Rapid identification using pyrolysis mass spectrometry and artificial neural networks of Propionibacterium acnes isolated from dogs

Curie-point pyrolysis mass spectra were obtained from reference Propionibacterium strains and canine isolates. Artificial neural networks (ANNs) were trained by supervised learning (with the back-propagation algorithm) to recognize these strains from their pyrolysis mass spectra; all the strains isolated from dogs were identified as human wild type P. acnes. This is an important nosological discovery, and demonstrates that the combination of pyrolysis mass spectrometry and ANNs provides an objective, rapid and accurate identification technique. Bacteria isolated from different biopsy specimens from the same dog were found to be separate strains of P. acnes , demonstrating a within-animal variation in microflora. The classification of the canine isolates by Kohonen artificial neural networks (KANNs) was compared with the classical multivariate techniques of canonical variates analysis and hierarchical cluster analysis, and found to give similar results. This is the first demonstration, within microbiology, of KANNs as an unsupervised clustering technique which has the potential to group pyrolysis mass spectra both automatically and relatively objectively.     

Phenotypic and Genotypic Comparison of Symbiotic and Free-Living Cyanobacteria from a Single Field Site

PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains.     

Cyanobacteria from benthic mats of Antarctic lakes as a source of new bioactivities

Aims:  To exploit the cyanobacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes for the discovery of novel antibiotic and antitumour activities.
Methods and results:  In all, 51 Antarctic cyanobacteria isolated from benthic mats were cultivated in the laboratory by optimizing temperature, irradiance and mixing. Productivity was generally very low (≤60 mg l⁻¹ d⁻¹) with growth rates ( μ ) in the range of 0·02–0·44 d⁻¹. Growth rates were limited by photosensitivity, sensitivity to air bubbling, polysaccharide production or cell aggregation. Despite this, 126 extracts were prepared from 48 strains and screened for antimicrobial and cytotoxic activities. Seventeen cyanobacteria showed antimicrobial activity (against the Gram-positive Staphylococcus aureus , the filamentous fungus Aspergillus fumigatus or the yeast Cryptococcus neoformans ), and 25 were cytotoxic. The bioactivities were not in accordance with the phylogenetic grouping, but rather strain-specific. One active strain was cultivated in a 10-l photobioreactor.
Conclusions:  Isolation and mass cultivation of Antarctic cyanobacteria and LC-MS (liquid chromatography/mass spectrometry) fractionation of extracts from a subset of those strains (hits) that exhibited relatively potent antibacterial and/or antifungal activities, evidenced a chemical novelty worthy of further investigation.
Significance and impact of the study:  Development of isolation, cultivation and screening methods for Antarctic cyanobacteria has led to the discovery of strains endowed with interesting antimicrobial and antitumour activities.     

Application of molecular genetic and microbiological techniques in ecology and biotechnology of cyanobacteria

The review discusses the advances and problems in biotechnology and ecology of cyanobacteria and considers the possibilities of molecular genetic and microbiological techniques in this field. Due to the ease of cultivation, high growth rate, availability of synchronous cultures, and existence of numerous molecular genetic and microbiological techniques for various cyanobacterial strains, cyanobacteria—prokaryotic organisms that are ancient relatives of the chloroplasts—are model organisms in the studies of photosynthesis, dinitrogen fixation, cell division, hydrogen production, and in a number of other areas of basic and applied science. These techniques make possible deeper understanding of the role of cyanobacteria in various ecosystems and utilization of their potential in numerous applied projects, including production of molecular hydrogen, phycobiliproteins, and cyanophycin; formation of nanoparticles; removal of heavy metals from the environment; substrate biodegradation; manufacture of products for medicine and food industry; and solution of the problem of cyanobacterial toxins in freshwater and marine environments.     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

Probing the in vivo biosynthesis of scytonemin, a cyanobacterial ultraviolet radiation sunscreen, through small scale stable isotope incubation studies and MALDI-TOF mass spectrometry

Scytonemin is a dimeric indole phenolic pigment found in the sheaths of many cyanobacteria. This pigment absorbs UV radiation protecting subtending cyanobacterial cells from harmful effects. Based on scytonemin's unique chemical structure, the pathway to its biosynthesis is uncertain, thus motivating the current investigation. Herein, we report the incorporation of both tyrosine and tryptophan into scytonemin, and provide in vivo data supporting the tryptophan origin of the ketone carbon involved in the condensation of the two biosynthetic precursors. This study also reports on the new use of a small-scale, MALDI-TOF mass spectrometry technique to monitor the incorporation of isotopically labeled tyrosine during scytonemin biosynthesis.     

