The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different...

Downloaded from www.microbiologyresearch.org by

IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

The 120592 bp IncF plasmid pRSB107 isolatedfrom a sewage-treatment plant encodes ninedifferent antibiotic-resistance determinants, twoiron-acquisition systems and other putativevirulence-associated functions

R. Szczepanowski, S. Braun, V. Riedel, S. Schneiker, I. Krahn, A. Puhlerand A. Schluter

Correspondence

A. Schluter

Andreas.Schlueter@Genetik.

Uni-Bielefeld.DE

Fakultat fur Biologie, Lehrstuhl fur Genetik, Universitat Bielefeld, Postfach 100131, D-33501Bielefeld, Germany

Received 19 November 2004

Accepted 23 December 2004

The antibiotic-multiresistance IncF plasmid pRSB107 was isolated by a transformation-based

approach from activated-sludge bacteria of a wastewater-treatment plant. It confers resistance

to ampicillin, penicillin G, chloramphenicol, erythromycin, kanamycin, neomycin, streptomycin,

sulfonamides, tetracycline and trimethoprim and against mercuric ions. Complete sequencing

of this plasmid revealed that it is 120592 bp in size and has a G+C content of 53?1 mol%.

The plasmid backbone is composed of three replicons, RepFIA, RepFIB and RepFII, which are

almost identical to corresponding regions located on the F-plasmid and on R100. The three

replicons encode replication initiation (rep) and replication control, multimer resolution (mrs),

post-segregational killing of plasmid-free cells (psk) and active plasmid partitioning (sopABC

locus). Part of the F-leading region and remnants of the F-homologous DNA-transfer (tra)

module complete the pRSB107 backbone. Plasmid pRSB107 contains a complex, highly

mosaic 35991 bp antibiotic-resistance region consisting of a Tn21- and a Tn10-derivative and

a chloramphenicol-resistance module. The Tn21 derivative is composed of a mercury-resistance

region (mer), a Tn4352B-like kanamycin/neomycin-resistance transposon, a streptomycin/

sulfonamide-resistance module, remnants of the b-lactam-resistance transposon Tn1, a

macrolide-resistance module flanked by copies of IS26 and IS6100, remnants of Tn402

integrating a class 1 integron and the Tn21-specific transposition module. A truncated version

of the tetracycline-resistance transposon Tn10 and the chloramphenicol acetyltransferase gene

catA complete the pRSB107 resistance region. In addition to antibiotic resistance, pRSB107

encodes the following putative virulence-associated functions: (i) an aerobactin iron-acquisition

siderophore system (iuc/iut); (ii) a putative high-affinity Fe2+ uptake system which was previously

identified on a pathogenicity island of Yersinia pestis and in the genome of the phytopathogen

Erwinia carotovora subsp. atroseptica SCRI1043; (iii) an sn-glycerol-3-phosphate transport

system (ugp); and (iv) the virulence-associated genes vagCD having a possible function in stable

plasmid inheritance. All the accessory modules are framed by insertion sequences, indicating

that pRSB107 was gradually assembled by integration of different horizontally acquired

DNA segments via transposition or homologous recombination.

INTRODUCTION

Plasmids are extra-chromosomal genetic elements, whichare normally not essential for their bacterial hosts. How-ever, under certain stress conditions, such as presence of

antibiotics or toxic compounds, nutrient limitation or highcell densities, and in the course of pathogenic interactions,plasmids can be advantageous for the host cell. Severalnaturally occurring plasmids encode resistance to anti-biotics, heavy metals and pollutants, additional metabolicpathways enabling utilization of particular carbon andenergy sources, or virulence and colonization factors(Burlage et al., 1990; Martinez et al., 2001; Pansegrauet al., 1994; Womble & Rownd, 1988). Plasmids also play

Abbreviation: DMP, dimethyl phosphate.

The GenBank/EMBL/DDBJ accession number for the sequencereported in this paper is AJ851089.

0002-7773 G 2005 SGM Printed in Great Britain 1095

Microbiology (2005), 151, 1095–1111 DOI 10.1099/mic.0.27773-0


IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

an important role in the evolution of bacteria by facilitat-ing vertical and horizontal gene transfer (Davies, 1994;Davison, 1999; Mazel & Davies, 1999).

Plasmids belonging to the incompatibility (Inc) group F areespecially versatile in providing advantageous traits to theirhosts. The prototype IncF plasmid is the fertility factor F ofEscherichia coli (Cavalli et al., 1953; Perez-Casal et al., 1989).It carries genes for its own conjugative transfer, is able tointegrate into the host chromosome via recombinationand can transfer chromosomal markers during conjugation(Boyd et al., 1996; Lawley et al., 2003). The IncF group isdivided into six subgroups: IncFI to IncFVI (Saadi et al.,1987). IncF plasmids mediate resistance to antibiotics[R100 (Womble & Rownd, 1988)], encode virulence func-tions [pColV-K30 (Herrero et al., 1988); pO157 (Burlandet al., 1998; Schmidt et al., 1997); p307 (Spiers et al., 1993)]or produce colicins [pColV plasmids (Gibbs et al., 1993);pColV3-K30 (Spiers et al., 1993)].

Since several IncF antibiotic-resistance and virulence plas-mids have been isolated from enteric bacteria of hospitalizedpatients (Burland et al., 1998; Di Lorenzo et al., 2003;Herrero et al., 1988; Schmidt et al., 1997; Womble & Rownd,1988) it is very likely that enteric bacteria containing suchplasmids reach wastewater-treatment plants with the sewagereleased from hospitals. It is generally believed that sewage-treatment plants represent hot-spots for horizontal genetransfer (Blazquez et al., 1996; Droge et al., 1998, 2000;Smalla & Sobecky, 2002) and contribute to the dissemina-tion of mobile genetic elements carrying antibiotic-resistance determinants. Several different multiresistanceplasmids have been isolated from bacteria residing in theactivated-sludge compartment and the final effluents ofwastewater-treatment plants (Blazquez et al., 1996; Drogeet al., 2000; Schluter et al., 2003; Smalla & Sobecky, 2002;Tennstedt et al., 2003). Promiscuous broad-host-rangeplasmids belonging to the IncP-1 group represent the mostabundant fraction of exogenously isolated plasmids fromsewage plants (Droge et al., 2000). Five different IncP-1resistance plasmids originating from activated-sludge bac-teria have so far been completely sequenced. These arethe IncP-1b plasmids pB2 and pB3 (Heuer et al., 2004), pB4(Tauch et al., 2003), and pB10 (Schluter et al., 2003), whichcarry different mobile genetic elements with antibiotic-resistance determinants, and the IncP-1a plasmid pTB11,which is closely related to the prototype IncP-1a ‘Birming-ham’ plasmids (Tennstedt et al., 2005).

In another study, 10 antibiotic-resistance plasmids con-ferring high-level erythromycin resistance were isolatedfrom activated-sludge bacteria by a transformation-basedapproach (Szczepanowski et al., 2004). One of theseplasmids, pRSB101, was completely sequenced. It confersresistance to 12 antibiotics and possesses a replicon relatedto plasmids residing in phytopathogenic bacteria. In con-trast to pRSB101, the replicon of pRSB107, which wasisolated in parallel with pRSB101, could be typed asbelonging to the IncF group. This plasmid confers resistance

to 10 antimicrobial compounds and is about 120 kb in size.Due to the size of pRSB107, we expected that the plasmid,in addition to antibiotic-resistance determinants, wouldcarry other accessory modules. Since many IncF plasmidsare known to encode virulence factors we wondered whetherpRSB107 also contains virulence-associated genes. To deter-mine the genetic organization of the pRSB107 replicon andits accessory elements the complete nucleotide sequenceof the plasmid was established. Detailed analysis of thesequence revealed new insights into conserved features andevolution of IncF plasmids and their accessory modules,including virulence-associated genes.

METHODS

Bacterial strains, growth conditions and genetic techniques.E. coli DH5a mcr (Grant et al., 1990), CSH61-HfrC (Miller, 1972),CV60 GFP (K. Smalla, Braunschweig, Germany), and S17-1 (Simonet al., 1983) containing the multiresistance plasmid pRSB107 weregrown at 37 uC in Luria Broth (LB) medium supplemented asneeded with antibiotics at the following final concentrations: 50 mgkanamycin ml21, 100 mg streptomycin ml21 or 5 mg tetracyclineml21. For solid media, 15 g agar per litre medium was added.Indicator medium for strains expressing an active b-galactosidasewas supplemented with 40 mg X-Gal ml21 (final concentration).

To test the transfer properties of pRSB107, this plasmid was transferredinto the mobilizer strain E. coli S17-1 and into the Hfr E. coli strainCSH61-HfrC and mated with the recipient E. coli CV60 GFP oncellulose acetate filters. Putative transconjugants were selected on LBmedium containing 30 mg rifampicin ml21 and 5 mg tetracycline ml21.

To test whether pRSB107 enables utilization of dimethyl phosphate(DMP) as the sole phosphorus source, E. coli DH5a was transformedwith the plasmid. To test the growth abilities of pRSB107-containingand plasmid-free E. coli DH5a cells with DMP as the sole phosphorussource, MOPS minimal medium (Neidhardt et al., 1974) supple-mented with 0?4 % glucose, 1 mg thiamine ml21 and 0?1 M DMP(McLoughlin et al., 2004) was used. Prior to the growth test, the E. coliDH5a test strains were inoculated from LB agar plates into MOPSminimal medium (containing glucose and thiamine) without DMPto ensure only a minor contamination with a phosphorus source.After growth for 8 h the cells were inoculated into fresh MOPS minimalmedium containing 0?1 M DMP, glucose and thiamine, and againincubated for approximately 12 h at 37 uC.

Standard DNA techniques. Plasmid DNA from plasmid-containingE. coli strains was isolated with the Nucleobond Kit PC100 on AX 100columns (Macherey-Nagel) in accordance with the manufacturer’sprotocol. Plasmid DNA for generation of a pRSB107 shotgun librarywas isolated with the Qiagen Large-Construct Kit according to themanufacturer’s instructions. The plasmid content of E. coli trans-formants was determined by Eckhardt-gel analysis as described byHynes et al. (1985). Restriction enzyme digestion, agarose gel electro-phoresis and transformation of E. coli DH5a were carried out accord-ing to Sambrook et al. (1989).

