Deteriogenic cyanobacteria on historic buildings in Brazil detected by culture and molecular techniques

There are few modern analyses of the cyanobacterial communities in biofilms on external building surfaces. As the classification of cyanobacteria is rapidly changing, we aimed to identify them on historic buildings in Brazil using both established and molecular techniques. In mature biofilms, cyanobacteria of subsections I and II were generally the major biomass; occasionally filamentous genera of the Scytonemataceae, Microchaetaceae and Rivularaceae were dominant. Filamentous organisms of subsections III and IV were more frequently isolated in culture. PCR products using cyanobacteria-specific 16S rDNA primers were sequenced from morphologically identified organisms. Homologies with deposited sequences were generally low. Phylogenetic analysis showed that many isolates were distant from their nearest neighbours, even though they grouped with their appropriate taxa. The majority of cyanobacterial DNA sequences deposited in data banks are aquatic; our results indicate that cyanobacteria from external walls are an ecologically isolated group.     

Algal and Cyanobacterial Biofilms on Calcareous Historic Buildings

Major microorganisms in biofilms on external surfaces of historic buildings are algae, cyanobacteria, bacteria, and fungi. Their growth causes discoloration and degradation. We compared the phototrophs on cement-based renderings and limestone substrates at 14 historic locations (47 sites sampled) in Europe and Latin America. Most biofilms contained both cyanobacteria and algae. Single-celled and colonial cyanobacteria frequently constituted the major phototroph biomass on limestone monuments (32 sites sampled). Greater numbers of phototrophs, and especially of algae and of filamentous morphotypes, were found on cement-based renderings (15 sites), probably owing to the porosity and small pore size of the latter substrates, allowing greater entry and retention of water. All phototrophic groups were more frequent on Latin American than on European buildings (20 and 27 sites, respectively), with cyanobacteria and filamentous phototrophs showing the greatest differences. The results confirm the influence of both climate and substrate on phototroph colonization of historic buildings. Received: 7 March 2002 / Accepted: 8 April 2002     

Cyanobacteria from Brazilian building walls are distant relatives of aquatic genera

The 16S-rDNA from 22 cyanobacteria isolated from biofilms on walls of modern and historic buildings in Brazil was partially sequenced (approximately 350 bp) using specific primers. The cyanobacteria with the closest matching sequences were found using the BLAST tool. The sequences were combined with 52 other cyanobacterial sequences already deposited in public data banks and a dendrogram constructed, after deletion from each sequence of one of the variable 16S rDNA regions (VI). The newly sequenced organisms fitted well within their respective families, but their similarities to other members of the groups were generally low, less than 96%. Close matches were found only with one other terrestrial (hot dry desert) cyanobacterium, Microcoleus sociatus, and with Anabaena variabilis. Phylogenetic analysis suggested that the deletion of the hypervariable regions in the RNA structure is essential for meaningful evolutionary studies. The results support the standard phylogenetic tree based on morphology, but suggest that these terrestrial cyanobacteria are distant relatives of their equivalent aquatic genera and are, indeed, a distinct population.     

Phylogenetic analysis and molecular signatures defining a monophyletic clade of heterocystous cyanobacteria and identifying its closest relatives

