Two Type VI Secretion Systems in Vibrio coralliilyticus RE22Sm exhibit differential target specificity for bacteria prey and oyster larvae

Vibrio coralliilyticus is an extracellular bacterial pathogen and a causative agent of vibriosis in larval oysters. Host mortality rates can quickly reach 100% during vibriosis outbreaks in oyster hatcheries. Type VI Secretion Systems (T6SS) are rapidly polymerizing, contact dependent injection apparatus for prey cell intoxication and play important roles in pathogenesis. DNA sequencing of V. coralliilyticus RE22Sm indicated the likely presence of two functional T6SSs with one on each of two chromosomes. Here, we investigated the antibacterial and anti-eukaryotic roles of the two T6SSs (T6SS1 and T6SS2) against E. coli Sm10 cells and Crassostrea virginica larvae, respectively. Mutations in hcp and vgrG genes were created and characterized for their effects upon bacterial antagonism and eukaryotic host virulence. Mutations in hcp1 and hcp2 resulted in significantly reduced antagonism against E. coli Sm10, with the hcp2 mutation demonstrating the greater impact. In contrast, mutations in vgrG1 or vgrG2 had little effect on E. coli killing. In eastern oyster larval challenge assays, T6SS1 mutations in either hcp1 or vgrG1 dramatically attenuated virulence against C. virginica larvae. Strains with restored wild type hcp or vgrG genes reestablished T6SS-mediated killing to that of wild type V. coralliilyticus RE22Sm. These data suggest that the T6SS1 of V. coralliilyticus RE22Sm principally targets eukaryotes and secondarily bacteria, while the T6SS2 primarily targets bacterial cells and secondarily eukaryotes. Attenuation of pathogenicity was observed in all T6SS mutants, demonstrating the requirement for proper assembly of the T6SS systems to maintain maximal virulence. Importance: Vibriosis outbreaks lead to large-scale hatchery losses of oyster larvae (product and seed) where Vibrio sp. associated losses of 80 to 100 percent are not uncommon. Practical and proactive biocontrol measures can be taken to help mitigate larval death by Vibrio sp. by better understanding the underlying mechanisms of virulence in V. coralliilyticus. In this study, we demonstrate the presence of two Type VI Secretion Systems (T6SS) in V. coralliilyticus RE22Sm and interrogate the roles of each T6SS in bacterial antagonism and pathogenesis against a eukaryotic host. Specifically, we show that the loss of T6SS1 function results in the loss of virulence against oyster larvae.

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A family of T6SS antibacterial effectors related to L,D-transpeptidases targets the Peptidoglycan

Access Microbiology ◽

10.1099/acmi.ac2020.po0033 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Stephanie Sibinelli de Sousa ◽

Julia Takuno Hespanhol ◽

Bruno Matsuyama ◽

Stephane Mesnage ◽

Gianlucca Nicastro ◽

...

Keyword(s):

Cell Envelope ◽

Point Mutations ◽

Time Lapse ◽

Secretion Systems ◽

Bacterial Antagonism ◽

New Family ◽

Type Vi Secretion ◽

Altered Cell ◽

Bacterial Effectors ◽

Insight Into

Type VI secretion systems (T6SSs) are contractile nanomachines widely used by bacteria to intoxicate competitors. Salmonella Typhimurium encodes a T6SS within the Salmonella pathogenicity island 6 (SPI-6) that is used during competition against species of the gut microbiota. We characterized a new SPI-6 T6SS antibacterial effector named Tlde1 (type VI L,D-transpeptidase effector 1). Tlde1 is toxic in target-cell periplasm and its toxicity is neutralized by co-expression with immunity protein Tldi1 (type VI L,D-transpeptidase immunity 1). Time-lapse microscopy revealed that intoxicated cells display altered cell division and lose cell envelope integrity. Bioinformatics analysis showed that Tlde1 is evolutionarily related to L,D-transpeptidases. Point mutations on conserved histidine121 and cysteine131 residues eliminated toxicity. Co-incubation of purified recombinant Tlde1 and peptidoglycan tetrapeptides showed that Tlde1 displays both L,D-carboxypeptidase activity by cleaving GM-tetrapeptides between meso-diaminopimelic acid3 and D-alanine4, and L,D-transpeptidase exchange activity by replacing D-alanine4 for a non-canonical D-amino acid. Tlde1 constitutes a new family of T6SS effectors widespread in Proteobacteria. This work increases our knowledge about the bacterial effectors used in interbacterial competitions and provides molecular insight into a new mechanism of bacterial antagonism.

