www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods 57 (2004) 197–206

Amplified ribosomal DNA restriction analysis for the

characterization of Azotobacteraceae: a contribution to the study of

these free-living nitrogen-fixing bacteria

Lucia Aquilantia,*, Ilaria Mannazzua, Roberto Papaa,Lucia Cavalcab, Francesca Clementia

aDipartimento di Scienze degli Alimenti, Universita Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, ItalybDipartimento di Scienze e Tecnologie Ambientali e Microbiologiche, Universita di Milano, via Celoria 2, 20133 Milan, Italy

Received 2 December 2003; received in revised form 8 January 2004; accepted 8 January 2004

Abstract

A 16S rRNA gene-based fingerprinting method was developed for the identification of Azotobacteraceae and tested onto 48

soil isolates and 28 reference strains belonging to the free-living nitrogen-fixing bacterial group and to the most common

species found in soil samples. According to this method, the 16S rRNA gene was amplified using universal primers for

Eubacteria and PCR products were subsequently digested with RsaI, HhaI, HpaII, FnuDII, and AluI. The analysis of the

restriction profiles obtained showed that the method is able to define a unique species-specific phylotype (SSP) for each of the

eight Azotobacteraceae species tested. Cluster analysis was successfully employed for the identification of members of the

family Azotobacteraceae, being assignation into species of the isolates confirmed by means of partial 16S rRNA gene

sequencing.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Azotobacter; Azotobacteraceae; ARDRA; Cluster analysis

1. Introduction within this family: Azotobacter and Azomonas. The

The family Azotobacteraceae belongs to the g-

subclass of the Proteobacteria (Tchan, 1984) and is

composed of free nitrogen-fixing bacteria (FNFB)

commonly living dispersed in soil, water and sedi-

ments. Studies on the rRNA cistrons (De Smedth et

al., 1980) and the application of molecular taxonomic

techniques led to the identification of two genera

0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.mimet.2004.01.006

* Corresponding author. Tel.: +39-712204985; fax: +39-

712204858.

E-mail address: anmicro@univpm.it (L. Aquilanti).

genus Azotobacter comprises seven species, namely

Azotobacter chroococcum, Azotobacter vinelandii,

Azotobacter beijerinckii, Azotobacter paspali, Azoto-

bacter armeniacus, Azotobacter nigricans (Tchan and

New, 1984a), and Azotobacter salinestris (Page and

Shivprasad, 1991), while genus Azomonas comprises

three species, namely Azomonas macrocytogenes,

Azomonas agilis, and Azomonas insignis (Tchan and

New, 1984b).

The positive interactions between members of the

Azotobacteraceae family and a variety of field grown

crops (Jackson et al., 1964; Rovira, 1965; Denarie and

L. Aquilanti et al. / Journal of Microbiological Methods 57 (2004) 197–206198

Blanchere, 1966), their capability to synthesize anti-

biotics and growth-promoting substances (Harper and

Lynch, 1979; Pandey and Kumar, 1990) and their

ability to fix atmospheric nitrogen non-simbiotically

(Gouri and Jagasnnatathan, 1995; Mrkovacki et al.,

1996; Zahir and Arshad, 1996; Zahir et al., 1996)

were widely taken under study. At present, strains

belonging to the species A. vinelandii and A. chroo-

coccum are proficiently employed as soil inoculants in

rainy areas and in warm and alkaline soils (Pandey et

al., 1998). Moreover, some species of Azotobactera-

ceae were exploited for the production of compounds

of commercial interest, such as polysaccharides

(Clementi, 1997; Sabra et al., 2001), pigments and

vitamins (Mishustin and Shilnikova, 1971; Pandey et

al., 1998).

All these evidences justify the interest recently

arisen on the development of analytical tools suitable

for a rapid and reliable identification of these bacteria

and for their study in soil microbial communities.

