Abstract:
Picocyanobacteria form most of the autotrophic picoplankton (APP) and are the major
contributors to primary production in both freshwater ecosystems and oligotrophic
oceans. Two related APP genera forming the picocyanobacterial clade are
Synechococcus sp. and Prochlorococcus sp. Several methods have been used for the
classification and quantification of picocyanobacteria including flow cytometry and
analysis of photosynthetic pigments. However, more recently, quantitative real-time
PCR (qPCR) has become the method of choice for quantification purposes as well as the
assessment of biodiversity in picoplanktonic environments, including studies on
cyanobacterial prevalence. Lately, both marine and freshwater picocyanobacteria were
found to harbor a gene, hcp, 100% conserved among all the strains isolated and
therefore representing a valuable target for quantification of picocyanobacteria by
qPCR, which is the aim of the present study. The approach used is summarized by the
creation of a plasmid that harbors the gene (based on one strain) to construct a standard
curve for qPCR, followed by qPCR of environmental samples. We have carried out
sequence alignment of hcp for Synechococcus sp. strains LS 0504, KD3a, ARC-11 and
ARC-21. Based on the sequence alignment, we have designed primers for part of the
hcp gene (186 bp). PCR reactions on both freshwater and marine DNA samples from
strains LS 0504, KD3a, ARC-11, ARC-21 and WH 8102 verified the successful primer
design. hcp, amplified from LS 0504 strain was then cloned and transformed into
plasmid DNA and absolute quantification of samples collected from the Sargasso Sea,
USA and from an artificial lake at Annaya, Lebanon was performed by qPCR using an external standard curve based on serial dilutions of the plasmid. The numbers of
picocyanobacteria from marine samples were in the range of 103 cells/mL and numbers
from freshwater samples were in the range of 105 cells/mL. We have thus successfully
designed an hcp-based assay that can be applied to quantify picocyanobacteria in local
freshwater and marine environments.
