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
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ABSTRACT |
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Abbreviations: DIG, digoxigenin
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INTRODUCTION |
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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.
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METHODS |
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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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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 antibodyalkaline 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.
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RESULTS |
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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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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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DISCUSSION |
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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
).
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ACKNOWLEDGEMENTS |
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Received 8 July 2002;
revised 24 September 2002;
accepted 1 October 2002.