Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

Stable transgenesis is a transformative tool in model organism biology. While the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2, piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression past metamorphosis. F0 animals were raised to sexual maturity and spawned to determine germline integration, transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrated transgene transmission through the germline, the first example of a germline transgenic sea urchin, and indeed of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility, and efficiency of sea urchin research.

Cotrimoxazole, the combined formulation of sulfamethoxazole and trimethoprim, is one of the treatments of choice for several infectious diseases, particularly urinary tract infections. Both components of cotrimoxazole are synthetic antimicrobial drugs, and their combination was introduced into medical therapeutics about half a century ago. In Gram-negative bacteria, resistance to cotrimoxazole is widespread, being based on the acquisition of genes from the auxiliary genome that confer resistance to each of its antibacterial components. Starting from previous knowledge on the genotype of resistance to sulfamethoxazole in a collection of cotrimoxazole resistant uropathogenic Escherichia coli strains, this work focused on the identification of the genetic bases of the trimethoprim resistance of these same strains. Molecular techniques employed included PCR and Sanger sequencing of specific amplicons, conjugation experiments and NGS sequencing of the transferred plasmids. Mobile genetic elements conferring the trimethoprim resistance phenotype were identified and included integrons, transposons and single gene cassettes. Therefore, strains exhibited several ways to jointly resist both antibiotics, implying different levels of genetic linkage between genes conferring resistance to sulfamethoxazole (sul) and trimethoprim (dfrA). Two structures were particularly interesting because they represented a highly cohesive arrangements ensuring cotrimoxazole resistance. They both carried a single gene cassette, dfrA14 or dfrA1, integrated in two different points of a conserved cluster sul2-strA-strB, carried on transferable plasmids. The results suggest that the pressure exerted by cotrimoxazole on bacteria of our environment is still promoting the evolution toward increasingly compact gene arrangements, carried by mobile genetic elements that move them in the genome and also transfer them horizontally among bacteria.

Clostridium acetobutylicum is a solventogenic, anaerobic, gram-positive bacterium that is commonly considered the model organism for studying acetone-butanol-ethanol fermentation. The need to produce these chemicals sustainably and with a minimal impact on the environment has revived the interest in research on this bacterium. The recent development of efficient genetic tools allows to better understand the physiology of this micro-organism, aiming at improving its fermentation capacities. Knowledge about gene essentiality would guide the future genetic editing strategies and support the understanding of crucial cellular functions in this bacterium. In this work, we applied a transposon insertion site sequencing method to generate large mutant libraries containing millions of independent mutants that allowed us to identify a core group of 418 essential genes needed for in vitro development. Future research on this significant biocatalyst will be guided by the data provided in this work, which will serve as a valuable resource for the community.
OBJECTIVE: Clostridium acetobutylicum is a leading candidate to synthesize valuable compounds like three and four carbons alcohols. Its ability to convert carbohydrates into a mixture of acetone, butanol, and ethanol as well as other chemicals of interest upon genetic engineering makes it an advantageous organism for the valorization of lignocellulose-derived sugar mixtures. Since, genetic optimization depends on the fundamental insights supplied by accurate gene function assignment, gene essentiality analysis is of great interest as it can shed light on the function of many genes whose functions are still to be confirmed. The data obtained in this study will be of great value for the research community aiming to develop C. acetobutylicum as a platform organism for the production of chemicals of interest.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

