Genome-scale fitness profile of Caulobacter crescentus grown in natural freshwater

ABSTRACTBacterial genomes evolve in complex ecosystems and are best understood in this natural context, but replicating such conditions in the lab is challenging. We used transposon sequencing to define the fitness consequences of gene disruption in the bacteriumCaulobacter crescentusgrown in natural freshwater, compared to axenic growth in common laboratory media. Gene disruptions in amino acid and nucleotide biosynthesis pathways and in metabolic substrate transport machinery impaired fitness in both lake water and defined minimal medium relative to complex peptone broth. Fitness in lake water was enhanced by insertions in genes required for flagellum biosynthesis and reduced by insertions in genes involved in biosynthesis of the holdfast surface adhesin. We further uncovered numerous hypothetical and uncharacterized genes for which disruption impaired fitness in lake water, defined minimal medium, or both. At the genome scale, the fitness profile of mutants cultivated in lake water was more similar to that in complex peptone broth than in defined minimal medium. Microfiltration of lake water did not significantly affect the terminal cell density or the fitness profile of the transposon mutant pool, suggesting thatCaulobacterdoes not strongly interact with other microbes in this ecosystem on the measured timescale. Fitness of select mutants with defects in cell surface biosynthesis and environmental sensing were significantly more variable in lake water than in defined medium, presumably owing to day-to-day heterogeneity in the lake environment. This study reveals genetic interactions betweenCaulobacterand a natural freshwater environment, and provides a new avenue to study gene function in complex ecosystems.

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Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation

Microbiology ◽

10.1099/mic.0.076091-0 ◽

2014 ◽

Vol 160 (6) ◽

pp. 1252-1266 ◽

Cited By ~ 28

Author(s):

Hassan B. Hartman ◽

David A. Fell ◽

Sergio Rossell ◽

Peter Ruhdal Jensen ◽

Martin J. Woodward ◽

...

Keyword(s):

Salmonella Enterica ◽

Energy Demand ◽

Drug Targets ◽

Minimal Medium ◽

Model Organism ◽

Metabolic Model ◽

Scale Model ◽

Glucose Minimal Medium ◽

A Genome ◽

Genome Scale

Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.

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USE OF KANAMYCIN FOR THE ISOLATION OF AUXOTROPHIC MUTANTS OF ACTINOBACILLUS MALLEI

Canadian Journal of Microbiology ◽

10.1139/m66-090 ◽

1966 ◽

Vol 12 (4) ◽

pp. 641-652 ◽

Cited By ~ 1

Author(s):

D. H. Evans

Keyword(s):

Amino Acids ◽

Nitrous Acid ◽

Minimal Medium ◽

Inhibitory Concentration ◽

Bactericidal Effect ◽

Defined Medium ◽

Chemically Defined Medium ◽

Auxotrophic Mutants ◽

Viable Cells ◽

Minimal Media

Growth of Actinobacillus mallei was inhibited by kanamycin; the minimal inhibitory concentration in a complex medium was 1.25 μg/ml and in a chemically defined medium 5 μg/ml. Higher concentrations of kanamycin had a pronounced bactericidal effect. When a suspension of cells containing 5 × 107 viable cells/ml was incubated in the presence of 20 μg/ml of kanamycin in a chemically defined medium, complete sterilization resulted after 6 hours. Cells irradiated with ultraviolet light were grown in complex or supplemental minimal media, washed, and exposed to 20 μg/ml of kanamycin in minimal medium for 4 hours. Auxotrophic mutants with requirements for tryptophane, phenylalanine, proline, and uracil were detected among the survivors of kanamycin treatment. After treatment with 0.01 M nitrous acid and growth in minimal medium supplemented with amino acids, cells were washed and then exposed to kanamycin in minimal medium. The proportion of autotrophs among the survivors varied from 1.3 to 75%. Mutants with requirements for each of the following amino acids were identified: methionine, methionine or cystine, arginine, leucine, tryptophane, histidme, and proline, with methionine-requiring mutants predominating. Exposure of mixtures of prototrophs and uracil-dependent and methionine-dependent auxotrophs to 20 μg/ml of kanamycin for 4 hours resulted in approximately 700- and 300-fold increases, respectively, in the ratio of auxotrophs to prototrophs.

