1 Institute for Molecular Biology, Nankai University, Tianjin, People's Republic of China
2 The Second Hospital of Tianjin Medical University, Tianjin, People's Republic of China
3 Department of Applied Chemistry and Microbiology, Fin-00014 University of Helsinki, Finland
Correspondence
Mingqianq Qiao
mingqiangqiao{at}yahoo.com.cn
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ABSTRACT |
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INTRODUCTION |
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The colonization and establishment of infection by P. aeruginosa are dependent on the production of a number of virulence factors, including lipases, proteases, exopolysaccharides, alkaline phosphatases and type IV pili (Beatson et al., 2002). Type IV pili are flexible surface filaments about 6 nm in diameter produced at the poles of the bacterial cell (Beatson et al., 2002
; Mattick, 2002
); they are essential for the attachment of the pathogen to host epithelial tissues and also mediate a form of surface translocation known as twitching motility (Bradley, 1980
; Mattick, 2002
). Twitching motility has been shown to be required for the initial attachment and development of a biofilm by P. aeruginosa (O'Toole & Kolter, 1998
; Costerton et al., 1999
; Mcbride, 2001
; Whiteley et al., 2001
; Whitchurch et al., 2002
). Once a biofilm is developed, cells growing in the biofilm can become 101000 times more resistant to the effects of antibiotics than their planktonic counterparts (Mah & O'Toole, 2001
). Biofilm bacteria embedded in an extracellular polymeric matrix cannot be eradicated even with the most aggressive antibiotics. Mutants that either lack type IV pili or are twitching-motility deficient show loss of ability for biofilm initiation (O'Toole & Kolter, 1998
) and reduced infectivity (Kang et al., 1997
; Whitchurch et al., 2002
).
P. aeruginosa pili are polymers of a single gene product, called PilA or pilin (Mattick, 2002), but their assembly and function require the products of almost 40 additional genes (Alm & Mattick, 1997
; Mattick, 2002
). The regulation of twitching motility, and the role and the signals of the signal transduction systems involved are still obscure.
Because twitching motility is crucial for systemic infection and biofilm formation inside a host, identification of genes involved in twitching motility may be helpful to develop new drugs in the future, which will help the host to eradicate P. aeruginosa more readily. In this study, we created a transposon mutant pool using Mu transposition complexes in P. aeruginosa PA68, and isolated mutants deficient in twitching motility. Analysis of these transposon insertion mutants identified two new genes required for twitching motility.
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METHODS |
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Identification of strain PA68.
The 16S rRNA gene of PA68 was PCR amplified (5 min at 94 °C; 30 cycles of 45 s at 94 °C, 45 s at 55 °C, 2 min at 72 °C; 10 min at 72 °C) from chromosomal DNA of PA68 using the universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-CGGYTACCTTGTTACGACTT-3') (Lane, 1991; Cai et al., 2003
), and the PCR product was cloned into cloning vector pMD18 (TaKaRa), following the manufacturer's instructions. The resulting plasmid, pMD18-16S, was used as the template for DNA sequencing to obtain the sequence of the 16S rRNA gene.
Construction of a mini-Mu insertion library in P. aeruginosa strain PA68.
The artificial mini-Mu transposon (KmMu) used in this study is defined as a segment of DNA that contains 50 bp of Mu R-end DNA as inverted repeats at each end, and a kanamycin resistance gene between the two ends; it was used for selection of transformants. A random insertion library was constructed by using Mu DNA transposition complexes, or MuA transpososomes, which were assembled with artificial mini-Mu transposons and MuA transposase in vitro and analysed by agarose gel electrophoresis, as previously reported (Lamberg et al., 2002). The MuA transpososomes were introduced into P. aeruginosa PA68 by electroporation. Mutants were selected on LB agar plates containing 50 µg kanamycin ml1.
Electroporation.
