Biomedical subjects

B A Neilan

Publications and source records attributed to B A Neilan.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of intergenic spacer region length polymorphisms to investigate the halophilic archaeal diversity of stromatolites and microbial mats.

Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel intergenic spacer profiles. ARISA-PCR, performed directly on extracted DNA from different sample sites, provided significant insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in this environment.

Archaea↗

PubMed2006

The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria.

Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin in Bacillus subtilis. The second family is typified by the Escherichia coli acyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partial Nodularia spumigena PPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products.

Amino Acid Sequence↗

PubMed2005

Detection of microcystin synthetase genes in health food supplements containing the freshwater cyanobacterium Aphanizomenon flos-aquae.

In this study we investigated the presence of toxin-producing cyanobacterial contaminants in food supplements manufactured from blooms of the non-toxic freshwater cyanobacterium Aphanizomenon flos-aquae. Previous reports investigating the contamination of health food supplements with toxin-producing cyanobacteria have used chemical and or biochemical methods such as HPLC, ELISA and protein phosphatase assays. Whilst these studies have drawn attention to the presence of hepatotoxic microcystins in some commercially available food supplements, the methods used do not provide any information on the source of the contaminant. Such information would be useful for the quality control of food supplements produced for human consumption. In this study we applied a molecular technique, involving the amplification of the 16s rRNA gene, the phycocyanin operon, and two genes of the microcystin synthetase gene cluster to show that all 12 food supplement samples, sourced from various internet distributors and containing non-toxic A. flos-aquae, also contained toxigenic cyanobacteria. Sequencing of the microcystin synthetase genes detected in all of the food supplements showed that M. aeruginosa was the organism responsible for the production of microcystins in the samples. The presence of microcystins in the food supplements was confirmed by ELISA, with concentrations within the range of 0.1--4.72 microgg(-1) (microcystin-LR equivalents). Given that the molecular methods applied here are highly sensitive, and show good agreement with the results obtained from ELISA, we believe that they could potentially be used as a quality control technique for food products that contain cyanobacteria.

Bacterial Proteins↗

PubMed2004

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis.

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.

Bacillus cereus↗

PubMed2003

Screening, identification and kinetic characterization of a bacterium for Mn(II) uptake and oxidation.

Following sample collection and screening at a number of Mn-associated mine sites in Northern Australia, a microbial strain was selected for its enhanced rate of Mn uptake. The strain was identified by phylogenetic analysis as a Rhizobium sp. Kinetic studies of Mn(II) uptake and oxidation by this strain in glucose-based media established that the uptake of Mn(II) was much greater than the conversion of Mn(II) to Mn oxide. Chemical analysis and scanning electron microscopy confirmed the production of significant amounts of polysaccharides by this strain. These polysaccharides may play a role both in enhancing Mn(II) accumulation and in minimizing Mn oxide production.

Cell Division↗

PubMed2003

Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.

Alkaloids↗

PubMed2001

Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis.

Toxic freshwater cyanobacteria can contaminate water supplies and adversely effect humans, agricultural livestock, and wildlife. Toxicity is strain-specific so morphological observations alone cannot predict the hazard level. Two microtiter plate based bioassays have emerged for measuring saxitoxin (STX) and its derivatives, commonly found in the freshwater cyanobacteria Anabaena and Aphanizomenon. They use radioactively labeled STX binding by sodium channels, STX's pharmacological target, or an unrelated protein, saxiphilin. These bioassays were challenged with extracts of toxic and nontoxic strains of Anabaena circinalis, and the results were compared with HPLC analysis. Both radioreceptor assays had detection limits of 2 microg STX equivalents (STXeq)/L, which is belowthe concentration proposed for a health alert, namely 3 microg STXeq/L. In all cases, statistically significant correlations existed between all toxicity measurements of the same extracts with the methods used herein. Sodium channel and saxiphilin assays however predicted less toxicity relative to HPLC analysis. The only exception to this was the equivalency observed between saxiphilin measurement and HPLC quantitation corrected for mammalian toxicity. Saxiphilin assay predicted toxicity in one strain was 3 orders of magnitude more than by sodium channel assay, and no STX was detected by HPLC. Lack of acetylcholinesterase inhibition showed this bioactivity was not anatoxin-a(S), a toxin also produced by this A. circinalis with some resemblance to the region of STX bound by saxiphilin. Presence of anatoxin-a(S) was predicted for another strain by this same acetylcholinesterase assay that, if confirmed by chemical analysis, would be the first report of anatoxin-a(S) in an Australian cyanobacterium.

Acetylcholinesterase↗

PubMed2001

On the presence of peptide synthetase and polyketide synthase genes in the cyanobacterial genus Nodularia.

