Recent Advances in Biological Functions of Thick Pili in the Cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria have evolved various strategies to sense and adapt to biotic and abiotic stresses including active movement. Motility in cyanobacteria utilizing the type IV pili (TFP) is useful to cope with changing environmental conditions. The model cyanobacterium Synechocystis sp. PCC 6803 (hereafter named Synechocystis) exhibits motility via TFP called thick pili, and uses it to seek out favorable light/nutrition or escape from unfavorable conditions. Recently, a number of studies on Synechocystis thick pili have been undertaken. Molecular approaches support the role of the pilin in motility, cell adhesion, metal utilization, and natural competence in Synechocystis. This review summarizes the most recent studies on the function of thick pili as well as their formation and regulation in this cyanobacterium.


INTRODUCTION
Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis and still significantly contribute to primary production on a global scale. They are adaptive in a wide range of ecological habitats. Certain species of cyanobacteria were observed to deal with changeable environment via type IV pili (TFP). For example, in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis), phototactic motility driven by TFP allows such cyanobacterium to respond to fluctuations in the intensity and spectral quality of light (Yoshihara and Ikeuchi, 2004).
The cells of Synechocystis are covered by two distinct types of pili as extracellular appendages. One morphotype is specified as thick pili with an external diameter of 5 nm and a length of more than 2 µm, and another morphotype is defined by thin pili with a diameter of 3-4 nm and a length of less than 1 µm (Bhaya et al., 2000;Yoshihara et al., 2001). Thin pili distribute along the entire cell surface and align in bundles (Yoshihara et al., 2001). So far, genes involved in the formation of thin pili and their roles are unknown. In contrast, Synechocystis thick pili have been well dissected.
Thick pili of Synechocystis, which belong to TFP, have much in common with that of heterotrophs . Synechocystis cells use thick pili for extension, adhesion to the substrate and retraction to pull the cell across the surface. Multiple motility-related genes have been identified for the function of pili in this model organism (Yoshihara and Ikeuchi, 2004;. Besides motility, TFP have been shown to be involved in a range of cellular processes, such as natural transformation (NT) (Bhaya et al., 2000;Yoshihara et al., 2001Yoshihara et al., , 2002, biofilm formation (Chandra et al., 2017;Allen et al., 2019), and metal acquisition (Lamb et al., 2014;Lamb and Hohmann-Marriott, 2017). The biogenesis of thick pili is regulated at multiple levels and has also been studied in Synechocystis (Kizawa et al., 2016;Gonçalves et al., 2018;Hu et al., 2018). This review summarizes recent advances on thick pili, including their function, biogenesis and regulation in this cyanobacterium.

MOTILITY AND PHOTOTAXIS
Motility, including both gliding and twitching motility, is a frequently observed and beneficial feature among cyanobacteria. The cells of Synechocystis sense and shift their location via twitching motility in response to environmental stimuli. TFP are extended by PilB, then adhere to the substrate. Next, pilus retraction is induced by PilT, and cells are pulled across the surface (Burrows, 2012). Thick pilus retraction drags Synechocystis cells in a jerky motion across moist surfaces (Burriesci and Bhaya, 2008;Wilde and Mullineaux, 2015). The direction of movement correlates with localization of PilB patches at the inner membrane . The identification of key genes involved in the motility of Synechocystis began from the observation of some spontaneous mutants with different motility (Tajima et al., 2011;Kanesaki et al., 2012;Trautmann et al., 2012;Morris et al., 2014;Ding et al., 2015). There are two distinct major groups of Synechocystis substrains: the motile PCC and non-motile GT-lineages (Morris et al., 2017). Genes critical for motility and pilus biosynthesis may have mutations among strains, for instance a frameshift mutation in pilC (Bhaya et al., 2000), insertion in spkA, deletion upstream and within slr2031, and/or deletion of a single nucleotide within slr0322 (Ding et al., 2015). Widespread genomic variations may contribute to the discovery of Synechocystis strain-specific mutations related to motility.
Phototactic movement, also known as phototaxis, mediated by thick pili allows Synechocystis to move either toward or away from a light source according to light wavelength and intensity. Synechocystis harbors a variety of photoreceptors to perceive and respond to the direction, quantity, and quality of illumination (Choi et al., 1999;Ng et al., 2003). Photoreceptors responsible for phototaxis include blue/green light-absorbing proteins PixJ1 and Cph2, blue-light receptor PixD, and UV-A receptor UirS (also known as ethylene receptor, SynEtr1) (Yoshihara et al., 2000;Wilde et al., 2002;Okajima et al., 2005;Song et al., 2011;Sugimoto et al., 2017). Analysis of phototactic movement on low concentration of agar or agarose plates can be achieved at macroscopic and microscopic scales, which represents group behavior and single-cell motility, respectively . Individual cells directly and accurately sense the position of light rather than respond to a spatiotemporal gradient in light intensity. During this process, Synechocystis cells act as spherical microlenses sensing and moving toward the light source (Schuergers et al., 2016). By fluorescent labeling of thick pili, quantitative analyses of cell tracking indicate asymmetric distribution of cells along the light axis for directional cell motility (Nakane and Nishizaka, 2017). However, the details of Synechocystis phototaxis need to be further explored.

