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Journal of Bacteriology, March 2007, p. 1689-1697, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01640-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Analysis of PilT from the Toxic Cyanobacterium Microcystis aeruginosa PCC 7806{triangledown}

Kenlee Nakasugi, Ralitza Alexova, Charles J. Svenson, and Brett A. Neilan*

School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia

Received 21 October 2006/ Accepted 8 December 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 ± 1.8 nmol Pi min–1 mg protein–1, with a requirement for Mg2+. Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Microcystis aeruginosa PCC 7806 is a naturally transformable, toxic-bloom-forming cyanobacterium. Putative type IV pilus (Tfp) genes and pilus-like structures in this strain have previously been identified (28). The cyanobacterium possesses a very large gene cluster encoding the production of the hepatotoxin microcystin (10, 43), the arrangement of which is very similar to the microcystin gene clusters of two other phylogenetically distant toxic cyanobacteria, Anabaena sp. strain 90 and Planktothrix agardhii (8, 37). However, within each of these species, non-toxin-producing strains also exist that do not possess microcystin biosynthesis genes. The cumulative gene loss theory has been proposed to explain the random distribution of microcystin toxigenicity in modern cyanobacteria (35). This theory suggests that a common toxic ancestral cyanobacterium gave rise to the toxic and nontoxic genera observed today through successive gene deletions, and not by acquisition via lateral gene transfer. Despite the phylogenetic evidence supporting this theory, lateral transfer of segments of the microcystin gene cluster between closely related species and strains of a given genus may still have occurred (35). An alternative hypothesis for the sporadic distribution of the microcystin gene clusters, at least between closely related species of cyanobacteria, is that of lateral gene transfer (26, 27, 30). In the context of massive cell lysis within a cyanobacterial bloom, lateral gene transfer via natural transformation is a very plausible scenario. The release of DNA provides enormous potential for the transformation of any naturally competent microorganism within the bloom population.

Natural transformation is a widely reported phenomenon in bacterial systems (19), and especially those which possess a Tfp system. Tfp proteins are also responsible for mediating a number of other bacterial physiological processes, such as twitching motility. Tfp proteins occur in a wide range of gram-negative bacteria, including Pseudomonas aeruginosa and Neisseria gonorrhoeae, the genetics of which have been well characterized (1, 12, 22, 23). Evidence for the association of the Tfp system and natural transformability is substantial, and often the presence of a Tfp system is an indication of natural competence (11, 19). In the gram-positive bacteria, accessories of the DNA uptake apparatus closely resemble Tfp components, including the ComG proteins of Bacillus subtilis and the Cil/Cgl proteins of Streptococcus pneumoniae (11).

One particularly important component of the Tfp system is the PilT protein, essential for twitching motility and for generating the pilus retraction force required for pulling the cell along a surface (21, 41). It possesses the nucleotide-binding Walker box motifs found in many NTPases (46) and is thought to cause the retraction of pili through the disassembly of subunits of the pilus filament at the expense of ATP hydrolysis (24, 47). The involvement of Tfp in transformation is based on the hypothesis that pilus retraction is concurrent with DNA uptake into the cell (13, 14). The dynamic retraction of pilus filaments into the cell membrane could create an open pore through which DNA could enter or interact with DNA-binding complexes. As such, the PilT component responsible for Tfp retraction can be considered a critical accessory for DNA uptake. In Synechocystis sp. strain PCC 6803, Tfp proteins are also responsible for mediating phototaxis via twitching motility. Tfp genes, such as pilA1, pilB1, and pilT1, have been shown to be essential for both motility and transformation (32, 51). Recent evidence has shown that the PilA subunit can bind to DNA (45). It is also postulated, therefore, that retraction of pili brings the cell into closer proximity to DNA, thereby increasing the chance of transformation.

