FEMS Microbiology Letters 127 (1995) 85-91

Identification of Azospirillum strains by restriction fragment

length polymorphism of the 16s rDNA and of the histidine
operon
Annamaria Grifoni, Marco Bazzicalupo, Claudia Di Serio, Silvia Fancelli,
Renato Fani *
Dipartimento di Biobgia Animale e Genetica “Leo Pardi”, Universith degli Studi di Firenze, Via Romana 17, I-50125 Firenze, Italy

Received 28 November 1994; revised 11 January 1995; accepted 25 January 1995

DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amuwnense, A. brasilense, A.
halopraeferens, A. irakense and A. lipoferum, were obtained by restriction analysis of the amplified 16s rDNA and by
restriction fragment length polymorphism of the histidiue biosynthetic genes. Data obtained showed that amplified rDNA
restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the
species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the
same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a
given species.

Keywords: Azospirillum; Restriction fragment length polymorphism; Histidine operon; 16s rDNA, Amplified rDNA
restriction analysis

1. Introduction methods of detection of these microorganisms in the

Bacteria belonging to the genus Azospirillum are
microaerophilic, nitrogen-fixing, Gram-negative rods,
often associated with roots of cereals and grasses;
some strains were previously demonstrated to be of
potential benefit in agriculture as a biological fertil-
izer [ll. The abundance and diversity of Azospiril-
lum isolates induced the search for new and reliable
?? Corresponding author. Tel.: +39 (55) 220 498; Fax:
(55) 222 565; e-mail: dibagefi@iCidg
environment and of their identification at species
and/or strain level. For this purpose, molecular fin-
gerprints of total DNA of Azospirillum strains by
restriction endonuclease analysis (REZA)[2] and ran-
dom amplified polymorphic DNA (RAPD) [3] were
previously developed. A third technique for identifi-
cation, restriction fragment length polymorphism
(RFLF’), which analyses only particular sequences of
the bacterial genome, is also a powerful tool in the
study of genetic similarity among strains. The most
+ 39 widely used sequences in FWLP are the rRNA genes
[4], although other sequences with known [5,6] or

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 86 A. Grifoni et al. /FEMS
unknown function (i.e. randomly cloned DNA) [7,8]
have been commonly utilized. The rRNA genes can
be easily obtained in high amounts by enzymatic
amplification via polymerase chain reaction (PCR);
the restriction analysis of rDNA amplified genes
(ARDRA) has also been utilized for bacterial identi-
fication [9,10].
In the present work we have used the restriction
analysis of amplified 16s rDNA to evaluate the
reliability of this technique for taxonomical and iden-
tification purposes in the genus Azospirillum, com-
paring the results with those obtained with RFLP on
total DNA probed with the histidine biosynthetic
genes. The latter technique has previously been used
successfully on a limited number of Azospirillum
strains [6].
2. Materials and methods
2.1. Bacterial strains and growth conditions
Bacterial strains are listed in Table 1. A. halo-
prueferens was grown at 41’C in Nutrient Broth [ll]
supplemented with 0.25% NaCI; all other Azospiril-
lum strains were grown in LB medium [ 1l] at 35°C.
2.2. Amplification and restriction analysis of 16s
rDNA (ARDRA)
Amplification of 16s rDNA was performed di-
rectly on a single bacterial colony as follows: a
whole colony, taken from an agar plate with a sterile
toothpick, was resuspended in 20 ~1 of sterile dis-
tilled water and heated to 95°C for 10 min to allow
cell lysis; 1 ~1 of lysed cell suspension was used for
PCR amplification. The reactions were performed as
described [12] with the two universal primers 27f
(~-GAGAGT-I-TGATCCTGGCI-CAG) and 1495r
(5’~CTACGGCTACCITGICGA), which allowed
the amplification of nearly the entire 16s rRNA
gene. A 4-6-~1 aliquot of each PCR reaction, con-
taining about 1.5 pg of DNA, was treated with 5
units of the restriction enzyme AluI (Boehringer,
Mannheim) in a total volume of 30 ~1 at 37°C for 3
h. The reaction products were analysed by agarose
gel (2.5% w/v) electrophoresis.
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Microbiology Letters 127 (1995) 85-91
2.3. RFLP analysis of the histidine operon
Total DNA of Azospirillum strains was purified
as described [2]. Restriction digestions using
Boehringer (Mannheim) enzymes and buffers, were
carried out by treating l-3 ,ug of DNA with lo-30
units of enzyme at 37°C for 5-6 h. Fragments were
separated by electrophoresis on a 0.8% (w/v) agarose
gel.
Probes, prepared according to standard protocols
[ll], were labeled and the hybridization signals de-
tected with the Digoxigenin Labeling and Detection
Kit (Boehringer, Mannheim) using the chemilumi-
nescence method following the instructions of the
supplier.
Southern blotting was carried out as described
1111 on nylon membranes (Hybond N, Amersham),
which were hybridized as described 161. The hy-
bridization patterns were analysed with a scanner
densitometer GDS2000 (Ultra-Violet Product Ltd.,
Cambridge, UK).
2.4. Cluster analysis
Pairwise comparison of the restriction patterns
was carried out and the coefficient of similarity S,
between pairs of strains was calculated according to
Fox et al. [13] from the ratio of twice the number of
common fragments to the total number of fragments.
These values were used to cluster the strains by the
unweighted pair group method with arithmetic mean
(UPGMA) using the CLUSTER and TREE proce-
dures of the SAS package [14].
3. Results and discussion
3.1. Restriction analysis of amplified Azospirillum
rDNA (ARDRA)
16s rDNA was amplified as described in Materi-
als and methods by means of PCR from twenty-nine
Azospirillum strains. Most of these strains had been
previously assigned, as shown in Table 1, on the
basis of phenotypic traits and/or molecular data, to
one of the five known species: A. amazonense, A.
brasilense, A. halopraeferens, A. irakense or A.
lipoferum; in contrast, other strains were not pre-
 A. Grifoni et al. / FEMS Microbiology Letters 127 (1995) 85-91 87

-- A./?. A.I. A.N.

___-- A.i.A.h.
1 2 3 4 5 6 7 X 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2X 29

Fig. 1. Agarose gel electrophoresis of amplified 16s rDNA of 29 Awspirillum strains digested with restriction endonuclease AluI. Lanes
1-16, A. brasilense (A. b.) strains Sp7, SpF94, Cdr, DSM 1859, DSM 2298, DSM 2287, DSM 1858, SpBrl7, SpF2, Tl, T2, F14, 129/l,
129/2, 129/3; lanes 17-24, A. Zipoferum(A. 1.) strains 133/l, 137/S, ATCC 29707, ATCC 29708, ATCC 29731, F, W03, PAl; lanes
25-27, A. amazonense (A. a.) strains: Yl, Y2, Y6; lane 28, A. irakense (A. i.) strain 103312; lane 29, A. halopraeferens (A. h.) strain
Au4. Numbers indicate the size of some of bands of the molecular mass marker (123 bp ladder, Boehringer, Mannheim).

ciscly identified (Table 1). A major strong amplifica- SpF94, Cdr, DSM 1859, DSM 2298, DSM
tion band of about 1450 bp from each Azospirillum 2787, DSM 1858, or A. amazonense strains
strain was obtained (not shown). The endonuclease Yl, Y2 and Y6).
Ah1 was chosen on the basis of a preliminary (ii) Strains SpP2, Tl, T2, F14, 129/l, 129/2,
screening of several four- or six-base cutting restric- 129/3 can be assigned to the species A.
tion endonucleases, performed on a limited number brasdense, and strains 133/l and 137/5 to
of strains. The reproducibility of ARDRA patterns, the species A. Zipoferum. This assignment is in
obtained after Ah1 digestion, was verified on PCR agreement with physiological tests (C. Scotti,
products of different independent amplifications. personal communication) and/or with DNA
The ARDRA from the 29 Azospirihm strains is fingerprints [2,15]. Strains SpBr17 and Sp242,
shown in Fig. 1. The following conclusions can be which had previously been assigned to the
drawn: species A. lipoferum, showed the typical
(i) Strains previously assigned to a given species ARDRA pattern of A. brasilense, which is in
share a very similar or identical restriction agreement with restriction endonuclease analy-
pattern (for example A. brusilense strains Sp7, sis of total DNA [2,15].

RI Pv Pv Pv Pv
1043 I 2150 1 739 1 590 1 1520 y%3~

ORF 978 ORF 548 F E2

P 7679 bp
P
I I
PV
L 750bp ;

Fig. 2. Restriction and genetic map of the hi&dine operon and the upstream region of A. brasilense strain SpF94. Symbols: Bg, Fv, P,
recognition sites for the restriction endonucleases BglII, PuuII, PstI, respectively; numbers between restriction sites indicate the length of
the restriction fragments ln bp; Bd, H, 1, A, F, E, 2, genes belonging to the histidine cluster hi&d, hti, ORFl, hM, hiss, his& ORF2,
respectively; black box, transcription terminator; the arrow with closed circle indicates the promoter and the direction of tmnscription of his
genes. 0RF978 and ORFS46 represent two putative ORFs with unknown function (unpublished results). Continuous lines under the genetic
map indicate the fragments used as probes.

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 88 A. Grifoni et al. /FEMS Microbiology Letters 127 (1995) 85-91

(iii) Four main groups of restriction profiles can be
recognized, three of which typical of the
species A. brusihzse (lanes l-161, A.
lipoferum (lane 17-24) or A. amazonense (lane
25-271, respectively. The fourth group in-
cludes strains A. in&we 103312 and A.
halopraeferens Au4, which show identical
Ah1 restriction patterns; these two strains also
share identical patterns with the enzyme
SadAI.
(iv) Only few differences are found in the restric-
tion patterns at intraqecific level, the most
relevant one being the presence of an addi-
tional band, in A. bradense strain DSM1859
(Fig. 1, lane 4). This fact can be explained by
the presence, within the DSM1859 genome, of
rDNA alleles with different sequences, as pre-
viously reported for Comamonadaceae [9].
The data obtained indicate that ARDRA could
represent a simple, extremely rapid, highly repro-
ducible and reliable tool for the identification of
Awspirillum species. The two species A. irukmse
and A. halopraejkrens, however, appear to be closely
related.

--

-3621

-2995
-2896

Fig. 3. Hybridization profiles on total DNA from Awspirillum strains digested with P&I and BgZII. The probe used was a 7679-bp PstI
DNA fragment from A. bradense (see Fig. 2). Identical patterns were found for strains SpF94, Cdr and Sp7 (not shown); for strains
129/l, 129/2, 129/3 (not shown) and for strains PAl, 137/S and 137/6 (not shown). Numbers indicate the size (in bp) of some
hybridization bands. Symbols: A.a., A.b., Ah., A.i., A.I., indicate species A. a mazmense, A. bradense, A. halopraeferens, A. irakeme
and A. lipoferum, respectively.

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 A. Grifoni et al. / FEMS Microbiology Letters 127 (1995) 85-91 89

Table 1 (Fig. 2) containing the complete histidine operon of

Bacterial strains A. brasilense SpF94 and its upstream region [16].
Species Strain Obtained from a Results obtained (Fig. 3) show that the 21 strains
A. amawnense Yl DSM 2787 gave 15 different restriction patterns. The DNA of
Y2 A. Hartmann strains belonging to the species A. amazonense, A.
Y6 A. Hartmann brasilense and A. lipoferum gave 6-8 strong hy-
A. brasilense spF94 [I81
G-h This laboratory
bridization bands, whereas the DNA from A. irak-
SP7 DSM 1690 ense strain 103312 and A. halopraeferens strain Au4
DSM I858 DSM showed only four and two bands, respectively, of
DSM 1859 DSM weaker intensity. The hybridization pattern of A.
DSM 2298 DSM
brasilense strain SpF94 is in agreement with the
F14 I21
DSM 2287 DSM
restriction map deduced by the nucleotide sequence
A. hdopraeferens Au4 A. Hartmann data [16,17]. The 15 hybridization patterns contain
A. irakense 103312 A. Hartmamr both polymorphic bands peculiar of a given strain
A. lipofemm SpRG2Oa ATCC 29708 and monomorphic bands characterizing one or more
SpRG6xx ATCC 29731
species. Analysis of the restriction patterns allowed
Sp59b ATCC 29707
F [191
the identification of species-specific bands: the 373-
wo3 WI bp and/or 883-bp band are specific for A. brasilense,
PA1 121 the 2896-bp or 2995bp band are specific for A.
SpBr17 ATCC 29709 lipoferum, and the 3621-bp band is specific for A.
sp242 J. Dobereiner
amazonense (Fig. 3). The presence of one or more of
Azaspirillum spp. 137/5 c. scotti
137/6 C. Sconi these bands in RFLP patterns could thus represent a
133/l C. Scotti signature for a given Azospirillum species.
129/l c. scotti In order to obtain information on the genetic
I29/2 C. Scotti relatedness among strains a pairwise comparison of
129/3 C. Scotti
the restriction patterns was carried out, enabling the
SPp2 AHartmann
Tl F. Favilli construction of the dendrogram shown in Fig. 4,
T2 F. Favilli whose topology corresponds to results previously
a DSM, Deutsche Sammhmg von Mikroorganismen, Braun-
reported concerning several Azospirillum strains
schweig, Germany; ATCC, American Type Culture Collection, [2,3,6]. Five groups were recognized with a cut-off
Rockville, MD, USA. F. Favilli, Dipartimento di Scienze e Tec- point for the S, value of 0.5.
nologie Alimentari e Microbiologiche, Universid di Firenze, Piaz- The first cluster contains the six A. brasilense
zale delle Cascine 17, 50144 Firenze, Italy; A. Hartmann, GSF- strains tested, which have already been shown to
Research Center for Envirormrent and Health/Institute of Soil
share DNA homology, although having rather differ-
Ecology, D-8042 Neuherberg, Germany; C. Scotti, Istituto Speri-
mentale per le Colture Foraggere, V.le Piacenza 29, 20075 Lodi, ent origins. Strains 129/l, 129/2 and 129/3, shar-
Italy. J. Dobereiner, EMPRAPA-CNPBS, Km 47, Seropedica, ing identical patterns, form an independent cluster,
23851 Rio de Janeiro, Brazil. although closer to A. brasilense than to other species,
according to results obtained by ARDRA (Fig. 1).
The six A. lipoferum strains and strains 137/5
3.2. RFLP of the h&dine operon in Azospirillum and 137/6 are grouped in a single cluster. This
cluster is the most branched one, in agreement with
The RFLPs were performed on 16 strains repre- physiological tests which showed a certain hetero-
sentatives of the five species A. amazonense, A. geneity among the A. lipoferum strains. Such hetero-
brasilense, A. halopraeferens, A. irakense and A. geneity did not appear from ARDRA patterns (Fig.
lipoferum and on five italian isolates 129/l, 129/2, 1).
129/3, 137/5 and 137/6. Total DNA from each The third cluster includes the two A. amazonense
strain was digested with BglII plus PuuII enzymes strains which are closer to A. lipoferum than to A.
and hybridized, after Southern blotting, to a probe brasilense.

