Significance Targeting of cellular components to a particular site in a cell is often a highly regulated process, even in cells as small as bacteria. Robust chemotactic signaling, which is used by motile bacteria to survey their environments and navigate in response to them, requires appropriate cellular distribution of a large chemosensory apparatus. Here, we report how polarly flagellated vibrios ensure polar localization of their chemotactic machinery by capturing signaling proteins at the pole. Polar localization is mediated by a tripartite protein interaction network in which one protein prevents disassociation of a key signaling component from chemotactic complexes and tethers the complexes to a polar anchor. Polar tethering and localization are prerequisites for proper chemotaxis.

Protein secretion typically involves translocation of unfolded polypeptides or transport of monomeric folded proteins. Here we provide, to our knowledge, the first experimental evidence for secretion of an intact multimeric complex requiring a signal formed by both members of the complex. Using systematic mutagenesis of a substrate involved in early secretory antigen 6 kDa (ESX) secretion in Bacillus subtilis, we demonstrate that export of the substrate requires two independent motifs. Using mixed dimers, we show that these motifs must form a composite secretion signal in which one motif is contributed by each subunit of the dimer. Finally, through targeted crosslinking we show that the dimer formed in the cell is likely secreted as a single unit. We discuss implications of this substrate recognition mechanism for the biogenesis and quality control of secretion substrates and describe its likely conservation across ESX systems.WXG protein | type VII secretion system | protein translocation | YukE P rotein secretion is critical for protein targeting in any living cell and for its communication with the environment. Bacteria use a wide range of secretion mechanisms to export proteins out of the cytoplasm. Signals for secretion are most commonly primary amino acid sequences, but in some cases also may be formed through interacting surfaces of a substrate and its delivery effector. Some secretion systems unfold their substrates to translocate them across the membrane and cell wall. Other systems export folded proteins, sometimes in complex with bound cofactors. For example, the general secretory machinery (Sec) denatures the tertiary and secondary structure of its substrates to thread the polypeptide through the narrow opening of the integral membrane translocon complex, SecYEG (1). Type III secretion system (T3SS) machinery is thought to unfold the tertiary structure of its substrates, while preserving the secondary structure elements for the substrate recognition (2, 3). In contrast, the twin-arginine transport (Tat) system exports folded substrates (4) and is hypothesized to be able to translocate protein oligomers and complexes via a "hitchhiking" mechanism (5). Overall, these and other secretion types differ in the nature of substrate recognition signal and the mode of substrate translocation.Early secretory antigen 6 kDa (ESX, or type VII) secretion systems are widespread in actinomycetes and Gram-positive bacteria and affect a range of bacterial processes including sporulation, conjugation, and cell wall stability (6-10). In two notorious human pathogens, Mycobacterium tuberculosis and Staphylococcus aureus, ESX secretion was found to be crucial for establishing and maintaining the infection (11-15). Despite the importance of the ESX secretion for human health, the mechanism of this type of secretion is still largely unknown.Recent characterization of the ESX system in Bacillus subtilis confirmed that a functional system is encoded by the yuk/yue operon (16,17). Importantly, the B. subtilis system codes for a...

Background: We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin-mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. Results: During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenind1 mRNAs with a splice-site morpholino caused lateral widening and anterior-posterior shortening of the presomitic mesoderm and somites and a shortened anterior-posterior axis. These phenotypes indicate a cell-migration effect. Co-injection of low amounts of wild-type Cdc42 or wild-type Rac1 or dominant-negative RhoA mRNAs, but not constitutively-active Cdc42 mRNA, rescued these p120 catenin d1-depleted embryos. Conclusions: These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development. Developmental Dynamics 241:1545-1561, 2012. V C 2012 Wiley Periodicals Inc.Key words: p120 Catenin (CTNND1); ARVCF; Delta-catenin (CTNND2b); Cdc42 GTPase; Rac1 GTPase, RhoA GTPase; gastrulation; presomitic mesoderm; somites; zebrafish Key findings p120 catetin is required for extension of the dorsal axis and normal migration of the presomitic mesoderm. Cdc42 and Rac1 GTPases are downstream of p120 catenin d1 signaling and require exchange of GTP for GDP. Local stimulation of the exchange of GTP for GDP in Cdc42 and Rac GTPases mediates directional migration of the presomitic mesoderm. A balance of the amount of p120 catenin d1 and localized activation or turnover of Cdc42, Rac1, and Rho GTPase are required for normal zebrafish cell migration. Accepted 31 July 2012 Developmental DynamicsABBREVIATIONS Ab antibody ARVCF armadillo repeat gene deleted in velo-cardio-facial syndrome CA constitutively active Chr chromosome C(t) relative amount of RT-PCR product hpf hours post-fertilization DN dominant negative d1 splice-MO antisense morpholino oligonucleotide to the 12 th splice site of zebrafish p120 catenin d1 p120 catenin d1 (CTNND1) also called p120 catenin, Xenopus p120 catenin is a CTNND1 p120 catenin d2b (CTNND2b) also called Delta-catenin Rok1 Rho kinase1 RT reverse transcriptase minus-RT controls without reverse transcriptase qRT-PCR quantitative real-time PCR WT wild-type Xp120 catenin mRNA Xenopus p120 catenin d1 mRNA.Additional Supporting Information may be found in the online version of this article.

