Improvement in the RFLP Procedure for Studying the Diversity of NifH Genes

7/27/2019 Improvement in the RFLP Procedure for Studying the Diversity of NifH Genes

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Res. Microbiol. 152 (2001) 95103 2001 ditions scientifiques et mdicales Elsevier SAS. All rights reservedS0923-2508(00)01172-4/FLA

Improvement in the RFLP procedure for studying the diversity ofnifHgenesin communities of nitrogen fixers in soil

Franck Poly, Lucile Jocteur Monrozier, Ren Bally

Laboratoire dcologie microbienne, UMR-CNRS 5557, UCB Lyon 1, 43, boulevard du 11 novembre 1918,69622 Villeurbanne cedex, France

Received 3 April 2000; accepted 8 August 2000

Abstract Several specific primers for the nifH gene were tested with different pure telluric N2-fixing strains.A PolF/PolR primer set provided successful amplification of 19 representative N2-fixing strains. Three restrictionenzymes, HaeIII, NdeII and MnlI, chosen for restriction fragment length polymorphism (RFLP) analyses, were the mostdiscriminating for the study of nifH gene diversity as they resulted in differences between strains at the species level.Amplification by selected primers and RFLP were applied to assess the genetic diversity of the nifH gene pool in soil.Pair soils, one under cultivation, the second under permanent pasture, were found to harbor a contrasting diversity ofnifHgenes. Pure strain profiles could not be recognized in the nifH soil patterns. Using the simple procedure described, it wasshown that the structure of nitrogen fixers in soil was influenced by soil functioning. 2001 ditions scientifiques etmdicales Elsevier SAS

PCR-RFLP /nifH / gene / soil / functional genetic diversity

1. Introduction

Nitrogen fixation is a process that enables re-duction of the atmospheric nitrogen N2 in ammo-nium (NH+4 ) by nitrogenase, a universal enzyme.This process introduces nitrogen into the biosphere.The natural fixing process is responsible for 65%

of annual fixation, while industrial processes repre-sent only 25% [16]. Natural fixation is accomplishedby fixing microorganisms only; these microorganismsbelong to the Archaea and bacteria. Among the bacte-ria, the ability to fix N2 has been shown in organismswith various metabolisms such as anaerobes and aer-obes, cyanobacteria and actinomycetes [28]. Such di-versity in physiology and ecology renders impossiblethe use of a universal selective culture medium for allnitrogen-fixing microorganisms.

These various microorganisms share the same ope-ron in which the nifH gene encodes for the Fe protein

subunit (=

nitrogenase reductase) of the nitrogenase.It has been shown by Young [28] that many featuresof a nifH-based phylogenic tree are entirely consistent

Correspondence and reprints.E-mail address: [email protected] (F. Poly).

with the 16S rRNA-based phylogeny of the nitrogen-fixing bacteria. Therefore, the diversity of the nifHgene permits bulk representation of the taxonomicdiversity of fixing bacteria [24], and can be used tostudy the diversity of the bacterial community thatcan fix nitrogen.

Through molecular biology techniques, the diver-

sity of nitrogen fixers in a complex environment suchas the soil can be estimated, avoiding the bias of cul-turing the organisms on synthetic media, a proce-dure known to select a low percentage of soil pop-ulations [26].

No recent work has been reported on the differentnifH primers, their specificity on pure cultures andtheir use for describing the diversity of nitrogen fix-ers in soils. However, Chelius and Lepo [8] report onapproaching nifH diversity of the plant rhizospherecommunity, using primers defined for cyanobacteria.

The objective of our study was to test several primerson different telluric bacterial genera well known tofix nitrogen (positive test) or on microorganisms thatfail to fix nitrogen (negative test). We suggest a sim-ple protocol for polymerase chain reaction-restrictionfragment length polymorphism (PCR-RFLP), limit-ing the number of cycles to minimize the possible bias


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96 F. Poly et al. / Res. Microbiol. 152 (2001) 95103

of PCR [25], so as to estimate the diversity of nitro-gen fixers through the diversity ofnifH genes.

