One ligand, two regulators and three binding sites: How KDPG controls primary carbon metabolism in Pseudomonas

Campilongo, Rosaria; Fung, Rowena; Little, Richard; Grenga, Lucia; Trampari, Eleftheria; Pepe, Simona; Chandra, Govind; Stevenson, Clare E. M.; Roncarati, Davide; Malone, Jacob G.

doi:10.1371/journal.pgen.1006839

Cited by 37 publications

(52 citation statements)

References 47 publications

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“…Cloning was carried out in accordance with standard molecular biology techniques. P. fluorescens deletion mutants were constructed by allelic exchange as described previously (109). Up- and downstream flanking regions (approx.…”

Section: Methodsmentioning

confidence: 99%

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

Stefanato

Trippel

Uszkoreit

et al. 2019

Preprint

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23Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, 24 including the inhibition of plant pathogens. Pseudomonas are one of the most abundant 25 bacterial genera in the soil and rhizosphere and play important roles in promoting plant growth 26 and preventing disease. However, the genetic determinants of this beneficial activity are only 27 partially understood, especially in relation to specialized metabolite production. Here, we 28 genetically and phenotypically characterize the Pseudomonas fluorescens population in 29 commercial potato field soils and identify strong correlations between specialized metabolite 30 biosynthetic pathways and antagonism of the potato pathogens Streptomyces scabies and 31 Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic 32 lipopeptides as key specialized metabolites associated with S. scabies inhibition. We show that 33 a single potato field contains a hugely diverse and dynamic population of Pseudomonas 34 bacteria, whose capacity to produce specialized metabolites is shaped both by plant 35 colonization and defined environmental inputs. 36 2 37 CFC agar and incubated overnight at 28 °C before streaking to single colonies on King's B (KB) 595 agar plates (96). Six isolates were selected at random per soil sample and subjected to 596 phenotypic/genomic analysis. 597 598 Amplicon sequencing 599 Genomic DNA was isolated from 3 g of pooled soil samples using the FastDNA™ SPIN Kit for 600 soil (MP Biomedicals, UK) following the manufacturer's instructions. Genomic DNA 601 concentration and purity was determined by NanoDrop spectrophotometry as above. Microbial 602 16S rRNA genes were amplified from soil DNA samples with barcoded universal prokaryotic 603 23 primers (515F/R806) targeting the V4 region, and then subjected to Illumina® MiSeq 604 sequencing (600-cycle, 2x300 bp) at the DNA Sequencing Facility, Department of Biochemistry, 605

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Section: Methodsmentioning

confidence: 99%

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

Stefanato

Trippel

Uszkoreit

et al. 2019

Preprint

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“…Given this model, we speculate that there are other ways to modulate behavior in Pseudomonas with primary metabolic intermediates to achieve antibiotic effects. In 2017, Malone and co‐workers discovered a new transcriptional regulator in P. fluorescens , which they dubbed RccR . Although this enzyme bears structural similarity to the known Entner–Duodoroff pathway (discussed further in the next section) regulator HexR, and both are controlled by the concentration of the Entner–Duodoroff intermediate 2‐keto‐3‐deoxy‐phosphogluconate (KDPG, Scheme ), they are responsible for regulating entirely different aspects of Pseudomonas primary metabolism.…”

Section: Metabolic Intermediates As Probes Into Antibiotic Adjuvationmentioning

confidence: 99%

“…The infection site could also be linked to the pathogenesis of P. aeruginosa , as higher flux through the shunt has been correlated with higher levels of virulence. In light of Malone's work (detailed in the previous section), high KDPG concentrations could conversely be linked to reduced levels of virulence in Pseudomonas , as this enables RccR to bind glyoxylate‐shunt genes more strongly, thereby reducing their transcription and reducing flux through the shunt . The glyoxylate shunt also plays an important role in the ability of P. putida to resist its own endogenously produced primary‐metabolism‐targeting antibiotics (detailed in next section).…”

Section: Metabolic Flexibility In Pseudomonasmentioning

confidence: 99%

“…In 2017, Malone and co-workersdiscovered an ew transcriptional regulator in P. fluorescens,w hich they dubbed RccR. [11] Although this enzyme bears structural similarity to the known Entner-Duodoroff pathway (discussed further in the next section) regulator HexR, and both are controlled by the concentration of the Entner-Duodoroff intermediate 2-keto-3-deoxy-phosphogluconate (KDPG, Scheme 2), they are responsible for regulating entirely different aspects of Pseudomonas primarym etabolism.I nt he presence of high KDPG concentrations, RccR has high affinity for glyoxylate-shunta nd gluconeogenesis genes, thereby downregulating transcription of these pathways, and low affinity for pyruvate metabolism genes, thereby upregulating them. This dual mode of action is enabled by two distinct "pseudopalindromic" binding sites that are differentially active depending on KDPG binding.…”

Section: Metabolic Intermediates As Probes Into Antibiotic Adjuvationmentioning

confidence: 99%

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From General to Specific: Can Pseudomonas Primary Metabolism Be Exploited for Narrow‐Spectrum Antibiotics?

