A minimized motile machinery for Mycoplasma genitalium

The cell wall‐less bacterium Mycoplasma genitalium uses specialized adhesins located at the terminal organelle to adhere to host cells and surfaces. The terminal organelle is a polar structure protruding from the cell body that is internally supported by a cytoskeleton and also has an important role in cell motility. We have engineered a M. genitalium null mutant for MG491 protein showing a massive downstream destabilization of proteins involved in the terminal organelle organization. This mutant strain exhibited striking similarities with the previously isolated MG_218 null mutant strain. Upon introduction of an extra copy of MG_318 gene in both strains, the amount of main adhesins P140 and P110 dramatically increased. These strains were characterized by microcinematography, epifluorescence microscopy and cryo‐electron microcopy, revealing the presence of motile cells and filaments in the absence of many proteins considered essential for cell adhesion and motility. These results indicate that adhesin complexes play a major role in the motile machinery of M. genitalium and demonstrate that the rod element of the cytoskeleton core is not the molecular motor propelling mycoplasma cells. These strains containing a minimized motile machinery also provide a valuable cell model to investigate the adhesion and gliding properties of this human pathogen.     

Electron cryotomography of Mycoplasma pneumoniae mutants correlates terminal organelle architectural features and function

The Mycoplasma pneumoniae terminal organelle functions in adherence and gliding motility and is comprised of at least eleven substructures. We used electron cryotomography to correlate impaired gliding and adherence function with changes in architecture in diverse terminal organelle mutants. All eleven substructures were accounted for in the prkC, prpC and P200 mutants, and variably so for the HMW3 mutant. Conversely, no terminal organelle substructures were evident in HMW1 and HMW2 mutants. The P41 mutant exhibits a terminal organelle detachment phenotype and lacked the bowl element normally present at the terminal organelle base. Complementation restored this substructure, establishing P41 as either a component of the bowl element or required for its assembly or stability, and that this bowl element is essential to anchor the terminal organelle but not for leverage in gliding. Mutants II‐3, III‐4 and topJ exhibited a visibly lower density of protein knobs on the terminal organelle surface. Mutants II‐3 and III‐4 lack accessory proteins required for a functional adhesin complex, while the topJ mutant lacks a DnaJ‐like co‐chaperone essential for its assembly. Taken together, these observations expand our understanding of the roles of certain terminal organelle proteins in the architecture and function of this complex structure.     

The EAGR box structure: a motif involved in mycoplasma motility

Mycoplasma genitalium is an emerging human pathogen with the smallest genome found among self‐replicating organisms. M. genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cell‐host adhesion. A well‐conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M. genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra‐subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra‐ and inter‐subunit symmetry axes, support a role for the EAGR box in protein–protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility.     

Functional domain analysis of the Mycoplasma pneumoniae co‐chaperone TopJ

Colonization of conducting airways of humans by the prokaryote Mycoplasma pneumoniae is mediated by a differentiated terminal organelle important in cytadherence, gliding motility and cell division. TopJ is a predicted J‐domain co‐chaperone also having domains unique to mycoplasma terminal organelle proteins and is essential for terminal organelle function, as well as stabilization of protein P24, which is required for normal initiation of terminal organelle formation. J‐domains activate the ATPase of DnaK chaperones, facilitating peptide binding and proper protein folding. We performed mutational analysis of the predicted J‐domain, central acidic and proline‐rich (APR) domain, and C‐terminal domain of TopJ and assessed the phenotypic consequences when introduced into an M. pneumoniae topJ mutant. A TopJ derivative with amino acid substitutions in the canonical J‐domain histidine–proline–aspartic acid motif restored P24 levels but not normal motility, morphology or cytadherence, consistent with a J‐domain co‐chaperone function. In contrast, TopJ derivatives having APR or C‐terminal domain deletions were less stable and failed to restore P24, but resulted in normal morphology, intermediate gliding motility and cytadherence levels exceeding that of wild‐type cells. Results from immunofluorescence microscopy suggest that both the APR and C‐terminal domains, but not the histidine–proline–aspartic acid motif, are critical for TopJ localization to the terminal organelle.     

