The nucleotide sequences of the broad-host-range antibiotic resistance plasmids pB2 (61 kb) and pB3 (56 kb), which were isolated from a wastewater treatment plant, were determined and analysed. Both have a nearly identical IncP-1β backbone, which diverged early from the sequenced IncP-1β plasmids R751, pB10, pJP4, pADP1 and pUO1. In contrast to the latter plasmids, the pB2 and pB3 backbone does not seem to have undergone any deletions. The complete partition gene parA is located downstream of the mating pair formation (trb) module. A 14·4 kb or 19·0 kb mobile genetic element is present between traC and parA of pB3 and pB2, respectively. This region is typical for insertions in IncP-1β plasmids, but the insertion site is unique. Both elements differ only by a duplication in pB2 of a tetA(C)–tetR–tnpAIS26 fragment. The 5 bp target site duplication and the 26 bp inverted repeats flanking the mobile genetic elements are still intact, indicating that the insertion occurred recently. The element consists of three nested transposable elements: (i) a relict of a Tn402-like transposon with a gene for a new class D β-lactamase (blaNPS-2); (ii) within that, another Tn402-like element with a class 1 integron harbouring the gene cassettes cmlA1 for a chloramphenicol efflux protein and aadA2 encoding a streptomycin/spectinomycin adenylyltransferase, and a copy of IS6100; (iii) into the integrase gene intI1 a tetracycline resistance module tetA(C)–tetR flanked by copies of IS26 is inserted. Interestingly, in contrast to all other IncP-1β plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted. The genes kluAB are also missing in that region and should thus be considered acquired genes. These findings, together with the fact that IncP-1β plasmids acquired accessory elements at various positions in the backbone, suggest that IncP-1β plasmids without any accessory genes exist in microbial communities. They must occasionally acquire accessory genes by transposition events, resulting in those plasmids that have been found based on selectable phenotypic traits.
BergstromC. T.,
LipsitchM.,
LevinB.
2000; Natural selection, infection transfer and the existence conditions for bacterial plasmids. Genetics 155:1505–1519
BingleL. E. H.,
ZatykaM.,
ManzoorS. E.,
ThomasC. M.
2003; Co-operative interactions control conjugative transfer of broad host-range plasmid RK2: full effect of minor changes in TrbA operator depends on KorB. Mol Microbiol 49:1095–1108[CrossRef]
BoydD.,
PetersG. A.,
CloeckaertA.,
BoumedineK. S.,
Chaslus-DanclaE.,
ImberechtsH.,
MulveyM. R.
2001; Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. J Bacteriol 183:5725–5732[CrossRef]
CoutureF.,
LachapelleJ.,
LevesqueR. C.
1992; Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases. Mol Microbiol 6:1693–1705[CrossRef]
DrögeM.,
PühlerA.,
SelbitschkaW.
2000; Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge. Mol Gen Genet 263:471–482[CrossRef]
EnneV. I.,
BennettP. M.,
LivermoreD. M.,
HallL. M. C.
2004; Enhancement of host fitness by the sul2-encoding plasmid p9123 in the absence of selected pressure. J Antimicrob Chemother 53:958–963[CrossRef]
EwingB.,
HillierL.,
WendlM.,
GreenP.
1998; Basecalling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res 8:175–185[CrossRef]
GrantS. G. N.,
JesseeJ.,
BloomF. R.,
HanahanD.
1990; Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 87:4645–4649[CrossRef]
HallT. A.
1999; BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
HeuerH.,
WellingtonE. M. H, KrögerrecklenfortE.8 other authors2002; Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42:289–302[CrossRef]
HillK. E.,
WeightmanA. J.,
FryJ. C.
1992; Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10. Appl Environ Microbiol 58:1292–1300
KlockgetherJ.,
RevaO.,
LarbigK., Tümmler B. 2004; Sequence analysis of the mobile genome island pKLC102 of Pseudomonas aeruginosa C. J Bacteriol 186:518–534[CrossRef]
MeyerF.,
GoesmannA.,
McHardyA. C.8 other authors2003; GenDB – an open source genome annotation system for prokaryote genomes. Nucleic Acids Res 31:2187–2195[CrossRef]
PartridgeS. R.,
BrownH. J.,
HallR. M.
2002; Characterization and movement of the class 1 integron known as Tn2521 and Tn1405. Antimicrob Agents Chemother 46:1288–1294[CrossRef]
PartridgeS. R.,
RecchiaG. D.,
StokesH. W.,
HallR. M.
