Insights into flagellar function and mechanism from the squid-vibrio symbiosis.

Flagella are essential and multifunctional nanomachines that not only move symbionts towards their tissue colonization site, but also play multiple roles in communicating with the host. Thus, untangling the activities of flagella in reaching, interacting, and signaling the host, as well as in biofilm formation and the establishment of a persistent colonization, is a complex problem. The squid-vibrio system offers a unique model to study the many ways that bacterial flagella can influence a beneficial association and, generally, other bacteria-host interactions. Vibrio fischeri is a bioluminescent bacterium that colonizes the Hawaiian bobtail squid, Euprymna scolopes. Over the last 15 years, the structure, assembly, and functions of V. fischeri flagella, including not only motility and chemotaxis, but also biofilm formation and symbiotic signaling, have been revealed. Here we discuss these discoveries in the perspective of other host-bacteria interactions.

Model-enabled gene search (MEGS) allows fast and direct discovery of enzymatic and transport gene functions in the marine bacterium Vibrio fischeri.

Whereas genomes can be rapidly sequenced, the functions of many genes are incompletely or erroneously annotated because of a lack of experimental evidence or prior functional knowledge in sequence databases. To address this weakness, we describe here a model-enabled gene search (MEGS) approach that (i) identifies metabolic functions either missing from an organism's genome annotation or incorrectly assigned to an ORF by using discrepancies between metabolic model predictions and experimental culturing data; (ii) designs functional selection experiments for these specific metabolic functions; and (iii) selects a candidate gene(s) responsible for these functions from a genomic library and directly interrogates this gene's function experimentally. To discover gene functions, MEGS uses genomic functional selections instead of relying on correlations across large experimental datasets or sequence similarity as do other approaches. When applied to the bioluminescent marine bacterium Vibrio fischeri, MEGS successfully identified five genes that are responsible for four metabolic and transport reactions whose absence from a draft metabolic model of V. fischeri caused inaccurate modeling of high-throughput experimental data. This work demonstrates that MEGS provides a rapid and efficient integrated computational and experimental approach for annotating metabolic genes, including those that have previously been uncharacterized or misannotated.

Transcriptional characterization of Vibrio fischeri during colonization of juvenile Euprymna scolopes.

The marine bacterium Vibrio fischeri is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes, and the establishment of this association involves a number of signaling pathways and transcriptional responses between both partners. We report here the first full RNA-Seq dataset representing host-associated V. fischeri cells from colonized juvenile E. scolopes, as well as comparative transcriptomes under both laboratory and simulated marine planktonic conditions. These data elucidate the broad transcriptional changes that these bacteria undergo during the early stages of symbiotic colonization. We report several previously undescribed and unexpected transcriptional responses within the early stages of this symbiosis, including gene expression patterns consistent with biochemical stresses inside the host, and metabolic patterns distinct from those reported in associations with adult animals. Integration of these transcriptional data with a recently developed metabolic model of V. fischeri provides us with a clearer picture of the metabolic state of symbionts within the juvenile host, including their possible carbon sources. Taken together, these results expand our understanding of the early stages of the squid-vibrio symbiosis, and more generally inform the transcriptional responses underlying the activities of marine microbes during host colonization.

Rhs proteins from diverse bacteria mediate intercellular competition.

Rearrangement hotspot (Rhs) and related YD-peptide repeat proteins are widely distributed in bacteria and eukaryotes, but their functions are poorly understood. Here, we show that Gram-negative Rhs proteins and the distantly related wall-associated protein A (WapA) from Gram-positive bacteria mediate intercellular competition. Rhs and WapA carry polymorphic C-terminal toxin domains (Rhs-CT/WapA-CT), which are deployed to inhibit the growth of neighboring cells. These systems also encode sequence-diverse immunity proteins (RhsI/WapI) that specifically neutralize cognate toxins to protect rhs(+)/wapA(+) cells from autoinhibition. RhsA and RhsB from Dickeya dadantii 3937 carry nuclease domains that degrade target cell DNA. D. dadantii 3937 rhs genes do not encode secretion signal sequences but are linked to hemolysin-coregulated protein and valine-glycine repeat protein G genes from type VI secretion systems. Valine-glycine repeat protein G is required for inhibitor cell function, suggesting that Rhs may be exported from D. dadantii 3937 through a type VI secretion mechanism. In contrast, WapA proteins from Bacillus subtilis strains appear to be exported through the general secretory pathway and deliver a variety of tRNase toxins into neighboring target cells. These findings demonstrate that YD-repeat proteins from phylogenetically diverse bacteria share a common function in contact-dependent growth inhibition.

Delivery of CdiA nuclease toxins into target cells during contact-dependent growth inhibition.

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA(UPEC536) were used to visualize transfer of CdiA from CDI(UPEC536+) inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA(UPEC536) protein is deposited onto the surface of target bacteria. CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA(UPEC536) is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA(UPEC536) prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI(+) inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.

Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems.

