A novel ColV plasmid encoding type IV pili

Uri Gophna1, Anat Parket1, Joerg Hacker2 and Eliora Z. Ron1

1 Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel
2 Institut für Molekulare Infektionsbiologie, 97070 Würzburg, Germany

Correspondence
Eliora Z. Ron
eliora{at}post.tau.ac.il


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many septicaemic Escherichia coli strains harbour ColV virulence plasmids. This paper describes pO78V, a conjugative ColV plasmid from an avian pathogenic E. coli strain that encodes type IV pili in addition to other virulence-related genes and tetracycline resistance. Plasmid location of type IV pili genes was demonstrated using Southern hybridization and expression of the pili was demonstrated using RT-PCR and phage sensitivity assays. This is a first report of a ColV plasmid encoding type IV pili. Plasmid pO78V is a mosaic plasmid containing replicons and other genes typical to both IncI1 and IncFII groups. As type IV pili of Gram-negative bacteria are involved in several stages of infection, their presence on a ColV virulence plasmid could expand the repertoire of pathogenesis-related genes.

Abbreviations: DIG, digoxigenin


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
ColV plasmids are large plasmids which encode the production of a colicin known as colicin V and are found primarily in virulent, mainly septicaemic, strains of Escherichia coli (Lafont et al., 1987; Warner et al., 1981; Waters & Crosa, 1991). Although the production of colicin V does not directly contribute to virulence (Quackenbush & Falkow, 1979), the introduction of a ColV plasmid significantly increases the virulence of the recipient bacteria (Smith & Huggins, 1976). This effect on virulence is attributed to the finding that members of this diverse group of plasmids harbour a variety of virulence properties, including the aerobactin iron uptake system, increased survival in serum, resistance to phagocytosis, temperature-sensitive haemagglutinin (Dozois et al., 2000) and adherence to intestinal epithelial cells. For a comprehensive review see Waters & Crosa (1991).

Type IV fimbriae are newly discovered pili present in several pathogenic Gram-negative bacteria. They are involved in dispersal of bacteria from autoaggregates in Neisseria (Pujol et al., 1999) and enteropathogenic E. coli (Bieber et al., 1998). In addition, type IV pili mediate adherence to host epithelial cells in Vibrio cholerae (Fullner & Mekalanos, 1999), Neisseria (Nassif et al., 1993) and Legionella pneumophila (Stone & Abu Kwaik, 1998). In Pseudomonas aeruginosa they play a role in multiple aspects of interaction with host cells: adherence (Chi et al., 1991), internalization and cytotoxicity (Comolli et al., 1999). In Neisseria and L. pneumophila, type IV pili are also required for natural transformation of DNA (Stone & Abu Kwaik, 1999; Wolfgang et al., 1999).

In enteric bacteria – Shigella sonnei and Salmonella typhimurium – type IV pili are encoded by the IncI1 group plasmids ColIb-P9 and R64, respectively (Kim & Komano, 1997) and are essential for conjugal transfer of these plasmids in liquid medium (Komano et al., 1995). These plasmids have not so far been associated with virulence.

This is a first report of a ColV plasmid encoding type IV pili. This ColV plasmid, which also encodes the aerobactin iron-uptake genes, may provide its host with an extended arsenal of virulence properties.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains used.
E. coli strain 789 was isolated from the bone marrow of a chicken with acute colisepticaemia (Yerushalmi et al., 1990). E. coli K-12 strain RBE23-17 was previously described (Babai & Ron, 1998).

PCR.
PCR reactions described were performed using either ExTaq proofreader thermostable polymerase (for products that underwent sequencing) or rTaq thermostable polymerase (Takara) using buffers and dNTPs supplied by the manufacturer. For amplification conditions and primers used see Table 1.


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Table 1. Primers and amplification conditions used in this work

 
Creation of a strain-specific mini-library by subtractive DNA–DNA hybridization.
A strain-specific mini library of strain 789 sequences was created by subtractive hybridization using the PCR-Select bacterial genome subtraction kit (Clontech), according to the manufacturer's instructions. Subtractive hybridization is a method used to enrich for strain-specific sequences. Briefly, genomic DNA from each strain – the driver (E. coli K-12 MG1655) and the tester (E. coli 789) – was digested with RsaI and two different PCR adaptors were ligated to two different aliquots of the tester DNA. Primary hybridization was performed with an excess of driver DNA using each of the tester ligation mixtures. Samples were heat denatured and allowed to anneal. After this hybridization, single-stranded DNA was enriched for tester-specific DNA, as DNA fragments that are not tester specific will form hybrid molecules with the driver DNA. A second hybridization was then performed where the two primary hybridization reaction mixtures were mixed together without denaturing. The result of this reaction was a reassociation of subtracted single-stranded tester-specific DNA. These double stranded DNA molecules had different adaptors on each end that could be used for specific amplification.

