Nutrient-limited Conditions Research Articles

Adaptation mechanisms of the soil microbial community under stoichiometric imbalances and nutrient-limiting conditions in a subtropical nitrogen-saturated forest

Adaptation mechanisms of the soil microbial community under stoichiometric imbalances and nutrient-limiting conditions in a subtropical nitrogen-saturated forest

Bursts in biosynthetic gene cluster transcription are accompanied by surges of natural compound production in the myxobacterium Sorangium sp.

A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains.

Identification of a broadly conserved family of enzymes that hydrolyze (p)ppApp

Bacteria produce a variety of nucleotide second messengers to adapt to their surroundings. Although chemically similar, the nucleotides guanosine penta- and tetraphosphate [(p)ppGpp] and adenosine penta- and tetraphosphate [(p)ppApp] have distinct functions in bacteria. (p)ppGpp mediates survival under nutrient-limiting conditions and its intracellular levels are regulated by synthetases and hydrolases belonging to the RelA-SpoT homolog (RSH) family of enzymes. By contrast, (p)ppApp is not known to be involved in nutrient stress responses and is synthesized by RSH-resembling toxins that inhibit the growth of bacterial cells. However, it remains unclear whether there exists a family of hydrolases that specifically act on (p)ppApp to reverse its toxic effects. Here, we present the structure and biochemical characterization of adenosine 3'-pyrophosphohydrolase 1 (Aph1), the founding member of a monofunctional (p)ppApp hydrolase family of enzymes. Our work reveals that Aph1 adopts a histidine-aspartate (HD)-domain fold characteristic of phosphohydrolase metalloenzymes and its activity mitigates the growth inhibitory effects of (p)ppApp-synthesizing toxins. Using an informatic approach, we identify over 2,000 putative (p)ppApp hydrolases that are widely distributed across bacterial phyla and found in diverse genomic contexts, and we demonstrate that 12 representative members hydrolyze ppApp. In addition, our in silico analyses reveal a unique molecular signature that is specific to (p)ppApp hydrolases, and we show that mutation of two residues within this signature broadens the specificity of Aph1 to promiscuously hydrolyze (p)ppGpp invitro. Overall, our findings indicate that like (p)ppGpp hydrolases, (p)ppApp hydrolases are widespread in bacteria and may play important and underappreciated role(s) in bacterial physiology.

Targeting dormant phenotype acquired mycobacteria using natural products by exploring its important targets: In vitro and in silico studies.

The dormant phenotype of Mycobacterium tuberculosis that develops during infection poses a major challenge in disease treatment, since these bacilli show tolerance to front-line drugs. An in vitro hypoxia dormancy model was established, which produced phenotypically dormant Mycobacterium smegmatis after prolonged incubation under conditions of low oxygen, low pH, and nutrient limitation. Bacilli in this model displayed the classical dormancy characters, including loss of acid fastness, altered morphology, and, most importantly, tolerance to front-line drugs. The dormant form of M. smegmatis was treated with drugs and phytomolecules. Three phytomolecules exhibited activity against dormant bacilli, as shown by lack of regrowth in solid and liquid media. Further investigation of dormancy-active hits was carried out using in silico approaches to understand the druggable targets of these phytomolecules in dormant bacilli. For this study, molecular docking, molecular dynamics simulations (MDS), and molecular mechanics-generalized born solvent accessibility (MM-GBSA)-based binding energy (ΔGbind) calculations were performed. Five different targets, namely, isocitrate lyase (ICL), GMP synthase, LuxR, DosR, and serine/threonine protein kinase (STPK), from M. smegmatis and M. tuberculosis were studied in details. DosR and STPK were found to be the common targets in both the species that were more prone to the phytomolecules. The standard DosR inhibitor, HC104A, showed a lower dock score and binding energy of -4.27 and -34.50 kcal/mol, respectively, compared to the natural products under study. The phytomolecule, icariin, showed better docking score (dock score = -5.92 kcal/mol with and binding energy ΔGbind= -52.96 kcal/mol) with DosR compared to known DosR inhibitor, HC104A (dock score = -4.27 kcal/mol and binding energy ΔGbind = -34.50 kcal/mol). Similarly, the known STPK inhibitor MRCT67127 showed a lower dock score and binding energy of -4.25 and -29.43 kcal/mol, respectively, compared to the phytomolecule, icariin (dock score = -5.74 kcal/mol and ΔGbind= -43.41 kcal/mol). These compounds might ultimately lead to new therapeutics or may be useful as adjuvants to the first-line drugs to reduce the lengthy anti-TB therapy in the future.

