Validity, sensitivity and resolution limit of the PCR-RFLP analysis of the rrs (16S rRNA gene) as a tool to identify soil-borne and plant-associated bacterial populations

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Validity, sensitivity and resolution limit of the

PCR-RFLP analysis of the rrs (16S rRNA gene) as a

tool to identify soil-borne and plant-associated bacterial

populations

Philippe Oger, Yves Dessaux, A. Petit, L. Gardan, Charles Manceau, C.

Chomel, Xavier Nesme

To cite this version:

Original

article

Validity, sensitivity

and

resolution limit

of the PCR-RFLP

_analysis

of

the

rrs

(16S

rRNA

_gene)

as a

tool

to

identify

soil-borne

and

_{plant-associated}

bacterial

_populations

Phil

_Oger

Yves Dessaux

Annick

Petit

Louis

Gardan

Charles Manceau

b

Cécil Chomel

Xavier Nesme

a

Institut des sciences

végétales, CNRS,

avenue de la

Terrasse,

91198 Gif-sur-Yvette

cedex,

France

b

Station

de

pathologie végétale,

Inra, 42, rue

_{Georges Morel,}

49071 Beaucouzé

cedex,

France

c

Laboratoire

_{d’écologie}

microbienne des

sols,

Université Claude

Bernard-Lyon-1,

UMR CNRS 5557,

Inra,

boulevard du 11

_novembre,

69622 Villeurbanne

_cedex,

France

Abstract - The value of the restriction

_{fragment length polymorphism}

_(RFLP)

analysis

of polymerase

chain reaction

_{(PCR)-amplified}

rrs

_(16S

ribosomal ribonucleic

acid

_{[rRNA] gene)}

for the

rapid

identification of bacteria isolated from soil or

_plants,

and the limits of this

technique,

were evaluated

_using

bacterial _genera characteristic of the above-mentioned _ecosystems. Results showed that _{up to two} restriction site differences _may occur between rrs _operons within the same bacterial _genome as well as between bacteria

_belonging

to the same

_genospecies.

In

_spite

of these

limited

_differences,

members of the same

_{genospecies yield}

_very similar rrs RFLP

patterns. The identification limit varies

according

to the

analyzed

taxa.

_Species

can

be differentiated amongst members of both the

_family

Rhizobiaceae and the genus

Stenotrophomonas,

while the

_{technique only}

allows

_grouping

of

_closely

related

_species

amongst Xanthomonas spp. and amongst

species

related to Psendomonas syringae.

On the basis of their rrs RFLP

_profiles,

all

_presently

known species of

_{Agrobacterium}

can be

_routinely

identified

_{using only}

the _enzymes

HpaII

(or MspI),

ALUI and HaeIII.

Moreover,

the method was also found to be valuable in

_assessing

the

biodiversity

of a bacterial

_community

isolated from the

rhizosphere.

From the

comparison

of

empiric

rrs

RFLPs,

published

_sequences and

deoxyribonucleic

acid

(DNA)

pairing studies,

we

suggest that the occurrence of five different restriction sites within two rrs _{genes is} *

the minimum difference

required

to

clearly

establish that two relevant bacteria

belong

to different

genospecies.

@

Inra/Elsevier,

Paris

PCR-RFLP

_/

16S

_/

rrs

_/

_{Agrobacterium}

_/

Pseudomonas

_/

Xanthomonas

Résumé - Examen _critique de _l’analyse par RFLP du gène déterminant l’ARN 16S. L’intérêt et les limites de l’identification bactérienne basée sur

_l’analyse

du

_gène

codant _pour l’ARN

_ribosomique

16S _par PCR-RFLP ont été évalués dans

_plusieurs

genres bactériens

typiques

des

écosystèmes

sol et

plante.

Les résultats montrent

qu’il

peut y avoir

jusqu’à

deux sites de restriction différents entre les

gènes

rrs de bactéries

appartenant à la même

espèce génomique

ou entre différents

opérons

d’un même

génome.

Les résultats permettent

cependant

d’identifier les bactéries

jusqu’au

niveau de

l’espèce

chez les Rhizobiaceae et chez

Stenotrophomonas,

et

_jusqu’au

niveau du

groupe

d’espèces apparentées

d’un même genre chez Xanthomonas et dans le groupe

apparenté

à P.

_syringae.

De

_plus,

la méthode s’est révélée

_{parfaitement adaptée}

pour identifier et classer les bactéries isolées d’un

peuplement rhizosphérique,

en permettant une identification

_approchée

du _{genre, voire} de

_l’espèce.

La

_comparaison

des données de PCR-RFLP avec les données de

_séquences

et

d’hybridations

ADN-ADN

_{suggèrent qu’un}

minimum de

_cinq

sites de restriction

_polymorphes

dans le

gène

16S est nécessaire pour affirmer que des bactéries

appartiennent

à des

espèces

génomiques

différentes.

_©

_{Inra/Elsevier,}

Paris

PCR-RFLP

_/

16S rrs _/ Agrobacterium / Pseudomonas / Xanthomonas

1. INTRODUCTION

Evaluating

the

_diversity

and

_defining

the structure and behaviour of micro-bial communities have been and still remain

_primary

_objectives

in microbial

ecology.

This assertion is

_particularly

valid when

_applied

to soil-borne and

plant-associated

bacteria,

which are involved in the

_major

biochemical

cycles

or exercise known beneficial or detrimental effects such as

_{biodepollution,}

sym-biotic

_nitrogen

fixation or induction of

human,

animal or

_plant

diseases.

Indeed,

soil and

_plant

microflora are

_probably

_amongst

the main reservoirs of

biodiver-sity. However,

this microbial world remains

_{largely unexplored}

and therefore

poorly

understood: Ward et al.

