ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2002 Vol. 45, Št. 1: 9 - 15 
tra region of the natural conjugative Escherichia coli plasmid 
pRKlOO is F-like 
Regija tra naravnega konjugativnega plazmida pRKIOO bakterije 
Escherichia coli je podobna plazmidu F 
Marjanca STARČIČ ERJAVEC1•2, Wim GAASTRA2, Darja ŽGUR-BERTOK1 
1 Department ofBiology, Biotechnical Faculty, University ofLjubljana, Večna pot 111, 1000 Ljubljana, 
Slovenia, and 
2 Department oflnfectious Diseases and Immunology, Utrecht University, P.O. Box 80. 165, 3508 TD 
Utrecht, The Netherlands; E-mail: darja.zgur@uni-lj.si 
Introduction 
Abstract. The aim of the presented study was to identify the similarity of the 
pRKI00 tra region with tra regions of other conjugative plasmids of 
Enterobacteriaceae . For this purpose severni tra genes were amplified with PCR and 
the nucleotide sequences of the obtained PCR products were determined. The pRKI OO's 
nucleotide sequences were compared to the nucleotide sequences deposited in GenBank 
and the nucleotide divergence between them was calculated. The obtained results 
clearly demonstrated, that the tra regi on of pRK 100 is the most similar to the tra 
regi on of plasmid F. 
Key words: conjugative plasmid, tra region, Enterobacteriaceae. 
Izvleček. Cilj raziskave je bil ugotoviti podobnost regije tra plazmi da pRK 100 z 
regijami tra drugih konjugativnih plazmidov enterobakterij. V ta namen smo več genov 
regije tra plazmida pRKIO0 pomnožili v PCR in določili nukleotidno zaporedje 
dobljenih produktov PCR. Nukleotidna zaporedja plazmida pRK I 00 srno primerjali z 
drugimi, deponiranimi, nukleotidnimi zaporedji in izračunali nukleotidno divergenco. 
Dobljeni rezultati so jasno pokazali, da je regija tra plazmi da pRKl 00 najbolj podobna 
regiji tra plazmida F. 
Ključne besede: konjugativen plazmid, regija tra, Enterobacteriaceae. 
Conjugative plasmids are extrachromosomal elements, that can promote their own DNA transfer, 
as well as of co-resident plasmids or even chromosomal DNA from a donor to a recipient celi in a 
process named conjugation (Firth & al. 1996). Ali information needed for this process is encoded in a 
Iarge, approximately 30 kb long, plasmid region, often denoted as the tra region. Conjugative plasmids 
facilitate the exchange and spread of resistances to antibiotics, chemicals, virulence factors and metabolic 
properties. 
One of the best studied tra regi on s is that of the Escherichia coli plasmid F (Fig. 1) The tra regi on 
of plasmid F contains approximately 40 genes that are organised into three operons with a complex 
network ofregulation of gene expression (Frost & al. 1994). The genes ofthe F-plasmid tra region can 
be divided, according to function, into 5 groups: i) regulatory genes (finP, tral, traY. andfinO); ii) 
genes for pilus biogenesis (traA, traL, traE, traK, traB, traV, traC, trbl, traW, traU, trbC, traF, traQ, 
10 Acta Biologica Slovenica, 45 (1), 2002 
traG, traH, traX); iii) genes for DNA metabolism (traM, traY, traD, tral); iv) genes for aggregate 
stabilisation (traN, traG); and v) genes for surface exclusion (traS, tra1) (Firth & al. 1996). 
Severa! conjugative plasmids of Enterobacteriaceae have been found to harbour F-like tra regions: 
pColV-K30, P307, R 100, R 1, pSLT. Even though these plasmids have many similarities at the leve! of 
nucleotide sequences, differences in regulation of conjugation are observed. Por example: plasmid Rl 
has two promoters upstream of the tral gene, while plasmid F has only one promoter (Dempsey 
1994). Therefore, characterisation of different conjugative plasmids and their tra regions is of interest. 