Characterization of Neisseria meningitidis capsular polysaccharides containing sialic acid by pyrolysis mass spectrometry

The group B, C, W-135, and Y capsular polysaccharides of Neisseria meningitidis which contain sialic acid were differentiated by Curie-point pyrolysis low-voltage mass spectrometry. A large series of partially purified group B polysaccharide preparations obtained from pathogenic as well as nonpathogenic strains were analyzed by the same technique. It was shown that the carbohydrate structure of these group B polysaccharides appears to be the same throughout the whole series. Slight immunogenicity of some of the group B polysaccharide preparations tested is probably due to protein impurities. Automated pyrolysis mass spectrometry coupled with multivariate analysis of the spectral data by computer turns out to be a rapid method of characterizing microgram samples of large series of polysaccharide preparations.     

Cyanobacterial diversity and activity in modern conical microbialites

Modern conical microbialites are similar to some ancient conical stromatolites, but growth, behavior and diversity of cyanobacteria in modern conical microbialites remain poorly characterized. Here, we analyze the diversity of cyanobacterial 16S rRNA gene sequences in conical microbialites from 14 ponds fed by four thermal sources in Yellowstone National Park and compare cyanobacterial activity in the tips of cones and in the surrounding topographic lows (mats), respectively, by high‐resolution mapping of labeled carbon. Cones and adjacent mats contain similar 16S rRNA gene sequences from genetically distinct clusters of filamentous, non‐heterocystous cyanobacteria from Subsection III and unicellular cyanobacteria from Subsection I. These sequences vary among different ponds and between two sampling years, suggesting that coniform mats through time and space contain a number of cyanobacteria capable of vertical aggregation, filamentous cyanobacteria incapable of initiating cone formation and unicellular cyanobacteria. Unicellular cyanobacteria are more diverse in topographic lows, where some of these organisms respond to nutrient pulses more rapidly than thin filamentous cyanobacteria. The densest active cyanobacteria are found below the upper 50 μm of the cone tip, whereas cyanobacterial cells in mats are less dense, and are more commonly degraded or encrusted by silica. These spatial differences in cellular activity and density within macroscopic coniform mats imply a strong role for diffusion limitation in the development and the persistence of the conical shape. Similar mechanisms may have controlled the growth, morphology and persistence of small coniform stromatolites in shallow, quiet environments throughout geologic history.     

Cyanobacteria in the Australian northern savannah detect the difference between intermittent dry season and wet season rain

This research was conducted in the northern Australian savannah at Boodjamulla National Park where cyanobacterial crusts dominate the soil and rock surfaces in between tussock grasses. It is widely accepted that terrestrial cyanobacteria are drought tolerant and rapidly recommence photosynthesis once moisture is available. Initial tests at the research site indicated that cyanobacteria did not respond to rehydration during the dry season, even after several days. We hypothesised that resurrection had not taken place and new growth from survival cells had to take place during the follow-up wet season. To further understand the desiccation–resurrection processes we tested photosystem II (PSII) responses both during the dry and wet seasons. In the 2009 dry season after 125 days without rain, crust samples were regularly rehydrated. Over the 10 day trial cyanobacteria did not recover PSII activity or CO₂-uptake. Although new colonies of Nostoc grew other cyanobacteria remained inactive, even though liverworts and lichens in the same crusts had responded within 24 h. Dry season cyanobacterial crusts were collected in 2010 then reintroduced into their natural environment and exposed to rainfall during the 2011 wet season. Within 24 h PSII in cyanobacteria from a range of crust types had resurrected and CO₂-uptake was verified, although different crust types responded at significantly different rates. These are the first studies that have demonstrated that PSII does not respond to rainfall during the dry season and cyanobacterial function appears controlled by other environmental conditions. It is likely that mass extracellular polysaccharide (EPS) production during the wet season, once dry, protects cyanobacteria from premature resurrection in the dry season. We propose that EPS regulates moisture penetration, thus the resurrection of PSII at the onset of the wet season, at which time moisture and humidity alters the rheological properties of EPS permitting rehydration.     

