Anna Konovalova

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.

Profiling the Outer Membrane Proteome during Growth and Development of the Social Bacterium Myxococcus xanthus by Selective Biotinylation and Analyses of Outer Membrane Vesicles

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins.

Significance In Escherichia coli, most outer membrane (OM) lipoproteins are thought to be soluble proteins that are simply tethered to the inner leaflet of this membrane by lipid moieties attached to the N terminus. Here we show that lipoprotein RcsF (regulator of capsule synthesis) adopts a transmembrane orientation with the lipidated N terminus on the cell surface and the folded C-terminal domain in the periplasm. The short, unstructured, polar linker domain spans the hydrophobic membrane by passing through the lumen of several different OM β-barrel proteins. This remarkable, interlocked structure is formed by the Bam complex, which folds and inserts all β-barrel proteins in the OM, suggesting that this assembly machine translocates the lipid moieties and then folds the β barrel around the RcsF linker. RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of β-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all β-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the β-barrel assembly machine.

Regulated Secretion of a Protease Activates Intercellular Signaling during Fruiting Body Formation in M. xanthus

In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. There are two morphogenetic events in this program, aggregation and sporulation, which are temporally and spatially coordinated by the contact-dependent intercellular C-signal protein (p17). p17 is generated by proteolytic cleavage of the p25 precursor protein, which accumulates in the outer membrane of vegetative and starving cells. However, p17 generation is restricted to starving cells. Here we identify the subtilisin-like protease PopC that is directly responsible for cleavage of p25. PopC accumulates in the cytoplasm of vegetative cells but is selectively secreted during starvation coinciding with the generation of p17. Consequently, p25 and PopC only encounter each other in starving cells. Thus, restriction of p25 cleavage to starving cells occurs by a compartmentalization mechanism that depends on selective secretion of PopC during starvation. Our results provide evidence for regulated proteolysis via regulated secretion.

Outer membrane lipoprotein biogenesis: Lol is not the end.

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.

Regulated proteolysis in bacterial development

Bacteria use proteases to control three types of events temporally and spatially during the processes of morphological development. These events are the destruction of regulatory proteins, activation of regulatory proteins, and production of signals. While some of these events are entirely cytoplasmic, others involve intramembrane proteolysis of a substrate, transmembrane signaling, or secretion. In some cases, multiple proteolytic events are organized into pathways, for example turnover of a regulatory protein activates a protease that generates a signal. We review well-studied and emerging examples and identify recurring themes and important questions for future research. We focus primarily on paradigms learned from studies of model organisms, but we note connections to regulated proteolytic events that govern bacterial adaptation, biofilm formation and disassembly, and pathogenesis.

Close encounters: contact‐dependent interactions in bacteria

Bacterial cells interact extensively within and between species. These interactions can be divided into those that rely on diffusible factors and those that depend on direct cell‐to‐cell contacts. An example of a contact‐dependent interaction is the transfer of lipoproteins between Myxococcus xanthus cells that leads to transient stimulation of motility in certain motility mutants. In this issue of Molecular Microbiology, Wei et al. (2011) provide mechanistic insights into this contact‐dependent transfer of lipoproteins. Briefly, a heterologous protein fused to a type II (lipoprotein) signal sequence that targets the protein to the outer membrane is required and sufficient for transfer. Moreover, evidence is provided that transfer may depend on specific contacts between donor and recipient cells. The data demonstrate that lipoprotein transfer in M. xanthus is not restricted to a few odd motility proteins but could be a wide‐spread phenomenon in M. xanthus and possibly other bacteria. Recent years have been fruitful in identifying contact‐dependent interactions between bacterial cells. These interactions can be grouped into those that involve delivery of cargo to a recipient and those that seem to be involved in cell‐to‐cell signalling. Several contact‐dependent interactions involve widely conserved proteins, suggesting that cell contact‐dependent processes may be widespread among bacteria.

A RelA‐dependent two‐tiered regulated proteolysis cascade controls synthesis of a contact‐dependent intercellular signal in Myxococcus xanthus

Proteolytic cleavage of precursor proteins to generate intercellular signals is a common mechanism in all cells. In Myxococcus xanthus the contact‐dependent intercellular C‐signal is a 17 kDa protein (p17) generated by proteolytic cleavage of the 25 kDa csgA protein (p25), and is essential for starvation‐induced fruiting body formation. p25 accumulates in the outer membrane and PopC, the protease that cleaves p25, in the cytoplasm of vegetative cells. PopC is secreted in response to starvation, therefore, restricting p25 cleavage to starving cells. We focused on identifying proteins critical for PopC secretion in response to starvation. PopC secretion depends on the (p)ppGpp synthase RelA and the stringent response, and is regulated post‐translationally. PopD, which is encoded in an operon with PopC, forms a soluble complex with PopC and inhibits PopC secretion whereas the integral membrane AAA+ protease FtsHD is required for PopC secretion. Biochemical and genetic evidence suggest that in response to starvation, RelA is activated and induces the degradation of PopD thereby releasing pre‐formed PopC for secretion and that FtsHD is important for PopD degradation. Hence, regulated PopC secretion depends on regulated proteolysis. Accordingly, p17 synthesis depends on a proteolytic cascade including FtsHD‐dependent degradation of PopD and PopC‐dependent cleavage of p25.

Two intercellular signals required for fruiting body formation in Myxococcus xanthus act sequentially but non-hierarchically

Starvation‐induced fruiting body formation in Myxococcus xanthus depends on intercellular signalling. A‐signal functions after 2 h of starvation and its synthesis depends on the asg genes. C‐signal functions after 6 h of starvation and is generated by proteolytic cleavage of a precursor by the protease PopC. Previous gene expression studies suggested that the A‐ and C‐signal lie on a hierarchical pathway. Here we explored the causal relationship between the A‐ and C‐signal. The asgA and asgB mutants have reduced popC expression, PopC accumulation and C‐signal accumulation. popC expression was shown not to depend on A‐signal but on the AsgA and AsgB proteins. Restored popC expression in the two mutants rescued PopC and C‐signal accumulation as well as C‐signalling and the developmental defects of the two mutants without restoring A‐signalling. Based on these results we suggest that A‐ and C‐signal do not lie on a hierarchical, dependent pathway. Instead the A‐ and C‐signal act sequentially and without a causal relationship suggesting that they are linked by a shared timing mechanism, which ensures the early and late onset of A‐signalling and C‐signalling, respectively, during starvation. This pathway topology represents a novel architecture for bacterial intercellular signalling systems involving more than one signal.

A lipoprotein/β-barrel complex monitors lipopolysaccharide integrity transducing information across the outer membrane

Lipoprotein RcsF is the OM component of the Rcs envelope stress response. RcsF exists in complexes with β-barrel proteins (OMPs) allowing it to adopt a transmembrane orientation with a lipidated N-terminal domain on the cell surface and a periplasmic C-terminal domain. Here we report that mutations that remove BamE or alter a residue in the RcsF trans-lumen domain specifically prevent assembly of the interlocked complexes without inactivating either RcsF or the OMP. Using these mutations we demonstrate that these RcsF/OMP complexes are required for sensing OM outer leaflet stress. Using mutations that alter the positively charged surface-exposed domain, we show that RcsF monitors lateral interactions between lipopolysaccharide (LPS) molecules. When these interactions are disrupted by cationic antimicrobial peptides, or by the loss of negatively charged phosphate groups on the LPS molecule, this information is transduced to the RcsF C-terminal signaling domain located in the periplasm to activate the stress response. DOI: http://dx.doi.org/10.7554/eLife.15276.001