Microcystin production has been extensively studied in M. aeruginosa PCC 7806 and was selected as a representative of the toxic Microcystis species for characterization of a functional Tfp system (28). Here, we show that the previously identified Tfp genes are transcribed in M. aeruginosa PCC 7806 cells. The functionality of the PilT protein (7806 PilT) was examined via its introduction into heterologous pilT mutant hosts by assessing its ATPase activity, as well as determining its theoretical protein structure. In addition to the characterization of 7806 PilT, further screening of the pilT gene in other toxic and nontoxic species of Microcystis was undertaken in order to assess its distribution within the genus.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and culture conditions. The positively phototactic strain Synechocystis sp. strain PCC 6803 was grown either in BG-11 liquid medium or on BGTS agar (1% [wt/vol]) plates {BG-11 supplemented with 10 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, pH 8.2, with KOH] and 0.3% (wt/vol) sodium thiosulfate} (50). Where appropriate, antibiotics were also added (7 µg/ml chloramphenicol, 5 µg/ml kanamycin, or 5 µg/ml spectinomycin). Cultures were grown under continuous light (25 µmol photons m–2 s–1) supplied by cool white fluorescent lamps at 28°C ± 1°C. Pseudomonas aeruginosa strain PAK and its pilT mutant R364 (49) were kind gifts from John Mattick (Institute for Molecular Bioscience, University of Queensland) and were grown on Luria-Bertani (LB) agar plates or in liquid medium at 37°C. Where necessary, LB medium was supplemented with 500 µg/ml of carbenicillin. Escherichia coli DH5{alpha} was cultured at 37°C in LB medium, and 100 µg/ml of ampicillin was added for cloning procedures. The BL21(DE3) strain of E. coli (Novagen) was used for expression studies and cultured as with DH5{alpha}.

DNA manipulations and plasmids. Genomic DNAs from all bacterial strains were extracted as described elsewhere (31). The pGEM T-Easy vector (Promega) was used for cloning procedures. All plasmids were maintained in E. coli DH5{alpha} under appropriate selection conditions. The vectors pKW1188 (50) and pHB956 were kind gifts from Xudong Xu (Institute of Hydrobiology, Chinese Academy of Sciences). The vector pKW1188 provides an integrative platform for the expression of desired genes in Synechocystis sp. strain PCC 6803 and facilitates this by double homologous recombination at the locus slr0168. It also possesses a kanamycin resistance cassette at its multiple cloning site. The vector pHB956 contains the glutamine synthetase A (glnA) promoter from Synechocystis sp. strain PCC 6803 in a pUC18 backbone. The vector pUCP22NotI (48) was a kind gift from Scott Rice (School of Biotechnology and Biomolecular Sciences, University of New South Wales). This vector is an E. coli-P. aeruginosa shuttle vector and enables the expression of desired genes extrachromosomally in P. aeruginosa. The expression vector pET43.1a(+) (Novagen) was used for protein expression studies.

Transformation of bacterial strains. Synechocystis sp. strain PCC 6803 was naturally transformed with constructed vectors as described elsewhere (29). Competent cells of P. aeruginosa were prepared and transformed as previously described (2).

Inactivation of the Synechocystis sp. strain PCC 6803 pilT1 gene. A 2,177-bp fragment containing the pilT1 gene of Synechocystis sp. strain PCC 6803 was disrupted by inserting a cassette conferring chloramphenicol resistance into a unique MunI site (position 1069 of the fragment). This fragment was amplified from genomic DNA using the primers 5'-GCCCCCAGTAATAAATCATC-3' and 5'-ATCATGACGGAAAACTGTC-G-3' and cloned into pGEM T-Easy for subsequent manipulations. The resulting construct, p0161Cm, was used to naturally transform Synechocystis sp. strain PCC 6803. Transformants were observed after 14 days, and complete segregation was confirmed by PCR.

Complementation of the Synechocystis sp. strain PCC 6803 pilT1 mutant. The EcoRI and PstI restriction sites were introduced into the 5' and 3' ends, respectively, of the pilT gene from M. aeruginosa PCC 7806 via PCR from genomic DNA with the primers 5'-TAGGAATTCCACTGATTTTACC-3' (primer 1) and 5'-TAGCTGCAGACTCTCCTA-TGC-3' (primer 2) and cloned into pGEM T-Easy. The pilT fragment with the new terminal restriction sites was subsequently isolated by EcoRI digestion and ligated to an EcoRI-linearized pHB956 vector. The pilT gene with the upstream glnA promoter was then digested from pHB956 using PstI and ligated into a PstI-linearized pKW1188 vector. The final construct, pKN7806, harbored the M. aeruginosa PCC 7806 pilT gene with an upstream glnA promoter attached to a kanamycin resistance cassette. This construct was then introduced into Synechocystis sp. strain PCC 6803 pilT1 mutants via electroporation, as previously described (29), for integration of the M. aeruginosa PCC 7806 pilT gene into the genome of Synechocystis sp. strain PCC 6803 at the locus slr0168. Complete segregation of the transformants was verified by PCR with primers targeting both the M. aeruginosa PCC 7806 pilT gene and slr0168.

Complementation of the P. aeruginosa R364 pilT mutant. The restriction sites EcoRI and PstI were introduced at the 5' and 3' ends, respectively, of the pilT gene from M. aeruginosa PCC 7806 via PCR from genomic DNA with primer 1 and primer 2, described above, and cloned into pGEM T-Easy. The pilT gene was then digested from pGEM T-Easy with EcoRI and PstI and ligated into pUCP22NotI, which had also been digested with these enzymes. The resulting construct, pUCP7806pilT, was transformed into competent R364 cells, which had been prepared as described above (2).

