Biology subjects

Martinez-Mendez, R.

Publications and source records attributed to Martinez-Mendez, R..

5 recordsLinked to original sources

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↗

bioRxiv2020

The conserved molting/circadian rhythm regulator NHR-23/NR1F1 serves as an essential co-regulator of C. elegans spermatogenesis

In sexually reproducing metazoans, spermatogenesis is the process by which uncommitted germ cells give rise to haploid sperm. Work in model systems has revealed mechanisms controlling commitment to the sperm fate, but how this fate is subsequently executed remains less clear. While studying the well-established role of the conserved nuclear hormone receptor transcription factor, NHR-23/NR1F1, in regulation of C. elegans molting, we discovered NHR-23/NR1F1 is also constitutively expressed in developing 1{degrees} spermatocytes and is a critical regulator of spermatogenesis. In this novel role, NHR-23/NR1F1 functions downstream of the canonical sex determination pathway. Degron-mediated depletion of NHR-23/NR1F1 within hermaphrodite or male germlines causes sterility due to an absence of functional sperm as depleted animals produce arrested primary spermatocytes rather than haploid sperm. These spermatocytes arrest in prometaphase I and fail to either progress to anaphase or attempt spermatid-residual body partitioning. They make sperm-specific membranous organelles (MOs) but fail to assemble their major sperm protein into fibrous bodies. NHR-23/NR1F1 appears to function independently of the known SPE-44 gene regulatory network, revealing the existence of an NHR-23/NR1F1-mediated module that regulates the spermatogenesis program. Summary StatementA well-characterized regulator of C. elegans molting also unexpectedly controls the spermatogenesis program; our work provides insights into the gene regulatory networks controlling spermatogenesis.

developmental biology↗

bioRxiv2020

Expanding the Caenorhabditis elegans auxin-inducible degron system toolkit with internal expression and degradation controls and improved modular constructs for CRISPR/Cas9-mediated genome editing

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell-types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying an equimolar co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. We have also constructed a set of plasmids to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These templates can be produced through frequently used cloning systems (Gibson assembly or SapTrap) or through ribonucleoprotein complex-mediated insertion of PCR-derived, linear repair templates. We have generated a set of sgRNA plasmids carrying modifications shown to boost editing efficiency, targeting standardized transgene insertion sites on chromosomes I and II. Together these reagents should complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID* tagging of factors of interest. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for facile assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

genetics↗