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The genome sequence of the incompatibility group Iγ plasmid R621a: Evolution of IncI plasmids
Plasmid 66 (2011) 112–121
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The genome sequence of the incompatibility group Ic plasmid R621a: Evolution of IncI plasmids Hiroyuki Takahashi, Ming Shao, Nobuhisa Furuya, Teruya Komano ⇑ Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Received 5 October 2010 Accepted 27 June 2011 Available online 8 July 2011 Communicated by Dr. W. Klimke Keywords: Plasmid R621a Genome IncIc plasmid Bacterial conjugation Plasmid evolution
a b s t r a c t We present the complete genome sequence of the tetracycline resistance plasmid R621a isolated from Salmonella typhimurium, which belongs to the incompatibility group Ic. In the 93,185 bp circular double-stranded R621a genome, 96 complete ORFs are predicted. In addition, one and six different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of four regions: replication, leading, transfer, and miscellaneous regions. The R621a genome is similar to those of IncI1 plasmids such as R64 and ColIb-P9 and particularly to those of pEK204 and pEC_Bactec. Three major differences including inc, parAB, and excA regions were noted between R621a and prototype IncI1 plasmids. Seven nucleotide replacements and one nucleotide deletion in the putative Inc RNA sequence are found between R621a and IncI1 plasmids irrespective of close similarity in the other parts of the rep system. The sequences of R621a parAB and excA genes are significantly different from those of R64 and ColIb-P9, while those of R621a parAB and excA genes exhibit close similarity to those of pEK204 and pEC_Bactec, respectively. The R621a genome is suggested to be formed by acquiring parAB and excA genes from pEK204 and pEC_Bactec genomes, respectively, and then novel inc function by the mutations. The insertions in the R621a, pEK204, and pEC_Bactec genomes are flanked by direct repeats, suggesting that insertions accompanied by long target duplications have also played an important role in the evolution of IncI plasmids. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Bacterial plasmids are extrachromosomal genetic elements, usually consisting of double-stranded circular DNA. They confer various phenotypes such as conjugation, resistance to antibiotics and metals, and production of bacteriocins. Plasmids are usually classified into different groups according to their incompatibility properties. If two plasmids are stably maintained within a single cell, they are regarded as belonging to distinct incompatibility groups. The replication mechanisms of plasmids belonging to the same incompatibility group interfere with each other and do not allow their stable maintenance within a
⇑ Corresponding author. Fax: +81 42 677 2559. E-mail address: [email protected] (T. Komano). 0147-619X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2011.06.004
single cell. Incompatibility is also expressed in the plasmid partitioning systems (Dam and Gerdes, 1994). We have recently reported the genome sequence of the incompatibility group I1 plasmid R64 (Sampei et al., 2010). The 121-kb R64 genome is highly organized into five major regions: replication, drug resistance, stability, leading, and transfer regions. Replication, leading, and transfer regions are highly conserved among IncI1 plasmids including R64, ColIb-P9, pSL476_91, pCVM29188_101, and pSE11-1. It was found that genetic recombination including the site-specific rfsF-ResD system have played an important role in diversity of IncI1 plasmids. The genome sequences of the other IncI1 plasmid pEK204 and pEC_Bactec have been also reported (Woodford et al., 2009; Smet et al., 2010). Replication, leading, and transfer regions of pEK204 and pEC_Bactec are highly similar to those of the other IncI1 plasmids, although they do not carry the
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ars1/ars2 sequence or colicin Ib determinants which are common in IncI1 plasmids. R621a is a tetracycline resistance conjugative plasmid that was originally isolated from Salmonella typhimurium and determines I pili (Hedges and Datta, 1973). Since R621a was transferred into and stably maintained in Escherichia coli cells harboring IncI1 plasmids, it was classified as the incompatibility group Ic. Here we describe the complete genome sequence of plasmid R621a.
Number AP011954. Accession Number and nucleotide number of plasmids used in this paper for comparison are: R64, AP005147, 120,826 bp; ColIb-P9, AB021078, 93,399 bp; pEK204, EU935740, 93,732 bp; and pEC_Bactec, GU371927, 92,970 bp.
2. Materials and methods
The genome of R621a consists of a 93,185 bp circular double-stranded DNA. The G + C content of the genome was estimated to be 48.8%. This value is close to that of the original host, S. typhimurium (52.2%), and the laboratory host, E. coli K-12 (50.8%). Gene organization of R621a is shown in Fig. 1 and comparison to those of IncI1 plasmids is shown in Fig. 2. The R621a genome is similar to those of IncI1 plasmids such as R64 and ColIb-P9 and particularly to those of pEK204 and pEC_Bactec. Major parts (79–88%) of R621a DNA sequence (including replication, leading, and transfer regions) exhibit 96–98% identity to the sequences of R64, ColIb-P9, pEK204, and pEC_Bactec. R621a carries Tn10-like transposon and insertion sequence IS2. At least 96 complete open reading frames (ORFs) were predicted within the R621a genome (Fig. 1). The sog gene is predicted to produce two kinds of proteins, SogL and SogS, by translational reinitiation as in R64 (Narahara et al., 1997). Six additional PilV proteins with different C-terminal segments could be created by shufflon multiple inversions as in R64 (Komano et al., 1987b). Thus, 103 different proteins, 82 of which exhibit >95% amino acid sequence identities to the corresponding proteins of R64, may be produced from R621a (Table 1). Two homologous genes in R64, yfeA and ygbA, were converted into pseudogenes in R621a. It is noteworthy that both genes are located on the repeat sequences R3 described below. The two genes, yfbA and ygaA, were interrupted by the insertion of a parAB segment and Tn10-like, respectively. Several kinds of repetitive sequence were found in the R621a genome in addition to two IS10 sequences of Tn10-like (Fig 1). Repeats R3, R4, and R5 were also found in R64 (Sampei et al., 2010). R6, R7, R8, and R9 may be formed during the insertions of the soj, parAB, ccgAII, and finQ segments, respectively, into the R621a genome (Fig. 2). The R621a genome was tentatively separated into four functional regions including the replication (coordinates 0–2.4 kb), miscellaneous (coordinates 2.4–7.1 kb), leading (coordinates 7.1–37.2 kb), and transfer (coordinates 37.3–93.2 kb) regions (Fig. 1).
2.1. Bacterial strains, plasmids and growth media Escherichia coli K-12 strains JM83 D(lac-proAB) rpsL thi ara u80 dlacZDM15 (Sambrook and Russell, 2001) and TN102 Nalr (Komano et al., 1990) were used. Plasmid R621a was used throughout this study. Mini-R64 plasmid pKK607 carried the replication and transfer regions of R64 together with a Kmr fragment (Komano et al., 1990). Plasmid vectors pCL1920 (Lerner and Inouye, 1990), pUC18, and pUC19 (Sambrook and Russell, 2001) were used for cloning and sequencing. Luria–Bertani (LB) medium was prepared as described previously (Sambrook and Russell, 2001). The solid medium contained 1.5% agar. Antibiotics were added to the liquid and solid media at the following concentrations: ampicillin, 100 lg/ml; kanamycin, 50 lg/ml; spectinomycin, 50 lg/ml; tetracycline, 12.5 lg/ml; and nalidixic acid, 20 lg/ml. 2.2. DNA manipulation and sequencing Preparation of plasmid DNA, transformation, construction of plasmids, and other methods of DNA manipulation were described previously (Sambrook and Russell, 2001). The nucleotide sequence was determined by the dideoxy chain-termination method (Sambrook and Russell, 2001). To clone R621a excA, the 2.47-kb BglII-EcoRI segment was inserted into the multicloning site of pCL1920, yielding pIG318. To clone R64 excAB gene, the 1.25-kb NsiI segment was inserted into pCL1920, yielding pKK318. 2.3. Conjugative transfer To avoid the effects of shufflon inversions on transfer frequencies in liquid media, surface mating was conducted as previously described (Komano et al., 1990). E. coli JM83 donor cells harboring R621a or pKK607 were grown to the log phase and mixed with an overnight culture of E. coli TN102 recipient cells. The mixture was filtered through a nitrocellulose membrane. The membrane was incubated on an LB agar plate for 90 min at 37 °C. Transconjugants were resuspended in saline and plated at various dilutions onto selective media. 2.4. Nucleotide sequence accession number The complete nucleotide sequence of R621a has been deposited in GenBank/EMBL/DDBJ under Accession
3. Results and discussion 3.1. General properties of genome sequence
3.2. Replication region The replication (rep) region of R621a is located at the coordinates 0–2.4 kb of the genome (Fig. 1). The DNA sequence of the R621a replication region is more than 94% identical to those of IncI1 plasmids such as R64, pEK204, pEC_Bactec, and ColIb-P9, in which replication function has been extensively studied (Asano and Mizobuchi, 1998; Asano et al., 1998; Hama et al., 1990). This region
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Fig. 1. Map of the entire R621a plasmid. Circular R621a map was linearized at a Sau3AI site, between the replication and transfer regions, from which bases are numbered. Genes identified on R621a are illustrated by bars together with gene names. Genes transcribed left-to-right and right-to-left are described above and below the thin lines, respectively. The functions of genes are classified into nine groups, and genes in the same groups are indicated by the same colors, which are shown at the bottom of the figure. Locations of IS2, Tn10-like, repeat sequences and shufflon, and oriV, ter, and oriT sites are also indicated. The names of four functional regions are presented at the top together with the names of four major parts of the transfer region. This figure represents one specific clone of many isomers produced by shufflon multiple inversions.
