding NiCl2. Soilmicrocosms were kept at room temperature for 30 days.Samples were taken over a period of 30 days (day 0, 3, 6,10, 14, 20 and 30). Triplicate microcosms were used for thedifferent analyses.

Bacterial counts and strains isolation

Soil (5 g) was blended with 45 ml of a 0.9% (w/v) sterile NaClsolution for 90 s in a Waring blender (Eberbach Corporation).Then, the soil suspension was serially diluted 10-fold in thesame NaCl solution. To evaluate nickel-resistant and total

Table 2. Structural and physico-chemical soil characteristics.

Acidicsoil

Ultramaficsoil

CSAsoil

Clay (%) 26.1 31.7 17Sand (%) 48.3 46.4 47.7Loam (%) 18.8 18.3 35.3Organic matter (%) 2.8 1.8 2CECa (mEq kg-1) 129 48 64pH (H2O) 6 7 7Initial nickel content (mg kg-1) 800 5100 20

a. Cation exchange capacity.

10 M. Héry et al.

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bacteria, 100 ml of appropriate dilutions was spread on nutri-tive broth containing 37.5% bacto beef extract and 62.5%bacto peptone (Difco) supplemented or not with 15 mM Ni(i.e. 2 g l-1 NiCl2). Three plates were inoculated per dilution.Bacterial colonies were counted after 8 days of incubation at28∞C.

Strains from colonies growing on plate agar supplementedwith NiCl2 were isolated on day 14. Isolated strains werechosen for their morphological characteristics; one colonycorresponding to one morphological type was isolated fromthe two neocaledonian soils (four different colony types persoil).

DNA extraction

Before DNA extraction, soils were washed to remove NiCl2.Three grams of soil was shaken in 30 ml of 1 M KCl for 1 hat room temperature. Soil templates were centrifuged for45 min at 6000 g, and the supernatants were discarded.Samples were dried at 35∞C overnight before DNA extrac-tion.

DNA was extracted from soil, purified and quantifiedaccording to a modification of the protocol initially describedby Ranjard et al. (1998). Two grams (equivalent dry weight)of soil was ground in liquid nitrogen (-172∞C) with a freezermill (Speek Industries) for 1 min at 50% of maximum power.The powdered soil was mixed with 3 ml of TES buffer(100 mM Tris-HCl, pH 8.0, 100 mM EDTA, pH 8.0, 100 mMNa2HPO4, pH 8.0, 1.5 M NaCl), 30 mg of lysozyme (Sigma)and 20 ml of proteinase K (10 mg ml-1; Boehringer Man-nheim) by shaking at 200 r.p.m. for 2 h at 37∞C. Hexadecylt-rimethylammonium bromide (CTAB; 30 mg; Sigma) wasadded, and the samples were incubated for 2 h at 65∞C.Samples were centrifuged at 6500 g for 30 min at 20∞C, andthe supernatants were collected in 12 ml centrifuge tubes.For DNA precipitation, isopropanol was added (v/v), and thesamples were kept at room temperature for 30 min. Thecrude DNA pellet was obtained by centrifugation at 6500 gfor 30 min at 20∞C. It was washed with cold 70% ethanol andsuspended in 200 ml of ultrapure water.

DNA was extracted from isolated strains by hot–coldcycles. Strains were resuspended in 100 ml of ultrapure ster-ile water, and the tubes were put in liquid nitrogen for 1 minand for 5 min at 65∞C, repeated three times. The tubes werethen centrifuged for 15 min at 10 000 r.p.m., and supernatantwas used for PCR.

PCR

The intergenic spacer region between the large and smallsubunit of ribosomal sequences was amplified by PCR using50 ng of purified template DNA. The universal bacterialprimers used were FGPS1490-72 and FGPL132-38(Normand et al., 1996). Amplification reactions were per-formed in a final volume of 50 ml containing 5 ml of 10¥ dilu-tion buffer, 200 mM each dNTP, 0.5 mM each primer, 1 mg ofT4 gene 32 protein and 2 units of Expand™ high fidelity Taqpolymerase (Boehringer Mannheim). Amplification was per-formed in a thermocycler (Applied Biosystems) as follows:hot start at 94∞C for 3 min, followed by 25 cycles consistingof 94∞C for 1 min, annealing at 55∞C for 30 s and elongation

at 72∞C for 1 min. A final elongation step at 72∞C for 5 minpreceded cooling at 4∞C. PCR was verified on a 2% (w/v)agarose gel.