2001; Family of class 1 integrons related to In4 from Tn1696. Antimicrob Agents Chemother 45:3014–3020[CrossRef]
RadströmP.,
SkoldO.,
SwedbergG.,
FlensburgJ.,
RoyP. H., SundströmL. 1994; Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J Bacteriol 176:3257–3268
SchlüterA.,
HeuerH.,
SzczepanowskiR.,
ForneyL. J.,
ThomasC. M.,
TopE. M, PühlerA.2003; The 64 508 bp IncP-1β antibiotic multiresistance plasmid pB10 isolated from a wastewater treatment plant provides evidence for recombination between members of different branches of the IncP-1β group. Microbiology 149:3139–3153[CrossRef]
SchnabelE. L.,
JonesA. L.
1999; Distribution of tetracycline resistance genes and transposons among phylloplane bacteria in Michigan apple orchards. Appl Environ Microbiol 65:4898–4907
SmithC. A.,
ThomasC. M.
1987; Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncPβ sub-group are closely related. Mol Gen Genet 206:419–427[CrossRef]
SmithC. A.,
PinkneyM.,
GuineyD. G.,
ThomasC. M.
1993; The ancestral IncP replication system consisted of contiguous oriV and trfA segments as deduced from a comparison of the nucleotide sequences of diverse IncP plasmids. J Gen Microbiol 139:1761–1766[CrossRef]
SorumH.,
L'Abee-LundT. M.,
SolbergA.,
WoldA.
2003; Integron-containing IncU R plasmids pRAS1 and pAr-32 from the fish pathogen Aeromonas salmonicida
. Antimicrob Agents Chemother 47:1285–1290[CrossRef]
SwoffordD. L.
1991; paup: Phylogenetic Analysis Using Parsimony, Version 3.1. Computer program distributed by the Illinois Natural History Survey. Champaign, Illinois:
TauchA.,
KalinowskiJ.,
ThierbachG, GötkerS.,
PühlerA.2002; The 27·8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100. Plasmid 48:117–129[CrossRef]
TauchA.,
KalinowskiJ.,
ThierbachG, PühlerA.2003a; Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics. J Biotechnol 104:27–40[CrossRef]
TauchA.,
BischoffN.,
GoesmannA.,
MeyerF, Schlüter A., PühlerA. 2003b; The 79,370-bp conjugative plasmid pB4 consists of an IncP-1β 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 Gen Genomics 268:570–584
ThomasC. M.,
SmithC. A.
1987; Incompatibility group P plasmids: genetics, evolution, and use in genetic manipulation. Annu Rev Microbiol 41:77–101[CrossRef]
ThorstedP. A.,
MacartneyD. P.,
AkhtarP.9 other authors1998; Complete sequence of the IncPβ plasmid R751: Implications for evolution and organisation of the IncP backbone. J Mol Biol 282:969–990[CrossRef]
TopE. M., Moënne-LoccozY., PembrokeT.,
ThomasC. M.
2000; Phenotypic traits conferred by plasmids. In The horizontal gene pool pp 249–286 Edited by
ThomasC. M.
Amsterdam: Harwood Academic Publishers;
TopE.,
De SmetI.,
MergeayM.,
VerstraeteW.
1994; Exogenous isolation of mobilizing plasmids from polluted soils and sludges. Appl Environ Microbiol 60:831–839
TopE.,
VanrolleghemP.,
MergeayM.,
VerstraeteW.
1992; Determination of the mechanism of retrotransfer by mechanistic mathematical modeling. J Bacteriol 174:5953–5960
TralauT.,
CookA. M.,
RuffJ.
2001; Map of the IncP-1β plasmid pTSA encoding the widespread genes (tsa) for p-toluenesulfonate degradation in Comamonas testosteroni T-2. Appl Environ Microbiol 67:1508–1516[CrossRef]
TrefaultN.,
De la IglesiaR.,
MolinaA. M.,
ManzanoM.,
LedgerT.,
StuardoM, Pérez-PantojaD.,
SánchezM. A., GonzálezB. 2004; Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways. Environ Microbiol 6:655–668[CrossRef]
Trieu-CuotP.,
CarlierC.,
MartinP.,
CourvalinP.
1987; Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria. FEMS Microbiol Lett 48:289–294[CrossRef]
ZatykaM.,
Jagura-BurdzyG.,
ThomasC. M.
1997; Transcriptional and translational control of the genes for the mating pair formation apparatus of promiscuous IncP plasmids. J Bacteriol 179:7201–7209