Fig. 2. Comparison of gene organization among R64, ColIb-P9, pEK204, pEC_Bactec and R621a. Conserved genes among each of five plasmids are indicated by the same colors, which are shown at the bottom of the figure. Insertions of IS elements, transposons, and gene segments flanked by direct repeats are described above the map. Locations of oriV, rfsF, and oriT sites are indicated by upward and downward arrowheads. Names of representative genes are indicated.
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H. Takahashi et al. / Plasmid 66 (2011) 112–121 Table 1 List of known or suggested genes in plasmid R621a. Lengtha
No.
Gene
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
inc repY repZ yafA yafB yagA soj impB impA impC yfaA yfaB yfbA parA parB yfbB yfcA yfcB yfdA yfeA yfeB yfeC yffA yffB ssb yfhA psiB psiA ygaA tnpR tetD tetC tetA tetR ybdA ybeA ybeB
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
ybfA tnpL ygbA ardA ygcA ygdA ccgAII ygdB ygeA ygfA ygfB yggA nikA nikB trbC trbB trbA finQ pndC pndA excA traY
166 144 88 129 307 83 111 282 111 110 899 763 356 402 361 67 50 204 745
59 60 61 62
traX traW traV traU
194 400 204 1014
29 343 169 200 448 208 423 145 82 89 308 326 140 227 73 144 256 141 140 63 104 175 652 144 239 402 138 197 401 207 228 162 106 401 402
Conserved domainb
pfam02387 cd00236 PHA02518 PRK03609 PRK10276 pfam06183 pfam06924 (intr) pfam06406 pfam10784 PRK13699 pfam07128 (pseudo) pfam03230
PRK13732 COG1475 PRK13701 PRK13704 (intr) pfam01609 PRK10219 pfam00440 pfam07690 PRK13756 cd00090 PRK06349 COG2329 pfam03616 pfam01609 (pseudo) pfam07275
pfam04754
pfam03432 cd03020
pfam01848
Function or product
Homologc
Inc RNA Regulator of repZ expression Replication initiation protein Hypothetical protein FinO bacterial conjugation repressor Hypothetical protein Type II bacterial partitioning protein DNA polymerase V subunit ImpB DNA polymerase V subunit ImpA DinI-like family protein Hypothetical protein DUF1281 family protein
R64 RepY (100%) R64 RepZ (96%) ColIb-P9 YafA (62%) ColIb-P9 YafB (92%) ColIb-P9 YagA (98%) R64 YefA (24%), pO113 LH0102 (94%) R64 ImpB (98%) R64 ImpA (100%) R64 ImpC (100%) R64 YfaA (98%) R64 YfaB (97%)
Type I plasmid partitioning protein Type I plasmid partitioning protein Putative methylase Hypothetical protein DUF1380 family protein Hypothetical protein
R64 R64 R64 R64 R64 R64
ParA (66%), pEK204 ParM (99%) ParB (38%), pEK204 StbB (100%) YfbB (97%) YfcA (79%) YfcB (92%) YfdA (95%)
Antirestriction protein Hypothetical protein Hypothetical protein Hypothetical protein Single-strand DNA-binding protein Type II bacterial partitioning protein Plasmid SOS inhibition protein B Plasmid SOS inhibition protein A
R64 R64 R64 R64 R64 R64 R64 R64
YfeB (94%) YfeC (89%) YffA (97%) YffB (93%) Ssb (97%) YfhA (95%) PsiB (98%) PsiA (99%)
Transposase_11 Regulator of tet operon (HTH_AraC) Regulator of tet operon (TetR_N) Major facilitator superfamily (MFS_1) Tetracycline repressor protein TetR Transcriptional repressor (HTH_ARSR) Homoserine dehydrogenase Biosynthesis of extracellular polysaccharides Sodium/glutamate symporter (GltS) Transposase_11
R64 R64 R64 R64 R64 R64 R64 R64
TnpR (100%) TetD (100%) TetC (100%) TetA (100%) TetR (100%) YbdA (100%) YbeA (100%) YbeB (100%)
Antirestriction protein Hypothetical protein Hypothetical protein Hypothetical protein Transposase_31 Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein NikA oriT-specific DNA binding protein NikB relaxase TrbC transfer protein Protein disulfide isomerase TrbA transfer protein Fertility inhibition protein Post-segregation killing protein Post-segregation killing protein Entry exclusion protein TraY integral membrane protein
R64 ArdA (97%) R64 YgcA (99%) R64 YgdA (98%) pEK204 CcgAII (100%), pSC138 CcgAII (100%) R64 YgdB (78%) R64 YgeA (97%) R64 YgfA (96%) R64 YgfB (98%) R64 YggA (99%) R64 NikA (100%) R64 NikB (99%) R64 TrbC (99%), Lpn IcmO/DotL (26%) R64 TrbB (99%) R64 TrbA (99%), Lpn IcmP/DotM (27%) pEK204 FinQ (99%), R802a FinQ (83%) R64 PndC (96%) R64 PndA (100%) R64 ExcA (56%), pEC_Bactec ExcA (100%) R64 TraY (87%), pEC_Bactec TraY (100%), Lpn DotA (23%) R64 TraX (96%) R64 TraW (99%) R64 TraV (99%) R64 TraU (99%), Lpn IcmB/DotO (28%)
TraX transfer protein TraW lipoprotein TraV transfer protein TraU nucleotide-binding protein
R64 YbfA (100%) R64 TnpL (99%)
(continued on next page)
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Table 1 (continued)
a b c
No.
Gene
Lengtha
Conserved domainb
Function or product
Homologc
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
insD insA traT traS traR traQ traP traO traN traM traL sogL sogS nuc traK traJ traI traH traG traF traE rci OrfB OrfB0 OrfD0 OrfC OrfC0 OrfA0 pilVA pilU pilT pilS pilR pilQ pilP pilO pilN pilM pilL pilK pilJ pilI traC traB traA
301 121 266 62 134 175 234 429 327 230 115 1255 844 183 96 382 272 152 194 400 274 384 81 83 95 83 72 69 474 218 186 204 365 517 150 431 560 145 355 197 136 84 227 177 95
PRK09409 PRK09413
IS2 transposase InsD IS2 repressor TnpA TraT transfer protein TraS transfer protein TraR transfer protein TraQ transfer protein TraP transfer protein TraO transfer protein TraN transfer protein (DUF3625) IcmL superfamily TraL transfer protein SogL DNA primase SogS transfer protein EDTA-resistant nuclease (NucT) TraK transfer protein TraJ transfer ATPase TraI lipoprotein TraH lipoprotein LPS heptose-phosphate phosphatase Hypothetical protein Hypothetical protein Shufflon-specific DNA recombinase PilVB C-terminal segment PilVB0 C-terminal segment PilVD0 C-terminal segment PilVC C-terminal segment PilVC0 C-terminal segment PilVA0 C-terminal segment PilVA type IV preadhesin PilU prepilin peptidase (peptidase_A24) PilT lytic transglycosidase PilS type IV prepilin PilR integral membrane protein PilQ type IV pilus ATPase PilP type IV pilus biogenesis protein PilO type IV pilus biogenesis protein PilN type IV pilus secretin PilM type IV pilus biogenesis protein PilL lipoprotein PilK type IV pilus biogenesis protein PilJ type IV pilus biogenesis protein PilI type IV pilus biogenesis protein DUF2913 family protein Transcription termination factor NusG Hypothetical protein
IS2 InsD (100%) IS2 InsA (100%) R64 TraT (99%) R64 TraS (100%) R64 TraR (95%) R64 TraQ (99%), Lpn IcmD/DotP (21%) R64 TraP (99%), Lpn IcmG/DotF (27%) R64 TraO (99%) R64 TraN (99%), Lpn IcmK/DotH (28%) R64 TraM (99%), Lpn IcmL/DotI (30%) R64 TraL (99%) R64 SogL (98%) R64 SogS (99%) R64 Nuc (98%) R64 TraK (99%), Lpn IcmT (34%) R64 TraJ (100%), Lpn DotB (33%) R64 TraI (99%), Lpn DotC (29%) R64 TraH (100%), Lpn DotD (31%) R64 TraG (100%) R64 TraF (99%) R64 TraE (99%) R64 Rci (99%) R64 OrfB (100%) R64 OrfB0 (100%) R64 OrfD0 (100%) R64 OrfC (100%) R64 OrfC0 (100%) R64 OrfA0 (100%) R64 PilVA (100%) R64 PilU (100%), BfpP (24%) R64 PilT (100%), BfpH (32%) R64 PilS (97%), BfpA (24%) R64 PilR (99%), BfpE (32%) R64 PilQ (98%), BfpD (31%) R64 PilP (96%) R64 PilO (96%) R64 PilN (100%), BfpB (29%) R64 PilM (100%) R64 PilL (99%) R64 PilK (89%), ColIb-P9 PilK (95%) R64 PilJ (77%), ColIb-P9 PilJ (57%) R64 PilI (90%), ColIb-P9 PilI (99%) R64 TraC (99%) R64 TraB (99%) R64 TraA (99%)
pfam12293 pfam11393 COG4643 PRK13878 PRK13912 TIGR02525
PRK15416
cd00796 (frag) (frag) (frag) (frag) (frag) (frag) pfam04917 pfam01478 pfam01464 pfam08805 COG1459 COG2804 TIGR03021 pfam06864 TIGR02520 pfam07419
pfam10623 pfam11140 pfam02357
Length in amino acid residues. NCBI conserved domain. intr, interrupted by the insertion of transposon or specific DNA segments; pseudo, pseudo genes; frag, gene fragment. Selected homologs: R64 and ColIb-P9 homologs and Legionella pneumophila (Lpn) icm/dot and enteropathogenic E. coli bfp homologs are listed. Amino