X , .. 
fino 
artA trbB 
Figure 1: Physical and genetic map of the F-plasmid tra regi on. Genes of the F tra region are depicted. 
To clearly show the region of a gene, some boxes representing genes are offline. Capital letters are 
shortened abbreviations for the genes named tra. The boxes designating the pRKI 00 genes analysed 
in this study are filled. The figure is based on Prost & al. 1994. 
Slika 1: Razpored genov tra plazmida F. Prikazani so geni tra plazmi da F. Kvadrati, ki označujejo 
gene, katerih nukleotidna zaporedja smo analizirali, so zapolnjeni. Slikaje pripravljena po Prost & al. 
1994. 
In the presented study the tra regi on of the natura! conjugati ve plasmid pRK 100 was characterised with 
regard to similarity with tra regions of related plasmids. pRK 100 is a -145-kb plasmid isolated from a 
uropathogenic Escherichia coli strain and it has been partially characterised (Ambrožič & al. 1998). It is a 
member of the IncF incompatibility group and encodes two antibiotic resistances, ampicillin and tetracycline, 
two colicins, Co!V and Colla, and the aerobactin iron uptake system (Žgur-Bertok & al. 1990). 
Por the purpose of defining the similarity of pRKlO0's tra region, PCR products of severa! tra 
genes were amplified and their nucleotide sequences were determined. The obtained (partial) nucleotide 
sequences of genes traM, finP, tral, traY, traD, tral, and finO were compared with the nucleotide 
sequences of the same genes of plasmids F, pCoIV-K30, P307, RlO0, Rl and pSLT and the nucleotide 
divergence between pRK 100 and the other plasmids was determined. From the obtained results it can 
be concluded that the tra region of pRK 100 is most similar to the tra regi on of plasmid F. 
Methods 
Bacterial strains, plasmids and growth conditions 
Bacterial strains and plasmids used in this study are presented in Tab. 1. Bacteria were grown in 
Luria-Bertani (LB) medi um with aeration at 37°C. Ampicillin (Ap, 100 µg/ml) and tetracycline (Te, 10 
µg/ml) were added to the growth media, when appropriate. 
Table 1: Bacterial strains and plasmids 
Tabela 1: Bakterijski sevi in plazmidi 
Strain or DNA Relevant features Reference or source* 
Strains 
HBIOI hsdR hsdM recA 13 supE44 leuB6 lacZ proA2 D. Ehrlich ' 
CL225 HBI0I harbouring pRK!00 Ambrožič & al. 1998 
DH5a <t>80dlacZ6Ml5 6(lacZYA-argF)Ul69 endAI recAI hsdR/7 BRL Life Technologies 
deoR thi-1 supE44 gyrA 96 re/Al 
Plasmids 
pGEM-TEasy T-vector for cloning of PCR products; Ap' Promega 
pRKI00 natura! plasmid; Ap', Te' Ambrožič & al. 1998 
*' Laboratoire Genetique Microbienne, INRA, Domain de Vilvert, Jouy en Josas , France 
M. Starčič Erjavec, W. Gaastra, D. Žgur-Bertok: tra region of the natura! conjugative Escherichia ... 11 
General DNA manipulation techniques 
Plasmid DNA isolation, ligation and transformation experiments were performed using standard 
methods (Sambrook & al. 1989). Restriction endonuclease digestions were carried out as specified by 
the manufacturer (Promega, Boehringer). DNA fragments were isolated from agarose gels using the 
QIAquick Gel Extraction kit (Qiagen). DNA sequencing was performed using a dye rhodamine 
terminator cycling reaction and an ABI PRISM™ 31 O Genetic Analyzer automated sequencer and ABI 
PRISM™ software. 
