Journal of Bacteriology, August 1998, p. 4017-4023, Vol. 180, No. 16
Centro Nacional de Biotecnología,
CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco
28049, Madrid, Spain,1 and
Centro de
Química Farmaceútica, Cubanacan, Ciudad Habana,
Cuba2
Received 25 February 1998/Accepted 3 June 1998
In the search for heterologous activators for actinorhodin
production in Streptomyces lividans, 3.4 kb of DNA from
Streptomyces rochei F20 (a streptothricin producer) were
characterized. Subcloning experiments showed that the minimal DNA
fragment required for activation was 0.4 kb in size. The activation is
mediated by increasing the levels of transcription of the
actII-ORF4 gene. Sequencing of the minimal activating
fragment did not reveal any clues about its mechanism; nevertheless, it
was shown to overlap the 3' end of two convergent genes, one of whose
translated products (ORF2) strongly resembles that of other genes
belonging to the ABC transporter superfamily. Computer-assisted
analysis of the 3.4-kb DNA sequence showed the 3' terminus of an open
reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2,
and ORFB). Searches in the databases with their respective gene
products revealed similarities for ORF1 and ORF2 with ATP-binding
proteins and transmembrane proteins, respectively, which are found in
members of the ABC transporter superfamily. No similarities
for ORFA and ORFB were found in the databases. Insertional inactivation
of ORF1 and ORF2, their transcription analysis, and their cloning in
heterologous hosts suggested that these genes were not expressed under
our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs:
oleandomycin, erythromycin, spiramycin, doxorubicin, and
tetracycline. Thus, this genetic system, named msr, is a
new bacterial multidrug ABC transporter.
The transport of molecules through
cellular membranes is essential for living cells and can involve a
significant part of the cell's genetic information. Most of the
systems involved in this process are classified into a small number of
families according to their sequences, the molecular arrangement of
their individual components, and their molecular mechanisms. Of these,
the so-called ABC transporter superfamily forms one of the largest
and most diverse groups (2, 8, 18). Members of this
superfamily are found in both prokaryotes and eukaryotes. They
normally have some specificity for their substrates despite the wide
diversity of compounds transported by each: peptides, amino acids,
sugars, ions, antibiotics, toxins, heavy metals, etc. (2, 8,
18). Proteins of this superfamily usually have four domains: two
hydrophilic domains facing the cytoplasmic side which are
involved in ATP binding and hydrolysis and two hydrophobic
domains (each with six membrane-spanning segments) involved in forming
the structure needed for the substrates to be transported across
the membrane. The four domains may be organized either in a
multifunctional polypeptide or in separate proteins. One of the
most relevant features of these transporters is the coupling of the
energy from ATP hydrolysis for pumping their substrates across the
cellular membrane against a concentration gradient. The importance and awareness of the ABC transporters has notably increased over the last
few years since some of their members were implicated in single
resistance or multiresistance to antibiotics in pathogenic bacteria and
in lactic bacteria, as well as in multidrug resistance in tumors
(2, 8, 18, 26, 44).
Here we describe the cloning and characterization from
Streptomyces rochei F20, a producer of streptothricin
(13), of a new member of the ABC transporter superfamily.
This new transport system is highly similar to those conferring
specific resistance to the anticancer drug doxorubicin and the
macrolide oleandomycin and can, in some conditions, induce resistance
to both drugs in addition to erythromycin, spiramycin, and
tetracycline. Thus, this ABC transporter behaves as a multidrug
resistance system, and we named it msr (multiresistance from
S. rochei).
Bacterial strains.
The Escherichia coli strains
used here were JM101 and JM110 (49). The Streptomyces
lividans 66 strain was TK21 (str-6 SLP2 Plasmids and bacteriophages.
The E. coli plasmids
used were pUC19 (49), pIJ2925, and pIJ2921 (21).
E. coli M13 derivative phages mp18 and mp19 (49) were used for DNA sequencing. The E. coli Media, culture conditions, and microbiological procedures.
Streptomyces manipulations were done as described previously
(20). For streptothricin production by S. rochei
F20, liquid FM medium was used (yeast extract 1%, Bacto Tryptone 1%,
25 mM TES buffer [pH 7.5]), at 30°C with good aeration. E. coli strains were grown on Luria-Bertani (LB) agar or in LB broth
(24). The streptothricin production assays from liquid
medium were made as previously described (13).
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Silent ABC Transporter Isolated from
Streptomyces rochei F20 Induces Multidrug
Resistance
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
SLP3) (20). The Streptomyces
coelicolor strains were J1501 (20) and the
actII ORF4 mutants B43, B58 (37), and JF1
(9).
phage-derived
vector EMBL4 (14) was used to prepare a chromosomal library.