Motility and competency assays. Motility assays for Synechocystis sp. strain PCC 6803 and P. aeruginosa were performed as described previously (2, 29). Competency assays were performed according to the natural transformation protocol described above. Exponentially growing wild-type and mutant Synechocystis sp. strain PCC 6803 cells were transformed with 600 ng of p0769Sp, a vector in which the gene slr0769 is disrupted by a spectinomycin cassette (29). Assays were performed in triplicate, and colonies were counted after 14 to 21 days of growth. Transformants were checked for complete segregation by PCR.

Transmission electron microscopy. Transmission electron microscopy was carried out as previously described (28).

Transcript analysis of Tfp genes. Fifty milliliters of mid-exponential-phase cultures at an optical density at 730 nm (OD730) between 0.5 and 0.6 (LKB Biochrom Ultrospec II) were harvested, snap-frozen in liquid nitrogen, and stored at –80°C. Total RNA was extracted within a week of harvesting, as described elsewhere (39). RNA extracts were treated with Turbo DNase (Ambion), and confirmation of successful DNase treatment was assessed by PCR. For transcript analysis in Synechocystis sp. strain PCC 6803, primers were designed to amplify regions of approximately 200 nucleotides of the pilA1 cDNA (sll1694 in CyanoBase) (forward, 5'-GCTATCCAAGACGCAACG-3'; reverse, 5'-ACTTCAGCACCACCACAATC-3') and 140 nucleotides of the 7806 pilT cDNA (forward, 5'-TTTTCCCCGCTAACCAAC-3'; reverse, 5'-CCACCATGATTTCCTGTACC-3'). For analysis of transcripts from M. aeruginosa PCC 7806, primers designed previously (28) were used to amplify a 100-bp region of the pilA cDNA, a 231-bp region of the pilB cDNA, a 226-bp region of the pilC cDNA, and a 100-bp region of the pilT cDNA. Reverse transcription-PCR of RNA was performed as previously described (29), but with 300 ng of starting total RNA template. Random nonamers (Sigma) were used for cDNA synthesis from M. aeruginosa PCC 7806 RNA. Two microliters of each cDNA reaction mixture was used for subsequent quantitative real-time PCRs (qPCR) or standard PCR. Transcript levels of the pilA1 gene in Synechocystis sp. strain PCC 6803 were quantified and analyzed by qPCR using the Rotor-Gene 3000 system (Corbett), as described elsewhere (29).

Expression and purification of M. aeruginosa PCC 7806 PilT. The BamHI and EcoRI sites of the pET43.1a(+) expression vector (Novagen) were used for introduction of pilT into the plasmid. The forward primer 5'-TAGGGATCCCTTATGGAATTAATG-3' (containing the BamHI restriction site) and the reverse primer 5'-TAGGAGCTCCCATGGGAAAC-3' were used to amplify pilT from M. aeruginosa PCC 7806 genomic DNA, which was then cloned into pGEM T-Easy (Promega). Amplification was achieved using a 2:1 mixture of Taq-Pfu DNA polymerases. The sequence of the insert was verified by DNA sequencing (data not shown). The M. aeruginosa PCC 7806 pilT was then digested from pGEM T-Easy with BamHI and EcoRI and ligated into pET43.1a(+) linearized with the same enzymes. The sequence of the insert was again checked by DNA sequencing. The resulting construct, pET43.1a-7806pilT, harbored the pilT gene fused to C-terminal Nus and His tags.

The M. aeruginosa PCC 7806 PilT (7806 PilT) protein was expressed in E. coli BL21(DE3). Cells transformed with pET43.1a-7806pilT were used to inoculate a 10-ml LB-ampicillin (100 µg/ml) culture, which was grown overnight at 37°C. Six milliliters of this overnight culture was used to inoculate 300 ml of LB-ampicillin (2% inoculum) medium. After growth to an OD600 of 0.5 at 37°C and 160 rpm, 0.5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) was added to induce expression. After 2.5 hours postinduction, cells were harvested at 4°C and stored at –20°C until lysis (usually within 1 week). The frozen cells were resuspended in 6 ml of cold HEPES buffer (50 mM HEPES, 150 mM NaCl, pH 7.4) with a 20.5-gauge needle and then sonicated on ice for four cycles at 30% amplitude and 25 0.5-s pulses and then once at 40% amplitude and 25 0.5-s pulses. The lysate was centrifuged at 25,000 x g for 30 min at 4°C, and the supernatant was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for verification of expression. Western blotting was carried out by standard methods (38) for confirmation of expression of the His-tagged PilT protein. Purification of the PilT protein was performed using a 1-ml HiTrap affinity column (Amersham Biosciences) connected to a BioLogic chromatography system (Bio-Rad Laboratories) and eluted by a stepwise gradient with 300 mM imidazole in HEPES buffer. The eluted fractions containing the PilT protein were pooled and concentrated by centrifugation using an Amicon Ultra-4 centrifugal filter device (Millipore), with five subsequent washes with 4 ml HEPES buffer. Purified and concentrated PilT protein was subjected to Western blotting (38) using colorimetric detection with nickel-nitrilotriacetic acid conjugated to alkaline phosphatase (QIAGEN) and nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate) (Promega).

