The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

Lee, Jaehyoun; Oh, Shi-Hwan; Bhattacharya, Saikat; Zhang, Ying; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

doi:10.1101/2020.06.02.130369

Cited by 3 publications

(4 citation statements)

References 53 publications

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“…Interestingly, all of these dehydrogenase multienzyme complexes sedimented as much smaller complexes than their suggested multimeric sizes. This observation is in agreement with a recently determined salt-labile structure of the PDC ( 38 ). The succinyl coenzyme A (succinyl-CoA)-synthetase α 2 β 2 complex, formed by the SucC and SucD proteins ( 39 ) from the TCA cycle, sedimented in fraction 3 (molecular weight [MW] of ∼140 kDa).…”

Section: Resultssupporting

confidence: 93%

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A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

Gerovac

Wicke

Chihara

et al. 2021

mBio

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The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.

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Section: Resultssupporting

confidence: 93%

“…For example, the PDH complex dissociated into stable smaller subcomplexes ( Fig. 5B ), which is in keeping with recent size exclusion experiments that revealed a salt-labile structure of the macromolecular complex ( 38 ). Nonetheless, in addition to the examples highlighted in Fig.…”

Section: Discussionsupporting

confidence: 89%

A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

Gerovac

Wicke

Chihara

et al. 2021

mBio

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show abstract

“…Interestingly, all of these dehydrogenase multi-enzyme complexes sedimented as much smaller complexes than by their suggested multimeric sizes. This observation is in agreement with a recently determined salt-labile structure of the PDC (38). The succinyl-CoA-synthetase α2β2 complex, formed by the SucC and SucD proteins (39) from the TCA cycle, sedimented in fraction 3 (MW of ~140 kDa).…”

Section: P Aeruginosa Proteins and Their Complexessupporting

confidence: 91%

A Grad-seq view of RNA and protein complexes inPseudomonas aeruginosaunder standard and bacteriophage predation conditions

Gerovac

Wicke

Chihara

et al. 2020

Preprint

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The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and post-transcriptional regulation through the RNA-binding proteins Hfq, RsmA and RsmN have been studied in detail, global information about stable RNA–protein complexes is currently lacking in this human pathogen. Here, we implement Gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated pRNAs. We observe specific clusters of non-coding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased co-sedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded non-coding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.IMPORTANCEStable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbour some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong co-localization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.

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“…Amino acid altering single nucleotide polymorphisms (SNPs) occurred in various unspecified oxidoreductases and known oxidases and dehydrogenases in metal-tdda-phen-treated strains. These enzymes are heavily involved in cellular respiration [ 69 , 70 ]. In situations, where electron acceptors in oxidative phosphorylation are unavailable, the protein D-lactate dehydrogenase may act as a temporary electron sink [ 71 ].…”

Section: Resultsmentioning

confidence: 99%

Antibacterial activity of metal–phenanthroline complexes against multidrug-resistant Irish clinical isolates: a whole genome sequencing approach

et al. 2022

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Antimicrobial resistance (AMR) is one of the serious global health challenges of our time. There is now an urgent need to develop novel therapeutic agents that can overcome AMR, preferably through alternative mechanistic pathways from conventional treatments. The antibacterial activity of metal complexes (metal = Cu(II), Mn(II), and Ag(I)) incorporating 1,10-phenanthroline (phen) and various dianionic dicarboxylate ligands, along with their simple metal salt and dicarboxylic acid precursors, against common AMR pathogens were investigated. Overall, the highest level of antibacterial activity was evident in compounds that incorporate the phen ligand compared to the activities of their simple salt and dicarboxylic acid precursors. The chelates incorporating both phen and the dianion of 3,6,9-trioxaundecanedioic acid (tdda) were the most effective, and the activity varied depending on the metal centre. Whole-genome sequencing (WGS) was carried out on the reference Pseudomonas aeruginosa strain, PAO1. This strain was exposed to sub-lethal doses of lead metal-tdda-phen complexes to form mutants with induced resistance properties with the aim of elucidating their mechanism of action. Various mutations were detected in the mutant P. aeruginosa genome, causing amino acid changes to proteins involved in cellular respiration, the polyamine biosynthetic pathway, and virulence mechanisms. This study provides insights into acquired resistance mechanisms of pathogenic organisms exposed to Cu(II), Mn(II), and Ag(I) complexes incorporating phen with tdda and warrants further development of these potential complexes as alternative clinical therapeutic drugs to treat AMR infections. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00775-022-01979-8.

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The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

Cited by 3 publications

References 53 publications

A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

A Grad-seq view of RNA and protein complexes inPseudomonas aeruginosaunder standard and bacteriophage predation conditions

Antibacterial activity of metal–phenanthroline complexes against multidrug-resistant Irish clinical isolates: a whole genome sequencing approach

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