Lotte Søgaard-Andersen

The RNA-Binding Protein Hfq of Listeria monocytogenes: Role in Stress Tolerance and Virulence

In gram-negative bacteria, the RNA-binding protein Hfq has emerged as an important regulatory factor in a variety of physiological processes, including stress resistance and virulence. In Escherichia coli, Hfq modulates the stability or the translation of mRNAs and interacts with numerous small regulatory RNAs. Here, we studied the role of Hfq in the stress tolerance and virulence of the gram-positive food-borne human pathogen Listeria monocytogenes. We present evidence that Hfq is involved in the ability of L. monocytogenes to tolerate osmotic and ethanol stress and contributes to long-term survival under amino acid-limiting conditions. However, Hfq is not required for resistance to acid and oxidative stress. Transcription of hfq is induced under various stress conditions, including osmotic and ethanol stress and at the entry into the stationary growth phase, thus supporting the view that Hfq is important for the growth and survival of L. monocytogenes in harsh environments. The stress-inducible transcription of hfq depends on the alternative sigma factor sigmaB, which controls the expression of numerous stress- and virulence-associated genes in L. monocytogenes. Infection studies showed that Hfq contributes to pathogenesis in mice, yet plays no role in the infection of cultured cell lines. This study provides, for the first time, information on the role of Hfq in the stress tolerance and virulence of a gram-positive pathogen.

Bacterial solutions to multicellularity: a tale of biofilms, filaments and fruiting bodies

Although bacteria frequently live as unicellular organisms, many spend at least part of their lives in complex communities, and some have adopted truly multicellular lifestyles and have abandoned unicellular growth. These transitions to multicellularity have occurred independently several times for various ecological reasons, resulting in a broad range of phenotypes. In this Review, we discuss the strategies that are used by bacteria to form and grow in multicellular structures that have hallmark features of multicellularity, including morphological differentiation, programmed cell death and patterning. In addition, we examine the evolutionary and ecological factors that lead to the wide range of coordinated multicellular behaviours that are observed in bacteria.

The FruA signal transduction protein provides a checkpoint for the temporal co-ordination of inter- cellular signals in Myxococcus xanthus development

During fruiting body morphogenesis in Myxococcus xanthus, the intercellular C‐signal induces aggregation, sporulation and developmental gene expression. To understand how a single signal system may induce temporally separated processes, we have focused on the class II gene, which codes for an essential component in the C‐signal transduction pathway. We report that class II is identical to fruA and codes for a DNA binding response regulator. Transcription of fruA is developmentally regulated and depends on the early acting intercellular A‐ and E‐signals. However, fruA transcription is independent of C‐signal. Rather, genetic evidence suggests that C‐signal controls FruA activity post‐translationally. Genetic evidence strongly indicates that FruA is activated by phosphorylation. We propose that C‐signalling results in the phosphorylation of FruA, thus activating FruA to interact with downstream targets. In the motility branch of the C‐signalling pathway, FruA interacts with the Frz motility system; in the sporulation branch, we show that FruA is required for transcription of the sporulation locus devRS. On the basis of the two levels of control of FruA activity, we propose that FruA serves as a control point for the temporal co‐ordination of intercellular signals during M. xanthus development.

Architecture of the type IVa pilus machine

How the bacterial pilus works Many bacteria, including important pathogens, move by projecting grappling-hook–like extensions called type IV pili from their cell bodies. After these pili attach to other cells or objects in their environment, the bacteria retract the pili to pull themselves forward. Chang et al. used electron cryotomography of intact cells to image the protein machines that extend and retract the pili, revealing where each protein component resides. Putting the known structures of the individual proteins in place like pieces of a three-dimensional puzzle revealed insights into how the machine works, including evidence that ATP hydrolysis by cytoplasmic motors rotates a membrane-embedded adaptor that slips pilin subunits back and forth from the membrane onto the pilus. Science, this issue p. 10.1126/science.aad2001 Structural models show how bacteria switch from pilus extension to retraction. INTRODUCTION Type IVa pili are bacterial cell surface structures that perform critical functions in motility, surface adhesion, virulence, and biofilm formation. Type IVa pili are anchored in the cell envelope and pull cells forward through cycles of extension, adhesion to surfaces, and retraction, all powered by the type IVa pilus machine (T4PM). Although the structures and connectivities of the 10 core T4PM proteins and minor pilins have already been determined, the overall architecture of the T4PM and its extension and retraction mechanisms have not. RATIONALE To elucidate the architecture of the intact T4PM, we directly imaged T4PMs within intact Myxococcus xanthus cells by cryo–electron tomography. Mutants that either lacked T4PM components or contained individual T4PM proteins fused to a tag were then imaged. Difference maps revealed the locations of all components of the T4PM machine. Hypothetical models were then built by fitting the known atomic structures of the components together in their relative positions. RESULTS Both piliated and nonpiliated T4PMs are multilayered structures that span the entire cell envelope. T4PMs include an outer membrane pore, three interconnected periplasmic ring structures and another in the cytoplasm, a cytoplasmic disc and dome, and a periplasmic stem. The PilQ secretin forms the outer membrane pore; TsaP forms a periplasmic ring around PilQ; periplasmic domains of PilQ together with PilP constitute the mid-periplasmic ring; and the globular domains of PilO and PilN constitute the lower periplasmic ring and connect via coiled coils across the inner membrane to PilM, which forms the cytoplasmic ring. The cytoplasmic domains of the inner membrane protein PilC form the cytoplasmic dome on the T4PM axis inside the PilM ring. The short stem in the nonpiliated state is composed of minor pilins and PilA, the major subunit of the pilus. In the piliated state, the pilus extends from the cell exterior through the PilQ pore and the periplasmic rings to PilC in the inner membrane. In the piliated structure, the hexameric adenosine triphosphatases (ATPases) PilB and PilT bind in a mutually exclusive manner to the base of the T4PM, where they appear as the cytoplasmic disc during extensions and retractions, respectively. Next, we asked whether the known atomic structures of the proteins could be fit within the map where our imaging results indicated, while still satisfying all known constraints of size, connectivities, and interfaces. This successful effort resulted in “pseudo-atomic” working models of both states of the T4PM. The models suggest that through ATP hydrolysis, PilB rotates PilC, incrementally moving it into positions that facilitate incorporation of new PilA subunits one by one from the inner membrane onto the base of the growing helical pilus. Pilus retraction is driven by replacement of PilB with PilT, which rotates PilC into positions that promote PilA departure from the base of the pilus back into the membrane. CONCLUSION We determined the architecture of the T4PM in the piliated and nonpiliated states and mapped all known components onto this architecture, producing a complete structural map of the T4PM. The results illustrate how the structure and function of macromolecular complexes that defy purification and traditional structural approaches can nonetheless be interrogated through cryo–electron tomography of intact cells and model building. Bacterial type IVa pilus machine (T4PM). Two T4PMs are depicted on the left, spanning the envelope of a Gram-negative bacterial cell. T4PMs extend and retract pili to pull cells forward. The structural data presented here support the hypothesis that ATP hydrolysis in the cytoplasm causes an adapter protein in the inner membrane to rotate, facilitating the transfer of pilin subunits from the inner membrane onto the growing pilus. The process is reversed during retraction. Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo–electron tomography of intact Myxococcus xanthus cells to visualize type IVa pili and the protein machine that assembles and retracts them (the type IVa pilus machine, or T4PM) in situ, in both the piliated and nonpiliated states, at a resolution of 3 to 4 nanometers. We found that T4PM comprises an outer membrane pore, four interconnected ring structures in the periplasm and cytoplasm, a cytoplasmic disc and dome, and a periplasmic stem. By systematically imaging mutants lacking defined T4PM proteins or with individual proteins fused to tags, we mapped the locations of all 10 T4PM core components and the minor pilins, thereby providing insights into pilus assembly, structure, and function.

Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins

Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus, T4P are unipolarly localized and undergo pole‐to‐pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non‐piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole‐to‐pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP–PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.

Extracellular biology of Myxococcus xanthus

Myxococcus xanthus has a lifecycle characterized by several social interactions. In the presence of prey, M. xanthus is a predator forming cooperatively feeding colonies, and in the absence of nutrients, M. xanthus cells interact to form multicellular, spore-filled fruiting bodies. Formation of both cellular patterns depends on extracellular functions including the extracellular matrix and intercellular signals. Interestingly, the formation of these patterns also depends on several activities that involve direct cell-cell contacts between M. xanthus cells or direct contacts between M. xanthus cells and the substratum, suggesting that M. xanthus cells have a marked ability to distinguish self from nonself. Genome-wide analyses of the M. xanthus genome reveal a large potential for protein secretion. Myxococcus xanthus harbours all protein secretion systems required for translocation of unfolded and folded proteins across the cytoplasmic membrane and an intact type II secretion system. Moreover, M. xanthus contains 60 ATP-binding cassette transporters, two degenerate type III secretion systems, both of which lack the parts in the outer membrane and the needle structure, and an intact type VI secretion system for one-step translocation of proteins across the cell envelope. Also, analyses of the M. xanthus proteome reveal a large protein secretion potential including many proteins of unknown function.

Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography

Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.

Collective Motion and Nonequilibrium Cluster Formation in Colonies of Gliding Bacteria

We characterize cell motion in experiments and show that the transition to collective motion in colonies of gliding bacterial cells confined to a monolayer appears through the organization of cells into larger moving clusters. Collective motion by nonequilibrium cluster formation is detected for a critical cell packing fraction around 17%. This transition is characterized by a scale-free power-law cluster-size distribution, with an exponent 0.88±0.07, and the appearance of giant number fluctuations. Our findings are in quantitative agreement with simulations of self-propelled rods. This suggests that the interplay of self-propulsion and the rod shape of bacteria is sufficient to induce collective motion.

PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus.

Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.

C‐signal: a cell surface‐associated morphogen that induces and co‐ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthus

In Myxococcus xanthus, morphogenesis of multicellular fruiting bodies and sporulation are co‐ordinated temporally and spatially. csgA mutants fail to synthesize the cell surface‐associated C‐signal and are unable to aggregate and sporulate. We report that csgA encodes two proteins, a 25 kDa species corresponding to full‐length CsgA protein and a 17 kDa species similar in size to C‐factor protein, which has been shown previously to have C‐signal activity. By systematically varying the accumulation of the csgA proteins, we show that overproduction of the csgA proteins results in premature aggregation and sporulation, uncoupling of the two events and the formation of small fruiting bodies, whereas reduced synthesis of the csgA proteins causes delayed aggregation, reduced sporulation and the formation of large fruiting bodies. These results show that C‐signal induces aggregation as well as sporulation, and that an ordered increase in the level of C‐signalling during development is essential for the spatial co‐ordination of these events. The results support a quantitative model, in which aggregation and sporulation are induced at distinct threshold levels of C‐signalling. In this model, the two events are temporally co‐ordinated by the regulated increase in C‐signalling levels during development. The contact‐dependent C‐signal transmission mechanism allows the spatial co‐ordination of aggregation and sporulation by coupling cell position and signalling levels.