Microcystin removal by microbial communities from a coastal lagoon: Influence of abiotic factors, bacterioplankton composition and estimated functions.

Toxic cyanobacterial blooms present a substantial risk to public health due to the production of secondary metabolites, notably microcystins (MCs). Microcystin-LR (MC-LR) is the most prevalent and toxic variant in freshwater. MCs resist conventional water treatment methods, persistently impacting water quality. This study focused on an oligohaline shallow lagoon historically affected by MC-producing cyanobacteria, aiming to identify bacteria capable of degrading MC and investigating the influence of environmental factors on this process. While isolated strains did not exhibit MC degradation, microbial assemblages directly sourced from lagoon water removed MC-LR within seven days at 25 ºC and pH 8.0. The associated bacterial community demonstrated an increased abundance of bacterial taxa assigned to Methylophilales, and also Rhodospirillales and Rhodocyclales to a lesser extent. However, elevated atmospheric temperatures (45 ºC) and acidification (pH 5.0 and 3.0) hindered MC-LR removal, indicating that extreme environmental changes could contribute to prolonged MC persistence in the water column. This study highlights the importance of considering environmental conditions in order to develop strategies to mitigate cyanotoxin contamination in aquatic ecosystems.

Microcystin drives the composition of small-sized bacterioplankton communities from a coastal lagoon.

Cyanobacterial blooms affect biotic interactions in aquatic ecosystems, including those involving heterotrophic bacteria. Ultra-small microbial communities are found in both surface water and groundwater and include diverse heterotrophic bacteria. Although the taxonomic composition of these communities has been described in some environments, the involvement of these small cells in the fate of environmentally relevant molecules has not been investigated. Here, we aimed to test if small-sized microbial fractions from a polluted urban lagoon were able to degrade the cyanotoxin microcystin (MC). We obtained cells after filtration through 0.45 as well as 0.22 µm membranes and characterized the morphology and taxonomic composition of bacteria before and after incubation with and without microcystin-LR (MC-LR). Communities from different size fractions (< 0.22 and < 0.45 µm) were able to remove the dissolved MC-LR. The originally small-sized cells grew during incubation, as shown by transmission electron microscopy, and changed in both cell size and morphology. The analysis of 16S rDNA sequences revealed that communities originated from < 0.22 and < 0.45 µm fractions diverged in taxonomic composition although they shared certain bacterial taxa. The presence of MC-LR shifted the structure of < 0.45 µm communities in comparison to those maintained without toxin. Actinobacteria was initially dominant and after incubation with MC-LR Proteobacteria predominated. There was a clear enhancement of taxa already known to degrade MC-LR such as Methylophilaceae. Small-sized bacteria constitute a diverse and underestimated fraction of microbial communities, which participate in the dynamics of MC-LR in natural environments.

Ecophysiological Aspects and sxt Genes Expression Underlying Induced Chemical Defense in STX-Producing Raphidiopsis raciborskii (Cyanobacteria) against the Zooplankter Daphnia gessneri.

Cyanobacteria stand out among phytoplankton when they form massive blooms and produce toxins. Because cyanotoxin genes date to the origin of metazoans, the hypothesis that cyanotoxins function as a defense against herbivory is still debated. Although their primary cellular function might vary, these metabolites could have evolved as an anti-predator response. Here we evaluated the physiological and molecular responses of a saxitoxin-producing Raphidiopsis raciborskii to infochemicals released by the grazer Daphnia gessneri. Induced chemical defenses were evidenced in R. raciborskii as a significant increase in the transcription level of sxt genes, followed by an increase in saxitoxin content when exposed to predator cues. Moreover, cyanobacterial growth decreased, and no significant effects on photosynthesis or morphology were observed. Overall, the induced defense response was accompanied by a trade-off between toxin production and growth. These results shed light on the mechanisms underlying zooplankton-cyanobacteria interactions in aquatic food webs. The widespread occurrence of the cyanobacterium R. raciborskii in freshwater bodies has been attributed to its phenotypic plasticity. Assessing the potential of this species to thrive over interaction filters such as zooplankton grazing pressure can enhance our understanding of its adaptive success.

Gut microbiota of adults with different metabolic phenotypes.

