Emergence of nontoxic mutants as revealed by single filament analysis in bloom-forming cyanobacteria of the genus Planktothrix

Abstract

Background: Bloom-forming cyanobacteria cause toxic algae outbreaks in lakes and reservoirs. We aimed to explore and quantify mutation events occurring within the large mcy gene cluster (55 kbp) encoding microcystin (MC) biosynthesis that inactivate MC net production. For this purpose we developed a workflow to detect mutations in situ occurring anywhere within the large mcy gene cluster as amplified from one single filament of the red-pigmented cyanobacterium Planktothrix rubescens. From five lakes of the Alps eight hundred Planktothrix filaments were isolated and each individual filament was analyzed for mutations affecting the mcy genes.

Results: Mutations inactivating MC synthesis were either through an insertion element ISPlr1 or the partial deletion of mcy genes. Neutral mutations not affecting MC biosynthesis occurred within two intergenic spacer regions, either through the insertion of a Holliday-junction resolvase RusA or ISPlr1. Altogether, the insertions affected a few mcy genes only and their location was correlated with regions similar to repetitive extragenic palindromic DNA sequences (REPs). Taking all of the filaments together, the mutations leading to the inactivation of MC synthesis were more rare (0.5-6.9%), when compared with the neutral mutations (7.5-20.6%). On a spatial-temporal scale the ratio of MC synthesis-inactivating vs. neutral mutations was variable, e.g., the filament abundance carrying partial deletion of mcyD (5.2-19.4%) and/or mcyHA (0-7.3%) exceeded the abundance of neutral mutations.

Conclusions: It is concluded that insertion events occurring within the Planktothrix mcy gene cluster are predictable due to their correlation with REPs. The frequency of occurrence of the REPs within the mcy gene cluster of Planktothrix relates to the rather common mutation of mcy genes in Planktothrix. Spatial-temporal variable conditions may favor the emergence of partial mcy deletion mutants in Planktothrix, in particular a higher proportion of genotypes resulting in inactivation of MC synthesis might be caused by increased ISPlr1 activity.

Fig. 1

Flow diagram showing steps of Planktothrix rubescens filament isolation and analysis (white boxes) and obtained results (grey boxes). For PCR conditions and primers see text and Additional file 6

Fig. 2

PCR amplification of the entire mcy gene cluster from single Planktothrix rubescens filaments to detect mutations by PCR size polymorphism. a Amplification of DNA fragments (approx. 3 kb) of the entire mcy gene cluster from DNA isolated from one individual filament (No 11, Mondsee, 15 Mar 2012). The nucleotide pos. represents the binding position of the forward primer according to the sequence of the mcy gene cluster from P. agardhii NIVA-CYA126/8 (AJ441056), (Additional file 6). b Amplification of DNA fragment using primer pair Fmcy2+/− from individual filaments No 59–76 isolated from Zürichsee (20 Jun 2012). Filament No 66 shows a deletion in mcyD. c Amplification of DNA fragment using primer pair Fmcy8+/Fmcym8- (1.75 kb) from individual filaments No 88–100 isolated from Wörthersee (3 Apr 2012). The larger PCR products (Filament No 88, 94, 96, 100) indicate the insertion into the IGS region (1423 bp) between mcyE and mcyG. M, PstI lambda DNA size marker. Positive control (+) was amplified from P. agardhii NIVA-CYA126/8 (AJ441056)

Fig. 3

a Schematic view of Planktothrix mcy gene cluster and location of mutations found in individual filaments and location of repetitive regions 1 – 7 (in green). Taking all filaments together the relative frequency of each mutation is given in parentheses. ISPlr1, P. rubescens IS element containing the conserved DDE domain for DNA transposition [9]; b Alignment of repetitive sequence regions within the P. agardhii NIVA-CYA126/8 mcy gene cluster (ASAK00000000). The framed boxes indicate the short directly repeated sequences (DR) of 10 bp in length resulting in insertion of transposable element ISPlr1. Bold letters indicate palindromic sequences

Fig. 4

Proportion of mcy gene cluster mutations found in the total Planktothrix population and in subpopulations. Relative frequency of various mcy gene cluster mutations in relation to the number of total filaments, the filaments carrying the long or short mcyA variant, the presence of the putative resolvase (inserted into the mcyTD-IGS) and the ISPlr1 inserted into mcyEG-IGS