Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation

A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35 % of the SybrGold-stained cells, which corresponded to 6.61 × 106 cells/ml and 1.76 ± 0.09 × 106 archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1.1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = –1.058, P = 0.308 and ANOVA t14 = 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free medium (ANOVA t14 = 12.12, P < 0.001). [Int Microbiol 2013; 16(3):177-189]Keywords: ammonia-oxidizing archaea and bacteria · ammonia monooxygenase alpha subunit (amoA) · Lake Kivu ·microcosm · multi-color CARD-FIS

The molar extinction coefficient of bacteriochlorophyll e and the pigment stoichiometry in Chlorobium phaeobacteroides

We have determined the molar extinction coefficient of bacteriochlorophyll (BChl) e, the main light-harvesting pigment from brown-coloured photosynthetic sulfur bacteria. The extinction coefficient was determined using pure[Pr,E]BChl eF isolated by reversed-phase HPLC from crude pigment extracts of Chlorobium (Chl.) phaeobacteroides strain CL1401. The extinction coefficients at the Soret and Qy bands were determined in four organic solvents. The extinction coefficient of BChl e differs from those of other related Chlorobium chlorophylls (BChl c and BChl d) but is similar to that of chlorophyll b. The determined extinction coefficient was used to calculate the stoichiometric BChl e to BChl a and BChl e to carotenoids ratios in whole cells and isolated chlorosomes from Chl. phaeobacteroides strain CL1401 using the spectrum-reconstruction method (SRCM) described by Naqvi et al. (1997) (Spectrochim Acta A Mol Biomol Spectrosc 53: 2229–2234). In isolated chlorosomes, BChl a content was ca. 1% of the total BChl content and the stoichiometric ratio of BChl e to carotenoids was 6. In whole cells,however, BChl a content was 3–4%, owing to the presence of BChl a-containing elements, i.e. FMO protein and reaction centre. An average of 5 BChl e molecules per carotenoid was determined in whole cells.EU(Contract No FMRX–CT96–0081). Ministerio de Educación y Ciencia (Ref. BIO96–1229–002–01)Peer reviewe

Nanosecond laser photolysis studies of chlorosomes and artificial aggregates containing bacteriochlorophyll e: Evidence for the proximity of carotenoids and bacteriochlorophyll a in chlorosomes from Chlorobium phaeobacteroides strain CL1401

Time-resolved, laser-induced changes in absorbance, ΔA(λ; t), have been recorded with a view to probing pigment–pigment interactions in chlorosomes (control as well as carotenoid-depleted) and artificial aggregates of bacteriochlorophyll e (BChle). Control chlorosomes were isolated from Chlorobium phaeobacteroides strain CL1401, whose chromophores comprise BChle, bacteriochlorophyll a (BChla) and several carotenoid (Car) pigments; Car-depleted chlorosomes, from cells grown in cultures containing 2-hydroxybiphenyl. Artificial aggregates were prepared by dispersing BChle in aqueous phase in the presence of monogalactosyl diglyceride. In chlorosomes ΔA(λ; t) shows, besides a signal attributable to triplet Car (with a half-life of about 4 μs), signals in the Qy regions of both BChl. The BChla signal decays at the same rate as the Car signal, which is explained by postulating that some Car are in intimate contact with some baseplate BChla pigments, and that when a ground-state Car changes into a triplet Car, the absorption spectrum of its BChla neighbors undergoes a concomitant change (termed transient environment-induced perturbation). The signal in the Qy-region of BChle behaves differently: its amplitude falls, under reducing conditions, by more than a factor of two during the first 0.5 μs (a period during which the Car signal suffers negligible diminution), and is much smaller under nonreducing conditions. The BChle signal is also attributed to transient environment-induced perturbation, but in this case the perturber is a BChle photoproduct (probably a triplet or a radical ion). The absence of long-lived BChle triplets in all three systems, and of long-lived BChla triplets in chlorosomes, indicates that BChle in densely packed assemblies is less vulnerable to photodamage than monomeric BChle and that, in chlorosome, BChla rather than BChle needs, and receives, photoprotection from an adjacent Car.Research Council of Norway. EU (contract FMRX-CT96-0081)Peer reviewe

Exploring the early stages of chemical unfolding of proteins at the proteome scale

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded←→unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding

Effect of urban wastewater discharge on the abundance of antibiotic resistance genes and antibiotic-resistant Escherichia coli in two Italian rivers

Background: Wastewater treatment plants (WWTPs) are microbial factories aimed to reduce the amount of nutrients and pathogenic microorganisms in the treated wastewater before its discharge into the environment. We studied the impact of urban WWTP effluents on the abundance of antibiotic resistance genes (ARGs) and antibiotic-resistant Escherichia coli (AR-E. coli) in the last stretch of two rivers (Arrone and Tiber) in Central Italy that differ in size and flow volume. Methods: Water samples were collected in three seasons upstream and downstream of the WWTP, at the WWTP outlet, and at sea sites near the river mouth, and analyzed for the abundance of ARGs by qPCR and AR-E. coli using cultivation followed by disk diffusion assays. Results: For all studied genes (16S rRNA, intI1, sul1, ermB, blaTEM, tetW and qnrS), absolute concentrations were significantly higher in the Tiber than in the Arrone at all sampling sites, despite their collection date, but the prevalence of target ARGs within bacterial communities in both rivers was similar. The absolute concentrations of most ARGs were also generally higher in the WWTP effluent with median levels between log 4 and log 6 copies per ml but did not show differences along the studied stretches of rivers. Statistically significant site effect was found for E. coli phenotypic resistance to tetracycline and ciprofloxacin in the Arrone but not in the Tiber. Conclusions: In both rivers, diffuse or point pollution sources other than the studied WWTP effluents may account for the observed resistance pattern, although the Arrone appears as more sensitive to the wastewater impact considering its lower flow volume

Surveilling the SARS -CoV-2 in sewage: the catalan case

Introduction. Shortly after the outbreak of the COVID-19 pandemic, scientists renewed their interest on the application of wastewater-based epidemiology (WBE) to track the communal circulation of SARS-CoV-2 through the quantification of its genetic traces in sewage. At national scale, such strategy was firstly implemented in The Netherlands in February 2020 and, following the Dutch example, similar sewage surveillance programs were later put into action by other countries at different scales and coverages. At the time of writing, 58 countries are currently monitoring the circulation of SARS-CoV-2 in wastewater as reported at the COVID-Poops19 website. The purpose of this work is to describe the roadmap for the implementation of a wastewater surveillance network at a national scale and to discuss the main challenges faced during its functioning. Material and methods. It should be noted that all data generated are free for scientific use, and it can be downloaded from a public repository at the Zenodo website. The database is weekly updated and contains all molecular data obtained from the samples analyzed. Results. In Catalonia, a Spanish region of 7,5 million inhabitants, the Public Health Agency of the Catalan government and the Catalan Water Agency promoted and funded the implementation of the Catalan Surveillance Network of SARSCoV-2 in Sewage (SARSAIGUA) to provide information on the circulation of SARSCoV-2 at community level that complement epidemiological & clinical data. SARSAIGUA started in 2020 by monitoring 56 WWTPs that assist 193 municipalities, representing 80% of the Catalan population. Within less than 72 hours, weekly samples are collected, analyzed, and results reported to Health authorities and finally published in an on-line dashboard. After 19 months of monitoring, the normalized daily loads of SARS-CoV-2 genes in the 56 WWTPs monitored, fairly matched the sum of COVID-19 cases along the successive pandemic waves. Moreover, a good fit was obtained between the aggregated viral load (gen copies/day/100.000 inhabitants) and the epidemiological evolution of diagnosed cases in the municipalities, served by the monitored WWTPs (rxy=0.59). In 2021, SARSAIGUA started the monitoring of SARS-CoV-2 variants by sequencing sewage samples every two weeks using Oxford nanopore technology and ARCTIC primers targeting the S gene. The deployment of this sequencing approach has allowed to track the introduction and spread of the Omicron variant and the concomitant wane of the Delta variant across the territory. Conclusions. Overall, and despite the difficulties and limitations associated to the inherent complexity of wastewater, the usefulness of WBE to rapidly detect viral transmission at community level is very helpful to Health authorities to better manage the pandemic situation. This is particularly relevant under the current scenario, where new emerging SARS-CoV-2 variants with higher fitness and transmission potential outcompete old ones in a weekly time scale. Acknowledgment. The research was realised in the JPIAMR projects: (PhageLand) – 22.80013.8007.1