Biofilm formation is correlated with low nutrient and simulated microgravity conditions in a Burkholderia isolate from the ISS water processor assembly

The International Space Station (ISS) Water Processor Assembly (WPA) experiences intermittent dormancy in the WPA wastewater tank during water recycling events which promotes biofilm formation within the system. In this work we aimed to gain a deeper understanding of the impact of nutrient limitation on bacterial growth and biofilm formation under microgravity in support of biofilm mitigation efforts in exploration water recovery systems. A representative species of bacteria that is commonly cultured from the ISS WPA was cultured in an WPA influent water ersatz formulation tailored for microbiological studies. An isolate of Burkholderia contaminans was cultured under a simulated microgravity (SμG) treatment in a vertically rotating high-aspect rotating vessel (HARV) to create the low shear modeled microgravity (LSMMG) environment on a rotating wall vessel (RWV), with a rotating control (R) in the horizontal plane at the predetermined optimal rotation per minute (rpm) speed of 20. Over the course of the growth curve, the bacterial culture in ersatz media was harvested for bacterial counts, and transcriptomic and nutrient content analyses. The cultures under SμG treatment showed a transcriptomic signature indicative of nutrient stress and biofilm formation as compared to the R control treatment. Further analysis of the WPA ersatz over the course of the growth curve suggests that the essential nutrients of the media were consumed faster in the early stages of growth for the SμG treatment and thus approached a nutrient limited growth condition earlier than in the R control culture. The observed limited nutrient response may serve as one element to explain a moderate enhancement of adherent biofilm formation in the SμG treatment after 24 h. While nutrients levels can be modulated, one implication of this investigation is that biofilm mitigation in the ISS environment could benefit from methods such as mixing or the maintenance of minimum flow within a dormant water system in order to force convection and offset the response of microbes to the secondary effects of microgravity.

Neisseria meningitidis Sibling Small Regulatory RNAs Connect Metabolism with Colonization by Controlling Propionate Use.

Neisseria meningitidis (meningococcus) colonizes the human nasopharynx, primarily as a commensal, but sporadically causing septicemia and meningitis. During colonization and invasion, it encounters different niches with specific nutrient compositions. Small noncoding RNAs (sRNAs) are used to fine-tune expression of genes, allowing adaptation to their physiological differences. We have previously characterized sRNAs (Neisseria metabolic switch regulators [NmsRs]) controlling switches between cataplerotic and anaplerotic metabolism. Here, we extend the NmsR regulon by studying methylcitrate lyase (PrpF) and propionate kinase (AckA-1) involved in the methylcitrate cycle and serine hydroxymethyltransferase (GlyA) and 3-hydroxyacid dehydrogenase (MmsB) involved in protein degradation. These proteins were previously shown to be dysregulated in a ΔnmsRs strain. Levels of transcription of target genes and NmsRs were assessed by reverse transcriptase quantitative PCR (RT-qPCR). We also used a novel gene reporter system in which the 5' untranslated region (5' UTR) of the target gene is fused to mcherry to study NmsRs-target gene interaction in the meningococcus. Under nutrient-rich conditions, NmsRs downregulate expression of PrpF and AckA-1 by direct interaction with the 5' UTR of their mRNA. Overexpression of NmsRs impaired growth under nutrient-limiting growth conditions with pyruvate and propionic acid as the only carbon sources. Our data strongly suggest that NmsRs downregulate propionate metabolism by lowering methylcitrate enzyme activity under nutrient-rich conditions. Under nutrient-poor conditions, NmsRs are downregulated, increasing propionate metabolism, resulting in higher tricarboxylic acid (TCA) activities. IMPORTANCE Neisseria meningitidis colonizes the human nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that coinhabit the human nasopharynx can be utilized by meningococci for replication in this environment. Here, we showed that sibling small RNAs, designated NmsRs, riboregulate propionic acid utilization by meningococci and, thus, colonization. Under conditions mimicking the nasopharyngeal environment, NmsRs are downregulated. This leads to the conversion of propionic acid to pyruvate and succinate, resulting in higher tricarboxylic acid cycle activity, allowing colonization of the nasopharynx. NmsRs link metabolic state with colonization, which is a crucial step on the trajectory to invasive meningococcal disease.

The phospholipid flippase ATP9A enhances macropinocytosis to promote nutrient starvation tolerance in hepatocellular carcinoma.

