Switching toxin production on and off: intermittent microcystin synthesis in a Microcystis bloom.

Toxic cyanobacterial blooms are increasing in prevalence. Microcystins are the most commonly produced cyanotoxin. Despite extensive research the variables regulating microcystin production remain unclear. Using a RT-QPCR assay that allowed the precise measurement of mcyE transcriptional gene expression and an ELISA that enabled small changes in total microcystin concentrations to be monitored, we demonstrate for the first time that microcystin production is not always constitutive and that significant up- and downregulation in microcystin synthesis can occur on time scales of 2-6 h. Samples were collected over 3 days from a small eutrophic lake during a dense microcystin-producing Microcystis bloom. McyE gene transcripts were detected in only four out of 14 samples. Vicissitudes in both microcystin quotas and extracellular microcystin levels corresponded with changes in mcyE expression. During the period of exalted microcystin synthesis Microcystis sp. cell concentrations increased from 70 000 cells ml(-1) to 4 000 000 cells ml(-1) . These data provide compelling evidence that changes in Microcystis cell concentrations influence microcystin production.

Hindcasting cyanobacterial communities in Lake Okaro with germination experiments and genetic analyses.

Cyanobacterial blooms are becoming increasingly prevalent worldwide. Sparse historic phytoplankton records often result in uncertainty as to whether bloom-forming species have always been present and are proliferating in response to eutrophication or climate change, or if there has been a succession of new arrivals through recent history. This study evaluated the relative efficacies of germination experiments and automated rRNA intergenic spacer analysis (ARISA) assays in identifying cyanobacteria in a sediment core and thus reconstructing the historical composition of cyanobacterial communities. A core (360 mm in depth) was taken in the central, undisturbed basin of Lake Okaro, New Zealand, a lake with a rapid advance of eutrophication and increasing cyanobacteria populations. The core incorporated a tephra from an 1886 volcanic eruption that served to delineate recent sediment deposition. ARISA and germination experiments successfully detected akinete-forming nostocaleans in sediment dating 120 bp and showed little change in Nostocales species structure over this time scale. Species that had not previously been documented in the lake were identified including Aphanizomenon issatschenkoi, a potent anatoxin-a producer. The historic composition of Chrococcales and Oscillatoriales was more difficult to reconstruct, potentially due to the relatively rapid degradation of vegetative cells within sediment.

Sources of edaphic cyanobacterial diversity in the Dry Valleys of Eastern Antarctica.

Cyanobacteria are major components of Antarctic Dry Valley ecosystems. Their occurrence in lakes and ponds is well documented, however, less is known about their distribution in edaphic environments. There has been considerable debate about the contribution of aquatic organic matter derived largely from cyanobacteria to terrestrial ecosystems. In this study, automated rRNA intergenic spacer analysis (ARISA) and 16S rRNA gene clone libraries were used to investigate cyanobacterial diversity in a range of soil environments within the Miers and Beacon Valleys. These data were used to elucidate the input of aquatic cyanobacteria to soil communities. Thirty-eight samples were collected from a variety of soil environments including dry and moist soils, hypoliths and lake and hydroterrestrial microbial mats. The results from the ARISA and 16S rRNA clone library analysis demonstrated that diverse cyanobacterial communities exist within the mineral soils of the Miers Valley. The soil samples from Beacon Valley were depauparate in cyanobacterial signals. Within Miers Valley, significant portions (29%-58%) of ARISA fragment lengths found in aquatic cyanobacterial mats were also present in soil and hypolith samples, indicating that lacustrine and hydroterrestrial cyanobacteria play a significant role in structuring soil communities. The influence of abiotic variables on the community structure of soil samples was assessed using BEST analysis. The results of BEST analysis of samples from within Miers Valley showed that total percentage of carbon content was the most important variable in explaining differences in cyanobacterial community structure. The BEST analyses indicated that four elements contributed significantly to species compositional differences between valleys. We suggest that the complete absence of lakes or ponds from Beacon Valley is a contributing factor to the low cyanobacterial component of these soils.

First report of homoanatoxin-a and associated dog neurotoxicosis in New Zealand.

