The Smallest Known Genomes of Multicellular and Toxic Cyanobacteria: Comparison, Minimal Gene Sets for Linked Traits and the Evolutionary Implications

Cyanobacterial morphology is diverse, ranging from unicellular spheres or rods to multicellular structures such as colonies and filaments. Multicellular species represent an evolutionary strategy to differentiate and compartmentalize certain metabolic functions for reproduction and nitrogen (N2) fixation into specialized cell types (e.g. akinetes, heterocysts and diazocytes). Only a few filamentous, differentiated cyanobacterial species, with genome sizes over 5 Mb, have been sequenced. We sequenced the genomes of two strains of closely related filamentous cyanobacterial species to yield further insights into the molecular basis of the traits of N2 fixation, filament formation and cell differentiation. Cylindrospermopsis raciborskii CS-505 is a cylindrospermopsin-producing strain from Australia, whereas Raphidiopsis brookii D9 from Brazil synthesizes neurotoxins associated with paralytic shellfish poisoning (PSP). Despite their different morphology, toxin composition and disjunct geographical distribution, these strains form a monophyletic group. With genome sizes of approximately 3.9 (CS-505) and 3.2 (D9) Mb, these are the smallest genomes described for free-living filamentous cyanobacteria. We observed remarkable gene order conservation (synteny) between these genomes despite the difference in repetitive element content, which accounts for most of the genome size difference between them. We show here that the strains share a specific set of 2539 genes with >90% average nucleotide identity. The fact that the CS-505 and D9 genomes are small and streamlined compared to those of other filamentous cyanobacterial species and the lack of the ability for heterocyst formation in strain D9 allowed us to define a core set of genes responsible for each trait in filamentous species. We presume that in strain D9 the ability to form proper heterocysts was secondarily lost together with N2 fixation capacity. Further comparisons to all available cyanobacterial genomes covering almost the entire evolutionary branch revealed a common minimal gene set for each of these cyanobacterial traits

Impact of Nitrogen Sources on Gene Expression and Toxin Production in the Diazotroph Cylindrospermopsis raciborskii CS-505 and Non-Diazotroph Raphidiopsis brookii D9

Different environmental nitrogen sources play selective roles in the development of cyanobacterial blooms and noxious effects are often exacerbated when toxic cyanobacteria are dominant. Cylindrospermopsis raciborskii CS-505 (heterocystous, nitrogen fixing) and Raphidiopsis brookii D9 (non-N2 fixing) produce the nitrogenous toxins cylindrospermopsin (CYN) and paralytic shellfish toxins (PSTs), respectively. These toxin groups are biosynthesized constitutively by two independent putative gene clusters, whose flanking genes are target for nitrogen (N) regulation. It is not yet known how or if toxin biosynthetic genes are regulated, particularly by N-source dependency. Here we show that binding boxes for NtcA, the master regulator of N metabolism, are located within both gene clusters as potential regulators of toxin biosynthesis. Quantification of intra- and extracellular toxin content in cultures at early stages of growth under nitrate, ammonium, urea and N-free media showed that N-sources influence neither CYN nor PST production. However, CYN and PST profiles were altered under N-free medium resulting in a decrease in the predicted precursor toxins (doCYN and STX, respectively). Reduced STX amounts were also observed under growth in ammonium. Quantification of toxin biosynthesis and transport gene transcripts revealed a constitutive transcription under all tested N-sources. Our data support the hypothesis that PSTs and CYN are constitutive metabolites whose biosynthesis is correlated to cyanobacterial growth rather than directly to specific environmental conditions. Overall, the constant biosynthesis of toxins and expression of the putative toxin-biosynthesis genes supports the usage of qPCR probes in water quality monitoring of toxic cyanobacteria

PSP toxin release from the cyanobacterium Raphidiopsis brookii D9 (Nostocales) can be induced by sodium and potassium ions