RISA

PCR products were loaded on a 5% (w/v) non-denaturingacrylamide gel containing (for 50 ml): 8.35 ml of bis-acryla-mide 37.5:1 (Bio-Rad); 10 ml of 5¥ TBE (Life Biotechnolo-gies); 31.65 ml of distilled water; 17.5 ml of temed (Bio-Rad);and 350 ml of 10% (w/v) persulphate (Bio-Rad). The volumeof PCR product loaded on the gel was calibrated to have asimilar intensity for all profiles. Separation by electrophoresiswas performed at constant temperature (18∞C) for 15 h at50 V. Gels were stained with SYBR green 1 (FMC Bioprod-ucts) according to the manufacturer’s instructions. The band-ing patterns were then photographed using Ilford FP4 filmand a 302 nm UV source.

Automated RISA (A-RISA) was performed according to themethod described by Ranjard et al. (2001).

Cloning and sequencing

The emerging bands detected in the RISA profiles of theNiCl2-spiked neocaledonian soils and the amplified intergenicspacer (IGS) of isolated strains were cloned and sequencedas described below.

PCR products were loaded on a 1.5% (w/v) Nusieve gel(FMC Bioproducts), and the fragments of interest wereextracted using a QIAquick gel extraction kit (Qiagen)according to the manufacturer’s instructions. DNA wasrecovered in 60 ml of elution buffer, concentrated by isopro-panol (v/v) precipitation and recovered in 20 ml of ultrapurewater. The fragments were cloned in the pUC19 cloning(Yanisch-Perron et al., 1985) vector using the Sure Cloneligation kit (Amersham Pharmacia Biotech) according to themanufacturer’s instructions. Transformations into Escheri-chia coli DH5a competent cells (Life Biotechnologies) werecarried out according to the manufacturer’s instructions.Transformed cells were grown in Luria–Bertani medium(Sambrook et al., 1989) supplemented with 100 mg l-1 ampi-cillin, 60 mg l-1 Xgal and 40 mg l-1 IPTG at 37∞C for 24 h.Positive clones were screened for complementation. Plas-mid DNA was isolated from positive clones using aQIAprep Spin miniprep kit (Qiagen) according to the manu-facturer’s instructions. Purified plasmids were digested byEcoRI and HindIII (Life Biotechnologies) to verify the pres-ence of the insert. Digestion products (5 ml) were loaded on1% (w/v) agarose gel to evaluate the insert size. DNA wasquantified by comparison with known quantities of standardcalf thymus DNA (Boehringer Mannheim). The sequencingreaction was performed with 300 ng of DNA, 10 pmol ofprimer M13r or M13f and a DYEnamic™ ET terminatorcycle sequencing kit (Amersham Pharmacia Biotech) for 50cycles consisting of 20 s at 95∞C and 1 min 15 s at 60∞C.PCR products were then purified using Sephadex G50 gel(Amersham Pharmacia Biotech). Sequencing was per-formed with a MegaBACE 1000 (Amersham PharmaciaBiotech) capillary sequencer, and results were analysedwith the SEQUENCE ANALYSER 2.1 software (AmershamPharmacia Biotech).

Bacterial adaptation to nickel in neocaledonian soils 11

© 2003 Blackwell Publishing Ltd, Environmental Microbiology, 5, 3–12

Sequence analysis and phylogenetic affiliation

Sequences obtained from clones were compared with data-bases available at the National Center for BiotechnologyInformation (NCBI: http://www.ncbi.nlm.nih.gov) using theBLASTN program (Altschul et al., 1990) for an initial phyloge-netic placement. Afterwards, sequences from clones corre-sponding to the 700 bp band were aligned with otherphylogenetically related sequences available at the NCBIusing SEQPUP (D. Gilbert, SeqPup@bio.indiana.edu). A phy-logenetic tree was constructed using the neighbour-joiningmethod (Saitou and Nei, 1987) as implemented in CLUSTAL

W (Thompson et al., 1994) and visualized using NJPLOT (Per-riere and Gouy, 1996). For the 350 bp band, only BLAST

results were considered.

Acknowledgements

We thank F. Gourbière for help with statistics, and T. Vogeland L. Jocteur-Monzorier (UCB, Lyon, France) for helpfuldiscussions. Thanks to Yohan Ambraisse and BenoitRemenant (UCB, Lyon, France) for technical assistance. Thiswork was supported by grants from the Ministère de l’Ecolo-gie et du développement durable.

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insert Figure 1 near hereinsert Figure 2 near hereinsert Figure 3 near hereinsert Figure 4 near hereinsert Figure 5 near hereinsert Figure 6 near hereplease insert Table 1 near here.insert Figure 7 near hereinsert Table 2 near herethe figures are availableas TIFF files on the disk. Please use if possible. If not, please scan hard copies.navarro@univ-lyon1.fr