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.     

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.     

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.     

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium

Mycoplasma genitalium, the causative agent of non‐gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo‐electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub‐tomogram averages of free and cell‐attached NAPs respectively, revealing a tetrameric complex with two‐fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and β lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The β lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative‐stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.     

Cryo‐electron tomography analyses of terminal organelle mutants suggest the motility mechanism of Mycoplasma genitalium

The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron‐dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryoelectron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, to systematically analyze the ultrastructural effects. These data were correlated with microcinematographies, from which the motility patterns can be quantitatively assessed. We visualized diverse phenotypes, ranging from mild to severe cell adhesion, motility and segregation defects. Based on our observations, we propose a double‐spring ratchet model for the motility mechanism that explains our current and previous observations. Our model, which expands and integrates the previously suggested inchworm model, allocates specific functions to each of the essential components of this unique bacterial motility system.     

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.     

P110 and P140 Cytadherence-Related Proteins Are Negative Effectors of Terminal Organelle Duplication in Mycoplasma genitalium

Background

The terminal organelle is a complex structure involved in many aspects of the biology of mycoplasmas such as cell adherence, motility or cell division. Mycoplasma genitalium cells display a single terminal organelle and duplicate this structure prior to cytokinesis in a coordinated manner with the cell division process. Despite the significance of the terminal organelle in mycoplasma virulence, little is known about the mechanisms governing its duplication.

Methodology/Principal Findings

In this study we describe the isolation of a mutant, named T192, with a transposon insertion close to the 3′ end of the mg192 gene encoding for P110 adhesin. This mutant shows a truncated P110, low levels of P140 and P110 adhesins, a large number of non-motile cells and a high frequency of new terminal organelle formation. Further analyses revealed that the high rates of new terminal organelle formation in T192 cells are a direct consequence of the reduced levels of P110 and P140 rather than to the expression of a truncated P110. Consistently, the phenotype of the T192 mutant was successfully complemented by the reintroduction of the mg192 WT allele which restored the levels of P110 and P140 to those of the WT strain. Quantification of DAPI-stained DNA also showed that the increase in the number of terminal organelles in T192 cells is not accompanied by a higher DNA content, indicating that terminal organelle duplication does not trigger DNA replication in mycoplasmas.

Conclusions/Significance

Our results demonstrate the existence of a mechanism regulating terminal organelle duplication in M. genitalium and strongly suggest the implication of P110 and P140 adhesins in this mechanism.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

Crystal structure of Helicobacter pylori MinE,a cell division topological specificity factor

In Gram‐negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid‐cell division site. Its structure consists of an anti‐MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long α‐helix and two anti‐parallel β‐strands, which mediate formation of a homodimeric α/β structure. Here we report the crystal structure of full‐length Helicobacter pylori MinE and redefine its TSD based on that structure. The N‐terminal region of the TSD (residues 19–26), previously defined as part of the anti‐MinCD domain, forms a β‐strand (βA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti‐parallel βA‐strands. Moreover, we observed serial dimer–dimer interactions within the crystal packing, resulting in the formation of a multimeric structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti‐parallel β‐strand interactions.     

Structure of the heme/hemoglobin outer membrane receptor ShuA from Shigella dysenteriae: Heme binding by an induced fit mechanism

Shigella dysentriae and other Gram‐negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 Å resolution the 3D structure of the TonB‐dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C‐terminal domain that folds into a 22‐stranded transmembrane β‐barrel, which is filled by the N‐terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 Å apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA‐Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N‐terminal TonB‐box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB‐box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB‐box. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Variation in assembly stoichiometry in non‐metazoan homologs of the hub domain of Ca2+/calmodulin‐dependent protein kinase II

The multi‐subunit Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is connected by a flexible linker to a C‐terminal hub domain that assembles into a 12‐ or 14‐subunit scaffold that displays the kinase domains around it. Studies on CaMKII suggest that the stoichiometry and dynamic assembly/disassembly of hub oligomers may be important for CaMKII regulation. Although CaMKII is a metazoan protein, genes encoding predicted CaMKII‐like hub domains, without associated kinase domains, are found in the genomes of some green plants and bacteria. We show that the hub domains encoded by three related green algae, Chlamydomonas reinhardtii, Volvox carteri f. nagarensis, and Gonium pectoral, assemble into 16‐, 18‐, and 20‐subunit oligomers, as assayed by native protein mass spectrometry. These are the largest known CaMKII hub domain assemblies. A crystal structure of the hub domain from C. reinhardtii reveals an 18‐subunit organization. We identified four intra‐subunit hydrogen bonds in the core of the fold that are present in the Chlamydomonas hub domain, but not in metazoan hubs. When six point mutations designed to recapitulate these hydrogen bonds were introduced into the human CaMKII‐α hub domain, the mutant protein formed assemblies with 14 and 16 subunits, instead of the normal 12‐ and 14‐subunit assemblies. Our results show that the stoichiometric balance of CaMKII hub assemblies can be shifted readily by small changes in sequence.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.     

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.     

Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD

KdpD/KdpE two‐component signaling system regulates expression of a high affinity potassium transporter responsible for potassium homeostasis. The C‐terminal module of KdpD consists of a GAF domain linked to a histidine kinase domain. Whereas certain GAF domains act as regulators by binding cyclic nucleotides, the role of the juxtamembrane GAF domain in KdpD is unknown. We report the high‐resolution crystal structure of KdpD GAF domain (KdpD^G) consisting of five α‐helices, four β‐sheets and two large loops. KdpD^G forms a symmetry‐related dimer, wherein parallelly arranged monomers contribute to a four‐helix bundle at the dimer‐interface, SAXS analysis of KdpD C‐terminal module reveals an elongated structure that is a dimer in solution. Substitution of conserved residues with various residues that disrupt the dimer interface produce a range of effects on gene expression demonstrating the importance of the interface in inactive to active transitions during signaling. Comparison of ligand binding site of the classic cyclic nucleotide‐binding GAF domains to KdpD^G reveals structural differences arising from naturally occurring substitutions in primary sequence of KdpD^G that modifies the canonical NKFDE sequence motif required for cyclic nucleotide binding. Together these results suggest a structural role for KdpD^G in dimerization and transmission of signal to the kinase domain.     

Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature

Motility is often a virulence factor of pathogenic bacteria. Although recent works have identified genes involved in gliding motility of mycoplasmas, little is known about the mechanisms governing the cell gliding behaviour. Here, we report that Mycoplasma genitalium MG217 is a novel protein involved in the gliding apparatus of this organism and it is, at least, one of the genes that are directing cells to move in narrow circles when they glide. In the absence of MG_217 gene, cells are still able to glide but they mainly move drawing erratic or wide circular paths. This change in the gliding behaviour correlates with a rearrangement in the terminal organelle disposition, suggesting that the terminal organelle operates as a guide to steer the mycoplasma cell in a specific direction. Immunogold labelling reveals that MG217 protein is located intracellular at the distal end of the terminal organelle, between the cell membrane and the terminal button. Such location is consistent with the idea that MG217 could act as a modulator of the terminal organelle curvature, allowing cells to move in specific directions.