Browsing by Subject "Cyanobacteria"

The rapid emergence of drug resistant pathogens prevents effective treatment of diseases and threatens the lives of millions of people. Similarly, resistance to chemotherapeutic agents has been observed in several types of cancer. Therefore, screening for novel antimicrobial and antileukemic substances is urgently needed. Screening microorganisms for bioactive molecules has resulted in the discovery of several substances that are currently used for disease treatment. Cyanobacteria represent an ancient group of oxygenic, photosynthetic prokaryotes that produce a variety of functionally diverse and structurally complex natural compounds, some of which have already been used as source of inspiration in drug development process. In this study, I evaluated the antimicrobial and antileukemic potential of filamentous cyanobacteria against Gram-positive, Gram-negative and fungal potential pathogens and acute myeloid leukemia (AML) MOLM-13 cell line. Extracts showing antibiotic and/or antileukemic activity were subjected to reversed-phase HPLC and individual fractions were re-evaluated for their ability to kill the abovementioned pathogens and/or induce cell death in MOLM-13 cell line. Metabolites present in the active HPLC fractions were analysed by UPLC/ESI/Q-TOF and elemental compositions were obtained. Identification of metabolites was accomplished by searching online databases for compounds with identical elemental composition. Chemical structures were further confirmed by comparing mass spectrometry data with publicly available data. New bioactive metabolites, new variants of known metabolites and a number of yet unidentified metabolites exhibiting antimicrobial and/or antileukemic activity are reported herein. In detail, novel metabolites belonging to aromatic polyketides and their new variants were present in the active fractions of Nostoc sp. CENA69. The cyanobacterium Aliinostoc sp. CENA513 produced the recently discovered metabolite nocuolin A, a compound with antimicrobial and antiproliferative properties, lipids and unidentified lipidic compounds that showed only bactericidal activity against B. cereus. Interestingly, none of the abovementioned strains had any effect on the growth of Gram-negative pathogens. Planktothrix agardhii UHCC 0018 and Anabaena sp. UHCC 0187 strains showed only antileukemic activity. The majority of the bioactive fractions deriving from these two strains contained either lipids or pigments and their derivatives. The remaining active HPLC fractions of these two strains contained a great number of unidentified compounds. Further studies are required to identify the unknown compounds and purify the novel metabolites and antibacterial lipids. The results presented herein clearly show that Cyanobacteria are an emerging source of bioactive metabolites that can be used in drug development process or act as a source of inspiration for the production of novel synthetic drugs.

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming species Nodularia spumigena, Aphanizomenon flosaquae, Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential toxin production in 40-to-175- year-old brackish water sediment archives in order to understand historical bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from northern Baltic Sea sediment up to 424 and 174 years old, at coastal and open-sea locations respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. Increases in sediment organic matter content and akinete abundance largely corresponded with the historical expansion of anthropogenic eutrophication-fueled blooms of cyanobacteria in the northern Baltic Sea, beginning in the mid-twentieth century. The detection of potential hepatotoxin production from akinetes and revived cultures was minimal and restricted to the coastal sediment core. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that the majority of strains likely belonged to benthic genera Anabaena. Findings also supported the notion that, in comparison with Nodularia and Aphanizomenon spp. akinetes, Anabaena/Dolichospermum spp. akinetes play a more significant role in their life cycle and bloom initiation strategies. Further research is recommended to accurately quantify akinetes and create a higher rate of toxin gene detection from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria. Overall, measuring cyanobacterial akinete abundance, germination experiments, and genetic methods can be effectively used to determine akinete persistence, viability, and potential toxin production in brackish water sediment samples. This study highlights the prolonged survival of cyanobacterial akinetes in northern Baltic Sea sediment samples, up to 174 years old.

Antibiotic resistance is a growing threat to global health due to overuse and misuse of antibiotics leading to untreatable or difficult to treat infections. Natural environments are an important reservoir of antibiotic resistance. The release of antibiotics into the environment promotes the development of antibiotic resistant bacteria and environmental occurrence of antibiotic resistance genes (ARGs). ARGs are common in nature and prevalent in aquatic environments such as surface waters and effluent. Cyanobacteria are widely found in marine, freshwater, and terrestrial environments. Since their ubiquitous presence in water environments cyanobacteria are exposed to antibiotic pollution and are in contact with resistant bacteria. The role of cyanobacteria in the antimicrobial resistome and dissemination of ARGs has only been studied recently. This work aimed to evaluate the antibiotic susceptibilities of 51 cyanobacterial strains against different classes of antibiotics, using liquid batch cultures, antibiotic discs, and bioinformatics approaches. Cyanobacterial strains used in this work were sensitive to most of the tested antibiotics. However, majority of the strains also showed resistance against trimethoprim and novobiocin. Overall, there was little variation in the antibiotic resistances observed between strains but differences in sensitivity to different antibiotics was observed between species and strains with most differences seen with Nostoc spp. According to bioinformatic tools used (CARD database and BLASTp) FosA protein was found only in strains showing resistance against fosfomycin but not in any sensitive phenotypes and therefore fosA gene was selected as the most promising putative resistance gene for subsequent assays. To determine whether the fosA from cyanobacteria could confer resistance to fosfomycin, the fosA gene from Nostoc sp. XPORK 5A was cloned into pET28a(+) expression vector under the control of T7 promoter and subsequently native cyanobacterial promoter. The ability of Escherichia coli BL21 (DE3) carrying each plasmid constructs to grow in the presence of fosfomycin was determined with agar plates and growth curve assay. E. coli transformants containing the fosA gene and T7 promoter conferred high-level resistance to fosfomycin showing ability to grow at the highest concentrations tested (1mg/ml) on agar plates and (500 µg/ml) in growth curve assay. FosA protein expression from the native cyanobacterial promoter appeared to be weaker and conferred lower-level resistance to fosfomycin (≥ 10 µg/ml). The results of this study provide more information about the antibiotic susceptibility of cyanobacteria. In addition, replicating a horizontal transfer of the fosA gene from cyanobacteria to proteobacteria conferred resistance to fosfomycin and these results may indicate that also nonpathogenic cyanobacteria could act as a source of fosA antibiotic resistance genes.