contains three genes, inc, repY and repZ. The repZ gene encodes replication initiation protein that may interact with oriV region located downstream of repZ to initiate DNA replication. DNA sequence of 417-bp regulatory region including inc is 99% identical to the previous report and expression of inc function in this region has been demonstrated (Nikoletti et al., 1988). The oriV and ter sequences of R621a are 95% and 100% identical to those of R64, respectively. repYZ mRNA is thought to form a unique structure consisting of three stable stem-loop structures, which is inactive for repZ expression. Translation of repY is required for the activation of repZ translation. Inc RNA, antisense RNA for repYZ mRNA, inactivates repY and repZ translation. Inc RNA sequences of R621a and IncI1 plasmids are significantly different from each other (Fig. 3a),
suggesting that this allows stable maintenance of both plasmids within a single cell. 3.3. Leading region R621a leading region from impB to yggA at the coordinates 7.1–37.2 kb is very similar to those of R64 and ColIb-P9 (Figs. 1 and 2) except for a few insertions in the R621a sequence. R621a carries ygaA followed by psiA as does R64, while ColIb-P9 carries ydcA at the corresponding site. The ardA, psiAB, and ssb genes of R621a leading region may play important roles in conjugation. The parAB genes, unlike those of R64 and ColIb-P9, are inserted into the yfbA gene in the R621a leading region (Fig. 1). The ccgAII gene is inserted into the intergenic
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Fig. 3. (a) Alignment of the nucleotide sequences of Inc RNA of R621a, R64, pMU720 (GenBank Accession No. M28718), R387 (M93063), pIE545 (M93064), pO113 (AY258503), pSERB1 (AY686591), and pECOED (CU928147). Conserved nucleotides are printed in boldface. Gaps, marked by dashes, are introduced to reveal maximal similarity among the sequences. Stem sequences are indicated by inverted arrows, and the highly conserved CGCCAA sequence is indicated by dots. (b) Alignment of the amino acid sequences of R621a ParA, pCoo StbA (GenBank Accession No. YP_424887), R64 ParA (NP_863404), pO113 StbA (YP_308777), R1 ParM (P11904), and pB171 StbA (NP_053130). Amino acid sequence of pEK204 ParM is 99% identical to that of R621a. (c) Alignment of the amino acid sequences of R621a ParB, pCoo StbB (YP_424888), R64 ParB (NP_863405), pO113 StbB (YP_308776), R1 ParR (P11906), and pB171 StbB (NP_053129). Amino acid sequence of pEK204 StbB is 100% identical to that of R621a. (d) Alignment of the amino acid sequences of ExcA homologs in R621a, R64 (NP_863442), ColIb-P9 (NP_52507), pO113 (YP_308714), and pECOED (CAQ87319). Amino acid sequences of pEC_Bactec and pEK204 ExcA are 100% and 99% identical to those of R621a and R64, respectively.
region between ygdA and ygdB. Insertion of ccgAII is also found in pEK204 and pEC_Bactec, but insertion of parAB is found only in pEK204 (Fig. 2). Tn10-like is inserted into ygaA gene in the R621a leading region. 3.4. Transfer region Gene organization of the R621a transfer region at the coordinates 37.3–93.2 kb is similar to those of R64, ColIbP9, pEK204, and pEC_Bactec except IS2 insertion (Figs. 1 and 2) (Komano et al., 2000; Sampei et al., 2010). The R621a transfer region exhibits a highly organized structure consisting of four major parts as in R64 (Fig. 1); (i) traABC regulatory genes, (ii) pil genes for type IV pilus biogenesis,
(iii) tra/trb genes for conjugation in general, and (iv) oriT operon for conjugative DNA processing. The R621a traABC genes are located immediately upstream of the replication region in the circular genome and may be involved in transfer gene expression as in R64 (Fig. 1). R621a and pEC_Bactec do not carry any homolog corresponding to the R64 traD gene or the ColIb-P9 and pEK204 trcD gene. The 14 pil genes of R621a may be responsible for the formation of a type IV pilus required only for conjugation in liquid media (Kim and Komano, 1997). The R621a shufflon with four invertible segments, A, B, C, and D, is located at the 30 region of the pilV gene (Gyohda et al., 2004; Komano, 1999). Multiple inversions of the four
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DNA segments in R621a shufflon were previously reported (Komano et al., 1987a). Shufflon inversions mediated by Rci shufflon-specific recombinase select one of seven PilV adhesins, in which the N-terminal region is constant while the C-terminal region is variable (Komano et al., 1987b). R621a, R64, and pEC_Bactec shufflon consists of four segments, while ColIb-P9 shufflon lacks segment D and pEK204 shufflon lacks segment C. PilV adhesins, located at the tip of the PilS polymer, determine the recipient specificity of liquid matings by recognition of the lipopolysaccharide of the recipient cells (Ishiwa and Komano, 2004; Shimoda et al., 2008). Three R621a genes, traEFG, might be dispensable for conjugation as in R64 (Komano et al., 1990). Twenty genes, traH to traY, may form another operon (Fig. 1 and Table 1). Amino acid sequence similarity of TraY between R621a and R64 is limited (87%). R621a carries insertion of IS2 between traT and traU, but this insertion does not affect its transfer ability (Table 2). The C-terminal 163 amino acids of the R621a ExcA exhibit 56% sequence identity to the corresponding region of R64 ExcA, while R621a and pEC_Bactec ExcA is identical (Fig. 3d). R621a ExcA is 204-amino-acid long, but in R64, ColIb-P9, and pEK204 it is 220-amino-acid long (Furuya and Komano, 1994). Although R64 excA gene produced ExcA and ExcB proteins by translational reinitiation, R621a excA gene lacks the sequence for the translational reinitiation. R621a, pEK204, and pEC_Bactec carry a finQ gene between pndCA and trbA, while R64 and ColIb-P9 do not. FinQ protein exhibits high sequence similarity to that of IncI plasmid R820a except for 16-amino-acid insertion (Ham and Skurray, 1989). pEC_Bactec carries ISCro1 insertion between finQ and pndCA. Processing of R621a DNA during conjugation may be conducted by the oriT operon (Fig. 1 and Table 1). The R621a oriT operon consists of the oriT sequence and the nikAB genes (Furuya et al., 1991). The 92-bp R621a oriT sequence, which is identical to that of R64, contains oriT nick site, repeat A, and repeat B sequences and 8-bp inverted repeats. R64 NikA, a ribbon–helix–helix DNAbinding protein (Yoshida et al., 2008), binds to the repeat A sequence (Furuya and Komano, 1995). NikB, a relaxase protein, binds to NikA-oriT to form a relaxation complex.
Table 2 Entry exclusion specificity of R621a and R64. Donor plasmid
Transfer frequency (%)a
Exclusion indexb
pCL1920
pIG318
pKK318
pIG318
PKK318
R621a pKK607
19 15
0.058 8.9
11 0.0032
330 1.7
1.7 4700
a Escherichia coli JM83 donor cells harboring R621a or mini-R64 plasmid pKK607 were grown to a log phase and then mixed with an overnight culture of E. coli TN102 recipient cells harboring pCL1920, pIG318 (R621a excA+), or pKK318 (R64 excA+). Transfer frequency of filter matings is expressed as a ratio (percentage) of the number of transconjugants to that of donor cells. The transfer frequency shown represents the average of three independent experiments. b The exclusion index is expressed as a ratio of transfer frequency of donor plasmid into the recipient cells harboring pCL1920 to that of donor plasmid into recipient cells harboring pIG318 or pKK318.
