Analysis of a ribosomal RNA operon in the

Gene, 111 (1992) 119-124
© 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.00
119
GENE 06287
Analysis of a ribosomal RNA operon in the actinomycete F r a n k i a
(Actinomycetales; 16S, 23S, 5S rRNA; nitrogen-fixing; Casuarina; recombinant DNA)
Philippe Normand, Benoit Cournoyer, Pascal Simonet and Syhie Nazaret
Laboratoire d'Ecologie Microbienne du Sol, U.R.A. C.N.R.S. 1450, Universitd Lyon I, F-69622, Villeurbanne Cedex (France)
Received by K.F. Chater: 29 August 1991
Revised/Accepted: 10 October/ll October 1991
Received at publishers: 22 November 1991
SUMMARY
The organisation of ribosomal RNA-encoding (rrn) genes has been studied in Frankia sp. strain ORS020606. The two
rrn clusters present in Frankia strain ORS020606 were isolated from genomic banks in phage ~EMBL3 by hybridization
with oligodeoxyribonucleotide probes. The 5'-3' gene order is the usual one for bacteria: 16S-23S-5S. The two clusters
are not distinguishableby restriction enzyme mapping inside the coding section, but vary considerably outside it. Sequencing
showed that the 16S-rRNA-encoding gene of ORS020606 is very closely related to that of another Alnus-infective Frankia
strain (Ag45/Mut 15) and highly homologous to corresponding genes of Streptomyces spp. Two possible promoter sequences
were detected upstream from the 16S gene, while no tRNA-encoding gene was detected in the whole operon. Regions with
a high proportion of divergence for the study of phylogenetic relationships within the genus were looked for and found in
the first intergenic spacer, in the 23S and in the 16S gene.
INTRODUCTION
The nitrogen-fixing symbiosis between the actinomycete
Frankia sp. and a number of woody dicotyledonous plant
genera play a major role in agroforestry. A better utilization of the Frankia-symbiosis will necessitate consideration
of such properties as persistence in the soil and competitiveness of released strains, which requires precise strain
identification, such as that afforded by oligo hybridization
(Simonet et al., 1990). The rRNA-encoding genes, with
Correspondence to: Dr. P. Normand, Laboratoire d'Ecologie Microbienne
du Sol, U.R.A.C.N.R.S. 1450, B[R. 741, Universit6 Lyon I, F-69622,
Villeurbanne Cedex (France) Tel. (33)72448289; Fax (33)72448466;
e-mail NORMAND @CISM.UNIV-LYON I.FR.
Abbreviations: bp, base pair(s); IGS, intergenic spacer; kb, kilobase(s) or
1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open
reading frame(s); PCR, polymerase chain reaction; rrn, rRNA-encoding
genes; rRNA, ribosomal RNA; tRNA, transfer RNA; Y, C or T (U).
their conserved areas that alternate with highly variable
zones, provide targets for oligo identification (Haun and
G0bel, 1987; Wilson et al., 1988).
Attempts at sequencing directly the 16S rRNA from
Frankia strains were successful in the case of two Alnusinfective strains (Hahn et al., 1989) but could not be
achieved in the case of Casuarina-infective strains due to
problems in the isolation of suitable rRNA (S.N. and E.
Stackebrandt, unpublished). For these reasons, the
Casuarina-infective Frankia strain ORS020606 from which
a gene bank had already been constructed (Normand et ai.,
1988), was deemed an appropriate candidate for the study
of rRNA-encoding genes in Frankia.
EXPERIMENTAL AND DISCUSSION
(a) Characterizationof rRNA-encoding gene clusters
There were clear signals when probes LSUll03 and
LSU2536' (Table I) labeled according to Zeffand Geliebter
120
TABLE I
Listofoligosused
Designation ~
5' ~ 3 ' sequence b
SSU413
SSU901
SSUllg0'
SSU1508
LSU20'
LSUII03
LSU2536'
AGC
GCC
GGG
TCT
AGA
GAA
GAA
GAC
TTG
GCA
AGA
TCT
AGA
CAG
GCC
GGA
TGA
GGT
GCC
GTG
CCC
GCG
GTA
TGA
GAA
AAG
CGT
AAC
TGA
CGG
CTT
GTC
GCA
AAT
CCT
GGG
CCG
GAC
GTA
TCC
AGC
TGG
ATG
CA
GTC
ACA
ACC
TCA
AAC
AGG
GTG
TAG
C
" The oligo designations include three letters describing whether they correspond to 16S (SSU) or to 23S (LSU) genes, a number that corresponds to
the E. coli sequence coordinates (Brosius et al., 1981), and a prime or no prime when the sequence is homologous or complementary, respectively, to the
RNA sequence.
