scholarly journals Activation of the type VI secretion system in the squid symbiont Vibrio fischeri requires the transcriptional regulator TasR and the structural proteins TssM and TssA

2021 ◽  
Author(s):  
Stephanie Smith ◽  
Fernanda Salvato ◽  
Aditi Garikipati ◽  
Manuel Kleiner ◽  
Alecia N. Septer
Keyword(s):  
Vibrio Fischeri ◽  
Transcriptional Regulator ◽  
Gene Clusters ◽  
Secretion System ◽  
Genomic Islands ◽  
Structural Proteins ◽  
Type Vi Secretion System ◽  
Differential Proteomics ◽  
Regulatory Factors ◽  
Type Vi Secretion

Bacteria have evolved diverse strategies to compete for a niche, including the type VI secretion system (T6SS), a contact-dependent killing mechanism. T6SSs are common in bacterial pathogens, commensals, and beneficial symbionts, where they affect the diversity and spatial structure of host-associated microbial communities. Although T6SS gene clusters are often located on genomic islands (GIs), which may be transferred as a unit, the regulatory strategies that promote gene expression once the T6SS genes are transferred into a new cell are not known. We used the squid symbiont, Vibrio fischeri , to identify essential regulatory factors that control expression of a strain-specific T6SS encoded on a GI. We found that a transcriptional reporter for this T6SS is active only in strains that contain the T6SS-encoding GI, suggesting the GI encodes at least one essential regulator. A transposon screen identified seven mutants that could not activate the reporter. These mutations mapped exclusively to three genes on the T6SS-containing GI that encode two essential structural proteins (a TssA-like protein and TssM) and a transcriptional regulator (TasR). Using T6SS reporters, RT-PCR, competition assays, and differential proteomics, we found that all three genes are required for expression of many T6SS components, except for the TssA-like protein and TssM, which are constitutively expressed. Based on these findings, we propose a model whereby T6SS expression requires conserved structural proteins, in addition to the essential regulator TasR, and this ability to self-regulate may be a strategy to activate T6SS expression upon transfer of T6SS-encoding elements into a new bacterial host. Importance Interbacterial weapons like the T6SS are often located on mobile genetic elements and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in Vibrio fischeri . These structural proteins also contain predicted GTPase and GTP binding domains, suggesting their role in promoting T6SS expression may involve sensing the energetic state of the cell. Such a mechanism would provide a direct link between T6SS activation and cellular energy levels, providing a “checkpoint” to ensure the cell has sufficient energy to build such a costly weapon. Because these regulatory factors are encoded within the T6SS gene cluster, they are predicted to move with the genetic element to activate T6SS expression in a new host cell.

scholarly journals Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host

2018 ◽  
Vol 115 (36) ◽  
pp. E8528-E8537 ◽  
Author(s):  
Lauren Speare ◽  
Andrew G. Cecere ◽  
Kirsten R. Guckes ◽  
Stephanie Smith ◽  
Michael S. Wollenberg ◽  
...  
Keyword(s):  
Intraspecific Competition ◽  
Vibrio Fischeri ◽  
Secretion System ◽  
Dependent Manner ◽  
Light Organ ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Natural Isolates ◽  
Physical Mixing

Intraspecific competition describes the negative interaction that occurs when different populations of the same species attempt to fill the same niche. Such competition is predicted to occur among host-associated bacteria but has been challenging to study in natural biological systems. Although many bioluminescentVibrio fischeristrains exist in seawater, only a few strains are found in the light-organ crypts of an individual wild-caughtEuprymna scolopessquid, suggesting a possible role for intraspecific competition during early colonization. Using a culture-based assay to investigate the interactions of differentV. fischeristrains, we found “lethal” and “nonlethal” isolates that could kill or not kill the well-studied light-organ isolate ES114, respectively. The killing phenotype of these lethal strains required a type VI secretion system (T6SS) encoded in a 50-kb genomic island. Multiple lethal and nonlethal strains could be cultured from the light organs of individual wild-caught adult squid. Although lethal strains eliminate nonlethal strains in vitro, two lethal strains could coexist in interspersed microcolonies that formed in a T6SS-dependent manner. This coexistence was destabilized upon physical mixing, resulting in one lethal strain consistently eliminating the other. When juvenile squid were coinoculated with lethal and nonlethal strains, they occupied different crypts, yet they were observed to coexist within crypts when T6SS function was disrupted. These findings, using a combination of natural isolates and experimental approaches in vitro and in the animal host, reveal the importance of T6SS in spatially separating strains during the establishment of host colonization in a natural symbiosis.