3.5. Miscellaneous region R621a lacks stability region which are conserved in IncI1 plasmids (Fig. 2). At the same time, R621a does not carry either arsenic resistance or colicin genes, which are characteristic of R64 or ColIb-P9, respectively. The remaining region (coordinates 2.4–7.1 kb) of the R621a genome was tentatively assigned as the miscellaneous region. It is practically identical to the corresponding regions of pEK204 and pEC_Bactec except for the insertion of a Tn3like transposon and IS1294-like insertion sequence or Tn3-like transposon, respectively (Fig. 2). Three genes, yafA, yafB, and yagA are similar to those of ColIb-P9. The amino acid sequence of the R621a soj product exhibits some similarity to that of R64 yefA product. R621a Soj protein may be involved in plasmid partition with YfhA (SpoOJ) protein encoded in the leading region, although putative parC locus for this system is not known. pEC_Bactec carries only soj-yfhA system as a putative partition system. R621a, pEK204, and pEC_Bactec do not carry R64/ColIb-P9 type parAB genes. Instead, a novel type of parAB genes is inserted into their leading region of R621a and pEK204 (Fig. 2). They also lack the resD and rfsF sitespecific recombination system, required for the resolution of a dimer DNA molecule. Although R621a lacks the resD and rfsF system, it is stably maintained in E. coli cells, suggesting a possibility of an unknown resolution system in R621a. 3.6. Three factors determining plasmid identities Incompatibilities of replication and partition systems and specificity of entry exclusion systems are thought to determine identities of conjugative plasmids. Nucleotide sequences of Inc RNA, and amino acid sequences of ParAB and ExcA proteins were compared between R621a and the related plasmids (Fig. 3). The incompatibility of replication system is based on Inc RNA that controls plasmid copy number within the cell. Nucleotide sequences of putative Inc RNA of R621a and the related plasmids are aligned in Fig. 3a. Inc RNA of IncI related plasmids was shown to fold into a single large stem-loop structure (Asano et al., 1998; Praszkier et al., 1991). Stems consisting of approximately 21 bp (inverted arrows in Fig. 3a), are relatively conserved. Most nucleotide replacements in stems still keep base complementarity. Loops consist of 20–22 nucleotides and base pairing within the loop might further stabilize the Inc RNA structure. Nucleotide sequence in the loop is relatively variable and might determine the incompatibility. However, it should be noted that the CGCCAA sequence (dots in Fig. 3a), complementary to the UUGGCG motif in the structure I within repY region of RepZ mRNA (Asano et al., 1998), is completely conserved among all the Inc RNA sequences in Fig. 3a. There are seven nucleotide replacements and one nucleotide deletion in the putative Inc RNA sequences between R621a and IncI1 plasmids (Fig. 3a). By contrast, the DNA sequences except for the inc region are highly conserved between R621a and IncI1 plasmids. Approximately 210-bp DNA sequence of R621a immediately downstream of the inc gene, carrying the
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Fig. 4. Duplication of the target sequences during insertion of (a) parAB, (b) ccgAII, (c) finQ, and (d) soj genes. The parAB, ccgAII, and finQ segments are thought to be inserted into the indicated R64 sequences, generating the indicated target duplication. Boldface letters represent nucleotides different from those of R64. (pEK204) and (pEC_Bactec) indicate exactly the same target duplication as that of R621a was found in pEK204 and pEC_Bactec, respectively. (pEK204) and (pEC_Bactec) indicate a target duplication with a few nucleotide replacements was found in pEK204 and pEC_Bactec, respectively. In the case of soj gene, the origin of target sequence is unknown. (e) Insertion of trcD segment into the indicated R64 sequence accompanied by the indicated target duplication (repeat G) and traD deletion formed ColIb-P9 and pEK204 sequences. Homologous recombination between the italicized sequences within the repeat G sequences results in deletion of trcD, to generate the R621a and pEC_Bactec sequence.
entire repY gene and repZ 50 region and playing important roles in replication control, is identical to those of R64, ColIb-P9, pEK204, and pEC_Bactec. In the ParMR/parC partition system of plasmid R1, a cis site, parC exhibits incompatibility (Dam and Gerdes, 1994; Gerdes et al., 2004). Ribbon-helix-helix DNA-binding protein, ParR (ParB in R621a) binds to parC to form a partition complex (Møller-Jensen et al., 2007). The partition complex is recognized by an actin-like partition protein ParM (ParA in R621a), which forms filaments required for the bidirectional movement of plasmid DNA. Partition proteins of R621a and pEK204 are significantly different from those of R64 and ColIb-P9. R621a ParA exhibits 99%, 52%, and 65% amino acid sequence identities to pEK204 and R1 ParM and R64 ParA, respectively (Fig. 3b). R621a ParB
exhibits 100%, 38%, and 36% sequence identity to pEK204 StbB, R1 ParR, and R64 ParB, respectively (Fig. 3c), and carries specific features for ribbon-helix-helix folds. R1 parC consists of ten 11-bp direct repeats, organized in two sets of five repeats flanking the parMR promoter sequence. Putative parC sequence of R64 and ColIb-P9 consists of six 20-bp direct repeats located upstream of parAB genes. The parC sequences of R1 and R64 were found to be ATrich. No homologous sequence to the putative R1 or R64 parC was found upstream of R621a and pEK204 parAB. However, approximately 200-bp sequence upstream of parAB carries the characteristic feature with a high A + T content, suggesting that this region functions as R621a and pEK204 parC. Highly similar partition systems to that of R621a were found in pSC138, pETEC35, and pCoo.
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Replacement of the parAB system might have been accomplished by loss of the R64-type and insertion of the R621a/pEK204-type parAB system into the R621a yfbB gene. A 60-bp direct repeat R7 was generated after insertion of the parAB system (Fig. 4a). Similarly, 36-bp R8 and 171-bp R9 could have been generated after insertions of ccgAII and finQ, respectively (Fig. 4b and c). In addition, the soj gene is flanked by a 42-bp direct repeat R6, although the origin of this sequence is unknown (Fig. 4d). Insertions accompanied by long target duplications were previously found in Helicobacter pylori, especially at the site of insertion of restriction and modification systems (Nobusato et al., 2000). The presence of direct repeats might be an indication that soj, parAB, ccgAII, and finQ have inserted into the R64-type genome. Following traC, R64 carries traD, and ColIb-P9 and pEK204 carry trcD, although R621a and pEC_Bactec do not carry any corresponding gene (Fig. 2). These findings could be explained as follows (Fig. 4e): First, R64 traD was replaced by trcD in ColIb-P9 and pEK204. The insertion of trcD was accompanied by the generation of repeat G. Second, homologous recombination between repeat G resulted in deletion of trcD to form R621a and pEC_Bactec. This mechanism might be important for genomes to delete nonessential genes from them. Since R621a was transmitted into the E. coli cells harboring IncI1 plasmid by conjugation (Hedges and Datta, 1973), the exclusion system of R621a is expected to be different from those of R64, ColIb-P9, and pEK204. The putative R621a excA gene exhibited only limited similarity at the C-terminal half to that of R64 (Fig. 3d). Conjugation experiments to determine their exclusion specificity were performed (Table 2). When donor E. coli cells harboring R621a were mated with the recipient cells harboring pIG318 (R621a excA+), transfer frequency of R621a was reduced 330-fold, whereas the reduction was only 1.7-fold in the case of the recipient cells harboring pKK318 (R64 excA+). In contrast, when donor E. coli cells harboring mini-R64 plasmid pKK607 was mated with the cells harboring pKK318 (R64 excA+), transfer frequency was reduced 4700-fold, whereas the reduction was only 1.7-fold in the case of the cells harboring pIG318 (R621a excA+). These results indicate that R621a excA gene does function in entry exclusion and that R621a and R64 belong to different exclusion groups. The DNA sequence similarity between R621a/pEC_Bactec and R64 of the excA gene was more than 95% in the 30 region. The DNA sequence similarity of the traY gene was more than 96% in the 50 region. In contrast, similarity of an approximately 1.5-kb segment containing traY 30 region and excA 50 region between R621a/pEC_Bactec and R64 was low. To generate the R621a/pEC_Bactec traY–excA region, the 1.5-kb DNA segment containing traY 30 and excA 50 region of R64-like genome has most likely been replaced by another DNA sequence, which encodes a similar function. Our model for R621a evolution is: deletion of colicin Ib gene and the stability region from a ColIb-P9-like plasmid might have produced a common ancestor of pEK204, pEC_Bactec, and R621a, into which soj, ccgAII, and finQ segments were inserted with formation of various direct re-
peats. To form pEK204, the shufflon segment C was deleted from the ancestor plasmid, and a novel parAB segment, a Tn3-like transposon, and IS66 were inserted. To form pEC_Bactec, a 1.5-kb traY–excA segment was replaced and a Tn3-like transposon (different from that of pEK204), IS1294-like and ISCro1 were inserted. Homologous recombination between pEK204 and pEC_Bactec ancestors might have formed R621a ancestor, into which Tn10-like and IS2 were inserted. Exchange of exclusion property allowed the R621a ancestor to enter into a cell harboring the other IncI1 plasmid. Then the nucleotide replacement and deletion occurred in the inc region, resulting in different incompatibility of replication function.
Acknowledgment This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References Asano, K., Mizobuchi, K., 1998. Copy number control of IncIa plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site. EMBO J. 17, 5201–5213. Asano, K., Niimi, T., Yokoyama, S., Mizobuchi, K., 1998. Structural basis for binding of the plasmid ColIb-P9 antisense Inc RNA to its target RNA with the 50 -rUUGGCG-30 motif in the loop sequence. J. Biol. Chem. 273, 11826–11838. Dam, M., Gerdes, K., 1994. Partitioning of plasmid R1. Ten direct repeats flanking the parA promoter constitute a centromere-like partition site parC, that expresses incompatibility. J. Mol. Biol. 236, 1289–1298. Furuya, N., Komano, T., 1994. Surface exclusion gene of IncI1 plasmid R64: nucleotide sequence and analysis of deletion mutants. Plasmid 32, 80–84. Furuya, N., Komano, T., 1995. Specific binding of the NikA protein to one arm of 17-base-bair inverted repeat sequences within the oriT region of plasmid R64. J. Bacteriol. 173, 6612–6617. Furuya, N., Nisioka, T., Komano, T., 1991. Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64. J. Bacteriol. 73, 2231–2237. Gerdes, K., Møller-Jensen, J., Ebersbach, G., Kruse, T., Nordström, K., 2004. Bacterial mitotic machineries. Cell 116, 359–366. Gyohda, A., Furuya, N., Ishiwa, A., Zhu, S., Komano, T., 2004. Structure and function of the shufflon in plasmid R64. Adv. Biophys. 38, 183–213. Ham, L.M., Skurray, R., 1989. Molecular analysis and nucleotide sequence of finQ, a transcriptional inhibitor of the F plasmid transfer genes. Mol. Gen. Genet. 216, 99–105. Hama, C., Takizawa, T., Moriwaki, H., Mizobuchi, K., 1990. Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid. J. Biol. Chem. 265, 10666–10673. Hedges, R.W., Datta, N., 1973. Plasmids determining I pili constitutes a compatibility complex. J. Gen. Microbiol. 77, 19–25. Ishiwa, A., Komano, T., 2004. PilV adhesins of plasmid R64 thin pili specifically bind to the lipopolysaccharides of recipient cells. J. Mol. Biol. 343, 615–625. Kim, S.-R., Komano, T., 1997. The plasmid R64 thin pilus identified as a type IV pilus. J. Bacteriol. 179, 3594–3603. Komano, T., 1999. Shufflons: multiple inversion systems and integrons. Annu. Rev. Genet. 33, 171–191. Komano, T., Funayama, N., Kim, S.-R., Nisioka, T., 1990. Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer. J. Bacteriol. 172, 2230–2235. Komano, T., Kim, S.-R., Nisioka, T., 1987a. Distribution of shufflon among IncI plasmids. J. Bacteriol. 169, 5317–5319. Komano, T., Kubo, A., Nisioka, T., 1987b. Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames. Nucleic Acids Res. 15, 1165–1172. Komano, T., Yoshida, T., Narahara, K., Furuya, N., 2000. The transfer region of IncI1 plasmid R64: similarities between R64 tra and Legionella icm/ dot genes. Mol. Microbiol. 35, 1348–1359.