b Oligo probes were end-labe!ed by phosphorylation using T4 polynucleotide kinase and [~,-32P]ATP (185 TBq/mmol) according to Zeff and Geliebter
(1997).
(1987) were hybridized with genomic DNA prepared as
described before (Normand et al., 1988)from Frankia strain
CeD isolated from Casuarina equisetifolia (ORS020606;
Diem et ai., 1983). One or two fragments, depending on the
restriction enzyme used, gave a positive signal (Fig. 1).
There are thus two rRNA-encoding gene clusters called
rrnA and n'nB, present as BamHI fragments of 6 + 6 and
3.5 + 2.2 kb, respectively (Fig. 1). It has been suggested that
in bacteria, the number of clusters could be directly related
to the growth rate. Frankia with a doubling rate of a few
days is certainly slow and the low number of clusters found
would thus be consistent with this explanation.
24kb-
~'~,~,,
9.5 -
tjjj~
(b) Cloning of the two rRNA-encoding gene clusters
Hybridization of the ORS020606 gene bank (Normand
et al., 1988) with oligo probe LSU1103 yielded five positive signals, two of which were chosen at random for further studies. These turned out to contain the two different
rRNA-encoding gene clusters, i.e., they had BamHI fragments of 6 and 3.5 kb that hybridized both with oligo probe
SSU413 (5' part ofl6Sgene) and LSU1103 (middle of the
23S gene). Two representative clones were named Olrnal
and Olrna2 containing clusters rrnA (containing the two
6-kb BamHI fragments) and rrnB (containing the 3.5 and
2.2-kb BamHI fragments), respectively. The two phages
~i,
O
6.",,
4.5 2,3
m
2.0 "
0.6
.
@
A
B
Fig. I. Genomic blots of Frankia total DNA. Frankia strain ORS020606 total DNA was cut with the indicated restriction enzymes. Electrophoresis was
carried out at 1 V/cm for 16 h in TAE buffer (Maniatis et ai., 1982), and the resulting gel was transfered onto a nylon filter and hybridized with ~2P-labeled
LSU1103 that corresponds to the first half of the 23S gene (panel A) or with LSU2536' that corresponds to the second half of the 23S gen0 (panel B).
Size markers were derived from phage ~. digested by HindIlI (sizes in kb marked on the left margin). Hybridizations were at 45 °C in 2 x S SC (Maniatis
et al. (1982)/1 u Denhardt's (1966)/1% (w/v) SDS/10~ (w/v) dextran/l mg per ml of denatured salmon sperm DNA.
121
were hybridized with purified, labeled 16S, 23S or 5S rRNA
p:epared from Frankia strain Tx30SAb (Normand and Lalonde, 1986) and partially hydrolyzed in 50 mM Tris-Cl pH
9.5 (pH at 25°C)/0.1 mM spermidine at 95°C for 15 min,
and with different oligos. The resulting genetic and restriction maps of the two clones are given in Fig. 2. This map
is totally consistent with the genomic blots (not shown).
The three rrn genes in the two operons were found to be
arranged in the order 16S-23S-5S usual for bacteria.
Restriction sites were identical within the rrn genes but
extensive sequence divergence started from around 700 bp
upstream from the EcoRI site present at the start of the 16S
genes (e.g., the Bglll site of O l r n a l is replaced by BamHl
6
3
?
4
5
cOR
8
9
6
indl
7
I0
aral
II
8
Cpnl
12
9
13
I0
II
pslt
-Kpnl
-
14
S alI
01rnal
01rna2
Fig. 2. Comparison of the two operons. Relevant parts of the inserts in
phages O l r n a l and Olrna2 are shown side by side with restriction maps.
Those sites mapping at similar points on the two dusters are connected.
