Revealing the microbiome diversity and biocontrol potential of field Aedes ssp.: Implications for disease vector management.

The mosquito Aedes spp. holds important relevance for human and animal health, as it serves as a vector for transmitting multiple diseases, including dengue and Zika virus. The microbiome's impact on its host's health and fitness is well known. However, most studies on mosquito microbiomes have been conducted in laboratory settings. We explored the mixed microbial communities within Aedes spp., utilizing the 16S rRNA gene for diversity analysis and shotgun metagenomics for functional genomics. Our samples, which included Ae. aegypti and Ae. albopictus, spanned various developmental stages-eggs, larvae, and adults-gathered from five semiurban areas in Mexico. Our findings revealed a substantial diversity of 8,346 operational taxonomic units (OTUs), representing 967 bacterial genera and 126,366 annotated proteins. The host developmental stage was identified as the primary factor associated with variations in the microbiome composition. Subsequently, we searched for genes and species involved in mosquito biocontrol. Wolbachia accounted for 9.6% of the 16S gene sequences. We observed a high diversity (203 OTUs) of Wolbachia strains commonly associated with mosquitoes, such as wAlb, with a noticeable increase in abundance during the adult stages. Notably, we detected the presence of the cifA and cifB genes, which are associated with Wolbachia's cytoplasmic incompatibility, a biocontrol mechanism. Additionally, we identified 221 OTUs related to Bacillus, including strains linked to B. thuringiensis. Furthermore, we discovered multiple genes encoding insecticidal toxins, such as Cry, Mcf, Vip, and Vpp. Overall, our study contributes to the understanding of mosquito microbiome biodiversity and metabolic capabilities, which are essential for developing effective biocontrol strategies against this disease vector.

A study of microbial diversity in a biofertilizer consortium.

Biofertilizers supply living microorganisms to help plants grow and keep their health. This study examines the microbiome composition of a commercial biofertilizer known for its plant growth-promoting activity. Using ITS and 16S rRNA gene sequence analyses, we describe the microbial communities of a biofertilizer, with 163 fungal species and 485 bacterial genera found. The biofertilizer contains a variety of microorganisms previously reported to enhance nutrient uptake, phytohormone production, stress tolerance, and pathogen resistance in plants. Plant roots created a microenvironment that boosted bacterial diversity but filtered fungal communities. Notably, preserving the fungal-inoculated substrate proves critical for keeping fungal diversity in the root fraction. We described that bacteria were more diverse in the rhizosphere than in the substrate. In contrast, root-associated fungi were less diverse than the substrate ones. We propose using plant roots as bioreactors to sustain dynamic environments that promote the proliferation of microorganisms with biofertilizer potential. The study suggests that bacteria grow close to plant roots, while root-associated fungi may be a subset of the substrate fungi. These findings show that the composition of the biofertilizer may be influenced by the selection of microorganisms associated with plant roots, which could have implications for the effectiveness of the biofertilizer in promoting plant growth. In conclusion, our study sheds light on the intricate interplay between plant roots and the biofertilizer's microbial communities. Understanding this relationship can aid in optimizing biofertilizer production and application, contributing to sustainable agricultural practices and improved crop yields.

Hen lysozyme fibrillogenesis, molten globule intermediate and effect of copper salts.

The amyloid fibres have been related to many diseases. The molten globule intermediate has been proposed to form part of the folding pathway of many proteins. In the present study, we investigated the mechanism of amyloid-fibres formation of hen egg-white lysozyme (HEWL) incubated in a potassium phosphate buffer, pH 11.8, 100 mM, at 37 °C for 30 h, and evaluated the influence of Cu(II) present in two salts (CuSO4 and CuCl2) during fibrillogenesis. Co-incubation and post-incubation of lysozyme with copper salts reduced the fluorescence signal of thioflavin T with an increment in the intrinsic fluorescence of the protein. The ANS fluorescence test showed that incubation of HEWL for 6 h generated a molten globule intermediate state that formed amyloid fibres when incubation was carried out for a 30-h timespan. Dynamic light scattering showed a heterogeneous population of states in samples incubated in the absence or the presence of salts during the fibrillation process. The existence of a reducing potential was verified during the formation of HEWL amyloid fibres with the bathocuproine disulphonate test. Transmission electron microscopy confirmed the presence and absence of fibres in solutions incubated with and without Cu(II). This work demonstrated that lysozyme formed amyloid fibres at 37 °C and copper inhibited its formation.Communicated by Ramaswamy H. Sarma.

Where environmental microbiome meets its host: Subway and passenger microbiome relationships.