2. Materials and methods

2.1. PCR primers

Primers were chosen taking into account threeparameters considered to be essential for the studyof diversity by RFLP: i) nifH amplification on thewidest range of N2-fixing species, ii) specificity ofamplification; and iii) an amplification size largeenough so as to obtain restriction fragment profilesrepresentative of the nitrogen fixer diversity.

Few regions of the nifH gene have been con-served among species of nitrogen-fixing bacteria(data not shown). This explains why, in previousstudies [13, 16, 18, 21, 23], all primers used were

defined from only three main regions. The for-ward primers were chosen between nucleotides 19and 47 (referring to Azotobacter vinelandii acces-sion number M20568 [13]) or between nucleotides112 and 134, with the corresponding amino acid se-quences being A(I/F)YGKGGIGK and GCDPKADS,respectively. The reverse primers were defined be-tween nucleotides 457 and 494 corresponding to theSGEMMA(M/L/T)YAANNI amino acid sequence.All primers had a similar sequence, with change inthe number and type of degeneracies only (table I).

First, we tried to amplify the selected nitrogen-

fixing strains Azospirillum brasilense strain Sp7,Azospirillum lipoferum strain CRT1 [10], Rhizobiumleguminosarum ATCC strain 14480, Sinorhizobiummeliloti ATCC strain 9930 and Frankia alni strainARI3 [7]) with already published primers (table I).These primers were modified (table I) to increasetheir specificity by reducing the degeneracy numberbut maintaining successful amplification over thewidest range of bacterial genera of the cluster I branchofnifH phylogeny [6].

PCR amplifications from pure strains (1 L ofcells in glycerol/water 40% or 50 ng of DNA) were

performed in a total volume of 50 L. The finalconcentrations were 0.5 M of each selected primer,and 4 M for highly degenerate primers defined byZehr and Mac Reynolds [29] (Zf and Zr) (table I) and469/470 (this study). Other reagents were 200 mMof each dNTP, 2 U of Expand High Fidelity DNApolymerase (Boehringer Mannheim), 1 PCR buffer

as specified by the manufacturer and 1 g T4 gene 32protein (Boehringer Mannheim) [15].

PCR conditions consisted of 30 cycles at 94C(1 min), 1 min for the annealing step (temperature ispresented in table II) and 72C (2 min), with a 5-minextension at 72C for the last cycle. All amplificationswere performed in a GeneAmp PCR System 2400

thermocycler (Perkin-Elmer, Cetus). Amplificationproducts were submitted to electrophoresis in a 2%agarose gel and stained with ethidium bromide.

Results of PCR amplification experiments areshown in table II. Depending on the primer combi-nations used, we obtained either: i) no PCR product;ii) no expected PCR product sizes; iii) expected prod-uct sizes only; or iv) expected product size with addi-tional unexpected bands. Amplification was consid-ered successful when only one PCR product with thecorrect size was detected. This was obtained with thePolF/PolR primer set (table I) successfully on DNA

from all five tested strains.To evaluate primer specificity and universality,this preliminary experiment was complemented withother reference nitrogen-fixing strains representativefor diverse bacterial sub-groups (table III) and non-nitrogen-fixing strains: Agrobacterium rhizogenesATCC 11325; Bacillus azotoformans ATCC 29788;Burkholderia cepacia ATCC 17759; Geodermatophi-lus obscurus ATCC 25080; Pseudomonas syringaevar. pisi strain 895A (Horticulture Research Inter-national, Wellesbourne, UK, 1975); Pseudomonasviridiflava ATCC 13222; Streptomyces lividans strain

TK24 [12]; Escherichia coli strain DH5 (Life Bio-technologies, France); and Xanthomonas populi strainSpm9 (ATCC 27642) were also assayed. The PolF/PolR primer set was able to amplify the nifH frag-ment out of the totality (19/19) of the tested fixingstrains. As expected, no amplification was obtainedwith negative control strains.

