1 Fakultät für Biologie, Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany
2 Center for Biotechnology (CeBiTec), Bioinformatics Resource Facility, Universität Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany
Correspondence
Andreas Schlüter
andreas.schlueter{at}genetik.uni-bielefeld.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL accession no. for the sequence reported in this paper is AJ698325.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Macrolides with the prototype drug erythromycin are clinically important antibiotics (Blondeau, 2002; Blondeau et al., 2002
). Their structures consist of 14-, 15- and 16-membered lactone ring systems with amino and/or neutral sugars attached via glycosidic bonds (Roberts et al., 1999
). Macrolides are applied in the treatment of upper and lower respiratory tract infections (including e.g. pneumonia and bronchitis), infections of the skin, the genito-urinary and intestinal tract caused by certain bacterial species belonging to the genera Pneumococcus, Legionella, Bordetella, Mycobacterium, Mycoplasma, Chlamydia and Corynebacterium (Stratton, 1998
; Roberts et al., 1999
; Zhanel et al., 2001
). Macrolides act by inhibition of protein biosynthesis at the large subunit (50S) of ribosomes. They dissociate the peptidyl-tRNA molecule from the ribosome resulting in termination of the growing peptide chain (Gaynor & Mankin, 2003
). Resistance to this class of antibiotics is mainly conferred by three different mechanisms: (i) modification of the 23S rRNA target site by different methyltransferases; (ii) efflux of the drug mediated by ATP-binding cassette (ABC)-type transport systems or by exporters belonging to the major facilitator superfamily (MFS); (iii) inactivation of the molecule by different enzymes such as esterases, hydrolases, transferases and phosphorylases (Sutcliffe et al., 1996
; Vester & Douthwaite, 2001
). Macrolide resistance genes were frequently found to be located on mobile plasmids which, to some extent, contribute to increased exchange of resistance to macrolide antibiotics (Matsuoka et al., 1998
; Lin & Chung, 1999
; Liebl et al., 2002
; Tauch et al., 2003
).
Several environmental hot-spots for the horizontal exchange of genetic material were discovered (Sèveno et al., 2002). Wastewater purification facilities are considered to play an important role for recombination, exchange and distribution of resistance determinants because they receive bacteria which were previously exposed to antimicrobial compounds with the inflow sewage water originating from hospitals, private households, industry and agriculture. Selective pressure might actually persist in sewage treatment plants since low concentrations of several antimicrobial drugs have been detected in sewage water (Ohlsen et al., 2003
). Wastewater treatment plants were found to be reservoirs for antibiotic-resistant bacteria and resistance plasmids (Mach & Grimes, 1982
; Blázquez et al., 1996
; Smalla & Sobecky, 2002
). Self-transmissible, broad-host-range plasmids belonging to the IncP-1 group and mediating diverse resistance spectra were frequently isolated from activated sludge bacteria of municipal wastewater treatment plants (Dröge et al., 2000
; Tennstedt et al., 2003
). Two such plasmids, namely the conjugative IncP-1
plasmids pB4 and pB10, were completely analysed at the DNA sequence level (Schlüter et al., 2003
; Tauch et al., 2003
). Plasmid pB4 possesses IncP-1
-specific backbone modules and is loaded with different mobile genetic elements carrying resistance genes. Among other resistance determinants pB4 encodes a tripartite antibiotic efflux system composed of a resistance-nodulation-division (RND) type transporter, a periplasmic membrane fusion protein (MFP) and an outer-membrane factor (OMF). Functional analysis showed that this efflux system conferred high-level resistance to the macrolide antibiotics erythromycin and roxythromycin on the host bacterium Pseudomonas sp. B13 (Tauch et al., 2003
). Since this was the first example of a macrolide resistance determinant of the RND-MFP-OMF-type to be found on a plasmid genome, the objective of the work described here was the isolation of other erythromycin-resistance plasmids from activated sludge bacteria of a wastewater treatment plant. Different erythromycin-resistance plasmids were characterized with respect to their replicon and erythromycin-resistance genes. Plasmid pRSB101 was selected for complete sequencing to learn more about the nature and organization of its replicon and resistance genes. In addition, we addressed the question of whether bacteria carrying erythromycin-resistance plasmids were released into the environment with the final effluents of the wastewater treatment plant.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antimicrobial susceptibility testing was carried out as described by Tennstedt et al. (2003). MIC tests were carried out as described by Dröge et al. (2000)
with minor modifications: E. coli DH5
with corresponding cloning vector without insert was used as negative control.
To test the transfer properties of pRSB101, this plasmid was transferred into the mobilizer strain E. coli S17-1 and mated with the recipients E. coli CV60 GFP, Pseudomonas sp. B13 GFP1 and Ralstonia eutropha GFP3 on cellulose acetate filters. Putative transconjugants were selected on LB media containing 30 µg rifampicin ml1 and 5 µg tetracycline ml1 for E. coli CV60 and 50 µg kanamycin ml1, 30 µg gentamicin ml1 and 5 µg tetracycline ml1 for Pseudomonas sp. B13 GFP1 and R. eutropha GFP3, respectively.
Standard DNA techniques.
Plasmid DNAs from the plasmid-containing E. coli DH5 mcr and Pseudomonas sp. B13 GFP1 derivatives were isolated with the Nucleobond PC100 kit on AX 100 columns (Macherey-Nagel) according to the protocol supplied by the manufacturer.
The pRSB101 plasmid DNA for the generation of a shotgun library was isolated with the Large-Construct kit (Qiagen) according to the manufacturers' instructions.
Total-plasmid-DNAs from bacteria of activated sludge and the final effluents were isolated with the Nucleobond PC100 kit on AX 100 columns according to the protocol supplied by the manufacturer. These total plasmid-DNAs were used to transform E. coli DH5 mcr by the CaCl2 method as described by Tennstedt et al. (2003)
or to transform Pseudomonas sp. B13 GFP1 by electroporation. Preparation of Pseudomonas sp. B13 GFP1 cells for electroporation was carried out as described by Artiguenave et al. (1997)
. Electroporation of Pseudomonas sp. B13 GFP1 cells was done according to the Gene Pulser (Bio-Rad) Electroprotocol as for Pseudomonas putida ATCC 12633T. Determination of the plasmid content of E. coli DH5
and Pseudomonas sp. B13 GFP1 transformants was done by Eckhardt-gel analysis as described by Hynes et al. (1985)
.
Recombinant pGEM-T-Easy (T-cloning vector; Promega), pBCKS (chloramphenicol resistant derivative of pBluescript), pUC18 (Yanisch-Perron et al., 1985), pK18mob (Schäfer et al., 1994
) and pBluescript (Stratagene) derivatives were isolated using the QIAprep Spin Miniprep kit according to the manufacturers' instructions. Restriction enzyme digestion, agarose gel electrophoresis, DNA cloning and transformation of E. coli DH5
was carried out according to Sambrook et al. (1989)
.
Construction of a shotgun library and DNA sequencing of pRSB101.
Purified pRSB101 plasmid DNA was partially restricted with the restriction enzyme Sau3A. Restriction fragments with sizes ranging from 1 to 3 kb were extracted from an agarose gel with the Sephaglas BandPrep kit (Amersham Pharmacia Biotech) according to the manufacturers' instructions and cloned into the BamHI-digested vector pZErO-2 (Invitrogen). Plasmid DNA was prepared from E. coli shotgun clones by an automated alkaline lysis with the RoboPrep 2500 (MWG) and BioRobot 9600 (Qiagen).
Sequencing reactions using dye-terminator chemistry were separated on MegaBACE 1000 capillar-sequencer (Amersham Biosciences) and ABI 377 (Applera; Applied Biosystems) DNA sequencers.
Sequencing reads were assembled using the Staden (GAP4) software package (Staden, 1996). Gap closure and polishing of the sequence was achieved by primer walking with walking primers designed on contig-DNA-sequences. This approach resulted in a single, circular molecule with a total length of 47 829 bp.
DNA sequence analysis and annotation.