Subways are urban transport systems with high capacity. Every day around the world, there are more than 150 million subway passengers. Since 2013, thousands of microbiome samples from various subways worldwide have been sequenced. Skin bacteria and environmental organisms dominate the subway microbiomes. The literature has revealed common bacterial groups in subway systems; even so, it is possible to identify cities by their microbiome. Low frequency bacteria are responsible for specific bacterial fingerprints of each subway system. Furthermore, daily subway commuters leave their microbial clouds and interact with other passengers. Microbial exchange is quite fast; the hand microbiome changes within minutes, and after cleaning the handrails, the bacteria are re-established within minutes. To investigate new taxa and metabolic pathways of subway microbial communities, several high-quality metagenomic-assembled genomes (MAG) have been described. Subways are harsh environments unfavorable for microorganism growth. However, recent studies have observed a wide diversity of viable and metabolically active bacteria. Understanding which bacteria are living, dormant, or dead allows us to propose realistic ecological interactions. Questions regarding the relationship between humans and the subway microbiome, particularly the microbiome effects on personal and public health, remain unanswered. This review summarizes our knowledge of subway microbiomes and their relationship with passenger microbiomes.

Metagenomics of mine tailing rhizospheric communities and its selection for plant establishment towards bioremediation.

Mining operations often generate tailing dams that contain toxic residues and are a source of contamination when left unconfined. The establishment of a plant community over the tailings has been proposed as a containment strategy known as phytostabilization. Previously, we described naturally occurring mine tailing colonizing plants such as Acacia farnesiana, Brickellia coulteri, Baccharis sarothroides, and Gnaphalium leucocephalum without finding local adaptation. We explored the rhizosphere microbes as contributors in plant establishment and described both the culturable and in situ diversity of rhizospheric bacteria using the 16S rRNA gene and metagenomic shotgun sequencing. We built a synthetic community (SC) of culturable rhizosphere bacteria from the mine tailings. The SC was then the foundation for a serial passes experiment grown in plant-derived nutrient sources, selecting for heavy metals tolerance, community cooperation, and competition. The outcome of the serial passes was named the 'final synthetic community' (FSC). Overall, diversity decreased from in situ uncultivable microbes from roots (399 bacteria genera) to the cultivated communities (291 genera), the SC (94 genera), and the lowest diversity was in the FSC (43 genera). Metagenomic diversity clustered into 94,245 protein families, where we found plant growth promotion-related genes such as the csgBAC and entCEBAH, coded in a metagenome-assembled genome named Kosakonia sp. Nacozari. Finally, we used the FSC to inoculate mine tailing colonizing plants in a greenhouse experiment. The plants with the FSC inocula observed higher relative plant growth rates in sterile substrates. The FSC presents promising features that might make it useful for phytostabilization tailored strategies.

Testing the Two-Step Model of Plant Root Microbiome Acquisition Under Multiple Plant Species and Soil Sources.

The two-step model for plant root microbiomes considers soil as the primary microbial source. Active selection of the plant's bacterial inhabitants results in a biodiversity decrease toward roots. We collected sixteen samples of in situ ruderal plant roots and their soils and used these soils as the main microbial input for single genotype tomatoes grown in a greenhouse. Our main goal was to test the soil influence in the structuring of rhizosphere microbiomes, minimizing environmental variability, while testing multiple plant species. We massively sequenced the 16S rRNA and shotgun metagenomes of the soils, in situ plants, and tomato roots. We identified a total of 271,940 bacterial operational taxonomic units (OTUs) within the soils, rhizosphere and endospheric microbiomes. We annotated by homology a total of 411,432 (13.07%) of the metagenome predicted proteins. Tomato roots did follow the two-step model with lower α-diversity than soil, while ruderal plants did not. Surprisingly, ruderal plants are probably working as a microenvironmental oasis providing moisture and plant-derived nutrients, supporting larger α-diversity. Ruderal plants and their soils are closer according to their microbiome community composition than tomato and its soil, based on OTUs and protein comparisons. We expected that tomato ß-diversity clustered together with their soil, if it is the main rhizosphere microbiome structuring factor. However, tomato microbiome ß-diversity was associated with plant genotype in most samples (81.2%), also supported by a larger set of enriched proteins in tomato rhizosphere than soil or ruderals. The most abundant bacteria found in soils was the Actinobacteria Solirubrobacter soli, ruderals were dominated by the Proteobacteria Sphingomonas sp. URGHD0057, and tomato mainly by the Bacteroidetes Ohtaekwangia koreensis, Flavobacterium terrae, Niastella vici, and Chryseolinea serpens. We calculated a metagenomic tomato root core of 51 bacterial genera and 2,762 proteins, which could be the basis for microbiome-oriented plant breeding programs. We attributed a larger diversity in ruderal plants roots exudates as an effect of the moisture and nutrient acting as a microbial harbor. The tomato and ruderal metagenomic differences are probably due to plant domestication trade-offs, impacting plant-bacteria interactions.

Passenger-surface microbiome interactions in the subway of Mexico City.

Interaction between hands and the environment permits the interchange of microorganisms. The Mexico City subway is used daily by millions of passengers that get in contact with its surfaces. In this study, we used 16S rRNA gene sequencing to characterize the microbiomes of frequently touched surfaces and compare regular and women-only wagons. We also explored the effect of surface cleaning on microbial resettling. Finally, we studied passenger behavior and characterized microbial changes after traveling. Most passengers (99%), showed some type of surface interaction during a wagon trip, mostly with the hands (92%). We found microbiome differences associated with surfaces, probably reflecting diverse surface materials and usage frequency. The platform floor was the most bacterial diverse surface, while the stair handrail and pole were the least diverse ones. After pole cleaning, the resettling of microbial diversity was fast (5-30 minutes); however, it did not resemble the initial composition. After traveling, passengers significantly increased their hand microbial diversity and converged to a similar microbial composition among passengers. Additionally, passenger hand microbiomes resembled subway surfaces in diversity. However, microbial fingerprints were preserved within passengers after traveling.

Station and train surface microbiomes of Mexico City's metro (subway/underground).

The metro is one of the more representative urban transportation systems of Mexico City, and it transports approximately 4.5 million commuters every day. Large crowds promote the exchange of microbes between humans. In this study, we determined the bacterial diversity profile of the Mexico City metro by massive sequencing of the 16S rRNA gene. We identified a total of 50,174 operational taxonomic units (OTUs) and 1058 genera. The metro microbiome was dominated by the phylum Actinobacteria and by the genera Cutibacterium (15%) (C. acnes 13%), Corynebacterium (13%), Streptococcus (9%), and Staphylococcus (5%) (S. epidermidis; 4%), reflecting the microbe composition of healthy human skin. The metro likely microbial sources were skin, dust, saliva, and vaginal, with no fecal contribution detected. A total of 420 bacterial genera were universal to the twelve metro lines tested, and those genera contributed to 99.10% of the abundance. The annual 1.6 billion ridership makes this public transport a main hub for microbe-host-environment interactions. Finally, this study shows that the microbial composition of the Mexico City metro comes from a mixture of environmental and human sources and that commuters are exposed to healthy composition of the human microbiota.

Marchantia liverworts as a proxy to plants' basal microbiomes.

Microbiomes influence plant establishment, development, nutrient acquisition, pathogen defense, and health. Plant microbiomes are shaped by interactions between the microbes and a selection process of host plants that distinguishes between pathogens, commensals, symbionts and transient bacteria. In this work, we explore the microbiomes through massive sequencing of the 16S rRNA genes of microbiomes two Marchantia species of liverworts. We compared microbiomes from M. polymorpha and M. paleacea plants collected in the wild relative to their soils substrates and from plants grown in vitro that were established from gemmae obtained from the same populations of wild plants. Our experimental setup allowed identification of microbes found in both native and in vitro Marchantia species. The main OTUs (97% identity) in Marchantia microbiomes were assigned to the following genera: Methylobacterium, Rhizobium, Paenibacillus, Lysobacter, Pirellula, Steroidobacter, and Bryobacter. The assigned genera correspond to bacteria capable of plant-growth promotion, complex exudate degradation, nitrogen fixation, methylotrophs, and disease-suppressive bacteria, all hosted in the relatively simple anatomy of the plant. Based on their long evolutionary history Marchantia is a promising model to study not only long-term relationships between plants and their microbes but also the transgenerational contribution of microbiomes to plant development and their response to environmental changes.

Exploring the cockatiel (Nymphicus hollandicus) fecal microbiome, bacterial inhabitants of a worldwide pet.