Construction of shotgun libraries and DNA sequencing ofpRSB107. Isolated pRSB107 DNA was partially digested with therestriction endonuclease Sau3A. Restriction fragments varying insize from 1 to 3 kb were extracted from an agarose gel by using theSephaglas BandPrep Kit (Amersham Pharmacia Biotech) accordingto the manufacturer’s instructions, and cloned into the BamHI-digested vector pZErO-2 (Invitrogen). A second library was gener-ated by hydro-shearing of pRSB107 plasmid DNA and subsequent

1096 Microbiology 151

R. Szczepanowski and others


IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

cloning of the 1?3–2 kb fragment fraction into the vector pGEM-T(MWG, Ebersberg, Germany). Plasmid DNA was prepared fromE. coli shotgun clones by automated alkaline lysis with the RoboPrep2500 (MWG) and BioRobot 9600 (Qiagen). Sequencing reactionsusing dye-terminator chemistry were separated on a MegaBACE1000 capillary sequencer (Amersham Biosciences) and an ABI 377(Applera, Applied Biosystems) DNA sequencer.

Sequencing reads were assembled using the Staden (GAP4) softwarepackage (Staden, 1996). Gap closure and polishing of the sequence wasachieved by primer walking with walking primers designed on contignucleotide sequences. The final gap was closed by sequencing a PCRproduct generated with the primers cem_pcr1_for (TTGGAAATT-AATGATAACAACGG) and cem_pcr1_rev (CAACGTTAACCGCA-ACAATTTT). These approaches resulted in a single, circular moleculewith a total length of 120 592 bp.

DNA sequence analysis and annotation. The finished pRSB107sequence was annotated by using the GenDB (version 2.0) Annota-tion Tool (Meyer et al., 2003). Repeat regions within the pRSB107sequence were identified and analysed by using the REPuter software(Kurtz et al., 2001). The annotated sequence of pRSB107 is availableunder EMBL accession number AJ851089.

RESULTS AND DISCUSSION

pRSB107 possesses F-like replicons and isloaded with resistance transposons andmodules encoding other accessory functions

Plasmid pRSB107 was isolated from activated-sludge bacteriaby using a transformation-based approach (Szczepanowskiet al., 2004). It confers resistance to the antimicrobial com-pounds ampicillin, chloramphenicol, erythromycin, kana-mycin, neomycin, penicillin G, streptomycin, sulfonamides,tetracycline and trimethoprim, and against inorganicmercury compounds. Hybridization analyses revealed thatpRSB107 possesses an IncF-like replicon. Because of itsbroad resistance spectrum, pRSB107 was completelysequenced to determine the nature of the resistance genesand the modular structure of its backbone.

In a shotgun sequencing approach 2982 reads could beassembled into one contig, which was circularized bysequencing of an appropriate amplicon generated onpRSB107 DNA by PCR. The finished pRSB107 sequenceis 120 592 bp in length and has a G+C content of53?1 mol%. Annotation of the sequencing data by usingthe GenDB (version 2.0) tool (Meyer et al., 2003) revealed135 coding regions. Twenty-eight genes encode replication,partitioning, multimer resolution, post-segregational kill-ing, DNA-transfer/mobilization and DNA-modificationfunctions. Fifteen genes are predicted to be involved inantibiotic, camphor, mercuric ion and copper resistance.Twenty-six genes have possible functions in transposition,DNA integration and site-specific recombination. Putativeregulators are encoded by twelve genes. Seven putativeiron-acquisition genes could be identified. Five genes arenecessary for sn-glycerol-3-phosphate uptake and metabo-lism. Further three genes encode putative beneficialfunctions, and four genes are involved in transport-related

functions. Hitherto unknown functions are encoded bythirty-five ORFs. Predicted functions for the pRSB107 genesare summarized in Table 1. The genetic map of the plasmidis shown in Fig. 1.

Two pRSB107 replicons are nearly identical toRepFIA and RepFIB of the F-plasmid

Plasmid pRSB107 possesses a replicon that is almostidentical to the primary replication region RepFIA of theE. coli F-plasmid.

A 6497 bp pRSB107 segment containing the origins ofvegetative replication oriV-1 and oriV-2, the genes ccdAB(post-segregational killing), resD (multimer resolution),repE (replication initiation), sopAB (active partitioning),the incompatibility iterons incC and the sopC-site (incD)is 99?9 % identical to the corresponding region of the F-plasmid (accession no. AP001918); see Fig. 2. The ccdABoperon encodes a post-segregation antitoxin (COG5302)and a protein toxin involved in killing of plasmid-freesegregants (Engelberg-Kulka & Glaser, 1999). A secondplasmid-stabilization system is specified by resD, locatedin the ccdAB–resD operon downstream of ccdB. The resDgene product is a site-specific recombinase (COG4973,XerC) for resolution of plasmid multimers containing therfsF sequence needed for site-specific recombination (Laneet al., 1986). A third stable maintenance mechanism isestablished by the products of the sopAB operon. SopA isan ATPase (Pfam00991, ParA) which is believed to pro-vide energy from ATP hydrolysis for active partition-ing of plasmid replicates, whereas SopB has a ParB-likenuclease domain (Pfam02195, ParBc) and interacts with acentromere-like site, termed sopC (incD), downstream ofsopAB (Gerdes et al., 2000). The sopC site of F is composedof twelve 43 bp direct repeats. The sopC site present onpRSB107 is 86 bp shorter than that on the F-plasmid:repeats 4 and 6 are truncated and repeat 5 is not present.The most important factor for plasmid replication is thereplication-initiation protein encoded by repE. This pro-tein binds to four 19 bp iterons (incB) located in oriV-2upstream of repE and initiates replication in cooperationwith host-encoded factors (Zzaman et al., 2004). Apart fromthe incB iterons, the oriV-2 of pRSB107 and F containstwo tandem DnaA-binding motifs, an AT-rich region anda consensus 13-mer sequence which is melted duringreplication initiation (Kawasaki et al., 1996; Zzaman et al.,2004). A single-strand-initiation-sequence motif (ssiA)which is required for priming of plasmid replication(Nomura et al., 1991) is located in the vicinity of oriV-2.The incC iterons (five directly repeated sequences) whichare conserved downstream of repE play a role in incom-patibility and copy number control (Uga et al., 1999)(Fig. 2). The second RepFIA-specific origin, designatedoriV-1, is completely identical on pRSB107 and F but thecorresponding replication-initiation gene, repC, is missingon pRSB107.

Another replicon which is homologous to RepFIB of the

http://mic.sgmjournals.org 1097

Complete sequence of the IncF-like plasmid pRSB107


IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

Table 1. Coding sequences (cds) of the multiresistance plasmid pRSB107 isolated from an unknown activated-sludge bacter-ium of a wastewater-treatment plant

Gene

no.

Coding

sequence

Identity/similarity (%) of pRSB107

at the gene-product level* to

Function PfamD COGd Reference

accession no.

1 ccdA 100, 100; CcdA from F-plasmid Post-segregational killing of

plasmid-free daughter cells

Pfam07362 COG5302 BAA97912

2 ccdB 100, 100; CcdB from F-plasmid Post-segregational killing of

plasmid-free daughter cells

Pfam01845 – BAA97913

3 resD 100, 100; ResD from F-plasmid Multimere resolution – COG4974,

COG9473

NP_061423

4 repE 100, 100; RepE from F-plasmid Replication initiation Pfam01051 COG5527 BAA97915

5 sopA 100, 100; SopA from F-plasmid Partitioning Pfam00991 COG1192 BAA97916

6 sopB 99, 99; SopB from F-plasmid Partitioning Pfam02192 – BAA97917

7 orf7 90, 92; YccB from ColIb-P9 Unknown Pfam06924 – NP_052478

8 orf8 96, 96; YcdA from ColIb-P9 Unknown – – NP_052479

9 orf9 98, 99; YcdA from pWR501 Unknown Pfam01555 COG0863 NP_085364

10 orf10 97, 100; Ecp042 from pO157 Unknown – – NP_958723

11 orf11 92, 95; YffA from F-plasmid Unknown Pfam07128 – BAA97924

12 orf12 97, 98; YcfA from ColIb-P9 Unknown – – NP_052483

13 orf13 100, 100; YciB from pC15-1a Unknown Pfam03230 – NP_957575

14 orf14 96, 97; YcjA from pC15-1a Unknown – – NP_957576

15 orf15 98, 100; Orf63 from F-plasmid Unknown – – AAD47185

16 ydaA 70, 70; YdaA from pC15-1a Unknown – – NP_957577

17 ydaB 100, 100; YdaB from R100 Unknown – – NP_052918

18 ydbA 99, 99; YdbA from R100 Unknown – – NP_052919

19 orf19 Represents the first 88 residues of YdcA

from R100

Unknown – – NP_052920

20 DtraI Represents the last 1209 residues of TraI

from R100


21 traX 97, 98; TraX from pCP301 Pilin acetylation Pfam05857 – NP_858383

22 finO 98, 99; FinO from pO157 Fertility inhibition Pfam05286 – T00302

23 orf23 94, 94; Ecp070 from pO157 Unknown – – NP_958725

24 srnB 79, 80; SrnB from F-plasmid Modulation of SrnB9 – – BAA97874

25 srnB9 85, 87; SrnB9 from F-plasmid Stable RNA degradation – – BAA97875

26 repA2 97, 98; RepA2 from R100 Negative regulation of repA1

expression

– – NP_052988

27 repA6 83, 87; RepA6 from R100 Positive regulation of repA1

expression

– – NP_052989

28 repA1 97, 98; RepA1 from R100 Replication initiation – – NP_052990

29 orf29 100, 100; Ecs1337 from pC15-1a Probably transposition Pfam01527 COG2963 NP_957590



32 tir 100, 100; Tir from R100 Transfer inhibition Pfam02517 – NP_052992

33 pemI 100, 100; PemI from R100 Plasmid stable inheritance Pfam04014 COG2336 NP_052993

34 pemK 100, 100; PemK from R100 Plasmid stable inheritance Pfam02452 COG2337 NP_052994

35 insAIS1-1 98, 100; InsA from R100 Transposition Pfam03811 COG3677 NP_052996

36 insBIS1-1 100, 100; InsB from R100 Transposition Pfam03400 COG1662 NP_052997

37 merR 100, 100; MerR from R100 Regulation of the mer operon Pfam00376 COG0789 NP_052881

38 merT 100, 100; MerT from R100 Mercury ion transport Pfam02411 – NP_052882

39 merP 100, 100; MerP from R100 Mercury ion transport Pfam00403 COG2608 NP_052883

40 merC 91, 91; MerC from R100 Mercury ion transport Pfam03203 – NP_052884

41 merA 100, 100; MerA from R100 Mercury ion reduction Pfam00403,

Pfam00070,

Pfam02852

COG2608 NP_052885

42 merD 100, 100; MerD from R100 Unknown Pfam00376 COG0789 NP_052886

43 merE 98, 98; MerE from R100 Unknown Pfam05052 – NP_052887




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

Table 1. cont.