In this context, the potential of amplified ribosomal

DNA restriction analysis (ARDRA) for the character-

ization and differentiation of Azotobacteraceae was

evaluated, and the more appropriate tool for the elab-

oration of data was assessed. This approach allowed a

sound method for the unambiguous identification of

Azotobacteraceae to be developed, also in view of

deepening knowledge on the ecology of these micro-

organisms and their biotechnological exploitations.

2. Materials and methods

2.1. Bacterial strains and culture conditions

We analysed 76 cultures, 28 of which were

reference strains. Among these: A. vinelandii

DSM576, DSM2289, DSM2290, DSM87; A. chroo-

coccum DSM2286, DSM377; A. armeniacus

DSM2284, A. beijerinkii DSM378, Azorhizophilus

paspali DSM2283, A. macrocytogenes DSM721, A.

agilis DSM375, A. insignis DSM1845, Azospirillum

brasilense DSM1690, Azospirillum lipoferum

DSM1691, Azospirillum amazonense DSM2787,

Azospirillum halopraeferens DSM3675, Beijerinckia

indica DSM1715, Beijerinckia mobilis DSM2326,

Beijerinckia fluminensis DSM2327, and Beijerinckia

derxii DSM2329 were obtained from Deutsche

Sammlung von Mikrorganismen und Zellkulturen

(Braunschweig, Germany); Agrobacterium tumefa-

ciens AT, Agrobacterium rhizogenes AR, Agrobacte-

rium radiobacter DCBA10, Sinorhizobium meliloti

DCBA8, Rhizobium leguminosarum DCBA11, Pseu-

domonas corrugata PC, Pseudomonas fluorescens

DCBA14, and Pseudomonas tolasii DCBA4 were

provided by the Department of Chemistry and Agri-

cultural Biotechnologies, University of Pisa, Italy.

The remaining 48 cultures were soil isolates that

originated from central Italian soils, preliminarily

characterized as described by Aquilanti et al. (sub-

mitted for publication).

Bacterial strains were cultured overnight on Tryptic

Soy Agar (TSA) or broth (TSB) (Oxoid, Basingstoke,

Hampshire, England) at 30 jC and stored frozen in

50% (w/v) glycerol in TSB at � 80 jC.

2.2. ARDRA

In order to choose the restriction enzymes to be used

for ARDRA, the 16S rRNA sequences of Azotobacter

and Azomonas retrieved from the GenBank were vir-

tually restricted using the Genetool software (Version

2.0) (Wishart et al., 2000). Crude DNAwas extracted as

previously described (Aquilanti et al., submitted for

publication). The 16S rRNA gene was amplified by

means of universal primers 27f and 1495r (Weisburg et

al., 1991) in 50 Al reaction mixture containing 1 U of

Taq DNA polymerase (Amersham Biosciences,

Uppsala, Sweden), 1� reaction buffer (10 mM Tris–

HCl pH 8.8, 1.5 mMMgCl2, 50 mM KCl, 0,1% Triton

X-100), 200 AM of the four dNTPs, and 2 AM of each

primer. The PCR reactions were run for 35 cycles on a

Applied Biosystem Gene Amp PCR System 9700

(Foster City, CA, USA) as follows: denaturation at 94

jC for 1 min, annealing at 55 jC for 1 min, elongation

at 72 jC for 2 min. An initial denaturation step at 95 jCfor 4 min and a final extension step at 72 jC for 15 min

were also performed. The PCR products were digested

with the tretrameric restriction enzymes (TRE) RsaI,

HhaI, HpaII, AluI, and FnuDII (Roche Diagnostics,

Mannheim, Germany). Five units of each enzyme were

added to 12.5 Al of the amplification product and

incubated for 5 h at 37 jC in a total volume of 25 Al.The digests were resolved by electrophoresis on a 2.5%