scholarly journals Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

2020 ◽  
Vol 202 (10) ◽  
Author(s):  
Yannick R. Brunet ◽  
Christophe S. Bernard ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Internal Promoter ◽  
E Coli ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

scholarly journals Vibrio cholerae Requires the Type VI Secretion System Virulence Factor VasX To Kill Dictyostelium discoideum

2011 ◽  
Vol 79 (7) ◽  
pp. 2941-2949 ◽  
Author(s):  
Sarah T. Miyata ◽  
Maya Kitaoka ◽  
Teresa M. Brooks ◽  
Steven B. McAuley ◽  
Stefan Pukatzki
Keyword(s):  
Vibrio Cholerae ◽  
Dictyostelium Discoideum ◽  
Virulence Factor ◽  
Membrane Lipids ◽  
Gene Clusters ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Enzymatic Function ◽  
Type Vi Secretion

ABSTRACTThe type VI secretion system (T6SS) is recognized as an important virulence mechanism in several Gram-negative pathogens. InVibrio cholerae, the causative agent of the diarrheal disease cholera, a minimum of three gene clusters—one main cluster and two auxiliary clusters—are required to form a functional T6SS apparatus capable of conferring virulence toward eukaryotic and prokaryotic hosts. Despite an increasing understanding of the components that make up the T6SS apparatus, little is known about the regulation of these genes and the gene products delivered by this nanomachine. VasH is an important regulator of theV. choleraeT6SS. Here, we present evidence that VasH regulates the production of a newly identified protein, VasX, which in turn requires a functional T6SS for secretion. Deletion ofvasXdoes not affect export or enzymatic function of the structural T6SS proteins Hcp and VgrG-1, suggesting that VasX is dispensable for the assembly of the physical translocon complex. VasX localizes to the bacterial membrane and interacts with membrane lipids. We present VasX as a novel virulence factor of the T6SS, as aV. choleraemutant lackingvasXexhibits a phenotype of attenuated virulence towardDictyostelium discoideum.

scholarly journals Promoter Swapping Unveils the Role of the Citrobacter rodentium CTS1 Type VI Secretion System in Interbacterial Competition

2012 ◽  
Vol 79 (1) ◽  
pp. 32-38 ◽  
Author(s):  
Erwan Gueguen ◽  
Eric Cascales
Keyword(s):  
Gene Clusters ◽  
Secretion System ◽  
Citrobacter Rodentium ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Artificial Induction ◽  
Release Assay ◽  
Promoter Swapping

ABSTRACT The type VI secretion system (T6SS) is a versatile secretion machine dedicated to various functions in Gram-negative bacteria, including virulence toward eukaryotic cells and antibacterial activity. Activity of T6SS might be followed in vitro by the release of two proteins, Hcp and VgrG, in the culture supernatant. Citrobacter rodentium , a rodent pathogen, harbors two T6SS gene clusters, cts1 and cts2 . Reporter fusion and Hcp release assays suggested that the CTS1 T6SS was not produced or not active. The cts1 locus is composed of two divergent operons. We therefore developed a new vector allowing us to swap the two divergent endogenous promoters by P tac and P BAD using the λ red recombination technology. Artificial induction of both promoters demonstrated that the CTS1 T6SS is functional as shown by the Hcp release assay and confers on C. rodentium a growth advantage in antibacterial competition experiments with Escherichia coli .

scholarly journals Redundancy and specificity of type VI secretion vgrG loci in antibacterial activity of Agrobacterium tumefaciens 1D1609 strain

2019 ◽  
Author(s):  
Mary Nia Santos ◽  
Shu-Ting Cho ◽  
Chih-Feng Wu ◽  
Chun-Ju Chang ◽  
Chih-Horng Kuo ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Agrobacterium Tumefaciens ◽  
Biological Significance ◽  
Gene Clusters ◽  
Functional Redundancy ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Multiple Copies ◽  
Close Relatives

