Rhizospheric bacteria from the Atacama Desert hyper-arid core: cultured community dynamics and plant growth promotion.

The Atacama Desert is the oldest and driest desert on Earth, encompassing great temperature variations, high ultraviolet radiation, drought, and high salinity, making it ideal for studying the limits of life and resistance strategies. It is also known for harboring a great biodiversity of adapted life forms. While desertification is increasing as a result of climate change and human activities, it is necessary to optimize soil and water usage, where stress-resistant crops are possible solutions. As many studies have revealed the great impact of the rhizobiome on plant growth efficiency and resistance to abiotic stress, we set up to explore the rhizospheric soils of Suaeda foliosa and Distichlis spicata desert plants. By culturing these soils and using 16S rRNA amplicon sequencing, we address community taxonomy composition dynamics, stability through time, and the ability to promote lettuce plant growth. The rhizospheric soil communities were dominated by the families Pseudomonadaceae, Bacillaceae, and Planococcaceae for S. foliosa and Porphyromonadaceae and Haloferacaceae for D. spicata. Nonetheless, the cultures were completely dominated by the Enterobacteriaceae family (up to 98%). Effectively, lettuce plants supplemented with the cultures showed greater size and biomass accumulation. We identified 12 candidates that could be responsible for these outcomes, of which 5 (Enterococcus, Pseudomonas, Klebsiella, Paenisporosarcina, and Ammoniphilus) were part of the built co-occurrence network. We aim to contribute to the efforts to characterize the microbial communities as key for the plant's survival in extreme environments and as a possible source of consortia with plant growth promotion traits aimed at agricultural applications.IMPORTANCEThe current scenario of climate change and desertification represents a series of incoming challenges for all living organisms. As the human population grows rapidly, so does the rising demand for food and natural resources; thus, it is necessary to make agriculture more efficient by optimizing soil and water usage, thus ensuring future food supplies. Particularly, the Atacama Desert (northern Chile) is considered the most arid place on Earth as a consequence of geological and climatic characteristics, such as the naturally low precipitation patterns and high temperatures, which makes it an ideal place to carry out research that seeks to aid agriculture in future conditions that are predicted to resemble these scenarios. Our main interest lies in utilizing microorganism consortia from plants thriving under extreme conditions, aiming to promote plant growth, improve crops, and render "unsuitable" soils farmable.

Characterization of biofilm formation by Exiguobacterium strains in response to arsenic exposure.

IMPORTANCE: In this work, we characterized the composition, structure, and functional potential for biofilm formation of Exiguobacterium strains isolated from the Salar de Huasco in Chile in the presence of arsenic, an abundant metalloid in the Salar that exists in different oxidation states. Our results showed that the Exiguobacterium strains tested exhibit a significant capacity to form biofilms when exposed to arsenic, which would contribute to their resistance to the metalloid. The results highlight the importance of biofilm formation and the presence of specific resistance mechanisms in the ability of microorganisms to survive and thrive under adverse conditions.

Exploring the Influence of Small-Scale Geographical and Seasonal Variations Over the Microbial Diversity in a Poly-extreme Athalosaline Wetland.

Microorganisms are the most diverse life form on the planet and are critical for maintaining the geochemical cycles, especially in extreme environments. Bacterial communities are dynamic and respond directly to changes in abiotic conditions; among these communities, poly-extremophiles are particularly sensitive to perturbations due to their high specialization. Salar de Huasco is a high-altitude wetland located on the Chilean Altiplano exhibiting several conditions considered extreme for life, including negative water balance, extreme variations in temperature and pH values, high UV radiation, and the presence of various toxic metal(oids). However, previous reports have revealed a diverse bacterial community that has adapted to these conditions, here, we aimed to determine whether microbial community diversity and composition changed in response to geographical and seasonal variations. We found that there are significant differences in diversity, abundance, and composition in bacterial taxa that could be attributed to local geographical and seasonal variations, which in turn, can be associated with microbial traits. In conclusion, in this poly-extreme environment, small-scale changes can trigger significant changes in the microbial communities that maintain basic biogeochemical cycles. Further in depth analysis of microbial functionality and geo-ecological dynamics are necessary to better understand the relationships between seasonal changes and bacterial communities.