2018

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The spread of antimicrobial resistance is a major threat to human health, and patients requiring prolonged antibiotic exposure are in desperate need of new therapeutic strategies. It has been hypothesized that tailoring our antibiotics to inhibit molecular targets specific to pathogens might stem the spread of resistance. A prime candidate for such a strategy is Pseudomonas aeruginosa, which can be found in the lungs of nearly all adult cystic fibrosis patients and, due to chronic exposure to antibiotics, has a high rate of multidrug-resistant strains. Although much research has been done on P. aeruginosa virulence factors as narrow-spectrum targets, less attention has been paid to primary carbon metabolism being leveraged for pathogen-specific mechanisms. However, early studies show that primary metabolic pathways, although shared amongst all organisms, contain intricacies specific to Pseudomonas species that have potential for antibiotic exploitation. Here we lay out some of this work in the hopes that it inspires researchers to continue developing a knowledge base for future antibiotic discovery to build upon and include a case study of a Pseudomonas primary metabolic pathway that has been targeted by small molecules in a species-specific manner.

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“…To enable effective rhizosphere colonisation, soil bacteria sense many different environmental inputs and translate them into an integrated phenotypic response. This requires an interconnected network of signal transduction systems functioning at multiple regulatory levels, including gene transcription (17), modulation of translational activity (18) and changes in protein function (19). The cyclic-di-GMP (cdG) signalling network mediates the switch between motile and sessile lifestyles in many bacterial species (20) and is a key regulator of rhizosphere colonisation in multiple plant-associated microbes (21–24).…”

Section: Introductionmentioning

confidence: 99%

Second messenger control of mRNA translation by dynamic ribosome modification

Grenga

Chandra

et al. 2020

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8Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their 9 environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal 10 protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In 11 this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed 12 phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, 13 biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger 14 cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: 15 indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with 16 translation of surface attachment factors, amino acid transporters and key secreted molecules linked 17 specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions 18 as a rapid-response mechanism that tunes global gene translation in response to environmental signals. 19 20 42 forms a highly complex, non-linear and pleiotropic network, with multiple connections to other signalling 43 systems and phenotypic outputs that vary profoundly in response to environmental cues (25, 26). The model 44 P. fluorescens strain SBW25, for example, contains over 40 cdG-metabolic enzymes (27) that influence 45 phenotypes at every regulatory level and whose expression varies throughout rhizosphere colonisation (22). 46Pseudomonas cdG signalling shows extensive overlap with other global gene regulators, such as Gac/Rsm 47 (28, 29) and the RNA-chaperone Hfq (30). 48The small hexameric protein Hfq facilitates binding between mRNA and regulatory sRNAs (31, 32) and 49 controls biofilm formation (33), carbon catabolite repression (34), amino-acid transport (35, 36), virulence 50 (37) and motility (36). In P. fluorescens, Hfq is important for niche adaptation, with deletion mutants 51 displaying strongly reduced motility, increased surface attachment, and severely compromised rhizosphere 52 colonisation (25, 30). The regulatory connections between cdG and Hfq are reflected in the close phenotypic 53 parallels between mutants in both pathways (30, 38, 39). 54We recently identified a further contributor to the post-transcriptional regulatory network in Pseudomonas 55 spp. (30). Similar to Hfq and cdG, the ribosomal modification protein RimK controls the transition between 56 active and sessile bacterial lifestyles, with an SBW25 rimK mutant affected in motility, root attachment and 57 amino-acid uptake, and showing reduced rhizosphere colonisation efficiency. Deletion of rimK in pathogenic 58Pseudomonas spp. leads to significantly reduced virulence and cytotoxicity (30). RimK is an ATP-dependent 59 glutamyl ligase that adds glutamate residues to the C-terminus of ribosomal protein RpsF, which in-turn 60 induces specific changes in the bacterial pro...

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One ligand, two regulators and three binding sites: How KDPG controls primary carbon metabolism in Pseudomonas

Cited by 37 publications

References 47 publications

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

From General to Specific: Can Pseudomonas Primary Metabolism Be Exploited for Narrow‐Spectrum Antibiotics?

Second messenger control of mRNA translation by dynamic ribosome modification

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