Paralytic shellfish poisoning (PSP) toxins are a group of naturally occurring neurotoxic alkaloids produced among several genera of primarily freshwater cyanobacteria and marine dinoflagellates. Although saxitoxin (STX) and analogs are all potent Na+ channel blockers in vertebrate cells, the functional role of these compounds for the toxigenic microorganisms is unknown. Based upon the known importance of monovalent cations (such as sodium) in the maintenance of cellular homeostasis and ion channel function, we examined the effect of high extracellular concentrations of these ions on growth, cellular integrity, toxin production and release to the external medium in the filamentous freshwater cyanobacterium, Raphidiopsis brookii D9; a gonyautoxins (GTX2/3) and STX producing toxigenic strain. We observed a toxin export in response to high (17 mM) NaCl and KCl concentrations in the growth medium that was not primarily related to osmotic stress effects, compared to the osmolyte mannitol. Addition of exogenous PSP toxins with the same compositional profile as the one produced by R. brookii D9 was able to partially mitigate this effect of high Na+ (17 mM). The PSP toxin biosynthetic gene cluster (sxt) in D9 has two genes (sxtF and sxtM) that encode for a MATE (multidrug and toxic compound extrusion) transporter. This protein family, represented by NorM in the bacterium Vibrio parahaemolyticus, confers resistance to multiple cationic toxic agents through Na+/drug antiporters. Conserved domains for Na+ and drug recognition have been described in NorM. For the D9 sxt cluster, the Na+ recognition domain is conserved in both SxtF and SxtM, but the drug recognition domain differs between them. These results suggest that PSP toxins are exported directly in response to the presence of monovalent cations (Na+, K+) at least at elevated concentrations. Thus, the presence of both genes in the sxt cluster from strain D9 can be explained as a selective recognition mechanism by the SxtF/M transporters for GTX2/3 and STX. We propose that these toxins in cyanobacteria could act extracellularly as a protective mechanism to ensure homeostasis against extreme salt variation in the environment

Draft genome sequences of Cylindrospermopsis raciborskii strains CS-508 and MVCC14, isolated from freshwater bloom events in Australia and Uruguay

Abstract Members of the genus Cylindrospermopsis represent an important environmental and health concern. Strains CS-508 and MVCC14 of C. raciborskii were isolated from freshwater reservoirs located in Australia and Uruguay, respectively. While CS-508 has been reported as non-toxic, MVCC14 is a saxitoxin (STX) producer. We annotated the draft genomes of these C. raciborskii strains using the assembly of reads obtained from Illumina MiSeq sequencing. The final assemblies resulted in genome sizes close to 3.6 Mbp for both strains and included 3202 ORFs for CS-508 (in 163 contigs) and 3560 ORFs for MVCC14 (in 99 contigs). Finally, both the average nucleotide identity (ANI) and the similarity of gene content indicate that these two genomes should be considered as strains of the C. raciborskii species

COVID-19 i ciutat: cap a un model integrat d'habitatge, microbiologia, ambient i urbanisme