The zones corresponding to the genes are shaded. The scales are in kb
and thek coordinates start at the BamHI junction site with the phage 2
leR arm.
and SalI sites in Olrna2) and about 2 kb downstream from
the HindllI site where a Xhol site present in Olrnal is
replaced by Sail and BamHI sites (Fig. 2). The unique
EcoRI site present in the first half of several bacterial 16S
genes is present in the two clusters.
(c) Sequencing of the rrnA gene cluster
The nt sequence ofgene cluster rrnA is shown in Fig. 3.
It has been deposited in GenBank and given accession No.
M55343. The whole sequence from the first of the two Bgill
sites up to theXhoI site is 6481 nt long. The restriction sites
found by computer search are completely consistent with
the restriction analysis of the O l r n a l phage (Fig. 2). The
allocation of 16S, 235 and 5S segments was determined
using the high sequence similarity of that region with
Streptomyces ambofaciens (Pernodet et al., 1989) with the
Bestfit comparison algorithm (Smith and Waterman, 1981).
The length of the 16S gene (1512 nt) is somewhat shorter
(by 16 nt) than the corresponding one in S. ambofaciens,
and more markedly so (by 30 nt) relative to Bacillus subtilis
(Green and Void, 1983) and E. coliRI (Brosius et al., 1981).
The G+C ofthe gene is 60~o, which is higher than most 16S
sequences with which it was compared but lower than total
DNA (71%) from Frankia according to Fernandez et al.
(1989). The 16S gene is the most conserved gene of the
operon, especially at its 3' end. The highest sequence similarity is with Frankia strain Ag45/Mut15 (95%) and with
S. ambofaciens (90 %). The highest sequence divergence is
not to be found in the first hypervariable region of Embley
et al. (1988) where only two mismatches with S. ambofaciens are present. However, the hypervariable zone of
Pernodet et al. (1989) at the beginning of the gene, and to
a lesser extent the second hypervariable zone of Emblcy
et al. (1988) are potential sites for species specific probes.
This last zone has been exploited by Nazaret et al. (1991)
for phylogonetic purposes in Frankia.
The 23S gene was found to extend from nt 2451 to 5549
for a length of 3099 nt, a length comparable to that of
S. ambofaciens (3120 nt) and thus amongst the longest bacterial 23S sequences determined so far (Pernodet et al.,
1989). The percentage G+C is lower (57%) than that of the
16S gene, which appears also to be the case in other bacteria studied. The highest sequence similarity with the
