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Aravena, P.

Publications and source records attributed to Aravena, P..

2 recordsLinked to original sources

The rhizosphere of Pappostipa frigida as a hotspot of active bacterial communities in the Andean steppe of the Atacama Desert

BackgroundThe rhizosphere is a resource-rich microenvironment where plants, soil nutrients, and microorganisms interact. In arid and semi-arid regions, this tripartite relationship must withstand long periods of drought followed by brief periods of rainfall. In the present study, we employed a combination of RNA/DNA metabarcoding and shotgun metagenomic sequencing to provide insights into the functional capabilities and activity levels of the bacterial communities present in the bulk soil and rhizosphere samples of Pappostipa frigida, a grass species endemic to the Andean steppe of the Atacama Desert. ResultsThe active bacterial community in the rhizosphere of P. frigida exhibited greater diversity and a higher Shannon index than the total bacterial community. In terms of beta diversity, the structures of the total and active communities differed markedly between the BS and the RZ. Furthermore, active bacteria in the RZ showed a stronger correlation with total bacterial populations than those in the BS. This finding is consistent with the low proportion of ASVs derived from RNA extractions detected in the BS. Notably, 64% of these putative inactive bacterial populations were identified as active RZ members and 73% grew in culture media, suggesting they were likely dormant. The bacterial communities of the BS exhibited higher abundances of sporulation genes. In contrast, active bacterial communities in the RZ consistently contained higher abundances of genes associated with halotolerance, siderophore synthesis, and resuscitation-promoting factors. ConclusionsThe results emphasize the importance of the conditions created by plants in recruiting bacterial populations from the soil and provide insights into how the rhizosphere of arid native plants influences the activity and functional traits of soil microorganisms in their natural habitat. Additionally, this study advances our understanding of the mechanisms employed by soil microorganisms to cope with desiccation in natural environments, establishing P. frigida as a model in plant science for studying grass traits and responses to extreme environments.

microbiology↗

Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is the causative agent of Salmonid Rickettsial Septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an IPTG-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous gene encoding green fluorescent protein (sfGFP), and the endogenous homolog of the fur gene, whose gene product regulates intracellular iron homeostasis in bacteria. The inducible expression of dcas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains, when the expression of the fur gRNA was induced with IPTG. By RNA-seq we detected a significant increase in the expression of genes encoding the Fe+2 and Fe+3 acquisition systems and iron mobilization in the fur1 knockdown, after IPTG induction. All the genes with over two-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The successful implementation of the Mobile-CRISPRi system in P. salmonis paves the way for systematic analysis of gene function in this pathogen We anticipate that these analyses will be very valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis. ImportanceSalmon Rickettsial Septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacteriums chromosome to ensure their constitutive and inducible expression, and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Furs relevance to the iron metabolism in the pathogen.

microbiology↗