Contact-dependent growth inhibition (CDI) systems encode polymorphic toxin/immunity proteins that mediate competition between neighboring bacterial cells. We present crystal structures of CDI toxin/immunity complexes from Escherichia coli EC869 and Burkholderia pseudomallei 1026b. Despite sharing little sequence identity, the toxin domains are structurally similar and have homology to endonucleases. The EC869 toxin is a Zn(2+)-dependent DNase capable of completely degrading the genomes of target cells, whereas the Bp1026b toxin cleaves the aminoacyl acceptor stems of tRNA molecules. Each immunity protein binds and inactivates its cognate toxin in a unique manner. The EC869 toxin/immunity complex is stabilized through an unusual ß-augmentation interaction. In contrast, the Bp1026b immunity protein exploits shape and charge complementarity to occlude the toxin active site. These structures represent the initial glimpse into the CDI toxin/immunity network, illustrating how sequence-diverse toxins adopt convergent folds yet retain distinct binding interactions with cognate immunity proteins. Moreover, we present visual demonstration of CDI toxin delivery into a target cell.

The toxin/immunity network of Burkholderia pseudomallei contact-dependent growth inhibition (CDI) systems.

Burkholderia pseudomallei is a category B pathogen and the causative agent of melioidosis--a serious infectious disease that is typically acquired directly from environmental reservoirs. Nearly all B. pseudomallei strains sequenced to date (> 85 isolates) contain gene clusters that are related to the contact-dependent growth inhibition (CDI) systems of γ-proteobacteria. CDI systems from Escherichia coli and Dickeya dadantii play significant roles in bacterial competition, suggesting these systems may also contribute to the competitive fitness of B. pseudomallei. Here, we identify 10 distinct CDI systems in B. pseudomallei based on polymorphisms within the cdiA-CT/cdiI coding regions, which are predicted to encode CdiA-CT/CdiI toxin/immunity protein pairs. Biochemical analysis of three B. pseudomallei CdiA-CTs revealed that each protein possesses a distinct tRNase activity capable of inhibiting cell growth. These toxin activities are blocked by cognate CdiI immunity proteins, which specifically bind the CdiA-CT and protect cells from growth inhibition. Using Burkholderia thailandensis E264 as a model, we show that a CDI system from B. pseudomallei 1026b mediates CDI and is capable of delivering CdiA-CT toxins derived from other B. pseudomallei strains. These results demonstrate that Burkholderia species contain functional CDI systems, which may confer a competitive advantage to these bacteria.

Crystallographic insights into the pore structures and mechanisms of the EutL and EutM shell proteins of the ethanolamine-utilizing microcompartment of Escherichia coli.

The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) of Escherichia coli is a polyhedral organelle that harbors specific enzymes for the catabolic degradation of ethanolamine. The compartment is composed of a proteinaceous shell structure that maintains a highly specialized environment for the biochemical reactions inside. Recent structural investigations have revealed hexagonal assemblies of shell proteins that form a tightly packed two-dimensional lattice that is likely to function as a selectively permeable protein membrane, wherein small channels are thought to permit controlled exchange of specific solutes. Here, we show with two nonisomorphous crystal structures that EutM also forms a two-dimensional protein membrane. As its architecture is highly similar to the membrane structure of EutL, it is likely that the structure represents a physiologically relevant form. Thus far, of all Eut proteins, only EutM and EutL have been shown to form such proteinaceous membranes. Despite their similar architectures, however, both proteins exhibit dramatically different pore structures. In contrast to EutL, the pore of EutM appears to be positively charged, indicating specificity for different solutes. Furthermore, we also show that the central pore structure of the EutL shell protein can be triggered to open specifically upon exposure to zinc ions, suggesting a specific gating mechanism.

Crystal structure of the EutL shell protein of the ethanolamine ammonia lyase microcompartment.

Bacterial microcompartments (BMCs) are specialized organelles that use proteinaceous membranes to confine chemical reaction spaces. The ethanolamine ammonialyase microcompartment of Escherichia coli represents such a class of cytosolic organelles that enables bacteria to survive on small organic molecules such as ethanolamine as the sole source for carbon and nitrogen. We present here the crystal structure of the shell protein EutL at 2.2-A resolution. With 219 residues, it is the largest representative of this BMC's shell proteins. In the crystal, EutL forms a trimer that exhibits a hexagonally shaped tile structure. The tiles arrange into a tightly packed 2D array that is likely to resemble the proteinaceous membrane of the intact BMC. In contrast to other BMC shell proteins, which have only 1 pore per tile, EutL exhibits 3 pores per tile, thereby significantly increasing the overall porosity of this protein membrane. Each of the individual pores is lined with negatively charged residues and aromatic residues that are proposed to facilitate passive transport of specific solutes. The characteristic shape of the hexagonal tile, which is also found in the microcompartments of carbon-fixating bacteria, may present an inherent and fundamental building unit that may provide a general explanation for the formation of differently sized microcompartments.

Preliminary structural investigations of the Eut-L shell protein of the ethanolamine ammonia-lyase metabolosome of Escherichia coli.

The ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes. Here, preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. Cloning, overexpression and purification resulted in highly pure protein that crystallized readily under many different conditions. In all cases the protein forms thin hexagonal plate-shaped crystals belonging to space group P3 that are of unusually high stability against different solvent conditions. The crystals diffracted to a resolution of 2.0 A using synchrotron radiation but proved to be radiation-sensitive. Preparations of heavy-atom-derivatized crystals for use in determining the three-dimensional structure are under way.