₍₁₉₉₀₎

estimated that no more than 10

%

of soil

bacteria have

_already

been identified. This lack of data _may be correlated with the

_shortage

of tools to

_{rapidly identify}

bacteria - and more

_{crucially -}

to assess the behaviour of

_given

bacterial

_{population(s)}

_amongst

microbial communities of

_complex

_ecosystems.

Presently,

the definition of bacterial

_species

relies

_essentially

on the results

of

deoxyribonucleic

acid

_(DNA)

pairing

studies. For

_Wayne

et al.

_(1987),

two bacteria

_belong

to the same

_species

when their DNA-DNA relatedness is

higher

than 70

%

_(with

ATm <

_{5 °C).}

_{Unfortunately, hybridisation-based techniques}

are

_generally

tedious and

_{time-consuming.}

The determination of the DNA

pairing

does not constitute an

_exception

to this rule.

_Furthermore,

it is

_solely

a

comparative

method which

always

calls for the

availability

of DNA from

reference strains.

_{Consequently,}

this method is not

_appropriate

to assess

rapidly

the

_{phylogenic position}

of new isolates. The _emergence of molecular

_techniques,

on these

questions

(Woese,

1987;

Normand et

al., 1992;

Stackebrandt et

al.,

1993).

DNA

_sequencing

of this

part

of the bacterial genome

provides

scientists with numerous data on the

_genetic

distance

separating bacteria,

a feature which

has led to a drastic revision of Aristotle’s fixed

_image

of the

’species’.

Moreover,

the

_availability

of sequence data from a

_growing

number of bacterial

species

ensures that _any new strain may

readily

be

positioned

somewhere in the life tree without the absolute

_requirement

of additional biochemical

_analysis.

This is of

_primary

_importance

when

_exploring

a world where most inhabitants are

still not cultivable.

At the

_beginning

of this

_work,

these

_techniques

_appeared

as

_promising

tools

for the

phylogenetic

identification of the

_{microorganisms,}

as

_{exemplified by}

various studies

performed

on the _sequence of the rrs _{gene, i.e.} the 16S rRNA

gene

according

to

Riley’s

nomenclature

(1993).

Unfortunately,

such a

_technique

remains

_{expensive, time-consuming}

and unavailable to field

laboratories,

even

though

the relevant sequences can be

successfully

obtained

by

polymerase

chain reaction

_(PCR)

_{amplification}

from numerous bacteria. It is also not

appropriate

to track soil-borne or

plant-associated

microbial communities. To solve these

problems,

we and others

(Gurtler

et

al.,

1991;

Navarro et

_{al., 1992;}

Vaneechoutte et

al., 1992, 1993;

Ponsonnet and

_{Nesme, 1994a;}

Nesme et

al.,

1995;

Laguerre

et

_{al., 1996;}

Latour et

_al.,

₁₉₉₆₎

have

_proposed

to evaluate the

_validity

of the

_analysis

of the restriction

_{fragment length polymorphism}

(RFLP)

of the 16S rRNA _gene

_following

its

_{amplification}

_by

a PCR-based

protocol.

Compared

to the

_{sequencing technique,}

the PCR-RFLP

_analysis

appears a

_{priori faster, cheaper}

and accessible to field laboratories.

However,

its

_{discriminating}

_capacity

has to be

_appreciated.

To address this

question,

we evaluated the

_validity,

sensitivity

and resolution limits of the PCR-RFLP

analysis

of the 16S RNA _gene with

_respect

to the identification of a limited number of bacterial taxa

_including

the _genera

_{Agrobacterium}

_amongst

the

alpha

subclass of

Proteobacteria,

and

_{Xanthomonas, Stenotrophomonas}

and Pseudomonas

_amongst

the _gamma subclass of Proteobacteria. We also

report

data that showed the

suitability

of the

_technique

in

_tracking

the

_biodiversity

of microbial communities from the

rhizosphere.

2. MATERIALS AND METHODS 2.1. Bacterial strains

The bacteria

_{investigated by}

the PCR-RFLP method

belonged

to

_readily

de-fined

_genospecies

of

_{Agrobacterium,}

Pseudomonas

_(of

the so-called P.

_syringae

group),

Xanthomonas and

_{Stenotrophomonas}

characterised

_by

biochemical

traits and DNA

_pairing

studies. Strains were from the collection of our

lab-oratories,

from the Collection

française

des bactéries

_{phytopathogènes}

_(CFBP,

Inra-Angers,

France)

and from the Laboratorium voor

Microbiologie,

Gent Cul-ture Collection

_(LMG,

Gent,

Belgium).

Strains were also collected from the root

_system

of the

_{plants by washing}

the roots in sterile distilled water

₍₁₀

mL of water

_g-

₁

of

_root)

with

_vigorous

shaking

for 1 min. Washed roots were eliminated and dilution series of the

bacterial

_suspensions

were

performed.

Aliquots

were

_plated

onto modified LB

400

_{¡.tg.mL -}

₁

_{cycloheximide.}

Plates were incubated for 1 week at 28 °C and

inspected

every

day

for the appearance of bacterial colonies. Colonies were

purified

on the same medium to

_homogeneity.

2.2.

_{Amplification}

of the 16S _gene

For

_{Agrobacterium}

and

_Xanthomonas,

the 16S rRNA gene was

amplified

exactly

as described

_by

Nesme et al.