Nodularin is a hepatotoxin produced by the bloom-forming cyanobacterial species Nodularia spumigena. Putative peptide synthetase and polyketide synthase genes were detected in toxic strains of Nodularia by degenerate PCR. Using specific primer sets, peptide synthetase and polyketide synthase gene homologues were detected in nodularin-producing strains indicating a possible role of peptide synthetase and polyketide synthase enzyme complexes in the biosynthesis of nodularin. Strains of Nodularia isolated from around the world were also analyzed for the production of nodularin by the protein phosphatase 2A inhibition assay. The protein phosphatase inhibition assay and the molecular detection of peptide synthetase and polyketide synthase genes in Nodularia may be useful techniques for the assessment of nodularin-producing cyanobacteria in the environment.

Amino Acid Sequence↗

PubMed2001

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYP020B were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii.

Alkaloids↗

PubMed2001

Molecular biology of peptide and polyketide biosynthesis in cyanobacteria.

Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular nonribosomal peptide and polyketide structures. Various bioactivities could be assigned to these compounds, and some may prove useful either for development into commercial drugs or as biochemical research tools. Microcystin, a worldwide common cyanobacterial hepatotoxin, was the first metabolite whose nonribosomal biosynthesis could be confirmed by knock-out mutagenesis. The microcystin synthetase complex consists of peptide synthetases, polyketide synthases, and hybrid enzymes, and reveals a number of novel enzymatic features, signifying the potential of cyanobacterial biosynthetic systems for combinatorial biochemistry. Recent studies have shown the presence of peptide synthetase genes and polyketide synthase genes within a number of cyanobacterial genomes. This knowledge may be very valuable for future screening projects aimed at the detection of new bioactive compounds.

Cyanobacteria↗

PubMed2001

A spontaneous mutant of microcystin biosynthesis: genetic characterization and effect on Daphnia.

Microcystis aeruginosa strain MRC is unique in its' possession of the mcyA-J gene cluster, which encodes microcystin synthetase, but its' inability to produce microcystins. M. aeruginosa strain MRD is genetically identical to MRC at numerous genomic loci examined, but produces a variety of microcystins, mainly with the amino acid tyrosine in the molecule. Zooplankton studies with Daphnia galeata and D. pulicaria, using the mutant (MRC) and its' wild type (MRD), showed for the first time that microcystins other than microcystin-LR can be responsible for the poisoning of Daphnia by Microcystis. Regardless of microcystin content, both Daphnia exhibited significantly reduced ingestion rates when fed with either strain of M. aeruginosa compared with the green alga Scenedesmus acutus. A disruption of the molting process in both Daphnia spp. was noted when these species were fed with MRC cells. Such symptoms on Daphnia have not been previously reported for cyanobacteria and may point to a bioactive compound, other than microcystin, which inhibits the hardening of protein-chitin complexes in Daphnia.

Animals↗

PubMed2001

rRNA sequences reflect the ecophysiology and define the toxic cyanobacteria of the genus Nodularia.

Nodularia, a member of the order Nostocales, is a bloom-forming filamentous cyanobacterium that possesses the ability to form toxic blooms. The toxin produced by Nodularia, nodularin, is a hepatotoxin, similar in structure to the heptapeptide toxin microcystin. Twenty-one strains of Nodularia, representing the species Nodularia spumigena, Nodularia harveyana and Nodularia sphaerocarpa, were analysed for toxin production by protein phosphatase inhibition assay and sequenced over the 16S rDNA region. Phylogenetic analysis of Nodularia 16S rDNA sequences found that Nodularia clustered into two main groups. An N. spumigena cluster was distinct from the benthic species N. harveyana and N. sphaerocarpa. There was no distinction between strains isolated from globally diverse locations. Nodularin-producing species were restricted to the single, evolutionally distinct cluster of N. spumigena. This observation has enabled the design of a specific 16S rRNA PCR for the rapid detection of nodularin-producing strains. Alignment of 16S rDNA sequences from toxic and non-toxic Nodularia with other members of the cyanobacteria allowed the design of both Nodularia generic and toxic N. spumigena-specific primers.

Bacterial Toxins↗

PubMed2001

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.

Algal Proteins↗

PubMed2001

Varied diazotrophies, morphologies, and toxicities of genetically similar isolates of Cylindrospermopsis raciborskii (nostocales, cyanophyceae) from Northern Australia.

