Christina Lyra

Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera

Toxic and non-toxic cyanobacterial strains from Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis, Planktothrix (Oscillatoria agardhii), Oscillatoria and Synechococcus genera were examined by RFLP of PCR-amplified 16S rRNA genes and 16S rRNA gene sequencing. With both methods, high 16S rRNA gene similarity was found among planktic, anatoxin-a-producing Anabaena and non-toxic Aphanizomenon, microcystin-producing and non-toxic Microcystis, and microcystin-producing and non-toxic Planktothrix strains of different geographical origins. The respective sequence similarities were 99.9-100%, 94.2-99.9% and 99.3-100%. Thus the morphological characteristics (e.g. Anabaena and Aphanizomenon), the physiological (toxicity) characteristics or the geographical origins did not reflect the level of 16S rRNA gene relatedness of the closely related strains studied. In addition, cyanobacterial strains were fingerprinted with repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR. All the strains except two identical pairs of Microcystis strains had different band profiles. The overall grouping of the trees from the 16S rRNA gene and the REP- and ERIC-PCR analyses was similar. Based on the 16S rRNA gene sequence analysis, four major clades were formed. (i) The clade containing filamentous heterocystous cyanobacteria was divided into three discrete groups of Anabaena/Aphanizomenon, Anabaena/Cylindrospermum/ Nodularia/Nostoc and Calothrix strains. The three other clades contained (ii) filamentous non-heterocystous Planktothrix, (iii) unicellular non-heterocystous Microcystis and (iv) Synechococcus strains.

Phylogenetic comparison of the cyanobacterial genera Anabaena and Aphanizomenon

Morphological analysis and sequencing of the 165 rRNA gene, the spacer region of the ribosomal operon (ITS1) and the rbcLX (RubisCO) region was performed on 26 Anabaena strains and 14 Aphanizomenon strains isolated from several lakes in Denmark, Finland and France. Based on their morphology, Anabaena strains differed from strains of Aphanizomenon: the vegetative cells, heterocysts and akinetes were significantly wider in Anabaena than in Aphanizomenon. Phylogenetic trees based on the 16S rDNA, ITS1 and rbcLX regions showed that the planktic Anabaena strains were not distinguishable from Aphanizomenon strains. The results of the clustering of Anabaena and Aphanizomenon strains based on 16S rDNA sequences showed that these two genera are not monophyletic. Sequence analysis of the 16S rDNA, ITS1-S and rbcLX regions of the planktic Anabaena strains showed that this genus is heterogeneous. In all methods, Anabaena strains that produced different toxic compounds (e.g. anatoxin-a, microcystin and an unknown neurotoxin) were clustered separately from each other but were grouped either with non-toxic Anabaena and/or Aphanizomenon strains. Our data suggest that the planktic Anabaena and Aphanizomenon isolates belong to the same genus, regardless of their morphological differences. Thus, a taxonomic revision of the two genera is required.

Detection of microcystin-producing cyanobacteria in Finnish lakes with genus-specific microcystin synthetase gene E (mcyE) PCR and associations with environmental factors.

ABSTRACT We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.

High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms

Cyanobacterial mass occurrences (water blooms) cause ecological, economic and health problems worldwide. Still, little is known about heterotrophic bacteria associated with cyanobacteria and the interactions between those organisms. We isolated 460 bacterial strains from more than 40 lakes and rivers (151 samples), Baltic Sea (32 samples) and treated drinking water of seven treatment plants (29 samples). The water bodies and the raw water of the treatment plants were frequently dominated by high numbers of cyanobacteria. Various growth media were used to isolate the strains. Analysis of partial 16S rRNA gene fragments (701–905 bp for 358 strains and 413–497 bp for 102 strains) classified the isolated bacteria as Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Deinococcus-Thermus. Some of these isolates represented possible new bacterial orders, families, genera or species. We isolated various potentially pathogenic bacteria, such as Aeromonas, Vibrio, Acinetobacter and Pseudomonas, that may cause adverse health effects in humans and animals and should be taken into consideration when assessing the risks caused by cyanobacterial blooms. Several strains also inhibited or enhanced the growth of cyanobacteria. Most of such strains had an enhancing effect on the cyanobacterial growth. Other isolates were affiliated with genera such as Sphingomonas or Flavobacterium, which include strains that are capable of degrading cyanobacterial toxins or other recalcitrant and problematic organic compounds. The isolated strains provide a large group of bacteria that could be used in assessing and controlling the harmful effects of cyanobacteria.

Characterization of Nodularia strains, cyanobacteria from brackish waters, by genotypic and phenotypic methods

An investigation was undertaken of the genetic diversity of Nodularia strains from the Baltic Sea and from Australian waters, together with the proposed type strain of Nodularia spumigena. The Nodularia strains were characterized by using a polyphasic approach, including RFLP of PCR-amplified 16S rRNA genes, 16S rRNA gene sequencing, Southern blotting of total DNA, repetitive extragenic palindromic- and enterobacterial repetitive intergenic consensus-PCR, ribotyping and phenotypic tests. With genotypic methods, the Nodularia strains were grouped into two clusters. The genetic groupings were supported by one phenotypic property: the ability to produce nodularin. In contrast, the cell sizes of the strains were not different in the two genetic clusters. 16S rRNA gene sequences indicated that all the Nodularia strains were closely related, despite their different origins. According to this study, two genotypes of Nodularia exist in the Baltic Sea. On the basis of the taxonomic definitions of Komarek et al. (Algol Stud 68, 1-25, 1993), the non-toxic type without gas vesicles fits the description of Nodularia sphaerocarpa, whereas the toxic type with gas vesicles resembles the species N. spumigena and Nodularia baltica.