_(1996),

_{by using genomic}

DNA isolated

by

the

_protocol

of Marmur

_(1961),

and the

prokaryote specific primers

used for PCR-FGPS6

_(5’-GGA

GAG TTA GAT CTT GGC TCA

_G-3’)

and FGPS1509’

(5’-AAG

GAG GGG ATC CAG CCG

_CA-3’),

_designed

from the conserved

regions

of the 16S and

_{allowing amplification}

of 99.5

%

of the gene. For Pseudomonas

_strains,

PCR was

performed

on frozen bacterial

_{pellets processed}

for 4 min at 94 °C

_(ca.

10

7

cells _per

_{amplification}

_reaction)

_using

the

_following

PCR reaction mixture:

_{Tris-HCI, pH}

_{9.0, 10}

_mM;

KCI 50

mM; MgCl

2

1.5

_mM;

dNTP 200

_vM

_(each);

_primers

1

_R

_M

_(each);

and

_Taq

DNA

_polymerase

0.5 U

(Appligène,

Illkirch,

France).

Amplification

cycles

were as follows: 1 min at

94 °C,

1 min at 55 °C and 2 min at 72 °C

completed

by

a 3-min

elongation

at 72 °C. Primers were rDl

(19-mer)

and fDl

(20-mer)

as described

_by

Weisburg

et al.

_(1991).

The 16S rRNA _gene from the root-associated bacteria

were

amplified

by adding

4

_R

_L

(ca.

10

5

cells _per

amplification

reaction)

of

a fresh bacterial cell

_suspension

to 100

_!tL

of the reaction mixture described later.

_Cycles

were as follows: bacterial cell

lysis

and DNA denaturation were

performed

for 5 min at

_{95 °C,}

followed

_by

five

_{amplification}

_cycles

₍₃₀

s at

95 °C;

45 s at

_{60 °C;}

2 min at

_{72 °C)}

and 30

_cycles

₍₁

min at

_{95 °C,}

1 min at 60 °C and 2 min at 72

_°C).

Reactions were terminated

_by

a 10-min incubation at 72 °C. The

_{amplification}

was

_{performed using}

the set of

_primers

FGPS6 and

FGP1509’

described earlier.

_Typically,

the PCR reaction mixture consisted of

’I!is-HCl, pH 9.0,

10

_mM;

KCI 50

_mM;

_MgCl

₂

1.5

_mM;

_gelatine

0.2

_mg/mL;

dNTP 200

_[tM

_(each);

_primers

0.1

_!M

_(each);

and

_Taq

DNA

_polymerase

0.4 U

(Appligène,

Illkirch,

France).

2.3. RFLP

_analysis

Restriction

_digests

were

_{performed using}

different endonucleases

_following

standard

_procedures

and the instructions of the _enzyme manufacturers. Re-actions were

_performed

in a final volume of 10 to 50

_R

_{L, using}

1 to _{30 vg} of PCR

_products

and the relevant amount of _{restriction enzyme.} Restriction

fragments

were

separated

in 1.5 to 3

%

standard _agarose

gels.

The sizes of the

_fragments

were

_{analysed by}

_comparison

with the 123

_bp

molecular

_weight

marker from Gibco BRL

_{(Gaithersburg,}

_USA)

or with a

₍

_D

_X174/Haelll

molec-ular marker

_(Eurogentec,

_Seraing,

_Belgium).

Sizes were deduced either

_by

vi-sual

_inspection

or

_by

_computer

_analysis

_using

the software Taxotron

(Institut

Pasteur, Paris,

France),

Bioprofil

and

_Biogene

_{(Vilbert-Lourmat,}

_France).

The restriction _maps of Xanthomonas and

_{Stenotrophomonas}

were determined from the observed RFLP

_{by comparison}

with those inferred from

published

rrs se-quences of reference strains. Restriction site differences were

_{analysed by}

the

Wagner parsimony

method

_using

the MIX software of the PHYLIP

_package

to construct

_dendrograms

_using

methods of Fitch and

_Margoliash,

UPGMA,

neighbour-joining

and maximum-likelihood as described

_by

Felsenstein

(1992).

Numerical

_analysis

based on

presence/absence

of restriction

fragments

were

performed by using

the Jaccard coefficient and UPGMA

_(Sneath

and

_Sokal,

1973).

2.4.

_Ribotyping

Standard

_ribotyping

was

_{performed essentially}

as

previously

described

by

Grimont and Grimont

_(1986).

Bacterial DNA was

_prepared

as described

_by

Glickmann et al.

_(unpublished

_report)

for

_{Gram-negative}

bacteria and

_by

the method of Dubnau and

_Cirigliano

₍₁₉₇₂₎

for those

_exhibiting

a variable

Gram reaction. Genomic DNA

preparations

of the clones were submitted

to endonuclease

_digestion

_using

either

EcoRI,

BamHI or HindIII restriction

enzymes. DNA

fragments

were

_separated

in standard agarose

gels.

Gels were

blotted onto an Amersham

Hybond

N membrane

using

a standard

_protocol.

Hybridisation

was carried out

_essentially

as described

by

Dessaux et al.

(1995)

using

a nonradioactive

probe

which

encompassed

the entire 16S rRNA

gene sequence of

Agrobacterium

strain C58.

Alternatively,

the

ribotyping

was

performed by

RFLP

_analysis

of

_{PCR-amplified intergenic}

spacers

lying

between the 16S

_(rrs)

and the 23S rRNA

_(rrl)

_genes as

_previously

described

(Ponsonnet

and

_{Nesme, 1994a,}

_1994b).

3. RESULTS AND DISCUSSION 3.1.

_Reliability

of the method

With all the

_{assayed bacteria,}

the

experimental

conditions for PCR allowed the

_{amplification}

of a

_single

band of the

expected

size. In

_particular,

this was

true for the unidentified bacteria isolated from the root

_system

of the

_plants

(data

not

_shown).