ATPase assay of the 7806 PilT protein. The ATPase activity of M. aeruginosa PCC 7806 PilT was measured as described previously, with modifications (6, 18, 32). Briefly, assays were carried out at 30°C for 30 min in 100 µl of reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM ATP) supplemented with either 1, 5, or 10 mM of MgCl2 or CaCl2. The reactions were terminated by the addition of 800 µl malachite green reagent (6) and stabilized by the addition of 100 µl of 34% (wt/vol) sodium citrate dihydrate (18). Absorbances were determined at 660 nm. Controls containing only ATP or protein were also included in the assays. The specific activity was defined as the nanomole amount of ATP hydrolyzed per minute per milligram of protein.

Protein modeling. Homology modeling was performed to determine the possible differences between the putative three-dimensional structures of the PilT protein of M. aeruginosa PCC 7806 and Synechocystis sp. strain PCC 6803 (6803 PilT). SWISS-MODEL (40; http://swissmodel.expasy.org) was utilized to search for suitable templates and to produce the theoretical structure. Swiss-PdbViewer (15) was used for visualization and reproduction of the models generated from SWISS-MODEL.

Screening for the pilT gene in Microcystis. Three categories of Microcystis strains were chosen: toxic strains possessing mcy genes, nontoxic species that possessed mcy genes, and nontoxic species without mcy genes (30, 44). A total of 13 strains of toxic and nontoxic Microcystis (see Table 2) were screened for the pilT gene with specific and degenerate primers (28). The forward specific primer 5'-CCTCGAAGAAGTAGAACG-3' and reverse specific primer 5'-AGCTGCTTTTAACTTGTGCG-3' were used. Where specific primers failed, degenerate primers amplifying highly conserved regions of pilT (forward, 5'-GTSACDGGVCMVACNGGYTC-3'; reverse, 5'-ACYAAGTGWCCDGTTTSNGCNGC-3') were utilized. These primers were originally used to detect the pilT gene in M. aeruginosa PCC 7806 (28).


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  		TABLE 2. Results for the screening of the pilT gene in toxic and nontoxic strains of Microcystisa

 
Nucleotide sequence accession numbers. All amplicons in this study were confirmed for identity by sequencing (data not shown) and deposited in GenBank under the accession numbers DQ666165, DQ666166, DQ666168, DQ666169, EF051581, EF051582, and EF058234, while those detected in a previous study had accession numbers AY973314 and AY973318 to AY973321 (28).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcript analysis of pilus genes in M. aeruginosa PCC 7806. Using primers that were designed to amplify the midregion or 3' end of the mRNA transcripts, transcription of the M. aeruginosa PCC 7806 pilA, pilB, pilC, and pilT genes was confirmed by PCR after reverse transcription (Fig. 1).


Figure 1
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  		FIG. 1. PCR analysis of cDNA from reverse transcribed total RNA of M. aeruginosa PCC 7806 with primers specific for pilA (lanes 1 and 2), pilB (lanes 3 and 4), pilC (lanes 5 and 6), and pilT (lanes 7 and 8). + indicates total RNA incubated with reverse transcriptase; – indicates total RNA incubated without reverse transcriptase.

 
Heterologous expression of M. aeruginosa PCC 7806 pilT in Synechocystis sp. strain PCC 6803. In contrast to the wild type (Fig. 2A), the pilT1 mutant of Synechocystis sp. strain PCC 6803 displayed hyperpiliation (Fig. 2B). When the pilT1 mutant was complemented with pKN7806 harboring 7806 pilT, reversion to the wild-type Tfp phenotype was expected. However, the complemented mutant still exhibited hyperpiliation (Fig. 2C). In addition, competency assays of the complemented pilT1 mutant, carried out to assess whether the 7806 pilT gene could restore transformability, yielded no transformants. The pilT1 mutant was also nonphototactic. At this point, it was also expected that motility could not be restored by the 7806 pilT gene, and this was also found to be true.


Figure 2
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  		FIG. 2. Transmission electron micrographs of wild-type Synechocystis sp. strain PCC 6803 (A), the pilT1 knockout (B), and the pilT1 knockout complemented with the M. aeruginosa PCC 7806 pilT gene (C). The scale bars indicate 2,000 nm. The cells were stained with 1% phosphotungstic acid (pH 7.0) for 30 s.

 
Reverse transcription performed on the total RNAs of three independent complemented mutants, followed by PCR on the cDNA with specific primers for 7806 pilT, revealed that it was being transcribed (Fig. 3A). This indicated that the nonfunctionality of 7806 PilT in Synechocystis sp. strain PCC 6803 was not due to a lack of transcription of the complemented gene. It was previously reported that a pilT1 knockout displayed up to a sevenfold increase in the pilA1 transcript compared to the wild type (3). Here, assessment of pilA1 transcript levels in the complemented mutants showed between 32- and 38-fold increase compared to the wild type (Fig. 3B). The pilT1 mutant displayed a 31-fold increase in pilA1 transcripts compared to the wild type. Thus, it also appeared that 7806 PilT could not function to restore the expression of pilA1 to wild-type levels.


Figure 3
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  		FIG. 3. (A) Expression of the M. aeruginosa PCC 7806 pilT gene in three independent pilT1 knockouts of Synechocystis sp. strain PCC 6803 (lanes 1 to 3). Total RNA was extracted and reverse transcribed, followed by PCR with specific primers for the 7806 pilT gene. Lane 4, PCR on reverse transcribed RNA from the pilT1 knockout; lane 5, positive control with genomic DNA. (B) Relative expression ratios of the pilA1 transcript in wild-type Synechocystis sp. strain PCC 6803 (bar 1), its pilT1 knockout (bar 2), and the pilT1 knockout complemented with 7806 pilT (bars 3 to 5) normalized to wild-type levels, as assessed by qPCR. The error bars indicate standard deviations.

 
Heterologous expression of M. aeruginosa PCC 7806 pilT in Pseudomonas aeruginosa. As with the pilT1 mutant of Synechocystis sp. strain PCC 6803, P. aeruginosa R364 was hyperpiliated and did not display twitching motility (Fig. 4). When complemented with the M. aeruginosa PCC 7806 pilT, motility was restored. In contrast to the wild-type P. aeruginosa, which appeared to disperse uniformly from the point of inoculation, R364 complemented with pUCP7806pilT dispersed into distinct colonies (Fig. 4A). Motility assays were carried out on eight individual R364 strains complemented with pUCP7806pilT, and in all cases, motility was restored. Strain R364 transformed with pUCP22NotI showed no dispersal from the point of inoculation, identical to the R364 mutant (Fig. 4A), indicating that the restored motility was due to the pUCP7806pilT construct and not the vector alone. All motility assays of P. aeruginosa cells transformed with pUCP22NotI and pUCP7806pilT were performed on 500 µg/ml carbenicillin LB plates. The altered form of motility of complemented cells could be due to unstable maintenance of the plasmid within some cells, the loss of which would stop growth on the selective plates and hence result in the distinct colonies observed. A survey of P. aeruginosa cells with transmission electron microscopy showed that the complemented mutant also regained wild-type piliation characteristics (Fig. 4B).


Figure 4
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  		FIG. 4. Heterologous expression of the M. aeruginosa PCC 7806 pilT gene in P. aerguinosa. (A) Twitching motility assay. The R364 pilT mutant complemented with pilT from M. aerguinosa PCC 7806 (R364 pilT mutant + 7806 pilT) regained twitching motility. (B) Representative transmission electron micrographs of cells stained with 1% phosphotungstic acid (pH 7.0) for 30 s. The R364 pilT mutant was hyperpiliated, while that complemented with pilT from M. aerguinosa PCC 7806 (R364 pilT mutant + 7806 pilT) regained normal wild-type (WT) piliation. The scale bars indicate 2,000 nm.

 
PilT expression and activity. The 7806 PilT protein was expressed as an N-terminal hexahistidine fusion protein. This fusion protein also contained a Nus tag upstream of the histidine tag, which promotes the cytoplasmic solubility of its fusion partner. The use of the pET43.1a(+) vector enhanced the solubility of 7806 PilT compared to expression from pET30a(+) (data not shown). Purification of 7806 PilT using the HiTrap affinity column charged with Ni2+ ions showed that the fusion protein eluted between 120 mM and 150 mM imidazole fractions from the column and separated at the expected mass of 106 kDa by SDS-PAGE (Fig. 5A). These fractions were pooled and further concentrated for ATPase assays (Fig. 5B). Western blotting confirmed that the concentrated fractions contained the fusion protein (Fig. 5C). Expression with BL21(DE3) cells containing only pET43.1a(+) was also performed (data not shown). No protein of the same size as 7806 PilT was detected between the 120 mM and 150 mM imidazole fractions, which was also confirmed by Western blotting after these fractions were concentrated.