Electrocompetent cells were prepared as described by Smith & Iglewski (1989). Electroporation was carried out at the following settings: capacitance, 25 µF; electrical field strength, 13 kV cm1; resistance, 200
. DNA used for electroporation was prepared by the alkaline lysis procedure (Sambrook et al., 1989
). For the construction of a mini-Mu transposon insertion library, 50100 ng MuA transpososomes were electroporated into electrocompetent P. aeruginosa PA68 cells. For gene replacement experiments, the plasmid DNA was linearized by a restriction enzyme (Arora et al., 2000
), and about 2 µg linear plasmid DNA was electroporated into electrocompetent P. aeruginosa PAO1 cells. For complementation experiments, 50100 ng supercoiled or covalently closed, circular plasmid DNA was electroporated into the target strains.
Twitching motility assay.
Twitching motility was assayed as described by Semmler et al. (1999). Briefly, cells were stab inoculated with a toothpick through a thin (approximately 3 mm) Difco LB agar (1 % Difco granulated agar) layer to the bottom of the Petri dish. After overnight growth at 37 °C, the zone of twitching motility between the agar and Petri dish interface was visualized by staining with Coomassie Brilliant Blue R250 (Sino-American Biotechnology).
Recombinant DNA techniques and sequence analysis.
The preparation of plasmid and genomic DNA, restriction endonuclease digestion and ligation reactions were carried out using standard protocols (Sambrook et al., 1989). Briefly, genomic DNA of each twitching-motility-deficient mutant was digested with BamHI (there is no BamHI site in the artificial mini-Mu transposon), generating a fragment with a transposon attached to its genomic DNA flanks. These fragments were then cloned into the BamHI site of pUC18. DNA sequences of transposon borders were determined from these recombinant plasmids by using transposon-specific primers reading sequences outwards from within the transposon. The primers used for DNA sequencing were primer1 (5'-GCAACTGTCCATACTCTGA-3') and primer2 (5'-CGCTGGGTTTATCGTCGA-3'). DNA sequencing was performed by Shanghai Sangon Biological Engineering Technology and Service Co. Ltd (Sangon). More than 500 nucleotides were sequenced on each flank of the insertion. The genomic locations of mini-Mu insertions were identified by using the flank sequences to do a BLAST search in the P. aeruginosa genome database (www.pseudomonas.com), or at the National Center for Biotechnology Information servers (www.ncbi.nlm.nih.gov). Domain analysis of genes was performed using SMART (http://smart.embl-heidelberg.de/) (Schultz et al., 2000
) and the Pfam database (http://pfam.wustl.edu/) (Bateman et al., 2000
). Function analysis of genes was performed using the Pseudomonas aeruginosa Community Annotation Project (PseudoCAP) (http://www.pseudomonas.com/GenomeSearchU.asp).
Southern blotting.
Chromosomal DNA isolated from twitching-defective mutants was digested with restriction enzymes, and electrophoretically separated in 0·8 % agarose gels; DNA was transferred onto a positively charged nylon membrane (Sino-American) and fixed by UV cross-linking. Southern analysis was performed using a PCR-generated 700 bp KmMu fragment as a probe. All probes were labelled with [-32P]dCTP (Beijing Yahui Biotechnology Company) using a random primer labelling kit (TaKaRa) according to the manufacturer's instructions.
PCR amplification and primers.
PCR was performed in a DNA Pelitre Thermal Cycler PTC-200 (MJ-research) to obtain specific amplification products. The reactions were performed in a final volume of 25 µl. Each reaction mixture contained 20 ng DNA template, 1·25 U LA Taq polymerase (TaKaRa), 1·5 mM MgCl2, 0·1 mM deoxynucleoside triphosphates mix and 0·2 µM primers. Thirty cycles were run, each consisting of incubation for 1 min at 94 °C, 1 min at 55 °C, and 14 min at 72 °C (depending on the length of the amplified segments). The primers used for PCR were purchased from Sangon. Restriction enzyme recognition sites were added to the ends of primers (shown in bold type, below) to facilitate subsequent cloning of the PCR products if desired. Additional nucleotides were added to the 5' ends (shown in italic) to ensure efficient cleavage. The following primers were used for PCR. Primer S1 (5'-AAGCTTCTTCCTAAAACACGGGGTC-3'), with a HindIII site, was used as the 5' end primer, and primer S2 (5'-GGATCCCATCCTGTCCTTCCGATTC-3'), with a BamHI site, was used as the 3' end primer to amplify the complete PA0171 gene from P. aeruginosa PA68 or PAO1. Primer F1 (5'-CCCAAAGGATCCGGGAGGCGCACTAGACC-3'), with a BamHI site, and primer F2 (5'-CCCAAAGGATCCCATCAGGCGGCCACCGG-3'), with a BamHI site, were used for the amplification of the complete PA1822 gene from P. aeruginosa PA68. Forward primer FP1 (5'-GGATCCTCTGCTCAAGGAACTGCT-3') (BamHI site in bold) and the reverse primer FP2 (5'-AAGCTTGCTTGTCGCCATTGGATT-3') (HindIII site in bold) were used to amplify a 620 bp PA1822 segment from P. aeruginosa PAO1. This fragment contains only one PstI site, while the complete PA1822 gene contains four PstI sites, which facilitated subsequent plasmid construction for insertional inactivation of the PA1822 gene. Primer Mu1 (5'-GCCGCTGATCATCTAGAGA-3') and primer Mu2 (5'-TCCCACCAGCTTATATACCT-3') were used for amplification of a 700 bp mini-Mu transposon segment from the plasmid carrying the artificial mini-Mu transposon. This fragment was used as probe in Southern hybridization.