_Digestion

of PCR

_{products generally}

_gave

_unambiguous

patterns

from which

_positions

of restriction sites could be inferred from the

published

sequences. In rare instances such as with

Stenotrophomonas

strains

LMG 11002 and LMG

10853,

complex

patterns

were obtained

_indicating

the occurrence of Alul and

_{HpaII dimorphic}

sites within a

single

_genome

(Nesme

et

_al.,

_1995).

This confirms the occurrence of RFLP detectable differences

within rrs _operons of a

_given

_genome as

_reported

in other bacterial

species

(Mevarech

et

_{al., 1989;}

Gurtler et

_al.,

_1991).

On the other

hand,

bacteria

belonging

to the same

_genospecies

_gave identical rrs

_{patterns except}

for strain LMG 852 of X. arboricola _pv.

_pruni,

which differed from other members of the

species

by

one restriction site

(table 1).

3.2.

_Validity

of the method

Results confirm that PCR

amplification

of rrs is a versatile method that

can be used to obtain

_large

copy numbers of this gene from various known and unknown soil-borne and

plant-associated

bacteria. The

_analysis

of the

polymorphism

of the restriction

_fragments

of rrs of various

_{organisms led,}

however,

to variable conclusions

_according

to the considered taxa. These are

3.3. Xanthomonas and

_{Stenotrophomonas}

Genera Xanthomonas and

_{Stenotrophomonas belong}

to the _gamma sub-class of the Proteobacteria

_(Woese,

_1987).

Xanthomonas spp. contains

species

causing

diseases on various

_plants,

while bacteria

_belonging

to the related genus

Stenotrophomonas

(formerly

Xanthomonas

maltophilia)

are not

_plant

pathogens

in

_spite

of their

frequent

isolation from

plant

material.

Twenty-one

genospecies

have been delineated _among the former _genus Xanthomonas

(Vau-terin et

al., 1990,

1995),

amongst

which 20 were retained in the _genus; the others

were transferred to the new _genus

Stenotrophomonas

(Palleroni

and

_Bradbury,

1993).

Results of rrs PCR-RFLP

_analysis

with 14 endonucleases showed that the 16S genes of the bacteria

belonging

to the genera

Xanthomonas,

Stenotro-phomonas

and

_Xylella

shared 69 common restriction sites. This is much more

than the number of sites shared with _any other _genus of the same subclass

(not shown).

This

result, therefore,

confirms the close relatedness of

Xylella

and Xanthomonas

_{(sensu lato)}

_already

shown

_by

Wells et al.

_(1987).

Howe-ver,

eight

restriction sites were found almost

_exclusively

in Xanthomonas and

_{Stenotrophomonas}

_spp. and ten

_exclusively

in

_{Xylella, allowing}

the clear differentiation of _{the latter genus}

_(Nesme

et

_al.,

_1995).

Numerous

_genospecies

of Xanthomonas could not be differentiated on the basis of their rrs RFLP

profiles,

and several exhibited

only

one or two restriction site differences. These results

_clearly

show that the PCR-RFLP method allows

_only

the

identification of _groups of related

_species

rather than that of well-defined

species

of Xanthomonas spp.

Conversely,

all nine strains of

Stenotrophomonas

yielded

distinguishable

patterns

(table 1).

Data were

_precise

_enough

to

_provide

unambiguous

trees of

pattern

relationships

with the

_parsimony

method as well as with distance-based

_{methods, Fitch-Margoliash,}

neighbour-joining,

UPGMA and maximum-likelihood.

_Furthermore,

_phylogenetic

distances calculated from rrs PCR-RFLP results

_strongly

suggested

that several

_genospecies

_{may exist} within the so-called

_species

S.

_maltophilia,

a result which

_exemplifies

the

discriminative power of the method within this taxon

_(Nesme

et

_al.,

_1995).

3.4. The Pseudomonas

_syringae

_group of

_species

Results similar to those obtained with Xanthomonas _spp. were found for Pseudomonas

_syringae,

a _group of oxidase

_negative

_species

that also

_belong

to the _gamma subclass of Proteobacteria

_{(Woese, 1987).}

The P.

_syringae

_group

gathers

essentially

plant

pathogenic

bacteria. The current classification of this

species

was

_originally

based on the nature of the host

plants

infected

by

the bacteria

_(Schaad,

_1982;

_Young

et

_al.,

_1992),

a criterion which allowed

pathologists

to sort isolates into

_pathovars

_(strains

of the same

_pathovar

were able to infect the same host

plants).

This classification has

recently

been re-examined in the

_light

of molecular data.

_Young

et al.

₍₁₉₉₆₎

list over 50

pathovars

of P.

_syringae,

three

_pathovars

of P. savastanoi and four related

species

(P.

amygdalii,

P.

_{caricapapayae,}

P.

_ficuserectae

and P.

_meliae).

DNA

RFLP of

_{PCR-amplified}

rrs was

_{analysed by}

_using

13 endonucleases. Eleven

enzymes

generated

near or identical

_patterns,

and

only

jffm

/1

and ALuI

generated

more variable

_patterns.

Analysis

of 42 strains

belonging

to the nine

_genospecies

revealed that strains

_belonging

to one

_genospecies

_may cluster with strains of

other

_genospecies

and may even share identical

_patterns

(figure 1).

Surprisingly,

however,

the rrs-based

_phenogram

is not

_congruent

with the DNA

_pairing

studies,

as

_{exemplified by}

the _pv.

_pisi

strain

_{(genospecies 1),}

which

_gathers

with most members of

_genospecies

3 in one

cluster,

and

by

the _pv.

