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Microbiology (1999),145,2355±2364 Printed in Great Britain
Dispensable ribosomal resistance to spiramycin
conferred by srmA in the spiramycin producer
Streptomyces ambofaciens
Jean-Luc Pernodet,
1
Anne Gourmelen,
1
Marie-He
!
le
'
ne Blondelet-Rouault
1
and Eric Cundliffe
2
Author for correspondence:Jean-Luc Pernodet.Tel:­33 1 69 15 46 41.Fax:­33 1 69 15 72 96.
e-mail:pernodet!igmors.u-psud.fr
1
Institut de Ge
!
ne
!
tique et
Microbiologie,UMR CNRS
8621,Ba
#
t.400,Universite
!
Paris-Sud XI,F-91405 Orsay
Cedex,France
2
Department of
Biochemistry,University of
Leicester,Leicester LE1
7RH,UK
Streptomyces ambofaciens produces the macrolide antibiotic spiramycin,an
inhibitor of protein synthesis,and possesses multiple resistance mechanisms
to the produced antibiotic.Several resistance determinants have been isolated
fromS.ambofaciens and studies with one of them,srmA,which hybridized
with ermE (the erythromycin-resistance gene fromSaccharopolyspora
erythraea),are detailed here.The nucleotide sequence of srmA was
determined and the mechanismby which its product confers resistance was
characterized.The SrmA protein is a methyltransferase which introduces a
single methyl group into A-2058 (Escherichia coli numbering scheme) in the
large rRNA,thereby conferring an MLS (macrolide±lincosamide±streptogramin
type B) type I resistance phenotype.A mutant of S.ambofaciens in which srmA
was inactivated was viable and still produced spiramycin,indicating that srmA
is dispensable,at least in the presence of the other resistance determinants.
Keywords:macrolide antibiotics,MLS resistance,rRNA methylation,spiramycin,
Streptomyces ambofaciens
INTRODUCTION
Streptomyces spp.are ®lamentous Gram-positive bac-
teria that are well-known for producing many important
antibiotics.Organisms that synthesize antibiotics to
which they are potentially sensitive must have a re-
sistance mechanism to protect themselves,at least
during the phase of antibiotic biosynthesis.In these
organisms,three different resistance strategies have been
reported:modi®cation of the antibiotic target,intra-
cellular inactivation of the antibiotic and limitation of
intracellular drug to subinhibitory levels via efflux
and}or exclusion (Cundliffe,1989).
Streptomyces ambofaciens produces spiramycin,a
macrolide antibiotic consisting of a 16-membered
lactone ring with three sugar or amino-sugar residues,
which speci®cally inhibits bacterial protein synthesis by
binding to the large subunit of the ribosome (Gale et al.,
.................................................................................................................................................
Abbreviations:MLS,macrolide±lincosamide±streptogramintype B;SAM,
S-adenosylmethionine.
The EMBL/GenBank accession number for the nucleotide sequence
described in this paper is AJ223970.
1981;Brisson-Noe
$
l et al.,1988).The most widespread
macrolide-resistance mechanism involves the so-called
MLS (macrolide±lincosamide±streptogramin type B)
resistance phenotype (Weisblum,1995).Despite struc-
tural dissimilarities,antibiotics belonging to these three
groups bind to the ribosomes in mutually competitive
fashion and inhibit protein synthesis.MLS resistance
involves N
'
-methylation of the amino group of a
particular adenosine residue (A-2058,Escherichia coli
numbering scheme) in the large rRNA,leading to
reduced binding of MLS antibiotics to the ribosome.
Such resistance was ®rst encountered in clinical strains
of Staphylococcus aureus and has since been detected in
a broad range of micro-organisms,including actino-
mycete producers or non-producers of MLS antibiotics.
Two varieties of MLS resistance have been described.
The phenotypes resulting fromN
'
-monomethylation of
A-2058 (MLS-I phenotype conferred by erm type I
genes) or N
'
,N
'
-dimethylation (MLS-II phenotype con-
ferredby ermtype II genes) differ mainly inthe resistance
levels to speci®c macrolides (Pernodet et al.,1996).