Cellular fatty acids as chemotaxonomic markers of the genera Anabaena, Aphanizomenon, Microcystis, Nostoc and Planktothrix (cyanobacteria)

The cellular fatty acid content of 22 cyanobacterial strains belonging to the genera Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis and Planktothrix were analysed. The identities of the major peaks were confirmed by MS. Correspondence analysis of the data revealed three distinct groups formed by the Microcystis strains, the Nostoc/Planktothrix strains and the Anabaena/Aphanizomenon/Cylindrospermum strains. The Calothrix strain did not cluster with the other heterocystous cyanobacteria, supporting its morphological classification separate from the Nostocaceae family. The presence of large amounts of the fatty acids 18:30omega6,9,12c and 18:0 iso distinguished the Microcystis strains from the other cyanobacteria studied. The high content of 16:1omega7c grouped the Nostoc strains with the Planktothrix strains. A free-living strain of Nostoc contained 16:1omegao5c and 16: 1omega7c (about 1: 1), separating it from the symbiotic Nostoc strain and the Planktothrix strains. the strains of Anabaena, Aphanizomenon and Cylindrospermum grouped tightly and were characterized by the presence of 16:1omega9c and 16:0 anteiso fatty acids. Correspondence analysis of Anabaena, Aphanizomenon and Cylindrospermum showed that all hepatotoxic Anabaena strains grouped together, whereas the non-toxic and neurotoxic Anabaena strains grouped with the non-toxic Aphanizomenon strains.

Quantitative real-time PCR detection of toxic Nodularia cyanobacteria in the Baltic Sea.

ABSTRACT A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml−1 of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.

Characterization of Cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene

Abstract Planktonic, filamentous cyanobacterial strains from different genera, both toxic and nontoxic strains, were characterized by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene. Total protein pattern analysis revealed the mutual relationships at the genus level. Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene with reference strains proved to be a good method for the cyanobacterial taxonomy. The nonheterocystous strains outgrouped from the nitrogen-fixing ones. With both methods, Aphanizomenon clustered with Anabaena, and Nodularia with Nostoc. In the RFLP study of Anabaena, the neurotoxic strains were identical, but the hepatotoxic ones formed a heterogeneous group. Genetic distances found in the RFLP study were short, confirming that close genotypic relationships underlie considerable diversity among cyanobacterial genera.

High-throughput quantification of antibiotic resistance genes from an urban wastewater treatment plant.

Antibiotic resistance among bacteria is a growing problem worldwide, and wastewater treatment plants have been considered as one of the major contributors to the dissemination of antibiotic resistance to the environment. There is a lack of comprehensive quantitative molecular data on extensive numbers of antibiotic resistance genes (ARGs) in different seasons with a sampling strategy that would cover both incoming and outgoing water together with the excess sludge that is removed from the process. In order to fill that gap we present a highly parallel quantitative analysis of ARGs and horizontal gene transfer potential over four seasons at an urban wastewater treatment plant using a high-throughput qPCR array. All analysed transposases and two-thirds of primer sets targeting ARGs were detected in the wastewater. The relative abundance of most of the genes was highest in influent and lower in effluent water and sludge. The resistance profiles of the samples cluster by sample location with a shift from raw influent through the final effluents and dried sludge to the sediments. Wastewater discharge enriched only a few genes, namely Tn25 type transposase gene and clinical class 1 integrons, in the sediment near the discharge pipe, but those enriched genes may indicate a potential for horizontal gene transfer.

Sulphonamide and Trimethoprim Resistance Genes Persist in Sediments at Baltic Sea Aquaculture Farms but Are Not Detected in the Surrounding Environment

Persistence and dispersal of antibiotic resistance genes (ARGs) are important factors for assessing ARG risk in aquaculture environments. Here, we quantitatively detected ARGs for sulphonamides (sul1 and sul2) and trimethoprim (dfrA1) and an integrase gene for a class 1 integron (intI1) at aquaculture facilities in the northern Baltic Sea, Finland. The ARGs persisted in sediments below fish farms at very low antibiotic concentrations during the 6-year observation period from 2006 to 2012. Although the ARGs persisted in the farm sediments, they were less prevalent in the surrounding sediments. The copy numbers between the sul1 and intI1 genes were significantly correlated suggesting that class 1 integrons may play a role in the prevalence of sul1 in the farm sediments through horizontal gene transfer. In conclusion, the presence of ARGs may limit the effectiveness of antibiotics in treating fish illnesses, thereby causing a potential risk to the aquaculture industry. However, the restricted presence of ARGs at the farms is unlikely to cause serious effects in the northern Baltic Sea sediment environments around the farms.