Polymerase chain reaction 
The primers used for the various PCR reactions are presented in Tab. 2. The polymerase chain 
reactions (PCR) were performed in a 50 µ! PCR reaction mixture with 20 pmol of the two primers, 5 
µl ligation mixture, 0.2 mM of dNTP mixture (Pharmacia), 0.625 U Taq DNA polymerase (Promega) 
and lx PCR buffer (Promega). The PCR programs used in this study are listed in Tab. 2. Each PCR 
amplification program started with a prolonged denaturation (94°C-4 min) step before 1 st cycle, and 
ended with a prolonged extension step (72°C- 1 O min) after the last cycle. The pGEM-T Easy system 
(Promega) was used for cloning of PCR products. 
Table 2: Oligonucleotide primers and PCR programs 
Tabela 2: Oligonukleotidni začetniki in programi verižne reakcije s polimerazo (PCR) 
Primer Nucleotide sequence PCRprogram 
Oligonucleotide primers 
for traM,finP, traJ PCR: 
FinP-1 5'-TATTGAGAAGCGTCGACAGG-3' (94 °C-1 :00, 55°C-1 :00, 
FinP-2 5'-TGACGAACATGAGCAGCATC-3' 72°C-1:00) 30x 
for traY PCR: 
TraY-f' 5'-GGAATTCAAGATTTGGTACACGTTCTGC-3' (94°C-1:00, 63°C-
TraY-r' 5'-GGAATTCCTTCCTCTTTATCTGCCTCCC-3' 1:00, 72°C-2:00) 30x 
for traD PCR: 
TraD-f' 5'-GGAATTCCAGATTGCGTCCATGCGTATCC-3' (94°C-1:00, 63°C-1:00, 
TraD-r' 5'-GGAATTCATCACCACACATATCACCGCGC-3' 72°C-1:00) 30x 
for tral PCR: 
Tral-1 5'-ACAGCGAATATACGTGACGG-3' (94°C-0:30, 57°C-0:30, 
Fin0-4 5'-CGTGGTGACA TTGATGATGG-3' 72°C-3:00) 30x 
for finO PCR: 
FinO-f' 5' -GGAA TTCGAAGCGACCGGTACTGACACTG-3' (94°C-l:OO, 63°C-l:OO, 
FinO-r' 5' -GGAATTCGCCTGAAGTTCTGCCTTTATCCG-3' 72°C-2:00) 30x 
1 Nucleotide sequence is based on Boyd & al. 1996 
1 Nukleotidno zaporedje z ozirom na Boyd & al. 1996 
Sequence analysis 
DNA sequences were compiled and analysed using the CLUSTAL W (Thompson & al. 1994) 
program for sequence alignment and the program DNADIST in the PHYLIP package (Felsenstein 
1993, Felsenstein 1989) for calculating distance matrixes. 
Results 
The compared nucleotide sequence of pRKlOO traM is most similar to traM of plasmids F and 
pCo!V-K30 
The traM gene ofF-like plasmids is approximately 380 bp long. Our obtained nucleotide sequence 
ofthe pRKlO0 traM gene is partial, covering 308 nucleotides (GenBank accession number AF237698). 
Subsequently, in nucleotide sequence analysis the obtained 308 nucleotides were compared with 
12 Acta Biologica Slovenica, 45 (1), 2002 
corresponding sequences of related plasmids. The phylogenetic distance between the obtained traM 
nucleotide sequence and traM of pColV-K30 and plasmid F is the smallest, namely 0.0098 (Fig. 2A). 
On the basis of the calculated nucleotide divergence, we can conclude, that the traM nucleotide 
sequence of pRKJO0 is most similar to traM of F and pColV-K30. 
The compared nucleotide sequence of pRKlOO finP is identical to finP of plasmid F 
ThefinP gene ofthe F-like plasmids is approximately 80 bp long and the full length pRKl00finP 
gene sequence (GenBank accession number AF237698) was compared with corresponding sequences 
of related plasmids. Since there is no nucleotide divergence between pRK 100 finP and F finP (Fig. 
