LMW RNA Profiles of Frankia Strains by Staircase Electrophoresis

8/10/2019 LMW RNA Profiles of Frankia Strains by Staircase Electrophoresis

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System. App . Microbio . 21, 539-545 (1998)

SYSTEI\IL TIC ND

__G_us_ta_v_Fi_sc_he_rV_e_rla_

g

APPLIED

MICROBIOLOGY

nalysis o LMW

RN

Profiles o

rankia

Strains

by Staircase Electrophoresis

ENCARNA

VELAzQUEZ , EMILIO CERVANTES

2

,

JOSE

MARIANO IGUAL2, ALVARO PEIX

2

,

PEDRO F

MATEos\

SAAD BENAMAR

3

, ANDRE MOIROUD\ CHRIS T. WHEELERS,

JEFF DAWSON

6

,

DAVID

LABEDA

7

,

CLAUDINO

RODRIGUEZ-BARRUEC0

2

, and EUSTOQUIO MARTINEZ-MoLINA

1 Departamento de Microbiologia y Genetica, Edificio Departamental, Salamanca, Spain

2

IRNA-CSIC, Salamanca, Spain

3 Laboratoire de Biologie et Physiologie Vegetales et Forestieres, Ecole Normale Superieure, Bensouda, Pes. Maroc

4 Universite Lyon 1 Ecologie Microbienne, Villeurbanne Cedex, France

5 Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow,

Scotland

Department of Natural Ressources and Environmental Sciences, University of Illinois, 1005a Plant Sciences Lab, Urbana, IL

7

National Center for Agricultural Utilization Research, 1815

North

University, Peoria,

IL USA

Received July 21 1998

Summary

An optimized technique of polyacrylamide gel electrophoresis, Staircase Electrophoresis (SCE), was ap

plied to determine the stable Low Molecular Weight RNA (LMW RNA) profiles of 25 Frankia strains

from diverse geographic origins and host specificity groups as well as species from other actinomycete

genera. Application of the technique permits the rapid identification of

Frankia

strains and their differ

entiation from other actinomycetes. The isolates used in this study were grouped in eight clusters, each

comprising strains with identical LMW RNA profiles. Comparison of these results with others obtained

from DNA sequences or DNA hybridization methods suggest a high degree of complexity

in

the genus

Frankia. Application of

SCE

to profile LMW RNA should in the future facilitate biodiversity studies of

Frankia and discrimination of new species.

Key words: Frankia - LMW RNA - Nitrogen fixation - Bacterial taxonomy

ntroduction

Bacteria belonging to the genus Frankia

are slow

growing actinomycetes

characterized

by

their capacity to

fix

atmospheric nitrogen

in symbiotic associations result

ing

in the

formation

of

nodule structures in

the

roots

of

many perennial woody dicotyledoneous plants.

Since

the

first reproducible report of isolation of a

Frankia strain in 1978 (CALLAHAN et a ., 1978) several

hundred of

isolates have been obtained for study in di

verse laboratories throughout the world

and

consider

able progress has been made in

understanding

many as

pects of the Biology of these polymorphic bacteria (BEN

SON and

SYLVESTER,

1993). As for Rhizobium

the

first at

tempts to establish a classification of

the

genus Frankia

were based on

their

host range (BEeKING, 1970)

and

di

verse

phenotypic

characteristics (LALONDE et a ., 1988).

More

recently, various techniques, based

on

molecular

biology protocols, have been used for this

purpose.

Studies of

DNA

relatedness, based

on

DNA-DNA re

association kinetics, revealed

the

existence

of at

least

nine genomic species

(FERNANDEZ

et a .,

1989).

Among

these, three contained strains

compatible

with the Alnus

specificity group five with the Elaeagnaceae and one

with

Casuarina. Genomic

species 1 was proposed to be

Frankia alnii the type species of the genus. These studies

are hampered by

the

difficulty to isolate and

grow

some

strains in laboratory media as well as by the low efficien

cy of DNA extraction

from

Frankia cultures.