Other macrolide-resistance mechanisms involving ex-
port or inactivation of the antibiotics have been charac-
terized,both in actinomycetes and in pathogenic bac-
0002-3228#1999 SGM
2355
J.-L.PERNODET and OTHERS
teria.Genes conferring macrolide resistance and whose
products belong to the superfamily of ATP-dependent
transport proteins have been found in pathogenic
bacteria such as Staph.aureus (Ross et al.,1990) and
in several macrolide-producing Streptomyces spp.
(Mendez & Salas,1998),including S.ambofaciens.
Macrolide inactivation can be due to different modi-
®cations,for instance phosphorylation (Marshall et al.,
1989;O'Hara et al.,1989),glycosylation (Kuo et al.,
1989;Cundliffe,1992;Vilches et al.,1992) or esteri-
®cation of the lactone ring (Ounissi &Courvalin,1985;
Arthur et al.,1986).
S.ambofaciens protects itself against spiramycin via at
least two different resistance mechanisms,one of which
involves ribosomal modi®cation (Pernodet et al.,1993).
Among the resistance determinants previously isolated
from this strain (Richardson et al.,1987),one of them,
srmB,is supposed to be involved in antibiotic export
(Schoner et al.,1992).We report here the cloning of
several resistance determinants fromS.ambofaciens,the
identi®cation of one that is involved in ribosomal
resistance and biochemical characterization of the
encoded resistance mechanism.
METHODS
Bacterial strains and plasmids.
Strains and plasmids used in
this study are listed in Table 1.
Media and microbiological methods.
Hickey±Tressner (HT)
(Pridham et al.,1957) or NE (Skeggs et al.,1985) complete
media were used for the growth and sporulation of Strepto-
myces strains at 30 °C.Liquid culture media for routine
growth were YEME (Hopwood et al.,1985) or tryptic soy
broth (TSB;Difco);MP5 (Pernodet et al.,1993) was used for
spiramycin production.The culture conditions and the assay
for spiramycin production (bioassay and HPLC) were as
described elsewhere (Gourmelen et al.,1998).E.coli strains
were grown at 37 °C,except when harbouring pKC505 or its
derivatives (Richardson et al.,1987),in which case they were
grown at 30 °C.Conjugal DNA transfer from E.coli to
Streptomyces was performed essentially as described by
Bierman et al.(1992) with slight modi®cation (Gourmelen et
al.,1998).MICs were determined on HT agar plates con-
taining antibiotics,and seeded with 5¬10
%
c.f.u.of spores per
plate.Growth was monitored after 3 d incubation.Antibiotics
were obtained from the following sources:apramycin,Eli
Lilly;ampicillin,Appligene;carbomycin,P®zer;chalcomycin,
erythromycin,geneticin (G418),nalidixic acid,oleandomycin
and tylosin,Sigma;hygromycin B,Boehringer Mannheim;
josamycin,Pharmuka;rosaramicin,Schering-Plough;nosi-
heptide,pristinamycin I and spiramycin,Rho
#
ne-Poulenc
Rorer.
DNA techniques and cloning procedures.
DNA extractions
and manipulations were performed according to well-es-
tablished procedures (Hopwood et al.,1985;Sambrook et al.,
1989).For the construction of the S.ambofaciens gene library,
genomic DNA of S.ambofaciens ATCC 23877 was partially
digested by Sau3AI to yield fragments in the range 30±40 kb.
Digested genomic DNA (3
l
g) was then ligated with 1
l
g
BamHI-digested cosmid vector pKC505,which confers apra-
mycin resistance in E.coli and Streptomyces.The ligation
mixture was packaged in vitro in
k
heads,using packaging
extracts obtained from Amersham,and the resulting phage
particles were used for infection of E.coli HB101.About
20000 apramycin-resistant E.coli clones were pooled and
their cosmid DNA was extracted.
Standard conditions were used for the transformation of E.
coli strains (Sambrook et al.,1989).Streptomyces protoplasts
were formed,transformed and regenerated as described
previously for Streptomyces griseofuscus (Rao et al.,1987)
and Streptomyces lividans (Hopwood et al.,1985).
DNA sequence determination and analysis.
Single-stranded
DNA templates were sequenced by the chain-termination
technique using restriction fragments clonedin M13 mp18}19.
We used a Deaza T7 sequencing kit from Pharmacia and
[
$&
S]dATP
a
S from Amersham.Sequence was analysed with
the frame program (Bibb et al.,1984) and the GCG package
(Wisconsin Package version 9.1,Genetics Computer Group).