Gene

no.

Coding

sequence




accession no.

44 urf2 100, 100; YaeA from R100 Unknown Pfam00563 COG2200 NP_052888

45 tniAD1 97, 97; TniAD1 from pRMH760 Transposition Pfam00665 COG4584 AAM89410

46 tnpAIS26-1 99, 100; Tnp26R from pTP10 Transposition Pfam00665 COG3316 NP_862253

47 aph 100, 100; Aph from pNGA2 Kanamycin and neomycin

resistance

Pfam01636 COG3231 NP_478145


49 strB 100, 100; StrB from pB10 Streptomycin resistance Pfam04655 COG3570 NP_858030

50 strA 99, 100; StrA from pB10 Streptomycin resistance Pfam01636 COG3231 NP_858029

51 sulII 100, 100; from pHCM1 Sulfonamide resistance Pfam00809 COG0294 CAD09805

52 repC 87, 87; RepC from pHCM1 Origin recognition Pfam06504 – NP_569413

53 repA 99, 99; RepA from Salmonella typhimurium Replication initiation – – AAS18378


55 blaTEM-1b 100, 100; BlaTEM-1b from pRMH760 b-Lactam resistance Pfam00144 COG2367 AAR91458


57 mph(A) 100, 100; Mph(A) from pTZ3509 Macrolide resistance Pfam01636 COG3173 BAB12239

58 mrx 100, 100; Mrx from pTZ3509 Unknown – – BAB12240

59 mphR(A) 100, 100; MphR(A) from pTZ3509 Mph(A) repression Pfam00440 – BAB12241

60 tnpAIS6100 100, 100; Tnp from pRAS1 Transposition Pfam00665 COG3316 CAD57187

61 mobC 19, 26; Orf15 from pSB102 Probably mobilization Pfam05713 – NP_361029

62 dhfR 100, 100; DhfR from pHCM1 Trimethoprim resistance Pfam00186 COG0262 NP_569370

63 intI1 99, 99; IntI1 from R100 Integration Pfam00589 COG4974 NP_052898

64 tnpMTn21 100, 100; TnpM from pTJ100 Modulation of Tn21 transposition – – AAT37601

65 tnpRTn21 100, 100; TnpR from R100 Resolution of Tn21 cointegrates Pfam00239,

Pfam02796

COG1961 NP_052900

66 tnpATn21 100, 100; TnpA from R100 Transposition Pfam01526 COG4644 NP_052901

67 ybjA 100, 100; YbjA from R100 Unknown Pfam00583 – NP_052902

68 catA 100, 100; CatA from R100 Chloramphenicol resistance Pfam00302 COG4845 NP_052903



71 orf71 100, 100; Orf17 from Shigella

flexneri 2a

Unknown – – AAL08442

72 jemC 100, 100; JemC from Tn10 Regulation Pfam01022 COG0640 AAD50245

73 tetR 100, 100; TetR from R27 Regulation Pfam02909,

Pfam00440

– NP_058294

74 tetA 100, 100; TetA from R27 Tetracycline resistance – COG2814 NP_058295

75 tetC 100, 100; TetC from R27 Probably regulation Pfam00440 COG1309 NP_058296

76 tetD 100, 100; TetD from R27 Probably regulation Pfam00165 COG2207 NP_058297

77 DyedAIS10 The first 27 residues are separated by

IS1 insertion from the remaining 375

residues, after rejoining 100, 100; YedA

from R100

Transposition Pfam01609 – NP_058298



80 scsD 70, 81; ScsD from Salmonella typhimurium Suppression of copper sensitivity Pfam00085 COG0526 AAC45603

81 nqrC 35, 49; NqrC from Vibrio cholerae 01

biovar eltor str. N16961

Na+ translocation Pfam04205 COG2869 NP_231924

82 orf82 70, 81; ECA1510 from Erwinia

carotovora subsp. atroseptica

Probably Fe2+ transport Pfam03239 COG0672 CAG74416

83 orf83 88, 96; Yp18 from Yersinia pestis Substrate binding – COG3470 CAA21359



Probably transport Pfam04945 COG4393,

COG3350

YP_049614



Probably transport Pfam02687 COG4591,

COG0577

YP_049615




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

Table 1. cont.

Gene

no.

Coding

sequence




accession no.



Probably transport – COG4591,

COG0577

YP_049616



ATP binding Pfam00005 COG1136 YP_049617



Unknown – COG1225 YP_049618

89 orf89 No significant similarity Unknown – – this work

90 orf90 Represents the first 180 residues of Agp Unknown – – NP_460090

91 tnpAIS26-5 99, 100; Tnp26L from pTP10 Transposition Pfam00665 COG3316 AAC64169

92 orf92 Represents the last 89 residues of OrfA

from IS629


93 orf93 Represents the first 148 residues of

OrfB from IS629


94 orf94 Represents the last 147 residues of

OrfB from IS629

Unknown Pfam00665 – NP_288506

95 orf95 Represents homology to residues 196–296

of Ecs3853 from Escherichia coli O157 : H7

Probably transposition Pfam02281 – BAB37276

96 ugpB 95, 97; UgpB from Enterobacter aerogenes Putative periplasmic binding of

sn-glycerol-3-phosphate

Pfam01547 COG1653 AAO83404

97 ugpC 91, 92; UgpC from Enterobacter aerogenes ATP binding Pfam00005 COG3839 AAO83403

98 phe 93, 94; Ph from Enterobacter aerogenes Phosphodiester hydrolysis Pfam00149 – AAO83402

99 ugpE 94, 97; UgpE from Enterobacter aerogenes Putative sn-glycerol-3-phosphate

transport

Pfam00528 COG0395 AAO83401

100 ugpA 94, 95; UgpA from Enterobacter aerogenes Putative sn-glycerol-3-phosphate

transport

Pfam00528 COG1175 AAO83400

101 orf101 32, 46; STM3532 from Salmonella

typhimurium LT2

Probably involved in lysine

synthesis

Pfam00701 COG0329 NP_462433

102 orf102 57, 71; CV0928 from Chromobacterium

violaceum ATCC 12472

Probably inhibition of translation

initiation

Pfam01042 COG0251 NP_900598

103 kdgT 62, 74; KdgT from Erwinia chrysanthemi 2-Keto-3-deoxygluconate transport Pfam03812 – JQ0113

104 orf104 47, 65; STM3533 from Salmonella

typhimurium LT2

Probably transcriptional regulation Pfam01614 COG1414 NP_462434

105 orf105 97, 100; L7010 from pO157 Probably transcriptional regulation – COG3905 AAC70078

106 orf106 95, 100; L7011 from pO157 Probably plasmid stabilization Pfam05016 COG3668 AAC70079

107 orf107 90, 92; Orf2 from pSERB1 Unknown – – AAT94181

108 orf108 No significant homology Unknown – – This work

109 int 99, 99; Int from p1658/97 Probably multimere resolution – – NP_863020

110 repB 99, 100; RepB from p1658/97 Replication Pfam01051 – NP_863021

111 orf111 99, 99; Orf46 from p1658/97 Unknown – – AAO49618

112 orf112 99, 100; Orf47 from p1658/97 Unknown – – NP_863024

113 insBIS1-4 100, 100; InsBcp3 from p1658/97 Transposition Pfam03400 COG1662 NP_863025

114 insAIS1-4 98, 100; InsAcp3 from p1658/97 Transposition Pfam03811 COG3677 NP_863026

115 iutA 99, 99; IutA from pLVPK Ferric aerobactin receptor Pfam00593 COG4771 NP_943364

116 iucD 100, 100; IucD from pColV-K30 Hydroxylation of L-lysine – COG3486 AAC00523

117 iucC 100, 100; IucC from pTJ100 Aerobactin synthase Pfam04183 COG4264 AAS66995

118 iucB 100, 100; IucB from pTJ100 Acetylation of N-hydroxylysine – COG1670 AAS66994

119 iucA 99, 99; IucA from pTJ100 Aerobactin synthase Pfam04183 COG4264 AAS66993

120 shiF 91, 95; ShiF from Escherichia coli CFT073 Unknown – – NP_755503

121 crcB 100, 100; CrcB-like from pAPEC-1 Probably camphor resistance Pfam02537 COG0239 AAT11270

122 orf122 99, 100; Orf2 from pAPEC-1 Unknown – – AAT11269

123 insAIS1-5 98, 100; InsA from pAPEC-1 Transposition Pfam03811 COG3677 AAT11263

124 insBIS1-5 100, 100; InsB from pAPEC-1 Transposition Pfam03400 COG1662 AAT11262




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

F-plasmid is located downstream of the pRSB107 aero-bactin-synthesis operon. A 2261 bp region containing thereplication-initiation gene repB flanked by iterons and theint gene for a site-specific recombinase is 99?7 % and 97?7 %identical to RepFIB of the E. coli plasmid p1658/97(accession no. AF550679) and the F-plasmid, respectively.The repB gene product belongs to the Rep_3 family(Pfam01051) of initiator replication proteins and is pre-dicted to possess topoisomerase I-like activity (Spiers et al.,1993). The repB gene is flanked by two sets of repeats(ABCD and EFGHIJK), which are believed to form part ofthe origin of replication (repeats BCD) and to sense andto set the plasmid copy number (repeats HIJ), respectively(Spiers et al., 1993). A single-strand-initiation-sequencemotif (ssiF) and a DnaA box are present between repeatsA and B. RepFIB is completed by the int gene, encoding apossible resolvase of the site-specific recombinase familyXerC (COG0582, COG4973), which most probably con-stitutes a plasmid multimer resolution system and thuscould be important for plasmid stability. Remnants of theinsertion sequence IS21 terminate RepFIB 644 bp upstreamof repB on pRSB107. IS21 is also inserted in the same targetsite on p1658/97. The RepFIB replicon has also beenfound on the E. coli IncF plasmids pRK100 (accessionno. AY230888), pO157 (accession no. NC_002128) andpO103-3 (accession no. AF246719). Presence of more thanone basic replicon has frequently been observed for IncF

plasmids which carry the replicons RepFIA, RepFIB andRepFIC in various combinations (Bergquist et al., 1986).

