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The Rcs signal transduction system is a phosphorelay responsible for sensing a wide variety of enterobacterial cell envelope stresses. In Escherichia coli, the Rcs system is required to survive A22 and mecillinam treatment, two drugs that perturb cell size. To test whether cell size changes might be correlated with envelope damage and thereby sensed by the Rcs system, we tuned E. coli cell size via drug inhibition with A22, point mutations to the cell-shape determinant MreB, and mechanically confined growth. In all conditions, cell width was strongly correlated with Rcs activation, with wider cells exhibiting more activation than wild-type. In all conditions, RcsF, the outer membrane-localized upstream component of the Rcs system, was essential for responding to cell width changes. Consistently, several envelope gene deletions known to induce the Rcs system via RcsF resulted in cells that were wider than wild-type. Cryo- electron microscopy revealed that the periplasm of a wide MreB mutant was on average ∼3 nm thinner than wild-type, thereby bringing RcsF closer to the downstream components of the signaling cascade in the inner membrane. Conversely, extending the flexible linker region of RcsF by ∼3 nm increased Rcs activity in wild-type cells. In summary, we propose that the Rcs system responds to changes in cell width because of altered periplasmic thickness.

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of PG synthases and hydrolases. InEscherichia coli, the activity of the two bifunctional PG synthases is driven by lipoproteins anchored in the outer membrane. However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form multimeric complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and cell division biosynthetic complexes based on its localization and genetic interactions. In line with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that PG regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.SignificanceThe activity of PG hydrolases may cause lysis of the bacterial cell if left unregulated. Hence, the cell must have ways of regulating and coordinating their activities. Our current understanding of how this occurs is incomplete. In this work, we present the outer membrane (OM) anchored lipoprotein, NlpI, as a scaffold of peptidoglycan hydrolases. We propose that NlpI facilitates the formation of multi-enzyme complexes and that, along with other regulators, it coordinates a safe enlargement and separation of the PG layer inE. coli.

SummaryUnderstanding how genetic variation contributes to phenotypic differences is a fundamental question in biology. Here, we set to predict fitness defects of an individual using mechanistic models of the impact of genetic variants combined with prior knowledge of gene function. We assembled a diverse panel of 696Escherichia colistrains for which we obtained genomes and measured growth phenotypes in 214 conditions. We integrated variant effect predictors to derive gene-level probabilities of loss of function for every gene across strains. We combined these probabilities with information on conditional gene essentiality in the reference K-12 strain to predict the strains’ growth defects, providing significant predictions for up to 38% of tested conditions. The putative causal variants were validated in complementation assays highlighting commonly perturbed pathways in evolution for the emergence of growth phenotypes. Altogether, our work illustrates the power of integrating high-throughput gene function assays to predict the phenotypes of individuals.HighlightsAssembled a reference panel ofE. colistrainsGenotyped and high-throughput phenotyped theE. colireference strain panelReliably predicted the impact of genetic variants in up to 38% of tested conditionsHighlighted common genetic pathways for the emergence of deleterious phenotypes

Understanding how genetic variation contributes to phenotypic differences is a fundamental question in biology. Combining high-throughput gene function assays with mechanistic models of the impact of genetic variants is a promising alternative to genome-wide association studies. Here we have assembled a large panel of 696 Escherichia coli strains, which we have genotyped and measured their phenotypic profile across 214 growth conditions. We integrated variant effect predictors to derive gene-level probabilities of loss of function for every gene across all strains. Finally, we combined these probabilities with information on conditional gene essentiality in the reference K-12 strain to compute the growth defects of each strain. Not only could we reliably predict these defects in up to 38% of tested conditions, but we could also directly identify the causal variants that were validated through complementation assays. Our work demonstrates the power of forward predictive models and the possibility of precision genetic interventions., eLife, 6, ISSN:2050-084X

International audience; Studying the evolution of macromolecular assemblies is important to improve our understanding of how complex cellular structures evolved, and to identify the functional building blocks that are involved. Recent studies suggest that the macromolecular complexes that are involved in two distinct processes in Myxococcus xanthus - surface motility and sporulation - are derived from an ancestral polysaccharide capsule assembly system. In this Opinion article, we argue that the available data suggest that the motility machinery evolved from this capsule assembly system following a gene duplication event, a change in carbohydrate polymer specificity and the acquisition of additional proteins by the motility complex, all of which are key features that distinguish the motility and sporulation systems. Furthermore, the presence of intermediates of these systems in bacterial genomes suggests a testable evolutionary model for their emergence and spread.

