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Draft genome of Prochlorothrix hollandica CCAP 1490/1T (CALU1027), the chlorophyll a/b-containing filamentous cyanobacterium
Standards in Genomic Sciences volume 11, Article number: 82 (2016)
Prochlorothrix hollandica is filamentous non-heterocystous cyanobacterium which possesses the chlorophyll a/b light-harvesting complexes. Despite the growing interest in unusual green-pigmented cyanobacteria (prochlorophytes) to date only a few sequenced genome from prochlorophytes genera have been reported. This study sequenced the genome of Prochlorothrix hollandica CCAP 1490/1T (CALU1027). The produced draft genome assembly (5.5 Mb) contains 3737 protein-coding genes and 114 RNA genes.
The majority of cyanobacteria use chl a as a sole magnesium tetrapyrrole and common phycobilisome functioning as the bulk LHC. The prochlorophytes are a unique pigment subgroup of phylum Cyanobacteria – besides chl a, they contain other chls (b; 2,4-divinyl a; 2,4-divinyl b; f; g) as antennal pigments and simultaneously do not depend on the PBP-containing photoreceptors . Prochlorophytes demonstrating these outgroup features are few and encompass three marine unicellular genera ( Prochloron , Prochlorococcus , Acaryochloris) and one freshwater filamentous ( Prochlorothrix ). Unicellular Prochlorococcus spp. dominate in phytoplankton of oligotrophic regions of the world’s ocean and they are of exceptional importance from the viewpoint of global primary productivity . Prochloron sp. and Acaryochloris sp. were isolated in symbiotic association with colonial ascidians [3, 4]. In contrast to other prochlorophytes distribution, P. hollandica is characterized by low abundance and patchy distribution ; more detailed genome analysis would explain the ecophysiological background of this microorganism.
The genus Prochlorothrix is represented by two cultivable free-living species: Prochlorothrix hollandica and Prochlorothrix scandica, as well as a number of unculturable strains, originating from environmental 16S rRNA sequences . The distinction between P. hollandica and P. scandica is predominantly based on the molecular-genetic characters: DNA reassociation less than 30 % and DNA GC mol% content difference more than 5 % .
P. hollandica was isolated from the water bloom of Loosdrecht lake (near Amsterdam, Nertherlands) and validly published under the rules of Bacteriological Code as the type strain CCAP 1490/1T [7, 8]. The strain CCAP 1490/1 was generously supplied in 1994 by Dr. Hans C.P. Matthijs (Amsterdam University) and since then stored as CALU1027 at the Collection of Cultures of Algae and Microorganisms of St. Petersburg State University, CALU . Prochlorothrix hollandica is also maintained as different strains under collection indexes CCMP34, CCMP682, NIVA-5/89, SAG10.89, and the strain PCC9006 was reported as well . Another filamentous strain Prochlorothrix scandica was isolated from the phytoplankton of Lake Mälaren (Sweden), and is maintained as NIVA-8/90 and CALU1205 .
Among prochlorophytes at first were sequenced small genomes of unicellular Prochlorococcus sp. strains from LL- and HL-clades [2, 12, 13]. Four sequenced genomes of symbiotic Prochloron didemni P1-P4 are second in number . Acaryochloris marina genomes were sequenced in the strains CCME5410 and MBIC11017 , but only one paper mentioned about P. hollandica PCC9006 genome sequenced by Shich et al. in the context of improving of global cyanobacterial phylogeny . Here we report that genomic DNA of P. hollandica CCAP 1490/1T (CALU1027) was sequenced and obtained draft genome was annotated in order to conduct investigations in the field of comparative genomics of cyanobacteria and prochlorophytes.