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The ChvG–ChvI and NtrY–NtrX two-component systems coordinately regulate growth of Caulobacter crescentus

10.1101/2021.04.17.440287 ◽

2021 ◽

Author(s):

Benjamin J. Stein ◽

Aretha Fiebig ◽

Sean Crosson

Keyword(s):

Gene Expression ◽

Caulobacter Crescentus ◽

Genetic Interaction ◽

Response Regulator ◽

Cell Envelope ◽

Defined Medium ◽

Growth Defect ◽

Phosphoryl Transfer ◽

Signaling Systems ◽

Two Component

AbstractTwo-component signaling systems (TCSs) regulate cellular homeostasis in response to changes in the environment. Typical TCSs comprise a sensor histidine kinase and a response regulator; the kinase senses environmental conditions and relays this information via phosphoryl transfer to its cognate response regulator, which controls gene expression. Bacteria often express many TCS gene pairs that control distinct physiological processes, but the regulatory connections between TCSs remain underexplored. We have identified regulatory links between the ChvG–ChvI (ChvGI) and NtrY–NtrX (NtrYX) TCSs, which control important and often overlapping processes in α-proteobacteria, including maintenance of the cell envelope. Deletion of chvG and chvI in Caulobacter crescentus limited growth in defined medium and a selection for genetic suppressors of this growth phenotype uncovered interactions among chvGI, ntrYX, and ntrZ. We found that NtrZ, a previously uncharacterized periplasmic protein, functions upstream of the NtrY sensor kinase. We observed significant overlap in the ChvI and NtrX transcriptional regulons, which provides support for the genetic connection between ntrYX and chvGI. Our analyses indicated that the growth defect of strains lacking ChvGI is determined by the phosphorylation state of NtrX and, to some extent, by the level of the TonB-dependent receptor ChvT. To explain the genetic interaction between these TCSs, we propose a model in which NtrZ functions in the periplasm to regulate the NtrY kinase, promoting phosphorylation of NtrX and modulating the regulatory overlap between NtrX and ChvI.ImportanceTwo-component signaling systems (TCSs) enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell-envelope function in many α-proteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and uncovered significant overlap with the NtrX regulon. We revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. Finally, we identified NtrZ as a novel NtrY regulator. Our work establishes C. crescentus as an excellent model to investigate multi-level regulatory connections between ChvGI and NtrYX in α-proteobacteria.

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Genome-scale fitness profile of Caulobacter crescentus grown in natural freshwater

The ISME Journal ◽

10.1038/s41396-018-0295-6 ◽

2018 ◽

Vol 13 (2) ◽

pp. 523-536 ◽

Cited By ~ 12

Author(s):

Kristy L. Hentchel ◽

Leila M. Reyes Ruiz ◽

Patrick D. Curtis ◽

Aretha Fiebig ◽

Maureen L. Coleman ◽

...

Keyword(s):

Caulobacter Crescentus ◽

Genome Scale

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Gene network analysis identifies a central post-transcriptional regulator of cellular stress survival

eLife ◽

10.7554/elife.33684 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 10

Author(s):

Matthew Tien ◽

Aretha Fiebig ◽

Sean Crosson

Keyword(s):

Gene Expression ◽

Caulobacter Crescentus ◽

Transcriptional Regulator ◽

Functional Roles ◽

Large Gene ◽

Stress Survival ◽

Gene Network Analysis ◽

Catalase Peroxidase ◽

Genome Scale ◽

And Oxidative Stress

Cells adapt to shifts in their environment by remodeling transcription. Measuring changes in transcription at the genome scale is now routine, but defining the functional significance of individual genes within large gene expression datasets remains a major challenge. We applied a network-based algorithm to interrogate publicly available gene expression data to predict genes that serve major functional roles in Caulobacter crescentus stress survival. This approach identified GsrN, a conserved small RNA that is directly activated by the general stress sigma factor, σT, and functions as a potent post-transcriptional regulator of survival across distinct conditions including osmotic and oxidative stress. Under hydrogen peroxide stress, GsrN protects cells by base pairing with the leader of katG mRNA and activating expression of KatG catalase/peroxidase protein. We conclude that GsrN convenes a post-transcriptional layer of gene expression that serves a central functional role in Caulobacter stress physiology.

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Development of a minimal chemically defined medium for Ketogulonicigenium vulgare WSH001 based on its genome-scale metabolic model

Journal of Biotechnology ◽

10.1016/j.jbiotec.2013.10.027 ◽

2014 ◽

Vol 169 ◽

pp. 15-22 ◽

Cited By ~ 10

Author(s):

Shicun Fan ◽

Zhenyu Zhang ◽

Wei Zou ◽

Zheng Huang ◽

Jie Liu ◽

...