28), thefinP of pRKlOO is completely the same as thefinP of plasmid F. 
The compared nucleotide sequence of pRKlOO traJ is most similar to traJ of plasmid F 
The traJ gene of F-like plasmids is 600-700 bp long. Our obtained nucleotide sequence of pRKl 00 
traJ gene is partial, covering approximately one half ofthe gene (363 bp) (GenBank accession number 
AF237698). Therefore, in nucleotide sequence analysis the obtained 363 bp were compared with 
corresponding sequences of related plasmids. The phylogenetic distance between the obtained traJ 
nucleotide sequence and traJ of plasmid F is the smallest, only 0.0028. The distance to traJ of plasmid 
pColV-K30 is also very small, only 0.0055 (Fig. 2C). We can conclude that, the tral nucleotide 
sequence of pRKl 00 is most similar to traJ of plasmid F. 
The compared nucleotide sequence of pRKlOO traY is most similar to traY of plasmids F and 
pCoIV-K30 
The traY genes ofF-like plasmids can be one oftwo lengths, approximately 230 bp, and probably 
due to duplication (Maneewannakul & al. 1996), approximately 400 bp. Our obtained nucleotide 
sequence of the pRKI00 traY gene is partial, covering 326 nucleotides (GenBank accession number 
AF237695). Due to the larger size, we can conclude that, the pRKI 00 traY is of the duplicated type. 
In our sequence analysis the obtained 326 bp were compared with corresponding sequences of related 
plasmids. The compared nucleotide sequences of pRK 100, pColV-K30 and plasmid F were completely 
identical with no nucleotide divergence among them (Fig. 2D) . 
The compared nucleotide sequence of pRKlOO traD is most similar to traD of plasmid F 
The traD gene ofF-like plasmids is approximately 2200 bp long. Our obtained nucleotide sequence 
of pRKJO0 traD gene is partial, covering 574 bp (GenBank accession number AF237693). Therefore, 
in nucleotide sequence analysis the obtained 574 nucleotides were compared with corresponding 
sequences of related plasmids. The phylogenetic distance between the obtained traD nucleotide sequence 
and traD of plasmid F is the smallest, only 0.0359 (Fig. 2E). We can conclude, that the traD nucleotide 
sequence of pRKlOO is most similar to traD of plasmid F. 
The compared nucleotide sequence of pRKlOO tral is most similar to tral of plasmid RlOO 
The tral gene ofF-like plasmids is approximately 5200 bp long. Our obtained nucleotide sequence 
of pRK 100 tral gene is partial, covering only 258 bp (GenBank accession number AY230887). In the 
nucleotide sequence analysis the obtained 258 bp were compared with corresponding sequences of 
related plasmids. The phylogenetic distance between the obtained tral nucleotide sequence and tral of 
plasmid RJO0 is the smallest, the nucleotide divergence is 0.0237 (Fig. 2F). The tral nucleotide 
sequence ofpRKIOO is therefore, most similar to tral ofplasmid RI00. 
The compared nucleotide sequence of pRKIOO finO is most similar to finO of plasmid F 
ThefinO gene of the F-like plasmids is approximately 560 bp long. The obtained pRKI00 finO 
gene sequence is partial, encompassing 486 nucleotides (GenBank accession number AF237696) and 
these were compared with corresponding sequences of related plasmids. The smallest nucleotide 
divergence, 0.0316, was found to be between pRKl 00 fin O and F fin O (Fig. 2G). Therefore, we can 
conclude thatfinO of pRKIOO is most similar tofinO of plasmid F. 
M. Starčič Erjavec, W. Gaastra, D. Žgur-Bertok: tra region of the natura) conjugative Escherichia ... 13 
A 
pColV-K30 
R100 
P307 
R1 
pSLT 
0,200 
0;21 4 
.. imM 
'------"-~~=~ ..... _..._._~__, 
o 0.2 0.4 0 ,6 0.8 
Nucleotide divergence 
B 
F [ O - f ----- - ·.· _. __ •.-.- ~• ~ .· .. 