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Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber

Molecules ◽

10.3390/molecules25041002 ◽

2020 ◽

Vol 25 (4) ◽

pp. 1002 ◽

Cited By ~ 1

Author(s):

Chunmeng Xu ◽

Lingjun Tang ◽

Youxiang Liang ◽

Song Jiao ◽

Huimin Yu ◽

...

Keyword(s):

Thermal Stability ◽

Large Scale ◽

Nitrile Hydratase ◽

Fusion Expression ◽

Solvent Tolerance ◽

Rhodococcus Ruber ◽

Bacterial Cells ◽

E Coli ◽

Nitrilase Activity ◽

Stability Enhancement

For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32–47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

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A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient

Journal of Bacteriology ◽

10.1128/jb.01678-08 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3712-3716 ◽

Cited By ~ 13

Author(s):

Vyacheslav Palchevskiy ◽

Steven E. Finkel

Keyword(s):

Escherichia Coli ◽

Bacterial Cells ◽

Wild Type ◽

Nutrient Source ◽

Term Survival ◽

Natural Competence ◽

E Coli ◽

Double Stranded Dna ◽

Better Than

ABSTRACT Nutritional competence is the ability of bacterial cells to utilize exogenous double-stranded DNA molecules as a nutrient source. We previously identified several genes in Escherichia coli that are important for this process and proposed a model, based on models of natural competence and transformation in bacteria, where it is assumed that single-stranded DNA (ssDNA) is degraded following entry into the cytoplasm. Since E. coli has several exonucleases, we determined whether they play a role in the long-term survival and the catabolism of DNA as a nutrient. We show here that mutants lacking either ExoI, ExoVII, ExoX, or RecJ are viable during all phases of the bacterial life cycle yet cannot compete with wild-type cells during long-term stationary-phase incubation. We also show that nuclease mutants, alone or in combination, are defective in DNA catabolism, with the exception of the ExoX− single mutant. The ExoX− mutant consumes double-stranded DNA better than wild-type cells, possibly implying the presence of two pathways in E. coli for the processing of ssDNA as it enters the cytoplasm.

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Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain

Microorganisms ◽

10.3390/microorganisms8050622 ◽

2020 ◽

Vol 8 (5) ◽

pp. 622 ◽

Cited By ~ 1

Author(s):

Mathilde Bouteiller ◽

Mathias Gallique ◽

Yvann Bourigault ◽

Artemis Kosta ◽

Julie Hardouin ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Sigma Factor ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Gene Encoding ◽

Prey Cell ◽

Over Expression ◽

Type Vi Secretion ◽

Flagellar Genes ◽

Class Iv

Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

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Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity inE. colivia Mutation of Free Cysteine Residues

BioMed Research International ◽

10.1155/2017/4817376 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12

Author(s):

Kun Zhang ◽

Yuejuan Zhang ◽

Jing Zi ◽

Xiaochang Xue ◽

Yi Wan

Keyword(s):

Large Scale ◽

Soluble Form ◽

Scale Production ◽

Superoxide Dismutase 1 ◽

Wild Type ◽

Lower Toxicity ◽

E Coli ◽

Promising Strategy ◽

Large Scale Production ◽

Cell Components

Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields inE. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 inE. coliwhereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it.Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

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Beyond dueling: roles of the type VI secretion system in microbiome modulation, pathogenesis and stress resistance

Stress Biology ◽

10.1007/s44154-021-00008-z ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Jinshui Lin ◽