The third pRSB107 replicon is closely related toRepFII

The pRSB107 region from coordinates 20901 to 27621 con-tains genes for replication initiation, control of replicationand post-segregational killing (see Fig. 2), which arehomologous to corresponding RepFII genes of differententerobacterial plasmids. RepFII is related to the RepFICrelict of the F-plasmid. The product of the replication-initiation gene repA1 is 97 % identical to RepA1 of theShigella flexneri resistance plasmid R100 (accession no.NC_002134) whereas the inc RNA (93 bp) encodedupstream of repA1 and transcribed divergently shows thehighest degree of similarity (89 %) to the inc RNA of thehaemolytic E. coli plasmid pSU316 (accession no. M26937).The small antisense inc RNA functions in controllingRepA1 protein synthesis at the posttranscriptional levelby forming a specific RNA duplex structure and it isassumed that differences in its nucleotide sequence giverise to different incompatibility properties (Ohtsubo et al.,1986). RepA2 (CopB) and RepA6, encoded upstreamof repA1, are homologous to corresponding products ofR100 and most probably play a role in negative and posi-tive regulation, respectively, of repA1 expression (Womble

Table 1. cont.

Gene

no.

Coding

sequence




accession no.

125 orf125 48, 63; Plu0597 from Photorhabdus

luminescens subsp. laumondii TTO1


126 orf126 46, 63; Plu0598 from Photorhabdus

luminescens subsp. laumondii TTO1

Unknown – COG4938 NP_927944

127 vagD-1 89, 93; Mck from R64 Probably involved in virulence

and plasmid maintenance

– COG1487 NP_863394

128 vagC-1 96, 97; Kor from R64 Probably involved in virulence


Pfam04014 COG4456 NP_863395

129 orf129 24, 38; BT4745 from Bacteroides

thetaiotaomicron VPI-5482


130 vagD-2 91, 93; Mck from R64 Probably involved in virulence


– COG1487 NP_863394

131 vagC-2 89, 93; Kor from R64 Probably involved in virulence


Pfam04014 COG4456 NP_863395



134 orf134 69, 77; LV092 from pLVPK Unknown – – NP_943348

135 orf135 45, 58; Orf16 from pKDSC50 Unknown – – NP_073240

*Complete amino acid sequences were compared.

DPfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families

(Bateman et al., 2004).

dThe database of Clusters of Orthologous Groups of proteins (COGs) is an attempt at phylogenetic classification of proteins (Tatusov et al.,

2001).




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

et al., 1985). The origin of vegetative replication oriV andthe repA4 locus, discussed to be important for stablemaintenance, have been disconnected from repA1 byinsertion of IS682 and show the highest degree of identity(97 %) to oriV-repA4 of the E. coli virulence plasmid pC15-1a (accession no. AY458016). A single-strand-initiation-sequence motif (ssiA) is located in the vicinity of repA4.The pRSB107 segment downstream of repA4 containingthe tir gene for inhibition of RP4 transfer and the stableinheritance genes pemI and pemK is 100 % identical to thecorresponding region of R100 (accession no. NC_002134).Tir possesses the Abi domain (Pfam02517) specific for aprotease family. PemI and PemK belong to the growth-regulator family MazE (COG2336) and growth-inhibitorfamily PemK/MazF (Pfam02452, COG2337), respectively,

and are predicted to represent the toxin and antitoxin of apost-segregational killing system (Engelberg-Kulka &Glaser, 1999). The pRSB107 RepFII replicon is terminatedby an IS1 insertion downstream of pemK.

RepFII basic replicons were previously found to be pre-sent on the virulence plasmids pWR100 (accession no.AL391753), pWR501 (accession no. NC_002698), pINV(accession no. AY206448) and pCP301 (accession no.NC_004851) of Shigella flexneri, pKDSC50 (accession no.NC_002638) of Salmonella enterica serovar Choleraesuis,pO157 (accession no. NC_002128) of the enterohaemor-rhagic Escherichia coli strain O157 : H7 and on the adherence-factor plasmid pB171 (accession no. NC_002142) of anenteropathogenic E. coli strain.

RepFIA, RepFIB, RepFII

Leading region

Conjugation functions

Aerobactin synthesis andtransport

sn-Glycerol 3-phosphatetransport

IS elements

Put. virulence-associatedgenes

Beneficial functions

Tn21, Tn10

Put. high-affinity Fe2+ uptakesystem (orf82-orf88)

Fig. 1. Genetic map of the multiresistance plasmid pRSB107. Coding regions are indicated by arrows. The modular structureof the plasmid is shown on the outer circle: plasmid replicons RepFIA, RepFIB and RepFII (dark blue); the leading region (lightgreen); a region encoding conjugative functions (magenta); the antibiotic-resistance transposon derivatives related to Tn21and Tn10 (lilac); a putative high-affinity Fe2+-uptake system (orange); a putative sn-glycerol-3-phosphate uptake operon(dark green); an aerobactin-synthesis and receptor operon (light blue); and putative virulence-associated genes, vagCD

(brown). Other genes with predicted functions are shown in grey. White arrows represent putative genes encoding unknownfunctions. The G+C plot is indicated on the inner circle, where a G+C content of >50% is shown in red and a G+Ccontent of <50% is shown in blue. The G+C plot was generated using the GenDB (version 2.0) Tool (Meyer et al., 2003).Coding sequences were numbered clockwise from 1 to 135.




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

Remnants of a conjugative transfer module arelocated upstream of RepFII on pRSB107

pRSB107 contains a relict of the conjugative-transfermodule, composed of a truncated DNA-helicase/nickasegene traI, the F-pilin acetylation gene traX and the fertilityinhibition gene finO. The pRSB107 traI gene starts with anATG start codon preceded by a putative ribosome-bindingsite and its deduced gene product has a length of 1209 aaas compared to 1756 aa for TraI of the F-plasmid (acces-sion no. BAA97974). Currently it is unknown whether thetruncated version of TraI retains residual activity. Attemptsto transfer pRSB107 to an E. coli recipient strain via con-jugation were not successful, which is consistent with theabsence of a complete tra module on pRSB107. Deletedconjugative transfer modules containing traI-specific se-quences, traX and finO were also found on the virulenceplasmids pWR100, pWR501, pINV, pCP301, pO157 andpKDSC50. For pWR101 it has been shown that it can bemobilized in the presence of conjugative helper plasmids(Sansonetti et al., 1982).

To test transferability of pRSB107, the plasmid was intro-duced into the E. coli strains S17-1 and CSH61-HfrC.Plasmid-containing derivatives of these strains were sub-sequently mated with E. coli CV60 GFP as recipient. Notransconjugants were obtained in mating experiments withE. coli S17-1, indicating that the RP4 plasmid integrated inthe chromosome of strain S17-1 was not able to mobilizepRSB107. Thirty-one green fluorescent putative trans-conjugants were obtained in mating experiments withE. coli CSH61-HfrC(pRSB107) as donor strain and E. coliCV60 GFP as recipient. Eckhardt-gel analysis revealed thatall transconjugants contained a plasmid which was largerthan the native pRSB107. Two of these transconjugantswere chosen for plasmid isolation and subsequent restric-tion analyses. These two plasmids displayed very similarrestriction patterns to the pRSB107-profile, but containedadditional restriction fragments. Most probably, the twoinvestigated plasmids acquired DNA segments from thechromosomally integrated F-plasmid via homologousrecombination. This hypothesis is supported by the factthat no nucleotide sequences with homology to the F origin

Fig. 2. The three replicons, RepFIA, RepFIB and RepFII, present on pRSB107. Genes for replication initiation (rep), multimereresolution (mrs), post-segregational killing (psk) and active plasmid partitioning (par) are differentiated by colour. The followingregulatory DNA sequence motifs are marked: site-specific resolution site for resolution of plasmid dimers (rfsF site, brown),origins of vegetative replication (oriV, lilac), DnaA-binding boxes (DnaA, yellow), AT-rich region (A/T, dark green), local DNA-melting-site (13mer, grey), single-strand-initiation sequences (ssi, pink), iteron repeats (incB, incC, red; sopC/incD, lightgreen; A-K, black), antisense RNA (inc, dark blue). Triangles indicate insertions of IS elements.




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

of transfer (oriT) could be identified on pRSB107, whichmeans that pRSB107 should not be mobilizable. Therefore,we speculate that oriT was acquired from the integratedF-plasmid, which would modify pRSB107 to becomemobilizable.

The pRSB107 traI gene is preceded by the coding sequencesydcA9, ydbA and ydaB, which are also present upstreamof yddA on plasmid R100 (accession no. NC_002134). Thefunction of these three genes remains unknown. The inter-vening segment containing other tra genes obviously wasdeleted in pRSB107.

The pRSB107 region downstream of RepFIA isclosely related to the F-plasmid leadingsegment

The pRSB107 segment downstream of RepFIA is verysimilar (94 % identity at the nucleotide sequence level) tothe distal part of the F-plasmid leading region, which isdefined as the first portion of F-DNA to enter the recipientcell during conjugative transfer. The leading region onpRSB107 contains nine coding sequences, seven of whichare homologous to F-plasmid orf227, -73, -144, -248, -141,-140 and -63 (Manwaring et al., 1999). In contrast tothe F-plasmid, orf168 and orf145 are fused to orf7 onpRSB107. The deduced product of orf8 downstream oforf7 is 96 % identical to YcdA of the Shigella sonneiplasmid ColIb-P9 (accession no. NC_002122) and doesnot correspond to orf101, which is present downstreamof orf145 on F. Functional predictions can only be madefor two coding sequences, namely orf9, which encodes aputative cytosine-specific or N-6 adenine-specific DNAmethylase (Pfam01555, COG0863) possibly involved inprotection of the transferred DNA from restriction endo-nucleases (Manwaring et al., 1999; Venkatesan et al., 2001),and orf13 for a putative antirestriction protein (Pfam03230),(Belogurov et al., 1993). The region containing the origin oftransfer (oriT) is deleted on pRSB107.

A Tn21 derivative integrating eight resistancedeterminants represents the core element ofthe pRSB107 antibiotic-resistance region

Downstream of the pRSB107 RepFII replicon a Tn21derivative has inserted. Tn21 is followed by a chlo-ramphenicol-resistance module and a derivative of thetetracycline-resistance transposon Tn10.

