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Must have a protocol. Your first 15 GB of storage are free with a Google account. Download on Android or iOS. Access Google Docs with a free Google account for personal use or Google Workspace account for business use. Briefly, they use combinations of many predictive tools to gather and add evidence, assigning possible functions with a good confidence level to those unclassified or unknown proteins. In a broad sense, most of the studied markers related to arsenic tolerance, oxidative and global stress are present in all strains Supplementary Figure S4.
However, there is also a clear differential pattern between the strains of the three environments SH, GSL and EI , which is consistent with their particular arsenic tolerance levels. Although our results imply that the presence of arsenic response genes such as arsC, acr3 and perhaps arsK and arsP indeed increases the resistance level SH strains regarding those from GSl and EI , this does not explain its heterogeneity among the SH strains.
Moreover, an explanation could be the regulation and expression impact on arsenic response, which has been discussed in other published works Cleiss-Arnold et al. Also, we support the participation of global and oxidative stress response systems role in strengthens this resistance capacity.
Moreover, enrichment in genes directly related to arsenic tolerance on SH strains is evidence of the selective pressure exerted by the presence of this toxic compound Slyemi and Bonnefoy, ; Andres and Bertin, Particularly, arsC, arsP and acr3 are only found in SH strains, additionally a second copy of arsR to regulate arsP is also present.
However, acr3 gene is missing in the three most sensitive SH strains from H1 site , which could be evidence of the important role of ACR3 expelling function in the arsenic resistance, which has been reported before Fu et al. ArsP is known to be an organoarsenical permease Shen et al.
Hence, resulting in the logical pattern observed, regarding the arsenic concentration between the three environments and the enrichment of genes displayed by these strains to face it. Since arsenic has been detected on the GSL basin in lower concentrations 0. Conversely, there are no reports for arsenic in EI. Taken together, most of SH strains have three clusters with arsenic response markers arsRDAB, arsP and arsK-acr3 , that could be under different regulations.
Since it is known that both ArsP and ArsK expel organic arsenics, it is necessary to find the missing piece As-methylase enzyme to promote this as a functional mechanism in Exiguobacterium Garbinski et al.
Nonetheless, these genes gave us some hints supporting the idea of a more varied repertoire that yields higher resistance. However, we have to consider that arsK is present in the GSL strains which were the most susceptible ones, so the role of this gene remains unclear.
Another interesting feature found in some of the studied genomes was the nitrate reductase gene cluster. Although being exclusively present in some SH strains, its distribution does not present a pattern of association by site or arsenic tolerance level, but it does show a clade specific pattern.
On the other hand, is important to take into consideration, that there could be a selection pressure that we are not considering, which pushes Exiguobacterium genomes toward convergence. However, we could not conclude that its presence has any relation with arsenic or environmental stress adaptation. In addition, a poorly characterized anaerobic arsenite oxidase arxA; molybdopterin containing enzyme was identified in the genome of a Mono Lake California, United States isolated that couples arsenite oxidation with nitrate reduction Zargar et al.
Furthermore, we were not able to detect an arxA sequence in any of the studied Exiguobacterium genomes, we cannot rule out the presence or function of some analog. But we did find within this cluster an uncharacterized 2Fe-2S protein that shares some characteristics with arsenite oxidases.
As we know the effects of lacking information and databases availability for the description of new mechanisms, we believe that this could be re-addressed when more and new information is generated.
Another finding that supports that a novel mechanism may be in play is the presence of eleven genes related to the molybdopterin biosynthesis are in this same genomic context, which is a well-known nitrate reductase co-factor Kelley, Also, this molecule is a co-factor for ArxA too Zargar et al. Besides, the presence of the transcriptional fumarate and nitrate reductase regulator fnr in this genetic cluster could be evidence of an anoxic related response Osorio et al. Finally, as most of our studied markers are part of the core genome compartment, we would imply that differences in regulation and expression levels among the strains could be the source of their variable arsenic tolerance levels.
On the other hand, we have to consider that these determining factors may be among the hypothetical and unknown function markers of the accessory genomes. Also, all these results further support the plasticity of the Exiguobacterium genus and suggest that environmental factors of each niche could shape the species by driving divergence.
Oxidative stress is an important arsenic toxicity mechanism and detoxifying enzymes has always been a target of interest Harrison et al.
Increase in catalases and superoxide dismutases expression and activity in response to arsenic, as well as the loss of this tolerance due to mutations in these enzymes are evidence of their role Parvatiyar et al. The results show that both arsenic tested conditions were able to promote intracellular ROS accumulation in the analyzed strains Figure 6. Which could be counteracted by catalase and superoxide dismutase activity. Detoxification of As V comprises an oxidation-reduction process that contributes to oxidative stress by depleting the thiols altering the cell redox balance Mukhopadhyay et al.
Generally, As III was not able to generate a significant increase of enzymatic activity on the three strains. Moreover, the lower increase in SOD activity was an unexpected result, since it has been reported that these enzymes respond to arsenic in H. Oxidative stress resistance could cause tolerance against other stresses, as was demonstrated in Deinoccocus radiodurans , in which high UV stress resistance is associated with its SOD efficiency Markillie et al. Most of the arsenic related genes showed a significative upregulation in response to both arsenic species, on the three strains Figure 7.