The selected primers PolF/PolR were tested onbacterial DNA extracted from soil. Soil samples werecollected from the upper layer (020 cm) of pairedsoils, i.e. soils of the same type under permanent pas-ture (LCSA-p), and under maize cultivation (LCSA-

c) in close field situation. One hundred nanograms ofDNA, extracted and purified as described [20], wereused for soil DNA amplification. A single band of360 bp was obtained from soil DNA (data not shown).

The specificity of this band was verified by hy-bridization of the PCR product with a probe for thenifH gene. For this the nifH gene of Azospirillum


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Table I. Sequences of various primers tested in previous work or in this study for nifH gene amplification.

References Name of Sequence Number of Forward primersprimer positiona degeneracies

[24] 19Fb 19 16 GCI WTY TAY GGI AAR GGI GG

[27] For A 19 128 GCI WTI TAY GGN AAR GGN GG

[21] FGPH19b 25 24 TAC GGC AAR GGT GGN ATH G

This study IGK 25 24 TAC GGY AAR GGB GGY ATC GG

[17] IGK 1b 31 384 AAR GGN GGN ATH GG

[27] For B 112 96 GGI TGT GAY CCN AAV GCN GA

This study 469b 112 128 GGN TGY GAY CCN AAR GC

[29] Zf b 115 128 TGY GAY CCN AAR GCN GA

This study Kadinob 115 8 TGY GAY CCI AAR GCI GA

This study PolFb

115 24 TGC GAY CCS AAR GCB GAC TCReverse primers

[21] FGPH273b 279 16 CTC CGG GCC RCC NGA YTC

[24] 407Rb 407 8 AAI CCR CCR CAI ACI ACR TC

This study AQEb 436 2 G ACG ATG TAG ATY TCC TG

[29] Zrb 460 96 ADN GC

This study PolRb 457 8 ATS GC

This study 470b 460 512 TAN ANN GC

This study Eminob 463 4 GCR TAI AII GC

[27] Rev 463 48 GCR TAI ABN GC

[17] YAA 478 256 YAA ATR TTR TTN GCN GCR TAa

Sequence position with reference to the A. vinelandii nifH coding sequence (Genbank accession number M20568).b

PrimUnion of Pure and Applied Chemistry Conventions was used to described DNA sequence degeneracies: Y = C/T; S = GH = T/C/A; N = A/G/C/T; W = A/T; I = inosine.


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Table II. Results of nifH amplifications with different primer sets using five reference strains of nitrogen-fixing bacteria.

Positive controls Final Annealing A. brasilense A. lipoferum R. leguminosarum S. meliloti F. alni ARI3 [7]

concen- temperat- ATCC [10] ATCC ATCC 9930

tration ure (C) 29145 14480

Primer sets

IGK1/FGPH273 0.5 M 55 ND +m ND

IGK1/AQE 0.5 M 48 + + w +

IGK1/PolR 0.5 M 55 + ND w +m ND

19F/407R 0.5 M 50 + +m ND ND ND

FGPH19/FGPH273 0.5 M 55 + + +

Kadino/Emino 0.5 M 50 ND ND ND

469/470 0.5 M 45 + ND

469/470 4 M 55 + +m w w

Zf/Zr 0.5 M 57 w +m w

Zf/Zr 4 M 57 +m ND +m ND ND

PolF/AQE 0.5 M 48 + ND +m ND ND

PolF/PolR 0.5 M 55 + + + + +ND, not determined; +, PCR product of the expected size; , no PCR product; w, no expected PCR products, only products of

unexpected and nonspecific size; +m, expected PCR products plus other products of unexpected and nonspecific size.

Table III. Bacterial strains.