Other approaches

to

allocating

Frankia

strains

to tax

onomic groups were based on

the

comparative analysis

of PCR amplified sequences, an approach which also fa-

Abbreviations:

LMW

RN

-

Low Molecular Weight RNA;

SCE

-

Staircase Electrophoresis


2/7

540 E. VELAzQUEZ et al.

cilitated the characterization of uncultured Frankia

strains, for which template DNA was directly obtained

from the nodules. NAZARET et al. (1991) developed a

protocol based on

DNA

amplification and sequencing of

the 268 base pairs corresponding to partial ribosomal

DNA sequences among eight of the previously described

genomic species. Their work included the elaboration of

a phylogenetic tree, showing a close relatedness among

strains belonging to the

Casuarina

and

Alnus

infectivity

groups, with both groups well separated from the

Elaeagnus

infectivity group. Atypical strains.

ORS020602 (Dll) and 55005 (DBB02060510) isolated

from, but unable to reinfect Casuarina plants, were

grouped close to Elaeagnus infective strains. In general,

strains from

Elaeagnus

groups showed less diversity than

those in the Alnus groups.

From an analysis of PCR amplified sequences corre

sponding to 23S rRNA, HONNERLAGE et al. (1994) de

scribed seven groups, four related to the

Alnus

compati

bility group, one including strains related

to

Casuarina

compatibility group, another including

Elaeagnus

related

strains and finally, a group consisting exclusively of the

uncultured endophyte of Coriaria

nepalensis.

These re

sults do not match exactly the results of the DNA-DNA

reassociation experiments. Thus, different strains sepa

rated by a short distance in the phylogenetic tree of HON

NERLAGE et al. (1994) are sometimes included in different

genomic species by FERNANDEZ et al. (1989), whereas

strains separated by longer distances are sometimes

placed together. However, a similar grouping of strains as

related to their host compatibilities

is

observed.

Based on the complete nucleotide sequences of the 16S

rRNA of eight

Frankia

strains, others in databases and

DNA

sequences amplified from nodules,

NORMAND

et al.

(1996) described the clustering of Frankia into four

groups: Cluster 1 included strains that infect Alnus and

Casuarina

species, Cluster 2 included the unisolated sym

biont

of

Dryas, related to the unisolated strains from

Co-

riaria and Datisca, Cluster 3 included Elaeagnus infec

tive Frankia strains and Cluster 4 included unclassified

strains Pdl and atypical strains Cn7, Dc2 and AgBl-9.

Given the present status of

Frankia

taxonomy, alter

native methods may make an important contribution

to

the classification of the genus and determination of the

phylogenetic relationships among diverse strains.

BEYA

ZOVA

and LECHEVALIER (1992) used LFRA to analyze the

phylogenetic relationship among more than 100

Frankia

strains. This technique

is

not particularly easy to master

in order to obtain satisfactory and reproducible analysis

and efforts to optimize other, simpler and faster methods,

may results in significant advances in these studies.

HOFLE

(1988) proposed to use the profiles of Low

Molecular Weight, stable RNA (LMW RNA) in studies

of bacterial taxonomy. The LMW RNAs include the

5S

rRNA and class 1 and 2 of tRNA

HOFLE,

1988). Recent

ly,

a new electrophoretic technique, Staircase Elec

trophoresis (SCE), has been developed that allows opti

mal separation of these molecules and their utilisation in

bacterial taxonomy CRuz-SANCHEZ et aI., 1997). This

new technique has been applied to the differentiation of

species in the family Rhizobiaceae, where different LMW

RNA profiles have been found to correspond well with

the described genera and species VELAZQUEZ et aI.,

1998). In the present work, the LMW RNA profiles of

Frankia isolates from diverse host plants and from di

verse geographic provenances have been analysed.