Mobile genetic elements drive the multidrug resistance and spread of Salmonella serotypes along a poultry meat production line.

The presence of mobile genetic elements in Salmonella isolated from a chicken farm constitutes a potential risk for the appearance of emerging bacteria present in the food industry. These elements contribute to increased pathogenicity and antimicrobial resistance through genes that are related to the formation of biofilms and resistance genes contained in plasmids, integrons, and transposons. One hundred and thirty-three Salmonella isolates from different stages of the production line, such as feed manufacturing, hatchery, broiler farm, poultry farm, and slaughterhouse, were identified, serotyped and sequenced. The most predominant serotype was Salmonella Infantis. Phylogenetic analyses demonstrated that the diversity and spread of strains in the pipeline are serotype-independent, and that isolates belonging to the same serotype are very closely related genetically. On the other hand, Salmonella Infantis isolates carried the pESI IncFIB plasmid harboring a wide variety of resistance genes, all linked to mobile genetic elements, and among carriers of these plasmids, the antibiograms showed differences in resistance profiles and this linked to a variety in plasmid structure, similarly observed in the diversity of Salmonella Heidelberg isolates carrying the IncI1-Iα plasmid. Mobile genetic elements encoding resistance and virulence genes also contributed to the differences in gene content. Antibiotic resistance genotypes were matched closely by the resistance phenotypes, with high frequency of tetracycline, aminoglycosides, and cephalosporins resistance. In conclusion, the contamination in the poultry industry is described throughout the entire production line, with mobile genetic elements leading to multi-drug resistant bacteria, thus promoting survival when challenged with various antimicrobial compounds.

Short-term characterisation of climatic-environmental variables and microbial community diversity in a high-altitude Andean wetland (Salar de Huasco, Chile).

Microbial community structures are shaped by geochemical factors and their interactions with the lithosphere, hydrosphere, and atmosphere through the processes of chemical mobilisation and mineralisation. High-altitude wetlands and salt flats in the central Andes are characterised by pronounced physicochemical gradients and extreme climatic conditions, representing hotspots of microbial diversity. We here hypothesise about the existence of direct relationships between the local microbiology and the climate cyclicity variables based on meteorological and biogeochemical patterns that develop over a short time scale (five years). We have here analysed the interactions between hydrometeorological and biogeochemical variables and the microbial communities of the Salar de Huasco. These results were obtained by correlating 16S cDNA and DNA gene Illumina sequences with meteorological/satellite data collected both at monitoring stations and by remote sensing between the years 2015 and 2020. The precipitation levels and flooded areas (i.e., areas covered and/or saturated with permanent water) detected in the Salar de Huasco revealed a marked hydric cyclicity that correlated seasonally with intra-annual wet and dry seasons. Overall, at this site, wet periods occurred from December to April, and dry periods from May to November. Meteorological variables such as solar radiation, air temperature, relative humidity, wind speed, atmospheric pressure, and wind direction were well-defined, showing a potential association with the hydrogeology of the area, which is directly related to the wetlands' flooded areas. Finally, the microbial presence and potentially active microbial communities were determined through the sequencing of the 16S gene (DNA and cDNA, respectively), this were associated with climatic seasonality and spatially distributed physical and chemical heterogeneity. Other non-local inter-annual scale processes, such as El Niño-Southern Oscillation (ENSO) events, modify the physical and chemical context of the wetland, thus forming unique ecological niches in the Andean Mountains.

Biochemical, genomic and structural characteristics of the Acr3 pump in Exiguobacterium strains isolated from arsenic-rich Salar de Huasco sediments.