Sequence comparisons with databases used the fasta (Pearson
&Lipman,1988) and blast programs (Altschul et al.,1997).
Preparation of mycelial extracts and salt-washed ribosomes.
Crude mycelial extracts (30000 g supernatant,S30) and salt-
washed ribosomes were prepared as described elsewhere
(Skeggs et al.,1985).Ribosomal wash fraction was prepared
as previously described (Zalacain & Cundliffe,1989).In S.
ambofaciens,expression of some resistance genes,probably
including srmA,could be induced by spiramycin (Pernodet et
al.,1993).Therefore,strains harbouring the cloned srmAgene
were grown in the presence of 5
l
g spiramycin ml

"
to select
for the presence of the clonedgene andtoensure its expression.
Coupled transcription±translation assay.
Coupled transcrip-
tion±translation was performed as described previously
(Calcutt & Cundliffe,1989).All components of the coupled
transcription±translation systemexcept the salt-washed ribo-
somes were prepared from S.lividans.Ribosomes were
prepared from S.griseofuscus containing various plasmids.
One microgram of the plasmid pUC18 was used as
template.Protein synthesis was measured as incorporation of
[
$&
S]methionine into TCA-precipitable material as described
by Thompson et al.(1984).
rRNA methylation in vitro.
Total rRNA was prepared by
extraction of 70S salt-washed ribosomes with LiCl and urea
(Fahnestock et al.,1974).Methylation assays were carried out
at 30 °C in a ®nal volume of 100
l
l and contained 40 pmol
rRNA as substrate,55
l
l S30 extract or ribosomal wash
fraction as methyltransferase enzyme,2±5
l
Ci S-adenosyl
[methyl-
$
H]methionine ([methyl-
$
H]SAM;500 mCi mmol

"
;
18±5 GBq mmol

"
) as methyl donor cofactor,in buffer
containing 50 mM HEPES}KOH (pH7±5 at 20 °C),7 mM
MgCl
#
,37±5 mM NH
%
Cl and 3 mM
b
-mercaptoethanol.
Methylation of rRNA was measured as incorporation of
[
$
H]methyl groups into TCA-precipitable material as pre-
viously described by Skeggs et al.(1985).To allow identi-
®cation of the radiolabelled residue,the assay mixture was
scaled up by a factor of three,and 15
l
Ci [methyl-
$
H]SAM
with higher speci®c activity (15 Ci mmol

"
;555 GBq mmol

"
)
was used.Incubation was for 45 min at 30 °Cand the labelled
rRNA was recovered by phenol extraction and ethanol
precipitation.
Identi®cation of the methylated residue.
Radiolabelled
rRNA obtained after bulk methylation was hydrolysed with
1 MHCl at 100 °C for 60 min and the products of hydrolysis
(purine bases,pyrimidine-3«-mononucleotides and ribose
phosphate) were separated by descending chromatography on
Whatman 3MM paper using as solvent either 2-propanol}
2356
Resistance to spiramycin in S.ambofaciens
Table 1.Strains and plasmids used in this study
Strain/plasmid Description Reference
Strains
Streptomyces
S.ambofaciens ATCC 23877 Wild-type strain,spiramycin producer Pinnert-Sindico (1954)
S.griseofuscus DSM40191 Restriction-less strain,naturally sensitive to
spiramycin
Cox &Baltz (1984)
S.ambofaciens OS41.102 Derivative of S.ambofaciens ATCC 23877,with
srmA inactivated by gene replacement
This study
S.lividans TK21 Strain devoid of plasmid Hopwood et al.(1983)
S.lividans OS456 Derivative of S.lividans TK21 with lrm and mgt
inactivated by gene replacement,highly sensitive
to macrolides
Pernodet et al.(1996)
Escherichia coli
HB101 Host strain for the cosmid library Boyer &Roulland-Dussoix (1969)
JM101 Host strain for subcloning experiments Yanisch-Perron et al.(1985)
S17.1 Donor strain used for E.coli}Streptomyces
conjugation
Simon et al.(1983)
Plasmids
pKC505 E.coli}Streptomyces shuttle cosmid Richardson et al.(1987)
pTZ19 High-copy-number E.coli vector Mead et al.(1986)
pIJ487 High-copy-number Streptomyces vector Ward et al.(1986)
