Microcystin Production in the Tripartite Cyanolichen Peltigera leucophlebia

We show that the cyanobacterial symbionts of a tripartite cyanolichen can produce hepatotoxic microcystins in situ. Microcystins were detected with high-performance liquid chromatography mass spectrometry both from cephalodia of the tripartite cyanolichen Peltigera leucophlebia and from a symbiotic Nostoc strain isolated from the same lichen specimen. Genetic identities of symbiotic Nostoc strains were studied by amplifying and sequencing the 16S rRNA gene. Also, the presence of the microcystin synthetase gene mcyE was confirmed by sequencing. Three highly toxic microcystins were detected from the lichen specimen. Several different Nostoc 16S rRNA haplotypes were present in the lichen sample but only one was found in the toxin-producing cultures. In culture, the toxin-producing Nostoc strain produced a total of 19 different microcystin variants. In phylogenetic analysis, this cyanobacterium and related strains from the lichen thallus grouped together with a previously known microcystin-producing Nostoc strain and other strains previously isolated from the symbiotic thalloid bryophyte Blasia pusilla. Our finding is the first direct evidence of in situ production of microcystins in lichens or plant–cyanobacterial symbioses. Microcystins may explain why cyanolichens and symbiotic bryophytes are not among the preferred food sources of most animal grazers.

Protein Expression Profiles in an Endosymbiotic Cyanobacterium Revealed by a Proteomic Approach

Molecular mechanisms behind adaptations in the cyano-bacterium (Nostoc sp.) to a life in endosymbiosis with plants are still not clarified, nor are the interactions between the partners. To get further insights, the proteome of a Nostoc strain, freshly isolated from the symbiotic gland tissue of the angiosperm Gunnera manicata Linden, was analyzed and compared with the proteome of the same strain when free-living. Extracted proteins were separated by two-dimensional gel electrophoresis and were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with tandem mass spectrometry. Even when the higher percentage of differentiated cells (heterocysts) in symbiosis was compensated for, the majority of the proteins detected in the symbiotic cyanobacteria were present in the free-living counterpart, indicating that most cellular processes were common for both stages. However, differential expression profiling revealed a significant number of proteins to be down-regulated or missing in the symbiotic stage, while others were more abundant or only expressed in symbiosis. The differential protein expression was primarily connected to i) cell envelope-associated processes, including proteins involved in exopolysaccharide synthesis and surface and membrane associated proteins, ii) to changes in growth and metabolic activities (C and N), including upregulation of nitrogenase and proteins involved in the oxidative pentose phosphate pathway and downregu-lation of Calvin cycle enzymes, and iii) to the dark, micro-aerobic conditions offered inside the Gunnera gland cells, including changes in relative phycobiliprotein concentrations. This is the first comprehensive analysis of proteins in the symbiotic state.

Evaluation of Nostoc Strain ATCC 53789 as a Potential Source of Natural Pesticides

ABSTRACT The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.

Expression of Cyanobacterial Genes Involved in Heterocyst Differentiation and Dinitrogen Fixation Along a Plant Symbiosis Development Profile

Members of the cyanobiont genus Nostoc, forming an endosymbiosis with members of the angiosperm genus Gunnera, undergo a number of characteristic phenotypic changes during the development of the symbiosis, the genetic background of which is largely unknown. Transcription patterns of genes related to heterocyst differentiation and dinitrogen fixation and corresponding protein profiles were examined, using reverse transcription-polymerase chain reaction and Western blots, along a developmental (apex to mature parts) sequence in Gunnera magellanica and G. manicata and under mimicked symbiotic conditions in a free-living Gunnera isolate (Nostoc strain 0102). The hetR gene was highly expressed and correlated positively with an increase in heterocyst frequency and with ntcA expression, whereas nifH expression was already high close to the growing apex and glnB (PII) expression decreased along the symbiotic profile. Although gene expression appeared to be regulated to a large extent in the same fashion as in free-living cyanobacteria, significant differences were apparent, such as the overexpression of both hetR and ntcA and the contrasting down-regulation of glnB, features indicating important regulatory differences between symbiotic and free-living cyanobacteria. The significance of these findings is discussed in a symbiotic context.