Annotation of the finished pRSB101 sequence was done by using the GenDB (version 2.0) Annotation Tool (Meyer et al., 2003) as recently described by Tauch et al. (2003)
. Repeat regions within the pRSB101 sequence were identified and analysed by using the REPuter software (Kurtz et al., 2001
). Global amino acid sequence similarities were determined by using the Align Plus 4 (version 4.10) software package incorporated in the Clone Manager Professional Suite (Scientific & Educational Software) with the scoring matrix Standard Linear. The annotated sequence of pRSB101 is available under the GenBank/EMBL accession number AJ698325.
Replicon typing by PCR.
Incompatibility (Inc)-group typing of resistance plasmids was done by a PCR-based approach with replicon-specific primers for the Inc-groups P, W, Q, N and A/C (Götz et al., 1996; Llanes et al., 1996
) and with pRSB101-repA specific primers (this work), repA-L409 (5'-GACGCTGACACAGACTTCCA) and repA-R854 (5'-GGCAAGTCCTTATCGAGCTG). The following Inc-group specific primer pairs were used: trfA2-1/-2 for IncP, oriV-1/-2 for IncQ, oriV-1/-2 for IncW, kikA-1/-2 for IncN and rep-1/-2 for IncA/C (Götz et al., 1996
; Llanes et al., 1996
).
Detection of the macrolide resistance operon genes mph(A), mrx and mphR(A) and the IS26- and IS6100-specific transposase genes (tnpA) by PCR.
Macrolide resistance genes on plasmids were detected by PCR using primer pairs specific for mph(A) (mphA-L 5'-CTTGGGCTCGACTATAGGAT and mphA-R 5'-CTCCGTGTTGTCGATGAG), mrx (mrx-L 5'-GCTGTTTGTAGATGCAGGAC and mrx-R 5'-GCCATTGTAGCAAATTGAAG) and mphR(A) (mphR-L 5'-AAGTCCGATGACGAGGTACT and mphR-R 5'-TCGGGAAACATTAAACACAG).
The IS26- and IS6100-specific transposase genes were detected by PCR with the following primer pairs: IS26-L (5'-TTGCAAATAGTCGGTGGTGA) and IS26-R (5'-CGTAAGCCGTCTTCATGGAT) for tnpAIS26 and IS6100-L (5'-CGCTGGTATTGTCGCTATCC) and IS6100-R (5'-CCAATGCCAAAAGCTCTCTC) for tnpAIS6100.
Subcloning of DNA fragments generated by PCR.
Amplification of repA-specific fragments on pRSB101, pRSB105 and total plasmid DNA isolated from bacteria of the final effluents as template DNAs was done by using the primers repA-L409 (see above) and repA-R854 (see above). The repA-specific amplicons were cloned into the vector pGEM-T-Easy (T-cloning vector; Promega) according to the pGEM-T-Easy Vector Systems protocol supplied by the manufacturer. Prior to cloning the PCR-products were purified on Sephacryl Microspin S-200 HR columns (Amersham Pharmacia Biotech). Recombinant pGEM-T-Easy derivatives were characterized by restriction analysis and by sequencing with standard sequencing primers.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Determination of the complete pRSB101 sequence was achieved by applying a shotgun sequencing approach. Gap closure and polishing by primer-walking resulted in a circularly closed DNA sequence of 47 829 bp (Fig. 1) with a mean G+C content of 56·4 %. Subsequent annotation of the finished pRSB101 DNA sequence revealed that the plasmid contains 42 complete and nine disrupted or partially deleted coding sequences (cds), respectively (see Table 2
). An inventory of the intact coding sequences showed that six have predicted functions in plasmid replication, maintenance and stable inheritance, three in plasmid mobilization, 11 in resistance and regulation of resistance, nine in transposition, recombination/inversion and DNA integration, three in transport processes and four in regulation. No function could be assigned to seven coding sequences. It appeared that approximately 18 % of the pRSB101 nucleotide sequence is occupied by genes for plasmid-specific functions whereas the rest (82 %) encodes accessory functions. The backbone region for plasmid replication (rep) and stable plasmid partitioning (par) during cell division is separated from a module for plasmid mobilization (mob) by insertion of a segment encoding a multidrug resistance (MDR) efflux system. A 20 kb antibiotic-resistance gene region is located downstream of the mob-module. This part of pRSB101 contains seven different resistance genes associated with mobile genetic elements. Another genetic load region adjacent to the 20 kb resistance region consists of four complete transposable elements, footprints of two more transposable elements and a putative integron. The characteristics of the pRSB101 genes and a comparison of the deduced gene products to corresponding proteins encoded by other plasmids are listed in Table 2
.
|
|
The replication module of pRSB101 is very similar to that of plasmid pXAC33 from the phytopathogen Xanthomonas axonopodis pv. citri
The replication module of pRSB101 consists of the replication gene repA and the partition genes parA and parC with the same organization as compared to the corresponding genes present on plasmid pXAC33 of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (accession no. NC_003921). RepA of pRSB101 shows the highest degree of identity (77 %) to the replication protein A of the Xanthomonas axonopodis pv. citri plasmid pXAC33 and is a member of the replicase family (Pfam03090) which includes bacterial plasmid DNA replication initiator proteins. RepA of pRSB101 is also closely related to RepA of pPT23A-like plasmids from the phytopathogen Pseudomonas syringae (Sesma et al., 1998, 2000
). The partition genes parC and parA were identified upstream of repA on pRSB101. The deduced gene products of both genes are also homologous to the partition proteins C and A of pXAC33, respectively. ParA, belonging to the ParA family of ATPases (Pfam00991), most probably is involved in stable partitioning of plasmid replicates into daughter cells during cell division. The origin of replication (oriV) seems to be located downstream of repA since two different types of repeat sequences, a putative DnaA box and an A/T-rich region were found in this region on pRSB101. The first repeat type has the consensus sequence NNNTAGCC and occurs four times on the forward strand and four times on the reverse strand. Three copies of the second repeat type were identified (two on the forward and one on the reverse strand), which have the conserved sequence CCGCAGG. The DnaA box has the sequence TTATCCAC and is located on the reverse strand. Finally, the A/T-rich region is 53 bp long and has an A+T content of approximately 79 %.
pXAC33 encodes the avirulence (avr) genes pthA1 and pthA2 which are not present on pRSB101. No other significant functions could be assigned to pXAC33 (da Silva et al., 2002). Therefore it has to be concluded that pRSB101 only shares its basic replicon-type with pXAC33.
The product of the adjacent gene downstream of repA is homologous (41 % identity) to the -helical coiled-coil protein TlpA of the Salmonella typhimurium virulence plasmid pEX102 and the Salmonella enterica subsp. enterica serovar Choleraesuis virulence plasmid pKDSC50. It has been suggested that TlpA has a possible virulence-associated regulatory function (Koski et al., 1992
; Haneda et al., 2001
). The TlpA-like protein of pRSB101 also shows significant similarity (42 %) to the regulatory protein KfrA of IncP-1
plasmid pADP-1 (accession no. NC_004956). KfrA was predicted to have a long
-helical tail which might form coiled-coil structures. It was hypothesized that KfrA has a function in the plasmid partitioning process during cell division (Adamczyk & Jagura-Burdzy, 2003
). In addition, KfrA of IncP-1 plasmids negatively autoregulates its own expression. A kfrA-like gene is also present downstream of repA on the endogenous plasmid pRA2 of Pseudomonas alcaligenes NCIB 9867 (Kwong et al., 2000
) which resembles the organization of repA and the kfrA-like gene on pRSB101. Several amino acid residues conserved between KfrA of pRA2 and IncP-1 plasmids are also conserved in the pRSB101 KfrA-like protein (Kwong et al., 1998
). Taking together the findings described above, it is very likely that the KfrA-homologue of pRSB101 and related plasmids plays a role in stable plasmid inheritance and/or plasmid-regulation. In summary, the replication region present on pRSB101 is composed of at least four genes having functions in replication and stable plasmid inheritance (partitioning) and is related to replicons from phytopathogenic bacteria.
