Geographical distribution of mobile genetic elements in microbial communities along the Yucatan coast.

Horizontal gene transfer (HGT) is a well-documented strategy used by bacteria to enhance their adaptability to challenging environmental conditions. Through HGT, a group of conserved genetic elements known as mobile genetic elements (MGEs) is disseminated within bacterial communities. MGEs offer numerous advantages to the host, increasing its fitness by acquiring new functions that help bacteria contend with adverse conditions, including exposure to heavy metal and antibiotics. This study explores MGEs within microbial communities along the Yucatan coast using a metatranscriptomics approach. Prior to this research, nothing was known about the coastal Yucatan's microbial environmental mobilome and HGT processes between these bacterial communities. This study reveals a positive correlation between MGEs and antibiotic resistance genes (ARGs) along the Yucatan coast, with higher MGEs abundance in more contaminated sites. The Proteobacteria and Firmicutes groups exhibited the highest number of MGEs. It's important to highlight that the most abundant classes of MGEs might not be the ones most strongly linked to ARGs, as observed for the recombination/repair class. This work presents the first geographical distribution of the environmental mobilome in Yucatan Peninsula mangroves.

Insights into coastal microbial antibiotic resistome through a meta-transcriptomic approach in Yucatan.

Antibiotic resistance (AR) is one of the greatest human and clinical challenges associated with different pathogenic organisms. However, in recent years it has also become an environmental problem due to the widespread use of antibiotics in humans and livestock activities. The ability to resist antibiotics comes from antibiotic resistance genes (ARGs) and our understanding of their presence in coastal environments is still limited. Therefore, the objective of the present study was to explore the presence and possible differences in the microbial resistome of four sites from the Yucatan coast through the evaluation of the composition and abundance of ARGs using a high-throughput analysis of metatranscriptomic sequences. In total, 3,498 ARGs were uncovered, which participate in the resistance to tetracycline, macrolide, rifamycin, fluoroquinolone, phenicol, aminoglycoside, cephalosporin, and other antibiotics. The molecular mechanisms of these ARGs were mainly efflux pump, antibiotic target alteration and antibiotic target replacement. In the same way, ARGs were detected in the samples but showing dissimilar enrichment levels. With respect to the sampling sites, the ARGs were present in all the samples collected, either from preserved or contaminated areas. Importantly, sediments of the preserved area of Dzilam presented the second highest level of ARGs detected, probably as a consequence of the antibiotics dragged to the coast by submarine groundwater discharge. In general, the resistance to a single antibiotic was greater than multiresistance, both at the level of gene and organisms; and multiresistance in organisms is acquired mainly by recruiting different monoresistance genes. To our knowledge, this is the first study that describes and compares the resistome of different samples of the Yucatan coast. This study contributes to generating information about the current state of antibiotic resistance on the Yucatan coasts for a better understanding of ARGs dissemination and could facilitate the management of ARGs pollution in the environment.

Identification of Novel Conotoxin Precursors from the Cone Snail Conus spurius by High-Throughput RNA Sequencing.

Marine gastropods of the genus Conus, comprising more than 800 species, have the characteristic of injecting worms and other prey with venom. These conopeptide toxins, highly diverse in structure and action, are highly potent and specific for their molecular targets (ion channels, receptors, and transporters of the prey's nervous system), and thus are important research tools and source for drug discovery. Next-generation sequencing technologies are speeding up the discovery of novel conopeptides in many of these species, but only limited information is available for Conus spurius, which inhabits sandy mud. To search for new precursor conopeptides, we analyzed the transcriptome of the venous ducts of C. spurius and identified 55 putative conotoxins. Seven were selected for further study and confirmed by Sanger sequencing to belong to the M-superfamily (Sr3.M01 and Sr3.M02), A-superfamily (Sr1.A01 and Sr1.A02), O-superfamily (Sr15.O01), and Con-ikot-ikot (Sr21.CII01 and Sr22.CII02). Six of these have never been reported. To our knowledge, this report is the first to use high-throughput RNA sequencing for the study of the diversity of C. spurius conotoxins.

