RESUMO
Diapause arrest in animals such as Caenorhabditis elegans is tightly regulated so that animals make appropriate developmental decisions amidst environmental challenges. Fully understanding diapause requires mechanistic insight of both entry and exit from the arrested state. Although a steroid hormone pathway regulates the entry decision into C. elegans dauer diapause, its role in the exit decision is less clear. A complication to understanding steroid hormonal regulation of dauer has been the peculiar fact that steroid hormone mutants such as daf-9 form partial dauers under normal growth conditions. Here, we corroborate previous findings that daf-9 mutants remain capable of forming full dauers under unfavorable growth conditions and establish that the daf-9 partial dauer state is likely a partially exited dauer that has initiated but cannot complete the dauer exit process. We show that the steroid hormone pathway is both necessary for and promotes complete dauer exit, and that the spatiotemporal dynamics of steroid hormone regulation during dauer exit resembles that of dauer entry. Overall, dauer entry and dauer exit are distinct developmental decisions that are both controlled by steroid hormone signaling.
Assuntos
Proteínas de Caenorhabditis elegans , Diapausa , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Hormônios/metabolismo , Larva/metabolismo , Mutação/genética , Esteroides/metabolismoRESUMO
Vibrio cholerae, the facultative pathogen responsible for cholera disease, continues to pose a global health burden. Its persistence can be attributed to a flexible genetic tool kit that allows for adaptation to different environments with distinct carbon sources, including the six-carbon sugar alcohol mannitol. V. cholerae takes up mannitol through the transporter protein MtlA, whose production is downregulated at the posttranscriptional level by MtlS, a cis antisense small RNA (sRNA) whose promoter lies within the mtlA open reading frame. Though it is known that mtlS expression is robust under growth conditions lacking mannitol, it has remained elusive as to what factors govern the steady-state levels of MtlS. Here, we show that manipulating mtlA transcription is sufficient to drive inverse changes in MtlS levels, likely through transcriptional interference. This work has uncovered a cis-acting sRNA whose expression pattern is predominantly controlled by transcription of the sRNA's target gene.IMPORTANCEVibrio cholerae is a bacterial pathogen that relies on genetic tools, such as regulatory RNAs, to adapt to changing extracellular conditions. While many studies have focused on how these regulatory RNAs function, fewer have focused on how they are themselves modulated. V. cholerae expresses the noncoding RNA MtlS, which can regulate mannitol transport and use, and here we demonstrate that MtlS levels are controlled by the level of transcription occurring in the antisense direction. Our findings provide a model of regulation describing how bacteria like V. cholerae can modulate the levels of an important regulatory RNA. Our work contributes to knowledge of how bacteria deploy regulatory RNAs as an adaptive mechanism to buffer against environmental flux.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Transporte de Monossacarídeos/genética , RNA Antissenso/genética , Transcrição Gênica , Vibrio cholerae/genética , Regiões Promotoras Genéticas , RNA Bacteriano/genética , RNA não Traduzido/genéticaRESUMO
The phosphoenopyruvate:carbohydrate phosphotransferase system (PTS) enables Vibrio cholerae - and other bacteria - to recognize and transport exogenous carbon sources for energy, including the six-carbon sugar alcohol, mannitol. The mannitol-specific PTS transporter is encoded by mtlA and its expression is expected to be regulated by the putative repressor encoded by the mtlR gene. Here, we show that mtlR overexpression inhibits V. cholerae growth in medium supplied with mannitol as the sole carbon source and represses MtlA-mediated biofilm formation. We demonstrate that when V. cholerae is grown in non-mannitol medium, knocking out mtlR leads to both increased MtlA protein and mtlA mRNA levels, with these increases being especially pronounced in non-glucose sugars. We propose that in non-mannitol, non-glucose growth conditions, MtlR is a major regulator of mtlA transcription. Surprisingly, with regard to mtlR expression, transcript and protein levels are highest in mannitol medium, conditions where mtlA expression should not be repressed. We further show that MtlR levels increase during growth of the bacteria and linger in cells switched from mannitol to non-mannitol medium. Our data suggests an expression paradigm for mtlA where MtlR acts as a transcriptional repressor responsible for calibrating MtlA levels during environmental transitions.
RESUMO
Decisions made over long time scales, such as life cycle decisions, require coordinated interplay between sensory perception and sustained gene expression. The Caenorhabditis elegans dauer (or diapause) exit developmental decision requires sensory integration of population density and food availability to induce an all-or-nothing organismal-wide response, but the mechanism by which this occurs remains unknown. Here, we demonstrate how the ASJ chemosensory neurons, known to be critical for dauer exit, perform sensory integration at both the levels of gene expression and calcium activity. In response to favorable conditions, dauers rapidly produce and secrete the dauer exit-promoting insulin-like peptide INS-6. Expression of ins-6 in the ASJ neurons integrate population density and food level and can reflect decision commitment since dauers committed to exiting have higher ins-6 expression levels than those of non-committed dauers. Calcium imaging in dauers reveals that the ASJ neurons are activated by food, and this activity is suppressed by pheromone, indicating that sensory integration also occurs at the level of calcium transients. We find that ins-6 expression in the ASJ neurons depends on neuronal activity in the ASJs, cGMP signaling, a CaM-kinase pathway, and the pheromone components ascr#8 and ascr#2. We propose a model in which decision commitment to exit the dauer state involves an autoregulatory feedback loop in the ASJ neurons that promotes high INS-6 production and secretion. These results collectively demonstrate how insulin-like peptide signaling helps animals compute long-term decisions by bridging sensory perception to decision execution.
RESUMO
The biological roles of the autofluorescent lysosome-related organelles ("gut granules") in the intestinal cells of many nematodes, including Caenorhabditis elegans, have been shown to play an important role in metabolic and signaling processes, but they have not been fully characterized. We report here a previously undescribed phenomenon in which the autofluorescence of these granules increased and then decreased in a rapid and dynamic manner that may be associated with nutrient availability. We observed that two distinct types of fluorophores are likely present in the gut granules. One displays a "flashing" phenomenon, in which fluorescence decrease is preceded by a sharp increase in fluorescence intensity that expands into the surrounding area, while the other simply decreases in intensity. Gut granule flashing was observed in the different life stages of C. elegans and was also observed in Steinernema hermaphroditum, an evolutionarily distant nematode. We hypothesize that the "flashing" fluorophore is pH-sensitive, and the fluorescence intensity change results from the fluorophore being released from the lysosome-related organelles into the relatively higher pH environment of the cytosol. The visually spectacular dynamic fluorescence phenomenon we describe might provide a handle on the biochemistry and genetics of these lysosome-related organelles.