Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-Synechococcus' clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon

Unité des Cyanobactéries (CNRS URA 2172), Département de Biochimie et Génétique Moléculaire, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France 1 Station Biologique de Roscoff (CNRS UPR 9042), BP 74, 29682 Roscoff Cedex, France 2 Author for correspondence: Isabelle Iteman. Tel: +33 1 4568 8416. Fax: +33 1 40613042. e-mail: iiteman{at}pasteur.fr PCR amplicons of the Internal Transcribed Spacer (ITS) of the rrn operon of three axenic OMF (oceanic, marine and freshwater) strains of ‘ Synechococcus ’ (WH7803, PCC 7001 and PCC 6307, respectively) differ greatly in length from that of the axenic Prochlorococcus marinus subsp. pastoris PCC 9511 T , although these four cyanobacteria cluster relatively closely in phylogenetic trees inferred from 16S rRNA gene sequences. The ITSs of three strains (PCC 9511 T , PCC 6307 and PCC 7001) were sequenced and compared with those available for strains Prochlorococcus MED4 (CCMP 1378) and MIT9313 from genome sequencing projects. In spite of large differences in length, sequence and mean DNA base composition, conserved domains important for transcriptional antitermination and folding of the rRNA transcripts were identified in all ITSs. A new group-specific primer permitted ITS amplification even with non-axenic isolates of Prochlorococcus and one OMF-‘ Synechococcus ’ strain. Prochlorococcus isolates of the high-light-adapted clade (HL) differed from representatives of the low-light-adapted clade (LL) by the length of their ITS. Restriction fragment length polymorphism (RFLP) of the ITS amplicons revealed three subclusters among the HL strains. Size, sequence data and RFLP of the ITS amplicons will therefore be valuable markers for the identification of different Prochlorococcus genotypes and for their discrimination from other cyanobacterial relatives with which they often co-exist in oceanic ecosystems. Keywords: Prochlorococcus marinus subsp. pastoris PCC 9511 T , cyanobacteria, group-specific ITS primers, oceanic, coastal marine and freshwater ‘ Synechococcus ’, secondary structure of the rrn operon Abbreviations: DV, divinyl; HL, high-light-adapted clade; LL, low-light-adapted clade; ITS, Internal Transcribed Spacer; OMF, oceanic, marine and freshwater; RFLP, restriction fragment length polymorphism The GenBank accession numbers for the ITS amplicon sequences reported in this paper are AF387607 (PCC 9511 T ), AF387610 (PCC 6307), AF387608 (PCC 7001) and AF387609 (PCC 7941). a Present address: Unité de Reche