Arsenic is a highly toxic metalloid of major concern for public safety. However, microorganisms have several resistance mechanisms, particularly the expression of arsenic pumps is a critical component for bacterial ability to expel it and decrease intracellular toxicity. In this study, we aimed to characterize the biochemical, structural, and genomic characteristics of the Acr3 pump among a group of Exiguobacterium strains isolated from different sites of the arsenic-rich Salar de Huasco (SH) ecosystem. We also determined whether the differences in As(III) resistance levels presented by the strains could be attributed to changes in the sequence or structure of this protein. In this context, we found that based on acr3 sequences the strains isolated from the SH grouped together phylogenetically, even though clustering based on gene sequence identity did not reflect the strain's geographical origin. Furthermore, we determined the genetic context of the acr3 sequences and found that there are two versions of the organization of acr3 gene clusters, that do not reflect the strain's origin nor arsenic resistance level. We also contribute to the knowledge regarding structure of the Acr3 protein and its possible implications on the functionality of the pump, finding that although important and conserved components of this family of proteins are present, there are several changes in the amino acidic sequences that may affect the interactions among amino acids in the 3D model, which in fact are evidenced as changes in the structure and residues contacts. Finally, we demonstrated through heterologous expression that the Exiguobacterium Acr3 pump does indeed improve the organisms As resistance level, as evidenced in the complemented E. coli strains. The understanding of arsenic detoxification processes in prokaryotes has vast biotechnological potential and it can also provide a lot of information to understand the processes of evolutionary adaptation.

Genetic Characterization of Salmonella Infantis with Multiple Drug Resistance Profiles Isolated from a Poultry-Farm in Chile.

Salmonella comprises over 2500 serotypes and foodborne contamination associated with this pathogen remains an important health concern worldwide. During the last decade, a shift in serotype prevalence has occurred as traditionally less prevalent serotypes are increasing in frequency of infections, especially those related to poultry meat contamination. S. Infantis is one of the major emerging serotypes, and these strains commonly display antimicrobial resistance and can persist despite cleaning protocols. Thus, this work aimed to isolate S. Infantis strains from a poultry meat farm in Santiago, Chile and to characterize genetic variations present in them. We determined their genomic and phenotypic profiles at different points along the production line. The results indicate that the strains encompass 853 polymorphic sites (core-SNPs) with isolates differing from one another by 0-347 core SNPs, suggesting variation among them; however, we found discrete correlations with the source of the sample in the production line. Furthermore, the pan-genome was composed of 4854 total gene clusters of which 2618 (53.9%) corresponds to the core-genome and only 181 (3.7%) are unique genes (those present in one particular strain). This preliminary analysis will enrich the surveillance of Salmonella, yet further studies are required to assess their evolution and phylogeny.

Living to the High Extreme: Unraveling the Composition, Structure, and Functional Insights of Bacterial Communities Thriving in the Arsenic-Rich Salar de Huasco Altiplanic Ecosystem.

Microbial communities inhabiting extreme environments such as Salar de Huasco (SH) in northern Chile are adapted to thrive while exposed to several abiotic pressures and the presence of toxic elements such as arsenic (As). Hence, we aimed to uncover the role of As in shaping bacterial composition, structure, and functional potential in five different sites in this altiplanic wetland using a shotgun metagenomic approach. The sites exhibit wide gradients of As (9 to 321 mg/kg), and our results showed highly diverse communities and a clear dominance exerted by the Proteobacteria and Bacteroidetes phyla. Functional potential analyses show broadly convergent patterns, contrasting with their great taxonomic variability. As-related metabolism, as well as other functional categories such as those related to the CH4 and S cycles, differs among the five communities. Particularly, we found that the distribution and abundance of As-related genes increase as the As concentration rises. Approximately 75% of the detected genes for As metabolism belong to expulsion mechanisms; arsJ and arsP pumps are related to sites with higher As concentrations and are present almost exclusively in Proteobacteria. Furthermore, taxonomic diversity and functional potential are reflected in the 12 reconstructed high-quality metagenome assembled genomes (MAGs) belonging to the Bacteroidetes (5), Proteobacteria (5), Cyanobacteria (1), and Gemmatimonadetes (1) phyla. We conclude that SH microbial communities are diverse and possess a broad genetic repertoire to thrive under extreme conditions, including increasing concentrations of highly toxic As. Finally, this environment represents a reservoir of unknown and undescribed microorganisms, with great metabolic versatility, which needs further study. IMPORTANCE As microbial communities inhabiting extreme environments are fundamental for maintaining ecosystems, many studies concerning composition, functionality, and interactions have been carried out. However, much is still unknown. Here, we sampled microbial communities in the Salar de Huasco, an extreme environment subjected to several abiotic stresses (high UV radiation, salinity and arsenic; low pressure and temperatures). We found that although microbes are taxonomically diverse, functional potential seems to have an important degree of convergence, suggesting high levels of adaptation. Particularly, arsenic metabolism showed differences associated with increasing concentrations of the metalloid throughout the area, and it effectively exerts a significant pressure over these organisms. Thus, the significance of this research is that we describe highly specialized communities thriving in little-explored environments subjected to several pressures, considered analogous of early Earth and other planets, that have the potential for unraveling technologies to face the repercussions of climate change in many areas of interest.

