Matching molecular diversity and ecophysiology of benthic cyanobacteria and diatoms in communities along a salinity gradient

The phylogenetic diversity of oxygenic phototrophic microorganisms in hypersaline microbial mats and their distribution along a salinity gradient were investigated and compared with the halotolerances of closely related cultivated strains. Segments of 16S rRNA genes from cyanobacteria and diatom plastids were retrieved from mat samples by DNA extraction and polymerase chain reaction (PCR), and subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Sequence analyses of DNA from individual DGGE bands suggested that the majority of these organisms was related to cultivated strains at levels that had previously been demonstrated to correlate with characteristic salinity responses. Proportional abundances of amplified 16S rRNA gene segments from phylogenetic groupings of cyanobacteria and diatoms were estimated by image analysis of DGGE gels and were generally found to correspond to abundances of the respective morphotypes determined by microscopic analyses. The results indicated that diatoms accounted for low proportions of cells throughout, that the cyanobacterium Microcoleus chthonoplastes and close relatives dominated the communities up to a salinity of 11% and that, at a salinity of 14%, the most abundant cyanobacteria were related to highly halotolerant cultivated cyanobacteria, such as the recently established phylogenetic clusters of Euhalothece and Halospirulina . Although these organisms in cultures had previously demonstrated their ability to grow with close to optimal rates over a wide range of salinities, their occurrence in the field was restricted to the highest salinities investigated.     

An Improved Method for Marine Cyanobacterial DNA Isolation

Summary The method of Bolch, Blackburn, Jones, Orr & Grewe [Phycologia, 36, 6 11, 1997] developed for isolation of DNA from freshwater cyanobacteria was suitably modified to yield a simple, efficient and reproducible protocol for the isolation of DNA from different morphological types of marine cyanobacteria. This method resulted in a high yield of quality DNA suitable for polymerase chain reaction (PCR) amplifications.     

A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.     

A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture,mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.     

Survey of extrachromosomal DNA found in the filamentous cyanobacteria.

Cleared lysates of 13 species of filamentous cyanobacteria were examined for the presence of extrachromosomal DNA by using agarose gel electrophoresis and ethidium bromide staining. Seven of the 13 species contained extrachromosomal covalently closed circular DNA, and all but 1 species contained multiple elements. There was no correlation between the presence of extrachomosomal DNA and either the range of metabolic activities found in the cyanobacteria or the differentiated cell types or structures elaborated by the morphologically complex filamentous cyanobacteria.     

Preparation of GDP-D-mannose specifically labeled with tritium in the D-mannosyl moiety

A method, called “bidirectional transfer”, has been described for the transfer of DNA and RNA from agarose or polyacrylamide gels onto diazobenzyloxymethyl (DBM)-paper or nitrocellulose filters. The gels were sandwiched between either two nitrocellulose filters or two diazobenzyloxymethyl-papers. Next, the nucleic acids were allowed to diffuse out of the gels onto the filters. In this way, duplicate blots were obtained from a single gel. The bidirectional transfer of DNA or RNA from 0.5 to 1% agarose gels was complete and nearly quantitative after 1 h of transfer. DNA fragments from 5% polyacrylamide gels were efficiently blotted after 36 h onto nitrocellulose filters using bidirectional transfer. The fragments were transferred with good resolution and were shown to be efficient substrates for homologous [³²P]DNA probes.     

Phylogenetic analysis of heterocystous cyanobacteria (Subsections IV and V) using highly iterated palindromes as molecular markers

Highly iterated palindromes (HIP) have been used as high resolution molecular markers for assessing the genetic variability and phylogenetic relatedness of heterocystous cyanobacteria (subsections IV and V) representing 12 genera of heterocystous cyanobacteria, collected from different geographical areas of India. DNA fingerprints generated using four HIP markers viz. HIP-AT, HIP-CA, HIP-GC, and HIP-TG showed 100 % polymorphism in all the heterocystous cyanobacteria studied and each marker produced unique and strain-specific banding pattern. Furthermore, phylogenetic affinities based on the dendrogram constructed using HIP DNA profiles of heterocystous cyanobacteria suggest the monophyletic origin of this entire heterocystous clade along with a clear illustration of the polyphyletic origin of the branched Stigonematalean order (Subsection V). In addition, phylogenetic affinities were validated by principal component analysis of the HIP fingerprints. The overall data obtained by both the phylogeny and principal component assessments proved that the entire heterocystous clade was intermixed, and there are immediate needs for classificatory reforms that satisfy morphological plasticity and environmental concerns.     

