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

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>_<i>IM</i> 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>_<i>IM</i> (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

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Analysis of IscU_IM<i>in vitro</i>.

<p>(A) Comparison of the CD spectra (expressed in mdeg) recorded in the region 190–250 nm between IscU_WT (filled line) and IscU_IM (dotted line). (B) Purified IscS, CyaY and IscU_WT (left panel) or IscU_IM (right panel) were mixed in 1:1:1 ratio (144 μM of each protein) in the presence of 4-fold excess of Fe(SO₄)₂(NH₄)₂, 10-fold excess of L-cysteine and 5 mM DTT and incubated for 40 minutes. The mixture was then loaded onto a QFF column equilibrated with 50 mM Tris pH 8. Proteins were eluted with 50 mM Tris pH 8 containing 1M NaCl. SDS-PAGE analyses have been performed on samples from the column on-put (0) and the peaks 1 and 2 for each mixture. (C) Reconstitution of [2Fe-2S] IscU_WT (filled line) and IscU_IM (dotted line) followed by UV-visible absorption spectroscopy. Apo-IscU_WT or apo-IscU_IM (144 μM) were incubated with 5 mM DTT, 1.44 μM IscS, 2 mM L-cysteine and 0.43 mM Fe(SO₄)₂(NH₄)₂ in 50 mM Tris-HCl pH 8. (D) Comparison of the kinetics of enzymatic Fe-S cluster formation on IscU_WT (black diamonds) and IscU_IM (white squares). Experiment was carried out using 25 μM IscU_WT or IscU_IM, 25 μM IscS, 100 μM Fe(SO₄)₂(NH₄)₂, 250 μM L-cysteine, 2 mM DTT. Fe-S cluster formation was followed by absorbance at 420 nm. The experiment was repeated at least three times. One representative experiment is shown.</p

The <i>iscU</i>_<i>IM</i> Δ<i>cyaY</i> strain is resistant to aminoglycosides.

<p>Survival of wt (DV901), <i>iscU</i>_<i>IM</i> (BR755) and their Δ<i>cyaY</i> derivatives (DV925 and BR756) without antibiotic (A) and after (B) Gentamicin (Gm) (5 μg/mL) and Kanamycin (Kan) (10 μg/mL) (C) treatment. Survival, measured by colony-forming units (CFU) per mL, was normalized relative to time zero at which the antibiotic was added (midexponential phase cells; ~5 ×10⁷ CFU/mL) and was plotted as log_<i>10</i> of % survival. Error bars represent the standard error from three independent experiments.</p

Functional Organization of a Multimodular Bacterial Chemosensory Apparatus

<div><p>Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium <i>Myxococcus xanthus</i>, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. <i>M. xanthus</i> contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on <i>M. xanthus</i> social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, <i>in vivo</i>, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.</p></div

Genetic clusters carrying <i>che</i> genes in <i>M. xanthus</i>.

<p>Genetic organization of the genes composing the eight <i>che</i> clusters encoding the putative components of the chemosensory apparatus in <i>Myxococcus xanthus</i>. Predicted genes are indicated with their locus_tag, and their annotations and assigned names. The color code indicates homologous genes.</p