pRSB101 has a three-Mob-protein type mobilization system and can be mobilized by a self-transmissible helper plasmid
Plasmid pRSB101 contains a mobilization (mob) module encoding proteins homologous to MobA, MobB and MobC of IncQ-like broad-host-range plasmids. The pRSB101 MobA shows the highest degree of similarity (37 %) to the MobA relaxase/mobilization nuclease domain (Pfam03442) of the Acidithiobacillus caldus IncQ-like cryptic plasmid pTC-F14. This domain is predicted to function in nicking the plasmid DNA at the origin of transfer (oriT). The pRSB101 mobB and mobC gene products are homologous to the corresponding proteins of pTC-F14, the Acidithiobacillus ferrooxidans IncQ-like plasmid pTF-FC2 and the Aeromonas salmonicida tetracycline resistance plasmid pRAS3, respectively. Interestingly, pRSB101 MobB is more than twofold larger as compared to other MobB proteins and seems to possess a two-domain structure. It should be noted that the amino acid sequences of the two domains of pRSB101 MobB are not identical. MobB and MobC were proposed to be accessory mobilization proteins with possible functions in enhancement of nic-site cleavage and relaxosome stability. MobB might also help to recognize the origin of transfer (oriT) which is localized in the mobCmobB intergenic region (Rawlings & Tietze, 2001). A possible nic-site motif (TCCTG
) was found upstream of the pRSB101 mobC gene which is divergently transcribed as compared to mobBmobA. Since the pRSB101 mobilization module is most similar to corresponding systems encoded by the IncQ-like plasmids pTC-F14, pTF-FC2 and pRAS3 of Acidithiobacillus caldus, Acidithiobacillus ferrooxidans and Aeromonas salmonicida, respectively (Rohrer & Rawlings, 1992
; Gardner et al., 2001
; L'Abée-Lund & Sørum, 2002
), it is tentatively assumed that the pRSB101 mob-module originates from a plasmid related to IncQ-like plasmids.
Plasmids of the IncQ-family can be mobilized in the presence of self-transmissible helper plasmids. Conjugative IncP-1 type plasmids were shown to be particularly efficient in the mobilization of IncQ plasmids (Rawlings & Tietze, 2001). To test mobilization, pRSB101 was transformed into the E. coli mobilizer strain S17-1 carrying a derivative of the IncP-1
plasmid RP4 integrated in its chromosome. E. coli S17-1 (pRSB101) was mated with the recipients E. coli CV60 GFP, Pseudomonas sp. B13 GFP1 and R. eutropha GFP3 and transconjugants were selected on media containing the following antibiotic concentrations: 30 µg rifampicin ml1, 50 µg kanamycin ml1 and 5 µg tetracycline ml1 or 100 µg spectinomycin ml1 (for E. coli CV60 GFP) and 30 µg gentamicin ml1, 50 µg kanamycin ml1 and 5 µg tetracycline ml1 or 100 µg spectinomycin ml1 (for Pseudomonas sp. B13 GFP1 and R. eutropha GFP3). Plasmid pRSB101 could be mobilized from E. coli S17-1 to E. coli CV60 GFP.
Incorporation of a mobilization module into the pRSB101 genome arguably was a prerequisite for acquisition of most of its accessory genes, which are of diverse origins (see below).
pRSB101 contains a tetracycline resistance module next to the Tn402-specific inverted repeat motif
A derivative of the transposon Tn402 constitutes the core element of a 20 kb resistance region present on plasmid pRSB101 (see Fig. 2a). A tetracycline resistance (tet) module was found at one end of this resistance region. The 25 bp inverted repeat (IR)-element of a Tn402-like transposon is conserved at the outermost end of the resistance region, but the Tn402 transposition gene tniA was separated from the IR by insertion of the tet module. A 2792 bp DNA segment containing the 3'-end of a pecM gene, tetA encoding a tetracycline efflux pump and tetR for a regulatory protein is 99·9 % identical to the corresponding region present on the Birmingham IncP-1
plasmids (Pansegrau et al., 1994
). Tetracycline resistance genes were frequently found in plant-associated and phytopathogenic bacteria since oxytetracycline is applied to protect plants from pathogens (McManus et al., 2002
). Schnabel & Jones (1999)
reported on the detection of the tetA determinant in fluorescent and non-fluorescent Pseudomonas spp. isolated from apple orchards which were treated with oxytetracycline. Likewise, derivatives of the transposon Tn1721, which usually carries tetAtetR, were found in Xylella fastidiosa, the causative agent of citrus variegated chlorosis (Ferreira et al., 2002
). The origin of the pRSB101 tetAR genes cannot be easily determined due to the wide dissemination of these genes among environmental and pathogenic bacteria (Aminov et al., 2002
; Chopra & Roberts, 2001
; Rhodes et al., 2000
).
|
The pRSB101 pecM gene was truncated by insertion of a 168 bp DNA element (designated DR1 in Fig. 2a) containing the 49 bp IR of IS3000 at one end and the outer part of this IR (26 bp) at the other end. This DR1-element might represent a deletion derivative of IS3000 or a distinct genetic element which possibly can be moved by a specific transposase provided in trans. Three identical copies of the DR1-element were found in the vicinity of the tet module. The second DR1 copy borders the tet module 532 bp downstream of tetR and the third copy is located at the 5'-end of tniA (see Fig. 2a
). The DNA segment between the second and third DR1 copy encodes two ORFs, the deduced gene products of which show the highest degree of identity to, respectively, a predicted transcriptional regulator (COG3905) of Xylella fastidiosa (accession no. ZP_00041599) (61 % identity) and a conserved hypothetical protein belonging to the group of plasmid stabilization proteins (ParE: COG3668, Pfam05016) of Xanthomonas axonopodis pv. citri (accession no. NP_642743) (62 % identity). On the resistance plasmid RK2 the parD gene is located upstream of parE. ParD belongs to a family of transcriptional repressors and has been shown to bind to DNA in the parD promoter region (Roberts & Helinski, 1992
). The predicted transcriptional regulator encoded upstream of the parE-like gene on pRSB101 seems to be the orthologue of ParD, although it exhibits only weak similarity (36 %) as compared to ParD of RK2. The gene products of the parD-like and parE-like genes are predicted to constitute a second system for plasmid stabilization involved in post-segregational killing (psk) of plasmid-free segregants (Roberts & Helinski, 1992
). In addition to the ParD/ParE system, pRSB101 also possesses the partitioning genes parA (ATPase), parC and kfrA (putative filamentous
-helical coiled-coil protein). These systems have predicted functions in active plasmid partitioning during cell division and in post-segregational killing (psk) of plasmid-free segregants (Roberts & Helinski, 1992
; Adamczyk & Jagura-Burdzy, 2003
). Presence of at least five putative stability genes could be the reason why we never observed loss of pRSB101, even under non-selective conditions, during our work with this plasmid.
In summary, the described region can formally be interpreted as two DR1-bounded cassettes' carrying tetAtetR and parDE-like genes, respectively, which were tandemly inserted into the terminal part of a Tn402-like transposon.