In November 2005, at least five dogs died rapidly after contact with water from the Hutt River (lower North Island, New Zealand). Necropsy performed 24h later on one of the dogs (a 20-month-old Labrador) revealed few findings of interest, except for copious amounts of froth in the respiratory tract down to the bifurcation of the trachea and large quantities of algal material in the dog's stomach. Low and relatively stable flows in the Hutt River during spring had resulted in the proliferation of benthic cyanobacteria that formed large black/brown mats along the river edge. Samples from the Labrador's stomach contents and cyanobacterial mats were analysed microscopically and screened using chemical and biochemical assays for cyanotoxins: anatoxin-a, homoanatoxin-a, cylindrospermopsins, saxitoxins and microcystins. Liquid chromatography-mass spectrometry (LC-MS) confirmed the presence of the neurotoxic cyanotoxins anatoxin-a and homoanatoxin-a and their degradation products, dihydro-anatoxin-a and dihydro-homoanatoxin-a. This is the first report of homoanatoxin-a and associated degradation product in New Zealand. Based on morphology, the causative species was identified as Phormidium sp. Subsequent phylogenetic analysis of 16S rRNA gene sequences demonstrated that the causative organism was most similar to Phormidium autumnale. Further investigations led to the detection of homoanatoxin-a and anatoxin-a in cyanobacterial mats from four other rivers in the Wellington region (lower North Island, New Zealand). Access restrictions were placed on over 60% of river catchments in the western Wellington region, severely affecting recreational users.

Removal of contaminating DNA from polymerase chain reaction using ethidium monoazide.

The presence of exogenous DNA in PCR reagents and DNA polymerase is a common occurrence. In particular, the amplification of 16S rRNA genes with universal primers for non-culture-based study is often hampered by the formation of false positives. Here, we describe the use of ethidium monoazide (EMA) to eliminate contaminating DNA in a polymerase chain reaction. The advantage of the proposed methodology is the retention of the highly sensitive nature of PCR with the ability to amplify template DNA at concentrations lower than those of contaminating DNA. The treatment of PCR master mix with EMA concentrations that exceeded those required to remove contaminating DNA can interfere with the amplification of low-template concentrations. The methodology presented is straightforward and can be accomplished within 10 min.

Development of a real-time PCR assay targeting the sporulation gene, spo0A, for the enumeration of thermophilic bacilli in milk powder.

Thermophilic bacilli, such as Anoxybacillus, Geobacillus and Bacillus, are common contaminants growing within the processing lines of milk powder producing factories. These contaminants are used as indicator organisms for plant hygiene and specification limits based on their numbers have been implemented to ensure milk powder quality. In this study, we present a SYBR Green-based real-time PCR assay for the rapid detection and enumeration of these thermophilic bacilli in milk powder using the spo0A sporulation gene as quantification target. With this method the detection of thermophilic bacilli in milk powder can be accomplished within 1 h. The detection limit for reconstituted and inoculated milk was 80 vegetative cfu ml(-1) and 640 spores ml(-1), respectively.

Survival of thermophilic spore-forming bacteria in a 90+ year old milk powder from Ernest Shackelton's Cape Royds Hut in Antarctica.

Milk powder taken to Antarctica on Shackelton's British Antarctic Expedition in 1907 was produced in New Zealand by a roller drying process in the first factory in the world dedicated to this process. Thermophilic bacilli are the dominant contaminants of modern spray-dried milk powders and the 1907 milk powder allows a comparison to be made of contaminating strains in roller-dried and spray-dried powders. Samples of milk powder obtained from Shackelton's Hut at Cape Royds had low levels of thermophilic contamination (< 500 cfu ml-1) but the two dominant strains (Bacillus licheniformis strain F and Bacillus subtilis) were typical of those found in spray-dried powders. Soil samples from the floor of the hut also contained these strains, whereas soils distant from the hut did not. Differences in the RAPD profiles of isolates from the milk powder and the soils suggest that contamination of the milk from the soil was unlikely. It is significant that the most commonly encountered contaminant strain in modern spray-dried milk (Anoxybacillus flavithermus strain C) was not detected in the 1907 sample.

Development of a rapid detection and enumeration method for thermophilic bacilli in milk powders.

Thermophilic strains of Geobacillus, Anoxybacillus and Bacillus that are able to grow at 55 degrees C and above are recognized as commonly occurring contaminants during the production of milk powders. In particular, Anoxybacillus flavithermus strain C and Bacillus licheniformis strain F are often the most prevalent. We describe here the development of a TaqMan-based real-time-PCR assay using a small amplicon of the ribosomal 16S rRNA gene for the selective and quantitative detection of thermophilic bacilli in milk powders. We further present an effective, rapid and inexpensive method for the isolation of total bacterial DNA from milk powder for quantitative PCR analysis within 20 min. With this method, the detection of thermophilic bacilli in milk powder can be accomplished within 1 h. The detection limit for reconstituted and inoculated milk was 8 vegetative cfu ml(-1) and 64 spores ml(-1), respectively.