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.

PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function inMyxococcus xanthus

“…His 6 -PilT remained soluble under the conditions used in this assay. Importantly, we observed a low but significant increase in accumulation of [␣- 32 P]ADP when His 6 -PilT was incubated with ␣-32 P-ATP (Fig. 3D).…”

“…PilT has been previously purified from A. aeolicus as a soluble C-terminal hexahistidine fusion (13) and as soluble Nterminal hexahistidine fusion proteins in the case of two cyanobacterial species (32,33). We cloned pilT of M. xanthus and attempted to overexpress the protein either with an N-terminal hexahistidine tag or with a C-terminal hexahistidine tag.…”

“…However, because His 6 -PilT did not follow standard reaction kinetics, the specific ATPase activity could not be determined. The specific activities of two PilT proteins have been reported; i.e., PilT of A. aeolicus has a specific activity of 15.7 nmol min Ϫ1 mg Ϫ1 (13), and PilT of M. aeruginosa has a specific activity of 37.5 nmol min Ϫ1 mg Ϫ1 (32). In vivo several factors may contribute to increasing the ATPase activity of secretion ATPases; i.e., for BfpD it was reported that ATPase activity is stimulated 1,200-fold by the interaction with cytoplasmic domains of the inner membrane proteins BfpC and BfpE (8), for EpsE of the TT2S in V. cholerae acidic phospholipids were reported to stimulate activity in combination with a cytoplasmic domain of the inner membrane protein EpsL (2), and for XpsE of the T2SS in X. campestris ATPase activity is stimulated by interaction with the cytoplasmic domain of the inner membrane protein XpsL (50).…”

“…For PilT orthologs, ATPase activity was reported in vitro for PilT of Synechocystis sp. strain PCC 6803 (33), for PilT of Microcystis aeruginosa (32), and for hexameric PilT from A. aeolicus (13). Moreover, for the last protein, replacement of the conserved lysine residue in the Walker A box abolishes ATP hydrolysis (13).…”

Cyanobacterial defense mechanisms against foreign DNA transfer and their impact on genetic engineering

“…The application of comparative genomics did not reveal further competence genes, probably due to the low sequence similarity with known competence genes, and the lack of clear operon structure in cyanobacterial genomes. Homologs to T4P genes were described in two other naturally competent cyanobacteria T. elongatus (Iwai et al, 2004) (Nakasugi et al, 2007). pilT knockouts of M. aeruginosa were not reported and whether T4P genes are involved in natural competence in Microcystis remains unknown.…”