Subgroup Organism References

(source)

Proteobacteria A. lipoferum CRT1 [10]

A. brasilense sp. 7 ATCC 29145

A. brasilense L4 [5]

A. brasilense sp. 245 [2]

A. irakense KAC5 [14]

A. irakense KBC1 [14]

A. caulinodans ATCC 43989

S. paucimobilis 5AJ [3]

Rhizobium legumirosarum ATCC 14480

M. loti LMG 6125

S. meliloti ATCC 9930

Proteobacteria A. tolulyticus ATCC 51758

B. vietnamiensis TVV75 [11]

Proteobacteria K. oxytoca 1ABI [3]

E. cloacae 7ATR [4]

Gram + high GC% F. alni ACN14 [9]

F. alni ARI3 [7]

Gram + low GC% P. polymyxa CF43 [1]

P. polymyxa PMD230 [1]

brasilense cloned in pAb1 plasmid, as described [19],was amplified with the primer set PolF/PolR. The

PCR product, 32P-labelled by random priming (Boe-hringer Mannheim kit), was used as the probe. Previ-ous work had already demonstrated probe specificityand reported that it did not hybridize with negativecontrols, chromosomal DNA of non-nitrogen-fixingbacteria and with non-nifH PCR products (data notshown). All PCR products of 360 bp obtained withPolF/PolR from DNA of reference strains or from

DNA of soil bacteria hybridized with the nifH geneprobe (data not shown).

2.2. Enzymes for nifHRFLP

To use PCR-RFLP analysis on nifH pool genesfrom nitrogen-fixing populations as a tool to study thecommunity structure, it was necessary to choose en-donucleases providing fragments in the range of 20360 bp (fragment sizes smaller than 20 bp are difficultto detect). For adequate separation, the resulting frag-ments must be distributed throughout the size range.Enzymes that generated the same size band from var-

ious nitrogen fixing species must be rejected as thismay hide the complexity of the nifHgene pool. Thus,the frequency of apparition for each fragment must below (


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F. Poly et al. / Res. Microbiol. 152 (2001) 95103 99

Figure 1. Frequencies (% of occurrence in tested strains or sequences) of nifH gene restriction fragments obtained with four enzymes(A, HinfI; B, HaeIII; C, NdeII; D, MnlI) from pure strain DNAs together with fragments obtained from theoretical digestions of databasesequences.

MnlI, Fnu4HI, HaeIII, TaqI, NdeII, HinfI (BoehringerMannheim). Digestions were performed overnight toensure that complete fragmentation was achieved. Di-gested DNAs were analyzed in a 5% polyacrylamidegel (19:1) (Bio-Rad, Ivry-sur-Seine, France) usingmanufacturers recommendations and staining with1 Syber Green (FMC BioProducts). This experi-ment was completed with theoretical digestions sim-ulated from 14 sequences of the nifH gene foundin the GenBank database: Anabaena sp. strain PCC7120 (X05595 accession number); Alcaligenes fae-

calis pBZ1 (X96609); Azoarcus sp BH72 (Y12545);Azospirillum brasilense Sp7 (X51500); Azotobac-ter chroococcum ATCC 4412 (M73020); Azotobac-ter vinelandii M20568 [13]; Bradyrhizobium japon-icum USDA 110 (K01620); Fischerella sp UTEX1931 (U49514); Frankia sp FaC1 (X73983); Kleb-siella pneumoniae (J01740) [22]; Rhizobium meliloti

(J01781) [23]; Rhodobacter sphaeroides 16PHC(AF031817); Thiobacillus ferrooxidans ATCC 33020(M15238); Trichodesmium thiebautii (U23507). Themain features of the occurrence (% of tested strainsand sequences) of the restriction fragments obtainedby four of the six enzymes are shown in figure 1.