Detailed phylogenetic and comparative genomic analyses are reported on 140 genome sequenced cyanobacteria with the main focus on the heterocyst-differentiating cyanobacteria. In a phylogenetic tree for cyanobacteria based upon concatenated sequences for 32 conserved proteins, the available cyanobacteria formed 8–9 strongly supported clades at the highest level, which may correspond to the higher taxonomic clades of this phylum. One of these clades contained all heterocystous cyanobacteria; within this clade, the members exhibiting either true (Nostocales) or false (Stigonematales) branching of filaments were intermixed indicating that the division of the heterocysts-forming cyanobacteria into these two groups is not supported by phylogenetic considerations. However, in both the protein tree as well as in the 16S rRNA gene tree, the akinete-forming heterocystous cyanobacteria formed a distinct clade. Within this clade, the members which differentiate into hormogonia or those which lack this ability were also separated into distinct groups. A novel molecular signature identified in this work that is uniquely shared by the akinete-forming heterocystous cyanobacteria provides further evidence that the members of this group are specifically related and they shared a common ancestor exclusive of the other cyanobacteria. Detailed comparative analyses on protein sequences from the genomes of heterocystous cyanobacteria reported here have also identified eight conserved signature indels (CSIs) in proteins involved in a broad range of functions, and three conserved signature proteins, that are either uniquely or mainly found in all heterocysts-forming cyanobacteria, but generally not found in other cyanobacteria. These molecular markers provide novel means for the identification of heterocystous cyanobacteria, and they provide evidence of their monophyletic origin. Additionally, this work has also identified seven CSIs in other proteins which in addition to the heterocystous cyanobacteria are uniquely shared by two smaller clades of cyanobacteria, which form the successive outgroups of the clade comprising of the heterocystous cyanobacteria in the protein trees. Based upon their close relationship to the heterocystous cyanobacteria, the members of these clades are indicated to be the closest relatives of the heterocysts-forming cyanobacteria.     

Endolithic Phototrophs in Built and Natural Stone

Lichens, algae and cyanobacteria have been detected growing endolithically in natural rock and in stone buildings in various countries of Australasia, Europe and Latin America. Previously these organisms had mainly been described in natural carbonaceous rocks in aquatic environments, with some reports in siliceous rocks, principally from extremophilic regions. Using various culture and microscopy methods, we have detected endoliths in siliceous stone, both natural and cut, in humid temperate and subtropical climates. Such endolithic growth leads to degradation of the stone structure, not only by mechanical means, but also by metabolites liberated by the cells. Using in vitro culture, transmission, optical and fluorescence microscopy, and confocal laser scanning microscopy, both coccoid and filamentous cyanobacteria and algae, including Cyanidiales, have been identified growing endolithically in the facades of historic buildings built from limestone, sandstone, granite, basalt and soapstone, as well as in some natural rocks. Numerically, the most abundant are small, single-celled, colonial cyanobacteria. These small phototrophs are difficult to detect by standard microscope techniques and some of these species have not been previously reported within stone.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Cyanobacteria-containing biofilms from a Mayan monument in Palenque,Mexico

Surfaces of buildings at the archaeological site of Palenque, Mexico, are colonized by cyanobacteria that form biofilms, which in turn cause aesthetic and structural damage. The structural characterization and species composition of biofilms from the walls of one of these buildings, El Palacio, are reported. The distribution of photosynthetic microorganisms in the biofilms, their relationship with the colonized substratum, and the three-dimensional structure of the biofilms were studied by image analysis. The differences between local seasonal microenvironments at the Palenque site, the bioreceptivity of stone and the relationship between biofilms and their substrata are described. The implications for the development and permanence of species capable of withstanding temporal heterogeneity in and on El Palacio, mainly due to alternating wet and dry seasons, are discussed. Knowledge on how different biofilms contribute to biodegradation or bioprotection of the substratum can be used to develop maintenance and conservation protocols for cultural heritage.     

Characterization of thylakoid lipid membranes from cyanobacteria and higher plants by molecular dynamics simulations

The thylakoid membrane is mainly composed of non-common lipids, so called galactolipids. Despite the importance of these lipids for the function of the photosynthetic reaction centers, the molecular organization of these membranes is largely unexplored. Here we use multiscale molecular dynamics simulations to characterize the thylakoid membrane of both cyanobacteria and higher plants. We consider mixtures of up to five different galactolipids plus phosphatidylglycerol to represent these complex membranes. We find that the different lipids generally mix well, although nanoscale heterogeneities are observed especially in case of the plant membrane. The fluidity of the cyanobacterial membrane is markedly reduced compared to the plant membrane, even considering elevated temperatures at which thermophilic cyanobacteria are found. We also find that the plant membrane more readily undergoes a phase transformation to an inverted hexagonal phase. We furthermore characterized the conformation and dynamics of the cofactors plastoquinone and plastoquinol, revealing of the fast flip-flop rates for the non-reduced form. Together, our results provide a molecular view on the dynamical organization of the thylakoid membrane.     

Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.     

DNA profiling of complex bacterial populations: toxic cyanobacterial blooms

Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Artificial optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocyst cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk

Toxic cyanobacteria pose a significant hazard to human health and the environment. The recent characterisation of cyanotoxin synthetase gene clusters has resulted in an explosion of molecular detection methods for these organisms and their toxins. Conventional polymerase chain reaction (PCR) tests targeting cyanotoxin biosynthesis genes provide a rapid and sensitive means for detecting potentially toxic populations of cyanobacteria in water supplies. The adaptation of these simple PCR tests into quantitative methods has additionally enabled the monitoring of dynamic bloom populations and the identification of particularly problematic species. More recently, DNA microarray technology has been applied to cyanobacterial diagnostics offering a high-throughput option for detecting and differentiating toxic genotypes in complex samples. Together, these molecular methods are proving increasingly important for monitoring water quality.     

Picobenthic cyanobacterial populations revealed by 16S rRNA-targeted in situ hybridization

We report on the morphological identification of a population of benthic cyanobacteria from microbial mats, known previously only from molecular analyses of field samples, based on the retrieval of environmental 16S rRNA sequences. We used in situ hybridization with horseradish peroxidase-labelled oligonucleotide probes designed to target the 16S rRNA of our unidentified population. Two probes were designed and checked for target binding ability and specificity using membrane hybridization against electroblotted bands from a denaturant gradient gel electrophoresis (DGGE) fingerprint of 16S rDNA gene fragments from the original cyanobacterial community. Under in situ hybridization, these probes bound specifically to extremely small, unicellular, colony-forming cyanobacteria, 0.75-1 microm in diameter, which were embedded in abundant mucilaginous investments. We propose the term picobenthos, by analogy with picoplankton, to describe those unicellular benthic microbes around or less than 1 microm in diameter. Although picoplanktonic cyanobacteria are abundant in ocean and freshwaters, picobenthic (<1 microm) unicellular cyanobacteria are not typically recognized as a major component of microbial mats. The small size and low levels of photopigment autofluorescence from these cells probably rendered them cryptic or indistinguishable from heterotrophic bacteria in routine microscopic observations. It is not known how widespread picobenthic cyanobacteria may be in other environments.     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocystous cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

Cyanobacterial bioactive molecules--an overview of their toxic properties

Allelopathic interactions involving cyanobacteria are being increasingly explored for the pharmaceutical and environmental significance of the bioactive molecules. Among the toxic compounds produced by cyanobacteria, the biosynthetic pathways, regulatory mechanisms, and genes involved are well understood, in relation to biotoxins, whereas the cytotoxins are less investigated. A range of laboratory methods have been developed to detect and identify biotoxins in water as well as the causal organisms; these methods vary greatly in their degree of sophistication and the information they provide. Direct molecular probes are also available to detect and (or) differentiate toxic and nontoxic species from environmental samples. This review collates the information available on the diverse types of toxic bioactive molecules produced by cyanobacteria and provides pointers for effective exploitation of these biologically and industrially significant prokaryotes.     

Shedding new light on the regulation of complementary chromatic adaptation

Complementary chromatic adaptation (CCA) is a light-dependent acclimation process that occurs in cyanobacteria and likely is related to increased fitness of these organisms in natural environments. Although CCA has been studied for over 40 years, significant advances in our understanding of the molecular foundations of CCA are still emerging. In this minireview, I explore recently reported developments that include novel insights into the molecular mechanisms utilized in the photoregulation of pigmentation and the molecular basis of light-dependent changes in cellular morphology, which are central elements of the process of CCA. I also discuss future avenues of study that are expected to lead to additional progress in our understanding of CCA and our general appreciation of light sensing and photomorphogenesis in cyanobacteria.     

