Transposons inactivate biosynthesis of the nonribosomal peptide microcystin in naturally occurring Planktothrix spp

Abstract

The filamentous cyanobacteria Planktothrix spp. occur in the temperate region of the Northern hemisphere. The red-pigmented Planktothrix rubescens bacteria occur in deep, physically stratified, and less eutrophic lakes. Planktothrix is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (mcy genes). Planktothrix spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of Planktothrix strains containing all mcy genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the mcy genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in mcyB; one strain) and 1,869 bp (in mcyHA; three strains), and three insertions (IS), spanning 1,429 bp (in mcyD; eight strains), 1,433 bp (in mcyEG; one strain), and 1,433 bp (in mcyA; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in mcyD and mcyA) and one deletion (in mcyHA) were found regularly in populations of P. rubescens in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the mcy gene pool in Planktothrix populations over several years.

FIG. 1.

mcy gene cluster of Planktothrix isolate CYA126/8 (2) and locations of mutations found in inactive mcy genotypes. Strains 110 and 40 were isolated from Lake Mondsee (Austria), strains 139, 145, 161, 166, 169, and 170 were isolated from Lake Grabensee (Austria), strain 178 was isolated from Lake Fuschlsee (Austria), strains 12, 62, and 65 were isolated from Lake Irrsee (Austria), and strain CCAP1459/36 was isolated from Lake Gjersjoen (Norway).

FIG. 2.

Sequence alignment of putative transposases (mcyDIS1 and mcyDIS2, observed in mcyD [at base pair 11,918 of the mcy gene cluster of Planktothrix; AJ441056], mcyEG [at base pair 23,822], and mcyA [at base pair 41,274]). The 5′ end (A) and the 3′ end (B) of each insertion (IS) and part of the mcy genes (bold) are shown. The short directly repeated sequences (DR) of 10 bp in length (straight lines) and the left inverted repeats (IRL) and right inverted repeats (IRR) (dotted lines) are indicated.