P65 truncation impacts P30 dynamics during Mycoplasma pneumoniae gliding

The cell wall-less prokaryote Mycoplasma pneumoniae is a major cause of community-acquired bronchitis and pneumonia in humans. Colonization is mediated largely by a differentiated terminal organelle, which is also the leading end in gliding motility. Cytadherence-associated proteins P30 and P65 appear to traffic concurrently to the distal end of developing terminal organelles. Here, truncation of P65 due to transposon insertion in the corresponding gene resulted in lower gliding velocity, reduced cytadherence, and decreased steady-state levels of several terminal organelle proteins, including P30. Utilizing fluorescent protein fusions, we followed terminal organelle development over time. New P30 foci appeared at nascent terminal organelles in P65 mutants, as in the wild type. However, with forward cell motility, P30 in the P65 mutants appeared to drag toward the trailing cell pole, where it was released, yielding a fluorescent trail to which truncated P65 colocalized. In contrast, P30 was only rarely observed at the trailing end of gliding wild-type cells. Complementation with the recombinant wild-type P65 allele by transposon delivery restored P65 levels and stabilized P30 localization to the terminal organelle.     

A novel type 3 secretion system effector,YspI of Yersinia enterocolitica,induces cell paralysis by reducing total focal adhesion kinase

Some of the world's most important diseases are caused by bacterial pathogens that deliver toxic effector proteins directly into eukaryotic cells using type III secretion systems. The myriad of pathological outcomes caused by these pathogens is determined, in part, by the manipulation of host cell physiology due to the specific activities of individual effectors among the unique suite each pathogen employs. YspI was found to be an effector, delivered by Yersinia enterocolitica Biovar 1B, that inhibits host cell motility. The action of YspI comes about through its specific interaction with focal adhesion kinase, FAK, which is a fulcrum of focal adhesion complexes for controlling cellular motility. The interaction was defined by a specific domain of YspI that bound to the FAK kinase domain. Further examination revealed that YspI–FAK interaction leads to a reduction of FAK steady‐state levels without altering its phosphorylation state. This collection of observations and results showed YspI displays unique functionality by targeting the key regulator of focal adhesion complexes to inhibit cellular movement.     

Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility

The human pathogen Mycoplasma genitalium is known to mediate cell adhesion to target cells by the attachment organelle, a complex structure also implicated in gliding motility. The gliding mechanism of M. genitalium cells is completely unknown, but recent studies have begun to elucidate the components of the gliding machinery. We report the study of MG312, a cytadherence-related protein containing in the N terminus a box enriched in aromatic and glycine residues (EAGR), which is also exclusively found in MG200 and MG386 gliding motility proteins. Characterization of an MG_312 deletion mutant obtained by homologous recombination has revealed that the MG312 protein is required for the assembly of the M. genitalium terminal organelle. This finding is consistent with the intermediate-cytadherence phenotype and the complete absence of gliding motility exhibited by this mutant. Reintroduction of several MG_312 deletion derivatives into the MG_312 null mutant allowed us to identify two separate functional domains: an N-terminal domain implicated in gliding motility and a C-terminal domain involved in cytadherence and terminal organelle assembly functions. In addition, our results also provide evidence that the EAGR box has a specific contribution to mycoplasma cell motion. Finally, the presence of a conserved ATP binding site known as a Walker A box in the MG312 N-terminal region suggests that this structural protein could also play an active function in the gliding mechanism.     

Structure of a putative ClpS N‐end rule adaptor protein from the malaria pathogen Plasmodium falciparum

The N‐end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP‐dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N‐terminal amino acids (N‐degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N‐end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N‐degrons. However, while the N‐degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal‐ClpS binds and discriminates peptides mimicking bona fide N‐end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal‐ClpS localizes to this plastid‐like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti‐malarial drugs aimed at disrupting parasite‐specific protein quality control pathways.     