H. Takahashi et al. / Plasmid 66 (2011) 112–121 Lerner, C.G., Inouye, M., 1990. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/ white insert screening capability. Nucleic Acids Res. 18, 4631. Møller-Jensen, J., Ringgaard, S., Mercogliano, C.P., Gerdes, K., Löwe, J., 2007. Structural analysis of the ParR/parC plasmid partition complex. EMBO J. 26, 4413–4422. Narahara, K., Rahman, E., Furuya, N., Komano, T., 1997. Requirement of a limited segment of the sog gene for plasmid R64 conjugation. Plasmid 38, 1–11. Nikoletti, S., Bird, P., Praszkier, J., Pittard, J., 1988. Analysis of the incompatibility determinants of I-complex plasmids. J. Bacteriol. 170, 1311–1318. Nobusato, A., Uchiyama, I., Ohashi, S., Kobayashi, I., 2000. Insertion with long target duplication: a mechanism for gene mobility suggested from comparison of two related bacterial genomes. Gene 259, 99– 108. Praszkier, J., Wei, T., Siemering, K., Pittard, J., 1991. Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids. J. Bacteriol. 173, 2393–2397. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Press, Cold Spring Harbor, NY.
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The genome sequence of the incompatibility group Ic plasmid R621a: Evolution of IncI plasmids Hiroyuki Takahashi, Ming Shao, Nobuhisa Furuya, Teruya Komano ⇑ Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Received 5 October 2010 Accepted 27 June 2011 Available online 8 July 2011 Communicated by Dr. W. Klimke Keywords: Plasmid R621a Genome IncIc plasmid Bacterial conjugation Plasmid evolution
a b s t r a c t We present the complete genome sequence of the tetracycline resistance plasmid R621a isolated from Salmonella typhimurium, which belongs to the incompatibility group Ic. In the 93,185 bp circular double-stranded R621a genome, 96 complete ORFs are predicted. In addition, one and six different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of four regions: replication, leading, transfer, and miscellaneous regions. The R621a genome is similar to those of IncI1 plasmids such as R64 and ColIb-P9 and particularly to those of pEK204 and pEC_Bactec. Three major differences including inc, parAB, and excA regions were noted between R621a and prototype IncI1 plasmids. Seven nucleotide replacements and one nucleotide deletion in the putative Inc RNA sequence are found between R621a and IncI1 plasmids irrespective of close similarity in the other parts of the rep system. The sequences of R621a parAB and excA genes are significantly different from those of R64 and ColIb-P9, while those of R621a parAB and excA genes exhibit close similarity to those of pEK204 and pEC_Bactec, respectively. The R621a genome is suggested to be formed by acquiring parAB and excA genes from pEK204 and pEC_Bactec genomes, respectively, and then novel inc function by the mutations. The insertions in the R621a, pEK204, and pEC_Bactec genomes are flanked by direct repeats, suggesting that insertions accompanied by long target duplications have also played an important role in the evolution of IncI plasmids. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Bacterial plasmids are extrachromosomal genetic elements, usually consisting of double-stranded circular DNA. They confer various phenotypes such as conjugation, resistance to antibiotics and metals, and production of bacteriocins. Plasmids are usually classified into different groups according to their incompatibility properties. If two plasmids are stably maintained within a single cell, they are regarded as belonging to distinct incompatibility groups. The replication mechanisms of plasmids belonging to the same incompatibility group interfere with each other and do not allow their stable maintenance within a
⇑ Corresponding author. Fax: +81 42 677 2559. E-mail address: [email protected] (T. Komano). 0147-619X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2011.06.004
single cell. Incompatibility is also expressed in the plasmid partitioning systems (Dam and Gerdes, 1994). We have recently reported the genome sequence of the incompatibility group I1 plasmid R64 (Sampei et al., 2010). The 121-kb R64 genome is highly organized into five major regions: replication, drug resistance, stability, leading, and transfer regions. Replication, leading, and transfer regions are highly conserved among IncI1 plasmids including R64, ColIb-P9, pSL476_91, pCVM29188_101, and pSE11-1. It was found that genetic recombination including the site-specific rfsF-ResD system have played an important role in diversity of IncI1 plasmids. The genome sequences of the other IncI1 plasmid pEK204 and pEC_Bactec have been also reported (Woodford et al., 2009; Smet et al., 2010). Replication, leading, and transfer regions of pEK204 and pEC_Bactec are highly similar to those of the other IncI1 plasmids, although they do not carry the
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ars1/ars2 sequence or colicin Ib determinants which are common in IncI1 plasmids. R621a is a tetracycline resistance conjugative plasmid that was originally isolated from Salmonella typhimurium and determines I pili (Hedges and Datta, 1973). Since R621a was transferred into and stably maintained in Escherichia coli cells harboring IncI1 plasmids, it was classified as the incompatibility group Ic. Here we describe the complete genome sequence of plasmid R621a.
Number AP011954. Accession Number and nucleotide number of plasmids used in this paper for comparison are: R64, AP005147, 120,826 bp; ColIb-P9, AB021078, 93,399 bp; pEK204, EU935740, 93,732 bp; and pEC_Bactec, GU371927, 92,970 bp.
2. Materials and methods
The genome of R621a consists of a 93,185 bp circular double-stranded DNA. The G + C content of the genome was estimated to be 48.8%. This value is close to that of the original host, S. typhimurium (52.2%), and the laboratory host, E. coli K-12 (50.8%). Gene organization of R621a is shown in Fig. 1 and comparison to those of IncI1 plasmids is shown in Fig. 2. The R621a genome is similar to those of IncI1 plasmids such as R64 and ColIb-P9 and particularly to those of pEK204 and pEC_Bactec. Major parts (79–88%) of R621a DNA sequence (including replication, leading, and transfer regions) exhibit 96–98% identity to the sequences of R64, ColIb-P9, pEK204, and pEC_Bactec. R621a carries Tn10-like transposon and insertion sequence IS2. At least 96 complete open reading frames (ORFs) were predicted within the R621a genome (Fig. 1). The sog gene is predicted to produce two kinds of proteins, SogL and SogS, by translational reinitiation as in R64 (Narahara et al., 1997). Six additional PilV proteins with different C-terminal segments could be created by shufflon multiple inversions as in R64 (Komano et al., 1987b). Thus, 103 different proteins, 82 of which exhibit >95% amino acid sequence identities to the corresponding proteins of R64, may be produced from R621a (Table 1). Two homologous genes in R64, yfeA and ygbA, were converted into pseudogenes in R621a. It is noteworthy that both genes are located on the repeat sequences R3 described below. The two genes, yfbA and ygaA, were interrupted by the insertion of a parAB segment and Tn10-like, respectively. Several kinds of repetitive sequence were found in the R621a genome in addition to two IS10 sequences of Tn10-like (Fig 1). Repeats R3, R4, and R5 were also found in R64 (Sampei et al., 2010). R6, R7, R8, and R9 may be formed during the insertions of the soj, parAB, ccgAII, and finQ segments, respectively, into the R621a genome (Fig. 2). The R621a genome was tentatively separated into four functional regions including the replication (coordinates 0–2.4 kb), miscellaneous (coordinates 2.4–7.1 kb), leading (coordinates 7.1–37.2 kb), and transfer (coordinates 37.3–93.2 kb) regions (Fig. 1).