pIJ903 E.coli}Streptomyces shuttle vector,low-copy
number in Streptomyces
Lydiate et al.(1985)
pOJ260 Replicative vector in E.coli,non-replicative in
Streptomyces,used for conjugation experiments
Bierman et al.(1992)
pLST391 Plasmid containing the cloned ermE gene Jenkins et al.(1989)
pHP45
X
hyg Plasmid containing the
X
hyg cassette Blondelet-Rouault et al.(1997)
pOS41.1 Cosmid from the S.ambofaciens ATCC 23877 gene
library in pKC505,hybridizing with ermE
This study
pOS41.3 PstI±KpnI fragment containing srmA (Fig.1),
cloned into pIJ487
This study
pOS41.44 BamHI±SplI fragment containing srmA (Fig.1),
cloned into pIJ903
This study
pOS41.102 BamHI±KpnI fragment containing srmA inactivated
by insertion of
X
hyg into the XhoI site (Fig.1);
cloned into pOJ260,used for gene replacement
experiment
This study
HCl}water (170:41:39,by vol.) or butanol}water}ammonia
(95:14:5,by vol.),as previously described (Zalacain &
Cundliffe,1989).After drying,markers were detected under
UV light,the chromatogram tracks were cut into strips 1 cm
wide and radioactivity was measured by liquid-scintillation
spectrometry.To con®rm the identi®cation radiolabelled
rRNA was digested with nuclease P1 to generate nucleoside-
5«-monophosphates,whichwere analysedby two-dimensional
TLC,as previously described by Jenkins et al.(1989).
RESULTS
Cloning of spiramycin-resistance determinants and
subcloning of srmA
Cosmid DNAwas extracted froma pool of about 20000
E.coli clones constituting the S.ambofaciens gene
library in pKC505 and introduced by protoplast trans-
formation into S.griseofuscus,a strain naturally sen-
sitive to spiramycin.Transformants containing the
vector were selected on apramycin and about 1300
apramycin-resistant colonies were picked onto HT
medium containing 5
l
g spiramycin ml

"
.Several
apramycin-resistant colonies also grew in the presence
of spiramycin and cosmids extracted fromthese colonies
were used to transformE.coli and S.griseofuscus.Five
cosmids able to confer apramycin resistance in E.coli
and co-resistance to spiramycin and apramycin in S.
griseofuscus were conserved.
As we had shown previously that a ribosomal resistance
mechanism was present in S.ambofaciens (Pernodet et
al.,1993),the conserved cosmids were screened by
hybridization analysis for the presence of MLS-resist-
ance genes,using as the probe ermE from Saccharo-
polyspora erythraea,which produces the macrolide
antibiotic erythromycin (Uchiyama &Weisblum,1985).
This gene had been successfully used as a probe in the
2357
J.-L.PERNODET and OTHERS
.................................................................................................................................................................................................................................................................................................................
Fig.1.(a) Restriction map of the chromosomal region of S.ambofaciens ATCC 23877 near srmA.Lines below the
restriction map represent the length of the inserts in various plasmids (see also Table 1).R and S indicate if the plasmid
conferred spiramycin resistance or not,respectively.(b) Restriction map of the chromosomal region of S.ambofaciens
OS41.102 in which srmA has been inactivated by gene replacement.The insert used in gene replacement experiments is
represented schematically.
cloning of ermSF (synonym,tlrA) from Strepto-
myces fradiae,which produces the macrolide tylosin
(Kamimiya & Weisblum,1988).One of the cosmids,
pOS41.1,hybridized with ermE (data not shown),
suggesting that the resistance determinant carried by
pOS41.1 could also encode an rRNAmethyltransferase.
Accordingly,this resistance determinant was subcloned
and the resistance mechanism was investigated.