_persicae

strain

(genospecies

3),

which

_gathers

with most members of the

_genospecies

1 in a remote cluster. This

_apparent

lack of _{congruency may} be caused

_by

horizontal transfers of rrs from one

_genospecies

to the other after their

_speciation,

or

_by

a

_{high degree}

of reverse mutations in this _gene. Neither

_hypothesis

fits with our view of bacterial evolution.

Furthermore,

if

_true,

our

_hypothesis

would

strongly

argue

against

the

_application

of rrs to

_ground

bacterial

_phylogeny

as

proposed by

Woese

_(1987).

_{However, in}

our

_opinion,

the

_apparent

_incongruency

of the

_present

results should be considered _very

_cautiously

because differences between

_patterns

were not

_analysed

in terms of

_polymorphic

restriction sites

(since

relevant rrs _sequences are

_lacking)

but in terms of restriction

_fragment

length using

an automated device.

Furthermore,

the UPGMA method used to construct

_dendrograms

is not

_fully

_appropriate

to

_phylogenetic

studies. The above

_{results, however,}

are consistent with the detection of a _very small

number

_{of polymorphic}

restriction sites in rrs and in rrl in the

analysed species

(Manceau

and

_Horvais,

_1997).

With these

_{reservations,}

results obtained with the

_genospecies

related to P.

_syringae

are identical to those described earlier with Xanthomonas spp. For both taxa, the rrs PCR-RFLP method can be

used

_only

to

_identify

a _group of

_closely

related

_genospecies

and not a

_given

genospecies.

That the

_genospecies

of P.

_syringae

and related

_species

defined on the basis

of DNA

_pairing

studies cannot be differentiated on the basis of distinctive

phenotypic

characters creates another

_discrepancy

in the classification of this group of bacteria. Taken

together,

these data and the results

generated by

rrs

PCR-RFLP

_analysis

_may

_impede

the

_recognition

of the

genospecies

defined

previously

as bona

fide

new

_{species. However,}

the current division in nine

genospecies

remains in

_agreement

with the results obtained

_by

classical

_ribotype

analysis

(Gardan

et

al., unpublished

data)

and

_by

PCR-RFLP

_analysis

of the

intergenic

spacer located between rrs and rrl

(Manceau

and

_Horvais,

1997).

It is

_tempting

to

_speculate

_that,

in contrast to the slow

_evolving

rrs or rrl _genes,

noncoding

sequences in the

vicinity

of these genes may constitute

’fast-running

molecular clocks’ suitable for

_analysis

of the relatednesses of

_closely

related

genospecies.

Finally,

the identification of six P.

_fluorescens

and three P.

_putida

biovars

(Laguerre

et

_{al., 1996;}

Latour et

_al.,

₁₉₉₆₎

_provides

additional information

on the method in the _genus Pseudomonas. These authors showed that rrs

PCR-RFLP

_analysis

and biovar classification is coherent. This

_suggests

_that,

contrary

to what we found with P.

_syringae,

a

_strong

polymorphism

can be

evidenced between members of P.

_fluorescens

and P.

_{putida. However,}

the

present

classification of the two latter

_species

is based _upon biochemical traits and remains uncertain.

Thus,

in our

hands,

DNA

_pairing

studies showed that

as low as 10

%

within the biovar IV alone

_{(unpublished data).}

_Thus,

we

assume that the resolution of the method is

_probably

similar whatever the

species

of Pseudomonas and that inconsistencies

_{merely originated}

from

_species

definitions,

which are based _upon DNA

_pairing

with P.

_syringae

and related

3.5.

_{Agrobacterium}

The _genus

_{Agrobacterium}

is a member of the Rhizobiaceae

_family

(Kersters

and De

_Ley,

_1984),

which has been included in the

_alpha

subclass of Proteobac-teria on the basis of ribosomal characteristics

(Woese

et

_al.,

_1984a).

Several members of the _genus induce

_hyperplasic

disorders _{in many} kinds of

_plants,

which are known as crown

_{gall, hairy}

root or cane

_gall

diseases. The

pathogenic-ity

is almost

_entirely

encoded

_by

transferable Ti or Ri

plasmids

(Ream,

1989),

and the delineation of

_species

was, until

recently,

based on

_pathogenic

traits

(Kersters

and De

_Ley,

₁₉₈₄₎

encoded

_{by plasmids}

that are transferable between strains

_(Van

Larebeke et

_al.,

_1975).

_{Independently}

of the

plasmid

content of the

_strains,

several biovars have been described on the basis of chromosomal

(and

therefore more

_stable)

characters

_(Keane

et

_{al., 1970;}

Kersters et

_{al., 1973;}

Kerr and

_{Panagopoulos, 1977;}

Holmes and

_Roberts,

_1981).

DNA

_pairing

studies showed that different biovars

_belong

to different

_genospecies

_(Heberlein

et

_al.,

1967;

Kersters and De

_{Ley, 1984;}

_Popoff

et

_{al., 1984;}

_Ophel

and

_Kerr,

_1990).

This observation led to a revised nomenclature of the _genus

(Ophel

and

_Kerr,

1990;

Sawada et

_{al., 1993; Bouzar,}

_1994),

in which biovar 1 is now

_designated

A.

_tumefaciens,

biovar 2 A.

_rhizogenes

and biovar 3 A. vitis. A fourth

_species

consisting

of

_only

three strains is known as A. rubi. In

_addition,

some strains

(like

NCPPB1650,

and

_AF3.10)

can be

_separated

from other

_agrobacteria

to constitute a nuclei around which future

Agrobacterium

isolates _may cluster

(Kersters

et

_{al., 1973;}

Bouzar et

_al.,

_1995).

