Egbert Hoiczyk

Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells

A number of pathogenic, Gram-negative bacteria are able to secrete specific proteins across three membranes: the inner and outer bacterial membrane and the eukaryotic plasma membrane. In the pathogen Yersinia enterocolitica, the primary structure of the secreted proteins as well as of the components of the secretion machinery, both plasmid-encoded, is known. However, the mechanism of protein translocation is largely unknown. Here we show that Y. enterocolitica polymerizes a 6-kDa protein of the secretion machinery into needles that are able to puncture the eukaryotic plasma membrane. These needles form a conduit for the transport of specific proteins from the bacterial to the eukaryotic cytoplasm, where they exert their cytotoxic activity. In negatively stained electron micrographs, the isolated needles were 60–80 nm long and 6–7 nm wide and contained a hollow center of about 2 nm. Our data indicate that it is the polymerization of the 6-kDa protein into these needles that provides the force to perforate the eukaryotic plasma membrane.

The junctional pore complex, a prokaryotic secretion organelle, is the molecular motor underlying gliding motility in cyanobacteria

BACKGROUND Whereas most bacteria move by means of flagella, some prokaryotes move by gliding. In cyanobacteria, gliding motility is a slow uniform motion which is invariably accompanied by a continuous secretion of slime. On the basis of these characteristics, a model has been proposed in which the gliding motility of cyanobacteria depends on the steady secretion of slime using specific pores, as well as the interaction of the slime with the filament surface and the underlying substrate. RESULTS The structures of the pore apparatus of two different filamentous cyanobacteria have been characterized. In both species, pores are formed by a hitherto uncharacterized type of prokaryotic organelle that spans the entire multilayered cell wall and possesses structural properties expected for an organelle that is involved in the rapid secretion of extracellular carbohydrates. Light microscopic observations of the secretion process provided direct evidence that the pore complexes are the actual sites of slime secretion, that the secreted slime fibrils are elongated at about the same rate as the filament glides (up to 3 micrometer s-1), and that gliding movements are caused directly by the secretion of slime. CONCLUSIONS It has been known for a long time that carbohydrate secretion has an important role in the gliding motility of various prokaryotes. Our results strongly suggest that slime secretion is not only a prerequisite for this peculiar type of motility in cyanobacteria, but also directly generates the necessary thrust for locomotion.

Viral Paratransgenesis in the Malaria Vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

OSCILLIN, AN EXTRACELLULAR, CA2+-BINDING GLYCOPROTEIN ESSENTIAL FOR THE GLIDING MOTILITY OF CYANOBACTERIA

Electron microscopic studies have demonstrated that various gliding filamentous cyanobacteria have trichome surfaces with a common structural organization. They contain an S‐layer attached to the outer membrane and an array of parallel fibrils on top of the S‐layer. In all species studied, the helical arrangement of these fibrils corresponds to the sense of rotation of the organism during the gliding movement. We have investigated the surface fibrils of Phormidium uncinatum using electron microscopic, spectroscopic and biochemical techniques. The fibrils consist of a single rod‐shaped protein, which we refer to as oscillin. Oscillin is a 646 amino acid residue protein (Mr 65 807; pI 3.63) and appears to be glycosylated. Sequence analysis reveals a two‐domain structure: a 554 residue domain contains 46 repeats of a Ca2+‐binding motif; it is followed by a 92 residue C‐terminal domain, which might mediate its export. Filaments that do not express oscillin lose their ability to move. Homology studies suggest that similar proteins play comparable roles in other motile cyanobacteria. The structure of oscillin appears to favour a passive role in gliding.

A virus capsid‐like nanocompartment that stores iron and protects bacteria from oxidative stress

Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane‐bound organelles, bacteria and archaea rely primarily on protein‐bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo‐electron microscopy, we determined that EncA self‐assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin‐like domains, attach to its inner surface. Native nanocompartments have dense iron‐rich cores. Functionally, they resemble ferritins, cage‐like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.

EzrA Contributes to the Regulation of Cell Size in Staphylococcus aureus

EzrA is a negative regulator of FtsZ in Bacillus subtilis, involved in the coordination between cell growth and cell division and in the control of the cell elongation–division cycle. We have now studied the role of the Staphylococcus aureus homologue of the B. subtilis EzrA protein and shown that it is not essential for cell viability. EzrA conditional and null mutants have an overall increase of the average cell size, compared to wild type strains. In the larger ezrA mutant S. aureus cells, cell division protein FtsZ and the cell wall synthesizing Penicillin Binding Proteins (PBPs) are not properly localized. This suggests that there may be a maximum cell diameter that allows formation of a Z-ring capable of recruiting the other components of the divisome and of driving cytokinesis. We propose that the major role of EzrA in S. aureus is in cell size homeostasis.