Within pOS41.1,ermE hybridized with a single PstI
fragment,a single KpnI fragment,and a single shorter
PstI±KpnI fragment.This latter fragment (4±8 kb) was
ligated into pTZ19R to give pOS41.2 and then intro-
duced into the Streptomyces cloning vector pIJ486 as an
EcoRI±HindIII fragment,yielding pOS41.3,which con-
ferred spiramycin resistance in S.griseofuscus.The
restriction map of the PstI±KpnI fragment (Fig.1a)
resembled strongly the one obtained previously for
srmA (Richardson et al.,1987),with the exception of a
single PvuII site that we did not detect,perhaps re¯ecting
the use of different S.ambofaciens strains.Moreover,
hybridization experiments (data not shown) con®rmed
that the DNAfragment we had cloned was similar to the
one previously isolated by these authors.Therefore,the
resistance determinant present in pOS41.3 was desig-
nated srmA.When restriction fragments derived from
the PstI±KpnI insert were introducedinto S.griseofuscus
and examined for their ability to confer spiramycin
resistance,they precisely located srmA within the
BamHI±SplI fragment (Fig.1a).
Nucleotide sequence of srmA
The nucleotide sequence of the BamHI±SplI fragment of
pOS41.3 was determined for both strands.Analysis of
the sequence for ORFs presenting the biased codon
usage typical of Streptomyces genes (Bibb et al.,1984)
revealed only one complete ORF,evidently correspond-
ing to srmA,with truncated ORFs on either side.The
deduced product of srmA is a protein of 259 aa
(molecular mass 28±6 kDa) and sequence comparisons
with databases showed end-to-end similarity to several
rRNA methyltransferases that confer resistance to MLS
antibiotics.For instance,SrmA presents 86%and 75%
identity with the Lrm protein (Jenkins & Cundliffe,
1991) and ErmSV (Kamimiya & Weisblum,1997),
respectively.Also,SrmA contains,between residues 40
and 48,a glycine-rich sequence that is shared by other
SAM-dependent methyltransferases (Kagan & Clarke,
1994) and has been shown by crystallographic analysis
to be part of the SAM-binding site (Schluckebier et al.,
1995).Since there is only one candidate start codon
upstream of this motif in the gene sequence,we were
able to assign the start of the coding sequence with
con®dence.
By alignment of SrmA with other deduced Erm-type
2358
Resistance to spiramycin in S.ambofaciens
.....................................................................................................
Fig.2.Dendrogram of the deduced MLS
resistance methyltransferase sequences from
actinomycetes.The name of the protein,
the MLS type (if known),the accession
number of the sequence in databases
(EMBL/GenBank or PIR),the name of the
organism and,for producers of macrolides
or lincosamides,the antibiotic produced are
indicated.
20
40
60
80
15 30 45 60
Time (min)
(a)
Control ribosomes
[35S]Methionine incorporated (% of the control at 60 min)
100
20
40
60
80
15 30 45 60
(b)
srmA ribosomes
100
.....................................................................................................
Fig.3.Effects of antibiotics on protein
synthesis in a coupled transcription±trans-
lation system.Ribosomes were from:(a) S.
griseofuscus harbouring pIJ487 (control);(b)
S.griseofuscus harbouring pOS41.3 (srmA).
All other components were derived from S.
lividans.Antibiotics added were:no drug
(+);1
l
g carbomycin ml

1
(D);1
l
g spira-
mycin ml

1
(E);50
l
g lincomycin ml

1
(y).
protein sequences from actinomycetes,the dendrogram
presented in Fig.2 was constructed.SrmA is most
closely related to Lrm and ErmSV,which mono-
methylate rRNA (Jenkins et al.,1989;Kamimiya &
Weisblum,1997),but as previously noted,it was not
possible todistinguishbetweenmonomethyltransferases
2359
J.-L.PERNODET and OTHERS
4
8
12
16
10 20 30 40
Time (min)
10–3
´[methyl-3H] Radioactivity (c.p.m.)
20
50
24
.................................................................................................................................................
Fig.4.Methylation of rRNA in vitro.Extract from S.
griseofuscus harbouring pOS41.3 (srmA) was used as the source
of methyltransferase together with [methyl-
3
H]SAM as co-
substrate.Total rRNA came from S.griseofuscus harbouring
pIJ487 (control) (+),pOS41.3 (srmA) (E) or pLST391 (ermE) (_).
(conferring the MLS type I resistance phenotype) and
dimethyltransferases (conferring the MLS type II re-
sistance phenotype) on the basis of sequence comparison
(Pernodet et al.,1996).Further investigations were
necessary to determine the precise mechanismby which
SrmA confers resistance.