Classification and features
A representative genomic 16S rDNA sequence of strain P. hollandica CCAP 1490/1T (CALU1027) was compared with another prochlorophytes and also with cyanobacterial type strains sequences obtained from GenBank. The tree was reconstructed using neighbor-joining with the Kimura-2 parameter substitution model in MEGA 6.0 [17, 18]. The phylogenetic position of P. hollandica CALU1027 represents in Fig. 1. Representatives of the genus Prochlorothrix are morphologically similar to other filamentous non-heterocystous cyanobacteria (Subsection III, Oscillatoriales) . In particular, P. hollandica CALU1027 produces long (>300 μm) straight, unbranched, non-motile trichomes (Fig. 2). Individual cells are 1.6 ± 0.1 μm wide and 11.8 ± 0.9 μm long that matches with the data reported [2, 4]. The opaque polar aggregates of gas vesicles resemble of those presented in Pseudanabaena type, but P. hollandica trichomes possess more slight intercellular constrictions (1/5 − 1/8 cell diameter). Trichomes multiply by means of occasional breakage without the resulting formation of hormogonia. Light- or electron microscopic-visible sheath and mucilaginous capsule were never observed; cell envelope demonstrates a typical Gram-negative triple-layer contour . A brief survey of P. hollandica CALU1027 properties according to MIGS recommendations  is given in Table 1.
Genome sequencing information
Genome project history
The WGS project AJTX02 has been deposited at DDBJ/EMBL/GenBank under accession AJTX00000000 (20.02.2013) and updated, in this research, as Draft Genome Project AJTX00000000.2 (29.04.2015). The assembled contigs have been deposited in NCBI. The project information and its association with the MIGS are summarized in Table 2.
Growth conditions and genomic DNA preparation
P. hollandica CALU1027 was grown in the BG-11 medium . The strain is a moderate mesophile, well growing at 20-22 °C under continuous flux of light. For DNA isolation cells were harvested by centrifugation and treated with 2 μg/mL Proteinase K in 0.1 M Tris-HCl (pH 8.5), 1.5 M NaCl, 20 mM Na2EDTA, and 2 % cetyltrimethylammonium bromide at 55 °C for 3-4 h. DNA was purified by standard protocol of organic extraction and ethanol precipitation.
Genome sequencing and assembly
For genome sequencing, DNA was randomly fragmented using Q800R sonicator system. After size selection, 500 bp DNA fragments were used for constructing sequence libraries and thereafter sequenced with a 250 bp paired-end reads method using the Illumina MiSeq platform according to the manufacturer’s protocol, resulting in 3,679,738 read pairs. Reads were processed via the Trimmomatic 0.32 tool  and after filtration there were 3,665,348 read pairs. The obtained reads were used for further genome assembly with SPAdes 3.5 . From the resulting assembly, the P. hollandica CALU1027 contigs was selected and scaffolded with Contiguator 2.7.4 , using assembly GCF_000332315.1 as a reference. The draft genome of P. hollandica CALU1027 contained about 5.5 Mbp in 286 contigs organized in 10 scaffolds; the N50 length of the contigs was 33,173 and N50 length of the scaffolds - 1,244,169 bp (Table 3).
Protein-coding genes of draft genome assembly were predicted using the NCBI Prokaryotic Genome Annotation Pipeline (v.2.10) and an annotation method of best-placed reference protein set with GeneMarkS+ . The annotated features were genes, CDS, rRNA, tRNA, ncRNA, and repeat regions. Functional assignments of the predicted ORFs were based on a BLASTP homology search against WGS of phylogenetically closest cyanobacteria and the NCBI non-redundant database. Functional assignment was also performed with a BLASTP homology search against the Clusters of Orthologous Groups (COG) database [25, 26]. As much as 2855 genes (66 %) were assigned as a putative function, and the remaining genes were annotated as either hypothetical proteins or proteins with unknown function.
Insights from the genome sequence
The assembly and analysis of P. hollandica CALU1027 genome annotation revealed a repertoire of genes necessary for the autonomous energy and substrate metabolism: 743 detected genes with relevance to 129 metabolic pathways have orthologs in P. hollandica CALU1027 and other cyanobacteria (Table 5). Comparative genomes analysis of P. hollandica CALU1027 with filamentious heterocystous cyanobacteria Anabaena variabilis ATCC29413 and unicellular prochlorophytes Prochlorococcus marinus CCMP1375 and Acaryochloris marina MBIC11017 revealed that the main differences were in the amino acids compounds, carbohydrates metabolism, membrane transport and stress response systems (data not shown).