Moreover, all transporters showed different induction levels, supporting the toxic expulsion as the main resistance mechanism used by these bacteria.
In addition, another strategy used by bacteria to tolerate the arsenic present in their environments is to block the influx, which correspond with glpF repression by As III Elias et al. On the other hand, the genes that code for catalase and superoxide dismutase did not show significant expression changes in response to arsenic among the three strains. Whereas, glutathione synthesis appears to be highly active due to gshAB significate overexpression, thus helping to restore cell redox balance.
Finally, we think that differential arsenic tolerance presented by these strains is the result or sum of a set of factors, markers and particular regulation, combining permeability, detoxification and homeostasis changes to ensure survival, as we described previously Castro-Severyn et al.
To sum up, the joint efforts and results of all our work led us to state that the main strategy used by the SH Exiguobacterium strains against arsenic is based on the toxic expulsion out of the cell. To this end, these strains have a wide variety of efflux proteins like ArsB, ArsP, ArsK and ACR3 to cope with any arsenic species, since arsenic could be reduced, oxidized or methylated.
Figure 8 shows a graphic summary which integrates our genomic, gene expression, proteomic and physiological findings This work; Castro-Severyn et al. Arsenic toxicity to the cell is known to generate oxidative and global stress states. In order to fight this, several responses are activated, particularly against oxidative stress, Cdr could be restoring the thiols depleted during As V reduction.
Furthermore, PdxS which is known to be involved in several types of stress resistance, is also a cysteine synthesis cofactor, aminoacid needed by the GshAB to form new thiols, counteracting the oxidative stress along with all of TrxA copies.
Also, our results support all the reported evidence promoting global stress markers such as LuxS, Hpf, DnaK and UspA as key players in arsenic resistance. Finally, we can suggest that the presence of ACR3 increases the level of resistance to the strains that possess it, such as the SH ones. However, the variability between the levels of these same strains may be due to the addition of other factors such as ArsK, ArsP and the global and oxidative stress markers and its regulation, that would be contributing to the resistant phenotype.
Salar de Huasco, as part of the Chilean altiplano, is indeed a very extreme and diverse environment in which arsenic appears to be one of the main communities shaping factors. As a whole, these results indicate that there are key elements at genome level that enable those bacteria to respond poly-stress and in particular As presence. Additionally, phylogenetic relationships and pan-genome composition found among the Exiguobacterium strains could imply that these adaptations emerge from their particular niche.
These observations are consistent with the transcriptional and enzymatic responses against arsenic of these bacteria. Finally, the increase in database information and the use of omics approaches is currently generating more data that is shedding light in understanding the evolutionary process of adaptation, especially on extreme environments. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We like to thank Dra. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U. Journal List Front Microbiol v. Front Microbiol. Published online Jul Katterinne N. Claudia P. Author information Article notes Copyright and License information Disclaimer. Saavedra, lc. This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology.
Received Apr 22; Accepted Jun The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.
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XLSX 11K. PDF K. PDF 9. PDF 75K. Abstract Polyextremophilic bacteria can thrive in environments with multiple stressors such as the Salar de Huasco SH. Keywords: Exiguobacterium genus, genomics, arsenic, niche, poly-extremophilic. Introduction Exiguobacterium is a bacterial genus initially described by Collins et al.
Materials and Methods Study Area and Sampling In January of during a field work in the Salar de Huasco National Park Chilean Altiplano , water and sediment samples were taken from five different sites previously described by Dorador et al. Exiguobacterium Genomic Data Sets Three different genomic data sets were used for the following analyzes: first we included the genomes of the 14 strains isolated from the five Salar de Huasco sites, including the previously described SH31 strain Castro-Severyn et al.
Pan Genome Analysis Pan genome was defined clustering the proteins families into ortholog groups based on their sequence similarity using the algorithm orthoMCL v1. Protein Searches Bi-directional best hit searches using Blast Altschul et al. Biochemical Reactive Oxygen Species Indicators and Antioxidant Activity Since oxidative stress has been described as a consequence of arsenic toxicity, we measured on the three selected strains some indicators of this process.
Open in a separate window. Bacterial Culture, Isolation and Exiguobacterium Identification Different bacterial morphotypes were isolated from sample enriched cultures and 16S rRNA molecular identification yielded 16 new Exiguobacterium strains from the SH sites. TABLE 2 Minimal inhibitory concentration of both As species for all the isolated strains; Accession number and assembly evaluation of the selected strains genomes.
Genomic Relationships Phylogeny, ANI and AAI were analyzed for all the sequenced genomes set and also within the complete set of 74 strains to consider their placement among the whole available diversity. Huasco Pan-Genome To determine the genotypic diversity among the SH strains as well as the differences that could be attributed to their specific niche we performed a Pan-genome analysis with only these 14 strains Figure 3.
Physiological Response to Arsenic Aiming to cover the most diversity considering arsenic tolerance and geographic origin among the studied bacteria, we selected three strains for experimental analyzes. Arsenic Response Gene Expression Our results show an active response to arsenic stress on the three strains, despite of different gene expression magnitudes among the strains. Discussion Altiplanic environments have extreme and variating conditions, even between geographically close areas Cabrol et al.
Conclusion Salar de Huasco, as part of the Chilean altiplano, is indeed a very extreme and diverse environment in which arsenic appears to be one of the main communities shaping factors. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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