(w/v) agarose gel in 1� Tris–borate–EDTA buffer

(TBE, 0.09MTris base, 0.09M sodium borate, 2.5mM

L. Aquilanti et al. / Journal of Microbiological Methods 57 (2004) 197–206 199

EDTA, pH 8.3) at 3.2 V/cm for 4 h, against a 50-bp

DNA ladder (Amersham Pharmacia, Uppsala, Swe-

den). Gels were stained in ethidium bromide for 15 min

and rinsed for 5 min in distilled water. Gel electronic

images were visualized by means of ImageMaster VDS

(Amersham Pharmacia), captured with LISCAP soft-

ware v.1.0 (Amersham Pharmacia), and stored as TIFF

files. Digital images were normalized with the above

mentioned 50-bp DNA size marker and analyzed with

GelCompar software v. 4.0 (Applied Maths, Kortrijk,

Belgium).

2.3. Statistical analyses of ARDRA patterns

For data scoring only fragments z 50-bp were

considered. A binary data matrix was constructed on

the basis of the presence or absence of each band

which was scored 1 or 0, respectively. The different

restriction patterns obtained were defined as different

haplotypes for each enzyme and all the strains char-

acterized by the same combination of haplotypes

obtained with the five enzymes were grouped togeth-

er, defining a phylotype, which is often used to term

the 16S rRNA clones differentiated by restriction

Table 1

Tetrameric restriction enzymes (TRE) employed in the ARDRA analysis

TRE Restriction fragments H Azotobacteraceae

No. %

RsaI 21 16 1.798 a 650–350–220–1

b 470–360–190–1

c 900–340–140–1

HhaI 27 24 2.496 a1 410–280–180–1

b1 580–410–280–2

e1 580–410–280–1

d1 580–410–280–1

c1 410–370–280–2

f1 410–380–280–2

g1 560–410–280–2

h1 410–370–280–2

AluI 27 23 2.347 b3 630–240–210–1

HpaII 27 22 2.374 a2 550–400–130–1

d2 600–400–130–1

c2 680–400–130–8

f2 550–450–1130–

g2 680–500–130–8

h2 680–500–170–1

FnuDII 23 15 2.298 c4 400–300–190–1

No.: number of restriction fragments obtained; %: percentage of polymorp

Shannon–Weaver index.

enzymes (Tiedje et al., 1999). Haplotype diversity

(H) was estimated using the Shannon–Weaver index

(Shannon and Weaver, 1949; Ludwig and Reynolds,

1988) which was calculated as: H =�Spilnpi, wherepi is the frequency of the ith haplotype.

All the strains characterized by the same combina-

tion of haplotypes obtained with the five enzymes

were grouped together, defining a phylotype.

Genetic distance between phylotypes was calculat-

ed using the Nei and Li (Nei and Li, 1979) coefficient

and the resulting distance matrix was used for the

UPGMA cluster analysis (Sokal and Sneath, 1963)

and the Neighbor-joining mid-point analysis (Rohlf,

1993; Saitou and Nei, 1987) using TREECON for

Windows (Version 1.2) (Van Der Peer and DeWachter,

1994) and NTSYS-PC (Version 1.6) (Rohlf, 1993),

respectively. The consistency of each node was esti-

mated by bootstrapping over markers (Felsenstein,

1985) using 1000 pseudoreplications.