Materials and Methods

Bacterial strains and media: A description of the Frankia

strains used in this study including the host plant from which

they were derived and their geographic origin is shown in Table

1.

Isolation

of

new strains for the present work was carried

out

by published protocols LECHEVALIER and LECHEVALIER, 1990).

Other actinomycetes obtained from culture colections are

Rhodococcus rhodochrous CECT3046 (ATCC4273), Dacty-

losporangium aurantiacum CEcn288

(ATCC2391),

Strepto-

myces cinammoneus

(former

Streptoverticillium cinammoneus)

CECT3258 (ATCCI11874), Streptomyces kentuckense (former

Streptoverticillium kentuckense) CEcn262

(ATCC12691),

Micromonospora melanosporea

CECn087

CBS 270.62),

Streptomyces halstedii

NRRL-2381 and

Streptomyces lividans

JI1326. The Frankia strains used in this study were cultivated in

Qmod medium LALONDE and CALVERT, 1979) without glucose

and with pyruvate

at 0,5 .

The cultures were kept

at 25C

for

three weeks. The other actinomycete strains were grown in

YEG medium (Yeast Extract

0,7 ,

Glucose 1 )

at 25C

for a

week.

RNA extraction and LMW RNA profile analysis: The RNA

of the strains studied was extracted as described by HOFLE

(1988). LMW RNA profiles were obtained using Staircase Elec

trophoresis in

14

polyacrylamide gels under denaturing con

ditions in steps

of

10 min, rising through a constant ramp with

50 V increases from 100 V to

2300

V as reported earlier

CRUZ

SANCHEZ

et aI., 1997).

The

following commercial molecules

from Boehringer Manheim (Manheim, Germany) and Sigma

St.

Louis, MO , USA) were used as reference: 5S rRNA from

Escherichia coli MRE 600

(120

and

115 nucleotides)

BIDLE

and

FLET

C

HER,

1995),

tRNA

specific for tyrosine from

E coli

(85 nucleotides) and tRNA specific for valine from E

coli 77

nucleotides)

SPRINZL

et aI., 1985). Samples were prepared as

reported elsewere

CRuz-SA

NCHEZ et aI., 1997). After elec

trophoresis, the gels were silver-stained as described by

HAAS

et

al. (1994).

Data analysis and construction of dendrograms: The bands

present in each profile were coded for input into a data base

that included all the strains studied and Jaccard's similarity

coefficient was calculated to construct the distance matrix. A

dendrogram was constructed from the distance matrix using the

Unweighted pair Group Arithmetic

Mean

(UPGMA).

Results

RNA

extraction

and LMW RNA profile analysis

The LMW RNA profiles of the strains used in this

study are shown in Figure 1. All contain the three expect

ed zones: 5S RNA, class 2 tRNA and class 1 tRNA

CRuz-SANCHEZ et aI., 1997). The number of bands pre

sent in the tRNA zone was as expected according to pre

vious results obtained for the

E.

coli

strain CECT99

(ATCC9637), using Staircase Electrophoresis

CRUZ-


3/7


4/7

542 E. VELAzQUEZ et al.

CECT3262 (Lane 5),

Streptomyces halstedii

NRRLB-

2382 (Lane

6)

and Streptomyces lividans JI1326 (Lane 7).

Data analysis and construction of dendrograms

Construction of a dendrogram (Figure 2) revealed two

major groups with a similarity coefficient of only 0.24.

One contains

Micromonospora

and

Dactylosporangium

and the other group contains the rest of the actino

mycetes (including Frankia strains) studied in this work.

This second group

is

further divided in two clusters with

a similarity coefficient of 0.3, one contammg all the

Frankia

strains and the other the remaining actino

mycetes.

