scholarly journals Paraburkholderia phymatum STM815 σ54 Controls Utilization of Dicarboxylates, Motility, and T6SS-b Expression

Nitrogen ◽  
2020 ◽  
Vol 1 (2) ◽  
pp. 81-98
Author(s):  
Martina Lardi ◽  
Yilei Liu ◽  
Sebastian Hug ◽  
Samanta Bolzan de Campos ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Nitrogenase Activity ◽  
Secretion Systems ◽  
Free Living ◽  
Major Life ◽  
Life Styles ◽  
Type Vi Secretion ◽  
Flagellar Biosynthesis ◽  
Free Living Conditions

Rhizobia have two major life styles, one as free-living bacteria in the soil, and the other as bacteroids within the root/stem nodules of host legumes where they convert atmospheric nitrogen into ammonia. In the soil, rhizobia have to cope with changing and sometimes stressful environmental conditions, such as nitrogen limitation. In the beta-rhizobial strain Paraburkholderia phymatum STM815, the alternative sigma factor σ54 (or RpoN) has recently been shown to control nitrogenase activity during symbiosis with Phaseolus vulgaris. In this study, we determined P. phymatum’s σ54 regulon under nitrogen-limited free-living conditions. Among the genes significantly downregulated in the absence of σ54, we found a C4-dicarboxylate carrier protein (Bphy_0225), a flagellar biosynthesis cluster (Bphy_2926-64), and one of the two type VI secretion systems (T6SS-b) present in the P. phymatum STM815 genome (Bphy_5978-97). A defined σ54 mutant was unable to grow on C4 dicarboxylates as sole carbon source and was less motile compared to the wild-type strain. Both defects could be complemented by introducing rpoNin trans. Using promoter reporter gene fusions, we also confirmed that the expression of the T6SS-b cluster is regulated by σ54. Accordingly, we show that σ54 affects in vitro competitiveness of P. phymatum STM815 against Paraburkholderia diazotrophica.

scholarly journals Differential Expression of Paraburkholderia phymatum Type VI Secretion Systems (T6SS) Suggests a Role of T6SS-b in Early Symbiotic Interaction

2021 ◽  
Vol 12 ◽  
Author(s):  
Sebastian Hug ◽  
Yilei Liu ◽  
Benjamin Heiniger ◽  
Aurélien Bailly ◽  
Christian H. Ahrens ◽  
...  
Keyword(s):  
Root Nodules ◽  
Rhizobial Strain ◽  
Secretion Systems ◽  
Phenotypic Analysis ◽  
Free Living ◽  
Symbiotic Interaction ◽  
Type Vi Secretion ◽  
Mutant Strains

Paraburkholderia phymatum STM815, a rhizobial strain of the Burkholderiaceae family, is able to nodulate a broad range of legumes including the agriculturally important Phaseolus vulgaris (common bean). P. phymatum harbors two type VI Secretion Systems (T6SS-b and T6SS-3) in its genome that contribute to its high interbacterial competitiveness in vitro and in infecting the roots of several legumes. In this study, we show that P. phymatum T6SS-b is found in the genomes of several soil-dwelling plant symbionts and that its expression is induced by the presence of citrate and is higher at 20/28°C compared to 37°C. Conversely, T6SS-3 shows homologies to T6SS clusters found in several pathogenic Burkholderia strains, is more prominently expressed with succinate during stationary phase and at 37°C. In addition, T6SS-b expression was activated in the presence of germinated seeds as well as in P. vulgaris and Mimosa pudica root nodules. Phenotypic analysis of selected deletion mutant strains suggested a role of T6SS-b in motility but not at later stages of the interaction with legumes. In contrast, the T6SS-3 mutant was not affected in any of the free-living and symbiotic phenotypes examined. Thus, P. phymatum T6SS-b is potentially important for the early infection step in the symbiosis with legumes.

scholarly journals The Bradyrhizobium Sp. LmicA16 Type VI Secretion System is Required for Efficient Nodulation of Lupinus Spp.