Cancer is a multifaceted genetic disease characterized by the acquisition of several essential hallmarks. Notably, certain cancers exhibit horizontal transmissibility, observed across mammalian species and diverse bivalves, the latter referred to as hemic neoplasia. Within this complex landscape, epigenetic mechanisms such as histone modifications and cytosine methylation emerge as fundamental contributors to the pathogenesis of these transmissible cancers. Our study delves into the epigenetic landscape of Cerastoderma edule, focusing on whole-genome methylation and hydroxymethylation profiles in heathy specimens and transmissible neoplasias by means of Nanopore long-read sequencing. Our results unveiled a global hypomethylation in the neoplastic specimens compared to their healthy counterparts, emphasizing the role of DNA methylation in these tumorigenic processes. Furthermore, we verified that intragenic CpG methylation positively correlated with gene expression, emphasizing its role in modulating transcription in healthy and neoplastic cockles, as also highlighted by some up-methylated oncogenic genes. Hydroxymethylation levels were significantly more elevated in the neoplastic samples, particularly within satellites and complex repeats, likely related to structural functions. Additionally, our analysis also revealed distinct methylation and activity patterns in retrotransposons, providing additional insights into bivalve neoplastic processes. Altogether, these findings contribute to understanding the epigenetic dynamics of bivalve neoplasias and shed light on the roles of DNA methylation and hydroxymethylation in tumorigenesis. Understanding these epigenetic alterations holds promise for advancing our broader understanding of cancer epigenetics.

We propose that several chromatin-mediated regulatory processes are dominated by source-sink relationships in which factors operate as \'sources\' to produce or provide a resource and compete with each other to occupy separate \'sinks\'. In this model, large portions of genomic DNA operate as \'sinks\', which are filled by \'sources\', such as available histone variants, covalent modifications to histones, the readers of these modifications and non-coding RNAs. Competing occupation for the sinks by different sources leads to distinct states of genomic equilibrium in differentiated cells. During dynamic developmental events, such as sexual reproduction, we propose that dramatic and rapid reconfiguration of source-sink relationships modifies chromatin states. We envision that re-routing of sources could occur by altering the dimensions of the sink, by reconfiguration of existing sink occupation or by varying the size of the source, providing a central mechanism to explain a plethora of epigenetic phenomena, which contribute to phenotypic variegation, zygotic genome activation and nucleolar dominance.

Cryptic conjugative plasmids lack antibiotic-resistance genes (ARGs). These plasmids can capture ARGs from the vast pool of the environmental metagenome, but the mechanism to recruit ARGs remains to be elucidated. To investigate the recruitment of ARGs by a cryptic plasmid, we sequenced and conducted mating experiments with Escherichia coli SW4848 (collected from a lake) that has a cryptic IncX (IncX4) plasmid and an IncF (IncFII/IncFIIB) plasmid with five genes that confer resistance to aminoglycosides (strA and strB), sulfonamides (sul2), tetracycline [tet(A)], and trimethoprim (dfrA5). In a conjugation experiment, a novel hybrid Tn21/Tn1721 transposon of 22,570 bp (designated Tn7714) carrying the five ARG mobilized spontaneously from the IncF plasmid to the cryptic IncX plasmid. The IncF plasmid was found to be conjugative when it was electroporated into E. coli DH10B (without the IncX plasmid). Two parallel conjugations with the IncF and the new IncX (carrying the novel Tn7714 transposon) plasmids in two separate E. coli DH10B as donors and E. coli J53 as the recipient revealed that the conjugation rate of the new IncX plasmid (with the novel Tn7714 transposon and five ARGs) is more than two orders of magnitude larger than the IncF plasmid. For the first time, this study shows experimental evidence that cryptic environmental plasmids can capture and transfer transposons with ARGs to other bacteria, creating novel multidrug-resistant conjugative plasmids with higher dispersion potential.
OBJECTIVE: Cryptic conjugative plasmids are extrachromosomal DNA molecules without antibiotic-resistance genes (ARGs). Environmental bacteria carrying cryptic plasmids with a high conjugation rate threaten public health because they can capture clinically relevant ARGs and rapidly spread them to pathogenic bacteria. However, the mechanism to recruit ARG by cryptic conjugative plasmids in environmental bacteria has not been observed experimentally. Here, we document the first translocation of a transposon with multiple clinically relevant ARGs to a cryptic environmental conjugative plasmid. The new multidrug-resistant conjugative plasmid has a conjugation rate that is two orders of magnitude higher than the original plasmid that carries the ARG (i.e., the new plasmid from the environment can spread ARG more than two orders of magnitude faster). Our work illustrates the importance of studying the mobilization of ARGs in environmental bacteria. It sheds light on how cryptic conjugative plasmids recruit ARGs, a phenomenon at the root of the antibiotic crisis.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens. Here we report sequence data of the STEC strain BfR-EC-18960, which has integrated IS elements in the B-subunit of the Shiga toxin Stx2b gene. The strain was isolated from deer meat at a local butchery in Germany in 2021.