Mycoplasma pneumoniae J-domain protein required for terminal organelle function

The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Colonization of the respiratory epithelium requires proper assembly of a complex, multifunctional, polar terminal organelle. Loss of a predicted J-domain protein also having domains unique to mycoplasma terminal organelle proteins (TopJ) resulted in a non-motile, adherence-deficient phenotype. J-domain proteins typically stimulate ATPase activity of Hsp70 chaperones to bind nascent peptides for proper folding, translocation or macromolecular assembly, or to resolve stress-induced protein aggregates. By Western immunoblotting all defined terminal organelle proteins examined except protein P24 remained at wild-type levels in the topJ mutant; previous studies established that P24 is required for normal initiation of terminal organelle formation. Nevertheless, terminal organelle proteins P1, P30, HMW1 and P41 failed to localize to a cell pole, and when evaluated quantitatively, P30 and HMW1 foci were undetectable in >40% of cells. Complementation of the topJ mutant with the recombinant wild-type topJ allele largely restored terminal organelle development, gliding motility and cytadherence. We propose that this J-domain protein, which localizes to the base of the terminal organelle in wild-type M. pneumoniae , functions in the late stages of assembly, positioning, or both, of nascent terminal organelles.     

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

Covalent attachment and Pro‐Pro endopeptidase (PPEP‐1)‐mediated release of Clostridium difficile cell surface proteins involved in adhesion

In the past decade, Clostridium difficile has emerged as an important gut pathogen. This anaerobic, Gram‐positive bacterium is the main cause of infectious nosocomial diarrhea. Whereas much is known about the mechanism through which the C. difficile toxins cause diarrhea, relatively little is known about the dynamics of adhesion and motility, which is mediated by cell surface proteins. This review will discuss the recent advances in our understanding of the sortase‐mediated covalent attachment of cell surface (adhesion) proteins to the peptidoglycan layer of C. difficile and their release through the action of a highly specific secreted metalloprotease (Pro‐Pro endopeptidase 1, PPEP‐1). Specific emphasis will be on a model in which PPEP‐1 and its substrates control the switch from a sessile to motile phenotype in C. difficile, and how this is regulated by the cyclic dinucleotide c‐di‐GMP (3′‐5′ cyclic dimeric guanosine monophosphate).     

Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/Wrch‐1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N‐ and C‐terminal domains. The N‐terminal extension promotes binding to SH3 (Src homology 3)‐domain‐containing adaptors, whereas the C‐terminal extension mediates membrane targeting through palmitoylation of its non‐conventional CAAX box. RhoU/Wrch‐1 possesses transforming activity, which is negatively regulated by its N‐terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c‐Src‐expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N‐terminal extension and the palmitoylation site were dispensable, whereas the C‐terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked‐down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C‐terminal domains are critical for these functions.     

Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility

The c‐di‐GMP‐binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c‐di‐GMP‐bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re‐annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ‐mediated downregulation of motility is fine‐tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c‐di‐GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c‐di‐GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c‐di‐GMP‐dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella‐driven swimming and swarming motility.     

The role of FlhF and HubP as polar landmark proteins in Shewanella putrefaciens CN‐32

Spatiotemporal regulation of cell polarity plays a role in many fundamental processes in bacteria and often relies on ‘landmark’ proteins which recruit the corresponding clients to their designated position. Here, we explored the localization of two multi‐protein complexes, the polar flagellar motor and the chemotaxis array, in Shewanella putrefaciens CN‐32. We demonstrate that polar positioning of the flagellar system, but not of the chemotaxis system, depends on the GTPase FlhF. In contrast, the chemotaxis array is recruited by a transmembrane protein which we identified as the functional ortholog of Vibrio cholerae HubP. Mediated by its periplasmic N‐terminal LysM domain, SpHubP exhibits an FlhF‐independent localization pattern during cell cycle similar to its Vibrio counterpart and also has a role in proper chromosome segregation. In addition, while not affecting flagellar positioning, SpHubP is crucial for normal flagellar function and is involved in type IV pili‐mediated twitching motility. We hypothesize that a group of HubP/FimV homologs, characterized by a rather conserved N‐terminal periplasmic section required for polar targeting and a highly variable acidic cytoplasmic part, primarily mediating recruitment of client proteins, serves as polar markers in various bacterial species with respect to different cellular functions.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Domain analysis of protein P30 in Mycoplasma pneumoniae cytadherence and gliding motility