The macrolide resistance operon present on pRSB101 confers high-level erythromycin resistance
Plasmid pRSB101 contains a macrolide resistance region flanked by copies of the insertion sequence elements IS26 and IS6100 (see Fig. 2a). A 5055 bp DNA segment containing the complete macrolide resistance operon, IS26 and IS6100 is 99·8 % identical to a corresponding segment of the Shigella flexneri Tn21-like transposon TnSF1 (accession no. AF188331). The macrolide resistance operon is composed of the genes mph(A), mrx and mphR(A) which encode a macrolide-2'-phosphotransferase I, a hydrophobic protein of unknown function and a negative transcriptional regulator, respectively. An identical macrolide resistance region was first cloned and sequenced from the clinical E. coli isolate Tf481A which exhibits high-level erythromycin resistance (Noguchi et al., 1995
, 2000
). Sequences upstream of E. coli mph(A) and downstream of mphR(A) suggest that this region is also flanked by IS26 (synonym: IS176) and IS6100. IS26 and IS6100 both belong to the insertion sequence family IS6 and possess almost identical 14 bp IR-elements. Therefore it is possible that these IS-elements, framing the macrolide resistance operon, constitute a composite transposon. The sequence upstream of the IS6100 tnpA gene on pRSB101 is nearly identical for 1309 bp to the corresponding region of Shigella flexneri TnSF1. The first 123 bp next to the insertion site of IS6100 are identical to one end of Tn402 and Tn1696 (IRt-end) including the 25 bp inverted repeat sequence (IRt) terminating these transposons (Partridge et al., 2001
). An IRt element is missing at the other end of IS6100. The DNA region between the IS6100 element and the 3'-conserved segment of a class 1 integron (see below) on pRSB101 contains two ORFs, orf1 and chrA, the first of which encodes a hypothetical protein that is conserved in various bacterial species (see Table 2
). It possesses the Pfam PadR-specific motif (Pfam03551) which is characteristic for transcriptional regulators belonging to the PadR-like family. The gene product of chrA shows 63 % identity to a probable chromate ion transporter (accession no. NP_252979) designated ChrA in other bacteria. Pseudomonas sp. B13 GFP1 and E. coli DH5
harbouring pRSB101 were checked for their growth properties in media containing potassium chromate (50800 µg ml1) and it was found that pRSB101 does not mediate chromate resistance to these host bacteria.
To determine the resistance level mediated by the pRSB101 macrolide resistance operon an appropriate restriction fragment was subcloned into the vector pUC18. The recombinant plasmid was shown to confer high level erythromycin resistance (up to 4500 µg ml1) to the host bacterium E. coli DH5 as compared to the basic vector (400 µg ml1). To our knowledge pRSB101 is the first example for a plasmid-borne mph(A)mrxmphR(A) macrolide resistance operon. Since the pRSB101 macrolide resistance operon and adjacent sequences are very closely related to a corresponding segment on the transposon TnSF1 (accession no. AF188331) of the human pathogen Shigella flexneri (strain SH595), which is the causative agent of bacillary dysentery and diarrhoea, we assume that pRSB101 captured part of a TnSF1-like transposon from an enteric bacterium sharing its mobile genetic element pool with Shigella flexneri.
Nine different erythromycin resistance plasmids were isolated in parallel with pRSB101. To determine whether these plasmids also contain the macrolide resistance operon mph(A)mrxmphR(A), specific primer pairs were designed for each gene. These primers were used to detect the respective genes by PCR on isolated plasmid DNAs as template. Plasmids pRSB101, pRSB102, pRSB103, pRSB104, pRSB106, pRSB107, pRSB108, pRSB109 and pRSB110 gave amplification products of the expected size for mph(A), mrx and mphR(A). In addition, internal fragments of IS6100 and IS26 which flank the mph(A)mrxmphR(A) operon of pRSB101 were also detected on the plasmids listed above. Plasmid pRSB105 does not contain a macrolide resistance operon and gave no PCR products with primers specific for the erythromycin esterification genes ereA, ereB and for the macrolide efflux genes mefA/E (Sutcliffe et al., 1996).
In summary, the mph(A)mrxmphR(A) operon seems to be the most abundant erythromycin resistance determinant on erythromycin-resistance plasmids isolated from activated sludge bacteria of the wastewater treatment plant under study.
pRSB101 contains a class 1 integron with an unusual composition of genes including a new class A -lactamase gene
An intact class 1 integron, potentially capable of integrating and disseminating resistance gene cassettes, was identified upstream of the putative chromate transporter gene chrA on pRSB101 (Fig. 2a). The variable region of this integron contains an unusual arrangement of gene cassettes and genes.
The integron-specific 5'-conserved segment on pRSB101, including the terminal inverted repeat (IRi), the intI1 integrase gene and the first recombination site (attI), is identical to the corresponding part of In0 on the Pseudomonas aeruginosa plasmid pVS1 (Bissonnette & Roy, 1992). Even the target site duplication (5 bp direct repeat, DR) next to the IRi-element is conserved in these integrons. The pRSB101 integron contains two resistance gene cassettes, namely dhfr1 for a dihydrofolate reductase conferring trimethoprim resistance and aadA2 encoding an aminoglycoside adenylyltransferase mediating streptomycin/spectinomycin resistance. The arrangement of these gene cassettes, including the 59-base elements, is essentially the same as described for the Klebsiella pneumoniae integron Int22AM (accession no. AY007807). Streptomycin resistance genes were frequently found in plant-associated and phytopathogenic bacteria since streptomycin has been widely used to control bacterial diseases in plants, especially in fruit orchards (Sundin & Bender, 1996
; Vivian et al., 2001
; McManus et al., 2002
; Sundin, 2002
). Some pPT23A-like plasmids of the phytopathogenic bacterium P. syringae as well as pRSB101 confer resistance to trimethoprim and streptomycin (Cooksey, 1990
). As outlined above the pPT23A RepA replication protein is related to RepA of pRSB101. Recently, the integron-specific streptomycin resistance gene cassette aadA2 which is present on pRSB101 was identified on the transposon Tn1404 from Pseudomonas sp. R9 which was isolated from a streptomycin-treated apple orchard (Schnabel & Jones, 1999
). Thus, we speculate that pRSB101 acquired the streptomycin resistance determinant during its evolution in a bacterium which lived in a streptomycin-contaminated environment. To determine spectinomycin and streptomycin resistance levels conferred by the aadA2 gene product, a 2282 bp SphI-fragment carrying aadA2 was cloned into the vector pUC18. The recombinant vector conferred high-level spectinomycin resistance (>2000 µg ml1) and streptomycin resistance (300 µg ml1) to the host bacterium E. coli DH5
as compared to the basic vector (100 µg spectinomycin ml1 and 6 µg streptomycin ml1, respectively).
Surprisingly, the composition of the DNA region downstream of the aadA2 cassette is puzzling because the genes acp-like, for a putative acyl-carrier protein, and blaTLA-2, encoding a new extended-spectrum class A -lactamase, do not seem to be located on integron-specific gene cassettes. Sequence motifs resembling the integron-specific 59-base element could not be found in the vicinity of acp and blaTLA-2, but it is noticeable that both genes are flanked by 145 bp direct repeats (termed DR2 in Fig. 2a
). Since these DR2-elements contain the consensus core-motif (GTTAAA) of integron-specific recombination sites we speculate that these DR-elements played a role in the integration of acp and blaTLA-2 into the variable region of the integron. The origin of these genes remains unknown. The deduced gene product of the acp-like gene is 29 % identical and 54 % similar to an acyl carrier protein of unknown function from Thermosynechococcus elongatus (accession no. NP_682662). The signature pattern for the acyl carrier protein phosphopantetheine domain (PROSITE no. PS50075) is conserved in the putative pRSB101 Acp protein.
The blaTLA-2 gene encodes a -lactamase of 304 amino acid residues. A putative ribosome-binding site (RBS) and possible 10/35 promoter motifs were found upstream of the ATG start codon. The blaTLA-2 gene was integrated into the variable region of the integron quite recently, since its G+C content of 38 % differs considerably from the mean G+C content of the whole plasmid (56 %). The deduced TLA-2 protein shows the highest degree of similarity to TLA-1 encoded by the conjugative plasmid RZA92 of the clinical E. coli isolate R170 (51 % identity and 64 % similarity) (Silva et al., 2000
). The motifs S70xxK, S130DN and K234TG, common to Ambler class A
-lactamases are conserved.
A 1771 bp SphI-fragment containing the complete blaTLA-2 gene and 359 bp of the upstream region was cloned into pZErO-2 and it was found that the recombinant plasmid pZErO-2-blaTLA-2 confers resistance to cefotaxime (30 µg ml1), cefuroxime (550 µg ml1) and cefpirome (30 µg ml1) to the host bacterium E. coli DH5. These resistance levels are, respectively, sixfold, 22-fold and sixfold above the intrinsic resistance of E. coli DH5
harbouring the basic vector. These results justify the classification of the pRSB101 TLA-2
-lactamase as an extended spectrum
-lactamase.