I

I

I

I

0 2 0 5

I

I

The

Frankia

strains used in this study are separated in

two groups with a similarity coefficient of 0.4. One

group includes strains NRRLB-16422 (isolated from

Myrica

gale

and NRRLB-16423 (isolated from

Purshia

tridentata , both strains with the same LMW RNA pro

file

Fig. lA lane 8). The second group

is

subdivided in

two with a coefficient of 0.5. One of these subgroups in

cludes two strains isolated from

Coriaria,

CN3 and

CN7

(Figure

lA

lanes 1 and 2). The remaining

Frankia

strains

are divided in two groups with a similarity coefficient of

0.6. One of them comprises the two strains obtained

from

Hippophae rhamnoides

(Hr 114.2 and

Hr

77.3),

the other

is

further subdivided into two groups with a

similarity coefficient of 0.8. One of these comprises

sev-

CN3

CN7

Hr1l4.2

Hr77.3

UGLOI0701

UGL013103

UGLOI3104

UGLOIl301

ENSOI07l2

ENSOI0714

SI4

AcNI4A

Ag67.5

Ag9S.2

Arll2.2

ORS020602

55005

Ea32.1

CS1020602

CS1020604

CS1020620

C81020621

UGL020603

8 6422

8 6423

RhodocOCCIIS

rhodocrholls

Streptoverticillium

cinamlllOnel 1Il

StreptoverlicilliulII Kenll/keme

Streptomyces haldstedii

Streptolllyces IMdans

Dactilosporangium aurantiacum

Micromonospora lIIonosporea

1

Fig. 2. UPGMA dendrogram

based on Jaccard s coefficient

derived from LMW RNA

profile characteristics for the

actinomycete strains.


5/7

eral strains isolated from

asuarina equisetifolia

UGL020603) and the isolates obtained in Salamanca

CS1020602, CS1020620 and CSI020621) . The remaining

strains are divided between a group

that

includes all the

tested strains from

Alnus

and a final group with two

atypical strains from

asuarina

and strain Ea32.1 from

Elaeagnus

similarity coefficient 0.86).

iscussion

In recent years new techniques based on molecular bi

ology protocols have been applied to the resolution of

traditional questions in microbial taxonomy. The well

known difficulties for isolating and growing in culture

Frankia

strains have led to the application of techniques

which do not require previous steps of isolation and cul

ture of microbial strains in the laboratory HONNERLAG

et al., 1994;

NAZARET

et al., 1991; NORMAND et al.,

1996). Taxonomic studies of this genus have shown in

general the relatedness among strains isolated from the

same host plant, but there are controversial results con

cerning the relative taxonomic position in dendrograms

of strains isolated from diverse host plants.

As

for other symbiotic, nitrogen-fixing bacteria,

namely those in the family Rhizobiaceae, attempts were

made initially to make the definition of species on the

bases of the capacity

to

nodulate a particular host plant

BECKING,

1970). This character in itself

is

not a criteri

um robust enough for classification TORREY, 1990) be

cause particular plants may be nodulated by strains

that

were derived from different host species or even genera.

Table 1

Characteristics

of Frankia

strains used in this study.

LMW RNA

from Frankia 543

In this sense, promiscuous hosts are Elaeagnus angustifo-

lia

and

Alnus glutinosa.

Also, most

Frankia

strains are

able

to

reinfect individuals of the host plant species from

which they were isolated. Nevertheless there are reports

of so called atypical strains,

i. e.

strains unable to infect

the host plant from which they are derived but able

to

in

fect other actinorhizal genera

BAKER,

1987). These re

sults emphasize the need for the application of new taxo

nomic techniques, that in the Rhizobiaceae were decisive

for the description of new genera and species.

The application of the technique of Staircase elec

trophoresis

to

obtain the LMW-RNA profiles in the Rhi

zobiaceae resulted in the reported observations that dif

ferent genera show differences in their 5S RNA whereas

different species of the same genus show differences in

the tRNAs and overall, the LMW-RNA profiles are char

acteristic of each species. Moreover, the analysis of the

corresponding data by UPGMA resulted in similar den

drograms as those obtained by 16S rRNA sequencing

and analysis in these bacteria VELAZQU EZ et al., 1998).

