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BONCAT (nascent) proteomes of starved Pseudomonas aeruginosa

ABSTRACT: We have characterized the responses of the opportunistic pathogen Pseudomonas aeruginosa to prolonged starvation for carbon or nitrogen sources, and to transitions between these states. We find that most cells survive both types of starvation for more than a week and maintain low but robustly detectable levels of protein synthesis in the absence of growth. Nitrogen-starved cells are larger, make more proteins and retain fewer ribosomes than carbon-starved cells, indicating that distinct physiological strategies are adopted during the two starvation types. We used bio-orthogonal non-canonical amino acid tagging (BONCAT) to enrich for proteins that were newly synthesised during prolonged starvation for nitrogen, during the transition from nitrogen to carbon starvation conditions, and during prolonged starvation for carbon. We found that the newly synthesized proteomes of each starvation type are distinct, although many of the most highly synthesized proteins are shared across conditions. Interestingly, we observed a temporary burst of protein synthesis as cells were transitioned between the two starvation conditions, which may reflect active remodelling of the proteome during growth arrest. Combining this dataset with quantification of total proteomes and identification of genes required for fitness using Tn-Seq, we highlight proteases and chaperones; flagellar motility; and the nitrogen-related phosphotransferase system as key fitness-impacting functions that are actively maintained by growth arrested Pseudomonas aeruginosa.

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ORGANISM(S): Pseudomonas Aeruginosa (strain Ucbpp-pa14) Bacteria

SUBMITTER: Megan Bergkessel

LAB HEAD: Megan Bergkessel

PROVIDER: PXD069190 | Pride | 2026-03-04

REPOSITORIES: Pride

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Project description:Amino acid assimilation and metabolism are crucial for bacterial growth and survival and this is particularly obvious for lactic acid bacteria (LAB) that are generally auxotroph for various amino acids. However, amino acid assimilation is poorly characterized and a complete description of the response during amino acid starvation is still lacking in LAB. In this context, the global response of the LAB model Lactococcus lactis was characterized during isoleucine starvation in batch culture. The stress was imposed by isoleucine natural consumption in an initially rich chemically defined medium. Dynamic analyses were performed both using transcriptomic and proteomic approaches. The response was found to occur gradually and could be divided into three major parts that were firstly deduced from transcriptomic analysis and generally corroborated by proteomic results: (i) a global repression of biogenic processes (transcription, translation, and carbon metabolism and transport), (ii) a specific response related to the limiting nutrient (numerous pathways belonging to carbon or nitrogen metabolism and leading to isoleucine supply were activated) and (iii) an additional response connected to oxidative stress (induction of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms such as growth rate regulation, stringent response, CodY, GlnR, and CcpA regulations, was discussed on the basis of transcriptomic data comparisons. Above the full description of L. lactis isoleucine starvation response, this work additionally provided a complex but realistic outlook of the regulation network involved in isoleucine starvation. Such integrated and comparative approach will allow, by its implementation to other regulations and environmental conditions, the whole regulatory network of L. lactis or any other microorganism to be deciphered. Batch cultivation of Lactococcus lactis IL1403 were carried out on a chemically defined medium and under controlled conditions (30 °C, pH 6.6, nitrogen atmosphere). Cell samples were harvested at steady state. Total RNA was extracted from these samples and radiolabelled cDNA were prepared and hybridized on nylon arrays. 1948 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. Samples corresponding to various growth rates were analyzed simultaneously and 3 independent repetitions were performed.