_{Nevertheless,}

the

_genospecies

sta-tus of the biovar 1

_{(characterised}

_by

the

_production

of

_{3-ketolactose)}

is more

complex

than

_generally

considered in recent taxonomic revisions. For

_instance,

biovar 1 includes strains like

_TT111,

which is

_genetically

_only

50

%

homolo-gous with the reference strain B6

(Kersters

et

_al.,

_1973).

The occurrence of nine

_genospecies

within biovar 1 was

_{unequivocally}

established

_{by Popoff}

et al.

(1984).

DNA

_pairing

studies

_{performed using}

nuclease Sl and ATm

_analysis

made it

possible

for these authors to show that strain B6 and bacteria related to TT111 indeed

_belong

to different

_genospecies.

The

_phylogenetic

relation-ships

of members of the _genus

_{Agrobacterium}

were studied

_by

rrs

_sequencing

in several research _groups

_(Sawada

et

_{al., 1993;}

Willems and

Collins,

1993;

Yanagi

and

_{Yamasato, 1993;}

Bouzar et

_al.,

_1995).

All identified

_genospecies

of

Agrobacterivm

could be

distinguished

on the basis of their rrs _sequences. Re-sults also showed that the two

_closely

related

_genospecies

defined

_by

strains B6 and TTIII differed

_by

ten nucleotides in their rrs _sequences

(table 11).

A sim-ilar distance has been detected between a

_{representative}

strain of A. rubi and strain

_NCPPB1650,

which

_{probably belong}

to different

_species

as

_judged

_by

the

analysis

of various

_phenotypical

traits

_(Kersters

et

_al.,

_1973).

This

_assertion,

however,

remains to be confirmed

by

DNA

_pairing

studies.

Interestingly,

that A.

_tumefaciens,

A.

_rhizogenes

and A. vitis are

_readily

identifiable

_using

rrs PCR-RFLP was demonstrated

_prior

to the

_availability

of the

_{corresponding}

rrs _sequences

(Ponsonnet

and

Nesme,

1994a).

Furthermore,

several rrs

_patterns

were obtained within members of biovar

_1,

_confirming

the

that all the tested _sequences can be

_{distinguished by}

means of rrs

_PCR-RFLP,

even when the number of nucleotide substitutions is as low as ten

_(e.g.

as

observed between

_closely

related B6 and T111

_genospecies

or between strain

NCPPB1650 and A.

_rubi;

table

_77).

The use of _{22 enzymes is} not

_possible

for the _purpose of routine identification.

Thus,

considering

both the resolution

properties

of _agarose

_gels

_(fragments

smaller than 75

_bp

cannot be

_easily

seen

in

_gels)

as well as the cost

_{of endonucleases,}

we found that

_HpaII

(or MspI),

Alul

and HaeIII would be the smallest and

_cheapest

set of four-base endonucleases

required

to

_identify

all the

_species

_{Agrobacterium}

on the basis of the RFLP

profiles

of their rrs _gene

(table 77).

With such a limited set _{of enzymes, the} average

price

for

_species

identification is about 4 ECU per

isolate,

which is

cheaper

than _every conventional

_gallery

method. Results of the simulation were

confirmed with all tested strains

_(data

not

_shown),

and this method is now of routine use in _many laboratories

_studying

wild isolates of

Agrobacterium

_spp.

In

_addition,

the PCR-RFLP

_analysis

of the ribosomal

_{region consisting}

of the rrs _gene

plus

the

intergenic

_spacer

(IGS,

located between rrs and

_rrl,

i.e. the

23S rRNA

_gene)

allowed us to obtain both a clear identification of the

_species

of

_{Agrobacterium -}

_by

the mean of rrs

_{fragments -}

and a _very accurate

_typing

of the isolates at the strain level -

by

means of IGS

fragments

(Ponsonnet

and

Nesme,

1994b).

This PCR-RFLP

_equivalent

of the classical

_ribotyping

coupled

to PCR-RFLP

analysis

of

_{pTi regions}

now constitutes a

_powerful

tool for

_{epidemiological}

studies on crown

gall

dissemination

(Pionnat

et

al.,

1996).

Thus,

for

_{Agrobacterium}

_spp., the results

_clearly

showed that this method allows the discrimination of strains

_belonging

to different

_genospecies.

The method should also

_permit

the identification of strains of the new

Agrobac-terium _sp., whose

_original

representatives

were isolated from

_{galled fig}

trees in Florida

_(Bouzar

et

_al.,

_1995;

_{Vaudequin-Dransart}

et

_al.,

_1995).

The

_sequencing

of the rrs of one of these

_’fig

strains’ indeed revealed that the rate of nucleotide substitutions

_ranged

from 1.8 to 2.7

%

when the 16S rRNA _gene of the

_fig

strain was

compared

to the

_{corresponding}

genes of A.

tumefaciens

and A.

rubi,

whereas the rate of nucleotide substitutions of the rrs _genes of these two

_closely

related

species

only

reached 1.5

%

_(Bouzar

et

_al.,

_1995).

This result is of inter-est since

_fig

strains and A,

_tumefaciens

strains were not

_{easily distinguishable}

on the basis of other

_{morpho-biochemical}

characters such as total

_fatty

acid

composition.

The

_validity

of the PCR-RFLP method was also

exemplified

by

the

_analysis

of the

_{Agrobacterium}

rrs _sequences available from the data base.

Amongst

those,

the

_digestion

in vitro and in silico revealed that two of these sequences were obtained from misidentified

Agrobacterium

_strains,

originally

identified as A.

_tumefaciens.