Characterization of the SrmAresistance mechanism
Ribosomes from S.griseofuscus (pIJ487;control) or
from S.griseofuscus harbouring pOS41.3 (srmA) were
introduced into a coupled transcription±translation
system in which all other components came from S.
lividans.In this system,the effects of various MLS
antibiotics onproteinsynthesis were studied.Ribosomes
from the strain containing the cloned srmA gene were
highly resistant to lincomycin and showed signi®cant
resistance to macrolides such as spiramycin and carbo-
mycin (Fig.3).This result clearly demonstrated that
SrmAacts by ribosome modi®cation.Experiments were
also performed with S.ambofaciens ribosomes.When
these came from young mycelium,not yet producing
spiramycin,they resembled those from S.griseofuscus
harbouring pIJ487,i.e.they were sensitive (data not
shown).However,when derived from young mycelium
in which resistance had been induced by subinhibitory
concentrations of spiramycin (Pernodet et al.,1993),or
from mycelium producing spiramycin,the ribosomes
behaved like those from S.griseofuscus harbouring
srmA (data not shown).Such behaviour was charac-
teristic of particles that had been monomethylated by
the products of ermtype I genes (Pernodet et al.,1996).
Methylation of rRNA
Since SrmA conferred resistance via ribosomal modi-
®cation and was highly similar to authentic rRNA
methyltransferases,we tested directly for the presence of
clone-speci®c rRNA methyltransferase activity,using
[methyl-
$
H]SAM as methyl donor and total rRNA as
substrate.Methyltransferase activity was detected in
S30 crude extracts and in ribosomal wash fractions from
S.griseofuscus harbouring the cloned srmA gene,when
total rRNA from S.griseofuscus harbouring pIJ487
(control strain) was used as substrate (Fig.4).The
stoichiometry of methylation was about 0±8.As a
negative control total rRNA from S.griseofuscus
harbouring srmA was used as substrate,and was not
signi®cantly methylated in vitro.Total rRNA from S.
griseofuscus harbouring pLST391,a plasmid containing
the ermE gene,was also not methylated by extracts
containing SrmA.This result suggested that the ErmE
and SrmAmethyltransferases might act at the same or at
closely related sites.
Identi®cation of the methylated residue
rRNA was radiomethylated in vitro by extracts con-
taining SrmA and submitted to acid hydrolysis or
enzymic digestion,following which the products
were separated by various chromatographic methods.
When the products of acid hydrolysis (purine bases,
pyrimidine-3«-mononucleotides and ribose phosphate)
were separated by descending paper chromatography,
using 2-propanol}HCl as solvent,a single radioactive
spot migrated ahead of adenine (data not shown),
together with N
'
-monomethyladenine and N
'
,N
'
-
dimethyladenine standards,which are poorly resolved
in this chromatographic system.However,when the
radiolabelled material was eluted from the paper and
analysed again with butanol}ammonia solvent,it co-
migrated with the N
'
-monomethyladenine standard,
whichwas clearly resolvedfromN
'
,N
'
-dimethyladenine
(data not shown,but see Fig.5).To con®rm the nature
of the modi®ed residue,radiomethylated rRNA was
completely digested by nuclease P1 and the nucleoside-
5«-monophosphates obtained were separated by two-
dimensional TLC.A single radioactive spot was
detected,co-migrating with the N
'
-monomethyl-
adenosine-5«-monophosphate standard (data not
shown).Collectively,these data indicated that SrmA
was a monomethyltransferase acting on adenine at
position N
'
.
Location of the methylated residue within rRNA
rRNA modi®ed by ErmE was a very poor substrate for
SrmA,suggesting that both methyltransferases might
act at the same site,although SrmA is a monomethyl-
transferase and ErmE a dimethyltransferase.ErmE can
2360
Resistance to spiramycin in S.ambofaciens
1
2
3
4
10 20 30 40
Distance migrated (cm)
10–3
´[methyl-3H] Radioactivity (c.p.m.)
0
m
6
A m
6
2
A
1
2
3
4
10 20 30 400
m
6
A m
6
2
A
(a)
(b)
.................................................................................................................................................