The 27 212 bp Tn21 transposon is terminated by 38 bpinverted repeats (IR) and has caused a 5 bp target-siteduplication (direct repeats: TATTA). The mercury-resistance (mer) and the transposition genes (tnpM-tnpR-tnpA) on pRSB107 Tn21 are identical to correspondinggenes on Tn21 of plasmid R100 (accession no. AP000342),but in contrast to the latter, pRSB107 Tn21 integrates thefollowing accessory elements (see Fig. 3): (i) a compositekanamycin/neomycin-resistance transposon flanked byIS26 elements; (ii) a streptomycin/sulfonamide-resistance

region with associated replication genes terminated by IS26;(iii) a Tn1 relict carrying a b-lactamase-resistance gene; (iv)a macrolide-resistance operon flanked by IS26 and IS6100;and (v) a Tn402 relict integrating a class 1 integron.

The pRSB107 Tn21 mercury-resistance (mer) regionencodes proteins responsible for binding of divalent Hg2+

ions in the periplasm (periplasmic protein MerP), thetransfer of Hg2+ into the cytoplasm (inner-membraneproteins MerT and MerC) and reduction of Hg2+ to Hg0,which diffuses from the cell (cytoplasmic flavin disulfideoxidoreductase MerA). MerR and MerD are responsiblefor transcriptional regulation of the merTPCAD operon.The function of the merE-encoded protein (Pfam05052) isstill unknown (Liebert et al., 1999). The urf2 gene pro-duct possesses an EAL domain (Pfam00563) that couldparticipate in metal binding.

The composite kanamycin/neomycin-resistance transposonTn4352B (IS26-aph-IS26) which truncated the Tn402-specific transposase gene tniA on pRSB107 has also beenfound on the transposon TnSF1 of Shigella flexneri (acces-sion no. AF188331), the multiresistance b-lactamasetransposon Tn1412 (accession no. L36547) of Pseudo-monas aeruginosa, the resistance plasmid NTP16 (accessionno. M20306) and the plasmid pRMH760 (accession no.AY123253) isolated from Klebsiella pneumoniae. Tn4352Bon pRMH760 and pRSB107 both inserted into exactly thesame target site, thus truncating tniA. Since Tn4352B onpRSB107 only shares its left-hand-side target-site dupli-cation (direct repeat: CGCCGATG) with Tn4352B ofpRMH760 (Partridge & Hall, 2003) we assume that thedownstream module consisting of strAB, sulII and 9repACentered pRSB107 by homologous recombination via IS26copies, thus removing the original right-hand-side directrepeat. It is noteworthy that the latter module is alsoterminated by an IS26 insertion. The incoming IS26-9repAC-sulII-strAB-IS26 module might originate from aplasmid similar to pHCM1 of Salmonella enterica subsp.enterica serovar Typhi strain CT18 (Wain et al., 2003), sinceits corresponding module is almost identical (99?9 %) tothe one on pRSB107. The genes strA and strB encodingthe streptomycin-resistance proteins A and B are pre-ceded by sulII for a dihydropteroate synthetase conferringsulfonamide resistance. The replication gene repC and the39 part of repA are located upstream of sulII. These geneswere originally found on the IncQ plasmid RSF1010 andnormally constitute the replication operon repAC (Scherzingeret al., 1991). Since the RSF1010-specific oriV and the 59 partof repA are missing on pRSB107 it is very unlikely that thisreplication region is functional.

The next module on pRSB107 is a remnant of Tn1 consist-ing of blaTEM-1b for a class A b-lactamase and part of theTn1-specific resolvase gene tnpR. This module is alsopresent in an equivalent position on pHCM1.

The Tn1 tnpR gene was truncated and tnpA most prob-ably was deleted by insertion of IS26, which borders the




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

macrolide-resistance operon mph(A)-mrx-mphR(A) at theleft-hand-side. A copy of IS6100 is located downstreamof mphR(A). The macrolide-resistance operon encodes amacrolide-29-phosphotransferase I (MphA), a hydrophobicprotein of unknown function (Mrx) and a negative transcrip-tional regulator (MphR), and mediates high-level erythro-mycin resistance. Nearly identical erythromycin-resistance

regions were previously identified on TnSF1 of Shigellaflexneri (accession no. AF188331) and on the multiresis-tance plasmid pRSB101 from a wastewater-treatmentplant (Szczepanowski et al., 2004). The macrolide-resistancemodule is separated from a class 1 integron by an inter-vening mobC-like sequence that encodes a putative mobili-zation protein of the relaxase group (Pfam05713).

R100-Tn21(19 763 bp)

pRMH760-Tn21(28 497 bp)

pHCM1-Tn21(19 249 bp)

TnSF1(26 652 bp)

pRSB107-Tn21(27 212 bp)

Fig. 3. Comparison of the genetic organization of the pRSB107 Tn21-derivative and related transposons present onplasmids R100, pRMH760 and pHCM1 or on the Shigella flexneri chromosome. The Tn21 derivative located on pRSB107shares homology with modules present either on related plasmid-borne Tn21-derivatives, or on TnSF1 of S. flexneri. Codingsequences are shown as arrows, indicating the direction of transcription. Boxes represent truncated genes. Modules sharingsequence homology are in the same colour. The following modules are differentiated by colour: mercury resistance (mer)and transposition genes (tnpMRA), lilac; Tn402 class 1 integron specific genes, red; IS1353, green; IS1326, yellow; Tn1transposon, light blue; Tn4352B transposon, dark blue; IS513, brown; repAC-sulII-strAB module present on plasmidspHCM1 and pRSB107 as well as on plasmid RSF1010, blue; IS4321, light yellow; mobilization gene, grey. Inverted repeats(IR, rectangles) have the same colour as the mobile element to which they belong. The target-site duplications (DR, circles)caused by a transposition event are shown in the same colour as the mobile element. The different DNA sequences weretaken from the GenBank database: R100-Tn21 (accession no. NC_002134), pRMH760-Tn21 (accession no. AY123253),pHCM1-Tn21 (accession no. AL513383), TnSF1 (accession no. AF188331).




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

The class 1 integron upstream of mobC is composed of theintegron-specific integrase gene intI1 and the resistancegene cassette dhfR for a dihydrofolate reductase conferringtrimethoprim resistance. Tn21 of pRSB107 is terminatedby the Tn21-specific transposition module (tnpM-tnpR-tnpA) responsible for transposition of this transposon.Class 1 integrons have frequently been found downstreamof Tn21 transposition modules, for example on R100,pRMH760 and on the transposon TnSF1 (see Fig. 3).

A chloramphenicol-resistance module and thetetracycline-resistance transposon Tn10complete the pRSB107 resistance region

A chloramphenicol-resistance module and a derivative ofthe tetracycline-resistance transposon Tn10 are locatednext to the pRSB107 Tn21 insertion.

Tn21 on pRSB107 is followed by ybjA, encoding a puta-tive acetyltransferase (Pfam00583), the chloramphenicol-resistance gene catA for a chloramphenicol acetyltransferase(COG4845, Pfam00302) and a copy of IS1. Identical seg-ments are also present on R100 (Womble & Rownd, 1988),pRMH760 (accession no. AY123253) and pHCM1 (Wainet al., 2003).

Finally, pRSB107 carries a truncated derivative of thetetracycline-resistance transposon Tn10 which insertednext to the chloramphenicol-resistance module. Tn10normally consists of IS10-L, jemA, jemB, jemC, tetR, tetA,tetC, tetD and IS10-R (Chalmers et al., 2000). IS10-L,jemA and the 59 part of jemB were deleted in the caseof pRSB107 Tn10. This deletion most probably occurredduring insertion of the chloramphenicol-resistance modulewhich is bordered by IS1 upstream of catA. The persistingTn10 genes encode JemC, a putative repressor of heavy-metal-resistance operons (Pfam01022, COG0640), thetetracycline repressor protein TetR, the tetracycline anti-porter protein TetA, the tetracycline transcriptionalregulator TetC of the AcrR family (COG1396), and TetD,a protein with a signature similar to AraC-type DNA-binding-domain-containing proteins (COG2207) whichprobably plays a role in transcriptional regulation. Ano-ther copy of IS1 disrupted the IS10-R transposase genetnpA. Collinearity between a chloramphenicol-resistancemodule and a truncated Tn10 derivative has also beenobserved in a pathogenicity island of Shigella flexneri 2a(Luck et al., 2001). The S. flexneri region is 99?6 % identicalto the corresponding region on pRSB107.

In summary, the pRSB107 antibiotic-resistance regionshows a highly mosaic structure consisting of resistancedeterminants carried by functional or nested transposableelements. It is also noticeable that several IS elements (fourcopies of IS26, IS6100 and three copies of IS1) frameresistance determinants. This observation leads to theassumption that IS elements played an important role inthe development and assembly of the pRSB107 resistanceregion.

pRSB107 encodes an aerobactin-synthesisoperon and a second putative iron-acquisitionsystem

An aerobactin-synthesis operon is located downstream ofRepFIB on pRSB107. An 8101 bp DNA fragment, contain-ing the genes iucA, iucB, iucC, iucD and iutA, shows 99?6 %identity to a corresponding sequence on plasmid pTJ100of the avian pathogenic E. coli strain A2363 (accession no.AY553855). The iuc (iron uptake chelate) genes encodeenzymes necessary for synthesis of the siderophore aero-bactin, whereas the iutA (iron uptake transport) geneproduct represents the TonB-dependent outer-membranereceptor for ferric aerobactin. The first step in aerobactinsynthesis is hydroxylation of L-lysine by IucD, whereasIucB is responsible for acetylation of N-hydroxylysine. TwoN-acetyl-N-hydroxylysine molecules are then attached tothe carboxylic groups of citric acid by IucC and probablyIucA, resulting in aerobactin (de Lorenzo & Neilands, 1986;de Lorenzo et al., 1986). The pRSB107 aerobactin operonmost probably is regulated by Fur (ferric uptake regulator),since a putative Fur box (GATAATGAGAATCATTATT) islocated upstream of iucA. IS1 elements could have played arole in the integration of the aerobactin synthesis operoninto pRSB107, since the iuc/iut region is framed by IS1copies.