_Using

a selected set of _{12 enzymes,} these two iso-lates were

_{unambiguously}

associated with other strains of the A.

rhizogerces

species

to which

_they

indeed

_belong

_(Oger

et

_{al., unpublished}

_data).

Laguerre

et al.

_(1994b)

extended the rrs PCR-RFLP

analysis

to most members of the Rhizobiaceae

_{by examining}

48 strains

_representing

the

_eight

recognised

Rhizobium

_species,

two new Phaseolus bean

_Rhizobium,

Bradyrhi-zobium spp.,

Agrobacterium

spp. and unclassified rhizobia from various host

plants. Twenty-one composite

patterns

were obtained from the combined data

detection of new data. It can thus be concluded that rrs PCR-RFLP allows the delineation of most

_species

_among Rhizobiaceae members.

3.6. Plant-associated bacteria

The

_previously

mentioned studies

_exclusively

involved known bacterial

species

and therefore

yielded

data on the

_validity,

_sensitivity

and resolution limit of the PCR- RFLP

_technique

with

_respect

to these

_species

_only.

Soil-borne and

_{plant-associated}

bacteria

_{being extremely}

diverse,

we feel that there is a need for a more

_general

evaluation of the

_validity

of the PCR-RFLP

_technique.

To address this

_question,

we took

_advantage

of

_previous

studies

_performed

on bacteria associated with the root

_system

of the

_{legume plant}

Lotus cornicu-latus

_(Oger

et

_al.,

_1997).

These bacteria were first identified

_using

morpho-logical

and biochemical criteria. Five

_{morpho-biochemical}

_(MB)

_groups were evidenced but their

_precise

identification met with limited success. We used

this material to

_investigate

whether the PCR-RFLP

_technique

could

_provide

us with more accurate data and whether these would be in

_agreement

with the results of other

molecular,

high-resolution

identification

_techniques.

Re-striction _enzymes

_enabling

the efficient discrimination of isolates of unrelated

species

were selected

by

a

_computer

simulation of the

_digestion

of rrs of four bacteria

_belonging

to the

_species

Escherichia

_coli,

Pseudomonas

_aeruginosa,

Frankia _sp. and

_{Agrobacterium tumefaciens.}

Twelve

_inexpensive

_enzymes were retained because

_they

appear to

generate

quite

different restriction

_patterns.

As described

_earlier,

this set of _enzymes also allowed the differentiation of four related

_species

of

_{Agrobacterium:}

A.

_tumefaciens,

A.

_rubi,

A.

_rhizogenes

and A.

vitis,

both in silico and in vitro

_(Oger

et

_{al., unpublished}

_data).

The set of _{12 enzymes} was used to sort 57 clones isolated from the

rhizo-sphere

of Lotus

_plants.

Seventeen RFLP groups were obtained. Most of them exhibited very distinct

patterns,

whereas others were

_{distinguished only by}

a

single

enzyme. In this latter case, the ’molecular

proximity’

of these bacteria was

_always

correlated with their

morpho-biochemical ’proximity’

(table III).

To validate the classification obtained

_by

the PCR-RFLP

_technique,

we sorted the 57

_rhizospheric

isolates on the basis of classical

_{ribotyping, using}

the enzymes EcoRI and HindIII. Results were

_compared

to those

_{generated by}

the PCR-RFLP method.

_Remarkably,

a

_{given ribotype}

was

_always

associated with a

single

PCR-RFLP _group and was never shared

_by

bacteria

_exhibiting

different

rrs

_patterns.

This was also true for the

_closely

related RFLP _groups whose de-lineation relies on the

_{profiles generated by}

a

single

restriction enzyme: a

_given

ribotype

was

_clearly

associated with each of these

_closely

related PCR-RFLP groups. The reverse

_{proposition, however,}

was not true. Isolates clustered in a

given

PCR-RFLP _{group may} exhibit one or more

_ribotypes.

This situation is in full

agreement

with the results described earlier with Pseudomonas _spp.

Some root-associated isolates were identified as Rhizobiaceae and

Corynebac-terium on the basis of their

_{respective morpho-biochemical}

characters.

Inter-estingly,

an excellent correlation was obtained between the

_computerised

and

experimental

data for the isolate of the

_{family Rhizobiaceae,}

_{which appears} to

_belong

to the _genus

_{Agrobacterium.}

_Conversely,

the rrs PCR-RFLP of the

This table shows the

_{relationships existing}

between the groups defined

_by

the various methods used in this paper. MB refers to the different

_{morphological/biochemical}

groups, 16S groups to those obtained

by

PCR-RFLP

analysis

of the 16S rRNA gene, R and

R’

groups to the EcoRI and HindIII

ribogroups, respectively. Group

numbers were

_{arbitrarily given}

and do not reflect the

possible

relatedness of the various

_profiles

except for groups G6a to

_G6e,

which are

_closely

related. nt.: not tested.

Within this

taxon,

however,

our

_experimental

_patterns

_appeared

related but still dissimilar to those obtained from members of the genus Clavibacter.

In-terestingly,

our isolate and most members of the _genus Clavibacter share some

morpho-biochemical

characters

_(such

as Gram variable

staining)

and the

prop-erty

of

_{being plant-associated}

bacteria

_(Oger

et

_al.,

_unpublished

_data).

Thus,

the PCR-RFLP

_technique

_proved

to be _very useful in

_sorting

unknown

plant-associated

bacteria.

_Contrary

to what was observed with the P.

_syringae

strains,

the results obtained

_by

the PCR-RFLP

_analysis

of the 16S _gene were

in excellent

agreement

with both

morpho-biochemical

data and

_ribotyping,

though

the PCR- RFLP method

_appeared

less discriminate than the

ribotyping

under our

_experimental

conditions.