S. ambofaciens sequence is to be found at both extremities,
especially at the 3' end of the gene (Fig. 3). Conversely, the
highest divergence was found in three regions (numbered
from the start of the Frankia 23S gene): at nt 310-450 (64
mismatches/140nt), at nt 1245-1350 (49 mismatches/
105 nt) and especially at nt 1510-1715 (81 mismatches/
210 nt).
The 5S gene extends from nt 5618 to 5736 for a length
of l l 9 n t . The last nt, a C, may not belong to the gene
itself since it is part of the perfect inverted repeat extend-
GTTGTGAGGG A T T ~
beginning of 16S qene
Itpal
GGTACGTGGG TAGGTTAACT
ACACGGTGGT GGGCCTGTGA G G ~ T G
TCGATGTCGG G G ~ C G A
GCC3~TT~IT ~fACCCCG~ CATGGCCTGG CTGGGTGGTG
GGrTC~TCGC TI~GAACGCG AACGTCCGGA GTGTGCAAGA
" -10"
~CCGAG
T~/%CTTAAAG AAGCACCAC4% ACG~AGCCCT
GTACGAGAGT G G ~ C A C C
TGGTGGTGTG GAAAGATTTA TCGGCTCGGG A ~ G G C C C G C GGCCTATCAG C~WGTTGGTG GGGTGATGGC
ACCGGTGCCG AAGGCGGGTC TCTGGGCCGG AACTGACGCT AA~AGCGAA AGCGTGGGGA GCGAA~AG~q TTAGATACCC TGGTAGT~A
Eagl
G~£GGGCGCT AG~TGT~GGG ~-ACCI~CCAC GGCCTCCGTG CCGCAGCT~A CGCATTAAGC GCCCCGCCTG GGGAGTACGG C C G C ~ G C T
TCGGCTGTGA
EcoRV
GCGCAGAT~T
/~GCACCGGC
AAC~CGACCC CGTGAAGTCG GAGTCGCTAG TAATC~AGA TCAGCJ~TGC TGCGG~G~T A~GTTCCCGG GCCTTGT/~A CACCGCCCGT
GCACGCTGTT G G G T C C ~ G
GGAGTGAGGC TCT~TCGGCT GGTGGGTTCC TGTGCTGTGG
GGG/~GTGAA ACATCTCATr ACCCGCAGGA GGAGRAAACA ACCGTGA~rC CGCG~GTAGT
GARTGGGCCG CCATAGA~GG TRATAGCCCT GTAGCCGARA GCAGTGTTCC TCCCGGACGT
C
~
GAATGAGL'CT GCGAGIT]'GC GGTGTGTGC.C GAGGrTAACC CGTGTGGGGT AGCCGTAGCG A~GcC_~AGTC CC~AGKGGGC ~ G C
Bali
CCA'IGGCCAG G~I'GAAGCGC GGGTAAGACC GTUTGGAC,GA CCGA~CCAU CAGGG'I'I'GAA ,~CCTGGGGG
C
GTCCGCCGGA TGACC~AGGG TrCC~GGGC AG~CTMTCC C-CCCAGGGTG
T A G ~
CAGGGCCGCC CGTACCCT~A N : C ~
GAGGTCAC.,GT AGAGAATACC GAGGCGTTCG
#ETG~ACTGT GGTTAAGC~A CTCGC-C~AAT GCCCCGTRAE TTCGGAC~ARG GGGGUCCGTT CTCCGTGTAG GGG'ITI'ACCT CCGRACEC~G G A ~ C G C
xcaI
AGAGACCAGG GGA~CGAC T~TTTAUrAA AAAUACAGCT CCGTGCT~G "23CGT~AGACG&~-T&TACG6 AG'DGREGCCT GCCCGGTC~T ~ G T T A A
GGffrGAT~CT AT~CTO~C~ C ~ q A A ~ T ~
CCAGC-CGGTG G'FrGTCCTGG GGCAI~GGTG TN;GACGAGG CGTN;GTAAA TCCGCGTCTr GTGTGTCTGA GkCCTGATGC CGAGCCGATT GCGGCGARGT
AGTCGGGACC TAAGGCGAGG CCGACAGGCG T ~ T C G A T G G ATAACGG~'I~ C~T~TrCCCG TACCGGCGTT GACGCGGCCA ~ C C T
G G ~ T
BamHI
4001 AACCATCTGA TCGGATGTGT CTCTTCGGAG GTG~A-A~GG C ~ T ~
~CCCGGCTGG TAGTAGGCAA GCGATUGGGT GACGAGGAAG GTA~CCAGC
CATACGAGTG AGAATGCAGG CATGAGTAGC GAATGACGGG ~
3501 TTCGTAGTCG AGAGGGA2~C AGCCCAGATC GCCAGCTAAG GCCCCTAI~GC G T G C U C T ~
TGTGGRGTCG CATAGACAAC CAGGTGGTGC
LSUl103
TrAGAAGCAG C C A C C ~
A~C,AGTC~q' AATAGC'IEAC T C ~ ' ~ G T G A T T U ~ G ~ C GA~A~TGTAG C G G G G C T C ~ GCGCACCGCC GAAGCTGCGG
SphI
CATGCACAAA ATrTCCCGGC ~ C ~ T
CCAGGTTTGT GTGTGGGTAG GGGAGCGTCG TGTGGCGTGT GAAGCGGCGG GGTGACCCAG CCGTGGATGC
ATGAGCTGTG GGTA~GGGTG A P ~ G C C A A T C ~ ' T C G G T GATRGCTGGT TCTCCCCG~A ATGC,qTTTA~ GTGCAGCGTC GCA'AUI'A~f TGCCGGAGGT
Hindl II
HlndlII
AGAGCACTGG ATGGCCT~J3G GGGCCCAC~d% GCTTACTGAA GTCAGCCRRA CTCCGAATGC CGGTA~GTGA AGTC~GGC~G TGAGACTC~G GGGGATAAGC
ATGTCCAAGA CCCGAAGCCG AGTGATCTAC
C
GTTCCCAAGT AC,CACGGI~C CCGTGTAA~T CCGTGTC~AT C'IV,GCGGGAC CACCCGCTRA GCCTAAATAC TCCCTGGTGA CCGATA~CGG ACTAGTACCG
Sinai
3001 TGAGC~A~%G GTGAAAAGTA CCCCGGGAGG GGAGI~AAT A G T A C ~
ACCGTGT~CC TACA~TCCGT GGGAGCTGGA CT~TGGTCTG GTG~CCGCGT
TCGGGGAGTC AGAAAGAGCA TrGTTAGGCG AAGGTCATGC
GGTGAGCGAA AGCGGAACAG C~TA~ACCAG TGTCGTGTGA GAGCCGGCAG GTGTTGCGAT GCTGGGG]WG TGGGATCGTC CAGGTGGAGC TGCCG'FFCUA
CACCCGTGCC TGAACACATA GGGCATGTGG AGGGAACGCG
TCC~GGT(3GG GATCGTGGTG GGGGGTCTGC TGGTGGAACG GACCGGTCGT GGTGGAGGAC TGCCTTCCCG T~GGGTGGGG GTGGGTTTCG CCGGGTCGGC
Ball/beglnnlng o f 235 qene
TGGGC,CCGTC CGTACGTTGA ~ T G C A C A
GTGGACGCGA GCATCTTTGT GG~CAAGTTA T T A ~ G G C ~ ACGGTGGATG CL"TTG~CAC~ ~ G ~ C C ~ T
DraI
25~I GAAGGACGT~ GGAGGCTGCG ATATGCCTCG GGGAGCTGTC ~ACCGAGCTG TGATCCGAGG ATrTCCGAAT GGGGARACCC GGCAGGGCTT TAAATCCTGT
TGC~GGGTGG AACC~TCCGG GTGGGTGGCT GGGGTCCGTG
CACGTCACGA A,~3TCGGTAA CACCCGAAGC CGGTGGCCTA ACCCIT~TGG GGGG~GCCGT CGAAGGTGGG ACCGGCGA~I GGGACGAAGT CGT~d~CAA~G
e n d o f 16s gene
SD
2001 T~C,CCGTACC GCJ%~GC:TC,CC, C~CTGGATCA~
GGCTGG'~'~ TCCTGTAGTG GGGTGGGCTG GTGCAGAGCC AGGGCCGGCT
Seal
GTGGATGCCG GTCTGGTTGC TCGTGGGTGG AACGCTCACG AT~GGGTT~C GGCTGGTTGT CCGGGGTCTA GTACTCCTCT GT~CTTATCT TCGGGI~GGG
CGGGGTCTGC
CCCTTACGTC C~V~3GCTGCA CACATGCT~J~ AATGGCCGGT ACAAAGGGCT GCGATGCCGT GAGGTGGAGC GAATCCCAPA gJ~CCGGTCT CAGT~CGGAT
CAACCCTCGT CCTATGTTGC CAGCGAG~CG TGTCGGGG~ TCATAGGAGA CTGCCGGGGT CAACTCGGAG C~AGGTC~GG AT~ACGTCAA GTCATCATGC
1501 ATCTCGTAGA GATACGGGGT CCGTAAGGGT CCTGCACAGG TGGTGCATGG CTGTCGTCAG CTCGTGTCGT GAGATG~rGG G ~ A A G T C C C CCP~CGAGCG
A ~ A C T C A A A GGAATTGACG GGGGCCCGCA CAAGCGGCGG AC~ATGTGGC T~AATTCGAT GCAACGCCAA C~u~CCTTACC AGGGCTTGAC ATCCAGGGAA
CGCCGTAAAC
CAGGAGGAAC
AGCCGGCCTG
C~GC~G~GCCT
CTACCAAGGC GACGACGGGT
AGAGGCEGAC CGGCCACACT GGGACTGACA CAEGGCCCAG A~TCCTACGG GAGGCAGCAG TGGGGAATAT TGCGCAATGG
5SU413
KpnI/Pst I