2.4. Sequence analyses

The nearly complete sequence of the 16S rRNA

gene of A. vinelandii DSM576, A. chroococcum

haplotypes Species

50–120-bp Azotobacter spp.; Azomonas macrocytogenes

40–120-bp Azomonas agilis

20-bp Azomonas insignis

60–120-bp Azotobacter vinelandii

60-bp Azotobacter chroococcum

90-bp Azotobacter beijerinckii

80–160-bp Azorizhophilus paspali

60–180–160-bp Azotobacter armeniacus

60–200-bp Azomonas macrocytogenes

60-bp Azomonas agilis

60–220-bp Azomonas insignis

60–80-bp Azotobacter armeniacus

10–80-bp Azotobacter vinelandii

10–80-bp Azorhizophilus paspali

0-bp Azotobacter armeniacus

110–80-bp Azomonas macrocytogenes

0-bp Azomonas agilis

40–80-bp Azomonas insignis

50–120-bp Azotobacter armeniacus

hic restriction fragments; H: diversity calculated on the basis of the

L. Aquilanti et al. / Journal of Microbiological Methods 57 (2004) 197–206200

DSM2286, A. beijerinckii DSM378, and A. macro-

cytogenes DSM721 was amplified using universal

primers 27f and 1495r (Weisburg et al., 1991) and the

internal primers Pi-1 (5V-AGC AGT GGG GAA TAT

TGG AC-3V) and Pi-2 (5V-GCTAAC TTC GTG CCA

GCA G-3V). A rRNA gene fragment of about 300-bp

was amplified with universal primers Y1 and/or Y2

(Young et al., 1991) from one isolate for each isolate

phylotype (IP) selected. PCR products were purified

using the GFX PCR DNA and Gel Band Purification

Kit (Amersham Pharmacia Biotech, Uppsala, Sweden)

according to the instructions and sent to MWG Biotech

(Ebersberg, Germany) for sequencing.

The partial rRNA gene sequences of the isolates

were preliminarily compared with those present in

the Basic BLAST search (Altschul et al., 1997),

and then aligned using the ClustalW program

(Higgins et al., 1992) available at the European

Bioinformatic Institute website (http://www.ebi.a-

c.uk/clustalw/) with the sequences retrieved from

the GenBank database (Benson et al., 2003) avail-

Table 2

Single haplotypes and species-specific phylotypes (SSPs) defined for the r

RsaI, HhaI, HpaII, AluI, and FnuDII

able at the NCBI website (http://www.ncbi.nlm.nih.

gov/) using the accession numbers: A. beijerinckii

AJ308319, A. paspali AJ308318, A. vinelandii

L40329, A. chroococcum AF035211, A. macrocyto-

genes AF035212 and with those obtained from our

reference strains.

2.5. Nucleotide sequence accession numbers

The GenBank accession numbers for the 16S rRNA

gene sequences are as follows: DSM576, AY336565;

DSM378, AY336566; DSM2286, AY353708.

3. Results

3.1. ARDRA

An about 1.5-kb portion of the 16S rRNA gene

from 76 bacterial cultures was amplified using the

universal primers 27f and 1495r.

eference strains by means of ARDRA, using the restriction enzymes

Table 3

Haplotypes and total isolates phylotypes (IPs) defined for soil

isolates by means of ARDRA, using the five restriction enzymes

RsaI, HhaI, HpaII, AluI, and FnuDII

Soil isolates Haplotype IP

RsaI HhaI HpaII AluI FnuDII

BB4, 6, 8;

AX16; AZ2, 8

a b1 b2 a3 a4 L7

BN16 a f1 b2 a3 a4 L35

AY7, 14 a b1 b2 b3 a4 L9

BN9; BZ55 a f1 b2 w3 a4 L12

AX8, BB3 a b1 b2 a3 b4 Ac2

BG8,18; BP11 a f1 b2 a3 d4 L13

BG10,13, 14, 22 a f1 b2 y3 l4 L10

BL46 h s1 ae2 m3 n4 L28

BD2; CH10 h w1 aa2 m3 t4 L27

CD20 l y1 ab2 r3 t4 L34

M1 r w1 ac2 z3 t4 L41

CM8 a b1 b2 a3 t4 L8

CH13 p j1 w2 u3 t4 L39

CH30 f v1 y2 t3 t4 L23

BH16 o x1 ad2 a3 u4 L38

BH23 a b1 ad2 j3 u4 L11

BH15 o x1 ad2 j3 u4 L37

BF15 s aa1 b2 k3 v4 L42

BY3, CA24 I ab1 b2 x3 z4 L31

BL22; BD2; BM33;