Macropinocytosis is an effective strategy to mitigate nutrient starvation. It can fuel cancer cell growth in nutrient-limited conditions. However, whether and how macropinocytosis contributes to the rapid proliferation of hepatocellular carcinoma cells, which frequently experience an inadequate nutrient supply, remains unclear. Here, we demonstrated that nutrient starvation strongly induced macropinocytosis in some hepatocellular carcinoma cells. It allowed the cells to acquire extracellular nutrients and supported their energy supply to maintain rapid proliferation. Furthermore, we found that the phospholipid flippase ATP9A was critical for regulating macropinocytosis in hepatocellular carcinoma cells and that high ATP9A levels predicted a poor outcome for patients with hepatocellular carcinoma. ATP9A interacted with ATP6V1A and facilitated its transport to the plasma membrane, which promoted plasma membrane cholesterol accumulation and drove RAC1-dependent macropinocytosis. Macropinocytosis inhibitors significantly suppressed the energy supply and proliferation of hepatocellular carcinoma cells characterised by high ATP9A expression under nutrient-limited conditions. These results have revealed a novel mechanism that overcomes nutrient starvation in hepatocellular carcinoma cells and have identified the key regulator of macropinocytosis in hepatocellular carcinoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Constitutive activation of TORC1 signalling attenuates virulence in the cross-kingdom fungal pathogen Fusarium oxysporum.

The filamentous fungus Fusarium oxysporum causes vascular wilt disease in a wide range of plant species and opportunistic infections in humans. Previous work suggested that invasive growth in this pathogen is controlled by environmental cues such as pH and nutrient status. Here we investigated the role of Target Of Rapamycin Complex 1 (TORC1), a global regulator of eukaryotic cell growth and development. Inactivation of the negative regulator Tuberous Sclerosis Complex 2 (Tsc2), but not constitutive activation of the positive regulator Gtr1, in F.oxysporum resulted in inappropriate activation of TORC1 signalling under nutrient-limiting conditions. The tsc2Δ mutants showed reduced colony growth on minimal medium with different nitrogen sources and increased sensitivity to cell wall or high temperature stress. Furthermore, these mutants were impaired in invasive hyphal growth across cellophane membranes and exhibited a marked decrease in virulence, both on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, invasive hyphal growth in tsc2Δ strains was rescued by rapamycin-mediated inhibition of TORC1. Collectively, these results reveal a key role of TORC1 signalling in the development and pathogenicity of F.oxysporum and suggest new potential targets for controlling fungal infections.

Cell Plasticity of Marine Mediterranean Diazotrophs to Climate Change Factors and Nutrient Regimes

Ocean acidification and warming are current global challenges that marine diazotrophs must cope with. Little is known about the effects of pH and temperature changes at elevated CO2 levels in combination with different nutrient regimes on N2 fixers, especially on heterotrophic bacteria. Here, we selected four culturable diazotrophs, i.e., cyanobacteria and heterotrophic bacteria, found in association with the endemic Mediterranean seagrass Posidonia oceanica. We tested different pH (from pH 4 to 8) and temperature levels (from 12 to 30 °C), under different nutrient concentrations of both phosphorus, P (0.1 µM and 1.5 mM), and iron, Fe (2 nM and 1 µM). We also tested different CO2 concentrations (410 and 1000 particles per million (ppm)) under different P/Fe and temperature values (12, 18, and 24 °C). Heterotrophic bacteria were more sensitive to changes in pH, temperature, and CO2 than the cyanobacterial species. Cyanobacteria were resistant to very low pH levels, while cold temperatures stimulated the growth in heterotrophic bacteria but only under nutrient-limited conditions. High CO2 levels (1000 ppm) reduced heterotrophic growth only when cultures were nutrient-limited, regardless of temperature. In contrast, cyanobacteria were insensitive to elevated CO2 levels, independently of the nutrient and temperature levels. Changes in N2 fixation were mainly controlled by changes in growth. In addition, we suggest that alkaline phosphatase activity (APA) and reactive oxidative species (ROS) can be used as biomarkers to assess the plasticity of these communities to climate change factors. Unlike other studies, the novelty of this work lies in the fact that we compared the responses of cyanobacteria vs. heterotrophic bacteria, studying which changes occur at the cell plasticity level. Our results suggest that the responses of diazotrophs to climate change may depend on their P and Fe status and lifestyle, i.e., cyanobacteria or heterotrophic bacteria.

Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosis

Macropinocytosis is an actin-dependent mode of nonselective endocytosis that mediates the uptake of extracellular fluid-phase cargoes. It is now well recognized that tumor cells exploit macropinocytosis to internalize macromolecules that can be catabolized and used to support cell growth and proliferation under nutrient-limiting conditions. Therefore, the identification of molecular mechanisms that control macropinocytosis is fundamental to the understanding of the metabolic adaptive landscape of tumor cells. Here, we report that the acetyl-CoA-producing enzyme, ATP citrate lyase (ACLY), is a key regulator of macropinocytosis and describes a heretofore-unappreciated association of ACLY with the actin cytoskeleton. The cytoskeletal tethering of ACLY is required for the spatially defined acetylation of heterodimeric actin capping protein, which we identify as an essential mediator of the actin remodeling events that drive membrane ruffling and macropinocytosis. Furthermore, we identify a requirement for mitochondrial-derived citrate, an ACLY substrate, for macropinocytosis, and show that mitochondria traffic to cell periphery regions juxtaposed to plasma membrane ruffles. Collectively, these findings establish a mode of metabolite compartmentalization that supports the spatiotemporal modulation of membrane-cytoskeletal interactions required for macropinocytosis by coupling regional acetyl-CoA availability with dynamic protein acetylation.

Protecting nature's most effective carbon sink: an important role for fungi in peatlands.

This article is a Commentary on Shao et al. (2023), 238: 80–95.

Identification of determinants for entering into a viable but nonculturable state in Vibrio alginolyticus by Tn-seq.

The viable but nonculturable (VBNC) state is a dormant state of nonsporulating bacteria that enhances survival in adverse environments. Systematic genome-wide research on the genetic basis of VBNC formation is warranted. In this study, we demonstrated that the marine bacterium Vibrio alginolyticus lost culturability but remained viable and entered into the VBNC state when exposed to low nutrient concentrations for prolonged periods of time. Using transposon-insertion sequencing (Tn-seq), we identified 635 determinants governing the formation of the VBNC state, including 322 genes with defective effects on VBNC formation and 313 genes contributing to entry into the VBNC state. Tn-seq analysis revealed that genes involved in various metabolic pathways were shown to have an inhibitory effect on VBNC formation, while genes related to chemotaxis or folate biosynthesis promoted entry into the VBNC state. Moreover, the effects of these genes on the formation of VBNC were validated with the growth of deletion mutants of eight selected genes under nutrient-limited conditions. Interestingly, fleQ and pyrI were identified as essential for entry into the VBNC state, and they affected the formation of the VBNC state independent of RpoE or ToxR regulation. Collectively, these results provide new insights into the mechanism of VBNC formation. KEY POINTS: • Vibrio alginolyticushas the ability to enter into the VBNC state under low nutrient conditions at lowtemperature. • The 635 determinants for entry into the VBNC state were systematically identified bytransposon-insertion sequencing. • PyrI and FleQ were validated to play significant roles in the formation of the VBNC state.

Role of Stress-Induced Proteins RpoS and YicC in the Persistence of Salmonella enterica subsp. enterica Serotype Typhimurium in Tomato Plants.

Understanding the functional role of bacterial genes in the persistence of Salmonella in plant organs can facilitate the development of agricultural practices to mitigate food safety risks associated with the consumption of fresh produce contaminated with Salmonella spp. Our study showed that Salmonella enterica subsp. enterica serotype Typhimurium (strain MDD14) persisted less in inoculated tomato plants than other Salmonella Typhimurium strains tested (JSG210, JSG626, JSG634, JSG637, JSG3444, and EV030415; P < 0.01). In-vitro assays performed in limited-nutrient conditions (growth rate, biofilm production, and motility) were inconclusive in explaining the in-planta phenotype observed with MDD14. Whole-genome sequencing combined with non-synonymous single nucleotide variations analysis was performed to identify genomic differences between MDD14 and the other Salmonella Typhimurium strains. The genome of MDD14 contained a truncated version (123 bp N-terminal) of yicC and a mutated version of rpoS (two non-synonymous substitutions, i.e., G66E and R82C), which are two stress-induced proteins involved in iron acquisition, environmental sensing, and cell envelope integrity. The rpoS and yicC genes were deleted in Salmonella Typhimurium JSG210 with the Lambda Red recombining system. Both mutants had limited persistence in tomato plant organs, similar to that of MDD14. In conclusion, we demonstrated that YicC and RpoS are involved in the persistence of Salmonella in tomato plants in greenhouse conditions and, thus, could represent potential targets to mitigate persistence of Salmonella spp. in planta. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Pseudomonas aeruginosa RpoH (σ32) Regulon and Its Role in Essential Cellular Functions, Starvation Survival, and Antibiotic Tolerance.