The high-copy-number Streptomyces plasmid vectors used were
pIJ486, pIJ487 (47), and pIJ702 (22). For
promoter activity assays in Streptomyces strains, plasmid
pIJ4083 (4) was used. The Streptomyces phage vector was the 
C31 derivative KC515 (33).
Nucleic acid manipulations. Isolation, cloning, and manipulation of nucleic acids were done as described for Streptomyces sp. (20) and for E. coli (24). For high-resolution S1 mapping, the method of Murray (25) was used. For RNA extraction from S. lividans and S. coelicolor, pregerminated spores were used to inoculate SY liquid medium (27), and mycelia were harvested after 48 h. RNA from S. rochei F20 was extracted from mycelia grown in FM (13) and harvested at several times of the cell cycle.
Construction of a chromosomal library.
For constructing a
chromosomal library of S. rochei F20 we used phage -EMBL4
as a vector and E. coli XL1-blue MRA as a host; both were
from Stratagene (catalog number 242201). Then 100 µg of S. rochei F20 chromosomal DNA was partially digested with
Sau3AI and fractionated on a 10 to 40% sucrose gradient
(24). Next, 5 µg of the fraction containing DNA fragments
of approximately 20 kb was ligated to 750 ng of vector previously
digested with BamHI and SalI. Packaging and
infection were performed according to the manufacturer's
recommendations.
DNA sequencing. DNA sequencing was carried out by the dideoxy-chain termination method (38). We used the 7-deaza-dGTP reagent kit from United States Biochemical Corp. (catalog number 70750) according to the manufacturer's recommendations.
Computer analysis of sequences. The DNA sequence was analyzed by using the software programs of the University of Wisconsin Genetics Computer Group (version 8.0-AXP) (5); analysis for open reading frames (ORFs) used CODONPREFERENCE with a codon usage table made from 100 Streptomyces genes (48). Comparisons of sequences were made against the EMBL Nucleic Acid Database and the Swissprot Data Base (both of them updated daily) by using FASTA, TFASTA, MOTIF, and BESTFIT. Protein alignments were made by using PILEUP and PRETTYBOX (32). Protein secondary structures were predicted with PEPTIDESTRUCTURE and PLOTSTRUCTURE. DNA secondary structure predictions were made with STEMLOOP.
Gene disruption.
For gene disruption of S. rochei
F20 we used insert-directed recombination of C31 derivatives as
described previously (3). In all cases, the chromosomal
arrangements of lysogens were confirmed by Southern blot analysis.
Engineering of msr ORFs. The promoter region of msr was replaced by the 444-bp MboII fragment containing the well-known actinorhodin polyketide synthase (PKS) promoter (12, 29); the resulting construct, named pMF1138.1 (see Fig. 4), thus carries msr-ORF1 and msr-ORF2 (nucleotides 464 to 2912) under the control of a heterologous promoter. No fusion proteins between the N terminus of ActI-ORF1 with Msr-ORF1 could have occurred because of an in-frame TGA codon upstream of ORF1. Both ORFs msr ORF1 and msr ORF2 would be translated by using their own ribosome binding site (RBS).
For a functional characterization of the cloned genes, several constructs were made (see Fig. 4). Thus, the entire ORFB (from the BstXI site at position 2532 to the right end) was removed from pMF1138.1, in several cloning steps, by replacement with the 155-bp HindIII/SphI fragment from the
fragment (30), leaving translation and transcription stop
signals immediately downstream of ORF2. This construction was named
pMF1147. To prevent ORF2 expression, the following recombinant plasmids
were made based on pMF1138.1 (see Fig. 4): pMF1139, in which the region
extending from nucleotide 1787 until the right end of the cloned
fragment was removed, leaving the whole ORF1 and 137 bp of ORF2;
pMF1145, in which the integron was inserted into the
NruI site of ORF2, introducing transcription and translation
stop signals; and pMF1146, a derivative of pMF1145 in which a
HindIII deletion removes the transcription stop signals,
leaving the translation stop codons in all frames within ORF2.
Likewise, to avoid the ORF1 expression, several pMF1138.1 derivatives
were made (see Fig. 4): pMF1140, in which most of ORF1 is deleted (up
to the NarI site, nucleotide 1577), leaving the last 75 bp
of ORF1 and the complete ORF2 including its own RBS; pMF1143, in which
an SmaI fragment containing the fragment was inserted
into the XmnI site of ORF1, introducing transcription and
translation stop signals; and finally pMF1144, in which an
HindIII fragment was deleted from pMF1143, leaving translation stop codons in all frames within ORF1.