BO7, 18, 27, 41;

BQ7; BY25;

BV62; BT1; CA28

h s1 r2 m3 n4 Agr25

BH27, 29, 34 h s1 r2 m3 n4-I Agr25-I

BO18; BQ7;

BT25; BY25

h s1 r2 m3 n4-II Agr25-II

L. Aquilanti et al. / Journal of Microbiological Methods 57 (2004) 197–206 201

The results of virtual restriction with the Genetool

software onto the sequences of A. paspali, A. chroo-

coccum, A. vinelandii, A. beijerinckii, and A. macro-

cytogenes (accession numbers: 18072914, 2661011,

790695, 18072421, and 2661012, respectively) led

to the selection of five restriction enzymes, namely

RsaI, HhaI, HpaII, AluI, and FnuDII, to be employed

for the characterization of members of the family

Azotobacteraceae. Thus, the amplification products

obtained by using the above mentioned primers

on all the cultures under study were subjected to

restriction analysis yielding a total of 125 fragments.

The percentages of polymorphic fragments obtained

with each enzyme are shown (Table 1).

The sum of the sizes of the restriction fragments

was consistent with the size of the undigested ampli-

fication products. Slight discrepancies were likely due

to the production of small restriction fragments (V 50

bp), undetectable on the agarose gels.

3.2. Haplotype analysis

In order to assess the existence of species-specific

restriction patterns the analysis of haplotypes was

performed for each of the enzymes utilized.

As reported (Table 1) the enzyme RsaI showed the

lowest diversity (H = 1.80). Among RsaI haplotypes,

a, b, and c referred uniquely to members of the family

Azotobacteraceae (Table 2). In particular, all the

Azotobacter species tested plus A. macrocytogenes

produced the haplotype a, while A. agilis and A.

insignis produced two species-specific haplotypes, b

and c, respectively (Table 1). Reference strains not

belonging to the Azotobacteraceae family produced

either species specific or nonspecific RsaI haplotypes

(Table 2).

Among the 48 soil isolates, 22 showed the RsaI

haplotype a, while none showed the haplotypes b or c

(Table 3).

HhaI and HpaII were characterized by high Shan-

non–Weaver index and showed the highest level of

resolution within the family Azotobacteraceae (Table

1).HhaI allowed the individuation of a species-specific

haplotype for each of the eight species within the

family of interest, while HpaII allowed six species-

specific haplotypes to be obtained (Table 1), being the

haplotype b2 common to the species A. chroococcum

and A. beijerinckii (Table 2). Both these two enzymes

revealed a high discriminatory power also within the

other genera considered (Table 1).

By contrast, AluI and FnuDII revealed to be the

less discriminating. In particular, restriction analysis

of the 16S rRNA gene of the reference strains with

AluI permitted to obtain 27 DNA restriction frag-

ments, combined in 14 haplotypes. Nine of them

were species-specific but only b3 was related to the

family Azotobacteraceae, being species-specific for

A. armeniacus (Table 1). Similarly, FnuDII produced

a total of 14 haplotypes, 2 of which, c4 and e4,

specific for A. armeniacus and A. insignis, respec-

tively (Table 2).