The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment and gliding motility are required for colonization of the respiratory epithelium and are mediated largely by a differentiated terminal organelle. P30 is a membrane protein at the distal end of the terminal organelle and is required for cytadherence and gliding motility, but little is known about the functional role of its specific domains. In the current study, domain deletion and substitution derivatives of P30 were engineered and introduced into a P30 null mutant by transposon delivery to assess their ability to rescue P30 function. Domain deletions involving the extracellular region of P30 severely impacted protein stability and adherence and gliding function, as well as the capacity to stabilize terminal organelle protein P65. Amino acid substitutions in the transmembrane domain revealed specific residues uniquely required for P30 stability and function, perhaps to establish correct topography in the membrane for effective alignment with binding partners. Deletions within the predicted cytoplasmic domain did not affect P30 localization or its capacity to stabilize P65 but markedly impaired gliding motility and cytadherence. The larger of two cytoplasmic domain deletions also appeared to remove the P30 signal peptide processing site, suggesting a larger leader peptide than expected. We propose that the P30 cytoplasmic domain may be required to link P30 to the terminal organelle core, to enable the P30 extracellular domain to achieve a functional conformation, or perhaps both.     

De novo design of signal sequences to localize cargo to the 1,2‐propanediol utilization microcompartment

Organizing heterologous biosyntheses inside bacterial cells can alleviate common problems owing to toxicity, poor kinetic performance, and cofactor imbalances. A subcellular organelle known as a bacterial microcompartment, such as the 1,2‐propanediol utilization microcompartment of Salmonella, is a promising chassis for this strategy. Here we demonstrate de novo design of the N‐terminal signal sequences used to direct cargo to these microcompartment organelles. We expand the native repertoire of signal sequences using rational and library‐based approaches and show that a canonical leucine‐zipper motif can function as a signal sequence for microcompartment localization. Our strategy can be applied to generate new signal sequences localizing arbitrary cargo proteins to the 1,2‐propanediol utilization microcompartments.     

The archaellum: a rotating type IV pilus

Microbes have evolved sophisticated mechanisms of motility allowing them to respond to changing environmental conditions. While this cellular process is well characterized in bacteria, the mode and mechanisms of motility are poorly understood in archaea. This study examines the motility of individual cells of the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Specifically, we investigated motility of cells producing exclusively the archaeal swimming organelle, the archaellum. Archaella are structurally and in sequence similar to bacterial type IV pili involved in surface motility via pilus extension‐retraction cycles and not to rotating bacterial flagella. Unexpectedly, our studies reveal a novel type of behaviour for type IV pilus like structures: archaella rotate and their rotation drives swimming motility. Moreover, we demonstrate that temperature has a direct effect on rotation velocity explaining temperature‐dependent swimming velocity.     

A Common Clathrin‐Mediated Machinery Co‐ordinates Cell–Cell Adhesion and Bacterial Internalization

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E‐cadherin, hijack the host adherens junction (AJ) machinery and invade non‐phagocytic cells by a clathrin‐dependent mechanism. Here, we investigate a potential role for clathrin in cell–cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell–cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E‐cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E‐cadherin. We demonstrate that non‐adherent cells expressing the InlA chimera, as bacteria, can be internalized by E‐cadherin‐expressing adherent cells. Together these results reveal that a common clathrin‐mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes.