Identification of Pilus-Like Structures and Genes in Microcystis aeruginosa PCC7806

Four putative type IV pilus genes from the toxic, naturallytransformable Microcystis aeruginosa PCC7806 were identified.Three of these genes were clustered in an arrangement whichis identical to that from other cyanobacterial genomes. TypeIV pilus-like appendages were also observed by electron microscopy.

INTRODUCTION

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Introduction

Microcystis aeruginosa PCC7806 is a toxic, unicellular cyanobacteriumthat is naturally transformable (4, 15). Microcystin (a hepatotoxin)production by this cyanobacterium involves a large gene clusterthat encodes its synthesis via a polyketide synthetase and nonribosomalpeptide pathway (4, 15). The arrangement of genes in this clusterand its primary structure show high similarity to those of twoother toxin clusters in cyanobacteria of different orders: thefilamentous Planktothrix and Nodularia (3, 11). The microcystingene cluster from Anabaena sp. strain 90 is also known, andthe individual genes show high sequence similarities to thecorresponding ones from the other cyanobacteria (14). The associationof transposases with this conserved gene cluster in all fourorganisms suggests that lateral gene transfer may have facilitatedthe distribution of an ancient genomic locus which, throughtime, has evolved to produce different toxin isoforms in theseorganisms.

Type IV pili (Tfp) are present in many gram-negative bacterialsystems and function in a number of physiological processessuch as cell adhesion, motility, and natural transformation(10). The apparent distribution of Tfp across vastly differentbacterial systems suggests that it may be an ancient apparatus.Since Tfp and natural transformation are linked in a numberof bacterial systems, including cyanobacteria (18), we haveinitiated studies of Tfp systems in the bloom-forming genusMicrocystis. This has led to the identification of four Tfp-likegenes and pilus-like structures in M. aeruginosa PCC7806. Becausecyanobacteria are believed to be an ancient group of organisms,the presence of a Tfp system in Microcystis (the third reportedcyanobacterial case after Synechocystis sp. strain PCC6803 and,putatively, Thermosynechococcus elongatus BP-1 [2, 9]) wouldfurther our knowledge regarding Tfp, lateral transfer of toxicgene clusters, and the evolution of prokaryotic genomes.

Identification of pilus-like structures by electron microscopy.

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Identification of pilus-like...

Transmission electron microscope (TEM) observations of M. aeruginosaPCC7806 cells led to the identification of numerous pilus-likestructures on the cell surface (Fig. 1). Exponential-phase cellsgrown in liquid BG-11 medium (17) as batch cultures under continuouslight (25 µmol photons m^–2 s^–1), suppliedby cool white fluorescent lamps at 28°C ± 1°C,were harvested at room temperature at 4,000 x g for 15 min,washed once in fresh BG-11, and resuspended in 100 µlof BG-11. Cells from BG-11 agar plate cultures were scrapedoff and resuspended in 100 µl of BG-11. Cell suspensionsdropped onto Formvar-coated copper grids were either air driedor wicked off with filter paper after standing for 10 min. Gridswere stained with 1% phosphotungstic acid (pH 7.0) for 30 seconds,excess stain was removed with filter paper, and grids were allowedto air dry before being observed under a Hitachi model H-7000TEM at 75 kV.

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FIG. 1. Transmission and scanning electron micrographs of M. aeruginosa PCC7806 cells displaying pilus-like structures. (A) TEM of representative sample of cells stained from liquid culture. (B and C) TEM of representative samples of cells stained from solid agar culture. (D) SEM of representative samples of cells from solid agar culture. Scale bars in panels A, B, C, and D represent 500 nm, 500 nm, 2,000 nm, and 2,000 nm, respectively. Cells for TEM were stained with 1% phosphotungstic acid (pH 7.0) for 30 seconds. Arrows in panel B indicate thick rigid pilus-like structures consisting of thinner filaments. The arrow in panel D shows an extracellular appendage connecting two cells.