Lei Xu ◽

Jianshe Yang ◽

Zhuo Wang ◽

Xihui Shen

Keyword(s):

Stress Resistance ◽

Secretion System ◽

Bacterial Cells ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Competitive Fitness ◽

Type Vi Secretion ◽

Protein Secretion Systems ◽

Extracellular Environment

AbstractBacteria inhabit diverse and dynamic environments, where nutrients may be limited and toxic chemicals can be prevalent. To adapt to these stressful conditions, bacteria have evolved specialized protein secretion systems, such as the type VI secretion system (T6SS) to facilitate their survival. As a molecular syringe, the T6SS expels various effectors into neighboring bacterial cells, eukaryotic cells, or the extracellular environment. These effectors improve the competitive fitness and environmental adaption of bacterial cells. Although primarily recognized as antibacterial weapons, recent studies have demonstrated that T6SSs have functions beyond interspecies competition. Here, we summarize recent research on the role of T6SSs in microbiome modulation, pathogenesis, and stress resistance.

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Identification of Escherichia coli ClpAP in regulating susceptibility to type VI secretion system-mediated attack by Agrobacterium tumefaciens

10.1101/734046 ◽

2019 ◽

Author(s):

Hsiao-Han Lin (林筱涵) ◽

Manda Yu (余文廸) ◽

Manoj Kumar Sriramoju ◽

Shang-Te Danny Hsu (徐尚德) ◽

Chi-Te Liu (劉啟德) ◽

...

Keyword(s):

Escherichia Coli ◽

Agrobacterium Tumefaciens ◽

Secretion System ◽

Gram Negative Bacteria ◽

Wild Type ◽

Type Vi Secretion System ◽

Competition Assay ◽

E Coli ◽

Type Vi Secretion ◽

In Trans

AbstractType VI secretion system (T6SS) is an effector delivery system used by gram-negative bacteria to kill other bacteria or eukaryotic host to gain fitness. In Agrobacterium tumefaciens, T6SS has been shown to kill other bacteria such as Escherichia coli. Interestingly, the A. tumefaciens T6SS killing efficiency differs when using different E. coli strains as recipient cells. Thus, we hypothesize that a successful T6SS killing not only relies on attacker T6SS activity but also depends on recipient factors. A high-throughput interbacterial competition assay was employed to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. From the 3909 E. coli Keio mutants screened, 16 candidate mutants were filtered out. One strain, ΔclpP::Kan, showed ten times more resistant to T6SS-mediating killing but restored its susceptibility when complemented with clpP in trans. ClpP is a universal and highly conserved protease that exists in both prokaryotes and eukaryotic organelles. In E. coli, ClpP uses either ClpA or ClpX as an adaptor for substrate specificity. Therefore, the susceptibility of the ΔclpA::Kan and ΔclpX::Kan was also tested. The T6SS attack susceptibility of ΔclpA::Kan is at the same level as ΔclpP::Kan, while ΔclpX::Kan showed no difference as compared to that of wild-type E. coli BW25113. The data also suggest that ClpA-ClpP interaction, rather than its protease activity, is responsible for enhancing susceptibility to T6SS killing. This study highlights the importance of recipient factors in determining the outcome of T6SS killing.

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Pantoea ananatis Utilizes a Type VI Secretion System for Pathogenesis and Bacterial Competition

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-14-0219-r ◽

2015 ◽

Vol 28 (4) ◽

pp. 420-431 ◽

Cited By ~ 28

Author(s):

Divine Y. Shyntum ◽

Jacques Theron ◽

Stephanus N. Venter ◽

Lucy N. Moleleki ◽

Ian K. Toth ◽

...