2.1. Bacterial strains, plasmids and growth media Escherichia coli K-12 strains JM83 D(lac-proAB) rpsL thi ara u80 dlacZDM15 (Sambrook and Russell, 2001) and TN102 Nalr (Komano et al., 1990) were used. Plasmid R621a was used throughout this study. Mini-R64 plasmid pKK607 carried the replication and transfer regions of R64 together with a Kmr fragment (Komano et al., 1990). Plasmid vectors pCL1920 (Lerner and Inouye, 1990), pUC18, and pUC19 (Sambrook and Russell, 2001) were used for cloning and sequencing. Luria–Bertani (LB) medium was prepared as described previously (Sambrook and Russell, 2001). The solid medium contained 1.5% agar. Antibiotics were added to the liquid and solid media at the following concentrations: ampicillin, 100 lg/ml; kanamycin, 50 lg/ml; spectinomycin, 50 lg/ml; tetracycline, 12.5 lg/ml; and nalidixic acid, 20 lg/ml. 2.2. DNA manipulation and sequencing Preparation of plasmid DNA, transformation, construction of plasmids, and other methods of DNA manipulation were described previously (Sambrook and Russell, 2001). The nucleotide sequence was determined by the dideoxy chain-termination method (Sambrook and Russell, 2001). To clone R621a excA, the 2.47-kb BglII-EcoRI segment was inserted into the multicloning site of pCL1920, yielding pIG318. To clone R64 excAB gene, the 1.25-kb NsiI segment was inserted into pCL1920, yielding pKK318. 2.3. Conjugative transfer To avoid the effects of shufflon inversions on transfer frequencies in liquid media, surface mating was conducted as previously described (Komano et al., 1990). E. coli JM83 donor cells harboring R621a or pKK607 were grown to the log phase and mixed with an overnight culture of E. coli TN102 recipient cells. The mixture was filtered through a nitrocellulose membrane. The membrane was incubated on an LB agar plate for 90 min at 37 °C. Transconjugants were resuspended in saline and plated at various dilutions onto selective media. 2.4. Nucleotide sequence accession number The complete nucleotide sequence of R621a has been deposited in GenBank/EMBL/DDBJ under Accession
3. Results and discussion 3.1. General properties of genome sequence
3.2. Replication region The replication (rep) region of R621a is located at the coordinates 0–2.4 kb of the genome (Fig. 1). The DNA sequence of the R621a replication region is more than 94% identical to those of IncI1 plasmids such as R64, pEK204, pEC_Bactec, and ColIb-P9, in which replication function has been extensively studied (Asano and Mizobuchi, 1998; Asano et al., 1998; Hama et al., 1990). This region
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Fig. 1. Map of the entire R621a plasmid. Circular R621a map was linearized at a Sau3AI site, between the replication and transfer regions, from which bases are numbered. Genes identified on R621a are illustrated by bars together with gene names. Genes transcribed left-to-right and right-to-left are described above and below the thin lines, respectively. The functions of genes are classified into nine groups, and genes in the same groups are indicated by the same colors, which are shown at the bottom of the figure. Locations of IS2, Tn10-like, repeat sequences and shufflon, and oriV, ter, and oriT sites are also indicated. The names of four functional regions are presented at the top together with the names of four major parts of the transfer region. This figure represents one specific clone of many isomers produced by shufflon multiple inversions.
Fig. 2. Comparison of gene organization among R64, ColIb-P9, pEK204, pEC_Bactec and R621a. Conserved genes among each of five plasmids are indicated by the same colors, which are shown at the bottom of the figure. Insertions of IS elements, transposons, and gene segments flanked by direct repeats are described above the map. Locations of oriV, rfsF, and oriT sites are indicated by upward and downward arrowheads. Names of representative genes are indicated.
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H. Takahashi et al. / Plasmid 66 (2011) 112–121 Table 1 List of known or suggested genes in plasmid R621a. Lengtha
No.
Gene
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
inc repY repZ yafA yafB yagA soj impB impA impC yfaA yfaB yfbA parA parB yfbB yfcA yfcB yfdA yfeA yfeB yfeC yffA yffB ssb yfhA psiB psiA ygaA tnpR tetD tetC tetA tetR ybdA ybeA ybeB
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
ybfA tnpL ygbA ardA ygcA ygdA ccgAII ygdB ygeA ygfA ygfB yggA nikA nikB trbC trbB trbA finQ pndC pndA excA traY
166 144 88 129 307 83 111 282 111 110 899 763 356 402 361 67 50 204 745
59 60 61 62
traX traW traV traU
194 400 204 1014
29 343 169 200 448 208 423 145 82 89 308 326 140 227 73 144 256 141 140 63 104 175 652 144 239 402 138 197 401 207 228 162 106 401 402
Conserved domainb
pfam02387 cd00236 PHA02518 PRK03609 PRK10276 pfam06183 pfam06924 (intr) pfam06406 pfam10784 PRK13699 pfam07128 (pseudo) pfam03230
PRK13732 COG1475 PRK13701 PRK13704 (intr) pfam01609 PRK10219 pfam00440 pfam07690 PRK13756 cd00090 PRK06349 COG2329 pfam03616 pfam01609 (pseudo) pfam07275
pfam04754
pfam03432 cd03020
pfam01848
Function or product
Homologc
Inc RNA Regulator of repZ expression Replication initiation protein Hypothetical protein FinO bacterial conjugation repressor Hypothetical protein Type II bacterial partitioning protein DNA polymerase V subunit ImpB DNA polymerase V subunit ImpA DinI-like family protein Hypothetical protein DUF1281 family protein
R64 RepY (100%) R64 RepZ (96%) ColIb-P9 YafA (62%) ColIb-P9 YafB (92%) ColIb-P9 YagA (98%) R64 YefA (24%), pO113 LH0102 (94%) R64 ImpB (98%) R64 ImpA (100%) R64 ImpC (100%) R64 YfaA (98%) R64 YfaB (97%)
Type I plasmid partitioning protein Type I plasmid partitioning protein Putative methylase Hypothetical protein DUF1380 family protein Hypothetical protein
R64 R64 R64 R64 R64 R64
ParA (66%), pEK204 ParM (99%) ParB (38%), pEK204 StbB (100%) YfbB (97%) YfcA (79%) YfcB (92%) YfdA (95%)
Antirestriction protein Hypothetical protein Hypothetical protein Hypothetical protein Single-strand DNA-binding protein Type II bacterial partitioning protein Plasmid SOS inhibition protein B Plasmid SOS inhibition protein A
R64 R64 R64 R64 R64 R64 R64 R64
YfeB (94%) YfeC (89%) YffA (97%) YffB (93%) Ssb (97%) YfhA (95%) PsiB (98%) PsiA (99%)
Transposase_11 Regulator of tet operon (HTH_AraC) Regulator of tet operon (TetR_N) Major facilitator superfamily (MFS_1) Tetracycline repressor protein TetR Transcriptional repressor (HTH_ARSR) Homoserine dehydrogenase Biosynthesis of extracellular polysaccharides Sodium/glutamate symporter (GltS) Transposase_11
R64 R64 R64 R64 R64 R64 R64 R64
TnpR (100%) TetD (100%) TetC (100%) TetA (100%) TetR (100%) YbdA (100%) YbeA (100%) YbeB (100%)
Antirestriction protein Hypothetical protein Hypothetical protein Hypothetical protein Transposase_31 Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein NikA oriT-specific DNA binding protein NikB relaxase TrbC transfer protein Protein disulfide isomerase TrbA transfer protein Fertility inhibition protein Post-segregation killing protein Post-segregation killing protein Entry exclusion protein TraY integral membrane protein
R64 ArdA (97%) R64 YgcA (99%) R64 YgdA (98%) pEK204 CcgAII (100%), pSC138 CcgAII (100%) R64 YgdB (78%) R64 YgeA (97%) R64 YgfA (96%) R64 YgfB (98%) R64 YggA (99%) R64 NikA (100%) R64 NikB (99%) R64 TrbC (99%), Lpn IcmO/DotL (26%) R64 TrbB (99%) R64 TrbA (99%), Lpn IcmP/DotM (27%) pEK204 FinQ (99%), R802a FinQ (83%) R64 PndC (96%) R64 PndA (100%) R64 ExcA (56%), pEC_Bactec ExcA (100%) R64 TraY (87%), pEC_Bactec TraY (100%), Lpn DotA (23%) R64 TraX (96%) R64 TraW (99%) R64 TraV (99%) R64 TraU (99%), Lpn IcmB/DotO (28%)
TraX transfer protein TraW lipoprotein TraV transfer protein TraU nucleotide-binding protein
R64 YbfA (100%) R64 TnpL (99%)
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Table 1 (continued)
a b c
No.
