Marcel Erhard

Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide–polyketide synthetase system

BACKGROUND Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the beta-amino polyketide moiety Adda (3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. RESULTS A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA-C and mcyD-J), has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA-E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. CONCLUSIONS This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a beta-amino-pentaketide precursor and the formation of beta- and gamma-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C- and N-methyltransferases, an integrated aminotransferase, and an associated O-methyltransferase and a racemase acting on acidic amino acids.

Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806

Several bloom‐forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non‐ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the microcystin‐producing strain Microcystis aeruginosa PCC 7806. Non‐hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloom‐forming cyanobacterium. Chemical and enzymatic analyses, including high‐performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins. Our results confirm that microcystins are synthesized non‐ribosomally and that a basic difference between toxic and non‐toxic strains of M. aeruginosa is the presence of one or more genes coding for microcystin synthetases.

Microcystin Biosynthesis in Planktothrix: Genes, Evolution, and Manipulation

Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.

Rapid Classification and Identification of Salmonellae at the Species and Subspecies Levels by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.

Determination of Oligopeptide Diversity within a Natural Population of Microcystis spp. (Cyanobacteria) by Typing Single Colonies by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Besides the most prominent peptide toxin, microcystin, the cyanobacteria Microcystis spp. have been shown to produce a large variety of other bioactive oligopeptides. We investigated for the first time the oligopeptide diversity within a naturalMicrocystis population by analyzing single colonies directly with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The results demonstrate a high diversity of known cyanobacterial peptides such as microcystins, anabaenopeptins, microginins, aeruginosins, and cyanopeptolins, but also many unknown substances in the Microcystis colonies. Oligopeptide patterns were mostly related to specificMicrocystis taxa. Microcystis aeruginosa(Kütz.) Kütz. colonies contained mainly microcystins, occasionally accompanied by aeruginosins. In contrast, microcystins were not detected in Microcystis ichthyoblabeKütz.; instead, colonies of this species contained anabaenopeptins and/or microginins or unknown peptides. Within a third group, Microcystis wesenbergii (Kom.) Kom. in Kondr., chiefly a cyanopeptolin and an unknown peptide were found. Similar patterns, however, were also found in colonies which could not be identified to species level. The significance of oligopeptides as a chemotaxonomic tool within the genus Microcystis is discussed. It could be demonstrated that the typing of single colonies by MALDI-TOF MS may be a valuable tool for ecological studies of the genus Microcystis as well as in early warning of toxic cyanobacterial blooms.

Distribution of microcystin-producing and non-microcystin-producing Microcystis sp. in European freshwater bodies: detection of microcystins and microcystin genes in individual colonies.

Microcystis is a well-known cyanobacterial genus frequently producing hepatotoxins named microcystins. Toxin production is encoded by microcystin genes (mcy). This study aims (i) to relate the mcy occurrence in individual colonies to the presence of microcystin, (ii) to assess whether morphological characteristics (morphospecies) are related to the occurrence of mcy genes, and (iii) to test whether there are geographical variations in morphospecies specificity and abundance of mcy genes. Individual colonies of nine different European countries were analysed by (1) morphological characteristics, (2) PCR to amplify a gene region within mcyA and mcyB indicative for microcystin biosynthesis, (3) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to detect microcystins. Almost one hundred percent of the colonies predicted to produce microcystins by PCR analysis were found to contain microcystins. A high similarity in microcystin variants in the different colonies selected from lakes across Europe was demonstrated. The different morphospecies varied in the frequency with which they contained mcy genes. Most colonies (>75%) of M. aeruginosa and M. botrys contained the mcy genes, whereas < or = 20% of the colonies identified as M. ichthyoblabe and M. viridis gave a PCR product of the mcy genes. No colonies of M. wesenbergii gave a PCR product of either mcy gene. In addition, a positive relationship was found between the size of the colony and the frequency of those containing the mcy genes. It is concluded that the analysis of morphospecies is indicative for microcystin production, although the quantitative analysis of microcystin concentrations in water remains indispensable for hazard control.

Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI-TOF mass spectrometry

Abstract:  Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI‐TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.

Isolation, Characterization, and Quantitative Analysis of Microviridin J, a New Microcystis Metabolite Toxic to Daphnia

This paper describes the purification and characterization of microviridin J, a newly discovered metabolite of Microcystis that causes a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. Microviridin J consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Unlike other known isoforms of microviridin, microviridin J contains arginine that imparts a unique solution conformation characterized by proximal hydrophobic interactions between Arg and other regions of the molecule. This eventually results in the formation and stabilization of an additional ring system. Microviridin J potently inhibits porcine trypsin, bovine chymotrypsin, and daphnid trypsin-like proteases. The activity against trypsin is most likely due to Arg and its distinctive conformational interactions. Overall, the data presented for microviridin J emphasize once again the ability of cyanobacteria to produce numerous and potent environmental toxins.

Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806.

Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.

Biomedical Mass Spectrometry in Today's and Tomorrow's Clinical Microbiology Laboratories

ABSTRACT Clinical microbiology is a conservative laboratory exercise where base technologies introduced in the 19th century remained essentially unaltered. High-tech mass spectrometry (MS) has changed that. Within a few years following its adaptation to microbiological diagnostics, MS has been introduced, embraced, and broadly accepted by clinical microbiology laboratories throughout the world as an innovative tool for definitive bacterial species identification. Herein, we review the current state of the art with respect to this exciting new technology and discuss potential future applications.