Comparative genomics of DNA-binding transcription factors in archaeal and bacterial organisms.

Archaea represent a diverse phylogenetic group that includes free-living, extremophile, mesophile, symbiont, and opportunistic organisms. These prokaryotic organisms share a high significant similarity with the basal transcriptional machinery of Eukarya, and they share regulatory mechanisms with Bacteria, such as operonic organization and DNA-binding transcription factors (TFs). In this work, we identified the repertoire of TFs in 415 archaeal genomes and compared them with their counterparts in bacterial genomes. The comparisons of TFs, at a global level and per family, allowed us to identify similarities and differences between the repertoires of regulatory proteins of bacteria and archaea. For example, 11 of 62 families are more highly abundant in archaea than bacteria, and 13 families are abundant in bacteria but not in archaea and 38 families have similar abundances in the two groups. In addition, we found that archaeal TFs have a lower isoelectric point than bacterial proteins, i.e., they contain more acidic amino acids, and are smaller than bacterial TFs. Our findings suggest a divergence occurred for the regulatory proteins, even though they are common to archaea and bacteria. We consider that this analysis contributes to the comprehension of the structure and functionality of regulatory proteins of archaeal organisms.

Comparing Sediment Microbiomes in Contaminated and Pristine Wetlands along the Coast of Yucatan.

Microbial communities are important players in coastal sediments for the functioning of the ecosystem and the regulation of biogeochemical cycles. They also have great potential as indicators of environmental perturbations. To assess how microbial communities can change their composition and abundance along coastal areas, we analyzed the composition of the microbiome of four locations of the Yucatan Peninsula using 16S rRNA gene amplicon sequencing. To this end, sediment from two conserved (El Palmar and Bocas de Dzilam) and two contaminated locations (Sisal and Progreso) from the coast northwest of the Yucatan Peninsula in three different years, 2017, 2018 and 2019, were sampled and sequenced. Microbial communities were found to be significantly different between the locations. The most noticeable difference was the greater relative abundance of Planctomycetes present at the conserved locations, versus FBP group found with greater abundance in contaminated locations. In addition to the difference in taxonomic groups composition, there is a variation in evenness, which results in the samples of Bocas de Dzilam and Progreso being grouped separately from those obtained in El Palmar and Sisal. We also carry out the functional prediction of the metabolic capacities of the microbial communities analyzed, identifying differences in their functional profiles. Our results indicate that landscape of the coastal microbiome of Yucatan sediment shows changes along the coastline, reflecting the constant dynamics of coastal environments and their impact on microbial diversity.

Mining the Yucatan Coastal Microbiome for the Identification of Non-Ribosomal Peptides Synthetase (NRPS) Genes.

Prokaryotes represent a source of both biotechnological and pharmaceutical molecules of importance, such as nonribosomal peptides (NRPs). NRPs are secondary metabolites which their synthesis is independent of ribosomes. Traditionally, obtaining NRPs had focused on organisms from terrestrial environments, but in recent years marine and coastal environments have emerged as an important source for the search and obtaining of nonribosomal compounds. In this study, we carried out a metataxonomic analysis of sediment of the coast of Yucatan in order to evaluate the potential of the microbial communities to contain bacteria involved in the synthesis of NRPs in two sites: one contaminated and the other conserved. As well as a metatranscriptomic analysis to discover nonribosomal peptide synthetases (NRPSs) genes. We found that the phyla with the highest representation of NRPs producing organisms were the Proteobacteria and Firmicutes present in the sediments of the conserved site. Similarly, the metatranscriptomic analysis showed that 52% of the sequences identified as catalytic domains of NRPSs were found in the conserved site sample, mostly (82%) belonging to Proteobacteria and Firmicutes; while the representation of Actinobacteria traditionally described as the major producers of secondary metabolites was low. It is important to highlight the prediction of metabolic pathways for siderophores production, as well as the identification of NRPS's condensation domain in organisms of the Archaea domain. Because this opens the possibility to the search for new nonribosomal structures in these organisms. This is the first mining study using high throughput sequencing technologies conducted in the sediments of the Yucatan coast to search for bacteria producing NRPs, and genes that encode NRPSs enzymes.