GACGCAGCGA CCCCGCGTGG CC,C~I~,~EGG CCfTCGGGTr GTAAF~CTCT TTCAGCAGGG P~GAAGCGAG AGTGACGGTA CCTGCAC~AG
Sstl
1001 CAACTACGTG CCAGCAGCCG CGGTAATACG TAGGGTGC~A GCG~TGTCCG GAATTA~33G GCGT~AAC~tG CTCGTAGGCG G C C T G ~ C G
Smal
Pstl
ECORI
A~ACCCGGGG CTCA%CTCCG GGCCTGCRGT CGATA~GGGC AGGCTAGAGT CCGGCAGGGG AGACTGG~AT TCCTGGTGTA GCGGTGRAAT
GCCGGGCATC
ATGGAGAGTT T ~ A T C ~
TCAGGACGJ~A CGCTGGCGGC GTGC~ITAACA CA~CCAAGTC GAGCCGGGAG
Sstl
CTTCGGCTCT CAGCGGCGAA CGGGT~AGTA ACACGTGGC-C A~CCTGCCCC GAGCTCTGGA ATAR~CTCGG GAARCCGGGG CTAATGCCGG ATATGAERTI•
501 TGZ'FL'A-J.I~-A TCGGCC'CTCT C ~ A T C - ~
TCCCACGTGG TGGGGTGGCT ~ T T G G
I]qlll
1 AGATCI~CTC CGAGGC,C,GCG TAC~C~CCCC GAGGAAA~GG GCCGCGGACG GTC~CATAAG
"-35"
"-lO"
ACCGGGCCGG AGTGATTTGA CACAATCA/~ CCPJ%AGCTCA T~ATCTAGGA / ~ A C G T T C C
Bgl I I
"-35"
AGGTAAGATC "IY2ITCGG~TG T C ~ T T D C
GC~CA~G
J%AGCGATI~G J%CACCGCCGG
RNA p r o c a s s l n q s l " a
GAC.nGTGC,~n GTCTGGTAGC G ~ C T ~ c " r
~
~;~:;CGTGCC GATTGTCAGT
~AGTAA
AUGG~GTUG TAALWATAAC CATCCTAAGG TA~£UAAATT cuTrffI~GGG
A
~
C
TGGGTCCGTG
A~TCA
GGGACAGTGT
CAC~TGGGrA GTITAACTGG
AGGGCT~£CC
GA~GGGATAA C C ~ G
AC~A~
CGCCACGCTG CKC.~_N:CAS CTCWI"CCGGT
Fig. 3. Nucleotide sequence ofoperon rrnA. Sequencing was by the chain termination method
of Sanger et al. (1977) using the deazaTaqTrack TM kit of Promega (Madison, WI) and
[35S]dATP on overlapping clones of both strands. The rRNA cluster was subcloned from
phage OIRNAI after digestion with Sail into pBR328 (Soberon et ai., 1980) yielding pFQ180,
from which further subclonings were then made. The sequencing strategy used was based on
single-stranded DNA clones in Bluescript (Stratagene, San Diego, CA) or in M13mpl8 and
19 derivatives (Yanisch-Pel"ron et al., 1985) propagated in DH5otF'IQ cells (BRL), using either universal primers on clones obtained with restriction enzymes or tailor-made primers
(Table l) so as to completely determine the sequence of both strands. The sequence coordinates start at the first of the two Bg/ll sites, upstream from the 16S gene. Some relevant restriction sites are shown above the sequence line. Putative promoter sites are highfighted by
double underlining. The putative RNA processing site is underlined. The beginning and end
of all three r r , genes are underlined. Relevant oligo sites are overlined. The messenger attachment site at the 3' end of the 16S gene (Shine and Dalgarno, 1974) is shown by a wavy line
(nt 2030-2034). The putative terminator downstream from the $S gene is highlighted by a wavy
line (nt 5737-5789).