Chl a/b-containing Prochlorothrix and Prochloron were long considered to have a common ancestry with chloroplasts of green algae and higher plants [27, 28]. However, P. hollandica and another prochlorophytes were shown to possess unique genes pcbA − pcbC coding chl a/b-LHC apoproteins and they are dissimilar from CAB apoprotein superfamily of chloroplast antenna [19–30]. It is notable that we found some PS II proteins commonly absent in cyanobacteria but usually belonging to chloroplast in green algae and higher plants: PsbW (6.1 kDa, nuclear encoded), PsbT (5 kDa, nuclear encoded), PsbR (10 kDa) and PsbQ (16 kDa, oxygen evolving complex protein). We also found that P. hollandica contains an ortholog of hetR gene (key regulator of heterocyst differentiation) although all these filamentous non-heterocystous cyanobacteria are devoid of nitrogenase and other prerequisites for diazotrophy [31, 32].
Partensky F, Garczarek L. The Photosynthetic apparatus of chlorophyll b- and d-containing cyanobacteria. In: Larkum AW, Douglas SE, Raven JA, editors. Photosynthesis in Algae. Dordrecht: Kluwer Academic Publishers; 2003. p. 29–62.
Rocap G, Larimer F, Lamerdin J, Malfatti S, Chain P, Ahlgren N, et al. Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature. 2003;424:1042–7.
Lewin RA. Prochloron, type genus of the Prochlorophyta. Phycologia. 1977;16:217.
Miyashita H, Ikemoto H, Kurano N, Miyachi S, Chihara M. Acaryochloris marina gen. et sp. nov. (Cyanobacteria), an oxygenic photosynthetic prokaryote containing chl d as a major pigment. J Phycol. 2003;39:1247–53.
Pinevich A, Velichko N, Ivanikova N. Cyanobacteria of the genus Prochlorothrix. Front Microbiol. 2012;3:1–13.
Velichko N, Averina S, Gavrilova O, Ivanikova N, Pinevich A. Probing environmental DNA reveals circum-Baltic presence and diversity of chlorophyll a/b-containing filamentous cyanobacteria (genus Prochlorothrix). Hydrobiol. 2014;736:165–77.
Burger-Wiersma T, Veenhius M, Korthals HJ, van de Wiel CCM, Mur LR. A new prokaryote containing chlorophylls a and b. Nature. 1986;320:262–4.
Burger-Wiersma T, Stal L, Mur LR. Prochlorothrix hollandica gen. nov., sp. nov., a filamentous oxygenic photoautotrophic prokaryote containing chlorophylls a and b: assignment to Prochlorothrichaceae fam. nov., and order Prochlorothrichales Florenzano, Balloni, and Materassi 1986, with emendation of the ordinal description. Int J Syst Bacteriol. 1989;39:250–7.
Pinevich A, Mamkaeva K, Titova N, Gavrilova O, Ermilova E, Kvitko K, et al. St. Petersburg Culture Collection (CALU): Four decades of storage and research with microscopic algae, cyanobacteria and other microorganisms. Nova Hedw. 2004;79:115–26.
Schyns G, Rippka R, Namane A, Campbell D, Herdman M, Houmard J. Prochlorothrix hollandica PCC 9006: genomic properties of an axenic representative of the chlorophyll a/b-containing oxyphotobacteria. Res Microbiol. 1997;148:345–54.
Velichko N, Averina S, Gavrilova O, Pinevich A, Ivanikova N, Skulberg O. Polyphasic emended description of the filamentous prochlorophyte Prochlorothrix scandica Skulberg 2008. Algol Stud. 2013;141:11–27.
Dufresne A, Salanoubat M, Partensky F, Artiguenave F, Axmann I, Barbe V, et al. Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimal oxyphototrophic genome. Proc Natl Acad Sci U S A. 2003;100:10020–5.
Biller SJ, Berube PM, Berta-Thompson J-W, Kelly L, Roggensack SE, Awad L, et al. Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus. Sci Data. 2014;1:140034.
Donia M, Fricke W, Partensky F, Coxe J, Elshahavi SI, White JR, et al. Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis. Proc Natl Acad Sci U S A. 2011;108:1423–32.