3.3. Phylotype analysis

Phylotypes were built up grouping together the

strains with the same combination of haplotypes

L. Aquilanti et al. / Journal of Microbiological Methods 57 (2004) 197–206202

(Table 2). The reference strains tested, belonging to 24

species, produced 22 different phylotypes. Of these,

all but one were species-specific, whereas the phylo-

type Agr25 was genus-specific for Agrobacterium,

including the three species A. tumefaciens, A. rhizo-

genes, and A. radiobacter (Table 2). Interestingly,

within the family Azotobacteraceae, eight SSPs were

found. More in detail, Av1, Ac2, Aa3, Ap4, and Ab5

defined the species belonging to the genus Azotobac-

ter, namely A. vinelandii, A. chroococcum, A. arme-

niacus, A. paspali, and A. beijerinckii, respectively,

while Am6, Aa14, and Ai15 were specific for the

three species of the genus Azomonas. On the basis of

the phylotype analysis, 2 out of the 48 isolates could

be ascribed to the species A. chrococcum and 12 to the

Fig. 1. UPGMA dendrogram of 42 phylotypes representing the 78 bacteri

strains and soil isolates, based on ARDRA. Phenetic relationships were der

numbers shown next to each bifurcation are bootstrap percent values base

genus Agrobacterium. On the contrary, the remaining

34 isolates produced 19 phylotypes which were dif-

ferent from all those produced by the reference strains

and therefore could not be unambiguously ascribed to

any of the 24 species considered (Table 3).

3.4. Cluster analysis

Both the UPGMA and NJ trees based on Nei and

Li genetic distances and UPGMA cluster exhibit the

same topology. For this reason only UPGMA den-

drogram is reported (Fig. 1).

The dendrogram reported in Fig. 1 is structured in

two main clusters, separating at a genetic distance of

0.82. Cluster I included SSPs related to genera Beijer-

al cultures taken under study, including Azotobacteraceae reference

ived from Nei and Li (1979) pairwise genetic distance estimates. The

d on 1000 pseudoreplications.

L. Aquilanti et al. / Journal of Microbiolo

inckia, Agrobacterium, Sinorhizobium, and Azospir-

illum along with three IPs, namely L41, L27, and L28.

Cluster II included all SSPs related to the family

Azotobacteraceae, and phylotypes related to genera

Pseudomonas (Pc32, Pf33, Pt36), B. derxii (Bd22),

and R. leguminosarum (Rl30). Fifteen soil IPs were

also included in this cluster (Fig. 1).

The eight Azotobacteraceae SSPs and seven IPs

were grouped in a single subcluster, named IIa

(d = 0.42; boot = 18%) (Fig. 1), separating from all

the other materials analysed. The Azotobacteraceae

subcluster was divided into two branches: one

(d = 0.30; boot = 64%) constituted by the two A. agilis

and A. insignis SSPs (Aa14, Ai15) and the other

(d = 0.35 boot = 28%) constituted by seven IPs (L7,

L9, L13, L12, L35, L8, and L10) plus all the Azoto-

bacter and A. macrocytogenes SSPs. Solidity within

the Azotobacteraceae subcluster was highlighted by

bootstrap values z 50% for half of the nodes and

50>xz 20 for most of the remaining ones.

The distance matrix, computed using the Nei and

Li coefficient on the basis of ARDRA restriction

profiles, was used to evaluate the genetic distance

between IPs and reference strains phylotypes. Seven

of the 19 phylotypes comprising soil isolates showed

a genetic distance V 0.20 to SSPs related to the

family under study (Table 4) and the corresponding

17 isolates were therefore ascribed to three species of

Azotobacteraceae. In particular, 13 isolates were

recognized as A. chroococcum, 2 as A. macrocyto-

genes, and 2 as A. beijerinckii.

Table 4

Assignation into species of soil isolates clustered in IPs included in

the Azotobacteraceae subcluster IIa

IP Soil strains Closest SSP Identification

Ac2 BB3, AX8 Ac2 (0.00) A. chroococcum

L7 BB4, BB6, BB8,

AX16, AZ2, AZ8

Ac2 (0.02) A. chroococcum

L8 CM8 Ac2 (0.12) A. chroococcum

L9 AY7, AY14 Ab5 (0.15) A. beijerinckii

L10 BG13 Am6 (0.26) A. macrocytogenes

L12 BN9, BZ55 Ac2 (0.17) A. chroococcum

L13 BG8, BG18, BP11 Am6 (0.04) A. macrocytogenes

L35 BN16 Ac2 (0.08) A. chroococcum

The closest SSP and the corresponding assigned species are shown.