Electron microscopy.
Carbon-coated copper grids were gently placed on the surface of the colony grown after 12 to 15 h at 37 °C on LB agar plates; after 1 min, the grids were carefully removed, rinsed twice with distilled water, and stained with 1 % phosphotungstic acid. The negatively stained cells were visualized with a Philips EM400-ST transmission electron microscope.
Construction of plasmids and mutant strains.
A 593 bp amplification product was obtained by PCR using primers S1 and S2 with PAO1 genomic DNA as template. This fragment, containing the complete PA0171 gene, was cloned to the PCR cloning vector pMD18, yielding pMD18S. pMD18S was linearized at the unique NaeI site present in the PA0171 gene, and a gentamicin-resistance gene cassette excised from pUC7G and blunt-ended by a fill-in reaction was inserted at that site, leading to the construction of pMD18SGm. This plasmid was utilized to generate a chromosomal mutation in the P. aeruginosa PA0171 gene by marker exchange in strain PAO1. Using the same strategy, a 620 bp PA1822 fragment was obtained by PCR with the primers FP1 and FP2, with PAO1 genomic DNA as template, and cloned into vector pMD18. This PA1822 fragment was subcloned into BamHI/HindIII site in pUC18, which removed a PstI site in pUC18, yielding pUC18FP. A gentamicin-resistance gene cassette excised from pUC7G was inserted into the unique PstI site in the PA1822 fragment, generating pUC18FPGm. This construct, which cannot replicate in P. aeruginosa, was used to generate a chromosomal mutant of PAO1 PA1822 by gene replacement. Plasmid pDN18S, which was obtained by cloning a 593 bp PCR fragment carrying the complete PA68 PA0171 gene using primers S1 and S2 into the BamHI and HindIII sites of the broad-host-range vector pDN18, was used for complementation of PA68 PA0171 mutant C54. Plasmid pDN18F, used for complementation of the PA1822 mutation in K2, was constructed by cloning a 1733 bp PCR fragment containing the PA68 PA1822 gene into the BamHI site of pDN18 in positive orientation relative to the T7 promoter. This fragment was amplified by PCR using primers F1 and F2 from the chromosomal DNA of PA68. Plasmids pDN18S and pDN18F were also used as templates for DNA sequencing of the PA68 PA0171 and PA1822 genes, respectively.
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RESULTS AND DISCUSSION |
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Mu transpososome mutagenesis is an efficient mutagenesis strategy, and has been used for the functional analysis of some bacterial genomes, such as E. coli and Yersinia enterocolitica (Lamberg et al., 2002). In this study, MuA transpososomes were electroporated into P. aeruginosa PA68 using the protocol described in Methods. High transformation efficiency was achieved (up to 3·66x104 transformants per µg transposon DNA), and a pool of approximately 6000 Mini-Mu insertion mutants of PA68 was made. These Mu transposon mutants were screened for defects in twitching motility, using the subsurface stab assay. When cells are stabbed through an agar layer to the bottom of the Petri dish, colony expansion at the interstitial surface between the agar and the plastic occurs by twitching motility (Semmler et al., 1999
; Rashid & Kornberg, 2000
). Eight mutants deficient in twitching motility were isolated, out of approximately 2000 insertion mutants (Fig. 1A
). Southern blotting analysis was performed and demonstrated that all insertions were unique and only one copy of the transposon had integrated into the genomes of the mutant strains (data not shown).
