Functional analysis of PilT from the toxic cyanobacterium Microcystis aeruginosa PCC 7806

Abstract

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 P(i) min(-1) mg protein(-1), with a requirement for Mg(2+). 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.

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.

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.

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.

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.

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).

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.