AbstractType VI secretion system (T6SS) is a contractile nanoweapon employed by many Proteobacteria to deliver effectors to kill or inhibit their competitors. One T6SS gene, vgrG, encodes a spike protein for effector translocation and is often present as multiple copies in bacterial genomes. Our phylogenomic analyses sampled 48 genomes across diverse Proteobacteria lineages and found ∼70% of them encode multiple VgrGs, yet only four genomes have nearly identical paralogs. Among these four, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Compared to A. tumefaciens model strain C58 which harbors two vgrG genes, 1D1609 encodes four vgrG genes (i.e. vgrGa-d) with each adjacent to different putative effector genes. Thus, 1D1609 was selected to investigate the functional redundancy and specificity of multiple vgrG genes and their associated effectors. Secretion assay of single and multiple vgrG deletion mutants demonstrated that these four vgrGs are functionally redundant in mediating T6SS secretion. By analyzing various vgrG mutants, we found that all except for the divergent vgrGb could contribute to 1D1609’s antibacterial activity. Further characterizations of putative effector-immunity gene pairs revealed that vgrGa-associated gene 2 (v2a) encodes an AHH family nuclease and serves as the major antibacterial toxin. Interestingly, C58’s VgrG2 shares 99% amino acid sequence identity with 1D1609’s VgrGa, VgrGc and VgrGd. This high sequence similarity allows 1D1609 to use an exogenous VgrG delivered from C58 to kill another competing bacterium. Taken together, Agrobacterium can use highly similar VgrGs, either produced endogenously or injected from its close relatives, for T6SS-mediated interbacterial competition.Author’s SummarySelective pressure drives bacteria to develop adaptive strategies, which include competitive and cooperative behaviors. Type VI secretion system (T6SS) is one powerful antibacterial and anti-host nanoweapon employed by many Gram-negative bacteria for growth advantages or pathogenesis. A T6SS-harboring bacterium can encode one to multiple VgrG proteins for delivery of cognate effector(s) but the prevalence and biological significance of having sequence redundant vgrGs have not been comprehensively explored. In this study, we investigated the extensiveness of having multicopy vgrG genes for effector delivery among diverse Proteobacteria with T6SS. Moreover, a plant pathogenic bacterium Agrobacterium tumefaciens strain 1D1609 with highest vgrG redundancy was selected for detailed characterization of the roles of multiple VgrGs in T6SS secretion and antibacterial activity. We revealed that the majority of Proteobacterial genomes harbor multiple copies of vgrG and the expansion of vgrG gene clusters contributed to effector diversity and functional redundancy. Furthermore, the near identical VgrG proteins between 1D1609 and its sibling strain C58 can be exchanged for effector delivery in killing another competing bacterium. Such strategy in using exchangeable effector carriers injected from its isogenic sibling or close relatives during T6SS attacks may be a beneficial strategy for agrobacteria to compete in their ecological niche.

scholarly journals VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

2021 ◽  
Vol 17 (12) ◽  
pp. e1010116
Author(s):  
Xiaoye Liang ◽  
Tong-Tong Pei ◽  
Hao Li ◽  
Hao-Yu Zheng ◽  
Han Luo ◽  
...  
Keyword(s):  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Structural Proteins ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Double Deletion ◽  
Type Vi Secretion ◽  
Structural Determinant

The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.

scholarly journals The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors

2018 ◽  
Vol 115 (49) ◽  
pp. 12519-12524 ◽  
Author(s):  
Panayiota Pissaridou ◽  
Luke P. Allsopp ◽  
Sarah Wettstadt ◽  
Sophie A. Howard ◽  
Despoina A. I. Mavridou ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Clusters ◽  
Secretion System ◽  
Supramolecular Complex ◽  
Type Vi Secretion System ◽  
Protein Protein Interaction ◽  
Bacterial Competition ◽  
Type Vi Secretion ◽  
C Terminus ◽  
Interstrain Competition

The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. Pseudomonas aeruginosa possesses three T6SS gene clusters and several hcp and vgrG gene islands, the latter encoding the spike at the T6SS tip. The vgrG1b cluster encompasses seven genes whose organization and sequences are highly conserved in P. aeruginosa genomes, except for two genes that we called tse7 and tsi7. We show that Tse7 is a Tox-GHH2 domain nuclease which is distinct from other T6SS nucleases identified thus far. Expression of this toxin induces the SOS response, causes growth arrest and ultimately results in DNA degradation. The cytotoxic domain of Tse7 lies at its C terminus, while the N terminus is a predicted PAAR domain. We find that Tse7 sits on the tip of the VgrG1b spike and that specific residues at the PAAR–VgrG1b interface are essential for VgrG1b-dependent delivery of Tse7 into bacterial prey. We also show that the delivery of Tse7 is dependent on the H1-T6SS cluster, and injection of the nuclease into bacterial competitors is deployed for interbacterial competition. Tsi7, the cognate immunity protein, protects the producer from the deleterious effect of Tse7 through a direct protein–protein interaction so specific that toxin/immunity pairs are effective only if they originate from the same P. aeruginosa isolate. Overall, our study highlights the diversity of T6SS effectors, the exquisite fitting of toxins on the tip of the T6SS, and the specificity in Tsi7-dependent protection, suggesting a role in interstrain competition.

scholarly journals Environmental Viscosity Modulates Interbacterial Killing during Habitat Transition

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lauren Speare ◽  
Stephanie Smith ◽  
Fernanda Salvato ◽  
Manuel Kleiner ◽  
Alecia N. Septer
Keyword(s):  
Intraspecific Competition ◽  
Physical Environment ◽  
Vibrio Fischeri ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Free Living ◽  
Host Colonization ◽  
Content Type ◽  
Type Vi Secretion ◽  
Habitat Transition

ABSTRACT Symbiotic bacteria use diverse strategies to compete for host colonization sites. However, little is known about the environmental cues that modulate interbacterial competition as they transition between free-living and host-associated lifestyles. We used the mutualistic relationship between Eupyrmna scolopes squid and Vibrio fischeri bacteria to investigate how intraspecific competition is regulated as symbionts move from the seawater to a host-like environment. We recently reported that V. fischeri uses a type VI secretion system (T6SS) for intraspecific competition during host colonization. Here, we investigated how environmental viscosity impacts T6SS-mediated competition by using a liquid hydrogel medium that mimics the viscous host environment. Our data demonstrate that although the T6SS is functionally inactive when cells are grown under low-viscosity liquid conditions similar to those found in seawater, exposure to a host-like high-viscosity hydrogel enhances T6SS expression and sheath formation, activates T6SS-mediated killing in as little as 30 min, and promotes the coaggregation of competing genotypes. Finally, the use of mass spectrometry-based proteomics revealed insights into how cells may prepare for T6SS competition during this habitat transition. These findings, which establish the use of a new hydrogel culture condition for studying T6SS interactions, indicate that V. fischeri rapidly responds to the physical environment to activate the competitive mechanisms used during host colonization. IMPORTANCE Bacteria often engage in interference competition to gain access to an ecological niche, such as a host. However, little is known about how the physical environment experienced by free-living or host-associated bacteria influences such competition. We used the bioluminescent squid symbiont Vibrio fischeri to study how environmental viscosity impacts bacterial competition. Our results suggest that upon transition from a planktonic environment to a host-like environment, V. fischeri cells activate their type VI secretion system, a contact-dependent interbacterial nanoweapon, to eliminate natural competitors. This work shows that competitor cells form aggregates under host-like conditions, thereby facilitating the contact required for killing, and reveals how V. fischeri regulates a key competitive mechanism in response to the physical environment.

scholarly journals The Bacterial Enhancer Binding Protein VasH Promotes Expression of a Type VI Secretion System in Vibrio fischeri during Symbiosis

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Kirsten R. Guckes ◽  
Andrew G. Cecere ◽  
Amanda L. Williams ◽  
Anjali E. McNeil ◽  
Tim Miyashiro
Keyword(s):  
Vibrio Fischeri ◽  
Binding Protein ◽  
Secretion System ◽  
Light Organ ◽  
Bacterial Symbionts ◽  
Type Vi Secretion System ◽  
Intercellular Interactions ◽  
Content Type ◽  
Type Vi Secretion ◽  
Enhancer Binding Protein

ABSTRACT Vibrio fischeri is a bacterial symbiont that colonizes the light organ of the Hawaiian bobtail squid, Euprymna scolopes. Certain strains of V. fischeri express a type VI secretion system (T6SS), which delivers effectors into neighboring cells that result in their death. Strains that are susceptible to the T6SS fail to establish symbiosis with a T6SS-positive strain within the same location of the squid light organ, which is a phenomenon termed strain incompatibility. This study investigates the regulation of the T6SS in V. fischeri strain FQ-A001. Here, we report that the expression of Hcp, a necessary structural component of the T6SS, depends on the alternative sigma factor σ54 and the bacterial enhancer binding protein VasH. VasH is necessary for FQ-A001 to kill other strains, suggesting that VasH-dependent regulation is essential for the T6SS of V. fischeri to affect intercellular interactions. In addition, this study demonstrates VasH-dependent transcription of hcp within host-associated populations of FQ-A001, suggesting that the T6SS is expressed within the host environment. Together, these findings establish a model for transcriptional control of hcp in V. fischeri within the squid light organ, thereby increasing understanding of how the T6SS is regulated during symbiosis. IMPORTANCE Animals harbor bacterial symbionts with specific traits that promote host fitness. Mechanisms that facilitate intercellular interactions among bacterial symbionts impact which bacterial lineages ultimately establish symbiosis with the host. How these mechanisms are regulated is poorly characterized in nonhuman bacterial symbionts. This study establishes a model for the transcriptional regulation of a contact-dependent killing machine, thereby increasing understanding of mechanisms by which different strains compete while establishing symbiosis.

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