Fig.5.Location of the methylated residue within rRNA.Total
rRNA was ®rst methylated by extracts containing SrmA,in the
presence of [methyl-
3
H]SAM.Then a large excess of cold SAM
was added and incubation was continued after addition of:(a)
buffer;(b) extract containing ErmE.The rRNA was then
subjected to acid hydrolysis and the products were separated by
paper chromatography.m
6
A,N
6
-monomethyladenine;m
6
2
A,
N
6
,N
6
-dimethyladenine.
also introduce a second methyl group at the N
'
position
of A-2058 that has already been N
'
-monomethylated.
Therefore,to know if SrmA acts on A-2058,a two-step
methylation experiment was performed as previously
described by Jenkins et al.(1989).Total rRNA was ®rst
radiolabelled in vitro by extracts containing SrmA,in
the presence of [methyl-
$
H]SAM;then a 150-fold excess
of non-radioactive SAMwas added and the reaction was
divided in two.Extract containing ErmE was added to
one half of the reaction and incubation was continued
for a further 45 min.As a control,buffer was added to
the other half and incubation was carried out under
similar conditions.Radiolabelled rRNA was then
extracted,submitted to acid hydrolysis and the products
were analysed by descending paper chromatography in
the butanol}ammonia solvent system (Fig.5).Since the
N
'
-monomethyladenine produced by SrmA was con-
verted into N
'
,N
'
-dimethyladenine after the action of
ErmE,a single adenosine is modi®ed by SrmA and this
being the one also modi®ed by ErmE,is A-2058 within
the large 23S rRNA.
Table 2.MICs for various antibiotics against strains of
S.lividans
Antibiotic MIC (
l
g ml

1
) for S.lividans OS456
harbouring:
pIJ903 pOS41.44 pLST391
(control) (srmA) (ermE)
Spiramycin 20"1000"1000
Carbomycin 5 500"1000
Rosaramicin 2 500"1000
Erythromycin 2 50"1000
Tylosin 2 20"1000
Chalcomycin 1 2"1000
Lincomycin 20"5000"5000
Pristinamycin I 200 500"1000
Resistance phenotype conferred by srmA
These experiments utilized S.lividans OS456,a
speci®cally deleted,antibiotic-sensitive strain that was
used previously to de®ne the MLS-I and MLS-II re-
sistance phenotypes (Pernodet et al.,1996).For com-
parison,pOS41.44 containing srmA and pLST391 con-
taining ermE were introduced separately into strain
OS456 and the MIC values of various MLS antibiotics
were determined.As seen in Table 2,srmAconferred the
MLS-I resistance phenotype characterized by high-level
resistance to lincomycin,moderate resistance to some
macrolides and streptogramin B antibiotics,and lower
resistance to chalcomycin,tylosin and erythromycin.In
comparison,ermE,which speci®es the MLS-II pheno-
type,conferred high resistance to all MLS antibiotics.
Generation of an srmA null mutant in S.ambofaciens
We attempted to determine the role played by srmA in
self-protection of S.ambofaciens against spiramycin by
inactivating the gene in the S.ambofaciens ATCC23877
wild-type strain.As shown in Fig.1(b),the
X
hyg cassette
(conferring hygromycin resistance in E.coli and
Streptomyces) was used to disrupt srmAat the XhoI site
within pOS41.102 (a conjugative vector derived from
pOJ260,which confers geneticin resistance in E.coli and
Streptomyces).Then,pOS41.102 was introduced into S.
ambofaciens via conjugal transfer fromE.coli S17.1 and
hygromycin selection was applied.Five days after
conjugation,hygromycin-resistant transconjugants
were picked and examined for geneticin resistance.
Hygromycin-resistant,geneticin-sensitive clones had
presumably undergone a double crossover recombi-
nation event,resulting in the replacement of srmAby its
disrupted counterpart from pOS41.102.This was
con®rmed by Southern blot hybridization analysis (data
not shown).One such disrupted strain was designated
OS41.102.This strain was not affected in its growth or
differentiation and,when grown in MP5 medium,it
produced spiramycin at a level comparable to the wild-
2361
J.-L.PERNODET and OTHERS
type,as checked by bioassay and con®rmed by HPLC
(data not shown).