As mentioned above, two lysine molecules are neededfor aerobactin synthesis. Lysine synthesis is feedback-controlled by lysine, which inhibits two enzymes involvedin this pathway, namely dihydrodipicolinate synthase andaspartokinase (Mirwaldt et al., 1995). Interestingly, orf101on pRSB107 encodes a putative dihydrodipicolinate syn-thase (COG0329, Pfam00701) which is, respectively, 32 %(46 % similarity) and 28 % (44 % similarity) identical tocorresponding gene products from Salmonella typhimuriumLT2 (accession no. NP_462433) and E. coli K-12 (accessionno. NP_418718). It might be speculated that higheramounts of dihydrodipicolinate synthase allow for sufficientproduction of aerobactin for efficient iron acquisition inaddition to the demand for lysine itself.

Importance of the iuc/iut region for pathogenesis could beshown for Shigella flexneri 2a strain 2457T, where aero-bactin synthesis plays a role in shigellosis (Wei et al., 2003).The iucABCD-iutA cluster has recently been found on the219 kb virulence plasmid pLVPK harboured in a bacter-aemic isolate of Klebsiella pneumoniae (Chen et al., 2004).Curing of pLVPK resulted in a decrease of virulence, butcurrently it is unknown whether this phenotype is due tothe absence of aerobactin synthesis.

The gene shiF upstream of iucA has also been found tobe conserved upstream of the aerobactin iron-acquisitionsiderophore system encoded in the S. flexneri pathogenicityisland SHI-2 (Shigella island 2) (Moss et al., 1999). Thetwo genes share 92 % sequence identity. ShiF is a puta-tive integral membrane protein containing 12 putativetransmembrane helices and a membrane lipoprotein lipid




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

attachment site, and shares weak similarity with proteinsof the tetracycline-resistance transporter family (Mosset al., 1999). The function of ShiF is currently unknown.Homology between pRSB107 and SHI-2 ends 9 bp down-stream of shiF. The pRSB107 segment downstream of shiFis nearly identical (99?7 %) to a locus on plasmid pAPEC-1of an avian pathogenic E. coli strain and contains the genescrcB and orf122. CrcB is a putative integral membraneprotein possibly involved in chromosome condensation andcamphor resistance (Pfam02537, COG0239), (Hu et al.,1996). Orf122 possesses the C-terminal part of an enolase-specific domain 9(Pfam00113,COG0148). Interestingly,part of the orf122 sequence has been found to be specificfor a novel virulence-associated locus in uropathogenic E.coli (SSH fragment SPL00265) (Sorsa et al., 2004).

Another gene region that encodes putative iron-acquisitiongenes is located next to the Tn10 insertion on pRSB107. Onthe other side, this region is flanked by a copy of IS26 (IS26-5, see Fig. 1). The gene product of orf82 is a putative high-affinity Fe2+/Pb2+ permease. In addition to eight probabletransmembrane helices Orf82 includes the FTR1 domain(COG0672, Pfam03239) of the yeast FTR1 gene which hasbeen shown to mediate high-affinity iron uptake (Stearmanet al., 1996). Further, a conserved domain, similar to that ofmammalian ferritin (REGAE) (Levi et al., 1994; Stearmanet al., 1996), could be identified within the amino acidsequence of Orf82. The glycine residue of the REGAE motifis thought to interact with iron. Orf83 possesses the Tpddomain (COG3470), which is present on uncharacterizedperiplasmic proteins, probably involved in high-affinityFe2+ transport. The genes downstream of orf83 encode aputative integral membrane protein with eight probabletransmembrane helices (Orf84, COG4393, Pfam04945),two ABC-type inner-membrane permeases with four puta-tive transmembrane helices each (Orf85, Orf86, COG4591,COG0577), an ABC-transporter ATP-binding protein(Orf87, COG1136) and a peroxiredoxin (Orf88, COG1225).Currently it is unknown whether these latter gene productsalso play a role in the context of iron uptake. The orf82–orf88region most probably is organized in two operons, sinceputative promoter sequences could be identified upstreamof orf82 and orf84. Gene regions homologous to orf82–orf88were previously identified in a 102 kb pathogenicity islandof the human pathogen Yersinia pestis (accession no.AL031866) and in the genome of the enterobacterialphytopathogen Erwinia carotovora subsp. atroseptica (acces-sion no. NC_004547). A comparison of the genetic organi-zation of the corresponding gene regions is shown inFig. 4. Strikingly, certain segments of the orf82–orf88region were also found to be specific for, respectively,neonatal meningitis-associated and uropathogenic Escheri-chia coli strains (Bonacorsi et al., 2000; Zhang et al., 2000).In detail, clones SauE15.B10, SauE4.A2 and SauE4.C10from neonatal meningitis-associated E. coli and P5 fromuropathogenic E. coli are identical or almost identicalto corresponding sequences of the pRSB107 orf82–orf88region. These observations led to the assumption that the

pRSB107-encoded iron-acquisition systems could enhancevirulence of pathogenic bacteria harbouring pRSB107.Many studies have demonstrated the important role ofiron in virulence. Pathogens have to compete for iron supplywith cells of the host organism. For example, Shigella flexneristrains that could not produce functional aerobactin, wereless infectious than the wild-type strain (Lawlor et al., 1987;Nassif et al., 1987). Likewise, derivatives of Pseudomonasaeruginosa PAO1 deficient in the production of the sidero-phores pyoverdin and pyochelin, resulting in decreased ironsupply, did not show lethal virulence in mice, as the wild-type strain does (Takase et al., 2000).

A putative virulence-associated gene region islocated upstream of RepFIA

Two copies of the virulence-associated genes vagCD werefound in a pRSB107 region upstream of the aerobactin-synthesis operon iuc/iut. The two copies of vagCD areseparated by an intervening sequence encoding orf129.

The pRSB107 vagCD copies are 90 % identical to each otherat the DNA-sequence level. Orthologues of these geneswere previously identified on plasmid R64 of Salmonellatyphimurium (accession no. NC_005014), the virulenceplasmid pLVPK of Klebsiella pneumoniae strain CG43(Chen et al., 2004) and a virulence plasmid of Salmonellaenterica subsp. enterica serovar Dublin (Pullinger & Lax,1992). For the latter plasmid it has been shown that amutation in vagC leads to reduced virulence. It wassuggested that the VagCD proteins play a role in coordina-tion of plasmid replication and cell division, thus ensuringplasmid maintenance. VagC contains the SpoVT/AbrB-likedomain (Pfam04014). One member of this protein family,namely AbrB, controls gene expression during the transi-tion between vegetative growth and the onset of stationaryphase. Growth-phase-dependent activity of VagC would bein accordance with a role in delaying cell division untilplasmid replication has been completed. Results obtainedby Pullinger & Lax (1992) indicated that VagC modulatesthe activity of vagD, the product of which is a predictednucleic-acid-binding protein containing a PIN (PilT N-terminus) domain (COG1487). The function of the PINdomain is unknown but a role in signalling is discussed.

The ugp operon of pRSB107 encodes ansn-glycerol-3-phosphate uptake system

A predicted sn-glycerol-3-phosphate uptake system (ugp:uptake of sn-glycerol-3-phosphate) is encoded fromcoordinates 78 420 to 83 475 on pRSB107. The 5372 bpugp operon shows 90 % identity to the correspondingoperon in the genome of Enterobacter aerogenes (accessionno. AY243367) and contains the genes ugpA, ugpE, phe,ugpC and ugpB. The products of ugpA, ugpB, ugpC andugpE are responsible for uptake of sn-glycerol-3-phosphatewhereas the phe product allows for growth on dimethylphosphate (DMP) as the sole phosphorus source(McLoughlin et al., 2004). The four Ugp proteins constitute




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

an ABC-type transporter including two permease com-ponents (UgpA and UgpE, COG0395), an ATPase com-ponent (UgpC, COG3839, Pfam00005) and a periplasmicsubstrate-binding protein (UgpB, COG1653). The Pheprotein belongs to the family of phosphodiesterases(Pfam00149) and is responsible for the hydrolysis ofDMP (McLoughlin et al., 2004). The sequence motifDXH(X)nGDXXD(X)nGNHD/E, which is specific for thelarge group of phosphoesterases (e.g. Ser/Thr proteinphosphatases and purple acid phosphatases, PAP) is alsopresent within Phe encoded on pRSB107 (Koonin, 1994).Presence of a putative Pho box (GCTTCATCAAACC-ATCGT) upstream of the ugp operon indicates that theoperon is controlled by the transcriptional regulator PhoB.

To test the ability of E. coli DH5a harbouring pRSB107to utilize DMP as the sole phosphorus source, plasmid-containing and plasmid-free cells were grown in MOPSminimal medium with 0?1 M DMP. In addition, MOPSminimal medium without DMP was used to ensure thatE. coli DH5a cells with and without pRSB107 are not ableto grow under phosphorus starvation. The results showedthat E. coli cells containing pRSB107 were able to utilize

DMP as the sole phosphorus source, whereas plasmid-freecells could not grow under these conditions.

The gene kdgT, which also has a function in hexosemetabolism, is located 2299 bp upstream of ugpA. Theencoded 2-keto-3-deoxygluconate permease (Pfam03812)is 62 % identical to KdgT of Erwinia chrysanthemi (acces-sion no. P15701) and to KdgT encoded by the uropatho-genic E. coli strain CFT073 (accession no. NP_756715). TheKdgT permease transports 2-keto-3-deoxygluconate intothe cell, which then can be degraded to pyruvate and 3-phosphoglyceraldehyde. Oxidation of the latter metaboliteyields ATP, NADH2 and pyruvate.

The pRSB107 accessory modules are flanked bydifferent IS elements

The pRSB107 accessory modules Tn21, Tn10, the putativehigh-affinity Fe2+-uptake locus, the sn-glycerol-3-phosphateuptake system (ugp) and the aerobactin-synthesis operon(iut/iuc) are framed by insertion sequence (IS) elements.The two transposons are flanked and separated from eachother by three IS1 copies (IS1-1, IS1-2 and IS1-3). The

Fig. 4. Genetic organization of the putative high-affinity Fe2+-uptake system present on pRSB107 and comparison withrelated chromosomal gene regions of Erwinia carotovora subsp. atroseptica SCRI1043 and Yersinia pestis. The arrowsindicate coding sequences and the direction of their transcription. Homologous genes are shown in the same colour.Predicted functions of gene products are given above the coding sequences. Identities and similarities (in %) betweencorresponding pRSB107 and Erwinia carotovora subsp. atroseptica SCGI1043 or Yersinia pestis gene products are shownbelow the coding sequences. The DNA sequences were taken from the GenBank database: Erwinia carotovora subsp.atroseptica SCGI1043 (accession no. NC_004547), Yersinia pestis (accession no. AL031866).