Molecular characterization and the effect of salinity on cyanobacterial diversity in the rice fields of Eastern Uttar Pradesh, India

Background

Salinity is known to affect almost half of the world's irrigated lands, especially rice fields. Furthermore, cyanobacteria, one of the critical inhabitants of rice fields have been characterized at molecular level from many different geographical locations. This study, for the first time, has examined the molecular diversity of cyanobacteria inhabiting Indian rice fields which experience various levels of salinity.

Results

Ten physicochemical parameters were analyzed for samples collected from twenty experimental sites. Electrical conductivity data were used to classify the soils and to investigate relationship between soil salinity and cyanobacterial diversity. The cyanobacterial communities were analyzed using semi-nested 16S rRNA gene PCR and denaturing gradient gel electrophoresis. Out of 51 DGGE bands selected for sequencing only 31 which showed difference in sequences were subjected to further analysis. BLAST analysis revealed highest similarity for twenty nine of the sequences with cyanobacteria, and the other two to plant plastids. Clusters obtained based on morphological and molecular attributes of cyanobacteria were correlated to soil salinity. Among six different clades, clades 1, 2, 4 and 6 contained cyanobacteria inhabiting normal or low saline (having EC < 4.0 ds m^-1) to (high) saline soils (having EC > 4.0 ds m^-1), however, clade 5 represented the cyanobacteria inhabiting only saline soils. Whilst, clade 3 contained cyanobacteria from normal soils. The presence of DGGE band corresponding to Aulosira strains were present in large number of soil indicating its wide distribution over a range of salinities, as were Nostoc, Anabaena, and Hapalosiphon although to a lesser extent in the sites studied.

Conclusion

Low salinity favored the presence of heterocystous cyanobacteria, while very high salinity mainly supported the growth of non-heterocystous genera. High nitrogen content in the low salt soils is proposed to be a result of reduced ammonia volatilization compared to the high salt soils. Although many environmental factors could potentially determine the microbial community present in these multidimensional ecosystems, changes in the diversity of cyanobacteria in rice fields was correlated to salinity.     

Photoresponsive cAMP signal transduction in cyanobacteria.

The molecular mechanism of cAMP-mediated signal transduction from light reception to the physiological response via regulation of gene expression in cyanobacteria is described based on our recent works. Cyanobacteria are known as the organisms that acquired oxygen-evolving, higher plant type photosynthesis. We have found that the cellular cAMP level in the filamentous cyanobacteria Anabaena was oppositely regulated by red and far-red light, i.e., decreasing and increasing, respectively, suggesting that a phytochrome-like red/far-red photoreversible pigment regulates the activity of a certain adenylate cyclase. On the other hand, in the unicellular cyanobacterium Synechocystis cellular cAMP content was increased by blue light irradiation, which led to stimulation of cell motility. The cAMP signaling pathway is known to play an important role in the regulation of various biological activities by altering enzyme activities or controlling gene expression levels in both prokaryotes and eukaryotes. We have isolated genes for adenylate cyclases and cAMP receptor proteins and characterized their molecular properties. Disruption of these genes resulted in the loss of cell motility. It is concluded that the light signal was transmitted by cAMP signal cascade in cyanobacteria.     

An update to modern taxonomy (2011) of freshwater planktic heterocytous cyanobacteria

It is essential for the modern taxonomic classification of cyanobacteria to be continually updated in accordance with revisions based on molecular sequence comparisons and combined with morphological features, ecophysiological characters and other biochemical and molecular markers (“polyphasic approach”). Several genera, which are characterized by their planktic life form and contain indicator species important for the evaluation of aquatic biocenoses in majority of water bodies are recognized in the monophyletic group of heterocytous cyanobacteria. Current taxonomic revisions (and nomenclatoric consequences) of the specific contents of these heterocytous cyanobacterial generic units are covered by this article. Among these genera, 12 contain only planktic species, three remaining genera contain both planktic and non-planktic species. Comments and suggestions for future research are stressed especially in the ecologically distinct genera, which includes species dominating in the plankton of various reservoir types.