DNA accession number. The DNA sequence reported here was submitted to EMBL-GenBank and was given accession number Y15759.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cloning and analysis of a DNA fragment inducing overproduction of actinorhodin in S. lividans. In order to gain further insight into the signals leading to activation of antibiotic biosynthetic genes, we used the ability of S. lividans to be converted to a blue phenotype when extra copies of a regulatory element are introduced by transformation. We used S. rochei F20 as a source of DNA since this strain is not an actinorhodin producer. Thus, any heterologous activation might well be due to general regulatory signals for secondary metabolism.
A library of S. rochei F20 chromosomal fragments was prepared in the pIJ702 vector, and protoplasts of S. lividans TK21 were transformed with the ligation mixture, selecting thiostrepton resistance. Plasmid DNA from one blue transformant (named pLE2032) was isolated and characterized. pLE2032 was found to contain a 0.8-kb DNA fragment (Fig. 1). By subcloning the original fragment, the minimal DNA region which was able to induce actinorhodin production was determined to be a 0.4-kb HincII fragment. This fragment was cloned in pIJ2925, then rescued as an EcoRI/HindIII fragment and cloned in the vectors pIJ486 and pIJ487 to yield plasmids pMF1141 and pMF1142, respectively. These recombinants differ only in insert orientation and were found to induce the same blue phenotype in S. lividans. Southern blot analysis confirmed that the cloned DNA represented an intact chromosomal fragment in S. rochei F20, while no hybridization was observed with DNA from the host strain.
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Actinorhodin induction in S. lividans is due to increased levels of transcription of the positive pathway-specific regulatory gene, actII-ORF4. To analyze the possible mechanism involved in the activation of actinorhodin by the cloned fragment, the plasmid pMF1141 was introduced into actII-ORF4 mutants B43, B58, and JF1. No complementation was observed, indicating that induction strictly requires the participation of actII-ORF4, the specific regulator for actinorhodin biosynthesis.
The effect of the activator fragment on actII-ORF4 transcription was examined in S. lividans TK21 strain by high-resolution S1 mapping. The probe was a 634-bp fragment (nucleotides 4825 to 5458) labeled on the 5' of the XhoI site at position 5458, extending therefore 354 nucleotides from the actII-ORF4 start codon into the coding region (11). Hybridization was done with total RNA from S. lividans TK21 containing the vector pIJ486 or plasmid pMF1141 and from S. coelicolor J1501. G+A and T+C Maxam and Gilbert sequencing reactions of the probe were run in parallel. A single S1-resistant hybridizing band was observed in S. lividans TK21(pMF1141) and in S. coelicolor, suggesting a unique transcription start point located at the C residue at position 5073 (Fig. 2).
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Subcloning and analysis of the region adjacent to the activator fragment. The DNA sequence of the activator fragment revealed that it contains part of a putative ABC transporter. To analyze if this overlapping arrangement involved a putative relationship between activation of antibiotic biosynthesis and a transport event, the adjacent region was isolated and characterized from a chromosomal library by using as a probe the 0.4-kb HincII DNA fragment from pMF1141. From a positive lambda clone, a 5.5-kb SacI fragment (overlapping the probe) was subcloned, yielding the plasmid pMF2036. Sequencing and computer-assisted analysis of 3.4 kb, covering the region coding for the putative ABC transporter, identified four putative ORFs (Fig. 1), which were named ORFA, ORF1, ORF2, and ORFB; the first three ORFs would be transcribed from left to right, whereas ORFB runs convergently. Thus, the activator fragment overlaps the 3' termini of ORF2 and ORFB. The most relevant features of this region, deduced from its DNA sequence, are summarized in Table 1.
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Deduced functions of the sequenced genes. Comparison of the deduced gene products of the truncated ORFA and ORFB with the databases showed no significant similarities and thus there are no clues about their possible roles. The ORF1 product showed strong resemblance with ATP-binding proteins belonging to the ABC transporter superfamily (18), particularly with several from Streptomyces species involved in the efflux of antibiotics, such as DrrA (for doxorubicin) (17), OleB and OleC-ORF4 (for oleandomycin) (28, 34), TlrC (for tylosin) (36), CarA (for carbomycin) (6) and SrmB (for spiramycin) (40), and from other genera, such as NodI from Bradyrhizobium japonicum, which presumably exports a lipooligosaccharide (45), and MsrA (for erythromycin) (35). Similarities varied from 50.8 to 81.6%, with identities from 25.3 to 66.6%, the highest values being found with DrrA from S. peucetius.