TCAGCTACCT CATCGAGCGC CTCCGTGCG~ GCGGI~CCGG C~ItCGTCCGG GTCTACGGt~ GCGC.~GGCGGGGTGATCCTG CCGGCGGAC~ TCGACCI~CT
BclI
Bcll
StuI
GC&CGCGCGC GGGGTCGCGC GCATCI'PC'IE CCCGCA~G~ GGGCAC.,CGGC TCGGTCTCGC A ~ A T C ~ T C AATA.~ATCA TCGN3G~CTG 1V.,ACGTCGAT
Smal
XhoI
CTGACGGAGG ACGGGCCGAA U~TCGATGCG ~ G C T G
~CACGG
CGCGCTCGC~ CGGGCGATCA CCGTGCTCGA G
GCC~CGRAGT GATCCAICIC
S¢a1
A ~ G C C C T G T CGTCCTATCA GGGCGGGCAT ~ A C T
Eagt
GCT~CGA~TA ACATCATG~ TCGCCT~TT~ CAGGOT~AGG
C.~CM,CCC,GC A C ~
GTCTCATI"CC C.~CCCCC,~A GCT~%GCTCI TCAGCGCCC~ TGGTACTGCA
Xcal
CTAG~GAGAC AGCCCCACA~ TCGTATACGC GTATTTrCCG
"-10"
TACATGGAT~ TC~?ACTAT~ ~ G ~ C ~ q A G Tb'I'~CTAGGT ATC'I~GC:'CG
~f~TCC
CCGACTAGAT GATCGGGTT~ ATAGGCCGGA GGTGGAAGTG
EagI
AAGGTGCT&C GCGTCCACTG TGCGGTIt'TC GC,G'~TACGG CCC,GTrCGGC
GGTAAGGCCC
GGGCATGACC GCAOCGTCGA GGAGGTCGTC ACCGCt~CGA TCCAGGAGGA CGCCCAGGGG
6001 CCGGATCGTG ACG~CGGCET C~CTCTTCC~ TG6GCATGAC
GGTCTCCCAC AGGGTAGCCT
e n d o f 23S gene
AGGCCC~.GGG CTI'GTCT~G
gene
TGGCGA~SG ~ G t ' % ' C G
qene XcaI
~
AD3AGTGTGG
"- 35"
ACTA~3GCT GGAGCACCCG CT1T~GCACC TTGGC~U~GG ~ A T ~ T A T
SmaI/SD
f-met
CCACATCGTC C ~ C
C ~ T
CCTC~T,ACGC G~TGTCCIEC
CC~GCGCAGG
TGGCTGGTrG GCTAPT[TCG
GTCGTGAUAC N3TI'CGGTCC CT&TCCGUCG
AGGACCGGGA CGGACC~ACC TCTGGTC/TC~ CAGTrGTTCT GCCAAGGGCA
CATCT~GCU GGAAGCCIGC ~TCG~EATGA
•
5501 CGGTGACGCA TC,~J~CTGAC ~
b e g l n n t n g o f 55
TGGTATATCT G C _ ~ T A ~
I~:C-,G'I~GTrl'
end o f 5S
T ~ r ~ ; ~ C ~ TGTGGC~GAG ~
CTAGTACGAG
I'GTTCGCCCA TTAAAGCGGT ~CGCGAGCTG GGTI~A~AAC
AGTUCAASTU CACA~TC.~AG CT'IV-,)ETGTG
Iq3nI/srraI
5001 AGACAGACAT GTCGAGCAGG TGCC~qAAGCA GGGACTAGTG ATCCGGCGGT G U ~
AGCGCCG~EG CTC~qUGG&T ~ G T R E C
CCGGGGATAA
IJJ2536 *
CAGGCTGATC TTGCCCAAGA GTCCATATCG ACGGCRAGGT TTGG~ACCTC ~ATGTCGGCT C TCGCATCC TGGGGC'IGGA GTaOTrC~CA .%~..C.n'I'TC~.r-.r-
GTTGTTC~AA TACCACTCIG GTCGTACTGG
GGCGGTTGCC TCCTAAN~G TAACGGAC,nC GOCCA~GGT TCCCT~QCC TGG'I'IV,CCM, T C A ~
TGGTGGAGTC
TAAGTI'CCGA CCTGCACGAA TC.,GCGTAAt'G ACIT~CCAC Tb'IT'Iv_A~C N~GACTCGG C ~ T I ~ : A
TTACC,AGTA~ AGAIT,CTCGT TACGOSCGGC
SmaI
&GGACGGAAA C4qCCCt'GGC~ CCITTACTAT AGCTIT~TAT TC,GI~TI'~]G ~
GTAG~ATKC,G ~
~ A G G
ACC,CCRGI'I'C
4 5 0 | GGGGACGGGT TAGCTL'T~G GGGCGAA~ ~ A A
123
ing slightly beyond the second XcaI site 52 nt downstream.