Gene
Lengtha
Conserved domainb
Function or product
Homologc
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
insD insA traT traS traR traQ traP traO traN traM traL sogL sogS nuc traK traJ traI traH traG traF traE rci OrfB OrfB0 OrfD0 OrfC OrfC0 OrfA0 pilVA pilU pilT pilS pilR pilQ pilP pilO pilN pilM pilL pilK pilJ pilI traC traB traA
301 121 266 62 134 175 234 429 327 230 115 1255 844 183 96 382 272 152 194 400 274 384 81 83 95 83 72 69 474 218 186 204 365 517 150 431 560 145 355 197 136 84 227 177 95
PRK09409 PRK09413
IS2 transposase InsD IS2 repressor TnpA TraT transfer protein TraS transfer protein TraR transfer protein TraQ transfer protein TraP transfer protein TraO transfer protein TraN transfer protein (DUF3625) IcmL superfamily TraL transfer protein SogL DNA primase SogS transfer protein EDTA-resistant nuclease (NucT) TraK transfer protein TraJ transfer ATPase TraI lipoprotein TraH lipoprotein LPS heptose-phosphate phosphatase Hypothetical protein Hypothetical protein Shufflon-specific DNA recombinase PilVB C-terminal segment PilVB0 C-terminal segment PilVD0 C-terminal segment PilVC C-terminal segment PilVC0 C-terminal segment PilVA0 C-terminal segment PilVA type IV preadhesin PilU prepilin peptidase (peptidase_A24) PilT lytic transglycosidase PilS type IV prepilin PilR integral membrane protein PilQ type IV pilus ATPase PilP type IV pilus biogenesis protein PilO type IV pilus biogenesis protein PilN type IV pilus secretin PilM type IV pilus biogenesis protein PilL lipoprotein PilK type IV pilus biogenesis protein PilJ type IV pilus biogenesis protein PilI type IV pilus biogenesis protein DUF2913 family protein Transcription termination factor NusG Hypothetical protein
IS2 InsD (100%) IS2 InsA (100%) R64 TraT (99%) R64 TraS (100%) R64 TraR (95%) R64 TraQ (99%), Lpn IcmD/DotP (21%) R64 TraP (99%), Lpn IcmG/DotF (27%) R64 TraO (99%) R64 TraN (99%), Lpn IcmK/DotH (28%) R64 TraM (99%), Lpn IcmL/DotI (30%) R64 TraL (99%) R64 SogL (98%) R64 SogS (99%) R64 Nuc (98%) R64 TraK (99%), Lpn IcmT (34%) R64 TraJ (100%), Lpn DotB (33%) R64 TraI (99%), Lpn DotC (29%) R64 TraH (100%), Lpn DotD (31%) R64 TraG (100%) R64 TraF (99%) R64 TraE (99%) R64 Rci (99%) R64 OrfB (100%) R64 OrfB0 (100%) R64 OrfD0 (100%) R64 OrfC (100%) R64 OrfC0 (100%) R64 OrfA0 (100%) R64 PilVA (100%) R64 PilU (100%), BfpP (24%) R64 PilT (100%), BfpH (32%) R64 PilS (97%), BfpA (24%) R64 PilR (99%), BfpE (32%) R64 PilQ (98%), BfpD (31%) R64 PilP (96%) R64 PilO (96%) R64 PilN (100%), BfpB (29%) R64 PilM (100%) R64 PilL (99%) R64 PilK (89%), ColIb-P9 PilK (95%) R64 PilJ (77%), ColIb-P9 PilJ (57%) R64 PilI (90%), ColIb-P9 PilI (99%) R64 TraC (99%) R64 TraB (99%) R64 TraA (99%)
pfam12293 pfam11393 COG4643 PRK13878 PRK13912 TIGR02525
PRK15416
cd00796 (frag) (frag) (frag) (frag) (frag) (frag) pfam04917 pfam01478 pfam01464 pfam08805 COG1459 COG2804 TIGR03021 pfam06864 TIGR02520 pfam07419
pfam10623 pfam11140 pfam02357
Length in amino acid residues. NCBI conserved domain. intr, interrupted by the insertion of transposon or specific DNA segments; pseudo, pseudo genes; frag, gene fragment. Selected homologs: R64 and ColIb-P9 homologs and Legionella pneumophila (Lpn) icm/dot and enteropathogenic E. coli bfp homologs are listed. Amino
contains three genes, inc, repY and repZ. The repZ gene encodes replication initiation protein that may interact with oriV region located downstream of repZ to initiate DNA replication. DNA sequence of 417-bp regulatory region including inc is 99% identical to the previous report and expression of inc function in this region has been demonstrated (Nikoletti et al., 1988). The oriV and ter sequences of R621a are 95% and 100% identical to those of R64, respectively. repYZ mRNA is thought to form a unique structure consisting of three stable stem-loop structures, which is inactive for repZ expression. Translation of repY is required for the activation of repZ translation. Inc RNA, antisense RNA for repYZ mRNA, inactivates repY and repZ translation. Inc RNA sequences of R621a and IncI1 plasmids are significantly different from each other (Fig. 3a),
suggesting that this allows stable maintenance of both plasmids within a single cell. 3.3. Leading region R621a leading region from impB to yggA at the coordinates 7.1–37.2 kb is very similar to those of R64 and ColIb-P9 (Figs. 1 and 2) except for a few insertions in the R621a sequence. R621a carries ygaA followed by psiA as does R64, while ColIb-P9 carries ydcA at the corresponding site. The ardA, psiAB, and ssb genes of R621a leading region may play important roles in conjugation. The parAB genes, unlike those of R64 and ColIb-P9, are inserted into the yfbA gene in the R621a leading region (Fig. 1). The ccgAII gene is inserted into the intergenic
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Fig. 3. (a) Alignment of the nucleotide sequences of Inc RNA of R621a, R64, pMU720 (GenBank Accession No. M28718), R387 (M93063), pIE545 (M93064), pO113 (AY258503), pSERB1 (AY686591), and pECOED (CU928147). Conserved nucleotides are printed in boldface. Gaps, marked by dashes, are introduced to reveal maximal similarity among the sequences. Stem sequences are indicated by inverted arrows, and the highly conserved CGCCAA sequence is indicated by dots. (b) Alignment of the amino acid sequences of R621a ParA, pCoo StbA (GenBank Accession No. YP_424887), R64 ParA (NP_863404), pO113 StbA (YP_308777), R1 ParM (P11904), and pB171 StbA (NP_053130). Amino acid sequence of pEK204 ParM is 99% identical to that of R621a. (c) Alignment of the amino acid sequences of R621a ParB, pCoo StbB (YP_424888), R64 ParB (NP_863405), pO113 StbB (YP_308776), R1 ParR (P11906), and pB171 StbB (NP_053129). Amino acid sequence of pEK204 StbB is 100% identical to that of R621a. (d) Alignment of the amino acid sequences of ExcA homologs in R621a, R64 (NP_863442), ColIb-P9 (NP_52507), pO113 (YP_308714), and pECOED (CAQ87319). Amino acid sequences of pEC_Bactec and pEK204 ExcA are 100% and 99% identical to those of R621a and R64, respectively.
region between ygdA and ygdB. Insertion of ccgAII is also found in pEK204 and pEC_Bactec, but insertion of parAB is found only in pEK204 (Fig. 2). Tn10-like is inserted into ygaA gene in the R621a leading region. 3.4. Transfer region Gene organization of the R621a transfer region at the coordinates 37.3–93.2 kb is similar to those of R64, ColIbP9, pEK204, and pEC_Bactec except IS2 insertion (Figs. 1 and 2) (Komano et al., 2000; Sampei et al., 2010). The R621a transfer region exhibits a highly organized structure consisting of four major parts as in R64 (Fig. 1); (i) traABC regulatory genes, (ii) pil genes for type IV pilus biogenesis,
(iii) tra/trb genes for conjugation in general, and (iv) oriT operon for conjugative DNA processing. The R621a traABC genes are located immediately upstream of the replication region in the circular genome and may be involved in transfer gene expression as in R64 (Fig. 1). R621a and pEC_Bactec do not carry any homolog corresponding to the R64 traD gene or the ColIb-P9 and pEK204 trcD gene. The 14 pil genes of R621a may be responsible for the formation of a type IV pilus required only for conjugation in liquid media (Kim and Komano, 1997). The R621a shufflon with four invertible segments, A, B, C, and D, is located at the 30 region of the pilV gene (Gyohda et al., 2004; Komano, 1999). Multiple inversions of the four
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DNA segments in R621a shufflon were previously reported (Komano et al., 1987a). Shufflon inversions mediated by Rci shufflon-specific recombinase select one of seven PilV adhesins, in which the N-terminal region is constant while the C-terminal region is variable (Komano et al., 1987b). R621a, R64, and pEC_Bactec shufflon consists of four segments, while ColIb-P9 shufflon lacks segment D and pEK204 shufflon lacks segment C. PilV adhesins, located at the tip of the PilS polymer, determine the recipient specificity of liquid matings by recognition of the lipopolysaccharide of the recipient cells (Ishiwa and Komano, 2004; Shimoda et al., 2008). Three R621a genes, traEFG, might be dispensable for conjugation as in R64 (Komano et al., 1990). Twenty genes, traH to traY, may form another operon (Fig. 1 and Table 1). Amino acid sequence similarity of TraY between R621a and R64 is limited (87%). R621a carries insertion of IS2 between traT and traU, but this insertion does not affect its transfer ability (Table 2). The C-terminal 163 amino acids of the R621a ExcA exhibit 56% sequence identity to the corresponding region of R64 ExcA, while R621a and pEC_Bactec ExcA is identical (Fig. 3d). R621a ExcA is 204-amino-acid long, but in R64, ColIb-P9, and pEK204 it is 220-amino-acid long (Furuya and Komano, 1994). Although R64 excA gene produced ExcA and ExcB proteins by translational reinitiation, R621a excA gene lacks the sequence for the translational reinitiation. R621a, pEK204, and pEC_Bactec carry a finQ gene between pndCA and trbA, while R64 and ColIb-P9 do not. FinQ protein exhibits high sequence similarity to that of IncI plasmid R820a except for 16-amino-acid insertion (Ham and Skurray, 1989). pEC_Bactec carries ISCro1 insertion between finQ and pndCA. Processing of R621a DNA during conjugation may be conducted by the oriT operon (Fig. 1 and Table 1). The R621a oriT operon consists of the oriT sequence and the nikAB genes (Furuya et al., 1991). The 92-bp R621a oriT sequence, which is identical to that of R64, contains oriT nick site, repeat A, and repeat B sequences and 8-bp inverted repeats. R64 NikA, a ribbon–helix–helix DNAbinding protein (Yoshida et al., 2008), binds to the repeat A sequence (Furuya and Komano, 1995). NikB, a relaxase protein, binds to NikA-oriT to form a relaxation complex.
Table 2 Entry exclusion specificity of R621a and R64. Donor plasmid
Transfer frequency (%)a
Exclusion indexb
pCL1920
pIG318
pKK318
pIG318
PKK318
R621a pKK607
19 15
0.058 8.9
11 0.0032
330 1.7
1.7 4700
a Escherichia coli JM83 donor cells harboring R621a or mini-R64 plasmid pKK607 were grown to a log phase and then mixed with an overnight culture of E. coli TN102 recipient cells harboring pCL1920, pIG318 (R621a excA+), or pKK318 (R64 excA+). Transfer frequency of filter matings is expressed as a ratio (percentage) of the number of transconjugants to that of donor cells. The transfer frequency shown represents the average of three independent experiments. b The exclusion index is expressed as a ratio of transfer frequency of donor plasmid into the recipient cells harboring pCL1920 to that of donor plasmid into recipient cells harboring pIG318 or pKK318.