Keyword(s):

Wild Type Strain ◽

Gene Clusters ◽

Wild Type ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Pantoea Ananatis ◽

Bacterial Competition ◽

Type Vi Secretion ◽

Pineapple Fruit

Type VI secretion systems (T6SSs) are a class of macromolecular machines that are recognized as an important virulence mechanism in several gram-negative bacteria. The genome of Pantoea ananatis LMG 2665T, a pathogen of pineapple fruit and onion plants, carries two gene clusters whose predicted products have homology with T6SS-associated gene products from other bacteria. Nothing is known regarding the role of these T6SS-1 and T6SS-3 gene clusters in the biology of P. ananatis. Here, we present evidence that T6SS-1 plays an important role in the pathogenicity of P. ananatis LMG 2665T in onion plants, while a strain lacking T6SS-3 remains as pathogenic as the wild-type strain. We also investigated the role of the T6SS-1 system in bacterial competition, the results of which indicated that several bacteria compete less efficiently against wild-type LMG 2665T than a strain lacking T6SS-1. Additionally, we demonstrated that these phenotypes of strain LMG 2665T were reliant on the core T6SS products TssA and TssD (Hcp), thus indicating that the T6SS-1 gene cluster encodes a functioning T6SS. Collectively, our data provide the first evidence demonstrating that the T6SS-1 system is a virulence determinant of P. ananatis LMG 2665T and plays a role in bacterial competition.

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BPSS1504, a Cluster 1 Type VI Secretion Gene, Is Involved in Intracellular Survival and Virulence of Burkholderia pseudomallei

Infection and Immunity ◽

10.1128/iai.01544-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 2006-2015 ◽

Cited By ~ 18

Author(s):

Verena Hopf ◽

André Göhler ◽

Kristin Eske-Pogodda ◽

Antje Bast ◽

Ivo Steinmetz ◽

...

Keyword(s):

Mutant Strain ◽

Burkholderia Pseudomallei ◽

Plaque Assay ◽

Giant Cells ◽

Host Cells ◽

Wild Type ◽

Secretion Systems ◽

Content Type ◽

Type Vi Secretion

ABSTRACTBurkholderia pseudomalleiis a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide.B. pseudomalleiharbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified aB. pseudomalleitransposon mutant defective in theBPSS1504gene that showed reduced plaque formation. TheBPSS1504locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis ofB. pseudomalleiin mammalian hosts. For further analysis, aB. pseudomalleiBPSS1504deletion (BpΔBPSS1504) mutant and complemented mutant strain were constructed.B. pseudomalleilacking theBPSS1504gene was highly attenuated in BALB/c mice, whereas thein vivovirulence of the complemented mutant strain was fully restored to the wild-type level. TheBpΔBPSS1504mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lackinghcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS componentsvgrG,tssA, andhcp1, as well as the T6SS regulatorsvirAG,bprC, andbsaN, was not dependent onBPSS1504expression. However, secretion of Hcp1 was not detectable in the absence ofBPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus.

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Structural biology of type VI secretion systems

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2011.0209 ◽

2012 ◽

Vol 367 (1592) ◽

pp. 1102-1111 ◽

Cited By ~ 140

Author(s):

Eric Cascales ◽

Christian Cambillau

Keyword(s):

Structural Information ◽

Cell Envelope ◽

Host Cells ◽

Target Cells ◽

Bacterial Cells ◽

Secretion Systems ◽

Host Interaction ◽

Type Vi Secretion ◽

And Function ◽

Role And Function

Type VI secretion systems (T6SSs) are transenvelope complexes specialized in the transport of proteins or domains directly into target cells. These systems are versatile as they can target either eukaryotic host cells and therefore modulate the bacteria–host interaction and pathogenesis or bacterial cells and therefore facilitate access to a specific niche. These molecular machines comprise at least 13 proteins. Although recent years have witnessed advances in the role and function of these secretion systems, little is known about how these complexes assemble in the cell envelope. Interestingly, the current information converges to the idea that T6SSs are composed of two subassemblies, one resembling the contractile bacteriophage tail, whereas the other subunits are embedded in the inner and outer membranes and anchor the bacteriophage-like structure to the cell envelope. In this review, we summarize recent structural information on individual T6SS components emphasizing the fact that T6SSs are composite systems, adapting subunits from various origins.

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