Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences

Abstract

In order to assess the appropriateness of the use of internal transcribed spacer (ITS) sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the 16S-23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphisms (T-RFLP). When using the standard application of these techniques, we obtained more than one band or terminal restriction fragment (T-RF) per strain or cloned sequence. Reports in literature have suggested that these anomalies can result from the formation of secondary structures. Secondary structures of the ITS sequences of Prochlorococcus and Synechococcus strains were computationally modelled at the different temperatures that were used during the polymerase chain reaction (PCR). Modelling results predicted the existence of hairpin loops that would still be present at the extension temperature; it is likely that these loops produced incomplete and single stranded PCR products. We modified the standard T-RFLP procedure by adding the labelled ITS primer in the last two cycles of the PCR reaction; this resulted, in most cases, in only one T-RF per ribotype. Application of this technique to a natural picoplankton community in marine waters off northern Chile, showed that it was possible to identify the presence, and determine the relative abundance, of several phylogenetic lineages within the genera Prochlorococcus and Synechococcus inhabiting the euphotic zone. Phylogenetic analysis of ITS sequences obtained by cloning and sequencing DNA from the same sample confirmed the presence of the different genotypes. With the proposed modification, T-RFLP profiles should therefore be suitable for studying the diversity of natural populations of cyanobacteria, and should become an important tool to study the factors influencing the genetic structure and distribution of these organisms.Key words: cyanobacteria, DGGE, T-RFLP, ITS, secondary structure. RESUMENCon el fin de evaluar la utilización de secuencias del espaciador interno transcrito (ITS) en estudios de genética de población de cianobacterias marinas, se amplificó y clonó la secuencia del gen ARNr 16S junto a la región espaciadora 16S-23S ARNr de seis cepas de Prochlorococcus y Synechococcus. Se analizaron los amplicones del ITS por electroforesis en gel de gradiente de desnaturalización (DGGE) y por polimorfismos de longitud de fragmentos de restricción terminal (T-RFLP). Al aplicar los métodos estándares de estas técnicas, se obtuvo más de una banda o fragmento de restricción terminal (T-RF) por cepa o secuencia clonada. Informes en la literatura han sugerido que estas anomalías podrían ser atribuidas a la formación de estructuras secundarias. Por consiguiente, la estructura secundaria de las secuencias de ITS de las cepas de Prochlorococcus y Synechococcus fue modelada a las diferentes temperaturas que se utilizaron durante la reacción en cadena de polimera...

Characterization of Cyanate Metabolism in Marine Synechococcus and Prochlorococcus spp

“…Tree topologies of cyanobacterial CynS matched phylogenies based on 16S rRNA, ITS and ntcA (12,21,28,30,33), and branching was supported by strong posterior probabilities. Based on these observations, we suggest that cynS was common in ancestral cyanobacteria and cynS was lost from many modern cyanobacteria.…”

Seasonal changes in picocyanobacterial diversity as revealed by pyrosequencing in temperate waters of the East China Sea and the East Sea

“…The barcoded primers consisted of adapter A (forward) or B (reverse) + Key (TCAG) + barcode (10 bp oligonucleotide, forward only) + picocyanobacteria-specific primers. The ITS-af (5'-GGA TCA CCT CCT AAC AGG GAG-3') and ITS-ar (5'-GGA CCT CAC CCT TAT CAG GG-3') primers were used as specific primers (Lavin et al 2008). The ITS-af and ITS-ar primers showed perfect matches to 99% of the 2389 picocyanobacterial ITS sequences obtained from cultures and clone libraries, indicating high specificity to picocyanobacteria.…”

“…These studies have revealed the existence of diverse Synechococcus and Prochlorococcus clades or ecotypes. Most studies of picocyanobacterial distribution in marine environments have been conducted using dotblot hybridization and real-time quantitative polymerase chain reaction (qPCR) methods using cladespecific probes (Fuller et al 2003, Zwirglmaier et al 2008, Tai & Palenik 2009, Post et al 2011, Ahlgren & Rocap 2012 or terminal restriction fragment length polymorphism (Lavin et al 2008). Recently, barcoded pyrosequencing methods that identify short taxonomic molecular markers via 454 sequencing technologies have been used to analyze microbial diversity in various environments (Binladen et al 2007, Qian et al 2011).…”

The influence of anoxia and substrate availability on N2O cycling by denitrification in the upper boundary of the oxygen minimum zone off northern Chile

“…Furthermore, the oxycline in the study area has the genetic potential to perform various processes, as has been indicated by several studies that show the presence of amo-A genes, coding for the active subunit of the AMO enzyme (Molina et al 2007), and nirS and nosZ genes, coding for NO − 2 and N 2 O reductase, respectively (Castro-González et al 2005; Castro-González, Ulloa, and Farías 2015). In addition, the oxycline base may coincide with the presence of a secondary fluorescence maximum where high relative abundances of Prochlorococcus and Synechococcus have been observed (Lavin et al 2008(Lavin et al , 2010. These cyanobacteria are able to assimilate NO − 2 via nitrite reductase, given that they lack nitrate reductase (Moore, Rocap, and Chisholm 1998;Partensky and Garczarek 2010;Zehr and Kudela 2011).…”

“…c. Effects of NO − 2 plus DOC addition on net N 2 Od cycling in the UBOMZ at the offshore station Table 2 presents the results for the experiments carried out under induced anoxic conditions at the base of the oxycline, where nutrients and/or DOC limitation may prevail. At that depth, low NO − 2 levels (0.22 μM) were registered onboard (Lavin et al 2008(Lavin et al , 2010. Furthermore, the addition of NO − 2 and DOC stimulated the denitrifying community and resulted in higher N 2 Opd (up to 78.8 nM d −1 ) and N 2 Ocd (58.71 nM d −1 ) rates than those observed at the UBOMZ at the coastal station.…”