Alzheimer\'s disease affects an average of 5 % of the population with a significant increase in prevalence with age, suggesting that the same mechanisms that underlie aging may influence this pathology. Investigation of these mechanisms is promising for effective methods of treatment and prevention of the disease. Possible participants in these mechanisms are transposons, which serve as drivers of epigenetic regulation, since they form species-specific distributions of non-coding RNA genes in genomes in evolution. Study of miRNA involvement in Alzheimer\'s disease pathogenesis is relevant, since the associations of protein-coding genes (APOE4, ABCA7, BIN1, CLU, CR1, PICALM, TREM2) with the disease revealed as a result of GWAS make it difficult to explain its complex pathogenesis. Specific expression changes of many genes were found in different brain parts of Alzheimer\'s patients, which may be due to global regulatory changes under the influence of transposons. Experimental and clinical studies have shown pathological activation of retroelements in Alzheimer\'s disease. Our analysis of scientific literature in accordance with MDTE DB revealed 28 miRNAs derived from transposons (17 from LINE, 5 from SINE, 4 from HERV, 2 from DNA transposons), the expression of which specifically changes in this disease (decreases in 17 and increases in 11 microRNA). Expression of 13 out of 28 miRNAs (miR-151a, -192, -211, -28, -31, -320c, -335, -340, -378a, -511, -576, -708, -885) also changes with aging and cancer development, which indicates the presence of possible common pathogenetic mechanisms. Most of these miRNAs originated from LINE retroelements, the pathological activation of which is associated with aging, carcinogenesis, and Alzheimer\'s disease, which supports the hypothesis that these three processes are based on the primary dysregulation of transposons that serve as drivers of epigenetic regulation of gene expression in ontogeny.
Болезнь Альцгеймера поражает в среднем 5 % населения со значительным увеличением распространенности с возрастом, что свидетельствует о возможном влиянии на данную патологию тех же механизмов, которые лежат в основе старения человека. Исследование этих механизмов перспективно для разработки эффективных методов лечения и профилактики заболевания. Возможными участниками этих механизмов являются транспозоны, которые служат драйверами эпигенетической регуляции, поскольку формируют в эволюции видоспецифические распределения генов некодирующих РНК в геноме человека. Изучение роли микроРНК в развитии болезни Альцгеймера актуально, поскольку по результатам проведенных GWAS ассоциаций белок-кодирующих генов (APOE4, ABCA7, BIN1, CLU, CR1, PICALM, TREM2) трудно объяснить сложный патогенез заболевания. Кроме того, в различных долях головного мозга при болезни Альцгеймера были обнаружены специфические изменения экспрессии множества генов, что может быть обусловлено глобальными регуляторными изменениями под влиянием транспозонов. Действительно, экспериментальные и клинические исследования показали патологическую активацию ретроэлементов при болезни Альцгеймера. Проведенный нами анализ научной литературы в соответствии с базой данных MDTE DB (microRNAs derived from transposable elements) позволил выявить 28 различных микроРНК, происходящих от мобильных элементов (17 – от LINE, 5 – от SINE, 4 – от HERV, 2 – от ДНК-транспозонов), экспрессия которых специфически изменяется при данном заболевании (снижается у 17 и повышается у 11 микроРНК). Экспрессия 13 из 28 микроРНК (miR-151a, -192, -211, -28, -31, -320c, -335, -340, -378a, -511, -576, -708, -885) меняется также при старении и развитии злокачественных новообразований, что подтверждает возможное наличие общих патогенетических механизмов. Большинство из этих микроРНК произошли от LINE-ретроэлементов, патологическая активация которых ассоциирована со старением, канцерогенезом и болезнью Альцгеймера, что свидетельствует в пользу гипотезы о том, что в основе этих трех процессов лежит первичная дисрегуляция транспозонов, которые служат драйверами эпигенетической регуляции экспрессии генов в онтогенезе.