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

high-affinity Fe2+-uptake locus is bordered by a relict ofIS26 and an intact copy of IS26 whereas the ugp region isterminated by remnants of IS629 downstream of ugpB.Finally, the iut/iuc operon is bounded by two IS1 copies(IS1-4 and IS1-5). In addition, the resistance determinantsintegrated in Tn21 are also framed by IS elements. Fourcopies of IS26 and one copy of IS6100 are located withinTn21. IS-specific sequences account for approximately13 300 bp (11 % of the total plasmid size). Some accessorymodules on pRSB107 represent composite transposons,although they did not enter the plasmid by transpositionsince corresponding target-site duplications (direct repeats,DR) could not be found next to the terminal invertedrepeats of the IS elements. It is assumed that differenthorizontally acquired DNA modules were assembled dur-ing plasmid evolution by using IS elements as homolo-gous DNA segments for recombination. Only Tn21 clearlyentered the plasmid by transposition. ConsequentlypRSB107 can be considered as a mosaic of different modulesderived from different sources, including other resistanceand virulence plasmids.

Concluding remarks

The IncF plasmid pRSB107 confers antibiotic and mercuryresistance and other beneficial properties upon its hostbacterium. IncF plasmids, in general, are known to encodeantibiotic-resistance determinants, haemolysins, toxins,invasins and colicins (Burland et al., 1998; Gibbs et al.,1993; Venkatesan et al., 2001; Womble & Rownd, 1988) andare widespread in the family Enterobacteriaceae. Thereforeit is very likely that the original host for pRSB107 also isan enterobacterium. Plasmid pRSB107 exhibits a mosaicstructure consisting of modules which were previouslyidentified on resistance and/or possible virulence plasmidsand in the chromosomes of human and plant pathogens.In detail, the multiresistance Tn21 transposon on pRSB107derives from other Tn21 elements present on differentresistance or virulence-associated plasmids. The putativehigh-affinity iron-acquisition system and the sn-glycerol-3-phosphate uptake operon were recently identified in thegenomes of Yersinia pestis and Enterobacter aerogenes,respectively. The pRSB107 iuc/iut gene region responsiblefor siderophore-dependent iron uptake has also beenfound on virulence plasmids and on the chromosomes ofpathogenic bacteria. Taking these observations together ithas to be concluded that pRSB107 is a chimera of antibiotic-resistance and virulence-associated plasmids which acquiredits accessory modules horizontally from different sources.Wastewater-treatment plants have previously been shownto be a reservoir for antibiotic-resistance plasmids and herewe provide evidence that these facilities also harbourbacteria carrying virulence-associated plasmids. It seemslikely that exchange of genetic material between resistanceplasmids and virulence plasmids occurs in sewage bacteria.Newly recombined plasmids promoting resistance andvirulence can then be released with the sewage treatmentplant’s final effluents and disseminated among bacteria in

the wider environment. Transfer of these plasmids to humanpathogens could clearly be of relevance for public health.

ACKNOWLEDGEMENTS

We thank the Bioinformatics Resource Facility (BRF) at the Centreof Biotechnology (CeBiTec, Bielefeld) for their support regardingbioinformatics, and especially for their help with the annotation toolGenDB. This work was supported by a grant from the Bundes-ministerium fur Bildung und Forschung (BMBF), Forderkennzeichen:0312384.

REFERENCES

Bateman, A., Coin, L., Durbin, R. & 10 other authors (2004). ThePfam protein families database. Nucleic Acids Res 32 (Databaseissue), D138–D141.

Belogurov, A. A., Delver, E. P. & Rodzevich, O. V. (1993). PlasmidpKM101 encodes two nonhomologous antirestriction proteins (ArdAand ArdB) whose expression is controlled by homologous regulatorysequences. J Bacteriol 175, 4843–4850.

Bergquist, P. L., Saadi, S. & Maas, W. K. (1986). Distribution ofbasic replicons having homology with RepFIA, RepFIB, and RepFICamong IncF group plasmids. Plasmid 15, 19–34.

Blazquez, J., Navas, A., Gonzalo, P., Martinez, J. L. & Baquero, F.(1996). Spread and evolution of natural plasmids harboringtransposon Tn5. FEMS Microbiol Ecol 19, 63–71.

Bonacorsi, S. P., Clermont, O., Tinsley, C., Le Gall, I., Beaudoin,J. C., Elion, J., Nassif, X. & Bingen, E. (2000). Identification ofregions of the Escherichia coli chromosome specific for neonatalmeningitis-associated strains. Infect Immun 68, 2096–2101.

Boyd, E. F., Hill, C. W., Rich, S. M. & Hartl, D. L. (1996). Mosaicstructure of plasmids from natural populations of Escherichia coli.Genetics 143, 1091–1100.

Burlage, R. S., Bemis, L. A., Layton, A. C., Sayler, G. S. & Larimer, F.(1990). Comparative genetic organization of incompatibility group Pdegradative plasmids. J Bacteriol 172, 6818–6825.

Burland, V., Shao, Y., Perna, N. T., Plunkett, G., Sofia, H. J. &Blattner, F. R. (1998). The complete DNA sequence and analysis ofthe large virulence plasmid of Escherichia coli O157 : H7. NucleicAcids Res 26, 4196–4204.

Cavalli, L. L., Lederberg, J. & Lederberg, E. M. (1953). An infectivefactor controlling sex compatibility in Bacterium coli. J Gen Microbiol8, 89–103.

Chalmers, R., Sewitz, S., Lipkow, K. & Crellin, P. (2000). Completenucleotide sequence of Tn10. J Bacteriol 182, 2970–2972.

Chen, Y. T., Chang, H. Y., Lai, Y. C., Pan, C. C., Tsai, S. F. & Peng,H. L. (2004). Sequencing and analysis of the large virulence plasmidpLVPK of Klebsiella pneumoniae CG43. Gene 337, 189–198.

Davies, J. (1994). Inactivation of antibiotics and the dissemination ofresistance genes. Science 264, 375–382.

Davison, J. (1999). Genetic exchange between bacteria in theenvironment. Plasmid 42, 73–91.

de Lorenzo, V. & Neilands, J. B. (1986). Characterization of iucA andiucC genes of the aerobactin system of plasmid ColV-K30 inEscherichia coli. J Bacteriol 167, 350–355.

de Lorenzo, V., Bindereif, A., Paw, B. H. & Neilands, J. B. (1986).Aerobactin biosynthesis and transport genes of plasmid ColV-K30 inEscherichia coli K-12. J Bacteriol 165, 570–578.




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

Di Lorenzo, M., Stork, M., Tolmasky, M. E. & 9 other authors(2003). Complete sequence of virulence plasmid pJM1 from themarine fish pathogen Vibrio anguillarum strain 775. J Bacteriol 185,

5822–5830.

Droge, M., Puhler, A. & Selbitschka, W. (1998). Horizontal genetransfer as a biosafety issue: a natural phenomenon of public

concern. J Biotechnol 64, 75–90.

Droge, M., Puhler, A. & Selbitschka, W. (2000). Phenotypic and

molecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activated sludge.

Mol Gen Genet 263, 471–482.

Engelberg-Kulka, H. & Glaser, G. (1999). Addiction modules andprogrammed cell death and antideath in bacterial cultures. Annu Rev

Microbiol 53, 43–70.

Gerdes, K., Moller-Jensen, J. & Bugge Jensen, R. (2000). Plasmid

and chromosome partitioning: surprises from phylogeny. MolMicrobiol 37, 455–466.

Gibbs, M. D., Spiers, A. J. & Bergquist, P. L. (1993). RepFIB: a basic

replicon of large plasmids. Plasmid 29, 165–179.

Grant, S. G., Jessee, J., Bloom, F. R. & Hanahan, D. (1990).Differential plasmid rescue from transgenic mouse DNAs intoEscherichia coli methylation-restriction mutants. Proc Natl Acad Sci

U S A 87, 4645–4649.

Herrero, M., de Lorenzo, V. & Neilands, J. B. (1988). Nucleotidesequence of the iucD gene of the pColV-K30 aerobactin operon and

topology of its product studied with phoA and lacZ gene fusions.J Bacteriol 170, 56–64.

Heuer, H., Szczepanowski, R., Schneiker, S., Puhler, A., Top, E. M.& Schluter, A. (2004). The complete sequences of plasmids pB2 and

pB3 provide evidence for a recent ancestor of the IncP-1b groupwithout any accessory genes. Microbiology 150, 3591–3599.

Hu, K. H., Liu, E., Dean, K., Gingras, M., DeGraff, W. & Trun, N. J.(1996). Overproduction of three genes leads to camphor resistanceand chromosome condensation in Escherichia coli. Genetics 143,

1521–1532.

Hynes, M. F., Simon, R. & Puhler, A. (1985). The development of

plasmid-free strains of Agrobacterium tumefaciens by using incom-patibility with a Rhizobium meliloti plasmid to eliminate Patc58.

Plasmid 13, 99–105.

Kawasaki, Y., Matsunaga, F., Kano, Y., Yura, T. & Wada, C. (1996).The localized melting of mini-F origin by the combined action of the

mini-F initiator protein (RepE) and HU and DnaA of Escherichiacoli. Mol Gen Genet 253, 42–49.

Koonin, E. V. (1994). Conserved sequence pattern in a wide variety ofphosphoesterases. Protein Sci 3, 356–358.

Kurtz, S., Choudhuri, J. V., Ohlebusch, E., Schleiermacher, C.,Stoye, J. & Giegerich, R. (2001). REPuter: the manifold applica-tions of repeat analysis on a genomic scale. Nucleic Acids Res 29,

4633–4642.

Lane, D., de Feyter, R., Kennedy, M., Phua, S. H. & Semon, D.(1986). D protein of miniF plasmid acts as a repressor of transcrip-tion and as a site-specific resolvase. Nucleic Acids Res 14, 9713–9728.

Lawley, T. D., Klimke, W. A., Gubbins, M. J. & Frost, L. S. (2003). F

factor conjugation is a true type IV secretion system. FEMS MicrobiolLett 224, 1–15.

Lawlor, K. M., Daskaleros, P. A., Robinson, R. E. & Payne, S. M.(1987). Virulence of iron transport mutants of Shigella flexneri and

utilization of host iron compounds. Infect Immun 55, 594–599.

Levi, S., Santambrogio, P., Cozzi, A., Rovida, E., Corsi, B.,Tamborini, E., Spada, S., Albertini, A. & Arosio, P. (1994). The

role of the L-chain in ferritin iron incorporation. Studies of homoand heteropolymers. J Mol Biol 238, 649–654.