BacM, an N-terminally processed bactofilin of Myxococcus xanthus, is crucial for proper cell shape

Bactofilins are fibre‐forming bacterial cytoskeletal proteins. Here, we report the structural and biochemical characterization of MXAN_7475 (BacM), one of the four bactofilins of Myxococcus xanthus. Absence of BacM leads to a characteristic ‘crooked’ cell morphology and an increased sensitivity to antibiotics targeting cell wall biosynthesis. The absence of the other three bactofilins MXAN_4637–4635 (BacN‐P) has no obvious phenotype. In M. xanthus, BacM exists as a 150‐amino‐acid full‐length version and as a version cleaved before Ser28. In the cell, native BacM forms 3 nm wide fibres, which assemble into bundles forming helix‐like cytoplasmic cables throughout the cell, and in a subset of cells additionally a polarly arranged lateral rod‐like structure. Isolated fibres consist almost completely of the N‐terminally truncated version, suggesting that the proteolytic cleavage occurs before or during fibre formation. Fusion of BacM to mCherry perturbs BacM function and cellular fibre arrangement, resulting for example in the formation of one prominent polar corkscrew‐like structure per cell. Immunofluorescence staining of BacM and MreB shows that their cellular distributions are not matching. Taken together, these data suggest that rod‐shaped bacteria like M. xanthus use bactofilin fibres to achieve and maintain their characteristic cell morphology and cell wall stability.

Lipid body formation plays a central role in cell fate determination during developmental differentiation of Myxococcus xanthus

Cell differentiation is widespread during the development of multicellular organisms, but rarely observed in prokaryotes. One example of prokaryotic differentiation is the Gram‐negative bacterium Myxococcus xanthus. In response to starvation, this gliding bacterium initiates a complex developmental programme that results in the formation of spore‐filled fruiting bodies. How the cells metabolically support the necessary complex cellular differentiation from rod‐shaped vegetative cells into spherical spores is unknown. Here, we present evidence that intracellular lipid bodies provide the necessary metabolic fuel for the development of spores. Formed at the onset of starvation, these lipid bodies gradually disappear until they are completely used up by the time the cells have become mature spores. Moreover, it appears that lipid body formation in M. xanthus is an important initial step indicating cell fate during differentiation. Upon starvation, two subpopulations of cells occur: cells that form lipid bodies invariably develop into spores, while cells that do not form lipid bodies end up becoming peripheral rods, which are cells that lack signs of morphological differentiation and stay in a vegetative‐like state. These data indicate that lipid bodies not only fuel cellular differentiation but that their formation represents the first known morphological sign indicating cell fate during differentiation.

Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition.

Myxococcus xanthus is a Gram‐negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation‐independent remodelling of the rod‐shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co‐polymerase 2a (OPX/PCP‐2a) machinery. Conversion of the capsule‐like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo or two other genetic loci encoding homologues of polysaccharide synthesis enzymes fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel Gram‐negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition.

Cell rejuvenation and social behaviors promoted by LPS exchange in myxobacteria

Significance Social organisms benefit from group behaviors that endow favorable fitness consequences among kin. We describe such a behavior in the bacterium Myxococcus xanthus in which damaged members of a population are repaired by their kin by exchange of outer membrane material. This behavior rescues lethal cellular damage, restores antibiotic resistance to a compromised cell membrane, and increases the overall fitness of a heterogeneous population. To our knowledge, we provide the first evidence that a social bacterium can use cell-content sharing to repair damaged siblings, leading to beneficial fitness outcomes for both the donor and recipient. Bacterial cells in their native environments must cope with factors that compromise the integrity of the cell. The mechanisms of coping with damage in a social or multicellular context are poorly understood. Here we investigated how a model social bacterium, Myxococcus xanthus, approaches this problem. We focused on the social behavior of outer membrane exchange (OME), in which cells transiently fuse and exchange their outer membrane (OM) contents. This behavior requires TraA, a homophilic cell surface receptor that identifies kin based on similarities in a polymorphic region, and the TraB cohort protein. As observed by electron microscopy, TraAB overexpression catalyzed a prefusion OM junction between cells. We then showed that damage sustained by the OM of one population was repaired by OME with a healthy population. Specifically, LPS mutants that were defective in motility and sporulation were rescued by OME with healthy donors. In addition, a mutant with a conditional lethal mutation in lpxC, an essential gene required for lipid A biosynthesis, was rescued by Tra-dependent interactions with a healthy population. Furthermore, lpxC cells with damaged OMs, which were more susceptible to antibiotics, had resistance conferred to them by OME with healthy donors. We also show that OME has beneficial fitness consequences to all cells. Here, in merged populations of damaged and healthy cells, OME catalyzed a dilution of OM damage, increasing developmental sporulation outcomes of the combined population by allowing it to reach a threshold density. We propose that OME is a mechanism that myxobacteria use to overcome cell damage and to transition to a multicellular organism.