Figure 5
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  		FIG. 5. (A) SDS-10% PAGE representing fractions of the M. aerguinosa PCC 7806 PilT protein expressed and purified from E. coli BL21(DE3)/pET43.1a-7806pilT by Ni2+ affinity column, eluted between 115 mM and 150 mM of imidazole. (B) Lanes 2 to 6 from panel A representing eluted fractions of 7806 PilT between 120 mM and 150 mM imidazole were pooled and concentrated (see the text). (C) Western blot of the concentrated 7806 PilT from panel B (lane 1), pooled and concentrated fractions of a pET43a(+)-only control expression (lane 2), and purified Nus tag from pET43a(+) as a positive control (lane 3).

 
ATPase assays carried out on the concentrated 7806 PilT showed that the protein displayed the highest specific activity (37.47 ± 1.76 nmol Pi min–1 mg protein–1) at 5 mM MgCl2 (Table 1) . Similar activities were observed at 1 mM and 10 mM MgCl2. It appeared that higher concentrations of CaCl2 may be required for higher activity of 7806 PilT, although even at 10 mM CaCl2, the specific activity was still approximately half that for 1 mM MgCl2. ATPase assays were also performed on the concentrated 120 mM to 150 mM fractions of the pET43.1a(+) control expression, with no activity detected, confirming that the specific activity observed was from 7806 PilT.


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  		TABLE 1. Specific ATPase activities of the M. aeruginosa PCC 7806 PilT protein under different concentrations of MgCl2 and CaCl2

 
PilT modeling. Due to the close phylogenetic relationship established between the PilT proteins of M. aeruginosa PCC 7806 and Synechocystis sp. strain PCC 6803 (28), a theoretical structural model of 7806 PilT was generated. Modeling was also performed to determine if the apparent nonfunctionality of 7806 PilT was due to structural differences between the two proteins. Database searches in the SWISS-MODEL repository presented the general secretion pathway NTPase E, EspE (1p9wA), from Vibrio cholerae as the best modeling template (5). As expected from their multiple-sequence alignment (28), the M. aeruginosa PCC 7806 PilT model was almost identical to that of Synechocystis sp. strain PCC 6803 (Fig. 6). The ATP-binding site and the Asp box domain were in close proximity in both PilT proteins (Fig. 6A and B). However, an additional region was present in the 6803 PilT protein (Fig. 6A) that was absent in 7806 PilT (Fig. 6B). The P. aeruginosa PilT (NP_249086), subjected to the same modeling approach, also did not generate this extra domain (Fig. 6C). Upon closer inspection of the 6803 PilT sequence, it was evident that at positions 269 and 277, alanine and serine residues were present, while the 7806 PilT sequence possessed Ser267 and Asn275 (Fig. 6D). In silico replacement of both 7806 PilT residues with those of 6803 PilT led to the generation of the extra region (Fig. 6F). Likewise, changing Ala269 to serine or Ser277 to asparagine in 6803 PilT resulted in the loss of this extra domain (Fig. 6E), indicating the requirement for alanine and serine at these positions for generation of this structure. Hydrogen bonds were computed in the reconstructed 7806 PilT model (Fig. 6G), and it was found that hydrogen bonds were formed between Ser261/Leu263 and Leu127, Ala264/Phe266 and Leu129, Phe266/Gln268 and Thr129, and Lys274/Pro276 and Ser275. Thus, it is presumed that Ser267 and Asn275 in the original sequence destabilized the formation of H bonds in the neighboring residues. These hydrogen bonds probably act to stabilize the formation of the additional domain in the 6803 PilT protein. Thus, there appeared to be a number of critical residues that influenced the conformation of the theoretical models of 7806 PilT and 6803 PilT, even though their overall sequences were highly identical (83% identity and 93% similarity).