Based on these considerations, the LMW RNA pro

files of

Frankia

strains from diverse host plants and geo

graphic provenances were analysed by Staircase Elec

trophoresis. The technique was simultaneously applied

to other actinomycetes, showing different LMW RNA

profiles among the diverse species and when compared to

Frankia

isolates Figure 1). The clustering of actinomyc

eta species was similar

to

the obtained with 16S rRNA

sequence analysis GOODFELLOW, 1989), thus

Dacty-

losporangium was more related to Micromonospora

being both separated from the other groups. Rhodococ-

cus remained isolated from the species of the genus

Designation Host source Location Source Reference

NRRLB-16422 MGI8) Myrica gale USA D. Labeda BAKER,

1987

NRRLB-16423 PtIl)

Purshia tridentata

USA D. Labeda

BAKER, 1987

ENS010712

S12)

Alnus glutino

sa

Morocco S.

Benamar Unpublished data)

ENS010714

S14)

Alnus glutinosa Morocco

S. Benamar Unpublished data)

UGLOI0701

Alnus glutino

sa Scotland C. T Wheeler

WHEELER

et al.,

1986

UGL013103

Alnus rubra

Scotland C. T Wheeler

HOOKER and WHEELER, 1987

UGL013104

Alnus rubra Scotland C.

T.

Wheeler

HOOKER

and WHEELER,

1987

UGL011301

Alnus inokumai S.

Korea

C.

T. Wheeler

SAYED et

al.,

1997

UGL020603

Casuarina equisetifolia

Egypt C. T Wheeler

Unpublished data)

Ea32.1

Eleagnus angustifolia

Francia A.

Moirud

Unpublisehd data)

Hr114.2

Hyppophae rhamnoides Francia

A.

Moirud

Unpublished data)

Hr77.3

Hyppophae rhamnoides

Francia A.

Moirud

Unpublished data)

Ag

67.5

Alnus glutinosa

Francia A.

Moirud

Unpublished data)

Ag 97.2

Alnus glutinosa

Francia A.

Moirud

Unpublished data)

Ar

112.2 Alnus rubra

Francia A.

Moirud

Unpublisehd data )

ACN 14a

Alnus cordata Canada

P. Normand NORMAND

and

LALONDE,

1982

ORS020602

Dl1)


Senegal

H. Ramirez GAUTHIER et al., 1981

Cn3

Coriaria nepalensis

Pakistan

H.

Ramirez MIRZA et al., 1992

Cn7

Coriaria nepalensis Pakistan

H.

Ramirez MIRZA et al., 1992

55005

DBB02060510)

Casuarina equisetifolia USA M.Igual

BAKER,

1987

CSI020602


Spain this study

CSI020604

Casuarina equisetifolia Spain

this study

CSI020620


Spain this study

CSI020621


Spain

this study


6/7

544 E. VELAZQUEZ et al.

Streptomyces that clustered toghether confirming the

finding of

WITT

and

STACKEBRANDT

(1990).

Our

results

support the unification of the genera Streptoverticillium

and

Streptomyces as

proposed

by

these authors because

their 5S rRNA show an identical profile.

Among the

Frankia

strains, eight LMW RNA profiles

were observed that, in general, correspond to groups of

strains isolated from different host plants, although

strains with different LMW RNA profiles may be ob

tained from the same host plant (Figure

lA

lanes 1 to

4)

and strains with the same LMW RNA may be obtained

from different hosts (Figure

lA

lane 8).

Strains isolated from

Alnus

consistently showed the

same LMW RNA profile independently of the host

species and their geographic origin (Table 1). Strain

ACN14a was in cluster 1 of NORMAND et

a .

(1996) and

closely associated to

CpIl

the strain designated

as

the

type strain for

Frankia

alnii in the analysis of

HONNER

LAGE et

a .