The bacterium can communicate identity through the secretion of the self-identity protein IdsD via the type VI secretion (T6S) system. IdsD secretion is essential for self-versus-nonself recognition behaviors in these populations. Here we provide an answer to the unresolved question of how the activity of a T6S substrate, such as IdsD, is regulated before secretion. We demonstrate that IdsD is found in clusters that form independently of the T6S machinery and activity. We show that the IdsC protein, which is a member of the proposed DUF4123 chaperone family, is essential for the maintenance of these clusters and of the IdsD protein itself. We provide evidence that amino acid disruptions in IdsC are sufficient to disrupt IdsD secretion but not IdsD localization into subcellular clusters, strongly supporting the notion that IdsC functions in at least two different ways: maintaining IdsD levels and secreting IdsD. We propose that IdsC, and likely other DUF4123-containing proteins, functions to regulate T6S substrates in the donor cell both by maintaining protein levels and by mediating secretion at the T6S machinery. Understanding the subcellular dynamics of self-identity proteins is crucial for developing models of self-versus-nonself recognition. We directly addressed how a bacterium restricts self-identity information before cell-cell exchange. We resolved two conflicting models for type VI secretion (T6S) substrate regulation by focusing on the self-identity protein IdsD. One model is that a cognate immunity protein binds the substrate, inhibiting activity before transport. Another model proposes that DUF4123 proteins act as chaperones in the donor cell, but no detailed molecular mechanism was previously known. We resolve this discrepancy and propose a model wherein a chaperone couples IdsD sequestration with its localization. Such a molecular mechanism restricts the communication of identity, and possibly other T6S substrates, in producing cells.

The opportunistic bacterial uropathogen Proteus mirabilis can communicate identity through the export of the self-identity protein, IdsD, via the type VI secretion (T6S) system. Expression of the ids genes provides a fitness advantage during polymicrobial infections in a mouse infection model. Here we provide an answer to the unresolved question of how the activity of a T6S substrate, such as IdsD, is regulated before export.We demonstrate that IdsD is found in clusters that form independently of the T6S machinery and activity. We show that the protein IdsC, which is a member of the proposed DUF4123 chaperone family, is essential for the stability of these clusters as well as the IdsD protein itself. And we provide evidence that amino acid disruptions in IdsC are sufficient to disrupt IdsD export but not IdsD localization into stable subcellular clusters, strongly supporting that IdsC functions in at least two different ways: IdsD stabilization and IdsD export. We propose that IdsC, and likely other DUF4123-containing proteins, function to regulate T6S substrates before export by both stabilizing the protein and mediating export at the T6S machinery.

The type VI secretion (T6S) system is a molecular device for the delivery of proteins from one cell into another. T6S function depends on the contractile sheath comprised of TssB/VipA and TssC/VipB proteins. We previously reported on a mutant variant of TssB that disrupts T6S-dependent export of the self-identity protein, IdsD, in the bacterium Proteus mirabilis. Here we determined the mechanism underlying that initial observation. We show that T6S-dependent export of multiple self-recognition proteins is abrogated in this mutant strain. We have mapped the mutation, which is a single amino acid change, to a region predicted to be involved in the formation of the TssB-TssC sheath. We have demonstrated that this mutation does indeed inhibit sheath formation, thereby explaining the global disruption of T6S activity. We propose that this mutation could be utilized as an important tool for studying functions and behaviors associated with T6S systems.

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The type VI secretion (T6S) system is a molecular device for the delivery of proteins from one cell into another. T6S function depends on the contractile sheath comprised of TssB/VipA and TssC/VipB proteins. We previously reported on a mutant variant of TssB that disrupts T6S-dependent export of the self-identity protein, IdsD, in the bacterium Proteus mirabilis. Here we determined the mechanism underlying that initial observation. We show that T6S-dependent export of multiple self-recognition proteins is abrogated in this mutant strain. We have mapped the mutation, which is a single amino acid change, to a region predicted to be involved in the formation of the TssB-TssC sheath. We have demonstrated that this mutation does indeed inhibit sheath formation, thereby explaining the global disruption of T6S activity. We propose that this mutation could be utilized as an important tool for studying functions and behaviors associated with T6S systems.