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.

The isolation of high-quality nucleic acids from cyanobacterial strains, in particular environmental isolates, has proven far from trivial. We present novel techniques for the extraction of high molecular weight DNA and RNA from a range of cultured and environmental cyanobacteria, including stains belonging to the genera Microcystis , Lyngbya , Pseudanabaena , Aphanizomenon , Nodularia , Anabaena , and Nostoc , based on the use of the nontoxic polysaccharide solubilizing compound xanthogenate. These methods are rapid, require no enzymatic or mechanical cell disruption, and have been used to isolate both DNA and RNA free of enzyme inhibitors or nucleases. In addition, these procedures have proven critical in the molecular analysis of bloom-forming and other environmental cyanobacterial isolates. Finally, these techniques are of general microbiological utility for a diverse range of noncyanobacterial microorganisms, including Gram-positive and Gramnegative bacteria and the Archea.

The relationship between toxigenicity and phylogeny within the cyanobacterial genus Microcystis is unclear. To investigate this issue, we have designed PCR primers for the N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA and have probed 37 Microcystis sp. cultures as well as several field samples. The NMT region was present in all 18 laboratory strains that gave positive reactions in the protein phosphatase inhibition assay for microcystin but was absent in 17 nontoxic strains. Two other nontoxic strains, one of which had previously been reported to produce microcystin, possessed the NMT region. Detection of NMTspecific DNA in field samples corresponded to periods of toxicity as assessed by protein phosphatase inhibition. The Microcystis strains formed a monophyletic cluster based on 16S rRNA gene sequences but comprised two groups with respect to phycocyanin intergenic spacer (PC-IGS) sequences. Toxic and nontoxic strains appeared to be erratically distributed within the PC-IGS and 16S rRNA trees. Sequence analysis of the NMT domain revealed two coherent groups. The genomic region immediately downstream of the mcyABC cluster in all 20 NMT-positive strains contained an open reading frame of unknown function (uma1) at a conserved distance from mcyC. All nontoxic strains also contained uma1, which is not cotranscribed with mcyABC. The consistent linkage of mcyC to uma1 suggests that mcyC has not been frequently transferred into nontoxic strains via any mechanism involving insertion at random chromosomal locations. These results are discussed with respect to various mechanisms that could explain the patchy distribution of toxigenicity among the various Microcystis clades.Microcystis spp., cyanobacteria that frequently occur as noxious blooms in eutrophic freshwaters, are of major concern because many strains produce cyclic heptapeptide toxins called microcystins (5). The microcystins are members of a family of more than 65 heptapeptides and share the common structure, where L-X and L-Z are variable L-amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decadienoic acid, DMeAsp is 3-methyl-aspartic acid, and Mdha is N-methyldehydroalanine (38). Toxicity is mediated through the active transport of microcystin into hepatocytes by the bile acid organic anion transport system, followed by inhibition of eukaryotic serine/threonine protein phosphatases 1 and 2A (12, 17). Acute poisoning, leading to death from massive hepatic hemorrhage, has been reported to occur in both animals and humans (3,19,37). Chronic ingestion of sublethal doses has been demonstrated to induce primary hepatocellular carcinoma in rodents (33) and has been epidemiologically linked to primary liver cancer in humans (56,57).Water resource management has been complicated by the inability to differentiate between toxic and nontoxic Microcystis blooms without isolation and testing for toxin production. Several morphological studies (25, 54) and molecular studies have attempted to resolve the ambiguous relationship...

The formation of stable foam in activated sludge plants is a global problem for which control is difficult. These foams are often stabilized by hydrophobic mycolic acid-synthesizing Actinobacteria, among which are Tsukamurella spp. This paper describes the isolation from activated sludge of the novel double-stranded DNA phage TPA2. This polyvalent Siphoviridae family phage is lytic for most Tsukamurella species. Whole-genome sequencing reveals that the TPA2 genome is circularly permuted (61,440 bp) and that 70% of its sequence is novel. We have identified 78 putative open reading frames, 95 pairs of inverted repeats, and 6 palindromes. The TPA2 genome has a modular gene structure that shares some similarity to those of Mycobacterium phages. A number of the genes display a mosaic architecture, suggesting that the TPA2 genome has evolved at least in part from genetic recombination events. The genome sequence reveals many novel genes that should inform any future discussion on Tsukamurella phage evolution.A common problem in activated sludge systems is the formation of stable foams on the aerated reactor, leading to major environmental, operational, cosmetic, and health problems (12,41,42). These foams are typically stabilized by members of the mycolic acid-containing Actinobacteria (the Mycolata), although other hydrophobic filamentous bacteria, including "Candidatus Microthrix parvicella," are also important (12,24,39). One potentially attractive approach for controlling such foaming events is the use of lytic bacteriophages targeting the problematic foam-stabilizing populations (46, 48). Similar "phage therapy" strategies have been proposed to treat infectious diseases (6) and decrease bacterial contaminants in food (50). Furthermore, Thomas et al. (46) showed that Mycolata lytic bacteriophages could be isolated readily from activated sludge, making the development of a phage-based foam control approach an environmentally safe and attractive option for this worldwide operational problem.Tsukamurella is in the suborder Corynebacterineae, and on the basis of its mycolic acid-containing hydrophobic cell surface it has been categorized as a Mycolata (18). Tsukamurella spp. have been isolated from activated sludge foams (12, 31), arthropods (44), and soil (18) and more recently in opportunistic clinical infections (23, 43). Although Tsukamurella spp. are a problem in environmental and medical contexts, little attention has been directed toward the isolation and characterization of bacteriophages specific for members of this genus. To our knowledge, only one partially characterized Tsukamurella phage (TPA1, targeting T. paurometabola) has been reported in the literature (46). With the aim of developing a biocontrol approach to manage foaming within activated sludge systems, we have sought to isolate and characterize new lytic phages for members of this genus.More fundamentally, the ecological role of bacteriophages in activated sludge communities has received relatively little attention. This is surprising given the ...

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