Project description:Rice blast is the most threatening disease to cultivated rice worldwide. Magnaporthe oryzae, its causal agent is likely to encounter environmental challenges during invasive growth in the host plants that require shifts in gene expression to establish a compatible interaction. Here, we tested the hypothesis that M. oryzae have similar gene expression patterns in planta, versus during in vitro stresses. Gene expression data was collected from in vitro experiments of heat shock, oxidative burst, and three different types of nutrient starvation. These data were compared to fungal gene expression during the invasive growth phase in compatible interactions on two grass hosts, rice and barley. We have identified 4,973 differentially expressed genes, from which 1,909 genes showed similar regulation patterns between at least one of the in vitro stresses and rice and/or barley. Hierarchical clustering of these 1,909 genes showed three major clusters in which growth in planta conditions closely grouped with the starving nutrient conditions. Out of these 1,909 genes, 55 and 129 genes were induced and repressed in all seven treatments, respectively. The functional categorization of those 55 induced genes revealed that most of them are either related to carbon metabolism, membrane proteins, or are involved in oxidoreduction reactions. The 129 repressed genes are putatively involved in vesicle trafficking, signal transduction, nitrogen metabolism, or molecular transport. These findings indicated that M. oryzae is likely coping with nutrient deprived environments in its hosts during the invasive growth stage about 72 hours post-inoculation, and not as much with host defense responses, or a temperature stress. Eight M. oryzae treatments: mycelia grown in liquid complete medium (set as the control); 72 hours post-incoulation (hpi) in rice leaves; 72 hours post-incoulation in barley leaves; mycelia under heat shock; mycelia under oxidative stress; mycelia under minimal medium; mycelia under nitrogen starvation; mycelia under carbon starvation. Three biological replicates had their total RNA pulled after RNA extraction. Dye-swaps used as technical replicates.

Project description:Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway leading to the expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive (pho7+) and negative (csk1+) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response for the ScPHO5 (pho1+), ScPHO84 (spbc8e4.01c+), and ScGIT1 (spbc1271.09+) orthologs. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. We provide a global analysis of the PHO pathway in S. pombe. Our results elucidate the conserved core regulon required for responding to phosphate starvation between distantly related ascomycetes and a better understanding of flexibility in environmental stress response networks. Schizosaccharomyces pombe 972 h- cells were starved for inorganic phosphate for 0, 30, 60, 120, or 240 minutes pior to microarray preparation to determine the extent and temporal resolution of the phosphate starvation response. At 120 minutes post-starvation we define a set of genes that are directly and specifically induced by phosphate starvation, providing a time-point at which all other experiments were performed. We characterize the pho7+- and csk1+- dependency of this PHO response at 120 minutes post-starvation in pho7+csk1+, pho7M-NM-^T, csk1M-NM-^T, and pho7M-NM-^Tcsk1M-NM-^T cells. In a seperate set of experiments we characterized the S. pombe stress response to copper limitation, iron limitation, and carbon switching at 120 minutes post-stress and osmotic shift at 20 minutes post-stress in both pho7+ and pho7M-NM-^T cells.

Project description:In order to provide information about the gene expression response that occurs when cells experience a change in carbon source, succinate limited chemostat cultures of Methylobacterium extorquens AM1 were grown to and maintained at an OD of ~0.63, transferred to flasks and methanol was added. Cells were harvested for RNA extraction at time: 0 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr post transition. At 30 min, a no methanol addition sample was extracted as a carbon starvation control. These data were used in conjunction with flux, enzymatic and metabolite measurements to assess the changes in central metabolism during this transition. Abstract from manuscript: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate to methanol growth by the methylotrophic bacterium Methylobacterium extorquens, analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylenetetrahydrofolate dehydrogenase as one control point. This “downstream priming” mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints. Gene expression in succinate limited chemostat cultures was compared to gene expression in cells transferred to flasks before and after methanol addition. As a control, a time = 0 sample (RNA prepared from cells harvested directly from the chemostat) was compared to a time = 0 sample immediately obtained after the cells were transferred to flasks, before methanol was added in order to identify changes due to flask transfer. A carbon starvation control was also done comparing expression from time = 0 (chemostat cells) to cells transferred to flasks for 30 min with no carbon source added. Two biological replicates each with two techinal replicates (dye swap) were analyzed for time = 0 (chemostat) vs 10 min, 30 min, 1 hr and 2 hr after methanol addition. One biological replicate with two technical replicates (dye swap) were analyzed for time = 0 (chemostat) vs time = 0 (flask transfer), and time = 0 (chemostat) vs time = 4 hr, 6 hr and 30 min no methanol addition.

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BONCAT (nascent) proteomes of starved Pseudomonas aeruginosa

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