3.5. Miscellaneous region R621a lacks stability region which are conserved in IncI1 plasmids (Fig. 2). At the same time, R621a does not carry either arsenic resistance or colicin genes, which are characteristic of R64 or ColIb-P9, respectively. The remaining region (coordinates 2.4–7.1 kb) of the R621a genome was tentatively assigned as the miscellaneous region. It is practically identical to the corresponding regions of pEK204 and pEC_Bactec except for the insertion of a Tn3like transposon and IS1294-like insertion sequence or Tn3-like transposon, respectively (Fig. 2). Three genes, yafA, yafB, and yagA are similar to those of ColIb-P9. The amino acid sequence of the R621a soj product exhibits some similarity to that of R64 yefA product. R621a Soj protein may be involved in plasmid partition with YfhA (SpoOJ) protein encoded in the leading region, although putative parC locus for this system is not known. pEC_Bactec carries only soj-yfhA system as a putative partition system. R621a, pEK204, and pEC_Bactec do not carry R64/ColIb-P9 type parAB genes. Instead, a novel type of parAB genes is inserted into their leading region of R621a and pEK204 (Fig. 2). They also lack the resD and rfsF sitespecific recombination system, required for the resolution of a dimer DNA molecule. Although R621a lacks the resD and rfsF system, it is stably maintained in E. coli cells, suggesting a possibility of an unknown resolution system in R621a. 3.6. Three factors determining plasmid identities Incompatibilities of replication and partition systems and specificity of entry exclusion systems are thought to determine identities of conjugative plasmids. Nucleotide sequences of Inc RNA, and amino acid sequences of ParAB and ExcA proteins were compared between R621a and the related plasmids (Fig. 3). The incompatibility of replication system is based on Inc RNA that controls plasmid copy number within the cell. Nucleotide sequences of putative Inc RNA of R621a and the related plasmids are aligned in Fig. 3a. Inc RNA of IncI related plasmids was shown to fold into a single large stem-loop structure (Asano et al., 1998; Praszkier et al., 1991). Stems consisting of approximately 21 bp (inverted arrows in Fig. 3a), are relatively conserved. Most nucleotide replacements in stems still keep base complementarity. Loops consist of 20–22 nucleotides and base pairing within the loop might further stabilize the Inc RNA structure. Nucleotide sequence in the loop is relatively variable and might determine the incompatibility. However, it should be noted that the CGCCAA sequence (dots in Fig. 3a), complementary to the UUGGCG motif in the structure I within repY region of RepZ mRNA (Asano et al., 1998), is completely conserved among all the Inc RNA sequences in Fig. 3a. There are seven nucleotide replacements and one nucleotide deletion in the putative Inc RNA sequences between R621a and IncI1 plasmids (Fig. 3a). By contrast, the DNA sequences except for the inc region are highly conserved between R621a and IncI1 plasmids. Approximately 210-bp DNA sequence of R621a immediately downstream of the inc gene, carrying the
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Fig. 4. Duplication of the target sequences during insertion of (a) parAB, (b) ccgAII, (c) finQ, and (d) soj genes. The parAB, ccgAII, and finQ segments are thought to be inserted into the indicated R64 sequences, generating the indicated target duplication. Boldface letters represent nucleotides different from those of R64. (pEK204) and (pEC_Bactec) indicate exactly the same target duplication as that of R621a was found in pEK204 and pEC_Bactec, respectively. (pEK204) and (pEC_Bactec) indicate a target duplication with a few nucleotide replacements was found in pEK204 and pEC_Bactec, respectively. In the case of soj gene, the origin of target sequence is unknown. (e) Insertion of trcD segment into the indicated R64 sequence accompanied by the indicated target duplication (repeat G) and traD deletion formed ColIb-P9 and pEK204 sequences. Homologous recombination between the italicized sequences within the repeat G sequences results in deletion of trcD, to generate the R621a and pEC_Bactec sequence.
entire repY gene and repZ 50 region and playing important roles in replication control, is identical to those of R64, ColIb-P9, pEK204, and pEC_Bactec. In the ParMR/parC partition system of plasmid R1, a cis site, parC exhibits incompatibility (Dam and Gerdes, 1994; Gerdes et al., 2004). Ribbon-helix-helix DNA-binding protein, ParR (ParB in R621a) binds to parC to form a partition complex (Møller-Jensen et al., 2007). The partition complex is recognized by an actin-like partition protein ParM (ParA in R621a), which forms filaments required for the bidirectional movement of plasmid DNA. Partition proteins of R621a and pEK204 are significantly different from those of R64 and ColIb-P9. R621a ParA exhibits 99%, 52%, and 65% amino acid sequence identities to pEK204 and R1 ParM and R64 ParA, respectively (Fig. 3b). R621a ParB
exhibits 100%, 38%, and 36% sequence identity to pEK204 StbB, R1 ParR, and R64 ParB, respectively (Fig. 3c), and carries specific features for ribbon-helix-helix folds. R1 parC consists of ten 11-bp direct repeats, organized in two sets of five repeats flanking the parMR promoter sequence. Putative parC sequence of R64 and ColIb-P9 consists of six 20-bp direct repeats located upstream of parAB genes. The parC sequences of R1 and R64 were found to be ATrich. No homologous sequence to the putative R1 or R64 parC was found upstream of R621a and pEK204 parAB. However, approximately 200-bp sequence upstream of parAB carries the characteristic feature with a high A + T content, suggesting that this region functions as R621a and pEK204 parC. Highly similar partition systems to that of R621a were found in pSC138, pETEC35, and pCoo.
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Replacement of the parAB system might have been accomplished by loss of the R64-type and insertion of the R621a/pEK204-type parAB system into the R621a yfbB gene. A 60-bp direct repeat R7 was generated after insertion of the parAB system (Fig. 4a). Similarly, 36-bp R8 and 171-bp R9 could have been generated after insertions of ccgAII and finQ, respectively (Fig. 4b and c). In addition, the soj gene is flanked by a 42-bp direct repeat R6, although the origin of this sequence is unknown (Fig. 4d). Insertions accompanied by long target duplications were previously found in Helicobacter pylori, especially at the site of insertion of restriction and modification systems (Nobusato et al., 2000). The presence of direct repeats might be an indication that soj, parAB, ccgAII, and finQ have inserted into the R64-type genome. Following traC, R64 carries traD, and ColIb-P9 and pEK204 carry trcD, although R621a and pEC_Bactec do not carry any corresponding gene (Fig. 2). These findings could be explained as follows (Fig. 4e): First, R64 traD was replaced by trcD in ColIb-P9 and pEK204. The insertion of trcD was accompanied by the generation of repeat G. Second, homologous recombination between repeat G resulted in deletion of trcD to form R621a and pEC_Bactec. This mechanism might be important for genomes to delete nonessential genes from them. Since R621a was transmitted into the E. coli cells harboring IncI1 plasmid by conjugation (Hedges and Datta, 1973), the exclusion system of R621a is expected to be different from those of R64, ColIb-P9, and pEK204. The putative R621a excA gene exhibited only limited similarity at the C-terminal half to that of R64 (Fig. 3d). Conjugation experiments to determine their exclusion specificity were performed (Table 2). When donor E. coli cells harboring R621a were mated with the recipient cells harboring pIG318 (R621a excA+), transfer frequency of R621a was reduced 330-fold, whereas the reduction was only 1.7-fold in the case of the recipient cells harboring pKK318 (R64 excA+). In contrast, when donor E. coli cells harboring mini-R64 plasmid pKK607 was mated with the cells harboring pKK318 (R64 excA+), transfer frequency was reduced 4700-fold, whereas the reduction was only 1.7-fold in the case of the cells harboring pIG318 (R621a excA+). These results indicate that R621a excA gene does function in entry exclusion and that R621a and R64 belong to different exclusion groups. The DNA sequence similarity between R621a/pEC_Bactec and R64 of the excA gene was more than 95% in the 30 region. The DNA sequence similarity of the traY gene was more than 96% in the 50 region. In contrast, similarity of an approximately 1.5-kb segment containing traY 30 region and excA 50 region between R621a/pEC_Bactec and R64 was low. To generate the R621a/pEC_Bactec traY–excA region, the 1.5-kb DNA segment containing traY 30 and excA 50 region of R64-like genome has most likely been replaced by another DNA sequence, which encodes a similar function. Our model for R621a evolution is: deletion of colicin Ib gene and the stability region from a ColIb-P9-like plasmid might have produced a common ancestor of pEK204, pEC_Bactec, and R621a, into which soj, ccgAII, and finQ segments were inserted with formation of various direct re-
peats. To form pEK204, the shufflon segment C was deleted from the ancestor plasmid, and a novel parAB segment, a Tn3-like transposon, and IS66 were inserted. To form pEC_Bactec, a 1.5-kb traY–excA segment was replaced and a Tn3-like transposon (different from that of pEK204), IS1294-like and ISCro1 were inserted. Homologous recombination between pEK204 and pEC_Bactec ancestors might have formed R621a ancestor, into which Tn10-like and IS2 were inserted. Exchange of exclusion property allowed the R621a ancestor to enter into a cell harboring the other IncI1 plasmid. Then the nucleotide replacement and deletion occurred in the inc region, resulting in different incompatibility of replication function.
Acknowledgment This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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