OBJECTIVE: The aim of this study was to investigate the gut microbiota of individuals with different metabolic phenotypes and to compare their characteristics. METHODS: This was a cross-sectional study with 109 adults, classified according to metabolic status, by body mass index, and homeostasis model assessment. Anthropometric and biochemical characteristics and blood pressure were evaluated. Level of physical activity was evaluated by means of a questionnaire, and dietary consumption was evaluated using a 3-d food record. Feces samples were collected from each participant, and gut microbiota profile was analyzed by sequencing of the 16S rRNA gene. The Kruskal-Wallis and χ2 tests were used, according to the scale of measurement of the variables, on the statistical program SPSS version 25, with significance level of 5%. RESULTS: Significantly lower values of saturated fat intake and fasting glycemia were observed in metabolically healthy individuals who were overweight and obese compared with their unhealthy counterparts. Diversity and richness of bacterial communities were lower in individuals who were obese and in the unhealthy phenotype. The genus Eubacterium rectale and genera of the family Prevotellaceae were more abundant in the metabolically healthy obese and metabolically healthy overweight groups than in the metabolically healthy normal weight one, whereas Bifidobacterium was more abundant in the metabolically healthy normal weight group. The genera Coprococcus and Ruminococcus were more abundant in the metabolically healthy overweight group than in the metabolically unhealthy overweight one. CONCLUSIONS: We observed distinct characteristics in the gut microbiota of different metabolic phenotypes. The intestines of individuals with unhealthy phenotypes hosted inflammation-associated microbiota, with lower butyrate production potential and reduced bacterial diversity.

Occurrence and diversity of viruses associated with cyanobacterial communities in a Brazilian freshwater reservoir.

As part of the phytoplankton of marine and freshwater environments around the world, cyanobacteria interact with viruses (cyanophages) that affect their abundance and diversity. Investigations focusing on cyanophages co-occurring with freshwater cyanobacteria are scarce, particularly in Brazil. The aim of this study was to assess the diversity of cyanophages associated with a Microcystis-dominated cyanobacterial bloom in a tropical reservoir. Samples were processed as viral fractions of water and cellular fractions, and temporal fluctuations in the abundance of Ma-LMM01-type cyanophages and their Microcystis hosts were determined by qPCR. We applied shotgun metagenomics to obtain a wider characterization of the cyanophage community. During the study period, Microcystis gene copies were quantified in all cellular fractions, and the copy number of the Ma-LMM01 phage gene tended to increase with host abundance. Metagenomic analysis demonstrated that Caudovirales was the major viral order associated with the cyanophage families Myoviridae (34-88%), Podoviridae (3-42%), and Siphoviridae (6-23%). The metagenomic analysis results confirmed the presence of Microcystis cyanophages in both viral and cellular fractions and demonstrated a high relative abundance of picocyanobacteria-related viruses and Prochlorococcus (36-52%) and Synechococcus (37-50%) phages. For other main cyanobacterial genera, no related cyanophages were identified, which was probably due to the scarce representation of cyanophage sequences in databanks. Thus, the studied reservoir hosted a diverse cyanophage community with a remarkable contribution of phages related to picoplanktonic cyanobacteria. These results provide insights that motivate future sequencing efforts to assess cyanophage diversity and recover complete genomes.

Influence of Iron on Physiological Parameters and Intracellular Microcystin in Microcystis Panniformis Strain Isolated from a Reservoir in the Amazon.

In the Amazon, the leaching from soil left unprotected by deforestation increases the entry of iron, among other elements, in aquatic ecosystems, which can cause cyanobacterial blooms. This study aimed to investigate the physiological response of a strain of Microcystis panniformis to iron variation. The strain was isolated from a reservoir located in the Western Amazon and produces microcystin-LR. After a period of iron deprivation, the cultures were submitted to three conditions: control (223 µgFe.L-1), treatment with 23 µgFe.L-1, and absence of iron. At regular intervals for eight days, the cell density, levels of chlorophyll a and microcystins were determined. On the second and fourth day, transcription of genes responsive to iron limitation was quantified. Starting on the fourth day of the experiment, the different iron concentrations affected growth, and on the eighth day in the iron-free condition cell density was 90% lower than in control. Chlorophyll cell quota in 23 µgFe.L-1 and control presented similar values, while without iron the cells became chlorotic as of the fourth day Toxin concentration in cells grow in 0 µgFe.L-1 in relation to the control. Higher transcription levels of the feo and fut genes were observed in the 0 µgFe.L-1 and 23 µgFe.L-1 treatments, indicating that the cells were activating high-affinity capture systems to reestablish an adequate concentration of intracellular iron. The increasing deforestation in the Jamari River Basin (Amazon region), can contribute to the occurrence of toxic cyanobacterial blooms due to the greater entrance of iron in water bodies.

Biotic and abiotic factors affect microcystin-LR concentrations in water/sediment interface.

Harmful cyanobacterial blooms are increasingly common in aquatic environments. This can lead to higher concentrations of cyanotoxins, such as microcystins (MCs), posing a great risk to diverse organisms, including humans. MCs are among the most commonly reported cyanotoxins in freshwater environments worldwide, where they may have different fates. MCs can adsorb to suspended particles into the water column and deposit onto the sediment where they can be affected by physical factors (e.g. winds in shallow lakes causing sediment resuspension) or biological factors (e.g. biodegradation). Here we focused on the conditions of a coastal shallow lagoon contaminated by MCs aiming to estimate the return of pre-existing MCs from the sediment to the water column, to evaluate the adsorption of dissolved MC-LR to the sediment and to verify the occurrence of biodegradation. In experiments with sediment, desorption and adsorption were tested under the influence of temperature, pH and aeration, reproducing conditions observed in the lagoon. MC-desorption was not detected under the tested conditions. Spiking MC-LR into lagoon water samples in the presence of sediment resulted in a 50 % reduction of soluble MC-LR concentration in control conditions (25 °C, pH 8.0, no aeration). Increasing temperature (45 °C) or introducing aeration further stimulated MC-LR removal from the water. Biodegradation was observed in sediment samples and interstitial water (even with tetracycline). The composition of the bacterial community differed in sediment and interstitial water: major phyla were Chloroflexi, Proteobacteria, Firmicutes, and OP3. From the assigned OTUs, we identified genera already described as MC degrading bacteria. Thus, the sediment is a key factor influencing the fate of MC-LR in this shallow coastal lake contributing to stable adsorption and biodegradation.

Intraspecific variability in response to phosphorus depleted conditions in the cyanobacteria Microcystis aeruginosa and Raphidiopsis raciborskii.

Phosphorus loading plays an important role in the occurrence of cyanobacterial blooms and understanding how this nutrient affects the physiology of cyanobacteria is imperative to manage these phenomena. Microcystis aeruginosa and Raphidiopsis raciborskii are cyanobacterial species that form potentially toxic blooms in freshwater ecosystems worldwide. Blooms comprise numerous strains with high trait variability, which can contribute to the widespread distribution of these species. Here, we explored the intraspecific variability in response to phosphorus depleted conditions (P-) testing five strains of each species. Strains could be differentiated by cell volume or genetic profiles except for those of the same species, sampling location and date, though these presented differences in their response to (P-). Although differently affected by (P-) over 10 days, all strains were able to grow and maintain photosynthetic activity. For most M. aeruginosa and R. raciborskii strains growth rates were not significantly different comparing (P+) and (P-) conditions. After ten days in (P-), only one M. aeruginosa strain and two R. raciborskii strains showed reduction in biovolume yield as compared to (P+) but in most strains chlorophyll-a concentrations were lower in (P-) than in (P+). Reduced photosystem II efficiency was found for only one R. raciborskii strain while all M. aeruginosa strains were affected. Only two M. aeruginosa and one R. raciborskii strain increased alkaline phosphatase activity under (P-) as compared to (P+). Variation in P-uptake was also observed but comparison among strains yielded homogeneous groups comprised of representatives of both species. Comparing the response of each species as a whole, the (P-) condition affected growth rate, biovolume yield and chlorophyll yield. However, these parameters revealed variation among strains of the same species to the extent that differences between M. aeruginosa and R. raciborskii were not significant. Taken together, these results do not support the idea that R. raciborskii, as a species, can withstand phosphorus limitation better than M. aeruginosa and also point that the level of intraspecific variation may preclude generalizations based on studies that use only one or few strains.

Quantitative gingival crevicular fluid proteome in type 2 diabetes mellitus and chronic periodontitis.

OBJECTIVE: The aim of this study was to investigate the proteome of the gingival crevicular fluid comparing the relative abundance of proteins from type 2 diabetes mellitus (2DM) individuals and chronic periodontitis (CP) affected sites, subjects affected by both conditions and healthy individuals. MATERIAL AND METHODS: Twenty individuals were equally allocated in four groups, 2DM with CP, 2DM periodontally healthy, CP without 2DM, and periodontally healthy without 2DM. The relative quantification of proteins was accessed with iTRAQ labeling and mass spectrometry. RESULTS AND CONCLUSION: A total of 104 proteins showed significant differences in abundance in pairwise comparisons. Some presented different levels in all diseased groups as compared to control, either increasing (rap guanine nucleotide exchange factor, S100A8, S100A9, and immunoglobulins) or decreasing (actins, myristoylated alanine-rich C-kinase substrate, and glutathione S-transferase). Other differences were specific for a given condition: Titin, neutrophil elastase, and myeloperoxidase levels were higher in the DP group, cathelicidin antimicrobial peptide decreased in CP, and annexin decreased in DH. These differences in the proteome can provide clues for further studies that will validate the variation in their levels and their role in both diseases.

Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR). Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk.

Growth and saxitoxin production by Cylindrospermopsis raciborskii (cyanobacteria) correlate with water hardness.

The cosmopolitan and increasing distribution of Cylindrospermopsis raciborskii can be attributed to its ecophysiological plasticity and tolerance to changing environmental factors in water bodies. In reservoirs in the semi-arid region of Brazil, the presence and common dominance of C. raciborskii have been described in waters that are considered hard. We investigated the response of a Brazilian C. raciborskii strain to water hardness by evaluating its growth and saxitoxin production. Based on environmental data, a concentration of 5 mM of different carbonate salts was tested. These conditions affected growth either positively (MgCO3) or negatively (CaCO3 and Na2CO3). As a control for the addition of cations, MgCl2, CaCl2 and NaCl were tested at 5 or 10 mM, and MgCl2 stimulated growth, NaCl slowed but sustained growth, and CaCl2 inhibited growth. Most of the tested treatments increased the saxitoxin (STX) cell quota after six days of exposure. After 12 days, STX production returned to concentrations similar to that of the control, indicating an adaptation to the altered water conditions. In the short term, cell exposure to most of the tested conditions favored STX production over neoSTX production. These results support the noted plasticity of C. raciborskii and highlight its potential to thrive in hard waters. Additionally, the observed relationship between saxitoxin production and water ion concentrations characteristic of the natural environments can be important for understanding toxin content variation in other harmful algae that produce STX.

The inhibitory effect of calcium on Cylindrospermopsis raciborskii (cyanobacteria) metabolism

Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba Raju is a freshwater cyanobacterium of worldwide distribution. In the North-eastern region of Brazil many eutrophic water reservoirs are characterized by the dominance of C. raciborskii, with recurrent occurrence of blooms. These water bodies have high conductivity due to a high ionic concentration, and are defined as hard (with high values of CaCO3). In this study, we investigated the long-term effect (12 days) of high calcium concentration (8 mM Ca2+) on C. raciborskii (T3 strain) growth, morphology, toxin content, and metabolism. Changes in protein expression profiles were investigated by proteomic analysis using 2D gel electrophoresis and mass spectrometry. A continued exposure to calcium had a pronounced effect on C. raciborskii (T3): it limited growth, decreased thricome length, increased chlorophyll-a content, altered toxin profile (although did not affect PST content, saxitoxin + neosaxitoxin), and inhibited the expression of proteins related to primary metabolism.

Fine-tuning control of phoBR expression in Vibrio cholerae by binding of phoB to multiple pho boxes.

The control of Vibrio cholerae phoBR expression by PhoB involves its binding to Pho boxes at -35 (box 1), -60 (box 2), and -80 (box 3) from the putative phoB translation start site. These loci were located in the sense (box 1) and antisense (boxes 2 and 3) strands of the phoBR regulatory region, and PhoB binds to these individual boxes with distinct affinities. Fusions of sequences containing different combinations of these boxes upstream of the lacZ reporter in a plasmid demonstrated that only those carrying boxes 1, 2, and 3, or 1 alone, activated transcription under inorganic phosphate (P(i)) limitation. When a fragment, including only boxes 1 and 2, was fused to lacZ, expression was no longer induced by low P(i), suggesting a repressive role for PhoB~box2 (PhoB bound to box 2) over the transcriptional activity induced by PhoB~box1. The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoB~box3 eliminated the repressive effect imposed by PhoB~box2 on phoBR transcription. Complementation assays with a phoBR-containing plasmid demonstrated that the 234-bp promoter fragment carrying the three boxes is absolutely required for operon expression in Vibrio cholerae ΔphoBR cells. This was observed under P(i) abundance, when phoBR was expressed at a basal level and, also in low P(i) conditions, when Pho regulon genes were fully expressed. Thus, under P(i) limitation, PhoB exerts dual regulatory functions by binding sequentially distinct Pho boxes to enable the fine-tuning and precise control of phoBR expression in V. cholerae cells.

