Quantitative detection of human- and canine-associated Bacteroides genetic markers from an urban coastal lagoon.

The contamination of water catchments by nonpoint source faecal pollution is a major issue affecting the microbial quality of receiving waters and is associated with the occurrence of a range of enteric illnesses in humans. The potential sources of faecal pollution in surface waters are diverse, including urban sewage leaks, surface runoff and wildlife contamination originating from a range of hosts. The major contributing hosts require identification to allow targeted management of this public health concern. In this study, two high-performing Microbial Source Tracking (MST) assays, HF183/Bac242 and BacCan-UCDmodif, were used for their ability to detect host-specific Bacteroides 16Sr RNA markers for faecal pollution in a 12-month study on an urban coastal lagoon in Sydney, Australia. The lagoon was found to contain year-round high numbers of human and canine faecal markers, as well as faecal indicator bacteria counts, suggesting considerable human and animal faecal pollution. The high sensitivity and specificity of the HF183/Bac242 and BacCan-UCDmodif assays, together with the manageable levels of PCR inhibition and high level DNA extraction efficiency obtained from lagoon water samples make these markers candidates for inclusion in an MST 'toolbox' for investigating host origins of faecal pollution in urban surface waters.

Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp.

The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. 'Macrozamia riedlei 65.1' and Nostoc sp. 'Macrozamia serpentina 73.1' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng µg-1 chlorophyll a in Nostoc sp. 'Macrozamia riedlei 65.1' and 12.5±8.4 ng µg-1 Chl a in Nostoc sp. 'Macrozamia serpentina 73.1' extracts. Further scans indicated the presence of the rare isoform [L-Har(2)] nodularin, which contains L-homoarginine instead of L-arginine. Nodularin was also present at 1.34±0.74 ng ml(-1) (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har(2)] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har(2)] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.