Inorganic phosphate (Pi) is the only readily utilizable form of phosphorus for toxic diazotrophic cyanobacterium Nodularia spumigena (N. spumigena). Pi is one of the limiting nutrients in the Baltic Sea where surprisingly N. spumigena are highly abundant especially during the summer. This indicates that N. spumigena possibly has an alternative pathway to fulfill its phosphorus requirement. The Baltic Sea, like most aquatic environments, is enriched with organic phosphorus compounds among which phosphonates may constitute a significant fraction. Interestingly, the Baltic Sea N. spumigena strains UHCC 0039 and CCY9414 have been found to carry phosphonate degrading gene cluster (phnC-M) implying that these cyanobacteria could assimilate phosphonates as a phosphorus source. However, the significance of the presence of phn gene cluster in N. spumigena for phosphonate utilization has not been investigated in detail. Here, I aimed to understand how N. spumigena copes with Pi limitation and utilizes phosphonates in laboratory conditions using biochemical assays, PCR-based methods and bioinformatics tools. This would aid in finding a suitable marker for Pi deficiency in cyanobacterial blooms in the Baltic Sea. In this study, bioinformatics and PCR screening showed that phn gene cluster was conserved in the Baltic Sea N. spumigena strains. The studied N. spumigena strains UHCC 0039 and UHCC 0060 were found to utilize naturally produced low molecular weight phosphonates, methylphosphonate (MPn), ethylphosphonate (EPn) and 2-aminoethylphosphonate (2APn). Among these phosphonates, MPn seemed to be the most preferred phosphorus source. Alkaline phosphatase activity, an indicator of Pi limitation, was found to be elevated in the media with Pi and 2APn questioning its suitability as a marker for phosphorus limitation. In addition, growth on MPn released methane indicating that massive blooms of N. spumigena might contribute to an elevated methane supersaturation in the Baltic Sea. Reverse transcriptase quantitative PCR (RT-qPCR) in N. spumigena strains did not show expected upregulation of high-affinity phosphate transporter pstS in Pi limitation. It demonstrated an induction of phosphonate transporter gene phnD in media lacking Pi and supplemented by 2APn. The phosphonate lyase gene phnJ was however, upregulated only in the presence of MPn suggesting that phnJ gene could be used as a marker for phosphonate bioavailability. The findings from this study suggest that the presence of phn gene cluster could provide N. spumigena a competitive advantage in Pi-limited cyanobacterial blooms in the Baltic Sea. The molecular detection methods designed in this study thus could be used in future to monitor the expression of genes induced during Pi limitation and the presence of phosphonates, and the method could be further optimized for screening natural water samples.

Anabaena is a common member of the phytoplankton in lakes, reservoirs and ponds throughout the world. This is a filamentous, nitrogen-fixing cyanobacterial genus and is frequently present in the lakes of Finland. Anabaena sp. strain 90 was isolated from Lake Vesijärvi and produces microcystins, anabaenopeptilides and anabaenopeptins. A whole genome shotgun sequencing project was undertaken to obtain the complete genome of this organism in order to better understand the physiology and environmental impact of toxic cyanobacteria. This work describes the genome assembly and finishing, the genome structure, and the results of intensive computational analysis of the Anabaena sp. strain 90 genome. Altogether 119,316 sequence reads were generated from 3 genomic libraries with 2, 6 and 40 kb inserts from high throughput Sanger sequencing. The software package Phred/Phrap/Consed was used for whole genome assembly and finishing. A combinatorial PCR method was used to establish relationships between remaining contigs after thorough scaffolding and gap-filling. The final assembly results show that there is a single 4.3 Mb circular chromosome and 4 circular plasmids with sizes of 820, 80, 56 and 20 kb respectively. Together, these 4 plasmids comprise nearly one-fifth of the total genome. Genomic variations in the form of 79 single nucleotide polymorphisms and 3 sequence indels were identified from the assembly results. Sequence analysis revealed that 7.5 percent of the Anabaena sp. strain 90 genome consists of repetitive DNA elements. The genome sequence of Anabaena sp. strain 90 provides a more solid basis for further studies of bioactive compound production, photosynthesis, nitrogen fixation and akinete formation in cyanobacteria.