Genome-Wide Association Study Reveals Candidate Genes for Litter Size Traits in Pelibuey Sheep.

The Pelibuey sheep has adaptability to climatic variations, resistance to parasites, and good maternal ability, whereas some ewes present multiple births, which increases the litter size in farm sheep. The litter size in some wool sheep breeds is associated with the presence of mutations, mainly in the family of the transforming growth factor ß (TGF-ß) genes. To explore genetic mechanisms underlying the variation in litter size, we conducted a genome-wide association study in two groups of Pelibuey sheep (multiparous sheep with two lambs per birth vs. uniparous sheep with a single lamb at birth) using the OvineSNP50 BeadChip. We identified a total of 57 putative SNPs markers (p < 3.0 × 10-3, Bonferroni correction). The candidate genes that may be associated with litter size in Pelibuey sheep are CLSTN2, MTMR2, DLG1, CGA, ABCG5, TRPM6, and HTR1E. Genomic regions were also identified that contain three quantitative trait loci (QTLs) for aseasonal reproduction (ASREP), milk yield (MY), and body weight (BW). These results allowed us to identify SNPs associated with genes that could be involved in the reproductive process related to prolificacy.

Evaluation of the Abundance of DNA-Binding Transcription Factors in Prokaryotes.

The ability of bacteria and archaea to modulate metabolic process, defensive response, and pathogenic capabilities depend on their repertoire of genes and capacity to regulate the expression of them. Transcription factors (TFs) have fundamental roles in controlling these processes. TFs are proteins dedicated to favor and/or impede the activity of the RNA polymerase. In prokaryotes these proteins have been grouped into families that can be found in most of the different taxonomic divisions. In this work, the association between the expansion patterns of 111 protein regulatory families was systematically evaluated in 1351 non-redundant prokaryotic genomes. This analysis provides insights into the functional and evolutionary constraints imposed on different classes of regulatory factors in bacterial and archaeal organisms. Based on their distribution, we found a relationship between the contents of some TF families and genome size. For example, nine TF families that represent 43.7% of the complete collection of TFs are closely associated with genome size; i.e., in large genomes, members of these families are also abundant, but when a genome is small, such TF family sizes are decreased. In contrast, almost 102 families (56.3% of the collection) do not exhibit or show only a low correlation with the genome size, suggesting that a large proportion of duplication or gene loss events occur independently of the genome size and that various yet-unexplored questions about the evolution of these TF families remain. In addition, we identified a group of families that have a similar distribution pattern across Bacteria and Archaea, suggesting common functional and probable coevolution processes, and a group of families universally distributed among all the genomes. Finally, a specific association between the TF families and their additional domains was identified, suggesting that the families sense specific signals or make specific protein-protein contacts to achieve the regulatory roles.

RNA-seq Transcriptome Analysis in Ovarian Tissue of Pelibuey Breed to Explore the Regulation of Prolificacy.