Electrophoresis of DNA restriction fragments in poly-N-acryloyl-tris gels

Poly-N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) gels were evaluated as a matrix for DNA electrophoresis. The resolution of DNA restriction fragments in three poly(NAT)-N,N'-methylenebisacrylamide (Bis) gels (4, 5, and 6%) was compared with the resolution in polyacrylamide (AA)-Bis gels of the same percentage. Poly(NAT) gels were found to give a substantially improved separation of DNA fragments larger than 200 bp. In contrast to poly(AA) gels, DNA fragments of up to 4 kbp were well resolved in the new matrix. By pulse-field electrophoresis the useful separation range of poly(NAT) gels was expanded to at least 23 kbp. For DNA fragments below 10 kbp, the resolution was better than that in a 0.7% agarose gel. Thus poly(NAT) gels are most suitable for the electrophoretic separation of DNA molecules whose size is out of the optimal fractionation range of poly(AA) or agarose gels.     

Improved recovery of DNA from polyacrylamide gels after in situ DNA footprinting

Methods used to date for the isolation of DNA from polyacrylamide gels are elution based, time-consuming and with low yield in DNA. This paper describes an improved system employing polyacrylamide gels made of a meltable matrix. The new system was successfully applied to in situ DNA footprinting following gel retardation assays.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

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.     

Photosynthetic production of fuels and chemicals in immobilized systems

Whole cells of algae, cyanobacteria and photosynthetic bacteria entrapped in alginate gels or polyurethane foams can retain their photo-synthetic activities for months and in some cases for years. Such immobilized cells can be used in bioreactors for the production of H₂, NH₃, NADPH₂, carbohydrates, hydrocarbons, etc., with sunlight as the energy source.     

Homologies in the Structural Genes Coding for Sulphate Reducing Enzymes from Higher Plants and Prokaryotes*

The structural genes of enterobacteria encoding for the enzymes involved in the assimilatory reduction of sulphate (“cys genes”) were used in order to identify homologous genes from phototrophic cyanobacteria and higher plants. By Southern hybridisation of genomic DNA from the higher organisms with the cys DNA-probes derived from Escherichia coli, discrete restriction fragments were found in higher plants and in cyanobacteria indicating the occurrence of related DNA. Two of the cyanobacterial genes were cloned and identified by DNA and amino acid sequence comparison as the structural genes encoding the PAPS-reductase (EC 1.8.4.-) and the ferredoxin: sulphite-reductase (EC 1.8.7.1). The nucleic acid of both genes showed stretches of highly conserved bases in regions of the sequences which are regarded as the functionally important domains of the gene products.     

High‐resolution melting analysis: a genotyping tool for population studies on Daphnia

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour‐ and time‐consuming. Here, we demonstrate high‐resolution melting analysis (HRMA) as a cost‐effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double‐stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.     

A simplified protocol for preparing DNA from filamentous cyanobacteria

The preparation of good quality genomic DNA from microalgae and plants is often time-consuming because of the need to remove contaminants that may interfere with the downstream enzymatic manipulation of the DNA. Simpler protocols have been reported but these are applicable only to a few species and in many cases are not effective for removing trace contaminants. In this report, we describe a modification of existing protocols that significantly simplified the preparation of genomic DNA from cyanobacteria and plants. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2–2.5 M NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. The preparation and enzymatic digestion of the DNA can be performed in a single day. The DNA was easily digested in 2 h at normal restriction enzyme concentrations, and is highly suitable for PCR and Southern hybridization. We successfully used this simplified protocol to prepare genomic DNA from several filamentous cyanobacteria, such asAnabaena sp. PCC 7120,Anabaena siamensis, andSpirulina strains M2 and Kenya. This protocol may also be useful for preparing genomic DNA from other algae and from higher plants.     

