Biology subjects

Zamorano-Sanchez, D.

Publications and source records attributed to Zamorano-Sanchez, D..

4 recordsLinked to original sources

bioRxiv2025

Insights on the regulation and function of the CRISPR/Cas transposition system located in the pathogenicity island VpaI-7 from Vibrio parahaemolyticus RIMD2210633.

CRISPR/Cas mediated transposition is a recently recognized strategy for horizontal gene transfer in a variety of bacterial species. However, our understanding of the factors that control their function in their natural hosts is still limited. In this work we report our initial genetic characterization of the elements associated with the CRISPR/Cas-transposition machinery (CASTm) from Vibrio parahaemolyticus (VpaCASTm), which are encoded within the pathogenicity island VpaI-7. Our results revealed that the components of the VpaCASTm and their associated CRISPR arrays (VpaCAST system) are transcriptionally active in their native genetic context. Furthermore, we were able to detect the presence of polycistrons and several internal promoters within the loci that compose the VpaCAST system. Our results also suggest that the activity of the promoter of the atypical CRISPR array is not repressed by the baseline activity of its known regulator VPA1391 in V. parahaemolyticus. Additionally, we found that the activity of the promoter of tniQ was modulated by a regulatory cascade involving ToxR, LeuO and H-NS. Since it was previously reported that the activity of the VpaCAST system was less efficient than that of the VchCAST system at promoting transposition of a miniaturized CRISPR-associated transposon (mini-CAST) in Escherichia coli, we analyzed if the transposition efficiency mediated by the VpaCAST system could be enhanced inside its natural host V. parahaemolyticus. We provide evidence that this might be the case suggesting that there could be host induction factors in V. parahaemolyticus that could enable more efficient transposition of CASTs. ImportanceMobile genetic elements such as transposons play important roles on the evolutionary trajectories of bacterial genomes. The success of transposon dissemination depends on their ability to carry selectable markers that improve the fitness of the host cell or loci with addictive traits such as the toxin-antitoxin systems. Here we aimed to characterize a transposon from Vibrio parahaemolyticus that carries and could disseminate multiple virulence factors. This transposon belongs to a recently discovered family of transposons whose transposition is guided by crRNA. We showed that the transposition machinery of this transposon is transcribed in V. parahaemolyticus and that there are likely host associated factors that favor transposition in the natural host V. parahaemolyticus over transposition in Escherichia coli.

microbiology↗

bioRxiv2024

Vibrio cholerae O1 El Tor A1552 encodes two functional ornithine lipid synthases and induces ornithine lipid formation under low phosphate and under low salinity growth conditions.

Ornithine lipids (OLs) are phosphorus-free membrane lipids that can be formed by a wide range of bacteria. The presence of OLs is frequently related to the resistance to abiotic stress conditions, and its synthesis is often induced as part of various stress responses. Two different pathways for synthesizing OLs are currently known: the OlsBA pathway first described in Sinorhizobium meliloti, and the OlsF pathway first described in Serratia proteamaculans. We identified in the genome of Vibrio cholerae O1 El Tor A1552 two genes encoding OlsF homologs, VC0489 is located on chromosome 1, whereas VCA0646 is located on chromosome 2. Both synthases, when expressed in Escherichia coli, caused the synthesis of OLs. Single mutants deficient in each of the OL synthases, double mutants deficient in both OL synthases, and mutants deficient in the transcriptional regulator PhoB were constructed and characterized. We corroborated that VC0489 is solely responsible for the synthesis of OLs under phosphate-limitation. The deletion of VC0489 reduced the growth velocity compared to the wildtype under phosphate-limiting conditions but not under phosphate-replete conditions. The expression of VCA0646 is favored under low salt growth conditions, and its deletion abrogates OL synthesis at low salinities. The absence of VCA0646 and, therefore, the lack of OLs under low salt conditions makes the respective mutant more susceptible to polymyxin than OL-forming strains. None of the mutants was affected in biofilm formation, swimming, or virulence assays using Caenorhabditis elegans or Galleria mellonella. Here, we describe two functional OL synthases present in a single bacterium for the first time, and we show evidence that OLs have an important function during the V. cholerae lifecycle.

microbiology↗

bioRxiv2023

OpaR exerts a dynamic control over c-di-GMP homeostasis and cpsA expression in V. parahaemolyticus through its regulation of ScrC and the trigger phosphodiesterase TpdA.

The second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) plays a central role in controlling decision making processes of vital importance for the environmental survival of the human pathogen Vibrio parahaemolyticus. The mechanisms by which c-di-GMP levels are dynamically controlled in V. parahaemolyticus are poorly understood. Here we report our findings regarding the involvement of OpaR in controlling c-di-GMP metabolism in planktonic and surface-attached cells through controlling the expression of the trigger phosphodiesterase (PDE) TpdA and other PDEs such as ScrC. Our results revealed that OpaR negatively modulates the expression of tpdA by maintaining a baseline level of c-di-GMP. The OpaR-regulated PDEs ScrC, ScrG and VP0117 enable the upregulation of tpdA, to a different degree, in the absence of OpaR. We also found that TpdA plays the dominant role in c-di-GMP degradation under planktonic conditions compared to the other OpaR-regulated PDEs. In cells growing over solid media the dominant c-di-GMP degrader role is played by ScrC for 72 hours and passes to TpdA after 96 hours of growth. We also report negative and positive effects of the absence of OpaR on cpsA expression in cells growing over solid media or forming biofilms over glass, respectively. These results suggest that OpaR can act as a double-edged sword to control c-di-GMP accumulation and cpsA expression positively or negatively in response to poorly understood environmental factors. Finally, through an in-silico analysis we point out outlets of the OpaR regulatory module that can impact decision making during the motile to sessile transition in V. parahaemolyticus. ImportanceThe second messenger c-di-GMP is extensively used by bacterial cells to control crucial social adaptations such as biofilm formation. Here we explore the role of the quorum-sensing regulator OpaR, from the human pathogen V. parahaemolyticus, on the dynamic control of c-di-GMP signaling. We found that OpaR can regulate positively or negatively c-di-GMP accumulation depending on the growth conditions. This dual role has not been reported for orthologues of OpaR, such as HapR from V. cholerae. OpaR controls c-di-GMP homeostasis through PDEs that are absent in V. cholerae, pointing toward further differences in c-di-GMP signaling in these two pathogens. It is important to investigate the origins and consequences of these differences to better understand pathogenic bacterial behavior and its evolution.

microbiology↗

bioRxiv2021

A trigger phosphodiesterase modulates the global c-di-GMP pool, motility and biofilm formation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells transit from free swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP were low or when the phosphodiesterase was catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called Trigger phosphodiesterases, hence we named the product of VP1881 TpdA, for Trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while its overexpression from an IPTG inducible promoter showed a positive effect on both swimming and swarming motility, and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes as well as the biofilm related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of this social behaviors. ImportanceC-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm-matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, hence not many examples of these type of signaling proteins have been reported in the literature. Here we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.

microbiology↗