Harmful Algae 75 (2018) 87–93
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The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis Liang Zhaoa,b , Yulong Songc, Lin Lia , Nanqin Gana , Jerry J. Brandd , Lirong Songa,* a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, PR China University of Chinese Academy of Sciences, Beijing, 100049, PR China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, PR China d The UTEX Culture Collection of Algae, University of Texas at Austin, Austin, TX, 78712, USA b c
A R T I C L E I N F O
Article history: Received 25 October 2017 Received in revised form 8 April 2018 Accepted 8 April 2018 Available online 26 April 2018 Keywords: Microcystis Cyanobacterial bloom DNA methylation modification SMRT Methyltransferase Epigenetics
A B S T R A C T
The occurrence of harmful Microcystis blooms is increasing in frequency in a myriad of freshwater ecosystems. Despite considerable research pertaining to the cause and nature of these blooms, the molecular mechanisms behind the cosmopolitan distribution and phenotypic diversity in Microcystis are still unclear. We compared the patterns and extent of DNA methylation in three strains of Microcystis, PCC 7806SL, NIES-2549 and FACHB-1757, using Single Molecule Real-Time (SMRT) sequencing technology. Intact restriction-modification (R-M) systems were identified from the genomes of these strains, and from two previously sequenced strains of Microcystis, NIES-843 and TAIHU98. A large number of methylation motifs and R-M genes were identified in these strains, which differ substantially among different strains. Of the 35 motifs identified, eighteen had not previously been reported. Strain NIES-843 contains a larger number of total putative methyltransferase genes than have been reported previously from any bacterial genome. Genomic comparisons reveal that methyltransferases (some partial) may have been acquired from the environment through horizontal gene transfer. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Cyanobacterial blooms, which are currently increasing in frequency and intensity, are a threat to ecosystems and public safety due to their production of noxious compounds, including potent toxins (Watson et al., 2000; Heisler et al., 2008; Pearson et al., 2010). Microcystis, the most commonly found and widely studied bloomforming cyanobacterium, is known for its phenotypic diversity and adaptive capacity (Wu et al., 2011; Harke et al., 2016; Sandrini et al., 2016), colonizing diverse eutrophic freshwaters and estuary ecosystems worldwide. Reasons for the proliferation of Microcystis in diverse niches are not well understood (D’Agostino et al., 2016; Harke et al., 2016), although features of its genome offer some insights. Microcystis genomes are extensively open and plastic (Yang et al., 2015). Comparative genomic studies demonstrate that different isolates display different numbers and arrangements of genes, and the core number of genes (approximately 2100) is less than half the number of genes in individual isolates (Humbert et al.,
* Corresponding author. E-mail address:
[email protected] (L. Song). https://doi.org/10.1016/j.hal.2018.04.005 1568-9883/© 2018 Elsevier B.V. All rights reserved.
2013; Yang et al., 2015). Genomic openness is also displayed by bacterial pathogens such as Helicobacter pylori (Alm et al., 1999), Escherichia coli (Hendrickson, 2009), and Pseudomonas (Özen and Ussery, 2012). Microcystis may share other characteristics with these pathogens, such as the features of their methylomes (Fang et al., 2012; Furuta and Kobayashi, 2012; Furuta et al., 2014; Murray et al., 2012; Krebes et al., 2014). DNA methylation is a mechanism of epigenetic control that is catalyzed by a system of DNA methyltransferases (MTases) (Jeltsch, 2002). It plays a crucial role in many bacterial pathogens, such as E. coli (Reisenauer et al., 1999; Low et al., 2001) and Salmonella typhimurium (Heithoff et al., 1999), and has expanded the genetic alphabet beyond G, A, T, and C (Fang et al., 2012). In prokaryotes, the most frequent DNA methylations are N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C) (Jeltsch, 2002; Wion and Casadesús, 2006). The role of DNA methylation in restriction-modification (R-M) systems is wellknown (Roberts et al., 2003; Tock and Dryden, 2005). DNA methylation is also important in replication, mismatch repair, phase variation, transcriptional regulation, and virulence (Reisenauer et al., 1999; Low et al., 2001; Wion and Casadesús, 2006; Marinus and Casadesús, 2009).
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Single Molecule Real-Time (SMRT) sequencing technology allows for rapid and reliable determination of sites of DNA modification in an entire bacterial genome at single-base resolution (Fang et al., 2012; Murray et al., 2012; Krebes et al., 2014; Beaulaurier et al., 2015; Blow et al., 2016). DNA methylation alters the kinetics of real-time DNA polymerase function during the SMRT sequencing process (Flusberg et al., 2010). The interpulse duration (IPD) information in raw SMRT data is interpreted to identify a DNA methylation profile called a methylome. Recent characterization of the methylomes of entire genomes has shown that the biological functions of DNA methylation extend far beyond what was previously known. For example, modulation of methylation patterns plays roles in global transcriptional regulation (Fang et al., 2012), epigenetics-driven adaptive evolution (Furuta and Kobayashi, 2012; Furuta et al., 2014), and bacterial survival under antibiotic stress (Cohen et al., 2016). As described herein, a comprehensive survey of the epigenomic landscape and R-M systems in several strains of Microcystis was conducted. These strains were shown to possess a large number of methylation motifs and R-M system components. An extensive examination of MTase genes in Microcystis and a comparison with homologous genes in other prokaryotes was performed. The results provide insight into how this epigenetic framework became established in Microcystis. 2. Material and methods 2.1. Strains and sequencing information Microcystis strains with well-assembled genomes were utilized in this study, including four Microcystis aeruginosa isolates (PCC 7806SL, NIES-2549 (Yamaguchi et al., 2015), NIES-843 (Kaneko et al., 2007) and TAIHU98 (Yang et al., 2013)) and one Microcystis panniformis strain (FACHB-1757 (Zhang et al., 2016)). Genomic and isolation information pertaining to these five strains are presented in Table S1. An axenic culture of PCC 7806 was obtained from the Pasteur Culture Collection of Cyanobacteria (Paris, France). The genome of this strain (labeled PCC 7806SL to distinguish it from the PCC 7806 sequence that was published previously (Frangeul et al., 2008)) was sequenced with the PacBio RS II platform according to previously described methods (LZ, and LS, submitted for publication). Briefly, >20 mg of high-quality purified genomic DNA was first sheared with a g-TUBE device (Covaris Inc., Woburn, MA, USA), then a 20-kb fragmented library was constructed using DNA Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). Upon completion of library preparation, the genomic DNA was sequenced using a single SMRT Cell with 240-min total collection time, using P6 DNA polymerase and C4 chemistry on the PacBio RS II platform by Nextomics Biosciences (Wuhan, Hubei, China). The unpublished raw SMRT sequencing data for NIES-2549, which was generated utilizing P6-C3 chemistry on the RS II platform, was provided by Dr. Haruyo Yamaguchi of the National Institute for Environmental Studies (NIES), Japan (Yamaguchi et al., 2015). DNA methylation information for PCC 7806SL and NIES-2549 was then analyzed with the SMRT raw data using the Modification Detection and MotifFinder (Motif Analysis) Module in the standard Pacific Biosciences SMRT Analysis System, Version 2.3 (http://www.pacb. com/wp-content/uploads/2015/09/SMRT-Pipe-Reference-Guide. pdf). The methylome information of FACHB-1757 was identified from previous work of Zhang et al. (2016). The method used for sequencing the three Microcystis strains used in this study did not have high enough coverage (100 X) for m5C determination (Table S2). Also there was no data on Tet-oxidation (He et al., 2011) for confident m5C identification. Thus, we collected DNA methylation data for m6A and m4C only.
2.2. Identification of R-M components in five Microcystis strains Sequences corresponding to all putative PCC 7806SL proteins were aligned to REBASE (Roberts et al., 2015) sequences in order to identify R-M components using BLAST (https://blast.ncbi.nlm.nih. gov/Blast.cgi) with an identity >50%, threshold e-value