Diversity of free-living nitrogen-fixing microorganisms in the rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings

The composition of free-living nitrogen-fixing microbial communities in rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings was studied by the presence of nifH genes using Polymerase Chain Reaction-Denatured Gradient Gel Electrophoresis (PCR-DGGE) approach. Eleven rhizosphere tailing samples and nine non-rhizosphere tailing samples from six plant communities were collected from two wastelands with different discarded periods. The nested PCR method was used to amplify the nifH genes from environmental DNA extracted from tailing samples. Twenty-two of 37 nifH gene sequences retrieved from DGGE gels clustered in Proteobacteria (α-Proteobacteria and β-Proteobacteria) and 15 nifH gene sequences in Cyanobacteria. Most nifH gene fragments sequenced were closely related to uncultured bacteria and cyanobacteria and exhibited less than 90% nucleotide acid identity with bacteria in the database, suggesting that the nifH gene fragments detected in copper mine tailings may represent novel sequences of nitrogen-fixers. Our results indicated that the non-rhizosphere tailings generally presented higher diversity of nitrogen-fixers than rhizosphere tailings and the diversity of free-living nitrogen-fixers in tailing samples was mainly affected by the physico-chemical properties of the wastelands and plant species, especially the changes of nutrient and heavy metal contents caused by the colonization of plant community.     

Diversity of diazotrophic unicellular cyanobacteria in the tropical North Atlantic Ocean

We present data on the genetic diversity and phylogenetic affinities of N2-fixing unicellular cyanobacteria in the plankton of the tropical North Atlantic Ocean. Our dinitrogenase gene (nifH) sequences grouped together with a group of cyanobacteria from the subtropical North Pacific; another subtropical North Pacific group was only distantly related. Most of the 16S ribosomal DNA sequences from our tropical North Atlantic samples were closely allied with sequences from a symbiont of the diatom Climacodium frauenfeldianum. These findings suggest a complex pattern of evolutionary and ecological divergence among unicellular cyanobacteria within and between ocean basins.     

Transfer of small DNA fragments from polyacrylamide gels to diazobenzyloxymethyl-paper and detection by hybridization with DNA probes.

A method for transferring small DNA fragments from composite polyacrylamide-agarose gels to diazobenzyloxymethyl (DBM)-paper is described. DNA fragments are separated by electrophoresis in polyacrylamide-agarose gels crosslinked with N,N′-diallyltartardiamide instead of N,N′-methylenebis-acrylamide. The crosslinks are cleaved by treating the gel with periodic acid after electrophoresis and the DNA is denatured with alkali. After neutralization, the single stranded DNA fragments are transferred to DBM-paper and detected by hybridization with labeled DNA probes. The procedure has been used to transfer and visualize unlabeled SV40 DNA fragments in the size range 28 to 1790 base pairs.     

Ribosomal RNA gene fragments from fossilized cyanobacteria identified in primary gypsum from the late Miocene,Italy

Earth scientists have searched for signs of microscopic life in ancient samples of permafrost, ice, deep‐sea sediments, amber, salt and chert. Until now, evidence of cyanobacteria has not been reported in any studies of ancient DNA older than a few thousand years. Here, we investigate morphologically, biochemically and genetically primary evaporites deposited in situ during the late Miocene (Messinian) Salinity Crisis from the north‐eastern Apennines of Italy. The evaporites contain fossilized bacterial structures having identical morphological forms as modern microbes. We successfully extracted and amplified genetic material belonging to ancient cyanobacteria from gypsum crystals dating back to 5.910–5.816 Ma, when the Mediterranean became a giant hypersaline brine pool. This finding represents the oldest ancient cyanobacterial DNA to date. Our clone library and its phylogenetic comparison with present cyanobacterial populations point to a marine origin for the depositional basin. This investigation opens the possibility of including fossil cyanobacterial DNA into the palaeo‐reconstruction of various environments and could also be used to quantify the ecological importance of cyanobacteria through geological time. These genetic markers serve as biosignatures providing important clues about ancient life and begin a new discussion concerning the debate on the origin of late Miocene evaporites in the Mediterranean.