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Construction of PA0171 and PA1822 mutants in the PAO1 strain
PA68 is a clinical P. aeruginosa strain. To confirm that PA0171 and PA1822 are also required for twitching motility in strain PAO1, a PA0171 mutant of strain PAO1 was constructed by gene replacement. The PAO1 PA0171 gene located on a 593 bp BamHI/HindIII PCR-generated fragment was inactivated by inserting a Gmr cassette into the unique NaeI site in this gene. The insertionally inactivated PA0171 gene on a non-replicating plasmid (pMD18SGm) was introduced into PAO1 by electroporation, where it replaced the corresponding chromosomal copy of the PA0171 gene by double reciprocal recombination, giving rise to a PA0171 mutant strain, PAO1-NS. The replacement of the wild-type PA0171 in PAO1-NS was confirmed by PCR. A 2·4 kb fragment was amplified using primers S1 and S2 combined with PAO1-NS genomic DNA as template (0·6 kb PA0171 gene with a 1·8 kb gentamicin-resistance gene insertion), while a 0·6 kb fragment was amplified using primers S1 and S2, and PAO1 genomic DNA as template. PAO1-NS was non-motile on 1 % LB agar (Fig. 1C). The PA1822 mutant strain PAO1-NF was constructed by using the same strategy and it was also deficient in twitching motility (Fig. 1C
). These results indicated that PA0171 and PA1822 are also needed for twitching motility in P. aeruginosa PAO1.
Alignment analysis and putative function of PA0171 and PA1822 genes
The complete nucleotide sequences of PA0171 were determined (GenBank accession number AY502957). Alignment of the sequences of this gene in PA68 and PAO1 using BLAST analysis showed that the two sequences are more than 99 % identical at the nucleotide level, and 92 % identical at the deduced amino acid level. The sequences of the deduced protein of PA0171 show 38 % identity with a conserved hypothetical protein of Geobacter sulfurreducens PCA.
PA0171 encodes a relatively short ORF (543 nucleotides). The gene and the putative protein have no significant homology with any gene or protein with known function in P. aeruginosa or other bacteria. However, a chemotaxis-like system, designated chemotaxis gene cluster 4 (Croft et al., 2000), is located upstream of this gene. This chemotaxis system is entirely novel and has no known function (Croft et al., 2000
); it is composed of PA0179, PA0178, PA0177, PA0176, PA0175, PA0174, PA0180 and PA0173, which encode homologues of CheY, CheA, CheW, CheR, CheD, CheB, and two associated MCPs, respectively (Fig. 2
). PA0172 is located directly upstream of the PA0171 gene, and is predicted to encode a protein containing a HAMP domain, which is found in bacterial sensor and chemotaxis proteins (Pfam); accordingly, we predict that PA0172 is involved in this chemotaxis system. PA0169 is located downstream of PA0171, and is predicted to encode a protein containing a DUF1 (or DDGFF) domain; other proteins with a similar domain structure include the Caulobacter crescentus protein PleD, which is part of a signal transduction pathway controlling cell differentiation (Hecht & Newton, 1995
; Aldridge & Jenal, 1999
). In P. aeruginosa, it probably participates in a phosphotransfer-dependent signal transduction pathway involving this chemotaxis-like system. In this study, the insertion in PA0171 not only caused twitching-motility deficiency, but also affected swimming and swarming motility, and all of the motility defects in the PA0171 mutant could be complemented by a plasmid harbouring the PA0171 gene (data not shown), suggesting that PA0171 could be part of this chemotaxis operon. Examination of morphology by transmission electron microscope showed that the type IV pili of the PA68 PA0171 mutant could not be detected, and that the type IV pili could be restored by trans-complementation (Fig. 3
). These data indicated that PA0171 is involved in the regulation of both motility and type IV pilus biogenesis.
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ACKNOWLEDGEMENTS |
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Received 3 March 2004;
revised 15 April 2004;
accepted 28 April 2004.
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