The potentially toxic freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in tropical and temperate water bodies worldwide. This paper investigates the effects of different nitrogen sources (NO3-, NH4+, and omission of a fixed form of nitrogen) on the growth rates, morphologies, and cylindrospermopsin (CYL) concentrations (expressed as a percentage of the freeze-dried weight) of seven C. raciborskii isolates obtained from a range of water bodies in northern Australia and grown in batch culture. In general, growth rates were lowest in the absence of a fixed-nitrogen source and highest with NH4+ as the nitrogen source. Conversely, the highest concentrations of CYL were recorded in cultures grown in the absence of a fixed-nitrogen source and the lowest were found in cultures supplied with NH4+. Cultures supplied with NO3- were intermediate with respect to both CYL concentration and growth rate. Different nitrogen sources resulted in significant differences in the morphology of C. raciborskii trichomes. Most notable were the loss of heterocysts and the tapering of end cells in cultures supplied with NH4+ and the statistically significant increase in vegetative cell length (nitrogen depleted < NO3- < NH4+). The morphological changes induced by different nitrogen sources were consistent for all isolates, despite measurable differences in vegetative-cell and heterocyst dimensions among isolates. Such induced morphological variation has implications for Cylindrospermopsis taxonomy, given that distinctions between species are based on minor and overlapping differences in cell lengths and widths. The close phylogenetic association among all seven isolates was confirmed by the high level (>99.8%) of similarity of their 16S rRNA gene sequences. Another genetic technique, analysis of the HIP1 octameric-palindrome repeated sequence, showed greater heterogeneity among the isolates and appears to be a useful method for distinguishing among isolates of C. raciborskii.

Alkaloids↗

PubMed2001

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.

Bacterial Proteins↗

PubMed2001

Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysis.

We report molecular analyses which identify cyanobacterial strains present in environmental samples. These analyses do not require the isolation and culture of strains. Identification of cyanobacteria used the polymerase chain reaction (PCR), based on the phycocyanin operon. Differentiation was either by restriction endonuclease digestion (restriction fragment length polymorphisms) or sequencing of the PCR products. Identification was based on sequence homology of the intergenic spacer region (IGS) between the beta- and alpha-phycocyanin subunits (PC-IGS) with database records. We have found that the length and sequence of the PC-IGS is capable of predicting the genus accurately, but not the species. Toxigenicity was determined with oligonucleotide probes for key steps in the microcystin toxin synthesis pathway. We have shown that it is possible to easily and routinely obtain PCR amplification products and differentiate the strains in bloom samples. The methods can detect even minor components in bloom samples, which may not be apparent on microscopic examination. Genetic probes for microcystin toxigenicity are effective on environmental samples, eliminating the need for isolation and culture of the organisms. The use of a suite of tests described here will allow water managers to determine the presence and the type of cyanobacteria and their microcystin toxigenicity.

Animals↗

PubMed2000

The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides.

Non-ribosomal peptides are a group of secondary metabolites with a wide range of bioactivities, produced by prokaryotes and lower eukaryotes. Recently, non-ribosomal synthesis has been detected in diverse microorganisms, including the myxobacteria and cyanobacteria. Peptides biosynthesized non-ribosomally may often play a primary or secondary role in the producing organism. Non-ribosomal peptides are often small in size and contain unusual or modified amino acids. Biosynthesis occurs via large modular enzyme complexes, with each module responsible for the activation and thiolation of each amino acid, followed by peptide bond formation between activated amino acids. Modules may also be responsible for the enzymatic modification of the substrate amino acid. Recent analysis of biosynthetic gene clusters has identified novel integrated, mixed and hybrid enzyme systems. These diverse mechanisms of biosynthesis result in the wide variety of non-ribosomal peptide structures and bioactivities seen today. Knowledge of these biosynthetic systems is rapidly increasing and methods of genetically engineering these systems are being developed. In the future, this may lead to rational drug design through combinatorial biosynthesis of these enzyme systems.

Bacteria↗

PubMed2000

The Helicobacter pylori pyrB gene encoding aspartate carbamoyltransferase is essential for bacterial survival.

The production of defined isogenic Helicobacter pylori pyrB mutants was undertaken to investigate the role of aspartate carbamoyltransferase (encoded by pyrB) in the survival of the bacterium. The complete structural gene for aspartate carbamoyltransferase from H. pylori strain RU1 was cloned into Escherichia coli by complementation of a pyrB auxotrophic mutant to facilitate the construction of a pyrB-disrupted copy in E. coli. The H. pylori pyrB gene had high similarity to other bacterial pyrB genes, and the phylogenetic clustering with different species was consistent with functional characteristics of the ACTase. The transcription initiation site for H. pylori pyrB-mRNA was mapped 25 bp upstream of the ATG start codon, and potential promoter regions were identified. In order to construct an isogenic pyrB H. pylori mutant by natural transformation and allelic exchange, the plasmid insert containing pyrB was disrupted by insertional mutagenesis of a chloramphenicol transferase gene cassette. In multiple transformations of H. pylori cells, no chloramphenicol-resistant pyrB mutants were isolated. Successful mutagenesis of other H. pylori genes and PCR amplification of the recombined gene demonstrated that the ACTase-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the pyrB gene with the chloramphenicol-pyrB-disrupted copy. These findings suggested that the ACTase enzyme is essential for the survival of H. pylori.

Alleles↗