Activation of regulator ArcA in the presence of hypochlorite in Salmonella enterica serovar Typhimurium.

Oxidative stress is the main mechanism behind efficient disinfectants, causing damage in bacterial macromolecules. Importantly, bacteria activate resistance mechanisms in response to damage generated by oxidative stress. Strategies allowing pathogens to survive oxidative stress are highly conserved among microorganisms. Many of these strategies entail genomic responses triggered by signals transduced through Two Component Systems (TCS). Recently, we demonstrated that the TCS ArcAB (specifically ArcA) participates in bacterial responses to hypochlorite, regulating the uptake of this toxic compound and being involved in resistance and survival inside neutrophils, where hypochlorous acid abounds. Here, we demonstrated that ArcA is required in the response to oxidative stress generated by hypochlorite, independent of its cognate sensor ArcB or the Asp54 of ArcA, the only phosphorylable residue in ArcA, which is required to function as a gene regulator. Our results suggest that ArcA could have additional functions to respond to oxidative stress, independent of its regulatory activity, which might require interaction with other unknown relevant proteins.

Genomic Variation and Arsenic Tolerance Emerged as Niche Specific Adaptations by Different Exiguobacterium Strains Isolated From the Extreme Salar de Huasco Environment in Chilean - Altiplano.

Polyextremophilic bacteria can thrive in environments with multiple stressors such as the Salar de Huasco (SH). Microbial communities in SH are exposed to low atmospheric pressure, high UV radiation, wide temperature ranges, salinity gradient and the presence of toxic compounds such as arsenic (As). In this work we focus on arsenic stress as one of the main adverse factors in SH and bacteria that belong to the Exiguobacterium genus due to their plasticity and ubiquity. Therefore, our aim was to shed light on the effect of niche conditions pressure (particularly arsenic), on the adaptation and divergence (at genotypic and phenotypic levels) of Exiguobacterium strains from five different SH sites. Also, to capture greater diversity in this genus, we use as outgroup five As(III) sensitive strains isolated from Easter Island (Chile) and The Great Salt Lake (United States). For this, samples were obtained from five different SH sites under an arsenic gradient (9 to 321 mg/kg: sediment) and isolated and sequenced the genomes of 14 Exiguobacterium strains, which had different arsenic tolerance levels. Then, we used comparative genomic analysis to assess the genomic divergence of these strains and their association with phenotypic differences such as arsenic tolerance levels and the ability to resist poly-stress. Phylogenetic analysis showed that SH strains share a common ancestor. Consequently, populations were separated and structured in different SH microenvironments, giving rise to multiple coexisting lineages. Hence, this genotypic variability is also evidenced by the COG (Clusters of Orthologous Groups) composition and the size of their accessory genomes. Interestingly, these observations correlate with physiological traits such as growth patterns, gene expression, and enzyme activity related to arsenic response and/or tolerance. Therefore, Exiguobacterium strains from SH are adapted to physiologically overcome the contrasting environmental conditions, like the arsenic present in their habitat.

The Transcription Factor ArcA Modulates Salmonella's Metabolism in Response to Neutrophil Hypochlorous Acid-Mediated Stress.

