Enumeration of Novel Lake Superior Cluster I (LSI) as a friction of the total picocyanobacteria in the autotrophic picoplankton (APP) Using quantitative real-time PCR. (c2009)

Abstract

Cyanobacteria known previously as blue-green algae are successful phototrophic organisms which are ubiquitously found in the environment. This organism contributes significantly to the global carbon and nitrogen budget. Classical classification of cyanobacteria is mainly based on morphology and subdivided into five categories. Molecular classification involves mainly 16S rRNA, 16S-23S rRNA intergenic spacer (IGS), and cpcBA IGS. Planktonic organisms are separated in literature based on size. We are primarily interested in picocyanobacteria (picos) as major fraction of the autotrophic picoplankton (APP) that are ubiquitously found in all aquatic ecosystems. They are major contributors of carbon and provide nutrients to their natural habitats through complex microbial food webs. Lake Superior is located in North America and has the largest surface area of any freshwater lake present in the world. The nature of this lake is ultraoligotrophic with very low concentrations of iron and phosphorous. Although 50% of Lake Superior chlorophyll is contributed by the APP, only few studies focused on studying the biodiversity of this lake. The phylogenetic diversity of Lake Superior picos was recently characterized. All samples were collected and filtered from several locations and at different depths including Sterner B (SB), Castle Danger 1 (CD1), Western Mid-lake (WM), and nearshore Keweenaw Waterway station Portage Deep (PD). Most of the 16S rRNA gene sequences clustered within the picocyanobacterial clade and two new groups pelagic Lake Superior clusters I (LSI) and II (LSII) were discovered. These groups do not cluster with any of the previously known freshwater Synechococcus. Also, these novel groups have never been isolated into culture. In our study, we assessed LSI and general picos by quantitative real-time PCR (qPCR). Specific PCR amplicons of LSI and picos were cloned into plasmid and transformed into E. coli competent cells for multiplication of the plasmid. Two separate standard curves were created for specific quantification. The concentration of general picos at CD1 (September, 2004) was found to be in the range of flow cytometry results shown by Ivanikova et al. (2007) of phycocyanin-rich and phycoerythrin-rich APP collected from the same location (August, 2006). Although Ivanikova et al. (2007) found that 16S rRNA sequences that clusters within LSI were less abundant at PD, similar and even higher concentrations of LSI were detected at this station compared to CD1, WM, and SB. WM samples collected from deep chlorophyll maximum (DCM) showed approximately the same concentrations of LSI as CD1 and PD samples collected from the epilimnion. Generally, LSI at PD and WM showed higher levels whereas picos were more concentrated at CD1 and WM. LSI proportions of picos at CD1, WM, and SB were 4.4 %, 24%, and 15.5% respectively. This study provides valuable information about the dynamics of the picos at the western arm of Lake Superior. Moreover, specific and sensitive qPCR protocol created in this study can be used in future work to monitor the spatial and temporal abundance of picos and LSI.