R100• o:~e 
pColV-K30 111111 0,{1a47 f . . 
pSLT. O;tje,\7 
P307 ,.. o:qas 
R1. oi1142 
o 0,2 
C 
0,4 0,6 0,8 
Nucleotide divergence 
............. ................................. T 
F 0,002i • _ i 
pColV-K30 0,00$1 
R100 
pSLT 
Rt 
P307 
o 0.2 0,4 0,6 0,8 
Nucleotide divergence 
Q; 1048 
0;9808 
1.2 
1.2 
• -1,0 39 
1. 491 
1,2 
D 
F O 
pColV-K30 O 
R1 
P307 
R100 
pSLT 
E 
o 0,2 0.4 
:· ! 
i 0.606 i 
07;011 
0!7232 
p,7647 
0,6 o.s 
Nucleolide divergence 
traY 
1,2 
. -~~=--~==--
F 
R100 
pColV-K30 
pSLT 
F 
o 0,2 
R 100 il 0,0237 
! . _; __ · 
0 ,4 0,6 0,8 
Nucleotide divergence 
F !III o.q1no 
pSLT - [O; 1584 
o 0,2 0,4 0,6 0,8 
Nucleotlde divergence 
G 
1raD 
traI 
F •• 0,031;5 fil10 
R100 1a 0,05~2 
1,2 
1,2 
···i··._,.,3·3· 79· .. . ... ......... , ......................... . 
{ .·" ·- 1 
pSLT ~~~~~~~L-~ _ _j _ _J 
o 0,2 0,4 0,6 0,8 1,2 
Nucleotide divergence 
Figure 2: Nucleotide divergence of the obtained pRK l 00 tra sequences and related plasmid sequences. 
The nucleotide sequences of pRK l 00 related plasmids used in the sequence analysis of tra pRKI 00 
nucleotide sequences are deposited in GenBank under the following accession numbers: F-plasmid -
UOl 159; pColV-K30 - AF237697 (traM, fin?, tral), AF237694 (traY), AF237692 (traD); P307 -
M62986; RlOO - AP000342; Rl - Ml 9710 and pSLT -AE00647 l. 
Nucleotide divergence, as the measurement of phylogenetic nucleotide distance, was calculated according 
to the Kimura-2 parameter. Nucleotide divergences between pRKlOO sequences and sequences of 
other related plasmids are plotted: Panel A - nucleotide divergence of traM, panel 8 - nucleotide 
divergence of finP, panel C - nucleotide divergence of traJ, panel D - nucleotide divergence of traY, 
panel E - nucleotide divergence of traD, panel F - nucleotide divergence of tral and panel G -
nucleotide divergence offinO. 
Slika 2: Divergenca nukleotidnih zaporedij genov tra plazmida pRKlOO in nukleotidnih zaporedij 
sorodnih plazmidov. Nukleotidna zaporedja plazmidov sorodnih pRKlOO, katera smo uporabili v 
analizi zaporedij tra genov pRKIOO, so shranjena v GenBank in označena s sledečimi številkami: 
Plazmid F - UOl 159; pColV-K30 - AF237697 (traM, fin?, tral) , AF237694 (traY), AF237692 
(traD); P307 - M62986; RlOO -AP000342; R 1 - Ml 9710 in pSLT -AE00647 l. 
14 Acta Biologica Slovenica, 45 (]), 2002 
Nukleotidna divergenca kot izračun filogenetske drugačnosti nukleotidov je bila preračunana z uporabo 
parametra Kimura-2. Divergence nukleotidnih zaporedij plazmi da pRKl 00 in nukleotidnih zaporedij 
sorodnih plazmidov so prikazane: panel A-nukleotidna divergenca gena traM, panel B - nukleotidna 
divergenca gena.fin?, panel C - nukleotidna divergenca gena traJ, panel D - nukleotidna divergenca 
gena traY, panel E- nukleotidna divergence gena traD, panel F - nukleotidna divergenca gena tra/ in 
panel G - nukleotidna divergence genafinO. 