There was a notable difference between the piliation characteristicsof cells from liquid preparations (Fig. 1A) and agar plates(Fig. 1B and 1C). Cells from liquid preparations exhibited thinand flexible pilus structures. The diameters of these structureswere 6 to 10 nm; however, it was difficult to discern theirlengths as they were highly intertwined and numerous. Thesepilus structures are comparable to the thick-pilus measurementsreported by Bhaya et al. (2). Cells from agar plate preparationsalso exhibited numerous pilus-like structures. However theseappeared more rigid and thicker, with diameters of 20 to 35nm. A dense network of these rigid filaments was observed betweenclumps of cells from agar plates, which appeared to span severaltens of micrometers (Fig. 1C). Interspaced between these rigidstructures, however, were filaments with diameters similar tothose observed with cells from liquid preparations (Fig. 1B).The rigid pilus-like structures appeared to consist of bundlesof thinner filaments (Fig. 1B, arrows), which could be an artifactof growth on solid media. In an aqueous environment, the thinner,more-flexible pilus structures may lack a solid support on whichstable interactions may facilitate bundling. Yoshihara et al.observed bundles of pili (diameters less than 45 nm) which appearedto consist of the more typical pili (diameter of 5 nm) in Synechocystissp. strain PCC6803, whereas Bhaya et al. observed only the latterpilus morphology (2, 18). The method of preparation of cellsfor electron microscopy in the former study was from agar platesin contrast to preparation from liquid culture in the latterstudy. From these observations, the method of preparation couldinfluence the morphology of the pili, and the results in thisstudy are comparable to those in previous reports.

Scanning electron microscopy (SEM) also revealed the presenceof extracellular structures that appeared to interconnect cells(Fig. 1D, arrow). For SEM, 1.3-mm-diameter coverslips coatedwith poly-L-lysine were touched against cells from agar platesand fixed overnight in 2.5% glutaraldehyde in 0.1 M sodium cacodylatebuffer (pH 7.2). Samples were washed three times for 5 min with0.1 M sodium cacodylate (pH 7.2) buffer and postfixed in 1%osmium tetroxide for 1 h, followed by another buffer wash for10 min. Samples were dehydrated in ethanol and critical pointdried with carbon dioxide. Dried samples were mounted onto stubs,sputter coated with gold, and examined with a FEI Company Quanta200 scanning electron microscope at 15 kV. Due to the resolutionof SEM, the structures seen in Fig. 1D probably representedthe thick rigid pilus-like structures observed from TEM, ratherthan the thinner flexible structures.

From TEM and SEM observations, a dense array of appendages appearedto exist between M. aeruginosa PCC7806 cells (Fig. 1C and 1D).Although these micrographs represent cells from solid agar insteadof a liquid suspension, they suggest that the pilus-like structuresmay be involved in the cohesion or aggregation of M. aeruginosaPCC7806 cells, especially if they are surrounded by a networkof these filaments which may restrain dispersion of clumpedcells. In Synechocystis sp. strain PCC6803, Tfp are often seento form links between cells, although the significance of theseconnections is not known (2).

Identification of putative pilus genes in M. aeruginosa PCC7806.

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Identification of putative pilus...

Degenerate PCRs, followed by partial-inverse and adaptor-mediatedPCR methods (11), led to the identification of four open readingframes (ORFs) from M. aeruginosa PCC7806 which showed high similarityto those encoding known PilA, PilB, PilC, and PilT proteins.Twenty-five primers were used, and these sequences are availableupon request. The four ORFs were putatively named pilA, pilB,pilC, and pilT, according to their similarity to the correspondingbacterial counterparts. Multiple alignments revealed conservedmotifs characteristic for each group of pilus proteins. ThePilA sequence possessed a putative cleavage site recognizedby PilD peptidases, followed by a phenylalanine, which is thefirst amino acid of the mature pilin (8) (Fig. 2A). The PilBand PilT (Fig. 2B and C) sequences both possessed nucleotide-bindingmotifs, known as the Walker A and B boxes, found in many ATPases(16). There were also two aspartate box domains between theWalker boxes, which are common for PilB and PilT homologs butnot found in other proteins with ATP-binding sites (5, 12).In addition, a distinguishing feature of the PilB class of proteinsis the presence of a tetracysteine motif, which resembles thezinc-binding motifs found in many zinc-dependent enzymes (13).Multiple alignments and motif searches in PilC revealed twogeneral secretion pathway F signature domains at positions 69to 192 and 272 to 395, which is typical of this group of pilusproteins.

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FIG. 2. Multiple alignments of the conserved regions of the deduced pilus proteins from M. aeruginosa PCC7806 to known pilus proteins. Identical residues are shown in the highlighted boxes. (A) Alignment of the PCC7806 PilA to known PilA proteins. The arrowhead indicates the putative site of cleavage by a leader peptidase. (B) Alignment of the PCC7806 PilB to characterized PilB proteins. Asterisks indicate conserved cysteine residues. (C) Alignment of the PCC7806 PilT to characterized PilT proteins. Abbreviations: 7806, M. aeruginosa PCC7806; 6803, Synechocystis sp. strain PCC6803; Pseae, P. aeruginosa PAO1; Ttherm, Thermus thermophilus HB27.

The pilB, pilC, and pilT genes are clustered, similar to othercyanobacterial genomes possessing Tfp genes (Fig. 3). Sequencingupstream of pilB in M. aeruginosa PCC7806 revealed a putativeheat shock gene, annotated as grpE here due to its high similarityto grpE in Synechocystis sp. strain PCC6803. There are alsoheat shock genes present at one end of the pilBCT cluster inother cyanobacterial genomes (Fig. 3). In Pseudomonas aeruginosa,the pilB and pilC genes are clustered with pilA and the leaderpeptidase gene pilD, instead of pilT. The organization of thepilBCT genes in other heterotrophic bacteria is similar to thatin P. aeruginosa (5). From these data, it is apparent that thearrangement of the cyanobacterial pilBCT gene cluster may bedifferent from that of other bacteria.

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FIG. 3. Comparison of the pilBCT gene cluster organization from cyanobacterial genomes and the organization of pilA, pilB, pilC, and pilD from P. aeruginosa.

Phylogenetic analysis of cyanobacterial and noncyanobacterialPilA, PilB, PilC, and PilT proteins showed that, within allpilus clusters, each of the putative M. aeruginosa PCC7806 pilusproteins was predicted to share a common ancestor with Synechocystissp. strain PCC6803 (Fig. 4). With the exception of PilA fromProchlorococcus marinus MIT9313, there was a clear distinctionbetween the cyanobacterial and noncyanobacterial pilus proteinsequences, suggesting that the organismal ancestor of cyanobacteriapossessed a progenote Tfp system. The PilA protein itself appearedto have undergone the most significant substitutions among thepilus proteins studied here. Apart from the leader peptide regionsharboring the cleavage sites, the remainders of the PilA sequencesdo not show much conservation (Fig. 2). Using primers designedfor structural analysis of the pilT genomic loci, amplificationof orthologs was also achieved from the toxigenic M. aeruginosaUWOCC MRC and UWOCC MRD, Microcystis wesenbergii NIES107, andthe nontoxigenic M. aeruginosa PCC7005 (data not shown). TheMicrocystis PilT sequences formed a monophyletic cluster (Fig.4).

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FIG. 4. Unrooted phylogenetic tree of cyanobacterial and noncyanobacterial PilA, PilB, PilC, and PilT proteins. The tree was generated using ClustalX 1.83 and reconstructed using TreeExplorer. The scale bar represents 1 substitution per 10 amino acids. The subtree of Microcystis PilTs is shown in the upper left corner (scale bar represents 5 substitutions per 1,000 amino acids). GenBank accession and CyanoBase accession numbers are indicated in parentheses. Abbreviations: 7806, M. aeruginosa PCC7806; MRC and MRD, M. aeruginosa UWOCC MRC and MRD, respectively; 7005, M. aeruginosa PCC7005; NIES107, M. wesenbergii NIES107; 6803, Synechocystis sp. strain PCC6803; 7120, Anabaena sp. strain PCC7120; BP-1, Thermosynechococcus elongatus BP-1; 9313, Prochlorococcus marinus MIT9313; 8102, Synechococcus sp. strain WH8102; 7421, Gloeobacter violaceus PCC7421; Pa, P. aeruginosa PAO1; Ps, Pseudomonas stutzeri JM300; Ng, Neisseria gonorrhoeae; Tt, Thermus thermophilus HB27.

This study describes the presence of putative Tfp genes in M.aeruginosa PCC7806, whose products show high sequence similarityto other cyanobacterial Tfp proteins. Numerous pilus-like appendageson the cell surface are also reported. The morphology resemblesthat seen in Synechocystis sp. strain PCC6803, the Tfp systemof which has been studied quite extensively in terms of phototacticmotility and natural transformation (1, 18). We have initiatedstudies of the M. aeruginosa PCC7806 pilT, homologs of whichare thought to be important for the DNA uptake process due toits retraction functionality (6, 7). DNA bound to the surfacesof cells around the pilus apparatus could concurrently be takenup when retraction of the pilus structure occurs. For thesereasons, this Tfp component may represent a critical step inthe process of lateral gene transfer, which is the uptake ofDNA from the environment. In addition to the presence of putativeTfp genes in M. aeruginosa PCC7806, putative pilT genes havealso been identified in other toxic and nontoxic M. aeruginosastrains. This finding is important in that it lends supportto the hypothesis that lateral gene transfer (natural transformationvia Tfp) may have facilitated the dissemination of toxin geneclusters among strains of the same species and may preempt theacquisition of toxigenicity by nontoxic strains.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

Nucleotide sequences of eight putative pilus genes reportedin this paper have been submitted to GenBank under the accessionnumbers AY973314 to AY973321. The putative heat shock gene isunder accession number AY987043.

ACKNOWLEDGMENTS

This work was financially supported by the Australian ResearchCouncil.

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.

REFERENCES

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