As for the ABC transporters, the typical consensus residues, known as "the signature of the family," are also present in ORF1 (residues 138 to 149), including the so-called loop 3, rich in glycine, which is postulated to interact with the phosphate group of the nucleotide (39, 46). Interestingly, a single Walker A and B motif is present in ORF1, as in DrrA, OleC, and NodI, whereas in MsrA, TlrC, CarA, SrmB, and OleB it is duplicated. The alignments of these domains are shown in Fig. 3. Based on these similarities, we postulated ATP-binding and hydrolysis activities for ORF1.
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-helical membrane-spanning segments and the N and C termini facing
the cytoplasmic side.
These similarities to other well-known gene products suggested that
ORF1 and ORF2 would presumptively constitute a two-component transport
system.
Functional characterization of ORF1 and ORF2. The functional characterization of msr was attempted in the original strain. Thus, recombinant phages containing DNA fragments internal to ORF1 and ORF2 were constructed and used to disrupt ORF1 and ORF2 by insertional inactivation (3) (Fig. 1).
To disrupt ORF1, an internal BalI fragment (nucleotides 715 to 1432) was first cloned in the HincII site of pIJ2925; the fragment was rescued with BglII and ligated to the phage KC515, which had been previously digested with BglII. Recombinant phagesAB29.1 and AB29.2, carrying
the insert in alternative orientations, were obtained. ORF2
disruptions were made in the same manner by using phages
AB28.1 and AB28.2 carrying the 507-bp
BssHII/DdeI fragment (nucleotides 1790 to 2297, previously blunt ended with Klenow enzyme) in either orientation.
Lysogens of S. rochei F20 were generated with the four
recombinant phages as previously described (13). No
phenotypic differences between the disruptants and the wild type
were observed and, therefore, no meaningful information about
the putative roles of these ORFs was obtained.
The expression of these genes was next explored at two levels: first,
transcription was analyzed by high-resolution S1 mapping in the
parental strain; second, the expression of their putative promoter
region in a promoter probe plasmid was tested in a heterologous host
(S. lividans) because S. rochei F20
could not be transformed with plasmid DNA by standard procedures
(13). For the former analysis, we determined the
transcription start point of ORF1 and ORF2 and the transcription
termination point of ORF2. After hybridization with total RNA, no S1
protected fragments were obtained, strongly suggesting that the genes
were not transcribed under our assay conditions. To analyze the
expression of the putative promoter region in S. lividans, the 247-bp BalI fragment (nucleotides 330 to
576, which includes the last 59 bp of ORFA and the first 16 bp of ORF1)
was cloned in the promoter probe vector pIJ4083, upstream of the
xylE reporter gene (50). No chromogenic
reaction with catechol was seen either in liquid medium or solid medium or on the plates containing a gradient of the agents which were previously tested as putative inducers (see above). These results are
in good agreement with the previous finding that no expression of these
genes occurred and did not allow the assignment of a function for the
cloned genes.
Since the apparent absence of expression of ORF1 and ORF2 might be due
to a silent or inducible promoter, which would not be expressed under
our experimental assay conditions, the cloned genes were engineered in
order to analyze their expression in a heterologous host: (i) the
SacI/XbaI fragment (sites 1 to 4 [Fig. 1],
nucleotides 1 to 2907) was cloned in the high-copy-number plasmid
pIJ486 to generate pMF1128; (ii) the promoter region for ORF1 and ORF2
was replaced by the well-known promoter that controls the expression of
the PKS for actinorhodin biosynthesis from S. coelicolor (12, 29), yielding plasmid pMF1138.1;
and (iii) pMF1138.1 was manipulated in order to allow selective
expression of ORF1 or ORF2 under the control of the PKS promoter (see
Materials and Methods and Fig. 4). The
resulting plasmids were used to transform S. lividans
TK21. The recombinant strains were tested by the disk assay method for
resistance to the following drugs and chemical agents: doxorubicin,
oleandomycin, erythromycin, tetracycline, spiramycin,
streptothricin, tobramycin, novobiocin, streptomycin, chloramphenicol,
phenol, pentachlorophenol, tetrachlorohydroquinone, and ethidium
bromide. Only strains containing ORF1 and ORF2 simultaneously under the
control of the PKS promoter showed a multiresistance phenotype to
erythromycin, spiramycin, oleandomycin, doxorubicin, and
tetracycline. As shown in Fig. 4, removing ORFB from pMF1138.1 (pMF1147) still conferred resistance to the same drugs, excluding a possible role for ORFB in the multiresistance phenotype.