Discussion 
In order to characterise the tra region of pRKIOO, nucleotide sequences of seven different tra 
regi on genes, amplified by PCR, were determined and compared with nucleotide sequences of related 
plasmids. The chosen tra region genes were: traM, finP. traJ, traY from one end the and traD, tral, 
finO from the other end of the pRK l 00 tra regi on. The obtained pRK l 00 nucleotide sequences were 
compared with nucleotide sequences of the following F-like plasmids: plasmid F, pColV-K30, P307 
and Rl from Escherichia coli, RIO0 from Shigellafiexneri and pSLT from Salmonella typhimurium. 
The choice of related plasmids was based on the fact, that pRK 100 seems to be an F-like plasmid, 
which evolved from at least two F-like plasmids. In our previous work it was discovered, that pRKl 00 
is an IncFI plasmid harbouring a RepFIB (Ambrožič & al. 1998) and RepFIIA replication regi on. The 
RepFIB replication regi on is similar to the RepFIB replication regi on of plasmid F, pColV-K30, P307, 
and the RepFIIA replication region is similar to RIO0 (our unpublished data). pRKIOO also encodes 
colicin V and the aerobactin uptake system as does plasmid pColV-K30 (Ambrožič & al. 1998). 
Further, it also carries IS/ , which is also present on pColV-K30 and on plasmid Rl 00. Apart from ISJ , 
pRKIO0 also encodes IS2 and ISJ insertion sequences (our unpublished data), which are also present 
on plasmid F, but not on RI00. 
A similar melange also emerges from results presented in this paper. The traM gene of pRKIO0 
has the same nucleotide divergence with traM ofF and traM of pColV-K30; thefinP gene of pRKlO0 
is exactly the same asfinP ofF; traY of pRKlOO has the same nucleotide sequence as traY ofF but also 
the same sequence as tra Y of pColV-K30; pRK l 00 traJ, traD and fin O are most similar to traJ, traD 
and finO of plasmid F; but tral of pRK 100 is most similar to tra/ from R 100. On the basi s of our 
results we can therefore conclude that, the tra regi on of pRK 100 is F-like and it is most similar to the 
tra region of plasmid F. 
Even though ali the plasmids, incorporated into this study, are known to be F-like plasmids, 
nucleotide sequence divergence among the different genes is not the same, some genes are more 
conserved than others. For example, the regulatory genes traJ and traY exhibit greater differences, than 
for example the traD gene, whose product is involved in transmembrane conveyance of nucleic acids 
(Firth & al. 1996). Plasmid genes are mosaic in structure due to multiple recombination events between 
diverse ancestral genes (Boyd & al. 1996). Conjugation by introducing horizontally transferred DNA 
into cells increases the opportunity of different plasmids to meet and exchange genetic information . 
Knowledge of plasmid structure and transfer genes is necessary to develop efficient means to reduce 
plasmid transfer and dissemination of antibiotic resistances as well as bacterial virulence factors. 
Conclusions 
To summarise and conclude: 
l. different tra genes of pRKIO0 show different levels of nucleotide divergence with different 
related plasmids ; 
2. the tra regi on of pRK 100 is F-like; 
3. the pRKIO0 tra region is pronouncedly most similar to the tra region of plasmid F 
4. the pRKIO0 tra region is mosaic. 
M. Starčič Erjavec, W. Gaastra, D. Žgur-Bertok: tra region of the natura! conjugative Escherichia ... 15 
Acknowledgement 
The authors are thankful to Peter Trontelj for his help with computer programs CLUSTAL W and 
PHYLIP. 
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