The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.

Nutritional and tissue-specific regulation of cytochrome P450 CYP711A MAX1 homologues and strigolactone biosynthesis in wheat.

Strigolactones (SLs) are a class of phytohormones regulating branching/tillering, and their biosynthesis has been associated with nutritional signals and plant adaptation to nutrient-limiting conditions. The enzymes in the SL biosynthetic pathway downstream of carlactone are of interest as they are responsible for structural diversity in SLs, particularly cytochrome P450 CYP711A subfamily members, such as MORE AXILLARY GROWTH1 (MAX1) in Arabidopsis. We identified 13 MAX1 homologues in wheat, clustering in four clades and five homoeologous subgroups. The utilization of RNA-sequencing data revealed a distinct expression pattern of MAX1 homologues in above- and below-ground tissues, providing insights into the distinct roles of MAX1 homologues in wheat. In addition, a transcriptional analysis showed that SL biosynthetic genes were systematically regulated by nitrogen supply. Nitrogen limitation led to larger transcriptional changes in the basal nodes than phosphorus limitation, which was consistent with the observed tillering suppression, as wheat showed higher sensitivity to nitrogen. The opposite was observed in roots, with phosphorus limitation leading to stronger induction of most SL biosynthetic genes compared with nitrogen limitation. The observed tissue-specific regulation of SL biosynthetic genes in response to nutritional signals is likely to reflect the dual role of SLs as rhizosphere signals and branching inhibitors.

In vivo characterisation of the Vibrio vulnificus stressosome: A complex involved in reshaping glucose metabolism and motility regulation, in nutrient- and iron-limited growth conditions.

Stressosomes are signal-sensing and integration hubs identified in many bacteria. At present, the role of the stressosome has only been investigated in Gram-positive bacteria. This work represents the first in vivo characterisation of the stressosome in a Gram-negative bacterium, Vibrio vulnificus. Previous in vitro characterisation of the complex has led to the hypothesis of a complex involved in iron metabolism and control of c-di-GMP levels. We demonstrate that the stressosome is probably involved in reshaping the glucose metabolism in Fe- and nutrient-limited conditions and mutations of the locus affect the activation of the glyoxylate shunt. Moreover, we show that the stressosome is needed for the transcription of fleQ and to promote motility, consistent with the hypothesis that the stressosome is involved in regulating c-di-GMP. This report highlights the potential role of the stressosome in a Gram-negative bacterium, with implications for the metabolism and motility of this pathogen.

4EBP1 senses extracellular glucose deprivation and initiates cell death signaling in lung cancer

Nutrient-limiting conditions are common during cancer development. The coordination of cellular glucose levels and cell survival is a fundamental question in cell biology and has not been completely understood. 4EBP1 is known as a translational repressor to regulate cell proliferation and survival by controlling translation initiation, however, whether 4EBP1 could participate in tumor survival by other mechanism except for translational repression function, especially under glucose starvation conditions remains unknown. Here, we found that protein levels of 4EBP1 was up-regulated in the central region of the tumor which always suffered nutrient deprivation compared with the peripheral region. We further discovered that 4EBP1 was dephosphorylated by PTPMT1 under glucose starvation conditions, which prevented 4EBP1 from being targeted for ubiquitin-mediated proteasomal degradation by HERC5. After that, 4EBP1 translocated to cytoplasm and interacted with STAT3 by competing with JAK and ERK, leading to the inactivation of STAT3 in the cytoplasm, resulting in apoptosis under glucose withdrawal conditions. Moreover, 4EBP1 knockdown increased the tumor volume and weight in xenograft models by inhibiting apoptosis in the central region of tumor. These findings highlight a novel mechanism for 4EBP1 as a new cellular glucose sensor in regulating cancer cell death under glucose deprivation conditions, which was different from its classical function as a translational repressor.

RNAi machinery regulates nutrient metabolism and fluconazole resistance in the pathogenic fungus Cryptococcus deneoformans.

The RNAi machinery has been extensively studied in plant and animal cells for their crucial roles in the regulation of genome function. However, the potential roles of RNAi in controlling fungal growth and development have been poorly studied, especially in the basidiomycetous yeast Cryptococcus deneoformans. To characterize the biological functions of RNAi in the pathogenic fungus, a comparative analysis of mRNA profiles using high-throughput sequencing technology was performed for the wild type and the RNAi mutants of C. deneoformans. The results revealed a clear difference in the expression of genes associated with metabolic processes in the RNAi mutants. Besides, the growth under nutrient-limited conditions was significantly reduced in the ago2Δ mutant, suggesting the essential roles of Ago2 in nutrient metabolism. Further investigations revealed the differentially expressed transporters in the RNAi mutants, in which transporters involved in fluconazole efflux were significantly up-regulated. More importantly, on account of the upregulated transporters, RNAi mutant strains developed resistance to fluconazole. By disrupting AFR1 gene using the 'suicide' CRISPR-Cas9 system, we verified that the upregulated ABC transporter Afr1 in the RNAi mutants contributed to the fluconazole resistance. In summary, our data demonstrate that in C. deneoformans the RNAi pathway participates in nutrient metabolism and plays a role in the repression of fluconazole resistance, which provides a deep insight into RNAi mechanisms in Cryptococcus and brings great hints for the clinical treatment of cryptococcosis.

Large-Scale CRISPRi and Transcriptomics of Staphylococcus epidermidis Identify Genetic Factors Implicated in Lifestyle Versatility.

Staphylococcus epidermidis is a ubiquitous human commensal skin bacterium that is also one of the most prevalent nosocomial pathogens. The genetic factors underlying this remarkable lifestyle plasticity are incompletely understood, mainly due to the difficulties of genetic manipulation, precluding high-throughput functional profiling of this species. To probe the versatility of S. epidermidis to survive across a diversity of environmental conditions, we developed a large-scale CRISPR interference (CRISPRi) screen complemented by transcriptional profiling (RNA sequencing) across 24 diverse conditions and piloted a droplet-based CRISPRi approach to enhance throughput and sensitivity. We identified putative essential genes, importantly revealing amino acid metabolism as crucial to survival across diverse environments, and demonstrated the importance of trace metal uptake for survival under multiple stress conditions. We identified pathways significantly enriched and repressed across our range of stress and nutrient-limited conditions, demonstrating the considerable plasticity of S. epidermidis in responding to environmental stressors. Additionally, we postulate a mechanism by which nitrogen metabolism is linked to lifestyle versatility in response to hyperosmotic challenges, such as those encountered on human skin. Finally, we examined the survival of S. epidermidis under acid stress and hypothesize a role for cell wall modification as a vital component of the survival response under acidic conditions. Taken together, this study integrates large-scale CRISPRi and transcriptomics data across multiple environments to provide insights into a keystone member of the human skin microbiome. Our results additionally provide a valuable benchmarking analysis for CRISPRi screens and are a rich resource for other staphylococcal researchers. IMPORTANCE Staphylococcus epidermidis is a bacteria that broadly inhabits healthy human skin, yet it is also a common cause of skin infections and bloodstream infections associated with implanted medical devices. Because human skin has many different types of S. epidermidis, each containing different genes, our goal is to determine how these different genes allow S. epidermidis to switch from healthy growth in the skin to being an infectious pathogen. Understanding this switch is critical to developing new strategies to prevent and treat S. epidermidis infections.

Sequestration of a dual function DNA-binding protein by Vibrio cholerae CRP

Although the mechanism by which the cyclic AMP receptor protein (CRP) regulates global gene transcription has been intensively studied for decades, new discoveries remain to be made. Here, we report that, during rapid growth, CRP associates with both the well-conserved, dual-function DNA-binding protein peptidase A (PepA) and the cell membrane. These interactions are not present under nutrient-limited growth conditions, due to post-translational modification of three lysines on a single face of CRP. Although coincident DNA binding is rare, dissociation from CRP results in increased PepA occupancy at many chromosomal binding sites and differential regulation of hundreds of genes, including several encoding cyclic dinucleotide phosphodiesterases. We show that PepA represses biofilm formation and activates motility/chemotaxis. We propose a model in which membrane-bound CRP interferes with PepA DNA binding. Under nutrient limitation, PepA is released. Together, CRP and free PepA activate a transcriptional response that impels the bacterium to seek a more hospitable environment. This work uncovers a function for CRP in the sequestration of a regulatory protein. More broadly, it describes a paradigm of bacterial transcriptome modulation through metabolically regulated association of transcription factors with the cell membrane.