43 research outputs found
Emergence and Modular Evolution of a Novel Motility Machinery in Bacteria
Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena
CnoX Is a Chaperedoxin: A Holdase that Protects Its Substrates from Irreversible Oxidation.
Bleach (HOCl) is a powerful oxidant that kills bacteria in part by causing protein aggregation. It inactivates ATP-dependent chaperones, rendering cellular proteins mostly dependent on holdases. Here we identified Escherichia coli CnoX (YbbN) as a folding factor that, when activated by bleach via chlorination, functions as an efficient holdase, protecting the substrates of the major folding systems GroEL/ES and DnaK/J/GrpE. Remarkably, CnoX uniquely combines this function with the ability to prevent the irreversible oxidation of its substrates. This dual activity makes CnoX the founding member of a family of proteins, the "chaperedoxins." Because CnoX displays a thioredoxin fold and a tetratricopeptide (TPR) domain, two structural motifs conserved in all organisms, this investigation sets the stage for the discovery of additional chaperedoxins in bacteria and eukaryotes that could cooperate with proteins from both the Hsp60 and Hsp70 families
The Chaperone and Redox Properties of CnoX Chaperedoxins Are Tailored to the Proteostatic Needs of Bacterial Species.
Hypochlorous acid (bleach), an oxidizing compound produced by neutrophils, turns the chaperedoxin CnoX into a powerful holdase protecting its substrates from bleach-induced aggregation. CnoX is well conserved in bacteria, even in non-infectious species unlikely to encounter this oxidant, muddying the role of CnoX in these organisms. Here, we found that CnoX in the non-pathogenic aquatic bacterium functions as a holdase that efficiently protects 50 proteins from heat-induced aggregation. Remarkably, the chaperone activity of CnoX is constitutive. Like CnoX, CnoX transfers its substrates to DnaK/J/GrpE and GroEL/ES for refolding, indicating conservation of cooperation with GroEL/ES. Interestingly, CnoX exhibits thioredoxin oxidoreductase activity, by which it controls the redox state of 90 proteins. This function, which CnoX lacks, is likely welcome in a bacterium poorly equipped with antioxidant defenses. Thus, the redox and chaperone properties of CnoX chaperedoxins were fine-tuned during evolution to adapt these proteins to the specific needs of each species. How proteins are protected from stress-induced aggregation is a crucial question in biology and a long-standing mystery. While a long series of landmark studies have provided important contributions to our current understanding of the proteostasis network, key fundamental questions remain unsolved. In this study, we show that the intrinsic features of the chaperedoxin CnoX, a folding factor that combines chaperone and redox protective function, have been tailored during evolution to fit to the specific needs of their host. Whereas CnoX needs to be activated by bleach, a powerful oxidant produced by our immune system, its counterpart in , a bacterium living in bleach-free environments, is a constitutive chaperone. In addition, the redox properties of and CnoX also differ to best contribute to their respective cellular redox homeostasis. This work demonstrates how proteins from the same family have evolved to meet the needs of their hosts
Turning Escherichia coli into a Frataxin-Dependent Organism
Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich’s ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the “eukaryotic-like” IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscUIM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold
Evolution and Design Governing Signal Precision and Amplification in a Bacterial Chemosensory Pathway
International audienceUnderstanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A-and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways
Activities of Fe-S proteins in <i>iscU</i><sub><i>IM</i></sub> and Δ<i>cyaY</i> strains.
<p>Repression of the IscR-regulated gene (<i>iscR</i>::<i>lacZ</i>) (A), Nuo (B) and Sdh (C) activities in the wt (DV901) (white bars), <i>iscU</i><sub><i>IM</i></sub> (BR755) (white bars), their <i>ΔcyaY</i> derivatives (DV925, BR756) (black bars), and Δ<i>iscU</i> (BR667) (grey bars) strains. The amount of IscR-dependent repression (fold repression) was determined by dividing the β-galactosidase activity present in the strain lacking IscR (DV915) by the β-galactosidase activity measured for each strain. Error bars represent the standard error from three independent experiments. (D) Cell extracts of indicated strains were subjected to immunoblot analysis using antibodies raised against IscU, IscR, NuoF and NuoC.</p
The <i>iscU</i><sub><i>IM</i></sub> Δ<i>suf</i> Δ<i>cyaY</i> strain is hypersensitive to oxidative stress.
<p>The wt (DV901), <i>iscU</i><sub><i>IM</i></sub> (BR755), <i>iscU</i><sub><i>IM</i></sub><i>ΔcyaY</i> (BR756), <i>iscU</i><sub><i>IM</i></sub><i>Δsuf</i> (BR763) and <i>iscU</i><sub><i>IM</i></sub><i>Δsuf ΔcyaY</i> (BR767) strains were grown overnight at 37°C in LB medium. Cultures were diluted in sterile PBS, and 5 μL were directly spotted onto LB medium plates containing either 1 mM H<sub>2</sub>O<sub>2</sub> or 250 μM paraquat. Growth was analysed after overnight incubation at 37°C. Each spot represents a 10-fold serial dilution.</p
Bacterial strains and plasmids used in this study.
<p>Bacterial strains and plasmids used in this study.</p
Model for CyaY protein evolution.
<p>Schematic representation of the universal tree of life, for which complete genome sequences are available. LUCA (Last Universal Common Ancestor), LECA (Last Eukaryotic Common Ancestor), LACA (Last Archaeal Common Ancestor) and LBCA (Last Bacterial Common Ancestor). For each prokaryotic phylum (whose color code is the same as the one used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005134#pgen.1005134.s005" target="_blank">S5 Fig</a>), the number of genomes encoding a CyaY and a IscU homolog with respect to the number of complete available genomes is given. The black arrow indicates the presence of a CyaY encoding gene in the ancestor of a given lineage. The evolutionary event at the origin of the <i>cyaY</i> gene in the Delta/Epsilon subgroup cannot be definitively inferred. One hypothesis is that the <i>cyaY</i> gene is originated in the common ancestor of the Proteobacteria which together with a probable massive loss of <i>cyaY</i> (#) in Delta/Epsilonproteobacteria subgroup explains the presence of CyaY in the species of the Delta/Epsilonproteobacteria subgroup. Dotted arrows indicate horizontal gene transfer events (HGT) (black) and the mitochondrial endosymbiosis (grey). Sequence-logo of the region 99–108 in IscU homologs is also represented using Phylo-mLogo. This region contains the LPPVK motif and amino acid residues at position 108.</p