Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (Phycocyanin Intergenic Spacer) phylogenies.

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 NMT-specific 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.

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.

Molecular strategies for overcoming antibiotic resistance in bacteria.

Overuse of antibiotics in humans and livestock has led to the rapid evolution of bacteria that are resistant to multiple drugs such that even vancomycin, the drug of last resort, is no longer effective against some strains. Apart from the discovery and exploitation of the natural peptide antimicrobial agents that form part of the innate immune systems of plants and animals, there have been few new antibiotics developed in recent years. Here we review strategies designed to exploit recent advances in molecular biology, including recombinant DNA technology, molecular modelling and genomics to develop new antibacterial agents that overcome antibiotic resistance.

Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.

Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites.

We describe a simple method for the cloning of PCR products without the need for post-amplification enzymatic treatment. Tailed PCR primer sets are used to create complementary staggered overhangs on both insert and vector by a post-PCR denaturation-hybridisation reaction. The single-stranded overhangs are designed to allow directional cloning in a ligase-free manner. This 'enzyme-free cloning' procedure is highly efficient, and is not constrained by the need for the presence of suitable restriction enzyme sites within the plasmid vector. The avoidance of post-amplification enzymatic procedures makes the technique rapid and reliable, avoiding the need for multiple sub-cloning steps.

A novel method of extracting plasmid DNA from Helicobacter species.

BACKGROUND: Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogenesis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. cinaedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter species is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for extracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms. MATERIALS AND METHODS: Eight H. pylori isolates, including the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effectiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories. RESULTS: High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) isolates. The size range of these plasmids was from 3 kb to >16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95-2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The relative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commercial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes. CONCLUSION: This study reports the development of an efficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helicobacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better understanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms.