RESUMEN
The bacterium Proteus mirabilis 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.IMPORTANCE 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteus mirabilis/metabolismo , Percepción de Quorum/fisiología , Sistemas de Secreción Tipo VI/fisiología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Chaperonas MolecularesRESUMEN
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.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Complejos Multiproteicos/metabolismo , Bacillus subtilis/metabolismo , Cromatografía en Gel , Dimerización , Electroforesis en Gel de Poliacrilamida , Mutagénesis , Transporte de Proteínas/fisiología , Colorantes de RosanilinaRESUMEN
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 catenin δ1 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 δ1-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.
Asunto(s)
Movimiento Celular/fisiología , Gastrulación/fisiología , Mesodermo/metabolismo , Pez Cebra/embriología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Western Blotting , Cateninas/genética , Cateninas/metabolismo , Clonación Molecular , Técnicas de Silenciamiento del Gen , Guanosina Trifosfato/metabolismo , Hibridación in Situ , Mesodermo/citología , Mesodermo/enzimología , Oligonucleótidos Antisentido/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Catenina deltaRESUMEN
Cancerous tissue is a largely unexplored microbial niche that provides a unique environment for the colonization and growth of specific bacterial communities, and with it, the opportunity to identify novel bacterial species. Here, we report distinct features of a novel Fusobacterium species, F. sphaericum sp. nov. ( Fs ), isolated from primary colon adenocarcinoma tissue. We acquire the complete, closed genome of this organism and phylogenetically confirm its classification into the Fusobacterium genus. Phenotypic and genomic analysis of Fs reveal that this novel organism is of coccoid shape, rare for Fusobacterium members, and has species-distinct gene content. Fs displays a metabolic profile and antibiotic resistance repertoire consistent with other Fusobacterium species. In vitro, Fs has adherent and immunomodulatory capabilities, as it intimately associates with human colon cancer epithelial cells and promotes IL-8 secretion. Analysis of the prevalence and abundance of Fs in â¼1,750 human metagenomic samples shows that it is a moderately prevalent member of the human oral cavity and stool. Intriguingly, analysis of â¼1,270 specimens from patients with colorectal cancer demonstrate that Fs is significantly enriched in colonic and tumor tissue as compared to mucosa or feces. Our study sheds light on a novel bacterial species that is prevalent within the human intestinal microbiota and whose role in human health and disease requires further investigation.
RESUMEN
We report the complete genome sequence of Morganella morganii CTX51T, a strain isolated from the resected tumor of a patient with cecal colorectal adenocarcinoma of the cecum. The genome comprises a circular chromosome of 4.19 Mbp, with an overall GC content of 50.4% and one circular plasmid of 8.48 kbp.
RESUMEN
We report the complete genome sequence of Clostridium cadaveris IFB3C5, a strain isolated from the resected tumor of a treatment naive colorectal cancer patient. This genome is comprised of a singular chromosome of approximately 3.63 Mbp in length, contains two plasmids, and has an overall mean GC content of 31.7%.
RESUMEN
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.