Swingley WD, Chen M, Cheung PC, Conrad AL, Dejesa LC, Hao J, et al. Niche adaptation and genome expansion in the chlorophyll d-producing cyanobacterium Acaryochloris marina. Proc Natl Acad Sci U S A. 2008;105:2005–10.
Shih P, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, et al. Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci U S A. 2013;110:1053–8.
Lewin RA. Prochloron and the theory of symbiogenesis. Ann NY Acad Sci. 1981;361:325–9.
Bruno WJ, Socci ND, Halpern AL. Weighted neighbor joining: a likelihood-based approach to distance-based phylogeny reconstruction. Mol Biol Evol. 2000;17(1):189–97.
Castenholz RW. Phylum BX Cyanobacteria. Oxygenic Photosynthetic Bacteria. In: Garrity G, Boone DR, Castenholz RW, editors. Bergey’s Manual of Systematic Bacteriology, vol. 1. 2nd ed. New York: Springer; 2001. p. 473–600.
Field D, Garrity G, Gray T, Morrison N, Slengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.
Bolger A, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.
Nurk S, Bankevich A, Antipov D, Gurevich AA, Korobeynikov A, Lapidus A, et al. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J Comp Biol. 2013;20:714–37.
Galardini M, et al. CONTIGuator: a bacterial genomes finishing tool for structural insights on draft genomes. Source Code Biol Med. 2011;6:11.
Besemer J, Lomsadze A, Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nuc Ac Res. 2001;29:2607–18.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.
Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000;28:33–6.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis v. 6.0. Mol Biol Evol. 2013;30:2725–9.
Turner S, Burger-Wiersma T, Giovannoni SJ, Mur LR, Pace NR. The relationship of a prochlorophyte Prochlorothrix hollandica to green chloroplasts. Nature. 1989;26:380–2.
La Roche J, van der Staay GM, Partensky F, Ducret A, Aebersold R, Li R, et al. Independent evolution of the prochlorophyte and green plant chlorophyll-a/b light-harvesting proteins. Proc Natl Acad Sci USA. 1996;93:15244–8.
Tomitani A, Okada K, Miyashita H, Matthijs H, Ohno T, Tanaka A. Chlorophyll b and phycobilins in the common ancestor of cyanobacteria and chloroplast. Nature. 1999;400:159–62.
Velichko N, Chernyaeva E, Averina S, Gavrilova O, Lapidus A, Pinevich A. Consortium of the «bichlorophylous» cyanobacterium Prochlorothrix hollandica and chemoheterotrophic partner bacteria: culture and metagenome-based description. Env Microbiol. 2015;7:623–33.
Zhang JY, Chen WL, Zhang CC. het R and patS, two genes necessary for heterocyst pattern formation, are widespread in filamentous nonheterocyst-forming cyanobacteria. Microbiology. 2009;155:1418–26.
Woese CR, Kandler O, Wheels ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.
Florenzano G, Balloni W, Materassi R. Nomenclature of Prochloron didemni (Lewin 1977) sp. nov., nom. rev., Prochloron (Lewin 1976) gen. nov., nom. rev., Prochloraceae fam. nov., Prochlorales ord. nov., nom. rev. in the class Photobacteria Gibbons and Murray 1978. Int J Syst Bacteriol. 1986;36:351–3.
This work was financially supported by Russian Foundation for Fundamental Research (grant № 16-04-00174) and by St. Petersburg State University (grant № 188.8.131.525). We gratefully acknowledge the technical help of St. Petersburg University «Resource Center for Microscopy and Microanalysis» and «Resource Center for Development of Molecular and Cell Technologies».
NV, EC and AL designed and carried out the experiments. NV, MR and AP performed the data analysis and drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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Velichko, N., Rayko, M., Chernyaeva, E. et al. Draft genome of Prochlorothrix hollandica CCAP 1490/1T (CALU1027), the chlorophyll a/b-containing filamentous cyanobacterium. Stand in Genomic Sci 11, 82 (2016). https://doi.org/10.1186/s40793-016-0204-4
- Prochlorothrix hollandica
- Comparative genomics