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 90 A. Grifoni et al. / FEMS Microbiology Letters I27 (1995) 8S-VI
Au4
I 103312
13716
13715
PA1
wo3
ATCC29708
F results of ARDRA suggested that the two strains A.
ATCC29731
ATCC29707
Yl
Y2
12913
12912
12911
DMS2287
DMS1859
DMS2298
“,$94
SP’
I rates of these two portions of the genome. However,
0.6 1.0
SAB
Fig. 4. Dendrogram of Azospirillum strains based on UPGMA
clustering of the S, values derived from the PuuII-&$I1
hybridization patterns reported in Fig. 2. S,, coefficient of
similarity. The dotted line represents the cut-off value.
A. irakense and A. halopraeferens form an inde-
pendent group, as the restriction patterns of these
strains did not show any band in common with other
species, including the two bands which were found
in the three other species. The first band, of 590 bp,
common to all strains but 129/l, 129/2 and 129/3,
corresponds to a PvuII fragment containing the 3’
region of the hisH gene and the 5’ region of ORFI
(the gene coding for open reading frame 1) (Fig. 2).
The second band, of 739 bp, present in all strains,
corresponds to a PvuII fragment including the 3’
portion of hisBd gene and the 5’ region of the hisH
gene (Fig. 2). The strong conservation of these PvuII
sites in the his operon of strains of the species A.
amazonense, A. brasilense and A. lipoferum can be
ascribed to the fact that in A. brasilense they code
for glutamine and leucine, both of which are highly
conserved in the corresponding positions of homolo-
gous proteins from different microorganisms
[ 16,20,21].
Taken together, the data presented here indicate
that ARDRA, being extremely rapid, easy to per-
form, highly reproducible and reliable, constitutes a
useful tool for rapid screening of Azospirillum popu-
lations and for identification of Azospirillum species.
Moreover, by using different restriction enzymes,
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this method could be extended to the identification
of Azospirillum strains at the subspecies level.
This analysis also gave interesting indications
about the genetic relationships existing among A.
irakense and the other Azospirillum species; in fact,
irakense 103312 and A. halopraeferens Au4 are
closely related.
These results were confirmed by RFLP analysis
with his probes which also enabled the identification
of Azospirillum at species and strain level. The
higher variability revealed by analysis of an enzy-
matic operon in comparison to 16s rDNA restriction
profiles is in line with the different evolutionary
in establishing relatedness, the two techniques gave
comparable results.
Acknowledgements
Part of this work was presented at NATO Ad-
vanced Research Workshop on Azospirillum and
related microorgamsms held in Sarvar, Hungary, 4-7
September 1994. The contribution of Cristina Indo-
rato and Francescopaolo Di Cello was greatly appre-
ciated. Research supported by Special Project
RAISA, Sub-project No. 2, paper no. 2065.
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