2021 ◽  
Author(s):  
Tighilt L. ◽  
Boulila F. ◽  
De Sousa BFS ◽  
Giraud E ◽  
Ruiz-Argüeso T ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Gene Clusters ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Free Living ◽  
Type Vi Secretion ◽  
Divergent Gene ◽  
Bradyrhizobium Sp ◽  
Symbiotic Phenotype ◽  
Free Living Conditions

Abstract Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and L. angustifolius . A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild type strain (wt) and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26 kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidy and motility than the wt strain in free living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

scholarly journals Two Functional Type VI Secretion Systems in Avian Pathogenic Escherichia coli Are Involved in Different Pathogenic Pathways

2014 ◽  
Vol 82 (9) ◽  
pp. 3867-3879 ◽  
Author(s):  
Jiale Ma ◽  
Yinli Bao ◽  
Min Sun ◽  
Wenyang Dong ◽  
Zihao Pan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chicken Embryo Fibroblast ◽  
Microvascular Endothelial Cell ◽  
Infection Model ◽  
Tail Fiber ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACTType VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. The VgrG protein, a core component and effector of T6SS, has been demonstrated to perform diverse functions. The N-terminal domain of VgrG protein is a homologue of tail fiber protein gp27 of phage T4, which performs a receptor binding function and determines the host specificity. Based on sequence analysis, we found that two putative T6SS loci exist in the genome of the avian pathogenicEscherichia coli(APEC) strain TW-XM. To assess the contribution of these two T6SSs to TW-XM pathogenesis, the crucialclpVclusters of these two T6SS loci and theirvgrGgenes were deleted to generate a series of mutants. Consequently, T6SS1-associated mutants presented diminished adherence to and invasion of several host cell lines culturedin vitro, decreased pathogenicity in duck and mouse infection modelsin vivo, and decreased biofilm formation and bacterial competitive advantage. In contrast, T6SS2-associated mutants presented a significant decrease only in the adherence to and invasion of mouse brain microvascular endothelial cell (BMEC) line bEnd.3 and brain tissue of the duck infection model. These results suggested that T6SS1 was involved in the proliferation of APEC in systemic infection, whereas VgrG-T6SS2 was responsible only for cerebral infection. Further study demonstrated that VgrG-T6SS2 was able to bind to the surface of bEnd.3 cells, whereas it did not bind to DF-1 (chicken embryo fibroblast) cells, which further proved the interaction of VgrG-T6SS2 with the surface of BMECs.

scholarly journals A new family of Type VI secretion system-delivered effector proteins displays ion-selective pore-forming activity

2019 ◽  
Author(s):  
Giuseppina Mariano ◽  
Katharina Trunk ◽  
David J. Williams ◽  
Laura Monlezun ◽  
Henrik Strahl ◽  
...  
Keyword(s):  
Effector Proteins ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
New Family ◽  
Type Vi Secretion ◽  
Outer Membrane Permeability ◽  
Polymicrobial Communities ◽  
Bacterial Effectors

AbstractType VI secretion systems (T6SSs) are nanomachines widely used by bacteria to compete with rivals. T6SSs deliver multiple toxic effector proteins directly into neighbouring cells and play key roles in shaping diverse polymicrobial communities. A number of families of T6SS-dependent anti-bacterial effectors have been characterised, however the mode of action of others remains unknown. Here we report that Ssp6, an anti-bacterial effector delivered by theSerratia marcescensT6SS, is an ion-selective pore-forming toxin.In vivo, Ssp6 inhibits growth by causing depolarisation of the inner membrane of intoxicated cells and also leads to increased outer membrane permeability, whilst reconstruction of Ssp6 activityin vitrodemonstrated that it forms cation-selective pores. A survey of bacterial genomes revealed that Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 represents a new family of T6SS-delivered anti-bacterial effectors, further diversifying the portfolio of weapons available for deployment during inter-bacterial conflict.

scholarly journals A family of Type VI secretion system effector proteins that form ion-selective pores

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Giuseppina Mariano ◽  
Katharina Trunk ◽  
David J. Williams ◽  
Laura Monlezun ◽  
Henrik Strahl ◽  
...  
Keyword(s):  
Opportunistic Pathogen ◽  
Effector Proteins ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
New Family ◽  
Type Vi Secretion ◽  
Genes Encoding ◽  
Outer Membrane Permeability ◽  
Bacterial Effectors

AbstractType VI secretion systems (T6SSs) are nanomachines widely used by bacteria to deliver toxic effector proteins directly into neighbouring cells. However, the modes of action of many effectors remain unknown. Here we report that Ssp6, an anti-bacterial effector delivered by a T6SS of the opportunistic pathogen Serratia marcescens, is a toxin that forms ion-selective pores. Ssp6 inhibits bacterial growth by causing depolarisation of the inner membrane in intoxicated cells, together with increased outer membrane permeability. Reconstruction of Ssp6 activity in vitro demonstrates that it forms cation-selective pores. A survey of bacterial genomes reveals that genes encoding Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 and similar proteins represent a new family of T6SS-delivered anti-bacterial effectors.

scholarly journals nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth

2016 ◽  
Vol 29 (10) ◽  
pp. 767-773 ◽  
Author(s):  
Jenjira Wongdee ◽  
Pongpan Songwattana ◽  
Nico Nouwen ◽  
Rujirek Noisangiam ◽  
Joel Fardoux ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Transcriptional Analysis ◽  
Main Role ◽  
Free Living ◽  
Living State ◽  
Bradyrhizobium Sp ◽  
Reporter Strains ◽  
Free Living Conditions

Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobium sp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity.

scholarly journals Production of Nitric Oxide and Nitrosylleghemoglobin Complexes in Soybean Nodules in Response to Flooding

2010 ◽  
Vol 23 (5) ◽  
pp. 702-711 ◽  
Author(s):  
Cristina Sánchez ◽  
Andrew J. Gates ◽  
Georgina E. Meakin ◽  
Toshiki Uchiumi ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrite Reductase ◽  
Nitrogenase Activity ◽  
Environmental Cues ◽  
Wild Type ◽  
Free Living ◽  
No Formation ◽  
Symbiotic Interactions ◽  
Negative Effect ◽  
Free Living Conditions

Nitric oxide (NO) has gained interest as a major signaling molecule during plant development and in response to environmental cues. Formation of NO during symbiotic interactions has been reported, but the role and sources of NO in nodules remain unclear. In this work, the involvement of denitrification, performed by the symbiont Bradyrhizobium japonicum, in NO formation in soybean nodules in response to flooding conditions has been investigated by inoculating plants with napA-, nirK-, or norC-deficient mutants. Levels of nitrosylleghemoglobin (LbNO) in flooded nirK and norC nodules were significantly higher than those observed in wild-type nodules. In addition, nirK and norC nodules accumulated more nitrite and NO, respectively, than wild-type nodules. By contrast, levels of LbNO, nitrite, and NO in flooded napA nodules were lower than in wild-type nodules. These results suggest that LbNO formation in soybean nodules in response to flooding conditions is caused by nitrite and NO generated from periplasmic nitrate reductase (Nap) and also containing nitrite reductase (NirK) denitrification enzymes. Flooding caused a decrease of nifH expression and nitrogenase activity in wild-type and norC nodules but not in napA or nirK nodules. Incubation of wild-type and norC nodules with a NO scavenger counteracted the effect of flooding. Under free-living conditions, β-galactosidase activity from a nifD′-′lacZ fusion decreased in a norC mutant, which also accumulated NO in the medium. These results suggest that NO formed by Cu-containing nitrite reductase in soybean nodules in response to flooding has a negative effect on expression of nitrogenase. We propose that Lb has a major role in detoxifying NO and nitrite produced by bacteroidal denitrification in response to flooding conditions.

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

2015 ◽  
Vol 28 (4) ◽  
pp. 420-431 ◽  
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.

scholarly journals The Cluster 1 Type VI Secretion System Is a Major Virulence Determinant inBurkholderia pseudomallei

2011 ◽  
Vol 79 (4) ◽  
pp. 1512-1525 ◽  
Author(s):  
Mary N. Burtnick ◽  
Paul J. Brett ◽  
Sarah V. Harding ◽  
Sarah A. Ngugi ◽  
Wilson J. Ribot ◽  
...  
Keyword(s):  
Growth Defect ◽  
Raw 264.7 ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Lethal Challenge ◽  
Hamster Model ◽  
Raw 264.7 Macrophages ◽  
Link Type ◽  
Type Vi Secretion

ABSTRACTTheBurkholderia pseudomalleiK96243genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology ofB. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge withK96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced byB. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exportedin vitrowhen the VirAG two-component regulatory system was overexpressed intrans. We also constructed sixhcpdeletion mutants (Δhcp1throughΔhcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD50s) for theΔhcp2throughΔhcp6mutants were indistinguishable fromK96243(<10 bacteria), but the LD50for theΔhcp1mutant was >103bacteria. Thehcp1deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. UnlikeK96243, theΔhcp1mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle ofB. pseudomallei.

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