BACKGROUND: Cockatiels (Nymphicus hollandicus) were originally endemic to Australia; now they are popular pets with a global distribution. It is now possible to conduct detailed molecular studies on cultivable and uncultivable bacteria that are part of the intestinal microbiome of healthy animals. These studies show that bacteria are an essential part of the metabolic capacity of animals. There are few studies on bird microbiomes and, to the best of our knowledge, this is the first report on the cockatiel microbiome. METHODS: In this paper, we analyzed the gut microbiome from fecal samples of three healthy adult cockatiels by massive sequencing of the 16S rRNA gene. Additionally, we compared the cockatiel fecal microbiomes with those of other bird species, including poultry and wild birds. RESULTS: The vast majority of the bacteria found in cockatiels were Firmicutes, while Proteobacteria and Bacteroidetes were poorly represented. A total of 19,280 different OTUs were detected, of which 8,072 belonged to the Erysipelotrichaceae family. DISCUSSION: It is relevant to study cockatiel the microbiomes of cockatiels owing to their wide geographic distribution and close human contact. This study serves as a reference for cockatiel bacterial diversity. Despite the large OTU numbers, the diversity is not even and is dominated by Firmicutes of the Erysipelotrichaceae family. Cockatiels and other wild birds are almost depleted of Bacteroidetes, which happen to be abundant in poultry-related birds, and this is probably associated with the intensive human manipulation of poultry bird diets. Some probable pathogenic bacteria, such as Clostridium and Serratia, appeared to be frequent inhabitants of the fecal microbiome of cockatiels, whereas other potential pathogens were not detected.

Comparative metagenomics of two microbial mats at Cuatro Ciénegas Basin I: ancient lessons on how to cope with an environment under severe nutrient stress.

The Cuatro Ciénegas Basin (CCB) is an oasis in the desert of Mexico characterized by low phosphorus availability and by its great diversity of microbial mats. We compared the metagenomes of two aquatic microbial mats from the CCB with different nutrient limitations. We observed that the red mat was P-limited and dominated by Pseudomonas, while the green mat was N-limited and had higher species richness, with Proteobacteria and Cyanobacteria as the most abundant phyla. From their gene content, we deduced that both mats were very metabolically diverse despite their use of different strategies to cope with their respective environments. The red mat was found to be mostly heterotrophic, while the green mat was more autotrophic. The red mat had a higher number of transporters in general, including transporters of cellobiose and osmoprotectants. We suggest that generalists with plastic genomes dominate the red mat, while specialists with minimal genomes dominate the green mat. Nutrient limitation was a common scenario on the early planet; despite this, biogeochemical cycles were performed, and as a result the planet changed. The metagenomes of microbial mats from the CCB show the different strategies a community can use to cope with oligotrophy and persist.

Comparative metagenomics of two microbial mats at Cuatro Ciénegas Basin II: community structure and composition in oligotrophic environments.

Microbial mats are self-sustained, functionally complex ecosystems that make good models for the understanding of past and present microbial ecosystems as well as putative extraterrestrial ecosystems. Ecological theory suggests that the composition of these communities might be affected by nutrient availability and disturbance frequency. We characterized two microbial mats from two contrasting environments in the oligotrophic Cuatro Ciénegas Basin: a permanent green pool and a red desiccation pond. We analyzed their taxonomic structure and composition by means of 16S rRNA clone libraries and metagenomics and inferred their metabolic role by the analysis of functional traits in the most abundant organisms. Both mats showed a high diversity with metabolically diverse members and strongly differed in structure and composition. The green mat had a higher species richness and evenness than the red mat, which was dominated by a lineage of Pseudomonas. Autotrophs were abundant in the green mat, and heterotrophs were abundant in the red mat. When comparing with other mats and stromatolites, we found that taxonomic composition was not shared at species level but at order level, which suggests environmental filtering for phylogenetically conserved functional traits with random selection of particular organisms. The highest diversity and composition similarity was observed among systems from stable environments, which suggests that disturbance regimes might affect diversity more strongly than nutrient availability, since oligotrophy does not appear to prevent the establishment of complex and diverse microbial mat communities. These results are discussed in light of the search for extraterrestrial life.

Additive effect of single amino acid replacements on the kinetic stability of ß-glucosidase B.

Previously, we applied in vitro evolution to generate the thermoresistant triple mutant H62R/N223Y/M319I of ß-glucosidase B (BglB) from Paenibacillus polymyxa. In order to dissect the energetic contributions to protein stabilization achieved by these mutations, we measured the kinetic constants of the heat denaturation of wild type BglB, the triple mutant and the three single mutants (H62R, N223Y, M319I) by circular dichroism at various temperatures. Our results show that all four mutants delayed the denaturation process. Based on the Transition State theory, the increase of the activation barrier for the thermal denaturation of the triple mutant (ΔΔG ( N→TS )) is equivalent to that produced by the sum of the contributions from the three single mutants, whose C ( ß ) s are located at least 18 Å apart. This analysis provides a formal demonstration of the generally accepted idea that protein thermal stability can be increased through sequential addition of individual mutations. Each of the mutations described here contribute in part to the overall effect, which in this case affects the unfolding barrier.