Results obtained in this

_part

of the

study

do not

_permit

us to determine

precisely

the resolution of the PCR-RFLP

_technique

with bacteria isolated from a

_community.

This

_question

is under further

investigation

and will be answered

_only

once the relevant

_{microorganisms}

have been identified

(by

the

analysis

of the rrs _sequence of a

_{representative}

member of each 16S

group).

identification of bacteria is not

_always

a

_prerequisite

in the assessment of the evolution of microbial communities.

3.7. Resolution limits of the method

Clearly,

these results showed that differences in rrs are

_great

_enough

to per-mit the identification of

_species

of

_{Rhizobiaceae,}

but

_only

of _groups of

_species

closely

related to Pseudomonas

_syringae

and Xanthomonas _sp. A

_question

then arises: do these results indicate that the rrs molecular clock runs faster in Rhi-zobiaceae than in Xanthomonadaceae?

_Perhaps

and

_probably

not.

_Genospecies

of

_{Stenotrophomonas}

and Rhizobiaceae are

_{efficiently distinguishable}

_by

the method and both

_{Stenotrophomonas}

and Rhizobiaceae are isolated from soil or from material which somehow

_{’trapped’}

the bacteria. The

_ecology

of

Agrobac-terium

_(and

that of other

_{Rhizobiaceae),}

which involves

_frequent

contacts with the soil

_microflora,

would favour the

_{diversity emphasised by}

the rrs PCR-RFLP method and

_by

_ribotyping,

a case identical to that of the fluorescent Pseudomonas isolated from soils which exhibit _very dissimilar rrs PCR-RFLPs

(Laguerre

et

al., 1996a; Latour,

1996).

On the other

_hand,

P.

_syringae

and

re-lated

_species

as well as

_pathogenic

xanthomonads are _very often isolated from

plant

aerial lesions caused

_by

clonal dissemination of the bacteria but not

_by

trapping

from the rich soil reservoir. This latter situation could lead to the

fre-quent

isolation of identical or

_closely

related bacterial clones as deduced from

the occurrence of bacteria of identical

_ribotype

in different

_biotopes.

In our

opinion,

the various levels of resolution observed between taxa should be at-tributed to

_sampling

_procedures

and bacterial

_origins

rather than to molecular clock differences between taxa. It is therefore

_tempting

to conclude that the

res-olution limit of the rrs PCR-RFLP method lies at the group of related

species.

These results

_might

be extended in the near future to the Rhizobiaceae in

_light

of the DNA

_pairing

studies

_{performed by Popoff}

et al.

₍₁₉₈₄₎

on

agrobacteria

belonging

to the biovar 1.

3.8.

_Sensitivity

of the method

The

_sensitivity

of the method could be evaluated

_by

the minimum number of

_polymorphic

restriction sites

_required

to ensure that two bacteria

belong

to different

_genospecies.

_Ideally,

this should not

_depend

upon the taxa consid-ered and should be based upon the fact that bacteria of the same

_genospecies

harbour _very similar rrs. Some conclusions can be drawn from our

_empirical

results.

_First,

the _presence of two

_polymorphic

sites within the rRNA operons of a

_single

bacterial genome

clearly

showed that a two restriction site

differ-ence between the rrs of two bacteria is not sufficient to demonstrate that

_they

belong

to different

_species.

The role of the rrs

(or

rrs

_product)

similarities in

taxonomy

was clarified

_by

Stackebrandt and Goebel

_(1994).

Their

_comparative

studies indicated that DNA-DNA association studies still remain the

_superior

method that needs to be

_performed

when rrs

_similarity

is 97

%

or

higher.

DNA

pairing

studies are therefore not

_required

when the rrs _sequences differ

_by

44 nucleotides or more. This also means that at least nine

_polymorphic

restriction sites are

_required

to discriminate

_{unambiguously}

_sequences

_differing

_by

44 bases

extremely

conservative

_since,

in our

_hands,

nine restriction site differences could be obtained between strains of Xanthomonas and

_{Stenotrophomonas}

or between

strains of remote

_species

of

_{Agrobacterium}

_{(table 11).}

A less conservative view could be based on our results

_(Nesme

et

_al.,

₁₉₉₅₎

as well as those of Stackebrandt and Goebel

_(1994),

which both show that rrs

_similarity

lower than 99.3

%

_(equivalent

to at least ten nucleotide

_{substitutions)}

_only

occurs

between members of different

_genospecies.

From our

empirical

results obtained

with _{14 enzymes,} five restriction site differences at least are _necessary to ensure

that two rrs differ

_by

at least ten nucleotides

_(figure

_1).

_Thus,

we _propose that five restriction site differences should be sufficient to establish

_by

PCR-RFLP of the 16S rRNA

_gene(s)

that two bacteria

_belong

to different

_{genospecies. Overall,}

our results

_strongly

_support

the view that the

_analysis

of the restriction

profile

of the 16S rRNA gene is a

_rapid

and versatile method to

_analyse

known and unknown bacterial

_populations.

The rrs PCR-RFLP

_study

is

_simpler

and faster than

_sequencing

and is suitable for use in almost _any

_laboratory.

If sufficient enzymes are used and the

_positions

of the

_recognition

sites are

_known,

the

technique

should also allow the accurate

_description

of

_phylogenetic

relatedness.

ACKNOWLEDGEMENTS

The authors wish to thank Marie-Andrée Poirier for skilful technical assistance. P.O. was

_{supported by}

a

fellowship

from the Minist6re de la recherche et de la

technologie.

This work was made possible

by

a _grant from the Bureau des Ressources

REFERENCES

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Request

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