In summary, the blaTLA-2 gene of pRSB101 encodes a new -lactamase belonging to Ambler class A and most probably originates from a clinical bacterium which was exposed to expanded spectrum cephalosporins. The mechanism by which the gene has integrated into the pRSB101 integron structure remains unknown.
The DNA sequence of the integron-specific 3'-segment present on pRSB101, including qacE1, sul1 and orf5
, is essentially the same as compared to the integron on TnSF1 (99·7 % identity at the DNA sequence level). The only difference is that the core-site of the 59-base element which is normally present upstream of qacE
1 is missing in the pRSB101 sequence. This sequence motif was replaced by a DR2 element (see Fig. 2a
) which might have played a role in the integration of the blaTLA-2 gene. Deletion of orf5
in TnSF1 and the pRSB101 integron most probably occurred by insertion of a Tn501-like transposon since a 38 bp IR element very similar to those of Tn21/Tn501 transposons was found at the 3'-end of orf5
(see Fig. 2a
). A 8384 bp fragment of pRSB101 containing IS26, the mph-operon, IS6100, chrA and the integron-specific 3'-conserved segment is also an integral part of the Shigella flexneri transposon TnSF1 (99·8 % identity at the DNA sequence level). Unfortunately, TnSF1 has not been published until now and the DNA-sequence of the database entry (accession no. AF188331) was only partially annotated.
pRSB101 encodes a putative MDR system of the ABC-type associated with a membrane fusion protein (MFP)
A putative MDR transport system consisting of three components and a regulatory protein is encoded between the replication and the mobilization module on pRSB101.
The first gene of the presumptive MDR region encodes a transcriptional regulator of the TetR/AcrR family (COG1309, Pfam00440). The acrR gene product shows 40 % similarity to the corresponding gene product of Geobacter sulfurreducens PCA (accession no. NP_952005) and 35 % similarity to the AcrR of E. coli K-12 (accession no. NP_414997). The repressor AcrR modulates the regulation of acrAB for a multidrug efflux pump in E. coli (Ma et al., 1996). The pRSB101 AcrR regulator possesses a DNA-binding helix-turn-helix motif near the N terminus (residues 33 to 54) which is typical for repressor proteins belonging to the TetR/AcrR family. A 28 bp palindromic sequence motif which might serve as a binding site for AcrR was found 12 bp upstream of the ATG start-codon of the acrR gene on pRSB101. The acrR gene and the downstream gene are most probably translationally coupled since the stop-codon of the first gene overlaps with the start-codon of the second one. It is therefore very likely that the AcrR regulator controls transcription of the complete MDR-operon including the regulatory gene. The gene downstream of the acrR gene encodes a protein that shows 45 % similarity to the probable RND efflux MFP of G. sulfurreducens (accession no. NP_952003) and 34 % to the MFP of E. coli K-12 (accession no. NP_414996) (see Fig. 3
). Classification of the pRSB101 MFP reveals that this protein clusters within the COG0845 group of putative membrane fusion proteins. The prototype of this group, AcrA, represents the accessory periplasmic protein of an RND-type multidrug efflux system which pumps out a wide variety of lipophilic and amphiphilic compounds (Nikaido & Zgurskaya, 2001
). MFP of pRSB101 possesses a signal-peptide which could be involved in guidance of the protein into the periplasmic space. The possibility also exists that the hydrophobic N-terminal segment anchors the protein in the cytoplasmic membrane. It is thought that MFPs physically connect the cytoplasmic transporter component (see below) to an outer-membrane factor (OMF) facilitating transport across the outer membrane of Gram-negative bacteria (Zgurskaya, 2002
). An OMF is not encoded in the pRSB101 MDR-region but the 3'-end of an oprM gene for an outer-membrane protein is located downstream of the tetracycline regulator gene tetR on pRSB101. It has been shown that OMFs function with more than one permease/MFP pair (Saier & Paulsen, 2001
; Paulsen et al., 1997
), so that we speculate that a heterologous, host-encoded OMF interacts with the pRSB101 MDR efflux components. The gene for the MFP is also translationally coupled to the downstream gene of the MDR-operon since its stop-codon overlaps with the start-codon of the following gene (combined ATGA start/stop motif). The deduced gene product of the third gene of the MDR-operon is 59 % similar to a possible ABC-type transporter transmembrane subunit of G. sulfurreducens (accession no. NP_952002). Analysis of the membrane topology using the TopPred tool revealed that the pRSB101 protein contains six putative transmembrane helices with the N and C termini being localized at the cytoplasmic site of the membrane. Similar membrane topologies were predicted for most ABC-type permeases (van Veen & Konings, 1998
; Higgins, 2001
). Another interesting feature of the pRSB101-encoded permease is that its C-terminal part gives a hit to the Pfam FtsX-family (Pfam02687) which includes predicted permeases some of which were shown to transport lipophilic substrates targeted to the outer membrane across the inner membrane. The transport process of ABC-transport systems is normally coupled to the hydrolysis of ATP. A putative ATP-binding protein is encoded by the last gene of the MDR-region. The corresponding gene product is 45 % identical to an ATP-binding component of an ABC-transporter of G. sulfurreducens (accession no. NP_952001). The amino acid sequence of the pRSB101 protein possesses a conserved Walker A motif (G42xxGxGKS), Q-loop (glutamine-loop, Q96), Walker B motif (V168ILAD) and a switch region (histidine-loop, H206). These sequence motifs are involved in forming a nucleotide-binding site (Schneider & Hunke, 1998
). In addition, a signature-conserved motif also known as linker peptide (L148SGGEQQR) specific for the ABC ATP-binding component was found in the pRSB101 protein. The possible function of this ATP-binding protein is to energize the transport process via ATP-hydrolysis.
|
To further analyse the transport function of the pRSB101 MDR system a 7214 bp SacII/XhoI-fragment containing the complete region was subcloned into the E. coli vector pBluescript-II-KS. The recombinant plasmid confers resistance to nalidixic acid (550 µg ml1) and low-level resistance to norfloxacin (1·25 µg ml1) to the host bacterium E. coli DH5. The basic vector confers resistance against 80 µg nalidixic acid ml1 and 0·5 µg norfloxacin ml1 to the host bacterium. Extrusion of antimicrobial drugs most probably is not the primary function of the pRSB107 efflux system. Very recently Burse et al. (2004)
provided strong evidence that the RND-type AcrAB transport system of the phytopathogen Erwinia amylovora is responsible for resistance to hydrophobic and amphiphilic toxins and to phytoalexins. Likewise, ABC exporters were shown to reduce the toxic effects of phytoalexins in phytopathogenic fungi (Del Sorbo et al., 2000
; Schoonbeek et al., 2001
; Fleißner et al., 2002
). Although the pRSB101 transporter is not completely homologous to the Erwinia amylovora system, it remains to be determined whether the primary functions of these systems are similar.
In summary, pRSB101 encodes a tripartite MDR efflux system composed of an ABC-type ATP-binding protein, a corresponding transmembrane permease and an RND-type MFP. This efflux system contributes to the resistance phenotype mediated by pRSB101.
The third genetic load region of pRSB101 contains four mobile genetic elements and a putative class 4 integron
Plasmid pRSB101 contains a third genetic load region located between the 20 kb resistance region and the replication module. This segment harbours four complete mobile genetic elements, relicts of an insertion sequence element and a transposon, and a putative integron which was interrupted by insertion of the ISRSB101-1 element (Fig. 2b).
The first putative mobile element possesses four genes and is bounded by 25 bp IRs, which show similarity to the IRi elements of class 3 and class 1 integrons. The deduced gene product of orf2 is 53 % identical to a product which was annotated as ISxac3 transposase on the Xanthomonas axonopodis pv. citri plasmid pXAC33 (accession no. NC_003921). The implied gene products are very short (110 and 90 amino acid residues, respectively) and are not homologous to any known transposases. Therefore the annotation of the ISxac3 transposase remains questionable. The products of orf3, orf4 and pin located upstream of orf2 show, respectively, 98, 81 and 89 % identity to conserved hypothetical proteins (designated XCC1632 and XCC1631) and an invertase/recombinase of Xanthomonas campestris pv. campestris (accession no. NC_003902). The organization of the corresponding genes is the same in Xanthomonas campestris pv. campestris and on pRSB101 and linkage of the region to an ISxac3 transposase gene also exists in Xanthomonas campestris pv. campestris. Orf3 is a predicted nucleic-acid binding protein containing a PIN-domain (PilT N terminus, COG5611, Pfam01850) of about 100 amino acids with two conserved aspartate residues. The function of this domain is unknown but a role in signalling has been proposed. The reference protein for these PIN-domain proteins is PilT, a putative NTPase playing a role in pilus-dependent surface motility and other processes (Herdendorf et al., 2002; Sakai & Komano, 2002
). Orf4 belongs to the group of AbrB-homologues with a regulatory function during the transition state between vegetative growth and the onset of stationary phase (COG2002, Pfam04014). AbrB of Bacillus subtilis regulates diverse and unrelated genes during periods of suboptimal growth conditions (Vaughn et al., 2001
). Finally, the pin gene product is an invertase/recombinase-like protein possessing the signature motifs present in the N terminus of the resolvase family (Pfam00239) and in the site-specific recombinases/DNA-invertase Pin homologues having functions in DNA-replication, recombination and repair (COG1961). It should be pointed out that the closest relatives of the pRSB101 Pin protein were found to be encoded by the Xanthomonas axonopodis pv. citri plasmids pXAC33 and pXAC64, the Acidithiobacillus ferrooxidans plasmid pTF5 and the Acidithiobacillus caldus IncQ-like plasmid pTC-F14. It might be supposed that Orf2, Orf3 and Pin play a role in transposition or regulation/modulation of transposition of a mobile genetic element related to ISxac3 of Xanthomonas species. Very recently, an element closely related to the one described above was identified on the large class I transposon TNCP23. This element is a composite of plasmid, integron and IS6100 elements and constitutes a genomic island in P. aeruginosa (Klockgether et al., 2004
).
A 969 bp DNA segment downstream of the pin gene is 98 % identical to a corresponding region present in the genome of P. aeruginosa and encodes a DNA-invertase-like enzyme and the N-terminal 65 amino acids of a putative modification methylase. The deduced amino acid sequences are 100 and 95 % identical to P. aeruginosa PaeR7IN (invertase) and the N terminus of PaeR7IM (modification methylase), respectively (Theriault et al., 1985; Vaisvila et al., 1995
). These findings indicate that the pRSB101 host bacterium and P. aeruginosa share a common gene pool.
Downstream of paeR7INpaeR7IM' a genetic element was identified which might represent an integron. The integrase gene (intI) of the putative integron was interrupted by insertion of the ISRSB101-1 element. The deduced gene product of the reconstructed intI open reading frame shows 57 % identity and 66 % similarity to the site-specific recombinase IntI4 of Vibrio cholerae and can be grouped into the XerC/XerD-family (COG4973, COG4974) of site-specific recombinases. Upstream of intI a putative site-specific recombination site (attI) with the inverse core-site motif -caaaAAC-, the core-site motif -GTTagcc- and imperfect 20 bp IRs in-between was detected. Therefore it might be speculated that orf6 located upstream of intI represents an integron-specific gene cassette which entered the variable region of the integron by site-specific recombination via the putative recombination site described above. The N terminus of the orf6 gene product contains the signature motifs COG2732 and Pfam01337 characteristic for barstar-like ribonuclease (barnase) inhibitors (Buckle et al., 1994) but a function for Orf6 cannot be predicted.
The intI coding region was interrupted by insertion of a 621 bp transposable element designated ISRSB101-1. The encoded transposase is 69 % identical and 77 % similar to the IS1004 transposase of V. cholerae O1 biovar eltor (accession no. NP_232602) which belongs to the transposase_17 family (Pfam01797, COG1943) of IS200-like elements. The IS present on pRSB101 represents a new member of the IS200 family. Insertion of the element into the intI gene caused a 5 bp target site duplication (5 bp direct repeats). Although the integrase gene intI has been inactivated, the integron in its functional state might have contributed to the acquisition of genetic material in the course of pRSB101 evolution.
Downstream of the integrase gene intI another IS has integrated. This element is 1196 bp in length, possesses 16 bp terminal IRs and encodes a transposase of the transposase_11 family (IS5-family; Pfam1609, COG3039). The DNA sequence of this element is almost identical (97 %) to IS5 present in the genome of E. coli K-12 (accession no. D90775).
The pRSB101 region adjacent to the IS5 element contains relicts of transposon Tn5710 originally found in K. pneumoniae subsp. ozenae (accession no. KPN011908) and an IS-element related to IS4 of E. coli (accession no. NP_418698).
The IS4-like element was truncated by insertion of another transposable element, designated ISRSB101-2, which has not been described before. This element is 1388 bp in length, carries 19 bp imperfect terminal IRs flanked by 10 bp DRs and encodes a transposase which is homologous to a transposase (accession no. CAC84124) of the marine psychrophilic bacterium Mst37. The implied transposases belong to the transposase_11 family (Pfam01609, COG3385) and contain the DDE-domain with three conserved carboxylate residues essential for catalytic activity of the enzymes.
In summary, the pRSB101 region described in this section contains four complete IS-elements, footprints of another IS-element and a transposon, and a new putative integron. This region might serve as matrix for the incorporation of other genetic material either by homologous recombination via IS-elements, site-specific recombination or transposition (illegitimate recombination).
Identification of pRSB101-like plasmids in different compartments of the wastewater treatment plant
Nine plasmids conferring erythromycin resistance were isolated in parallel with pRSB101 (see Table 1). To test whether these plasmids possess a similar replicon-type as compared to pRSB101, we tried to amplify internal repA-fragments by using primers (designated repA-L409 and repA-R854) specific for the pRSB101 repA replication gene.
An amplification product of the expected size (446 bp) was detected for plasmid pRSB105. Sequencing of this repA-specific amplicon revealed that it differs only in 9 bp as compared to the homologous pRSB101 repA-fragment.
To address the question of whether bacteria carrying pRSB101-like plasmids are released from the wastewater treatment plant into the environment, total plasmid-DNA preparations isolated from erythromycin-resistant bacteria of the final effluents were used as template DNAs in pRSB101 repA-specific PCR. This approach resulted in the amplification of an approximately 450 bp product which was cloned into the vector pGEM-T-Easy. Twenty-five insert-amplicons of the resulting repA-library were analysed for their RFLP-profiles by digestion with the restriction enzymes Sau3A and HhaI. It was found that 22 amplicons displayed identical restriction profiles as compared to the corresponding pRSB101 repA-fragment. The restriction patterns for the three other amplicons are identical to each other but differ to that of the pRSB101 repA-fragment. Sequencing of one of these amplicons revealed a difference in 7 bp as compared to pRSB101. These findings indicate that pRSB101-like plasmids are released with bacteria residing in the final effluents into the environment.
Conclusion
It is very likely that the pRSB101 progenitor plasmid accounted for an adaptive advantage to the host bacterium, for example low-level resistance to plant-borne or other toxic compounds. The presence of a multi-drug resistance determinant might have facilitated initial survival of the host bacterium and provided the opportunity to incorporate other resistance genes. Accordingly, plasmid pRSB101 might be regarded as a plasmid whose primary function was not related to antibiotic resistance. Later on in evolution, pRSB101 acquired antibiotic resistance determinants from environmental and/or pathogenic bacteria. The pRSB101 antibiotic resistance region was only recently extended by integration of genes which confer resistance to currently clinically important antimicrobial drugs such as cephalosporins and macrolides. Since pRSB101-like plasmids were also identified in the final effluents of the wastewater treatment plant it cannot be excluded that resistance determinants carried by these plasmids will be disseminated widely among environmental bacteria.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aminov, R. I., Chee-Sanford, J. C., Garrigues, N., Teferedegne, B., Krapac, I. J., White, B. A. & Mackie, R. I. (2002). Development, validation, and application of PCR primers for detection of tetracycline efflux genes of Gram-negative bacteria. Appl Environ Microbiol 68, 17861793.