Degradation of a benzene-toluene mixture by hydrocarbon-adapted bacterial communities.

We examined the rate of degradation of a benzene-toluene mixture in aerobic microcosms prepared with samples of an aquifer that lies below a petrochemical plant (SIReN, UK). Five samples exposed to different concentrations of benzene (from 0.6 to 317 mg l(-1)) were used. Fast degradation (approx. 1-6 mg l(-1) day(-1)) of both contaminants was observed in all groundwater samples and complete degradation was recorded by the seventh day except for one sample. We also identified the microbial community in each of the samples by culture-independent techniques. Two of the less impacted samples harbour the aerobic benzene degrader Pseudomonas fluorescens, while Acidovorax and Arthrobacter spp. were found in the most polluted sample and are consistent with the population observed in situ. Hydrogenophaga was found in the deepest sample while Rhodoferax spp. were recovered in an alkaline sample (pH 8.4) and may also be implicated in benzene degradation. Time series analysis shows that each of the samples has a different community but they remain stable over the degradation period. This study provides new information on a well not previously studied (no. 309s) and confirms that adapted communities have the ability to degrade hydrocarbon mixtures and could be used in further bioaugmentation approaches in contaminated sites.

Thermal denaturation of ß-glucosidase B from Paenibacillus polymyxa proceeds through a Lumry-Eyring mechanism.

ß-glucosidase B (BglB), 1,4-ß-D: -glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T ( m ), the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min(-1)) or temperatures (T ≥ T ( m )), the measurable activation energy involves only the elementary step of denaturation.

Hydrophobic repacking of the dimer interface of triosephosphate isomerase by in silico design and directed evolution.

Triosephosphate isomerase from Saccharomyces cerevisiae (wt-TIM) is an obligated homodimer. The interface of wt-TIM is formed by 34 residues. In the native dimer, each monomer buries nearly 2600 A(2) of accessible surface area (ASA), and 58.4% of the interface ASA is hydrophobic. We determined the thermodynamic and functional consequences of increasing the hydrophobic character of the wt-TIM interface. Mutations were restricted to a cluster of five nonconserved residues located far from the active site. Two different approaches, in silico design and directed evolution, were employed. In both methodologies, the obtained proteins were soluble, dimeric, and compact. In silico-designed proteins are very stable dimers that bind substrate with a wild-type-like K(m); albeit, they exhibited a very low k cat. Proteins obtained from directed evolution experiments show wild-type-like catalytic activity, while their stability is decreased. Hydrophobic replacements at the interface produced a remarkable shift in the dissociation step. For wt-TIM and for TIMs obtained by directed evolution, dissociation was observed in the first transition, with C(1/2) values ranging from 0.58 to 0.024 M GdnHCl, whereas for TIMs generated by in silico design, dissociation occurred in the last transition, with C(1/2) values ranging form 3.01 to 3.65 M GdnHCl. For the latter mutants, the stabilization of the interface changed the equilibrium transitions to a novel four-state process with two dimeric intermediates. The change in the intermediate nature suggests that the relative stabilities of different folding units are similar so that subtle alterations in their stability produce a total transformation of the folding pathway.

Evolutionary engineering of a beta-Lactamase activity on a D-Ala D-Ala transpeptidase fold.

The beta-Lactamase hydrolytic activity has arisen several times from DD-transpeptidases. We have been able to replicate the evolutionary process of beta-Lactamase activity emergence on a PBP2X DD-transpeptidase. Some of the most interesting changes, like modifying the catalytic properties of an enzyme, may require several mutations in concert; therefore it is essential to explore efficiently sequence space by generating the right diversity. We designed a biased combinatorial library in which biochemical and structural information were incorporated by site directed mutagenesis on relevant residues and then subjected to random mutagenesis to allow for mutations in unforeseen positions. We isolated mutants from this library conferring 10-fold higher cefotaxime resistance levels than the background wild-type through mutations exclusively in the coding sequence. We demonstrate that only three substitutions in the DD-transpeptidase active site, two produced by the directed and one by the random mutagenesis, are sufficient to acquire this activity. The purified product of one mutant (MutE) had a 10(5)-fold increase in cefotaxime deacylation rate allowing it to hydrolyze beta-Lactams yet it has apparently conserved DD-peptidase activity. This work is the first to show a possible evolutionary intermediate between a beta-Lactamase and a DD-transpeptidase necessary for the development of antibiotic resistance.