Fossilization of the cells of natronophilic endoevaporite cyanobacterium ‘<Emphasis Type="Italic">Euhalothece natronophila</Emphasis>’ in a modelling system

Laboratory simulation of fossilization of cyanobacterial cells in the high-carbonate medium in the presence of calcium was carried out for the haloalkaliphilic natronophilic cyanobacterium ‘Euhalothece natronophila’ Z-M001. This organism was isolated from the Magadi soda lake, where the bioherms consisting of mineralized coccoid cyanobacteria were found in the Quaternary sediments. The structural and chemical heterogeneity of the minerals produced during this process was established, with calcium carbonate and trona being the main products. The differences in the process of cyanobacterial cell carbonatization in soda lakes and marine or freshwater systems were determined. Initial precipitation of calcium carbonate was shown to occur due to a chemical reaction not involving cyanobacteria. At the subsequent stages, amorphous CaCO₃ is sorbed and crystallized on the surface of some of the cells within a cyanobacterial population, resulting in formation of a shell-like mineral layer. The cells embedded in trona in the same system were shown to undergo deformation and destruction. In both cases the mineralized cells were shown to lose their photosynthetic activity.     

Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to ?stensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the fur- ther study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Ana- baena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.     

A single cyanobacterial ribotype is associated with both red and black bands on diseased corals from Palau

Filamentous cyanobacteria forming red and black bands (black band disease, BBD) on 3 scleractinian corals from Palau were molecularly identified as belonging to a single ribotype. Red cyanobacterial mats sampled from infections on Pachyseris speciosa and a massive Porites sp. yielded red strains RMS1 and RMS2 respectively; the black cyanobacterial mat sampled from an infection on Montipora sp. yielded black strain BMS1. Following trials of a range of specialized media and culture conditions, 2 media, Grund and ASN-III, were identified as the best for successful isolation and culturing. Cultured cyanobacteria were examined under a light microscope to establish purity, color and morphological appearance. DNA extraction and partial sequencing of the 16S rDNA gene of both red and black cyanobacterial isolates demonstrated 100% sequence identity. These isolated strains were also found to have 99% sequence identity with an uncultured cyanobacterial strain previously identified by molecular techniques as belonging to a cyanobacterial ribotype associated with BBD-infected corals in the Caribbean. This is the first report of the successful isolation and culture of cyanobacterial strains derived from both red bands and BBD. Based on these findings, it is suggested that the classification of these 2 syndromes as separate coral diseases be postponed until further evidence is collected.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high‐value chemicals and next‐generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO₂, H₂O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357–1371, 2016     

Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers

The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.     

Evidence of high Ca uptake by cyanobacteria forming intracellular CaCO3 and impact on their growth