Strains selected for sequencing are in bold, while in brackets are

shown the genetic distance values calculated on the basis of the Nie

and Li coefficient.

3.5. Sequence alignment

In order to confirm the efficacy of the identification

hypothesized by combining ARDRA and cluster anal-

ysis, we proceeded with the sequence analysis of one

representant for each IP assigned to the Azotobacter-

aceae family (Ac2, L7, L8, L9, L10, L12, L13, and

L35). Thus, an about 300-bp 16S rRNA gene fragment

was sequenced with universal primers Y1 and Y2 from

the eight isolates selected (Table 4). Due to the low

number of Azotobacteraceae rRNA gene sequences in

GenBank and EMBL databases, the 16S rRNA gene of

reference strains belonging to the four species identi-

fied in the population of soil isolates was also se-

quenced. Thus almost the entire rRNA gene of A.

vinelandii DSM576, A. chroococcum DSM2286, A.

beijerinckii DSM378, and A. macrocytogenes

DSM721 was amplified. The sequences obtained were

deposited in GenBank. The rRNA partial gene sequen-

ces of isolates and reference strains were subjected to

BLAST analysis and multiple alignment by means of

ClustalW. Assignation into species obtained by means

of ARDRA and cluster analysis was confirmed for

each of the eight isolates investigated, with a percent-

age of homology z 97% to the species A. chroococ-

cum, A. beijerinckii, and A. macrocytogenes. Indeed,

even though there is no threshold value of 16S rRNA

homology for species recognition (Stackebrandt and

Goebel, 1994), organisms sharing more than 97%

rRNA similarity are considered as belonging to a

single species (Vandamme et al., 1996).

gical Methods 57 (2004) 197–206 203

4. Discussion

Seventy-six bacterial cultures were taken under

study by means of ARDRA analysis. Of these, 48

constitute a collection of soil strains isolated from

different areas of central Italy (L. Aquilanti, F. Favilli,

and F. Clementi, submitted for publication). The

remaining 28 were reference strains of either species

considered within the family Azotobacteraceae or

species of the most common soil bacterial genera,

among which: (i) Azospirillum and Beijerinckia,

which comprise FNFB commonly living in soil; (ii)

Rhizobium whose members are symbiotic N-fixers but

can occur in soil also as free living forms; (iii) Agro-

bacterium which, surprisingly, constituted more than

L. Aquilanti et al. / Journal of Microbiological Methods 57 (2004) 197–206204

50% of the isolates retrieved on free nitrogen media in

our previous study (L. Aquilanti, F. Favilli, and F.

Clementi, submitted for publication). In addition,

three strains ascribed to the genus Pseudomonas were

included in this study, being easily isolated from soil

samples and strictly related to the genus Azotobacter

for what concerns the 16S rRNA sequence (De

Smedth et al., 1980).

ARDRA is commonly utilized as an alternative to

more laborious and expensive methods for the

identification of eubacteria, being the analysis of

the rRNA cistron a good criterion for microbial

classification at both genus and species level (Gri-

mont and Grimont, 1986; Massol-Deya et al., 1995).

However, as far as we know, this molecular tool has

never been utilized on Azotobacteraceae whose

identification, traditionally based on conventional

biochemical tests, is often tentative. Therefore, the

development of a simple identification method yield-

ing reliable and unambiguous results appears to be

useful, even necessary, for azotobacters assignation

into species.

In the present work, we propose a biphasic ap-

proach to Azotobacteraceae spp. identification. We

shall demonstrate that this approach, consisting of

haplotypes definition and cluster analysis, can be

utilized as an alternative to more complex and time-

consuming methods such as Single Strain Conforma-

tion Polymorphism (SSCP) (Schwieger and Tebbe,

1998) and Denaturing Gradient Gel Electrophoresis

(DGGE) (Lovell et al., 2001; Rosado et al., 1998)

recently applied to FNFB identification.

The first step in developing this method was the

selection of the enzymes to be used for the 16S

rRNA gene restriction analysis. In order to avoid

time-consuming and low-yielding experimental

work, the sequences of Azotobacteraceae, available

in ribosomal databases, were subjected to computer

analysis of digestion sites. Other studies have shown

that at least four enzymes are necessary to resolve

the 16S rRNA gene of different species (Tchan,

1984; Moyer et al., 1996). It is well known that

the combined use of four or more tetrameric restric-

tion enzymes has the effect of increasing the degree

of taxa discrimination rendering the type of TRE

used of no importance. All these evaluations promp-

ted us to select and utilize five enzymes, namely,

AluI, HpaII, FnuDI, RsaI, and HhaI.

On the other hand, it has been reported that

identification at the species level by ARDRA can be

achieved even with three or fewer TREs (Cheneby et

al., 2000; Ventura et al., 2001; Jang et al., 2002),

provided that a careful choice of the TRE is prelim-

inary performed. Our results showed that the use of

RsaI, HpaII, and HhaI on reference strains leads to the

clear discriminate between the different Azotobacter-

aceae species. This is in agreement with the results

obtained by Tiedje et al. (1999) which evidenced that

HhaI, RsaI, and HpaII gave the greatest resolution in

terms of philogenetical dissection of soil natural

communities, and by Moyer et al. (1996) which

described HhaI and RsaI as optimal for the detection

and differentiation of bacterial taxa, on the basis of the

average number of restriction sites.

However, utilization of RsaI, HhaI, and HpaII on

our soil isolates resulted not sufficient for their

unequivocal identification, possibly due to the pres-

ence of one or more restriction sites on all of the

rRNA gene copies present on a single genome, or to

the presence of rRNA gene alleles with different

sequences within a single genome, as described by

Vaneechoutte et al. (1992). In fact, only a limited

number of isolates could be identified on the basis of

the combination of the haplotypes obtained with the

three enzymes. Therefore, we considered advisable

to analyze the population of soil isolates with the

complete set of enzymes in order to identify them on

the basis of cluster analysis and assess whether this

statistic tool could disclose the data obtained by

ARDRA.

The similarity dendrograms, constructed by the

UPGMA method, confirmed the phylogenetic rela-

tionships between the genera Azotobacter, Azomo-

nas, Azospirillum, and Beijerinckia proposed by De

Smedth et al. (1980). Moreover, all Azotobactera-

ceae SSPs were clustered together, thus confirming

the phylogenetic relatedness of species within this

taxon. The closer genetic relatedness of the Pseudo-

monadaceae rRNA cistrons to those of Azotobacter

and Azomonas other than to those of the remaining

reference species was also shown. This is in accor-

dance with the results of De Smedth et al. (1980),

who described these three groups as close branches

of the same phylogenetic tree. The ARDRA-based

dendrogram obtained confirmed also that A. vinelan-

dii and A. paspali were the closest species within

L. Aquilanti et al. / Journal of Microbiological Methods 57 (2004) 197–206 205

genus Azotobacter (De Smedth et al., 1980). Within

genus Azomonas, A. insignis and A. agilis clustered

at a 0.32 genetic distance, forming a well-defined

subcluster separated both from A. macrocytogenes

and the other Azotobacter species. The closer relat-

edness observed for A. macrocytogenes with respect

to the genus Azotobacter, rather than Azomonas

argues in favour of the classification proposed by

Skerman et al. (1980) in contrast to that of New and

Tchan (1982).

Finally, the analysis of ribosomal sequences con-

firmed the identification obtained for 17 isolates by

the combination of ARDRA and cluster analysis.

Thus the biphasic approach proposed proved to be

suitable for the identification of members of the

Azotobacteraceae family at the species level and

represents a contribution to the disclosure and study

of the microbial diversity, also in view of biotech-

nological exploitation of free-living nitrogen-fixing

bacteria.

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