Persistence of low pathogenic avian influenza virus in artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate

Altres ajuts: acords transformatius de la UABInstituto Nacional de Investigación y Tecnologı́a Agraria y Alimentaria PID2020-114060RR-C33-INFLUOMAAvian influenza viruses (AIVs) can affect wildlife, poultry, and humans, so a One Health perspective is needed to optimize mitigation strategies. Migratory waterfowl globally spread AIVs over long distances. Therefore, the study of AIV persistence in waterfowl staging and breeding areas is key to understanding their transmission dynamics and optimizing management strategies. Here, we used artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate (day/night cycles of photosynthetic active radiation and temperature, low water velocity, and similar microbiome to lowland rivers and stagnant water bodies) and then manipulated temperature and sediment presence (i.e., 10-13 °C vs. 16-18 °C, and presence vs. absence of sediments). An H1N1 low pathogenic AIV (LPAIV) strain was spiked in the streams, and water and sediment samples were collected at different time points until 14 days post-spike to quantify viral RNA and detect infectious particles. Viral RNA was detected until the end of the experiment in both water and sediment samples. In water samples, we observed a significant combined effect of temperature and sediments in viral decay, with higher viral genome loads in colder streams without sediments. In sediment samples, we didn't observe any significant effect of temperature. In contrast to prior laboratory-controlled studies that detect longer persistence times, infectious H1N1 LPAIV was isolated in water samples till 2 days post-spike, and none beyond. Infectious H1N1 LPAIV wasn't isolated from any sediment sample. Our results suggest that slow flowing freshwater surface waters may provide conditions facilitating bird-to-bird transmission for a short period when water temperature are between 10 and 18 °C, though persistence for extended periods (e.g., weeks or months) may be less likely. We hypothesize that experiments simulating real environments, like the one described here, provide a more realistic approach for assessing environmental persistence of AIVs

Does ALS-FUS without FUS mutation represent ALS-FET? Report of three cases

Altres ajuts: This study was partially funded by Fundacio Marató de TV3 (grant no. 20143810 to RSV, no. 20141610 to EG and no. 201437.10 to RRG) and Fondo Europeo de Desarrollo Regional (FEDER) (PI16/01673 to JG and PI15/01618 to RRG). We are indebted to the Neurological Tissue Bank of the Biobanc-Hospital Clinic-IDIBAPS, Barcelona, Spain, for data and sample procurement. We thank Sara Charif, Veronica Santiago, Carmen Schweiger, Leire Etxarri and Abel Muñoz for technical assistance

Dynamics of SARS-CoV-2 Alpha (B.1.1.7) variant spread: The wastewater surveillance approach

Wastewater based epidemiology (WBE) offers an overview of the SARS-CoV-2 variants circulating among the population thereby serving as a proper surveillance method. The variant of concern (VOC) Alpha was first identified in September 2020 in the United Kingdom, and rapidly became dominant across Europe. Our objective was to elucidate the Alpha VOC outcompetition rate and identify mutations in the spike glycoprotein (S) gene, indicative of the circulation of the Alpha VOC and/or other variants in the population through wastewater analysis. In the period covered by this study (November 2020-April 2021), forteen wastewater treatment plants (WWTPs) were weekly sampled. The total number of SARS-CoV-2 genome copies per L (GC/L) was determined with a Real-Time qPCR, targeting the N gene. Surveillance of the Alpha VOC circulation was ascertained using a duplex RT-qPCR, targeting and discriminating the S gene. Our results showed that in a period of 6 weeks the Alpha VOC was present in all the studied WWTPs, and became dominant in 11 weeks on average. The outcompetition rates of the Alpha VOC were estimated, and their relationship with different parameters statistically analyzed. The rapid spread of the Alpha VOC was influenced by its initial input and by the previous circulation of SARS-COV-2 in the population. This latter point could be explained by its higher transmissibility, particularly advantadgeous when a certain degree of herd immunity exists. Moreover, the presence of signature mutations of SARS-COV-2 variants were established by deep-sequencing of the complete S gene. The circulation of the Alpha VOC in the area under study was confirmed, and additionally two combinations of mutations in the S glycoprotein (T73A and D253N, and S477N and A522S) that could affect antibody binding were identified