A partir de mayo de 2020, la crisis sanitaria global causada por el virus SARS-CoV-2 traslada su epicentro hacia Latinoamérica, con foco en ciudades que presentan altos índices de pobreza, segregación y hacinamiento. Los avances en microbiología posibilitan comprender en profundidad las relaciones entre ciudad, COVID-19 y otros microorganismos, pero falta establecer un marco conceptual que las articule, especialmente en contextos donde las determinantes sociales son tan relevantes. Este artículo tiene como objetivo el proponer una aproximación integrada de microbiología, vivienda, ambiente y urbanismo, a partir de un modelo de interacciones y un análisis empírico para Santiago de Chile. En base a esto, se pudo analizar cómo el proceso de propagación en la ciudad se ve potenciada por vulnerabilidades de índole socio espacial, sanitaria intradomiciliaria y urbana, más una aproximación desde el concepto de pobreza energética. Al mismo tiempo, se pudo comprobar como las variables asociadas a estas vulnerabilidades permitían explicar la tasa de incidencia de casos confirmados por cada 100 000 habitantes a través de las distintas comunas en el área metropolitana de Santiago de Chile. Dentro de éstas, destacan el nivel de hacinamiento de las viviendas, la cantidad de hogares con jefes/as de hogar en trabajo precario y los viajes hacia el distrito central de negocios de la ciudad. Finalmente, se establece la necesidad de proponer una agenda de investigación para este nuevo equipo multidisciplinario de “Microbioma Urbano” en relación con la necesidad de realización de muestreos microbiológicos que permitan mejorar las condiciones de viviendas, barrios y ciudades, aportando en la superación de las vulnerabilidades identificadas en este estudio.A partir de maig de 2020, la crisi sanitària global causada pel virus SARS-CoV-2 trasllada el seu epicentre cap a Llatinoamèrica, amb focus en ciutats que presenten alts índexs de pobresa, segregació i amuntegament. Els avenços en microbiologia permeten entendre en profunditat les relacions entre ciutat, COVID-19 i altres microorganismes, però cal establir un marc conceptual que les articuli, especialment en contextos on les determinants socials són tan rellevants. Aquest article té com a objectiu el proposar una aproximació integrada de microbiologia, habitatge, ambient i urbanisme, a partir d'un model d'interaccions i una anàlisi empírica per a Santiago de Xile. En base a això, es va poder analitzar com el procés de propagació a la ciutat es veu potenciada per vulnerabilitats d'índole soci espacial, sanitària intra-domiciliaria i urbana, més una aproximació des del concepte de pobresa energètica. Al mateix temps, es va poder comprovar com les variables associades a aquestes vulnerabilitats permetien explicar la taxa d'incidència de casos confirmats per cada 100 000 habitants a través de les diferents comunes a l'àrea metropolitana de Santiago de Xile. Dins d'aquestes, destaquen el nivell d'amuntegament dels habitatges, la quantitat de llars amb caps de la llar en treball precari i els viatges cap al districte central de negocis de la ciutat. Finalment, s'estableix la necessitat de proposar una agenda de recerca per a aquest nou equip multidisciplinari de "Microbioma Urbà" en relació amb la necessitat de realització de mostrejos microbiològiques que permetin millorar les condicions d'habitatges, barris i ciutats, aportant en la superació de les vulnerabilitats identificades en aquest estudi.As of May 2020, the global health crisis caused by the SARS-CoV-2 virus moves its epicentre to Latin America, with cities showing high rates of poverty, segregation, and overcrowding. Current advances in microbiology make it possible to understand in depth the relationships between cities, COVID-19, and other microorganisms, but a conceptual framework to articulate them is lacking, especially in contexts where social determinants are so relevant. This article proposes an integrated approach to microbiology, housing, environment, and urbanism, based on a model of interactions and an empirical analysis applied to Santiago de Chile. It was possible to analyse how the propagation of COVID-19 in the city is enhanced by vulnerabilities of socio-spatial, residential and urban health, including an approach from the concept of energy poverty. At the same time, it was possible to verify how the variables associated with these vulnerabilities allowed to explain the incidence rate per 100 000 inhabitants through the different communes of Santiago de Chile. Among these, the level of housing overcrowding, the number of households with heads of household in precarious employment, and travel to the central business district stand out. Finally, the need for microbiological sampling to improve housing conditions, neighbourhoods, and cities propose a new research agenda for this Urban Microbiome" multidisciplinary team, contributing to overcoming the vulnerabilities identified in this research.Este artículo se ha realizado con financiamiento de la Agencia Nacional de Investigación y Desarrollo (ANID), por medio del proyecto FONDECYT Regular Nº1201332 “NEXO POBREZA-ENERGÍA-VIVIENDA: Lineamientos de política pública para abordar la pobreza energética desde la relación con la vivienda en áreas metropolitanas”. También ha contado con el apoyo del Centro de Desarrollo Urbano Sustentable (CEDEUS), Proyecto ANID/FONDAP Nº15110020; del Centre of Applied Ecology and Sustainability (CAPES) y del MillenniumInitiative for Collaborative Research on Bacterial Resistance (MICROB-R).Peer Reviewe

Binding energies of the PST analogues and putative binding sites in SxtF and SxtM.

*<p>Interactions obtained from docking calculations to Grid 2.</p><p>Raph: <i>Raphidiopsis brookii</i>; CR: <i>C. raciborskii</i>; AC: <i>A. circinalis</i>.</p><p>NP: Analogue is not present.</p><p>(−): System was not analyzed.</p

Predicted protein structure of SxtF of <i>R. brookii</i> D9, lateral view.

<p>The model was built based on the X-ray structure of the MATE transporter NorM of <i>Vibrio cholerae</i> (ID code: 3MKT; Resolution: 3,65 Å; R-Value: 0,312). The location in which we obtained the most favorable binding energies (domain ³⁹⁰VGLRD³⁹⁵ in licorice representation) is shown circled in blue. STX corresponds to the yellow colored molecule.</p