Salmonella Typhimurium, a bacterial pathogen with high metabolic plasticity, can adapt to different environmental conditions; these traits enhance its virulence by enabling bacterial survival. Neutrophils play important roles in the innate immune response, including the production of microbicidal reactive oxygen species (ROS). In addition, the myeloperoxidase in neutrophils catalyzes the formation of hypochlorous acid (HOCl), a highly toxic molecule that reacts with essential biomolecules, causing oxidative damage including lipid peroxidation and protein carbonylation. The bacterial response regulator ArcA regulates adaptive responses to oxygen levels and influences the survival of Salmonella inside phagocytic cells. Here, we demonstrate by whole transcriptomic analyses that ArcA regulates genes related to various metabolic pathways, enabling bacterial survival during HOCl-stress in vitro. Also, inside neutrophils, ArcA controls the transcription of several metabolic pathways by downregulating the expression of genes related to fatty acid degradation, lysine degradation, and arginine, proline, pyruvate, and propanoate metabolism. ArcA also upregulates genes encoding components of the oxidative pathway. These results underscore the importance of ArcA in ATP generation inside the neutrophil phagosome and its participation in bacterial metabolic adaptations during HOCl stress.

Arsenic Response of Three Altiplanic Exiguobacterium Strains With Different Tolerance Levels Against the Metalloid Species: A Proteomics Study.

Exiguobacterium is a polyextremophile bacterial genus with a physiology that allows it to develop in different adverse environments. The Salar de Huasco is one of these environments due to its altitude, atmospheric pressure, solar radiation, temperature variations, pH, salinity, and the presence of toxic compounds such as arsenic. However, the physiological and/or molecular mechanisms that enable them to prosper in these environments have not yet been described. Our research group has isolated several strains of Exiguobacterium genus from different sites of Salar de Huasco, which show different resistance levels to As(III) and As(V). In this work, we compare the protein expression patterns of the three strains in response to arsenic by a proteomic approach; strains were grown in absence of the metalloid and in presence of As(III) and As(V) sublethal concentrations and the protein separation was carried out in 2D electrophoresis gels (2D-GE). In total, 999 spots were detected, between 77 and 173 of which showed significant changes for As(III) among the three strains, and between 90 and 143 for As(V), respectively, compared to the corresponding control condition. Twenty-seven of those were identified by mass spectrometry (MS). Among these identified proteins, the ArsA [ATPase from the As(III) efflux pump] was found to be up-regulated in response to both arsenic conditions in the three strains, as well as the Co-enzyme A disulfide reductase (Cdr) in the two more resistant strains. Interestingly, in this genus the gene that codifies for Cdr is found within the genic context of the ars operon. We suggest that this protein could be restoring antioxidants molecules, necessary for the As(V) reduction. Additionally, among the proteins that change their expression against As, we found several with functions relevant to stress response, e.g., Hpf, LuxS, GLpX, GlnE, and Fur. This study allowed us to shed light into the physiology necessary for these bacteria to be able to tolerate the toxicity and stress generated by the presence of arsenic in their niche.

Correction: The ArcAB two-component regulatory system promotes resistance to reactive oxygen species and systemic infection by Salmonella Typhimurium.

[This corrects the article DOI: 10.1371/journal.pone.0203497.].

Characterization and Salt Response in Recurrent Halotolerant Exiguobacterium sp. SH31 Isolated From Sediments of Salar de Huasco, Chilean Altiplano.

Poly-extremophiles microorganisms have the capacity to inhabit hostile environments and can survive several adverse conditions that include as variations in temperature, pH, and salinity, high levels UV light and atmospheric pressure, and even the presence of toxic compounds and the formation of reactive oxygen species (ROS). A halotolerant Exiguobacterium strain was isolated from Salar de Huasco (Chilean Altiplano), a well-known shallow lake area with variable salinity levels, little human intervention, and extreme environmental conditions, which makes it ideal for the study of resistant mechanisms and the evolution of adaptations. This bacterial genus has not been extensively studied, although its cosmopolitan location indicates that it has high levels of plasticity and adaptive capacity. However, to date, there are no studies regarding the tolerance and resistance to salinity and osmotic pressure. We set out to characterize the Exiguobacterium sp. SH31 strain and describe its phenotypical and genotypical response to osmotic stress. In this context, as a first step to characterize the response to the SH31 strain to salinity and to establish the bases for a molecular study, we proposed to compare its response under three salt conditions (0, 25, and 50 g/l NaCl). Using different physiology, genomic, and transcriptomic approaches, we determined that the bacterium is able to grow properly in a NaCl concentration of up to 50 g/l; however, the best growth rate was observed at 25 g/l. Although the presence of flagella is not affected by salinity, motility was diminished at 25 g/l NaCl and abolished at 50 g/l. Biofilm formation was induced proportionally with increases in salinity, which was expected. These phenotypic results correlated with the expression of related genes: fliG and fliS Motility); opuBA and putP (transport); glnA, proC, gltA, and gbsA (compatible solutes); ywqC, bdlA, luxS y pgaC (biofilm and stress response); and therefore, we conclude that this strain effectively modifies gene expression and physiology in a differential manner when faced with different concentrations of NaCl and these modifications aid survival.