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, no resistance;
+, resistance.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A new multiresistance determinant has been identified in Streptomyces rochei by isolating an overlapping trans-acting transcriptional activator for actinorhodin biosynthetic genes in S. lividans. It was shown that this activation is mediated by increasing the transcription of the pathway-specific positive regulator of the act genes, actII-ORF4. Although we have no clue about the precise mechanism, this could involve either the neutralization of a putative repressor (by direct binding or by an antisense mechanism) or be the response to a putative stress induced by the high copy number of this DNA. In any case, there is not enough experimental information to establish a correlation between this activator and the process which leads to the multiresistance phenotype. This will be the subject of future studies.
Interestingly, in our studies of the activator fragment we have identified a new member of the ABC transporter superfamily. This transporter, which is flanked by stem-loop structures, shows a high degree of similarity to the other members of the family, particularly with those involved in the resistance to the antitumor drug doxorubicin in the producer organism, S. peucetius. Based on the sequence similarities, we believe that this new transporter would be structurally formed by two homodimeric constituents as in many other bacterial ABC transporters: two Msr-ORF1 proteins facing the cytoplasmatic side (presumptively involved in ATP binding and hydrolysis) and two Msr-ORF2 molecules included in the cell membrane (presumptively forming the structure needed for extrusion of the corresponding substrate).
The failure to detect expression of msr, which has been demonstrated by gene disruption and by expression analysis both in the original strain and in heterologous hosts, suggests the existence of an inducer that might activate its promoter. Attempts to identify these putative inducers failed. Moreover, even those chemicals which were shown to be substrates for the engineered functional genes did not behave as inducers. Further experimental analysis will shed light on the existence and nature of the putative inducer of this new ABC transporter. Thus, the in vivo role of msr in S. rochei F20 is unclear: it could be involved in self-resistance to any bioactive metabolite produced by the strain (which would imply either an alternative resistance mechanism or the lack of production of such a metabolite in the disruptant strains); it could induce multiresistance as a selective advantage against biocide agents; or perhaps removing toxic chemicals from the cell could be the biochemical function of this system.
Despite the large diversity of substrates for ABC transporters described in the literature, the specificity of each system is relatively high and only a few members belonging to the ABC transporter superfamily induce multidrug resistance. All of them are of eukaryotic origin, such as the P glycoproteins, and only one characterized system is of bacterial origin (44).
From the experimental information presented in this paper, it is clear that msr-ORF1 and msr-ORF2 are simultaneously required for the multidrug resistance phenotype (Table 2). Neither msr-ORF1 nor msr-ORF2 can be replaced by any putative homologous gene in the host strain unless, if present, it is not expressed. This raises interesting questions: how widespread are msr or its analogs among actinomycetes, and how widespread are silent multidrug resistance systems such as the one described in this work?
Studies on efflux pumps such as the one described here are relevant because in the last few years it has been shown that pumping activities are involved in a large number of multiresistance phenomena by pathogenic bacteria and tumor cells. Particularly, tumor cells use ATP hydrolysis mediated by members of the ABC transporter superfamily to obtain the energy to efflux the chemotherapeutic agents. In addition, the unique bacterial multiresistance pump belonging to the ABC transporters is able to complement the human multidrug resistance P-glycoprotein gene (43), supporting the clinical and academic value of studying these mechanisms of transport.
As far as we know, the system described here is the first reported ABC transporter from a Streptomyces sp. conferring multidrug resistance. This system may be an important tool in the search for new drugs where resistance genes, with relaxed requirements for substrates, are needed. Likewise, it will offer a useful model for further studies on multidrug resistance mechanisms from either bacterial or eukaryotic cells.
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ACKNOWLEDGMENTS |
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We thank D. A. Hopwood for critically reading the manuscript and L. Servin for technical suggestions.
This work was supported by grants from the Spanish CICYT (BIO96-1168 and BIO-2072-E), the Ministerio de Educación y Ciencia (Programa de Cooperación con Iberoamérica), and the Consejo Superior de Investigaciones Científicas/Agencia de Ciencia y Tecnologia para el desarrollo de Cuba and by an institutional grant from Repsol Petróleo S.A. to Centro Nacional de Biotecnología.
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FOOTNOTES |
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* Corresponding author. Mailing address: Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34-1-5854548. Fax: 34-1-5854506. E-mail: fmalpart{at}cnb.uam.es.
Present address: Departamento de Bioquímica, Facultad de
Medicina, Universidad Autónoma de Madrid/Instituto de
Investigaciones Biomédicas del CSIC, 28029 Madrid, Spain.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Bacteriology, August 1998, p. 4017-4023, Vol. 180, No. 16
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