Amplification products were cloned into a pGEM-T vector (Promega) and transformed into competent E. coli XL-1 Blue cells. White colonies were picked and used as templates for PCR amplification (using Takara ExTaq DNA polymerase and its standard dNTP mix, with primers pUCF and pUCR (see Table 1). PCR products from all the reactions were purified using Concert rapid PCR purification kit and sequenced as previously described (Babai et al., 2000) using universal primers and when required, specific primers as detailed in Table 1.

Sequence analysis.
Sequences were compared with the databases using BLASTN and BLASTX (Altschul et al., 1997) at NCBI (http://www.ncbi.nlm.nih.gov).

Southern hybridization.
DNA of pO78V was isolated as previously described (Kado & Liu, 1981) using conditions recommended for E. coli. DNA was loaded onto four lanes and electrophoresed through an agarose gel (0·75 %). Digoxigenin (DIG)-labelled probes were generated by PCR, using DIG-labelled dNTPs (Roche) according to the manufacturer's instructions. Following transfer, membrane was split into four parts, each containing plasmid DNA and each lane was hybridized with a different probe: one specific for each of pilN, cvaC, iucD, and 23S rRNA at 100 % stringency. For detection, anti-DIG antibody–alkaline phosphatase conjugate was used and visualization was performed using the CDP-star chemiluminescent substrate for alkaline phosphatase.

Conjugation.
Mating was performed using E. coli K-12 strain RBE23-17 as a recipient strain. This strain does not ferment lactose and is kanamycin resistant. Both donor (E. coli strain 789, lactose fermenting) and recipient were grown to mid-exponential phase with moderate aeration. Liquid mating was performed by mixing equal amounts of recipient and donor cells, and incubating them at 37 °C for 2 h. Conjugation on solid medium was performed by filtering equal amounts of donor and recipient cells onto filters with 0·2 mm pore size, and incubating on LB plates for 2 h. Conjugants were selected on MacConkey agar plates containing tetracycline (17·5 mg l-1) and kanamycin (30 mg l-1).

RT-PCR.
Bacterial total RNA was isolated from strain 789 using the Promega SV total RNA isolation kit and reverse transcribed using Expand reverse transcriptase (Roche) according to the manufacturer's instructions. DNA obtained from reverse transcription was used as a template for PCR, using non-reverse transcribed RNA as a negative control.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
DNA sequences homologous to IncI plasmid genes
E. coli strain 789 is an avian pathogen which harbours one ColV plasmid of at least 80 kb in size (Ron et al., 1991), referred to in this work as pO78V. To identify new virulence-related genes in this strain, we used subtractive hybridization with a non-virulent K-12 strain, to construct a strain-specific mini library (see Methods). In this mini library we identified 14 sequences homologous to sequences previously characterized in large plasmids (Table 2). Two of these were identical to genes involved in F-pilus biogenesis and another to a gene involved in production of aerobactin. This finding is compatible with our previous data, indicating that the one large plasmid present in our strain – pO78V – is a ColV plasmid, encoding aerobactin (Babai et al., 1997; Ron et al., 1991). However, the remaining 11 sequences showed a high degree of similarity to sequences from various regions of IncI1 plasmids R64 and ColIb-P9.


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Table 2 Sequences obtained from subtractive hybridization showing homology to genes found in large plasmids

Numbers indicate positions in referenced GenBank entries

 
Analysis of orthologues
The identified sequences showing homology to a typical IncI1 plasmid included maintenance genes such as those encoding stable inheritance proteins (parA, parB), the exc gene encoding a surface exclusion protein and the impA gene, encoding an error-prone DNA repair system. Another group of genes with high similarity between pO78V and the IncI1 plasmids contains genes involved in conjugal DNA transfer: nikB, traB, traC, traQ, traT and traU (Komano et al., 2000). The putative TraE homologue in pO78V is truncated after 160 aa, due to the presence of an insertion sequence (IS1). This truncation, which probably reflects a relatively recent transposition event, may not be detrimental, since plasmids without a functional TraE have been shown to be unaffected in liquid mating (Komano et al., 2000).

The data also indicate that pO78V contains genes involved in type IV pili biogenesis, including the shufflon-specific recombinase rci, responsible for gene shuffling, the pilL and pilN genes encoding lipoproteins required for pili biogenesis (Sakai & Komano, 2000) and pilK which is also essential for formation of pili. Taken together, the sequence data indicate that pO78V is a mosaic plasmid consisting of genes originating from ColV plasmids and IncI plasmids.

