A cAMP Receptor Protein, SYCRP1, is Responsible for the Cell Motility of Synechocystis sp. PCC 6803

Abstract

Disruption of the sycrp1 gene encoding a cyanobacterial cAMP receptor protein makes cells of Synechocystis sp. PCC 6803 non-motile. Electron microscopy showed that the sycrp1-deficient strain had a reduced number of thick pili on the cell surface compared with the wild-type strain. It is suggested that cAMP-SYCRP1 complex controls the biogenesis of pili.

. (Received November 30, 2001; Accepted January 30, 2002).

Many species of cyanobacteria show motility in a form of gliding, twitching or swimming. Unlike Escherichia coli, which uses flagella for motility, cyanobacteria use a pili apparatus for movement (Diehn et al. 1979, Häder 1987, Waterbury et al. 1985). Gliding occurs in filamentous cyanobacteria such as Spirulina (Ohmori et al. 1992), Oscillatoria, and Anabaena species (Hoiczyk 2000). The marine unicellular cyanobacterium, Synechococcus sp. WH8102, moves via swimming motility. SwmA, which is a cell-surface associated protein, is required for this motility (Waterbury et al. 1985, Brahamsha 1996). The unicellular cyanobacterium Synechocystis sp. PCC 6803 shows sporadic motility via twitching (Stanier et al. 1971). It was reported previously that the alternative sigma factor, SigF, the sigma factor responsible for the biogenesis of pili, and PilA1, a structural component of type IV pili, is essential for the twitching motility observed in Synechocystis sp. PCC 6803 (Bhaya et al. 1999, Bhaya et al. 2000). It was also reported that the ATPase, PilT, is required for motility in Synechocystis sp. PCC 6803 (Okamoto and Ohmori 1999). Despite the recent advances in understanding the components required for motility in cyanobacteria, the molecular mechanism of motility in cyanobacteria has not yet been clearly elucidated.

Type IV pili responsible for cell motility are well studied in, Gram-negative bacteria such as Myxococcus xanthus, Pseudomonas aeruginosa, and Neisseria gonorrhoeae (Wall and Kaiser 1999, Spormann 1999, Mattick et al. 1996, Fussenegger et al. 1997). Type IV pili are associated with a form of translocation called twitching motility (Henrichsen 1983). They are comprised of a protein subunit, called pilin, which is formed by cleaving the N-terminal region of prepilin using a specific peptidase, PilD (Mattick et al. 1996, Filloux et al. 1998). The N-terminal region of prepilin is highly conserved, while the cleaved leader sequence and C-terminal region of the protein are not conserved. Type IV pili are responsible not only for cell motility but also various cellular functions such as transformation, infection, and adhesion (Strom and Lory 1993). Synechocystis sp. PCC 6803 has multiple genes encoding type IV prepilin-like proteins (Bhaya et al. 2001, Yoshihara et al. 2001).

Cyclic AMP signaling pathways play an important role in the regulation of various biological activities by altering enzyme activities or controlling gene expression levels in both prokaryotes and eukaryotes (Botsford and Harman 1992, Skalhegg and Tasken 2000). It is well known that cAMP stimulates gliding motility of the filamentous cyanobacterium Spirulina platensis (Ohmori et al. 1992) and twitching motility of Synechocystis sp. PCC 6803 (Terauchi and Ohmori 1999). Recent studies have shown that cAMP binds to the cAMP receptor protein, SYCRP1, in Synechocystis sp. PCC 6803 (Yoshimura et al. 2000), and this cAMP-SYCRP1 complex functions as a transcription factor (Yoshimura et al. 2002). However, the physiological function of SYCRP1 remains unknown. In this report, we describe possible role of the cAMP-SYCRP1 complex in the biogenesis of pili in Synechocystis sp. PCC 6803.

The sycrp1 gene was disrupted in Synechocystis sp. PCC 6803 as described previously (Yoshimura et al. 2002).

The cell motility was estimated by colony shape on 0.8% (w/v) agar (Bradley 1980). Cells were spread on a BG11 plate containing 0.8% (w/v) agar and 0.3% (w/v) sodium thiosulfate and cultivated at 30°C under continuous illumination provided by fluorescent lamps at light intensity of 30 µmol m^–2 s^–1 for 4 d.

Electron microscopy was performed according to the methods described by Vaara and Vaara (1988) and Yoshihara et al. (2001). Cells of each strain growing on agar plates were gently suspended in BG11 medium and examined after staining with 0.8% (w/v) phosphotungstic acid (pH 7.0) by transmission electron microscope (model 1200EX, JEOL, Tokyo, Japan).

The wild-type cells formed spreading colonies on agar plate (Fig. 1A). It has been reported that cya1 (encoding adenylate cyclase) disruptant cells show non-motile phenotype and thus Cya1 and cAMP are involved in cell motility in Synechocystis sp. PCC 6803 (Terauchi and Ohmori 1999). The sycrp1 disruptant cells formed dome-shaped colonies indicating the non-motile phenotype (Fig. 1B), similar to the phenotype of the cya1 disruptant cells. It is suggested that sycrp1 plays an important role in cell motility via the cAMP signal transduction pathway.

The structures of pili on the cell surface of the sycrp1 disruptant were determined by electron microscopy after negative staining with phosphotungstic acid. There are three types of pili on the cell surface of Synechocystis sp. PCC 6803, thin pili, bundles of thin pili and thick pili (Yoshihara et al. 2001). Bundles of thin pili are invariant in wild-type cells and sycrp1 disruptant cells (Fig. 2). Major differences were observed in the thick pili. The lengths of the thick pili of the wild-type cells on average (more than 2 µm) were longer than those of the sycrp1 disruptant (Fig. 2A). And, although the sycrp1 disruptant retained the thick pili, they were present in a reduced quantity (Fig. 2B).

In the previous report, target genes for SYCRP1 were surveyed in Synechocystis sp. PCC 6803 by DNA microarray analysis (Yoshimura et al. 2002). A putative operon which included the following open reading frames (slr2015, slr2016, slr2017 and slr2018 in Cyanobase) was down regulated among the target gene candidates of SYCRP1. The previous experiments, however, have failed to show that SYCRP1 binds to the putative promoter of slr2015 gene (Yoshimura et al. 2002). The N-terminal hydrophobic regions of the predicted slr2015, slr2016, and slr2017 gene products show significant homology to those of type IV prepilin from the Gram-negative pathogen Pseudomonas aeruginosa (Filloux et al. 1998), a soil bacterium Myxococcus xanthus (Wall and Kaiser 1999), and Synechocystis sp. PCC 6803 (Fig. 3). Therefore, the slr2015, slr2016, and slr2017 genes were designated pilA9, pilA10, and pilA11, respectively. The pilA1-8 genes were assigned previously (Yoshihara et al. 2001). The conserved N-terminal region of type IV prepilin was not found in that of the predicted slr2018 gene product. Slr2018 is not homologous to other known proteins, nor does it contain any known functional domains. However, slr2018 disruptant cells generated by in vitro transposon mutagenesis showed non-motile phenotype similar to pilA10 and pilA11 disruptants (Bhaya et al. 2001). The essential structural components of thick pili for cell motility might be composed of not only PilA1 but also all the other putative pil operon products or a combination of some of these pil gene products.

Results of DNA microarray analysis (http://www.genome.ad.jp/kegg/expression) show that the pilA1 expression level of the sycrp1 disruptant was the same as that of the wild type. Nevertheless, the sycrp1 disruptant displayed reduction in the length and the number of thick pili and a non-motile phenotype. It is considered that the lack of cell motility of the sycrp1 disruptant is due to the reduction in the length and the number of thick pili. These results suggest that cAMP-SYCRP1 complex controls the biogenesis of pili required for cell motility.

Acknowledgment

This work was supported by a Grant-in-aid for General Scientific Research (12206002) from the Ministry of Education, Science, Sports and Culture of Japan and also by a grant from the Program for the Promotion of Basic Research Activities for Innovative Biosciences of Japan.

1

Corresponding author: E-mail, [e-mail]; Fax, [phone].

Fig. 1 Colony morphology of wild-type strain (A) and sycrp1 disruptant (B) on the 0.8% agar plates for 4 d under illumination at 30 µmol m^–2 s^–1. Bars show 50 µm.

Fig. 2 Electron micrographs of negatively stained cells of wild type (A) and sycrp1 disruptant (B). Bars show 1 µm. Black arrows indicate representative thick pili. White arrows indicate representative thin pili or bundles of thin pili.

Fig. 3 Sequence alignment of the conserved N-termini of pilA-like gene products with other bacterial PilA. Black boxes indicate identical residues at the same position in all seven sequences while dark gray and light gray boxes indicate conserved residues in six of the seven and five of the seven sequences, respectively. The GenBank accession numbers for the M. xanthus and P. aeruginosa sequences are L39904 and L37109, respectively. Comparisons were performed by using CLUSTAL.