Artiguenave, F., Vilagines, R. & Danglot, C. (1997). High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain. FEMS Microbiol Lett 153, 363369.[CrossRef][Medline]
Bateman, A., Coin, L., Durbin, R. & 10 other authors (2004). The Pfam protein families database. Nucleic Acids Res 32, database issue: D138D141.
Bennett, P. M. (1999). Integrons and gene cassettes: a genetic construction kit for bacteria. J Antimicrob Chemother 43, 14.
Bissonnette, L. & Roy, P. H. (1992). Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. J Bacteriol 174, 12481257.[Abstract]
Blázquez, J., Navas, A., Gonzalo, P., Martinez, J. L. & Baquero, F. (1996). Spread and evolution of natural plasmids harboring transposon Tn5. FEMS Microbiol Ecol 19, 6371.[CrossRef]
Blondeau, J. M. (2002). The evolution and role of macrolides in infectious diseases. Expert Opin Pharmacother 3, 11311151.[Medline]
Blondeau, J. M., DeCarolis, E., Metzler, K. L. & Hansen, G. T. (2002). The macrolides. Expert Opin Investig Drugs 11, 189215.[Medline]
Buckle, A. M., Schreiber, G. & Fersht, A. R. (1994). Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2·0-Å resolution. Biochemistry 33, 88788889.[Medline]
Burse, A., Weingart, H. & Ullrich, M. S. (2004). The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora. Mol PlantMicrobe Interact 17, 4354.[Medline]
Chopra, I. & Roberts, M. (2001). Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65, 232260.
Cooksey, D. A. (1990). Genetics of bactericide resistance in plant pathogenic bacteria. Annu Rev Phytopathol 28, 201219.[CrossRef]
da Silva, A. C., Ferro, J. A., Reinach, F. C. & 62 other authors (2002). Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417, 459463.[CrossRef][Medline]
Davies, J. (1994). Inactivation of antibiotics and the dissemination of resistance genes. Science 264, 375382.[Medline]
Davison, J. (1999). Genetic exchange between bacteria in the environment. Plasmid 42, 7391.[CrossRef][Medline]
Del Sorbo, G., Schoonbeek, H. & De Waard, M. A. (2000). Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal Genet Biol 30, 115.[CrossRef][Medline]
Dröge, M., Pühler, A. & Selbitschka, W. (2000). Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge. Mol Gen Genet 263, 471482.[CrossRef][Medline]
Ferreira, L. P., Lemos, E. G. & Lemos, M. V. (2002). Transposon Tn1721 distribution among strains of Xylella fastidiosa. FEMS Microbiol Lett 208, 163168.[CrossRef][Medline]
Fleißner, A., Sopalla, C. & Weltring, K. M. (2002). An ATP-binding cassette multidrug-resistance transporter is necessary for tolerance of Gibberella pulicaris to phytoalexins and virulence on potato tubers. Mol PlantMicrobe Interact 15, 102108.[Medline]
Gardner, M. N., Deane, S. M. & Rawlings, D. E. (2001). Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon. J Bacteriol 183, 33033309.
Gaynor, M. & Mankin, A. S. (2003). Macrolide antibiotics: binding site, mechanism of action, resistance. Curr Top Med Chem 3, 949961.[Medline]
Götz, A., Pukall, R., Smit, E., Tietze, E., Prager, R., Tschäpe, H., van Elsas, J. D. & Smalla, K. (1996). Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl Environ Microbiol 62, 26212628.[Abstract]
Grant, S. G., Jessee, J., Bloom, F. R. & Hanahan, D. (1990). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 87, 46454649.[Abstract]
Hall, R. M. & Collis, C. M. (1995). Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol Microbiol 15, 593600.[Medline]
Haneda, T., Okada, N., Nakazawa, N., Kawakami, T. & Danbara, H. (2001). Complete DNA sequence and comparative analysis of the 50-kilobase virulence plasmid of Salmonella enterica serovar Choleraesuis. Infect Immun 69, 26122620.
Herdendorf, T. J., McCaslin, D. R. & Forest, K. T. (2002). Aquifex aeolicus PilT, homologue of a surface motility protein, is a thermostable oligomeric NTPase. J Bacteriol 184, 64656471.
Higgins, C. F. (2001). ABC transporters: physiology, structure and mechanism an overview. Res Microbiol 152, 205210.[CrossRef][Medline]
Hynes, M. F., Simon, R. & Pühler, A. (1985). The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58. Plasmid 13, 99105.[Medline]
Klockgether, J., Reva, O., Larbig, K. & Tummler, B. (2004). Sequence analysis of the mobile genome island pKLC102 of Pseudomonas aeruginosa C. J Bacteriol 186, 518534.
Koski, P., Saarilahti, H., Sukupolvi, S., Taira, S., Riikonen, P., Österlund, K., Hurme, R. & Rhen, M. (1992). A new -helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid. J Biol Chem 267, 1225812265.
Kurtz, S., Choudhuri, J. V., Ohlebusch, E., Schleiermacher, C., Stoye, J. & Giegerich, R. (2001). REPuter: the manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res 29, 46334642.
Kwong, S. M., Yeo, C. C., Chuah, D. & Poh, C. L. (1998). Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region. FEMS Microbiol Lett 158, 159165.[CrossRef][Medline]
Kwong, S. M., Yeo, C. C., Suwanto, A. & Poh, C. L. (2000). Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer. J Bacteriol 182, 8190.
L'Abée-Lund, T. M. & Sørum, H. (2002). A global non-conjugative Tet C plasmid, pRAS3, from Aeromonas salmonicida. Plasmid 47, 172181.[CrossRef][Medline]
Lèvesque, C., Piche, L., Larose, C. & Roy, P. H. (1995). PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 39, 185191.[Abstract]
Liebl, W., Kloos, W. E. & Ludwig, W. (2002). Plasmid-borne macrolide resistance in Micrococcus luteus. Microbiology 148, 24792487.[Medline]
Lin, C. F. & Chung, T. C. (1999). Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors. Plasmid 42, 3141.[CrossRef][Medline]
Llanes, C., Gabant, P., Couturier, M., Bayer, L. & Plesiat, P. (1996). Molecular analysis of the replication elements of the broad-host-range RepA/C replicon. Plasmid 36, 2635.[CrossRef][Medline]
Ma, D., Alberti, M., Lynch, C., Nikaido, H. & Hearst, J. E. (1996). The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol Microbiol 19, 101112.[Medline]
Mach, P. A. & Grimes, D. J. (1982). R-plasmid transfer in a wastewater treatment plant. Appl Environ Microbiol 44, 13951403.[Medline]
Matsuoka, M., Endou, K., Kobayashi, H., Inoue, M. & Nakajima, Y. (1998). A plasmid that encodes three genes for resistance to macrolide antibiotics in Staphylococcus aureus. FEMS Microbiol Lett 167, 221227.[CrossRef][Medline]
Mazel, D. & Davies, J. (1999). Antibiotic resistance in microbes. Cell Mol Life Sci 56, 742754.[CrossRef][Medline]
McManus, P. S., Stockwell, V. O., Sundin, G. W. & Jones, A. L. (2002). Antibiotic use in plant agriculture. Annu Rev Phytopathol 40, 443465.[CrossRef][Medline]
Meyer, F., Goesmann, A., McHardy, A. C. & 8 other authors (2003). GenDBan open source genome annotation system for prokaryote genomes. Nucleic Acids Res 31, 21872195.
Nikaido, H. & Zgurskaya, H. I. (2001). AcrAB and related multidrug efflux pumps of Escherichia coli. J Mol Microbiol Biotechnol 3, 215218.[Medline]
Noguchi, N., Emura, A., Matsuyama, H., O'Hara, K., Sasatsu, M. & Kono, M. (1995). Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Escherichia coli. Antimicrob Agents Chemother 39, 23592363.[Abstract]
Noguchi, N., Takada, K., Katayama, J., Emura, A. & Sasatsu, M. (2000). Regulation of transcription of the mph(A) gene for macrolide 2'-phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A). J Bacteriol 182, 50525058.