Localization of type IV pili genes to the plasmid
Southern hybridization was carried out to determine whether the type IV pili genes are located on the ColV plasmid pO78V. The native plasmid DNA was hybridized with probes specific for pilN (a gene encoding a lipoprotein involved with type IV pili biogenesis in R64), cvaC (from the biosynthetic pathway of colicin V), iucD (from the aerobactin synthesis pathway) and a 23S rDNA probe to identify chromosomal location. The results demonstrated that pil specific probes (pilN) and ColV-plasmid specific probes (cvaC, iucD) hybridized with the same DNA band (Fig. 1b), corresponding with plasmid DNA (Fig. 1a). The negative control – 23S rDNA probe – did not hybridize with the plasmid band. The data support the assumption that the IncI pil genes are indeed localized on the ColV plasmid.



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Fig. 1. Southern hybridization of pO78V to ColV-related genes and genes encoding type IV pili. DNA of pO78V was isolated as described in the text. Samples of 10 µl were electrophoresed on 0·75 % agarose gels (a). DNA was subjected to DIG Southern hybridization (Roche Molecular Biochemicals), as described in the manufacturer's instructions (b). Lanes represent hybridization with the following probes: pilN, a gene encoding a lipoprotein involved with type IV pili biogenesis in R64; cvaC, from the biosynthetic pathway of colicin V; iucD, from the aerobactin synthesis pathway; and 23S rRNA, a chromosomal gene used as negative control.

 
Conjugation of pO78V
Plasmids belonging to the IncI group, as well as ColV plasmids, are usually conjugative. To further characterize pO78V we determined its ability to be transferred by conjugation. As many IncI1 plasmids carry the gene for tetracycline resistance, and as strain 789 is tetracycline resistant, we examined whether this resistance could be transferred by conjugation to a tetracycline sensitive K-12 strain. Resistant colonies were obtained by conjugation both in liquid and on solid medium. In liquid medium the frequency of tetracycline resistant colonies per recipient colony was about 10-5. All of the tetracycline resistant colonies exhibited the Lac- phenotype of the recipient and contained the plasmid genes iucD and pilN, as determined by PCR. These genes were probably retained on the original plasmid, which could be detected by standard procedures. There were no spontaneous tetracycline resistant colonies in the recipient-only control, and we could not detect either iucD or pilN in this control strain by PCR. None of the recombinant colonies contained the fyuA gene, which is part of the chromosomally encoded high pathogenicity island, previously identified in strain 789 (Gophna et al., 2001a). These data indicate that pO78V is a conjugative plasmid that encodes tetracycline resistance in addition to aerobactin biosynthesis and pil genes.

Plasmid encoded type IV pili are expressed in strain 789
Production of type IV pili in the IncI plasmid R64 involves 14 genes (pilIpilV) which are organized in a single operon. Twelve of these genes (pilK to pilV) are required for biogenesis of the pili (Yoshida et al., 1999), the pilS gene encodes the major prepillin, processed by the pilU product, and pilV encodes the minor component of the pili (Yoshida et al., 1999). In addition, the gene cluster contains a shufflon-specific recombinase rci (Gyohda & Komano, 2000), which mediates recombination events between four DNA segments resulting in several PilV variants. We demonstrated by PCR the presence of the pilS and pilV structural genes (for primers used see Table 1) in pO78V, suggesting the presence of an intact pil gene cluster. To examine whether these genes are transcribed we performed RT-PCR and confirmed the transcription of the major prepilin gene pilS (Fig. 2).



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Fig. 2. Agarose gel electrophoresis of RT-PCR products amplifying the prepilin gene pilS. Experiment was carried out as described in the text using primers detailed in Table 1. As a negative control, the RT reaction was performed without the antisense primer. Lane 1: pilS product obtained from amplification of genomic DNA; lane 2: pilS product obtained from amplification of RT reaction; lane 3: negative control; lane 4: DNA ladder (Epicenter).

 
Further demonstration of the expression of IncI pili was obtained by using the IncI1 specific phage PR64FS, to which the wild-type bacteria carrying pO78V were extremely sensitive (Fig. 3), even more than strain JM83(pCD641-A) which expresses IncI pili constitutively (kindly provided by T. Komano, Tokyo Metropolitan University, Japan). The same bacteria were not sensitive to the IncI2 specific phage I2-2 (phages kindly provided by H. W. Ackermann, Quebec, Canada).