Keyword(s):

Metabolic Model ◽

Defined Medium ◽

Chemically Defined Medium ◽

Ketogulonicigenium Vulgare ◽

Genome Scale

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First Genome-Scale Metabolic Model of Dolosigranulum pigrum Confirms Multiple Auxotrophies

Metabolites ◽

10.3390/metabo11040232 ◽

2021 ◽

Vol 11 (4) ◽

pp. 232

Author(s):

Alina Renz ◽

Lina Widerspick ◽

Andreas Dräger

Keyword(s):

Minimal Medium ◽

Laboratory Experiments ◽

Carbon Sources ◽

Metabolic Model ◽

High Quality ◽

High Importance ◽

Manual Curation ◽

A Genome ◽

Severe Infections ◽

Genome Scale

Dolosigranulum pigrum is a quite recently discovered Gram-positive coccus. It has gained increasing attention due to its negative correlation with Staphylococcus aureus, which is one of the most successful modern pathogens causing severe infections with tremendous morbidity and mortality due to its multiple resistances. As the possible mechanisms behind its inhibition of S. aureus remain unclear, a genome-scale metabolic model (GEM) is of enormous interest and high importance to better study its role in this fight. This article presents the first GEM of D. pigrum, which was curated using automated reconstruction tools and extensive manual curation steps to yield a high-quality GEM. It was evaluated and validated using all currently available experimental data of D. pigrum. With this model, already predicted auxotrophies and biosynthetic pathways could be verified. The model was used to define a minimal medium for further laboratory experiments and to predict various carbon sources’ growth capacities. This model will pave the way to better understand D. pigrum’s role in the fight against S. aureus.

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Comparative genome-scale modelling ofStaphylococcus aureusstrains identifies strain-specific metabolic capabilities linked to pathogenicity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1523199113 ◽

2016 ◽

Vol 113 (26) ◽

pp. E3801-E3809 ◽

Cited By ~ 104

Author(s):

Emanuele Bosi ◽

Jonathan M. Monk ◽

Ramy K. Aziz ◽

Marco Fondi ◽

Victor Nizet ◽

...

Keyword(s):

Core Genome ◽

Systems Approach ◽

Vitamin B1 ◽

Bacterial Pathogen ◽

Ecological Niches ◽

Vitamin B3 ◽

Nucleotide Biosynthesis ◽

Scale Modelling ◽

Multiple Strains ◽

Genome Scale

Staphylococcus aureusis a preeminent bacterial pathogen capable of colonizing diverse ecological niches within its human host. We describe here the pangenome ofS. aureusbased on analysis of genome sequences from 64 strains ofS. aureusspanning a range of ecological niches, host types, and antibiotic resistance profiles. Based on this set,S. aureusis expected to have an open pangenome composed of 7,411 genes and a core genome composed of 1,441 genes. Metabolism was highly conserved in this core genome; however, differences were identified in amino acid and nucleotide biosynthesis pathways between the strains. Genome-scale models (GEMs) of metabolism were constructed for the 64 strains ofS. aureus. These GEMs enabled a systems approach to characterizing the core metabolic and panmetabolic capabilities of theS. aureusspecies. All models were predicted to be auxotrophic for the vitamins niacin (vitamin B3) and thiamin (vitamin B1), whereas strain-specific auxotrophies were predicted for riboflavin (vitamin B2), guanosine, leucine, methionine, and cysteine, among others. GEMs were used to systematically analyze growth capabilities in more than 300 different growth-supporting environments. The results identified metabolic capabilities linked to pathogenic traits and virulence acquisitions. Such traits can be used to differentiate strains responsible for mild vs. severe infections and preference for hosts (e.g., animals vs. humans). Genome-scale analysis of multiple strains of a species can thus be used to identify metabolic determinants of virulence and increase our understanding of why certain strains of this deadly pathogen have spread rapidly throughout the world.

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Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program

10.1101/2020.01.29.924274 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rajalakshmi Srinivasan ◽

Adhish S. Walvekar ◽

Aswin Seshasayee ◽

Sunil Laxman

Keyword(s):

Amino Acid ◽

Ribosomal Biogenesis ◽

Promoter Regions ◽

Nucleotide Biosynthesis ◽

Translation Machinery ◽

Metabolic Genes ◽

Genome Wide ◽

Genome Scale ◽

Global Growth

AbstractGrowth and starvation are considered opposite ends of a spectrum. To sustain growth, cells must manage biomolecule supply to balance constructive metabolism with high translation, through coordinated gene expression programs. Global growth programs couple increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis, and how this is collectively managed is a fundamental question. Conventionally, the role of the Gcn4/ATF4 transcription factor has been studied only in the context of amino acid starvation. However, high Gcn4/ATF4 has been observed in contexts of rapid cell proliferation, and the specific role of Gcn4 in growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum through which metabolic supply dependent sustenance of translation outputs is maintained. Integrating time-matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lys/arg enriched transcripts, which include the translation machinery. The sustained Gcn4 dependent lys/arg supply is required to maintain sufficient translation capacity, by allowing the synthesis of the translation machinery itself. Gcn4 thereby enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controled using the same Gcn4 transcriptional outputs, in entirely distinct contexts.

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