DISCUSSION
At least ®ve genes conferring resistance to macrolides
have been isolated from S.ambofaciens (Richardson et
al.,1987;Gourmelen et al.,1998;present work).The
presence of several genes conferring resistance to the
autogenous antibiotic is not unusual in antibiotic-
producing actinomycetes.Among macrolide producers,
pairs of resistance genes have been isolated from the
carbomycin producer Streptomyces thermotolerans
(Epp et al.,1987),and from`Micromonospora
griseorubida',the mycinamicin producer (Inouye et al.,
1994).Four tylosin-resistance genes have been cloned
from the tylosin producer Streptomyces fradiae
(Birmingham et al.,1986;Baltz &Seno,1988;Rosteck
et al.,1991;Zalacain & Cundliffe,1991),and at least
®ve genes are supposed to be involved in oleandomycin
resistance in Streptomyces antibioticus (Hernandez et
al.,1993;Rodriguez et al.,1993;Olano et al.,1995;
Quiros et al.,1998).In contrast,only single resistance
genes have been isolated from`Streptomyces mycaro-
faciens',the midecamycin producer (Hara &
Hutchinson,1990),and fromthe erythromycin producer
Sacch.erythraea (Uchiyama &Weisblum,1985;Dhillon
&Leadlay,1990).
The gene srmAencodes a monomethyltransferase acting
on A-2058 in the large rRNA.Other macrolide-pro-
ducing actinomycetes in which genes conferring re-
sistance to the autogenous antibiotic have been sought
also contain at least one erm-type gene (see Fig.2),
except in the case of S.antibioticus,the oleandomycin
producer.In this organism,no ribosomal resistance
mechanism seems to be present (Fierro et al.,1987).
Therefore,although many macrolide producers modify
their ribosomes,at least during the antibiotic-
production phase,this is often not the only protection
mechanism used,and ribosomal resistance is not com-
pulsory in all macrolide producers.Here,when srmA
was inactivated in S.ambofaciens,the resulting strain
was viable and produced spiramycin,revealing that this
particular resistance gene is dispensable.However,in
addition to srmA,S.ambofaciens possesses another
erm-type gene,srmD,which also confers ribosomal
resistance to MLS antibiotics (unpublished data).There-
fore,the inactivation of srmA does not imply that
S.ambofaciens ribosomes are sensitive during the
spiramycin-production phase.
The MLS-I resistance phenotype is characterized by a
high level of resistance to lincosamides and variable
resistance to macrolides,lower than that observed with
the MLS-II phenotype.Type I erm genes are found in
strains producing lincosamides or macrolides such as
carbomycin or spiramycin (Fig.2;also Calcutt &
Cundliffe,1990),where the monomethylation of A-2058
appears to confer an adequate level of resistance.In
contrast,type II erm genes are found in strains pro-
ducing tylosin and erythromycin,against which mono-
methylation of A-2058 does not confer high-level re-
sistance.In summary,although the paradigm for MLS
resistance involves ermC,encoding an MLS-II pheno-
type in Staph.aureus,most erm-type genes yet charac-
terized in strains that produce macrolides or
lincosamides encode type I mechanisms.Interestingly,
no erm-type gene (of either variety) has been isolated
from streptogramin producers.
S.ambofaciens is not the only macrolide producer that
harbours multiple erm-type genes.In the tylosin pro-
ducer S.fradiae,ribosomes are constitutively mono-
methylated by the product of tlrD (Zalacain &
Cundliffe,1991),and the appearance of glycosylated
macrolides induces expression of the erm type II gene
tlrA (Kelemen et al.,1994).In contrast,A-2058 is not
constitutively methylated in S.ambofaciens and the
interplay between srmA and srmD remains to be
elucidated.
ACKNOWLEDGEMENTS
We are very grateful to M.Zalacain for discussion,advice and
kind help.We thank J.Gagnat for skilful technical assistance.
We thank M.Richardson and R.Nagaraja Rao for the gift of
cosmids (pKC505 and pKC534),M.Bibb for the gift of the
ermE probe,and colleagues in the pharmaceutical industry for
the gift of antibiotics.We also thank EMBO for a short-term
fellowship awarded to J.-L.P.A.G.received a PhDfellowship
fromthe Ministe
'
re de l'Education Nationale,de la Recherche
et de la Technologie,and from the Fondation pour la
Recherche Me
!
dicale.
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.................................................................................................................................................
Received 18 January 1999;accepted 2 March 1999.
2364