3. Results and discussion

All six enzymes discriminated pure strains, exceptfor HinfI, which gave similar patterns with the various

reference strains (figure 1A). Obviously, HinfI restric-tion targeted two sites highly conserved in nifH genesequences and, in most cases, produced three frag-ments ( 15 bp, 130 bp and 210 bp) from am-plicons (figure 1A). This endonuclease could be usedto confirm the nifHorigin of a PCR product, but not todiscriminate between strains. Three enzymes, HaeIII,


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Figure 2. HaeIII RFLP of nifH PCR products from pure strains and soil DNA.

NdeII and MnlI (figure 1B, C, D) were selected on thebasis of the above arguments to study nifH pool di-versity: HaeIII was used in their study on the nifHgene pool by Widmer et al. [27] and by Chelius andLepo [8], while NdeII and MnlI were applied here forthe first time to the nifH study.

Pure strains from N2 fixers from various generawere discriminated by RFLP resulting from the threeselected enzymes HaeIII (figure 2), NdeII (figure 3)and MnlI (figure 4). Slight differences between nifHprofiles of (i) Azospirillum irakense and Azoarcustolulyticus, and (ii) Rhizobium leguminosarum and

Mesorhizobium loti resulted from the presence ofsmall fragments (


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Figure 3. NdeIII RFLP of nifH PCR products from pure strains and soil DNA.

The sum of the molecular weights of all frag-

ments higher than the PCR products was obtained inSphingomonas paucimobilis and Enterobacter cloa-cae RFLP profiles. The presence of only one bandin the PCR product and the positive hybridizationwith nifH probes excluded a nonspecific amplifica-tion. Such a discrepancy would indicate a substan-tial contamination of DNA from S. paucimobilis andE. cloacae by DNA of other nitrogen fixers.

3.1. RFLP results on soil DNA

RFLP data concerning the amplification products

from soil DNA digested by either endonucleaseHaeIII, MnlI or NdeII were reproducible (data notshown). Each enzyme provided a specific profile foreach soil DNA (figures 24). Comparison of soiland strain profiles showed that some strain profileswere found in soils such as A. brasilense Sp7 andA. tolulyticus with MnlI restriction in LCSA-c soil

or S. meliloti and Paenibacillus polymixa (CF43 and

PMD230) with NdeII restriction in LCSA-p soil.However, soil DNA RFLP never resulted in profilesidentical to a pure strain on the three restrictionenzymes. For instance, A. brasilense Sp 245 profileswere found in LCSA-c soil with HaeIII and NdeIIbut not with MnlI. Moreover, several bands in soilDNA profiles did not correspond with any bandof the pure strains. These observations could leadto the conclusion that the pure strains used werenot dominant in the environment and that thereexisted a number of nitrogen fixers still unknown

(nonculturable) [24, 27]. The protocol used revealedthat the nifH gene pool, and probably the nitrogen-fixing community, were different under cultivationand under permanent pasture, even in the same soiltype.

A simple protocol with specific primers of thenifH gene and with three discriminating endonucle-


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Figure 4. MnlI RFLP of nifH PCR products from pure strains and soil DNA.

ases was described here which allows rapid assess-ment of the structure of nitrogen fixers in an ecosys-

tem through the diversity of nifH genes. This mole-cular tool revealed that long-term cultivation withtillage, fertilization, pesticide treatments or specificplant cover would result in a structure of the nifHgene pool different from permanent pasture. This ap-proach could be used to compare the diversity of ni-trogen fixers according to soil characteristics suchas texture, structure, plant cover, etc. It should be apowerful tool for monitoring the community of ni-trogen fixers in soil in order to investigate seasonalchanges, variations due to modification in the plantcover or effects of the input of chemical products in

soils.

Acknowledgements

This investigation was supported by the Ecocom-patibility of Solid Wastes program (grant 9674056/

DIMT/mfb) from the Agence de lEnvironnemmentet de la Matrise de lEnergie (ADEME). We are

grateful to Thierry Heulin and Wafa Achouak forkindly providing P. polymixa strains.

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Improvement in the RFLP Procedure for Studying the Diversity of NifH Genes