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Fig. 3. Plaques obtained with phage specific for IncI pili. Drops of phage PR64FS (serial dilutions) were applied to soft agar containing about 107 ml-1 of the following bacteria: 789, K-12 strain JM83(pCD641-A) constitutively expressing IncI type IV pili (positive control), wild-type K-12 strain not expressing type IV pili (negative control).

 
Replication control genes of pO78V
Since pO78V appeared to contain genes typical of the IncF (ColV) and IncI incompatibility groups we examined its replication control regions, including the genes that determine incompatibility. Several sets of primers were used to amplify replication-related genes from a K-12 strain carrying the pO78V plasmid (see above). The repYZpO78V genes, involved in replication initiation, were amplified by PCR and were found to be 100 % identical to those of pColIb-P9 and R64, as was the IncIpO78V gene encoding the small countertranscript RNA involved in copy number control of IncI plasmids, placing pO78V in the IncI group. However, by sequence analysis we could show that the plasmid also contains a typical IncF replication region, including an origin of replication and control genes (repA1, repB and copA). The predicted hairpin structure formed by the copApO78V is similar to that of other IncF plasmids and is identical in sequence to that of ColV2-K94 (accession M13472) as illustrated in Fig. 4. Moreover, the IncF-like replication region of pO78V has more than 94 % identity with the Shigella flexneri large virulence plasmid pWR501 (accession AF348706), which has a RepFIIA-like replicon and belongs to the IncFII group (Venkatesan et al., 2001). Thus, it appears that pO78V is a multi-replicon plasmid containing replication genes from the IncI and IncF groups.



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Fig. 4. Predicted structure of the major hairpin formed by copA from pO78V compared to the R100 homologue, as predicted by Zuker's mfold algorithm (Mathews et al., 1999; http://www.bioinfo.rpi.edu/applications/mfold/).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Here we describe plasmid pO78V, which is a conjugative ColV plasmid carrying genes encoding virulence factors. However, although ColV plasmids belong to the IncF incompatibility group, pO78V contains many genes characteristic of plasmids of the IncI1 group. Moreover, the plasmid contains two replicons, one of IncF and one of IncI1 and is therefore a co-integrate which could have originated from one or more recombination events, as was previously suggested (Kato & Mizobuchi, 1994). In the case of pO78V, the IncI replicon is highly conserved whereas the IncF replicon is divergent. This is in agreement with previous findings on replicon evolution in mosaic plasmids, where the existence of one conserved replicon relaxes the selective pressure on the other replicon, resulting in a faster rate of evolution (Osborn et al., 2000). Exchange of large amounts of genetic material can be expected in strains that carry a variety of plasmids, including conjugal plasmids, which contribute to virulence and fitness. One such possible case is the presence of genes encoding aerobactin in an unclassified, non-ColV plasmid (Ginns et al., 2000). In pathogenic strains, the mobility of plasmids and genes is probably a driving evolutionary force that accelerates their adaptation to new ecological niches as well as their resistance to antibacterial agents and the immune response.

An unexpected finding was the existence of type IV pili genes in a ColV virulence plasmid. To the best of our knowledge, this is the first report of such a combination of genes, which could broaden the spectrum of plasmid-encoded virulence properties. Since pO78V is a co-integrate of IncI and IncF plasmids, it seems likely that it maintained its IncI-associated pili whose original role was primarily plasmid conjugation, because they conferred some evolutionary advantage – to either the spread of the plasmid or the virulence or fitness of the bacteria carrying it.

As written above, one potential role of type IV pili could be to promote adhesion to host tissue. However, transfer of pO78V to a non-piliated K-12 strain did not improve its adherence to epithelial cells (data not shown). This finding is in agreement with recent data demonstrating that similar pili from a shiga-toxigenic E. coli strain did not contribute to adhesion (Srimanote et al., 2002). The presence of the IncI1 elements could also contribute to pathogenicity by mobilizing bacterial virulence factors, since previous research suggested the possibility that the IncI1 transfer system delivers proteins as well as DNA (Wilkins & Thomas, 2000). This mechanism could lead to a delivery of an effector molecule to host cells as part of bacterial host interaction, as has been previously suggested for L. pneumophila (Segal et al., 1998). In strain 789, the proximity of bacteria to host cells, which would be essential for the execution of such a delivery, could also be facilitated by additional adherence pili, such as the previously characterized AC/I pili (Babai et al., 2000) or curli fibres (Gophna et al., 2001b).


   ACKNOWLEDGEMENTS
 
This work was supported by the Israel Center of Emerging Infectious Diseases and by the Manja and Morris Leigh Chair for Biophysics and Biotechnology. We thank Orlev N. Levy for his invaluable assistance in RT-PCR.


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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 8 July 2002; revised 24 September 2002; accepted 1 October 2002.