The ArcAB two-component regulatory system promotes resistance to reactive oxygen species and systemic infection by Salmonella Typhimurium.

Salmonella enterica Serovar Typhimurium (S. Typhimurium) is an intracellular bacterium that overcomes host immune system barriers for successful infection. The bacterium colonizes the proximal small intestine, penetrates the epithelial layer, and is engulfed by macrophages and neutrophils. Intracellularly, S. Typhimurium encounters highly toxic reactive oxygen species including hydrogen peroxide and hypochlorous acid. The molecular mechanisms of Salmonella resistance to intracellular oxidative stress is not completely understood. The ArcAB two-component system is a global regulatory system that responds to oxygen. In this work, we show that the ArcA response regulator participates in Salmonella adaptation to changing oxygen levels and is also involved in promoting intracellular survival in macrophages and neutrophils, enabling S. Typhimurium to successfully establish a systemic infection.

The transcription factor SlyA from Salmonella Typhimurium regulates genes in response to hydrogen peroxide and sodium hypochlorite.

Salmonella Typhimurium is an intracellular pathogen that is capable of generating systemic fever in a murine model. Over the course of the infection, Salmonella faces different kinds of stressors, including harmful reactive oxygen species (ROS). Various defence mechanisms enable Salmonella to successfully complete the infective process in the presence of such stressors. The transcriptional factor SlyA is involved in the oxidative stress response and invasion of murine macrophages. We evaluated the role of SlyA in response to H2O2 and NaOCl and found an increase of slyA expression upon exposure to these toxics. However, the SlyA target genes and the molecular mechanisms by which they influence the infective process are unknown. We hypothesised that SlyA regulates the expression of genes required for ROS resistance, metabolism, or virulence under oxidative stress conditions. Transcriptional profiling in wild type and ΔslyA strains confirmed that SlyA regulates the expression of several genes involved in virulence [sopD (STM14_3550), sopE2 (STM14_2244), hilA (STM14_3475)] and central metabolism [kgtP (STM14_3252), fruK (STM14_2722), glpA (STM14_2819)] in response to H2O2 and NaOCl. These findings were corroborated by functional assay and transcriptional fusion assays using GFP. DNA-protein interaction assays showed that SlyA regulates these genes through direct interaction with their promoter regions.

Comparative Genomics Analysis of a New Exiguobacterium Strain from Salar de Huasco Reveals a Repertoire of Stress-Related Genes and Arsenic Resistance.

The Atacama Desert hosts diverse ecosystems including salt flats and shallow Andean lakes. Several heavy metals are found in the Atacama Desert, and microorganisms growing in this environment show varying levels of resistance/tolerance to copper, tellurium, and arsenic, among others. Herein, we report the genome sequence and comparative genomic analysis of a new Exiguobacterium strain, sp. SH31, isolated from an altiplanic shallow athalassohaline lake. Exiguobacterium sp. SH31 belongs to the phylogenetic Group II and its closest relative is Exiguobacterium sp. S17, isolated from the Argentinian Altiplano (95% average nucleotide identity). Strain SH31 encodes a wide repertoire of proteins required for cadmium, copper, mercury, tellurium, chromium, and arsenic resistance. Of the 34 Exiguobacterium genomes that were inspected, only isolates SH31 and S17 encode the arsenic efflux pump Acr3. Strain SH31 was able to grow in up to 10 mM arsenite and 100 mM arsenate, indicating that it is arsenic resistant. Further, expression of the ars operon and acr3 was strongly induced in response to both toxics, suggesting that the arsenic efflux pump Acr3 mediates arsenic resistance in Exiguobacterium sp. SH31.