Figure 6
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  		FIG. 6. Theoretical model of the PilT proteins from (A) Synechocystis sp. strain PCC 6803, (B) M. aeruginosa PCC 7806, and (C) P. aerguinosa (PaeruPilT), using SWISS-MODEL, based on the V. cholerae NTPase EspE (1p9wA) template. Only residues Leu115 to Phe283 of 6803 PilT (BAA18564), and Asn112 to Phe246 of the original 7806 PilT (AAY51448) sequence were able to be modeled. The gray regions are the Walker box A/B domains and the Asp box, which presumably forms the catalytic site. In 6803 PilT, an additional region was generated (panel A, white). When Ser267 and Asn275 of 7806 PilT were mutated to alanine and serine, respectively (D), the extra region was generated (F). Conversely, when Ala269 or Ser277 of 6803PilT was mutated to serine and asparagine, respectively (D), the extra region was lost (E). Hydrogen bonds (dashed lines) in the modified 7806 PilT sequence (G) showed that they are critical for generation of the extra region. The images were generated using Swiss-PdbViewer.

 
Screening for pilT in Microcystis. The pilT gene was present in the majority of microcystin-producing and -nonproducing species, which presumably possess all the genes required for microcystin production. It was also detected in four of the non-mcy-possessing Microcystis species (Table 2). However, the pilT gene was truncated at the 3' end by 350 bp in two of the strains (UWOCC Q and C3-11), which could imply that it is not functional. Despite the fact that the pilT gene is highly conserved among Tfp-containing species (4, 47), it could not be detected in the remaining nontoxigenic strains, even when degenerate primers and a range of PCR conditions were used. Degenerate primers for pilT were designed based on the Walker box A and His box motifs, which are highly conserved in the PilT group of proteins (5, 32). The degenerate PCR also detected pilB (28) and a second copy of the pilT gene, homologous to pilT2 of Synechocystis sp. strain PCC 6803, from M. aeruginosa PCC 7806 (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it is known to be naturally transformable, M. aeruginosa PCC 7806 is notoriously difficult to transform, requiring the addition of at least 10 µg of methylated DNA to exponential cells (OD730, 0.35) to achieve 10 to 20 transformants per 1.9 x 108 cells (data not shown). This has been attributed to its stringent restriction modification system, as well as possible extracellular nucleases that degrade incoming DNA (10, 20, 42). Although mutants of Tfp genes in M. aerguinosa PCC 7806, including pilT, are currently under investigation to confirm the functionality of its Tfp system, subsequent confirmation of transformability is a substantial hurdle given the low transformation efficiency of M. aeruginosa PCC 7806, as well as its relatively low growth rate (division time, approximately 24 h). In this study, we have initiated an alternative approach, which was to characterize the functionality of the PilT protein in an attempt to provide evidence of a functional Tfp system in M. aeruginosa PCC 7806. Transcript analysis by reverse transcription of total RNA has indicated that the Tfp genes pilA, pilB, pilC, and pilT are expressed in exponentially growing M. aeruginosa PCC 7806 (Fig. 1).

The functionality of 7806 PilT was demonstrated in vitro, displaying a maximal ATPase specific activity of 37.5 nmol Pi min–1 mg protein–1 under the conditions tested. This value is in comparison to the maximal activities observed with 6803 PilT (29.9 nmol Pi min–1 mg protein–1 at 30°C, pH 7.5, 5 mM MgCl2) and the PilT of Aquifex aeolicus (15.7 nmol Pi min–1 mg protein–1 at room temperature, pH 7.0, 5 mM MgCl2) under similar but not identical assay conditions (16, 32). The ATPase activity of 7806 PilT appeared to be more dependent on Mg2+ rather than Ca2+ (Table 1), although higher concentrations of Ca2+ may result in higher activities. Divalent cations function as essential activators of numerous ATPases (9). Since it has been postulated that the Asp box domain could function as an Mg2+ binding site (34), the proximity of the Asp box domain and the ATP-binding domains (Walker boxes) in the 7806 PilT and 6803 PilT proteins (Fig. 6) could form a catalytic site where ATP-Mg2+ complexes bind. The ATPase activities of 6803 PilT and the PilT of A. aeolicus are also dependent on Mg2+ (16, 32), and the EspE ATPase of V. cholerae, on which 7806PilT and 6803PilT were modeled, is also dependent on Mg2+ for activity (5).

While there have been several studies highlighting the importance of the PilT group of ATPases in generating the force for pilus retraction (21, 25, 41), little is known of the interactions of PilT with other Tfp components at the molecular level (7). A few reports describe the role of PilT in the Tfp system of Synechocystis sp. strain PCC 6803 (3, 32). It is therefore difficult to speculate as to why 7806 PilT did not function in Synechocystis sp. strain PCC 6803 but complemented P. aeruginosa. Based on structural modeling, however, it appears that a few different key residues could produce conformational disparities between the two cyanobacterial PilT proteins and result in differences in compatibility with other native Tfp components. By using a simple modeling approach, two key residues were identified in 6803 PilT (Ala269 and Ser277) that allowed the stabilization of an extra structural domain within the protein that was not present in 7806 PilT (Fig. 6). This extra region corresponded to a minor part of the N-terminal section of the Cm domain in the EspE protein (36). This Cm domain is found in members of the GspE and PilB subfamilies of proteins but does not occur in the PilT group of proteins (36). In fact, the closest homolog of EspE, the type IV secretion protein HP0525 of Helicobacter pylori, does not possess this Cm domain. Given the sequence dissimilarity after the second Walker box motif between 6803 PilT and EspE (data not shown), it is likely that the structural similarity between these two proteins is limited to this additional region and the N terminus.

The Cm domain of EspE contains a tetracysteine motif, absent in 6803 PilT and 7806 PilT, that is prevalent in zinc-dependent enzymes (5). This Cm domain was shown to coordinate Zn2+ via the tetracysteine motif, and removal of Zn2+ reduced the ATPase activity of EspE by less than 50%. Thus, the Cm domain is dispensable for ATPase activity, which is supported by the fact that two EspE homologs from Xylella fastidiosa and Xanthomonas campestris do not possess the tetracysteine motif (5). The Cm domain was thus suggested to facilitate folding/oligomerization, or protein-protein interactions, due to its position in the overall structure of the protein, which is favorable for protein interactions. Whether the extra region of 6803 PilT renders specific protein-protein interactions unable to be complemented by 7806 PilT is not clear. The protein-modeling approach used here highlighted the likelihood that 7806 PilT and 6803 PilT, despite their high sequence identity (83%), could have subtle conformational differences that other native Tfp components require for interaction.

7806 PilT could complement the pilT mutant of P. aeruginosa. However, unlike 6803 PilT, neither of these PilT proteins could be modeled to the Cm domain. Attempts to generate the Cm domain by mutating the PilT of P. aeruginosa in silico were not successful (data not shown). This was due to much larger sequence differences between 7806 PilT and P. aeruginosa PilT (48% identity; 68% similarity), from which critical residues were not able to be detected. Nonetheless, the functionality of 7806 PilT in P. aeruginosa suggests that the Cm domain may play an important role in 6803 PilT and further emphasizes that the conformational difference between 7806 PilT and 6803 PilT may be due to a few critical residues. Thus, the 6803 PilT sequence and structure, and perhaps also its function in the Tfp system, may be unique. Indeed, the pilT1 mutant of Synechocystis sp. strain PCC 6803 exhibits a high level of pilA1 transcript, while mutants of other Tfp genes, such as pilT2, pilC, and pilD, exhibited wild-type levels (3). This implied a regulatory function for PilT1, directly or indirectly, toward pilA1, which to our knowledge has been observed only in the Synechocystis sp. strain PCC 6803 Tfp system.

The general lack of the pilT gene in the non-mcy-possessing Microcystis strains (including the truncated pilT from UWOCC Q and C3-11) is interesting. It should also be noted that the pilA gene was detected in the majority of strains listed in Table 2 (data not shown), implying a specific lack of the pilT gene in the non-mcy strains of Microcystis. There are also other cyanobacteria that appear to be missing several Tfp genes despite possessing almost a full complement of other genes for this system (17, 33). A much larger number of both toxic and nontoxic strains of Microcystis are required for screening to elucidate any trends or correlations between the absence of the pilT gene and non-mcy strains of Microcystis. The detection of pilT in the non-mcy-possessing M. aerguinosa PCC 7005 and Microcystis sp. strain HUB 5-3, and not in the toxic M. aerguinosa UWOCC E7, raises the question of potential for natural transformation in currently nontoxigenic strains. However, the screening performed here provides another physiological focus for the study of microcystin evolution in terms of the impact of lateral gene transfer, in addition to the characterization of pilT from M. aerguinosa PCC 7806.

Despite the phylogenetic evidence for or against the impact of lateral gene transfer on microcystin evolution, there is a noticeable lack of experimental studies regarding the actual mechanisms that could facilitate this process. Here, we have shown that 7806 PilT is functional in vitro, and in vivo using P. aeruginosa as a host. Further elucidation of the DNA uptake system of M. aeruginosa PCC 7806 will enable this cyanobacterium to be used as a model organism and will provide physiological evidence for the role of genetic exchange in the evolution of cyanobacterial toxicity.


   ACKNOWLEDGMENTS
 
This work was financially supported by the Australia Research Council.


   FOOTNOTES
 
* Corresponding author. Mailing address: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia. Phone: 61 2 9385 3235. Fax: 61 2 9385 1591. E-mail: b.neilan{at}unsw.edu.au. Back

{triangledown} Published ahead of print on 15 December 2006. Back


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Journal of Bacteriology, March 2007, p. 1689-1697, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01640-06
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