Liebert, C. A., Hall, R. M. & Summers, A. O. (1999). Transposon

Tn21, flagship of the floating genome. Microbiol Mol Biol Rev 63,

507–522.

Luck, S. N., Turner, S. A., Rajakumar, K., Sakellaris, H. & Adler, B.

(2001). Ferric dicitrate transport system (Fec) of Shigella flexneri

2a YSH6000 is encoded on a novel pathogenicity island carrying

multiple antibiotic resistance genes. Infect Immun 69, 6012–6021.

Manwaring, N. P., Skurray, R. A. & Firth, N. (1999). Nucleotide

sequence of the F plasmid leading region. Plasmid 41, 219–225.

Martinez, B., Tomkins, J., Wackett, L. P., Wing, R. & Sadowsky,M. J. (2001). Complete nucleotide sequence and organization of

the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain

ADP. J Bacteriol 183, 5684–5697.

Mazel, D. & Davies, J. (1999). Antibiotic resistance in microbes. Cell

Mol Life Sci 56, 742–754.

McLoughlin, S. Y., Jackson, C., Liu, J. W. & Ollis, D. L. (2004).

Growth of Escherichia coli coexpressing phosphotriesterase and

glycerophosphodiester phosphodiesterase, using paraoxon as the

sole phosphorus source. Appl Environ Microbiol 70, 404–412.

Meyer, F., Goesmann, A., McHardy, A. C. & 8 other authors (2003).GenDB – an open source genome annotation system for prokaryote

genomes. Nucleic Acids Res 31, 2187–2195.

Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory.

Mirwaldt, C., Korndorfer, I. & Huber, R. (1995). The crystal structure

of dihydrodipicolinate synthase from Escherichia coli at 2?5 A

resolution. J Mol Biol 246, 227–239.

Moss, J. E., Cardozo, T. J., Zychlinsky, A. & Groisman, E. A. (1999).The selC-associated SHI-2 pathogenicity island of Shigella flexneri.

Mol Microbiol 33, 74–83.

Nassif, X., Mazert, M. C., Mounier, J. & Sansonetti, P. J. (1987).Evaluation with an iuc : : Tn10 mutant of the role of aerobactin

production in the virulence of Shigella flexneri. Infect Immun 55,

1963–1969.

Neidhardt, F. C., Bloch, P. L. & Smith, D. F. (1974). Culture medium

for enterobacteria. J Bacteriol 119, 736–747.

Nomura, N., Masai, H., Inuzuka, M. & 7 other authors (1991).

Identification of eleven single-strand initiation sequences (ssi) for

priming of DNA replication in the F, R6K, R100 and ColE2

plasmids. Gene 108, 15–22.

Ohtsubo, H., Ryder, T. B., Maeda, Y., Armstrong, K. & Ohtsubo, E.

(1986). DNA replication of the resistance plasmid R100 and its

control. Adv Biophys 21, 115–133.

Pansegrau, W., Lanka, E., Barth, P. T. & 7 other authors

(1994). Complete nucleotide sequence of Birmingham IncP alpha

plasmids. Compilation and comparative analysis. J Mol Biol 239,

623–663.

Partridge, S. R. & Hall, R. M. (2003). In34, a complex In5 family class

1 integron containing orf513 and dfrA10. Antimicrob Agents

Chemother 47, 342–349.

Perez-Casal, J. F., Gammie, A. E. & Crosa, J. H. (1989). Nucleotide

sequence analysis and expression of the minimum REPI replication

region and incompatibility determinants of pColV-K30. J Bacteriol

171, 2195–2201.

Pullinger, G. D. & Lax, A. J. (1992). A Salmonella dublin virulence

plasmid locus that affects bacterial growth under nutrient-limited

conditions. Mol Microbiol 6, 1631–1643.

Saadi, S., Maas, W. K., Hill, D. F. & Bergquist, P. L. (1987).Nucleotide sequence analysis of RepFIC, a basic replicon present in

IncFI plasmids P307 and F, and its relation to the RepA replicon of

IncFII plasmids. J Bacteriol 169, 1836–1846.




IP: 54.157.13.203

On: Thu, 11 Feb 2016 04:04:35

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:a Labolatory Manual. Cold Spring Harbor, NY: Cold Spring HarborLaboratory.

Sansonetti, P. J., Kopecko, D. J. & Formal, S. B. (1982). Involvementof a plasmid in the invasive ability of Shigella flexneri. Infect Immun35, 852–860.

Scherzinger, E., Haring, V., Lurz, R. & Otto, S. (1991). PlasmidRSF1010 DNA replication in vitro promoted by purified RSF1010RepA, RepB and RepC proteins. Nucleic Acids Res 19, 1203–1211.

Schluter, A., Heuer, H., Szczepanowski, R., Forney, L. J., Thomas,C. M., Puhler, A. & Top, E. M. (2003). The 64 508 bp IncP-1bantibiotic multiresistance plasmid pB10 isolated from a waste-watertreatment plant provides evidence for recombination betweenmembers of different branches of the IncP-1b group. Microbiology149, 3139–3153.

Schmidt, H., Henkel, B. & Karch, H. (1997). A gene cluster closelyrelated to type II secretion pathway operons of gram-negativebacteria is located on the large plasmid of enterohemorrhagicEscherichia coli O157 strains. FEMS Microbiol Lett 148, 265–272.

Simon, R., Priefer, U. & Puhler, A. (1983). A broad host rangemobilization system for in vivo genetic-engineering-transposonmutagenesis in gram-negative bacteria. Bio/Technology 1, 784–791.

Smalla, K. & Sobecky, P. A. (2002). The prevalence and diversity ofmobile genetic elements in bacterial communities of differentenvironmental habitats: insights gained from different methodolo-gical approaches. FEMS Microbiol Ecol 42, 165–175.

Sorsa, L. J., Dufke, S. & Schubert, S. (2004). Identification of novelvirulence-associated loci in uropathogenic Escherichia coli by suppres-sion subtractive hybridization. FEMS Microbiol Lett 230, 203–208.

Spiers, A. J., Bhana, N. & Bergquist, P. L. (1993). Regulatory inter-actions between RepA, an essential replication protein, and the DNArepeats of RepFIB from plasmid P307. J Bacteriol 175, 4016–4024.

Staden, R. (1996). The Staden sequence analysis package. MolBiotechnol 5, 233–241.

Stearman, R., Yuan, D. S., Yamaguchi-Iwai, Y., Klausner, R. D. &Dancis, A. (1996). A permease-oxidase complex involved in high-affinity iron uptake in yeast. Science 271, 1552–1557.

Szczepanowski, R., Krahn, I., Linke, B., Goesmann, A., Puhler, A. &Schluter, A. (2004). Antibiotic multiresistance plasmid pRSB101isolated from a wastewater treatment plant is related to plasmidsresiding in phytopathogenic bacteria and carries eight differentresistance determinants including a multidrug transport system.Microbiology 150, 3613–3630.

Takase, H., Nitanai, H., Hoshino, K. & Otani, T. (2000). Impact ofsiderophore production on Pseudomonas aeruginosa infections inimmunosuppressed mice. Infect Immun 68, 1834–1839.

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, 22–28.

Tauch, A., Schluter, A., Bischoff, N., Goesmann, A., Meyer, F. &Puhler, A. (2003). The 79,370-bp conjugative plasmid pB4 consists

of an IncP-1beta backbone loaded with a chromate resistance

transposon, the strA-strB streptomycin resistance gene pair, the

oxacillinase gene bla(NPS-1), and a tripartite antibiotic efflux system

of the resistance-nodulation-division family. Mol Genet Genomics

268, 570–584.

Tennstedt, T., Szczepanowski, R., Braun, S., Puhler, A. &Schluter, A. (2003). Occurrence of integron-associated resistance

gene cassettes located on antibiotic resistance plasmids isolated from

a wastewater treatment plant. FEMS Microbiol Ecol 45, 239–252.

Tennstedt, T., Szczepanowski, R., Krahn, I., Puhler, A. &Schluter, A. (2005). Sequence of the 68,869 bp IncP-1a plasmid

pTB11 from a waste-water treatment plant reveals a highly conserved

backbone, a Tn402-like integron and other transposable elements.

Plasmid doi:10.1016/j.plasmid.2004.09.004 <http://dx.doi.org/

10.1016/j.plasmid.2004.09.004>

Uga, H., Matsunaga, F. & Wada, C. (1999). Regulation of DNA

replication by iterons: an interaction between the ori2 and incC

regions mediated by RepE-bound iterons inhibits DNA replication of

mini-F plasmid in Escherichia coli. EMBO J 18, 3856–3867.

Venkatesan, M. M., Goldberg, M. B., Rose, D. J., Grotbeck, E. J.,Burland, V. & Blattner, F. R. (2001). Complete DNA sequence and

analysis of the large virulence plasmid of Shigella flexneri. Infect

Immun 69, 3271–3285.

Wain, J., Diem Nga, L. T., Kidgell, C. & 9 other authors (2003).Molecular analysis of IncHI1 antimicrobial resistance plasmids from

Salmonella serovar Typhi strains associated with typhoid fever.

Antimicrob Agents Chemother 47, 2732–2739.

Wei, J., Goldberg, M. B., Burland, V. & 14 other authors (2003).Complete genome sequence and comparative genomics of Shigella

flexneri serotype 2a strain 2457T. Infect Immun 71, 2775–2786.

Womble, D. D. & Rownd, R. H. (1988). Genetic and physical map of

plasmid NR1: comparison with other IncFII antibiotic resistance

plasmids. Microbiol Rev 52, 433–451.

Womble, D. D., Sampathkumar, P., Easton, A. M., Luckow, V. A. &Rownd, R. H. (1985). Transcription of the replication control region

of the IncFII R-plasmid NR1 in vitro and in vivo. J Mol Biol 181,

395–410.

Zhang, L., Foxman, B., Manning, S. D., Tallman, P. & Marrs, C. F.(2000). Molecular epidemiologic approaches to urinary tract

infection gene discovery in uropathogenic Escherichia coli. Infect

Immun 68, 2009–2015.

Zzaman, S., Abhyankar, M. M. & Bastia, D. (2004). Reconstitution of

F factor DNA replication in vitro with purified proteins. J Biol Chem

279, 17404–17410.



The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different...

Documents

Transcript of The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different...