CELL ADHESION
Synechocystis may transit from motile states to cell adhesive states, like flocculation and sessile biofilm. These processes include cell-cell and cell-substrate adhesion through cell surface fractions, such as TFP. TFP in Synechocystis have recently been revealed to be responsible for biofilm and floc formation (Allen et al., 2019;Conradi et al., 2019). Mutants lacking TFP were unable to aggregate, and loss of pilC was shown to significantly reduce biofilm formation and prevent flocculation as well. Except for PilC, the PilB1 and PilT1 proteins are required for flocculation, but PilA1 may be completely absent, indicating the active cycles of thick pilus extension and retraction is dispensable for flocculation. Besides, the minor pilins encoded by the pilA9-slr2019 operon have been implicated in cell-cell adhesion in flocculation and the processes switching between cell-cell adhesion and cell surface adhesion depend on cyclic AMP (cAMP) levels (Chandra et al., 2017). It was speculated that alteration of surface-attached behavior responsible by TFP might confer Synechocystis the resistance to adverse stimuli during the environmental adaptation.

NATURAL COMPETENCE
Natural transformation is a generally conserved mechanism of horizontal gene transfer among bacteria (Lorenz and Wackernagel, 1994). Natural competence in Synechocystis was reported in the late 1980s (Williams, 1988), making this alga as an important model cyanobacterial strain for molecular genetic studies. The process of NT in bacteria mainly includes DNA uptake, transport, processing, and recombination (Johnston et al., 2014). Furthermore, genomic analysis in cyanobacteria has cataloged the genes that are involved in NT post entry of the DNA into the periplasm in a similar manner (Cassier-Chauvat et al., 2016). Synechocystis TFP have been proven to be crucial for the uptake of extracellular DNA from the extracellular milieu (Yoshihara et al., 2001).
Subsequently, PilQ is found to bind DNA for transport into the periplasm, one strand of the dsDNA is then degraded by an endonuclease, and the other strand is transported across the inner membrane via the competence gene products (Com) ComA (Slr0197), ComE (Sll1929), and ComF (Yoshihara et al., 2001;Nakasugi et al., 2006). After internalization, the ssDNA strand may be bound by recombination mediator proteins, which would recruit recombinases into a complex to promote homologous recombination (Salleh et al., 2019). Nevertheless, many details of these processes are still unclear in Synechocystis.
Recently, this conserved mechanism of horizontal gene transfer by TFP competence pili during NT has been revealed in Vibrio cholerae (Ellison et al., 2018). The surface pili directly binding to DNA via their tips and then internalizing DNA through retraction could be observed using fluorescent probes. It is likely that similar processes occur during DNA uptake in Synechocystis. Besides TFP, NT of Synechocystis is also affected by the physiological factors, including phase of cell growth, foreign DNA concentration and incubation time of cells and DNA (Zang et al., 2007). Genome analysis deciphered that pilus structural and assembly proteins and Com proteins are highly conserved across the known species of competent cyanobacteria species (Wendt and Pakrasi, 2019). For instance, the introduction of TFP assembly protein PilN from Synechococcus elongatus PCC 7942 into S. elongatus UTEX 2973 successfully recovered its natural transformability . Whereas, mutants defective in comF were not transformable, confirming its role in natural transformation. The transcripts of comA in such mutants was not significantly affected, indicating an alternative pathway independent of ComA may exist in Synechocystis . Therefore, to a large extent, the presence of these conserved proteins may explain why some cyanobacterial species are naturally competent whereas others are not.

METAL UTILIZATION
Being considered as microbial nanowires, TFP may enable metal acquisition for microbial cells and facilitate electron donation to extracellular electron acceptors in some bacteria such as Shewanella oneidensis MR-1 (Gorby et al., 2006). Nevertheless, Pirbadian et al. (2014) found that S. oneidensis does not produce conductive pili. Recently, it was revealed that microbial nanowires in G. sulfurreducens that were earlier thought to be TFP were assembled by polymerized chains of the hexaheme cytochrome OmcS . Later, Lovley and Walker (2019) evaluate the available evidence on the in vivo expression of electrically conductive pili and OmcS filaments and support that both of these two proteins are required for electron transfer in G. sulfurreducens. Electrically conductive Synechocystis nanowires have also been investigated using scanning tunneling microscopy (Gorby et al., 2006) and subsequently conductive atomic force microscopy (Sure et al., 2015). Further evidences should be provided to determine whether nanowires in this strain are assembled by PilA1. In addition, Synechocystis pilA1 mutants exhibit slower growth rates than wild type on oxidized iron minerals, indicating the role of PilA1 in electron transport to iron oxides. Physiological and spectroscopic data suggested the role of Synechocystis PilA1 in oxidization of iron minerals (Lamb et al., 2014), enhancement of manganese acquisition (Lamb and Hohmann-Marriott, 2017) and non-metallic element arsenic deposition (Sure et al., 2016).

GENES INVOLVED IN PILIN REGULATION
Comparative genomic analysis clearly showed that genes encoding core components of TFP display high sequence similarity among divergent bacterial groups (Pelicic, 2008). The Synechocystis genome contains pilin and pilin-like genes that are organized mainly in polycistronic operons (Kaneko et al., 1996). Table 1 summarizes genes involved in motility, including regulatory pilin genes (Supplementary Figure S1A).
In addition, the RNA chaperone Hfq (Ssr3341) was predicted to play important roles in phototaxis in post-transcriptional regulation in Synechocystis (Schuergers et al., 2014). The antisense RNA PilR acts as a direct negative regulator of PilA11 and cell motility . Disruption in the second ethylenebinding domain of ethylene receptor, named SynEtr1 TM2 cells, showed a large increase in both transcriptional and translational levels of PilA1 (Lacey and Binder, 2016). The Deg (slr1204/sll1679/sll1427) mutant appeared to be hyperpiliated with thick pili, indicating the regulation of PilA1 stability by Deg proteases (Barker et al., 2006).
Moreover, post-translational modifications (PTMs) have been characterized as one of the most important factors in Synechocystis pilus function over the past two decades. Various PTMs of pilin in Synechocystis, have also been proposed or verified to be important factors in phototactic movement (Sergeyenko and Los, 2000;Kim et al., 2004Kim et al., , 2009Kim et al., , 2011. For instance, Kamei et al. (2001b) found that six Serine/Threonine protein kinases participate in cell motility control, and phosphorylation participates in the phototactic movement process. Later, it was interesting to identify pilin phosphorylated at Serine 59  and acetylated at lysine 58 (Mo et al., 2015), implying cross-talk between serine phosphorylation and lysine acetylation. PilD (Slr1120) has been predicted to be responsible for pre-pilin leader peptide cleavage as well as N-terminal methylation (Bhaya et al., 2001). PilD was also identified to play a role in PilA1 glycosylation (Linhartová et al., 2014). Pilin has been confirmed to be glycosylated in Neisseria (Chamot-Rooke et al., 2007) and Pseudomonas (Voisin et al., 2007), and several pilin-glycosylation genes have also been characterized in Synechocystis. Notably, Kim et al. (2011) discovered trimethylation at the C-terminal lysine and O-glycosylation within the pilus peptide in Synechocystis, indicating an indispensable role of PTMs for pilus assembly and pilus-mediated motility. sll0899 is involved in O-glycosylation between amino acids 67 and 75 in pilin and inactivation of sll0899 produces increased molecular mass of pilins (Kim et al., 2009). sll0141, sll0180, and slr0369 genes, which encoded putative inner membrane translocase components of TolCmediated secretion, are also responsible for pilus glycosylation.  The superscript lowercase letters of a, b, c, d, and e represent genes involved in transcriptional, post-transcriptional, translational, protein stability, post-translational levels in the regulation of pilin, and/or pilin-like proteins, respectively.
Nevertheless, motility assays confirmed that Sll0141 and Slr0369 are not essential for motility (Gonçalves et al., 2018). The three Deg mutants with less fucose in cells were found to have impaired motility, implying impairment of PilA1 glycosylation (Cheregi et al., 2015). Additionally, other types of PTMs and functional associations with PilA1 may exist in Synechocystis, similar to other bacteria (Stimson et al., 1996;Naessan et al., 2008). Moreover, it will be more meaningful to dissect the specific role of such modification sites on the mature pili in this model organism.

MOTILITY SIGNALING PATHWAY
Currently, increasing evidence suggest that Synechocystis motility is associated with several signaling pathways (Supplementary Figure S1B), for example the cAMP and/or cyclic-di-GMPmediated pathway (Bhaya et al., 2006;Yoshimura et al., 2010;Savakis et al., 2012;Xie et al., 2018). Two cAMP receptor-like proteins, named as Sycrp2 and Sycrp1, are known to be involved in twitching motility (Song et al., 2018). Sycrp1 has binding affinity for cAMP and directly binds to the upstream region of slr1667, and positively regulates the expression of pilA9-pilA10-pilA11-slr2018 gene cluster (Yoshimura et al., 2002a). Sycrp2 does not bind cAMP (Yoshimura et al., 2000(Yoshimura et al., , 2002b but may interact and work with Sycrp1 without functional redundancy (Song et al., 2018). Photoreceptor Cph2 with the GGDEF domain acts as a blue-light triggered c-di-GMP producer and thereby inhibits cell motility in blue light. Furthermore, Cph2 modulates motility by interacting with Cip1 (Slr1143, Cph2interacting protein 1) under red light (Angerer et al., 2017). MCP-CheA-CheY systems include sll0038-sll0043, slr1041-slr1044 and slr0322, slr0073, sll1291-sll1296 three gene clusters (Chung et al., 2001;Yoshihara et al., 2002). Jakob et al. (2019) have confirmed the direct interaction of the PixD-PixE complex with PilB1, suggesting that blue-light dependent negative phototaxis is controlled by the PixD-PixE signal transduction system. Using a computer-assisted video microscope motion analysis system, researchers found that Ca 2+ plays a significant role in regulating Synechocystis photo-orientation and motility (Moon et al., 2004). Additionally, histidine kinases (Hik18, Hik36, and Hik43) were predicted or demonstrated to be involved in phototaxis (Xu and Wang, 2019). Hik18 regulates positive phototaxis by suppressing pilus biosynthesis and expression of regulatory genes through the interplay with positive phototaxis/motility two-component proteins (Shin et al., 2008). An ethyleneresponsive signaling pathway affecting phototaxis was also characterized in Synechocystis (Kuchmina et al., 2017). Endogenous ethylene produced by heterologous expression of the Pseudomonas syringae ethylene-forming enzyme accelerates positive phototaxis. Ethylene mainly inactivates transcription from the csiR1/lsiR promoter. This promoter is under the control of UirS and its response regulator UirR. Synechocystis might use ethylene as an environmental signal in aquatic environments (Lacey and Binder, 2016). Further details of signal transduction regulating motility in Synechocystis remain to be explored.

PERSPECTIVE
There is substantial investigation of biological function of the thick pili in the model cyanobacterium Synechocystis. For instance, emerging evidence suggest the signaling pathway of motility is mediated by thick pili, which actively sense and respond to several environmental conditions. Apart from the subunit of the thick pilus apparatus, some regulatory proteins and small RNAs involved in these processes are likely to directly or indirectly influence thick pilus biogenesis and function. To uncover these regulation of pilus genes or their coding products, we may resort to mutant library and high-throughput "-omics" to facilitate elucidation of pilus-involved signaling pathways. In addition, thick pili of Synechocystis may also be significantly used in large-scale applications for biofuel biotechnology owing to their characteristics of biofilm formation and adhesion, which will render cell immobilization, biomass harvesting, and product purification more convenient. Although there is a contentious debate over microorganisms which can produce conductive pili, the studies on the Synechocystis thick pili may also have practical applications in the sustainable composite materials, and further bioavailability of metals in the future.

AUTHOR CONTRIBUTIONS
ZC designed and wrote the manuscript. XL revised the figures. YZ, XT, and BW made major revisions of the manuscript.

ACKNOWLEDGMENTS
We thank Christina Croney, Ph.D., from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2020.00241/ full#supplementary-material FIGURE S1 | Schematic illustration of genes involved in pilin regulation at different levels (A) and three potential signal pathways involved in motility in Synechocystis (B).