(1994). The uniformity in the LMW RNA

profiles obtained for the

Alnus

strains, including

ACN14a, supports the designation of

Frankia alnii

as the

species type of the genus and this technique provides a

rapid and efficient method to identify strains belonging

to this species.

The typical strains isolated from Casuarina that were

used in this study have an identical LMW RNA profile

and constitute the same species. Atypical strains, isolated

but unable to re-infect Casuarina, but able to infect

Elaeagnus (BAKER,

1987;

NAzARET

et a ., 1991) 55005

and ORS020602 share their LMW RNA profile with

strain Ea32.1 isolated from Elaeagnus and may thus be a

separate species.

The two Frankia species comprising the Alnus isolates

and the typical

Casuarina

isolates are close to each other

phylogenetically, in agreement with the results of other

authors

(HONNERLAGE

et a ., 1990;

NAZARET

et a ., 1991;

NORMAND et a ., 1996).

The low similarity coefficient among many clusters of

Frankia strains makes it likely to consider the existence

of different bacterial species (or even genera) in the genus

Frankia. This possibility

is

supported by the observed

variability in the region corresponding to

5S

rRNA.

Sev

eral authors have shown before that differences in the 5S

rRNA reflect differences among bacterial genera (HOFLE,

1988;

HOFLE,

1990;

VELAzQUEZ

et a ., 1998).

Strains Hr114.2 and Hr77.3 isolated from Hippophae

grouped in the same cluster but the coefficient of similar

ity (0,8) indicates that each of the strains may belong to a

different species.

Strains Cn3 and Cn7, isolated from Coriaria grouped

separately from the other Frankia strains. Similar results

were obtained for strain Cn7 by

RAMIREZ-SAAD

et

a .

(1998) using sequence data comparisons. Our results,

showing a low coefficient of similarity between Cn3 and

Cn7 (0.6), probably indicate that both strains may be

long to different species.

In summary, our results show that the grouping of

Frankia isolates in taxonomic groups broadly reflects the

host plants from which they were derived. This conclu

sion confirms previous analyses based on diverse tech-

niques (BEYAZOVA and

LECHEVALIER,

1992;

FERNANDEZ

et

aI., 1989;

LALONDE

et aI., 1988;

NAZARET

et aI., 1991;

NORMAND et a ., 1996) but presents interesting excep

tions that may

be

grouped in two classes:

1)

Strains from

very divergent host plants that share a very similar or

identical LMW RNA profile, as for example MgI8 and

Pd1 and 2) Strains isolated from the same host plant that

have very divergent LMW RNA profiles, as several iso

lates obtained from

Coriaria

and Hippophae in our

study.

The results presented indicate the usefulness of LMW

RNA profiles to differentiate species in the genus

Frankia

and to establish taxonomic groups among strains isolat

ed from diverse host plants and from diverse geographic

origins. The technique will allow in the future easy and

rapid identification of

Frankia

strains and their alloca

tion to the described species of the genus. This will im

prove significantly knowledge of the taxonomy of this

bacterial group and help in studies of the biodiversity of

these ecologically and economically important bacteria.

Acknowledgements

We

thank

PHILIPPE NORMAND, HUGO RAMIREZ SAAD and

EMETERIO IGLESIAS JIMENEZ for suppling Frankia

strains

and

their commentaries

to this

work.

This

work

was

supported by

Grant

CS101-97

of Junta de Castilla y

Leon

and the

DGICYT

(Direccion General

de Investigacion

Cientffica Tecnica)

Grant

PB92/097.

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Corresponding author:

EMILIO CERVANTES, IRNA-CSIC, Apartado 257 37080 Sala

manca

Spain

Phone:

011-34-923-219606 Fax: 011-34-923-219609

Documents

LMW RNA Profiles of Frankia Strains by Staircase Electrophoresis