Advances in our ability to systematically introduce and track controlled genetic variance in microorganisms have, in the past decade, fuelled high-throughput reverse genetics approaches. When coupled to quantitative readouts, such approaches are extremely powerful at elucidating gene function and providing insights into the underlying pathways and the overall cellular network organization. Yet, until now, all efforts to quantify microbial macroscopic phenotypes have been restricted to monitoring growth in a small number of model microorganisms. We have developed an image analysis software named Iris, which allows for systematic exploration of a number of orthogonal-to-growth processes, including biofilm formation, colony morphogenesis, envelope biogenesis, sporulation and reporter activity. In addition, Iris provides more sensitive growth measurements than currently available software and is compatible with a variety of different microorganisms, as well as with endpoint or kinetic data. We used Iris to reanalyse existing chemical genomics data in Escherichia coli and to perform proof-of-principle screens on colony biofilm formation and morphogenesis of different bacterial species and the pathogenic fungus Candida albicans. We thereby recapitulated existing knowledge but also identified a plethora of additional genes and pathways involved in both processes.

Bacterial motility is ubiquitous and essential to complex lifestyles including host infection and biofilm formation. While a tremendous amount of work has been dedicated to understand the mechanism of bacterial swimming in liquid with a rotary flagellum, much less is understood about mechanisms allowing bacteria to colonize surfaces. One such mechanism, called gliding motility, allows the smooth translocation of rod-shaped bacteria along their long axis in the absence of visible external organelles. While the mechanism of gliding has long been mysterious, recent work in two model systems, Myxococcus xanthus and Flavobacterium johnsoniae have shed new light on the propulsion mechanism. In both systems, key features of the motility processes were revealed by imaging the dynamics of critical motility proteins in real-time. Generally, these studies suggest that both Myxococcus and Flavobacterium appear to use dynamic helical envelope assemblage for their movement, but there are also important differences in the motility mechanisms. In this chapter, we review recent progress on the molecular characterization of the Myxococcus and Flavobacterium motility machineries. Beyond motility, this research will likely impact our understanding of dynamic processes in the bacterial cell envelope.

The Myxococcus Agl-Nfs machinery, a type of bacterial transport system, is modular and is seen to also rotate a carbohydrate polymer directionally at the spore surface to assist spore coat assembly., Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories., Author Summary Many living cells use processive cytoskeletal motors to transport proteins and subcellular organelles to specific subcellular sites. In bacteria, this type of transport has yet to be identified and it is generally thought that random protein collisions underlie most biochemical processes. In recent years, our view of the bacterial cell was changed by the discovery of subcellular compartmentalization and a cytoskeleton, suggesting that processive motors might also operate in prokaryotes. We previously characterized a mechanism of intracellular transport that drives cell motility across solid surfaces in the gram-negative bacterium Myxococcus xanthus. Since the transport apparatus was also found in bacterial species that do not move on surfaces, we postulated that intracellular transport underlies other cellular processes in bacteria. Indeed, we show here that the Myxococcus motility motor can be adapted to transport sporulation-specific proteins around the nascent spore surface. Because the transported proteins are linked to the main spore coat, this motion assists the assembly of a protective spore coat. In conclusion, the Myxococcus motility/sporulation transport machinery defines an emerging class of versatile transport systems, suggesting that processive transport has been overlooked and may well orchestrate many processes in bacteria.

Fungi are important actors in the ecological processes occurring in mangroves and are thought to play a role in organic matter decomposition pathways, despite the fact that a large fraction of the carbon processing in mangroves happens in the anoxic bulk sediment. The fungal diversity in these strata is, however, poorly known. Here, using microscopic observations of calcofluor white stained samples, we detected the presence of fungal hyphae indicating that fungal growth was occurring under these anoxic and sulfidic conditions. To assess the fungal diversity we used 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer land 2 (ITS-1 and ITS-2). We analysed approximately 5 000 reads for each ITS, corresponding to around 115 molecular operational taxonomic units. The taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The most abundant operational taxonomic units were affiliated to: a taxon with affinity to the genus Sistotremastrum (Trechisporales), Dipodascus australiensis (Saccharomycetales), an Alternaria species (Pleosporales) and an unknown Lecanoromycete. This study indicates the presence of rich fungal communities in anoxic mangroves sediments. It also raises the question of the adaptation of these organisms to the environmental conditions and of the roles they play in organic matter decomposition processes. (C) 2011 Elsevier Ltd and The British Mycological Society. All rights reserved.

Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility.

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., PLoS Genetics, 7 (9), ISSN:1553-7390, ISSN:1553-7404