The Pelibuey sheep (Ovis aries) is an indigenous breed distributed in the tropical regions of Mexico. The prolificacy of this sheep is on average from 1 to 1.5 lambs, being an important breeding characteristic that owners seek to increase with the purpose of economic improvements. New-generation RNA sequencing technology has been used to identify the genes that are expressed in the ovarian tissue of sheep that have two or more lambs per parturition, as well as to elucidate the metabolic pathways that are affected by the expression of these genes, with the purpose of better understanding the prolificacy in the sheep. In the present study, the transcriptional expression of multiparous and uniparous sheep was compared using RNA sequencing. Multiparous (M group) and uniparous (U group) sheep that had a genealogical record for three generations (M, n = 5 and U, n = 5) were selected. RNA was extracted from ovarian tissue and subsequently used to prepare the libraries that were sequenced using the Illumina NextSeq500 platform. A total of 31,575 genes were detected from the transcriptomic analysis of which 4908 were significantly expressed (p-value ≤ 0.001) in the ovary of sheep. Subsequently, a second filter was carried out to evaluate the false discovery rate (FDR) and select those genes with p-values ≤ 0.05 and values of expression ≥ 1 (log2), obtaining 354 differential expressed genes (DEG): 120 genes up-regulated and 234 genes down-regulated in the group M with respect to the group U. Through Gene Ontology (GO) and metabolic analysis, we obtained information on the function of differentially expressed genes, and its importance in the reproduction of multiparous sheep. This result suggest that genes identified in the present study participate in the development of the final stages of follicles.

Dissecting the Repertoire of DNA-Binding Transcription Factors of the Archaeon Pyrococcus furiosus DSM 3638.

In recent years, there has been a large increase in the amount of experimental evidence for diverse archaeal organisms, and these findings allow for a comprehensive analysis of archaeal genetic organization. However, studies about regulatory mechanisms in this cellular domain are still limited. In this context, we identified a repertoire of 86 DNA-binding transcription factors (TFs) in the archaeon Pyrococcus furiosus DSM 3638, that are clustered into 32 evolutionary families. In structural terms, 45% of these proteins are composed of one structural domain, 41% have two domains, and 14% have three structural domains. The most abundant DNA-binding domain corresponds to the winged helix-turn-helix domain; with few alternative DNA-binding domains. We also identified seven regulons, which represent 13.5% (279 genes) of the total genes in this archaeon. These analyses increase our knowledge about gene regulation in P. furiosus DSM 3638 and provide additional clues for comprehensive modeling of transcriptional regulatory networks in the Archaea cellular domain.

Functional Prediction of Hypothetical Transcription Factors of Escherichia coli K-12 Based on Expression Data.

The repertoire of 304 DNA-binding transcription factors (TFs) in Escherichia coli K-12 has been described recently, with 196 TFs experimentally characterized and 108 proteins predicted by sequence comparisons. Based on 303 expression profile patterns retrieved from the Colombos database 12 clusters were identified, including hypothetical and experimentally characterized TFs, using a spectral clustering algorithm based on a 3NN graph built using 14 principal components that represent 65% of the variance of the expression data. In a posterior step, clusters were characterized in terms of their associated overrepresented functions, based on KEGG, Supfam annotations and Pfam assignments among other functional categories using an enrichment test, reinforcing the notion that the identified clusters are functionally similar among them. Based on these data, the we identified 12 clusters in which hypothetical and known TFs share similar regulatory and physiological functions, such as module associations of toxin-antitoxin (TA) systems with DNA repair mechanisms, amino acid biosynthesis, and carbon metabolism/transport, among others. This analysis has increased our knowledge about gene regulation in E. coli K-12 and can be further expanded to other organisms.

Abundance, diversity and domain architecture variability in prokaryotic DNA-binding transcription factors.