The inhibitory effect of calcium on Cylindrospermopsis raciborskii (cyanobacteria) metabolism.

Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba Raju is a freshwater cyanobacterium of worldwide distribution. In the North-eastern region of Brazil many eutrophic water reservoirs are characterized by the dominance of C. raciborskii, with recurrent occurrence of blooms. These water bodies have high conductivity due to a high ionic concentration, and are defined as hard (with high values of CaCO3). In this study, we investigated the long-term effect (12 days) of high calcium concentration (8 mM Ca(2+)) on C. raciborskii (T3 strain) growth, morphology, toxin content, and metabolism. Changes in protein expression profiles were investigated by proteomic analysis using 2D gel electrophoresis and mass spectrometry. A continued exposure to calcium had a pronounced effect on C. raciborskii (T3): it limited growth, decreased thricome length, increased chlorophyll-a content, altered toxin profile (although did not affect PST content, saxitoxin + neosaxitoxin), and inhibited the expression of proteins related to primary metabolism.

Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5.

BACKGROUND: Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins. RESULTS: Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat. CONCLUSION: The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.

Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Rio de Janeiro city, Brazil.

Enterotoxigenic Escherichia coli (ETEC) strains have been implicated as important etiological agents of diarrheal disease, especially in developing countries. This group of microorganisms has been associated with a diverse range of genotypic and phenotypic markers. In the present study, 21 ETEC isolates previously defined according to the toxigenic genotypes, were characterized on the basis of O:H typing, cell adherence patterns, and colonization factors (CFs) antigens. Genetic diversity was investigated by random amplification polymorphic DNA (RAPD-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE). LT-I probe-positive isolates belonged to serotypes ONT:HNT, O7:H24, O48:H21, O88:H25, O148:H28, O159:H17 and O159:H21. ST-h probe-positive isolates belonged to serotypes O159:H17, O148:H28 and O6:H-. Serotypes O148:H28, O159:H17 and O6:H- were associated with the CS6, CFA/I and CS1 CS3 antigens, respectively. Most ETEC strains exhibited a diffuse pattern of adherence to cultured epithelial cells. In general, phenotypic and genotypic characteristics correlated well. RAPD-PCR, PFGE and MLEE showed reproducibility and good discriminatory potential. The application of molecular typing systems allowed the detection of significant diversity among the isolates, indicating a non-clonal origin and revealing intra-serotype variation overlooked by classical epidemiological approaches. The phenotypic and genotypic diversity observed lead us to recommend the use of different typing systems in order to elucidate the epidemiology of ETEC infection.

Autoregulation of Helicobacter pylori Fur revealed by functional analysis of the iron-binding site.

The ferric uptake regulator protein Fur regulates iron-dependent gene expression in bacteria. In Helicobacter pylori it has been shown to regulate iron-activated and iron-repressed genes. In this study, we show that H. pylori Fur protein regulates transcription from its own sigma 80 promoter P fur in response to iron. Footprinting analysis shows that Fur binds at three distinct operators at P fur overlapping and proximal to the promoter elements. Site-directed mutagenesis of the proposed iron-binding site of the protein results in derepression of P fur and the loss of iron regulation. In vivo oligomerization assays reveals that the C-terminus of Fur is necessary for multimerization of the protein and that the mutations do not affect this activity. Molecular and phenotypic analysis of the mutant proteins provides evidence that the iron-binding site controls the specific affinity of Fur for the operators at P fur and hence its repressive ability. In summary, the data presented are consistent with a model in which Fur acts as a rheostat of transcription to autoregulate its own expression in response to iron, which in turn controls expression of iron-induced and iron-repressed genes, providing maintenance of homeostasis.

Site-directed mutagenesis of the foot-and-mouth disease virus RNA-polymerase gene

O gene da RNA-polimerase do vírus da febre aftosa foi mutagenizado dentro do seu sítio de atividade. Utilizando o cDNA da cepa viral CS8 C1 - Santa Pau, o gene foi digerido com Pst I gerando um fragmento contendo a seqüência crítica de mutagênese. Este fragmento foi subclonado em M13mp8 e quatro mutaçöes foram geradas in vitro através do método de mutagênese sítio-dirigida por oligonucleotídeo. O gene da polimerase foi entäo reconstruído e subclonado em pUC19. Estes mutantes seräo usados em estudos de estrutura e atividade da enzima e no desenvolvimento da técnica de "imunizaçäo intracelular" em células eurcariontes