The first 411-nt IGS between the 16S and the 23S genes
is markedly longer than spacers in S. ambofaciens (303 nt)
and in B. subtilis (169 nt) where no tRNA-encoding genes
have been detected. It is of a length comparable to that
in E. coli (440 nt) which must accommodate a 75-nt
tRNA °lu. In Frankia, a search using the Staden (1980)
program revealed no tRNA. An 83-nt tRNA-like structure
was detected in the last part of the IGS at nt 2337-2419
but the lack of a GTT(or ~)C in the side arm and the
presence of mismatches in all arms make it a poor candidate for tRNA. The search for tRNAs reve',ded no other
such structure in the zone upstream from the 16S gene.
The second IGS is 68 nt long, comparable to the 82 nt
in S. ambofaciens, 55 nt in B. subtilis and to the 92 nt in
E. coll. The similarity found with S. ambofaciens between
the 16S and the 23S genes is low: less than 50% similarity. Together with the three regions in the 23S gene, the first
IGS is potentially the most interesting region to probe with
oligos for discrimination between closely related strains.
There were two potential promoters upstream from the
16S gene, one situated at nt 114-147 and the other at nt
245-276 (Fig. 3). The consensus sequence of these two was
TTGACA for the --35 motif and TAAYYT for the -10
motif, separated by 18 and 17 nt respectively. The presence
of two or more promoters is a common feature in E. coii
(Brosius et al., 1981), B. subtilis (Green and Void, 1983)
and Streptomyces spp. (Baylis and Bibb, 1988; Pernodet
et al., 1989).
Furthermore, a comparison with the corresponding sequence of S. ambofaciens yielded a 75% similar 99 nt sequence (with one gap) in common between the two organisms (nt 280-378), containing the motif 5'-GCTCCTTGAGAACTCAACA. The motif, which corresponds to the
RNA processing site of the primary transcript postulated
by Baylis and Bibb (1988), is situated 175 nt upstream
from the S. ambofaciens 16S gene and 185 nt upstream
from the Frankia corresponding sequence. Downstream
from the 5S gene, a 53-nt sequence (5'-CGTATACGAGTGTGGGGCTGTCTCCCTAGGGAGACAGCCCCACACTCGTATAC_GCGTATTIT) of inverted symmetry (underlined) was found bordered by two Xcal sites
(doubly underlined), which are otherwise rare (only one
other site in the whole rrn operon). The free energy of the
stem would be low (AG = -45.6 kcal/mol) as calculated
using the model of Zucker and Stiegler (1981). The repeat
is perfect suggesting a strong terminator, followed by a
block of four Ts, characteristic of eubacterial Rho-independent terminators (Platt, 1986).
Further downstream we found a putative ORF of more
than 555 nt with a putative promoter (-35:TGGCAT,
[N]t7,-10:TACTAT), a ribosome-binding site and an
ATG. The ORF has a > 95 ~o probability of coding for a
protein according to Fickett's (1982) statistic. No homologous region was found in other published sequences.
(d) Conclusions
(1) There are two rRNA operons in Frankia strain
ORS020606.
(2) Both were cloned and found to be very conserved in
the coding regions and to differ outside it. The gene order
is the usual one for bacteria: 16S-23S-5S.
(3) The nt sequence of one operon was determined. Sequence conservation is high with S. ambofaciens. No tRNA
could be detected in the spacer.
ACKNOWLEDGEMENTS
Thanks are expressed to Claude Lemieux, Jean Boulanger and Christian Otis (D6partement de Biochimie,
Universit6 Laval) for help with cloning into Bluescript
phagemids and synthesis of oligos, to P.H. Roy (D6partemerit de Biochimie, Universit6 Laval) and Guy Perri6re
(Biom6trie, Universit6 Lyon I) for help with the sequence
analysis programs, to Luc Simon (CRBF, Universit6 Laval)
for helpful discussions about primer design and to Laurence
Vergnaud for technical help with sequencing (CRBF, Universit6 Laval).
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