Gene regulation at the transcriptional level is a central process in all organisms, and DNA-binding transcription factors, known as TFs, play a fundamental role. This class of proteins usually binds at specific DNA sequences, activating or repressing gene expression. In general, TFs are composed of two domains: the DNA-binding domain (DBD) and an extra domain, which in this work we have named "companion domain" (CD). This latter could be involved in one or more functions such as ligand binding, protein-protein interactions or even with enzymatic activity. In contrast to DBDs, which have been widely characterized both experimentally and bioinformatically, information on the abundance, distribution, variability and possible role of the CDs is scarce. Here, we investigated these issues associated with the domain architectures of TFs in prokaryotic genomes. To this end, 19 families of TFs in 761 non-redundant bacterial and archaeal genomes were evaluated. In this regard we found four main groups based on the abundance and distribution in the analyzed genomes: i) LysR and TetR/AcrR; ii) AraC/XylS, SinR, and others; iii) Lrp, Fis, ArsR, and others; and iv) a group that included only two families, ArgR and BirA. Based on a classification of the organisms according to the life-styles, a major abundance of regulatory families in free-living organisms, in contrast with pathogenic, extremophilic or intracellular organisms, was identified. Finally, the protein architecture diversity associated to the 19 families considering a weight score for domain promiscuity evidenced which regulatory families were characterized by either a large diversity of CDs, here named as "promiscuous" families given the elevated number of variable domains found in those TFs, or a low diversity of CDs. Altogether this information helped us to understand the diversity and distribution of the 19 Prokaryotes TF families. Moreover, initial steps were taken to comprehend the variability of the extra domain in those TFs, which eventually might assist in evolutionary and functional studies.

Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea.

Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell's metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein-protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

Tracing the Repertoire of Promiscuous Enzymes along the Metabolic Pathways in Archaeal Organisms.

The metabolic pathways that carry out the biochemical transformations sustaining life depend on the efficiency of their associated enzymes. In recent years, it has become clear that promiscuous enzymes have played an important role in the function and evolution of metabolism. In this work we analyze the repertoire of promiscuous enzymes in 89 non-redundant genomes of the Archaea cellular domain. Promiscuous enzymes are defined as those proteins with two or more different Enzyme Commission (E.C.) numbers, according the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. From this analysis, it was found that the fraction of promiscuous enzymes is lower in Archaea than in Bacteria. A greater diversity of superfamily domains is associated with promiscuous enzymes compared to specialized enzymes, both in Archaea and Bacteria, and there is an enrichment of substrate promiscuity rather than catalytic promiscuity in the archaeal enzymes. Finally, the presence of promiscuous enzymes in the metabolic pathways was found to be heterogeneously distributed at the domain level and in the phyla that make up the Archaea. These analyses increase our understanding of promiscuous enzymes and provide additional clues to the evolution of metabolism in Archaea.

The lifestyle of prokaryotic organisms influences the repertoire of promiscuous enzymes.

The metabolism of microbial organisms and its diversity are partly the result of an adaptation process to the characteristics of the environments that they inhabit. In this work, we analyze the influence of lifestyle on the content of promiscuous enzymes in 761 nonredundant bacterial and archaeal genomes. Promiscuous enzymes were defined as those proteins whose catalytic activities are defined by two or more different Enzyme Commission (E.C.) numbers. The genomes analyzed were categorized into four lifestyles for their exhaustive comparisons: free-living, extremophiles, pathogens, and intracellular. From these analyses we found that free-living organisms have larger genomes and an enrichment of promiscuous enzymes. In contrast, intracellular organisms showed smaller genomes and the lesser proportion of promiscuous enzymes. On the basis of our data, we show that the proportion of promiscuous enzymes in an organism is mainly influenced by the lifestyle, where fluctuating environments promote its emergence. Finally, we evidenced that duplication processes occur preferentially in metabolism of free-living and extremophiles species.

Increments and duplication events of enzymes and transcription factors influence metabolic and regulatory diversity in prokaryotes.

In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected.

The repertoire of DNA-binding transcription factors in prokaryotes: functional and evolutionary lessons.

The capabilities of organisms to contend with environmental changes depend on their genes and their ability to regulate their expression. DNA-binding transcription factors (TFs) play a central role in this process, because they regulate gene expression positively and/or negatively, depending on the operator context and ligand-binding status. In this review, we summarise recent findings regarding the function and evolution of TFs in prokaryotes. We consider the abundance of TFs in bacteria and archaea, the role of DNA-binding domains and their partner domains, and the effects of duplication events in the evolution of regulatory networks. Finally, a comprehensive picture for how regulatory networks have evolved in prokaryotes is provided.