Ohlsen, K., Ternes, T., Werner, G., Wallner, U., Loffler, D., Ziebuhr, W., Witte, W. & Hacker, J. (2003). Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage. Environ Microbiol 5, 711716.[CrossRef][Medline]
Pansegrau, W., Lanka, E., Barth, P. T. & 7 other authors (1994). Complete nucleotide sequence of Birmingham IncP plasmids. Compilation and comparative analysis. J Mol Biol 239, 623663.[CrossRef][Medline]
Partridge, S. R., Brown, H. J., Stokes, H. W. & Hall, R. M. (2001). Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins. Antimicrob Agents Chemother 45, 12631270.
Paulsen, I. T., Park, J. H., Choi, P. S. & Saier, M. H., Jr (1997). A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol Lett 156, 18.[CrossRef][Medline]
Rawlings, D. E. & Tietze, E. (2001). Comparative biology of IncQ and IncQ-like plasmids. Microbiol Mol Biol Rev 65, 481496.
Rhodes, G., Huys, G., Swings, J., McGann, P., Hiney, M., Smith, P. & Pickup, R. W. (2000). Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant tetA. Appl Environ Microbiol 66, 38833890.
Roberts, R. C. & Helinski, D. R. (1992). Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. J Bacteriol 174, 81198132.[Abstract]
Roberts, M. C., Sutcliffe, J., Courvalin, P., Jensen, L. B., Rood, J. & Seppala, H. (1999). Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrob Agents Chemother 43, 28232830.
Rohrer, J. & Rawlings, D. E. (1992). Sequence analysis and characterization of the mobilization region of a broad-host-range plasmid, pTF-FC2, isolated from Thiobacillus ferrooxidans. J Bacteriol 174, 62306237.[Abstract]
Rowe-Magnus, D. A. & Mazel, D. (1999). Resistance gene capture. Curr Opin Microbiol 2, 483488.[CrossRef][Medline]
Saier, M. H., Jr & Paulsen, I. T. (2001). Phylogeny of multidrug transporters. Semin Cell Dev Biol 12, 205213.[CrossRef][Medline]
Sakai, D. & Komano, T. (2002). Genes required for plasmid R64 thin-pilus biogenesis: identification and localization of products of the pilK, pilM, pilO, pilP, pilR, and pilT genes. J Bacteriol 184, 444451.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schäfer, A., Tauch, A., Jäger, W., Kalinowski, J., Thierbach, G. & Pühler, A. (1994). Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 6973.[CrossRef][Medline]
Schlüter, A., Heuer, H., Szczepanowski, R., Forney, L. J., Thomas, C. M., Pühler, A. & Top, E. M. (2003). The 64 508 bp IncP-1 antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1
group. Microbiology 149, 31393153.[CrossRef][Medline]
Schnabel, E. L. & Jones, A. L. (1999). Distribution of tetracycline resistance genes and transposons among phylloplane bacteria in Michigan apple orchards. Appl Environ Microbiol 65, 48984907.
Schneider, E. & Hunke, S. (1998). ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol Rev 22, 120.[CrossRef][Medline]
Schoonbeek, H., Del Sorbo, G. & De Waard, M. A. (2001). The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil. Mol PlantMicrobe Interact 14, 562571.[Medline]
Sesma, A., Sundin, G. W. & Murillo, J. (1998). Closely related plasmid replicons coexisting in the phytopathogen Pseudomonas syringae show a mosaic organization of the replication region and altered incompatibility behavior. Appl Environ Microbiol 64, 39483953.
Sesma, A., Sundin, G. W. & Murillo, J. (2000). Phylogeny of the replication regions of pPT23A-like plasmids from Pseudomonas syringae. Microbiology 146, 23752384.[Medline]
Sèveno, N. A., Kallifidas, D., Smalla, K., van Elsas, J. D., Collard, J. M., Karagouni, A. D. & Wellington, E. M. H. (2002). Occurrence and reservoirs of antibiotic resistance genes in the environment. Reviews Med Microbiol 13, 1527.
Silva, J., Aguilar, C., Ayala, G., Estrada, M. A., Garza-Ramos, U., Lara-Lemus, R. & Ledezma, L. (2000). TLA-1: a new plasmid-mediated extended-spectrum -lactamase from Escherichia coli. Antimicrob Agents Chemother 44, 9971003.
Smalla, K. & Sobecky, P. A. (2002). The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: insights gained from different methodological approaches. FEMS Microbiol Ecol 42, 165175.[CrossRef]
Staden, R. (1996). The Staden sequence analysis package. Mol Biotechnol 5, 233241.[Medline]
Stratton, C. W. (1998). Macrolides, lincosamides, and streptogramins: new agents and new roles. Antimicrob Infect Dis Newsl 17, 8992.[CrossRef]
Sundin, G. W. (2002). Distinct recent lineages of the strA-strB streptomycin-resistance genes in clinical and environmental bacteria. Curr Microbiol 45, 6369.[CrossRef][Medline]
Sundin, G. W. & Bender, C. L. (1996). Dissemination of the strA-strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants. Mol Ecol 5, 133143.[Medline]
Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 40, 25622566.[Abstract]
Tatusov, R. L., Natale, D. A., Garkavtsev, I. V. & 7 other authors (2001). The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res 29, 2228.
Tauch, A., Schlüter, A., Bischoff, N., Goesmann, A., Meyer, F. & Pühler, A. (2003). The 79,370-bp conjugative plasmid pB4 consists of an IncP-1 backbone loaded with a chromate resistance transposon, the strA-strB streptomycin resistance gene pair, the oxacillinase gene blaNPS-1, and a tripartite antibiotic efflux system of the resistance-nodulation-division family. Mol Genet Genomics 268, 570584.[Medline]
Tennstedt, T., Szczepanowski, R., Braun, S., Pühler, A. & Schlüter, A. (2003). Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. FEMS Microbiol Ecol 45, 239252.[CrossRef]
Theriault, G., Roy, P. H., Howard, K. A., Benner, J. S., Brooks, J. E., Waters, A. F. & Gingeras, T. R. (1985). Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products. Nucleic Acids Res 13, 84418461.[Abstract]
Tschäpe, H. (1994). The spread of plasmids as a function of bacterial adaptability. FEMS Microbiol Ecol 15, 2331.[CrossRef]
Vaisvila, R., Vilkaitis, G. & Janulaitis, A. (1995). Identification of a gene encoding a DNA invertase-like enzyme adjacent to the PaeR7I restriction-modification system. Gene 157, 8184.[CrossRef][Medline]
van Veen, H. W. & Konings, W. N. (1998). The ABC family of multidrug transporters in microorganisms. Biochim Biophys Acta 1365, 3136.[Medline]
Vaughn, J. L., Feher, V. A., Bracken, C. & Cavanagh, J. (2001). The DNA-binding domain in the Bacillus subtilis transition-state regulator AbrB employs significant motion for promiscuous DNA recognition. J Mol Biol 305, 429439.[CrossRef][Medline]
Vester, B. & Douthwaite, S. (2001). Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob Agents Chemother 45, 112.
Vivian, A., Murillo, J. & Jackson, R. W. (2001). The roles of plasmids in phytopathogenic bacteria: mobile arsenals? Microbiology 147, 763780.[Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103119.[CrossRef][Medline]
Zgurskaya, H. I. (2002). Molecular analysis of efflux pump-based antibiotic resistance. Int J Med Microbiol 292, 95105.[Medline]
Zhanel, G. G., Dueck, M., Hoban, D. J., Vercaigne, L. M., Embil, J. M., Gin, A. S. & Karlowsky, J. A. (2001). Review of macrolides and ketolides: focus on respiratory tract infections. Drugs 61, 443498.[Medline]
Received 10 May 2004;
revised 12 July 2004;
accepted 3 August 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |