We report the first complete genome of Microcystis aeruginosa from North America. A harmful bloom that occurred in the Caloosahatchee River in 2018 led to a state of emergency declaration in Florida. Although strain FD4 was isolated from this toxic bloom, the genome did not have a microcystin biosynthetic gene cluster

Dahedl, Elizabeth K.

Hancock, Taylor L.

Krausfeldt, Lauren E.

Lopez, Jose V.

Ndungu, Luka K.

Rosen, Barry H.

Steele, Jacob H.

Urakawa, Haruka E.

Urakawa, Hidetoshi

English

NSU Works

Nova Southeastern University NSUWorks Biology Faculty Articles Department of Biological Sciences 9-1-2020 Complete genome sequence of microcystis aeruginosa FD4, isolated from a subtropical river in Southwest Florida Hidetoshi Urakawa Florida Gulf Coast University Taylor L. Hancock Florida Gulf Coast University Jacob H. Steele Florida Gulf Coast University Elizabeth K. Dahedl Florida Gulf Coast University Haruka E. Urakawa Florida Gulf Coast University See next page for additional authors Follow this and additional works at: https://nsuworks.nova.edu/cnso_bio_facarticles  Part of the Biology Commons NSUWorks Citation Urakawa, Hidetoshi; Taylor L. Hancock; Jacob H. Steele; Elizabeth K. Dahedl; Haruka E. Urakawa; Luka K. Ndungu; Lauren E. Krausfeldt; Barry H. Rosen; and Jose V. Lopez. 2020. "Complete genome sequence of microcystis aeruginosa FD4, isolated from a subtropical river in Southwest Florida." Microbiology Resource Announcements 9, (38). doi:10.1128/MRA.00813-20. This Article is brought to you for free and open access by the Department of Biological Sciences at NSUWorks. It has been accepted for inclusion in Biology Faculty Articles by an authorized administrator of NSUWorks. For more information, please contact nsuworks@nova.edu. Authors Hidetoshi Urakawa, Taylor L. Hancock, Jacob H. Steele, Elizabeth K. Dahedl, Haruka E. Urakawa, Luka K. Ndungu, Lauren E. Krausfeldt, Barry H. Rosen, and Jose V. Lopez This article is available at NSUWorks: https://nsuworks.nova.edu/cnso_bio_facarticles/1154 Complete Genome Sequence of Microcystis aeruginosa FD4,Isolated from a Subtropical River in Southwest FloridaHidetoshi Urakawa,a Taylor L. Hancock,a Jacob H. Steele,a Elizabeth K. Dahedl,a Haruka E. Urakawa,b Luka K. Ndungu,aLauren E. Krausfeldt,c Barry H. Rosen,a Jose V. LopezcaDepartment of Ecology and Environmental Studies, The Water School, Florida Gulf Coast University, Fort Myers, Florida, USAbDepartment of Marine and Earth Sciences, The Water School, Florida Gulf Coast University, Fort Myers, Florida, USAcDepartment of Biological Sciences, Nova Southeastern University, Dania Beach, Florida, USAABSTRACT We report the first complete genome of Microcystis aeruginosa fromNorth America. A harmful bloom that occurred in the Caloosahatchee River in 2018led to a state of emergency declaration in Florida. Although strain FD4 was isolatedfrom this toxic bloom, the genome did not have a microcystin biosynthetic genecluster.Lake Okeechobee is the largest lake in the southeastern United States (1,900 km2)and serves as a hub for water flow from the north to the Everglades in southernFlorida. When flood control releases are necessary, water is directed to the Atlantic andGulf coasts through two waterways, the St. Lucie River and the Caloosahatchee River,respectively (1). Since the 1980s, Lake Okeechobee and its waterways have sufferedfrom chronic eutrophication problems and harmful cyanobacterial blooms (2). A recentMicrocystis aeruginosa bloom that occurred in the Caloosahatchee River in 2018 led toa state of emergency declaration in Florida (3).M. aeruginosa FD4 was isolated from surface water collected from the Caloosa-hatchee River at the Fort Denaud Bridge (26.7444N, 81.5103W) on 27 June 2018 duringthe bloom (3). The water quality parameters determined were as follows: watertemperature, 32.4°C; pH 7.7; dissolved oxygen, 3.0 mg/liter; total iron, 1.32 mg/liter;chlorophyll a, 72.5 g/liter; microcystin, 450.5 g/liter; and hydrogen peroxide, 9.2 g/liter (3). Surface scum was originally incubated with ultrapure water with pyruvic acid(8.8 g/liter) at room temperature and then transferred to pyruvic acid-amended 10%BG-11 with germanium dioxide (10 mg/liter) and cycloheximide (100 mg/liter) to inhibitthe growth of diatoms and other eukaryotes, respectively. Once we microscopicallyconfirmed that the culture was unialgal, strain FD4 was maintained in BG-11 mediumat 25°C under fluorescent light with a 12:12-h light/dark cycle. Genomic DNA wasextracted using the Quick-DNA miniprep plus kit (Zymo Research). The DNA was madeinto SMRTbell libraries using the Express Template prep kit 2.0 (Pacific Biosciences). Thesample was multiplexed with other samples into a single library and size selected usingBluePippin (Sage Sciences) according to the manufacturer’s recommendations usingthe 0.75% DF Marker S1 high-pass 6-kb to 10-kb v3 run protocol and S1 marker (BPstartvalue of 8,000). The size-selected SMRTbell library was annealed and bound accordingto the SMRT Link setup and sequenced with the PacBio Sequel II system (PacificBiosciences). Raw PacBio reads were converted to FASTA format with samtools fastaand then assembled with Flye version 2.6 (4). Default parameters were used for allsoftware. The assembled genome was annotated with Prokka version 1.11 (5). Thegenome was annotated with SEED Viewer (6) and the NCBI Prokaryotic GenomeAnnotation Pipeline (GeneMark S-2 version 4.10) (7). The final assembly of thegenome comprised 5.45 Mbp at 125-fold coverage (N50 and N90, 10,352 and 6,453 bp,Citation Urakawa H, Hancock TL, Steele JH,Dahedl EK, Urakawa HE, Ndungu LK, KrausfeldtLE, Rosen BH, Lopez JV. 2020. Completegenome sequence of Microcystis aeruginosaFD4, isolated from a subtropical river insouthwest Florida. Microbiol Resour Announc9:e00813-20. https://doi.org/10.1128/MRA.00813-20.Editor Frank J. Stewart, Georgia Institute ofTechnologyCopyright © 2020 Urakawa et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to HidetoshiUrakawa, hurakawa@fgcu.edu.Received 14 July 2020Accepted 26 August 2020Published 17 September 2020GENOME SEQUENCEScrossmVolume 9 Issue 38 e00813-20 mra.asm.org 1Downloaded from https://journals.asm.org/journal/mra on 29 October 2021 by 137.52.38.11.respectively) and consisted of two completely closed contigs, one chromosome, andone plasmid (Table 1).Although strain FD4 was isolated from the toxic algal bloom (3), the genome did nothave a microcystin biosynthetic gene cluster. It was confirmed by the annotations ofthe predicted open reading frames and homology searches against the genome. Thepresence of nine secondary metabolite gene clusters, including piricyclamide (8),micropeptin, and aeruginosin, were identified using antiSMASH version 5.1.2 (9). Thesenumbers were quite small in comparison with those of M. aeruginosa NIES-2481(GenBank accession number CP012375.1), in which 28 secondary metabolite geneclusters were found (10). Haft et al. (11) found that all bacteria with a short C-terminalhomology domain that includes a highly conserved motif proline-glutamate-prolinetriad (PEP-CTERM) have both an outer membrane and exopolysaccharide productiongenes. Notably, 62 clusters of PEP-CTERM sorting domain-containing protein werefound along with genes of exopolysaccharide biosynthesis polyprenyl glycosylphos-photransferase, polysaccharide pyruvyl transferase (CsaB), and WecB/TagA/CpsF familyglycosyltransferase in the genome, suggesting the possible association of these genesfor Microcystis colony formation (11). Consistent with the colony-forming ability ofstrain FD4, we found a coding gene of gas vesicle protein GvpC and psb and apcphotoregulation clusters, which confer an ecological advantage to M. aeruginosa FD4to compete with other phytoplankton through surface scum formation (12). Kardinaaland colleagues (13) reported that nontoxic strains of Microcystis were better compet-itors for light than toxic strains. Further genome annotation and genome comparisonswith other strains of M. aeruginosa will provide additional insights into the ecologicaladaptation of this cyanobacterium.Data availability. The genome sequence information has been deposited underBioProject number PRJNA595771 (GenBank accession numbers CP046973.1 [chromo-some] and CP046974.1 [plasmid]). The PacBio reads have been deposited in the SRAunder the accession number SRR12188899.ACKNOWLEDGMENTSWe thank Rick D. Bartleson at Sanibel-Captiva Conservation Foundation for fieldassistance.This research was supported by the NSF Division of Environmental Biology grantDEB-1664052 and the Aquatic Nuisance Species Research Program, U.S. Army Corps ofEngineers (cooperative agreement number W912HZ-19-2-0014). A part of this work wasalso supported via funding provided by FGCU via The Water School, the Blair Founda-tion, the Seidler Fund, and the FGCU Center for Environmental and SustainabilityEducation.TABLE 1 Genome statistics of Microcystis aeruginosa FD4aAttributeb ValueTotal genome size (bp) 5,493,112Chromosome size (bp) 5,449,501Plasmid size (bp) 43,611GC content (%) 42.59Total no. of genes 5,455Total no. of CDSs 5,403No. of coding genes 4,679No. of CDSs with protein 4,679No. of RNA genes 52No. of rRNA sets (5S, 16S, 23S) 2No. of tRNAs 42No. of ncRNAs 4Total no. of pseudogenes 724No. of CRISPR arrays 3a Annotation is based on the total genome and the NCBI Prokaryotic Genome Annotation Pipeline.b CDS, coding sequences; ncRNAs, noncoding RNAs; CRISPR, clustered regularly interspaced short palindromicrepeat.Urakawa et al.Volume 9 Issue 38 e00813-20 mra.asm.org 2Downloaded from https://journals.asm.org/journal/mra on 29 October 2021 by 137.52.38.11.REFERENCES1. 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Complete genome sequence of microcystis aeruginosa FD4, isolated from a subtropical river in Southwest Florida

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Complete genome sequence of microcystis aeruginosa FD4, isolated from a subtropical river in Southwest Florida

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