Several species of cyanobacteria biomineralizing intracellular amorphous calcium carbonates (ACC) were recently discovered. However, the mechanisms involved in this biomineralization process and the determinants discriminating species forming intracellular ACC from those not forming intracellular ACC remain unknown. Recently, it was hypothesized that the intensity of Ca uptake (i.e., how much Ca was scavenged from the extracellular solution) might be a major parameter controlling the capability of a cyanobacterium to form intracellular ACC. Here, we tested this hypothesis by systematically measuring the Ca uptake by a set of 52 cyanobacterial strains cultured in the same growth medium. The results evidenced a dichotomy among cyanobacteria regarding Ca sequestration capabilities, with all strains forming intracellular ACC incorporating significantly more calcium than strains not forming ACC. Moreover, Ca provided at a concentration of 50 μM in BG‐11 was shown to be limiting for the growth of some of the strains forming intracellular ACC, suggesting an overlooked quantitative role of Ca for these strains. All cyanobacteria forming intracellular ACC contained at least one gene coding for a mechanosensitive channel, which might be involved in Ca influx, as well as at least one gene coding for a Ca²⁺/H⁺ exchanger and membrane proteins of the UPF0016 family, which might be involved in active Ca transport either from the cytosol to the extracellular solution or the cytosol toward an intracellular compartment. Overall, massive Ca sequestration may have an indirect role by allowing the formation of intracellular ACC. The latter may be beneficial to the growth of the cells as a storage of inorganic C and/or a buffer of intracellular pH. Moreover, high Ca scavenging by cyanobacteria biomineralizing intracellular ACC, a trait shared with endolithic cyanobacteria, suggests that these cyanobacteria should be considered as potentially significant geochemical reservoirs of Ca.     

Cyanobacterial formation of intracellular Ca‐carbonates in undersaturated solutions

Cyanobacteria have long been thought to induce the formation of Ca‐carbonates as secondary by‐products of their metabolic activity, by shifting the chemical composition of their extracellular environment to conditions favoring mineral precipitation. Some cyanobacterial species forming Ca‐carbonates intracellularly were recently discovered. However, the environmental conditions under which this intracellular biomineralization process can occur and the impact of cyanobacterial species forming Ca‐carbonates intracellularly on extracellular carbonatogenesis are not known. Here, we show that these cyanobacteria can form Ca‐carbonates intracellularly while growing in extracellular solutions undersaturated with respect to all Ca‐carbonate phases, that is, conditions thermodynamically unfavorable to mineral precipitation. This shows that intracellular Ca‐carbonate biomineralization is an active process; that is, it costs energy provided by the cells. The cost of energy may be due to the active accumulation of Ca intracellularly. Moreover, unlike cyanobacterial strains that have been usually considered before by studies on Ca‐carbonate biomineralization, cyanobacteria forming intracellular carbonates may slow down or hamper extracellular carbonatogenesis, by decreasing the saturation index of their extracellular solution following the buffering of the concentration of extracellular calcium to low levels.     

Exogenous Hexoses Cause Quantitative Changes of Pigment and Glycerolipid Composition in Filamentous Cyanobacteria

Cyanobacteria Spirulina platensis and Nostoc linckia were grown in the presence of 5 mM and 50 mM glucose or 5 mM mannose, non-metabolisable glucose analogue that effectively triggers the repression of photosynthesis. Glucose evoked active cyanobacterial growth but chlorophyll (Chl) content decreased to some extent and porphyrins were excreted. The content of monogalactosyldiacylglycerol decreased in glucose-grown cyanobacteria and that of phosphatidylglycerol increased substantially. Mannose inhibited cyanobacteria growth as well as Chl synthesis, however, phosphatidylglycerol contents were higher than in respective control samples. In cyanobacterial cells glucose may not only inhibit photosynthetic processes, but also cause structural transformations of membranes which may be necessary for the activity of respiratory electron transport chain components under heterotrophic conditions.     

Cyanobacterial diversity in the rhizosphere of rice and its ecological significance

This investigation was undertaken to characterize the abundance and genera-wise diversity of cyanobacteria in the rice rhizosphere and nitrogen-fixing ability of the isolated strains. The cyanobacterial strains belonging to the genera Nostoc and Anabaena comprised 80% of the rhizosphere isolates, which were also efficient in enhancing the germination and growth of wheat seeds and exhibited significantly high protein accumulation and IAA production. Distinct profiles for the cyanobacterial strains were obtained on amplification with extended Hip 1 primer — HipTG, indicative of the diversity among these strains. Our investigation helped in identifying promising cyanobacterial isolates from the rhizosphere of rice, which can be utilized in developing efficient plant growth promoting cyanobacterial inoculants.     

The Effects of Cyanobacterial Exudates on Bacterial Growth and Biodegradation of Organic Contaminants

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.