1,721,125 research outputs found
Gene amplification and qRT-PCR
No full textThis chapter includes methods for the use of the polymerase chain reaction (PCR) with Pseudomonas, and several specific tips for their successful application in this organism. The first part of the chapter includes methods for purifying genomic DNA from, and amplifying genes from, Pseudomonas, in addition to methods which describe how to prepare a cell lysate from Pseudomonas species for colony PCR reactions. The chapter continues with a switch in focus from DNA to RNA, describing methods for RNA isolation from Pseudomonas, cDNA generation, and finally q-RT-PCR to investigate relative changes in gene expression
Take it of leave it: Mechanisms underlying bacterial bistable regulatory networks
No full textBistable switches occur in regulatory networks that can exist in two distinct stable states. Such networks allow distinct switching of individual cells. In bacteria these switches coexist with regulatory networks that respond gradually to environmental input. Bistable switches play key roles in high investment processes such as cellular differentiation in which only the end-result of the process is functional. Bistable switches are involved in development of phenotypical traits, such as virulence, bacterial persistence, sporulation and production of valuable compounds like antibiotics. The phenomenon of bistable networks is here explained and discussed. Additionally, natural and evolutionary solutions for creating all-or-none regulatory decisions are described as well as the use of bistable networks in synthetic biology
Contribution of Cyclic di-GMP in the Control of Type III and Type VI Secretion in Pseudomonas aeruginosa
No full textBacteria produce toxins to enhance their competitiveness in the colonization of an environment as well as during an infection. The delivery of toxins into target cells is mediated by several types of secretion systems, among them our focus is Type III and Type VI Secretion Systems (T3SS and T6SS, respectively). A thorough methodology is provided detailing how to identify if cyclic di-GMP signaling plays a role in the P. aeruginosa toxin delivery mediated by T3SS or T6SS. This includes in vitro preparation of the samples for Western blot analysis aiming at detecting possible c-di-GMP-dependent T3SS/T6SS switch, as well as in vivo analysis using the model organism Galleria mellonella to demonstrate the ecological and pathogenic consequence of the switch between these two secretion systems
Characterising the role of TssA proteins in the assembly and dynamics of the type VI secretion system
Full text linkThe gram-negative, opportunistic pathogen Pseudomonas aeruginosa encodes many weapons for virulence and interbacterial warfare, including the type VI secretion system (T6SS), a contractile nanomachine delivering effector proteins and antibacterial toxins either into the extracellular environment or directly into neighbouring cells. The T6SS component TssA is variable in size, domain organisation, symmetry and structure. Some TssA proteins are proposed to coordinate the assembly of the T6SS contractile sheath, but the exact role of TssA proteins remains unclear, and understanding is complicated by this relatively low level of conservation of TssA proteins. This project aimed to characterise the three TssA proteins of P. aeruginosa to assess their contribution to, and positioning within, their respective T6SSs and determine whether these were influenced by TssA variation. To achieve this, functional assays were optimised to monitor the activity of the three P. aeruginosa T6SSs and assess the requirement for TssA proteins. Interactions of both full length TssA proteins and individual domains with other T6SS proteins were also characterised and in silico approaches allowed the position of TssA proteins within the T6SS to be modelled. It was identified that each P. aeruginosa TssA protein was specifically required for the activity of its respective T6SS. Interactions of these TssA proteins with T6SS components were identified including specific interactions with the cognate contractile sheath, where this specificity could be altered by the exchange of domains. This work conclusively demonstrated specificity determinants for TssA proteins, which allowed the generation of functional chimeric TssA proteins, even with components from different TssA sub-groups. From this work a new docking has been proposed for TssA proteins with the contractile sheath and a model has been developed for how sheath assembly is coordinated by TssA proteins from this position.Open Acces
Hcp-dependent delivery of type VI secretion system effectors by Pseudomonas aeruginosa
Full text linkPseudomonas aeruginosa, a Gram-negative opportunistic pathogen, utilises the type VI secretion system (T6SS) for virulence and bacterial competition by firing toxic effectors into eukaryotes and prokaryotes. The T6SS is a macromolecular complex that propels an effector-loaded spear into target cells. The effectors are attached to the VgrG/PAAR tip or the Hcp tube of the spear. There are three P. aeruginosa T6SSs, H1-H3-T6SS, delivering seventeen characterised effectors - most are delivered by the VgrG tip and usually encoded next to the vgrG gene. We further contributed to understanding the delivery of VgrG effectors, determining the importance of particular PAAR proteins and the preference for VgrG-PAAR-effector combinations at the spear tip.
The few known Hcp tube-delivered effectors, Tse1-3, were previously identified through secretome screening. We performed a protein pull-down screen and identified several putative effectors that interact with the P. aeruginosa Hcp tubes (Hcp1, Hcp2 or Hcp3). Three of these putative effectors cause toxicity in bacteria. Previous studies suggest the Tse1-3 effectors travel inside the Hcp1 tube, since the Hcp1 inner tube residues were essential for Tse1-3 secretion. We revealed that Hcp2 and Hcp3 inner tube residues were also important for interaction with two newly identified effectors, suggesting a common delivery mode for multiple effectors via the inside of the Hcp tube. Whilst the known diameter of the Hcp inner tube is 40 Å, whether effectors have to conform to this size limit was until now unknown. We identified a size limit where, using a large Tse1 chimera, secretion and H1-T6SS-dependent killing was inhibited, since the Hcp1-Tse1 chimeric complex blocked tube formation and subsequent firing.
These findings advance our understanding of Hcp-delivered effectors. The approach used to identify and characterise new effectors is directly applicable to other organisms, wherein antibacterial effectors discovered could be used to design future antimicrobials.Open Acces
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Using the Pseudomonas aeruginosa T6SS as a protein shuttle to deliver effectors
Full text linkPseudomonas aeruginosa is a dreadful opportunistic pathogen that can cause both acute and chronic infections in humans. This bacterium exhibits great abundance of virulence factors and has the ability to flourish in a wide range of environmental niches, partly due to its remarkable arsenal of protein secretion systems. The type VI secretion system (T6SS), is a contractile injection apparatus that translocates a spike loaded with various effectors directly into eukaryotic and prokaryotic target cells. P. aeruginosa can load either one of its three T6SSs with a variety of bullets by specific but different modes. This dissertation reveals work focussed on the molecular details of how effector delivery relies on the appropriate attachment onto the T6SS apparatus and spike.
Our work provides details on how the spike components of the T6SS can be manipulated to deliver effector proteins, which highlights an exceptional modularity for loading the T6SS nanoweapon. The spike, which punctures the bacterial cell envelope allowing effector transport, consists of a torch-like VgrG trimer on which sits a PAAR sharpening the VgrG tip. We show how various effectors can be fused directly to either of the two spike components and reach the supernatant or are delivered into target prey cells. Further, we reveal that effectors specifically bind to different parts of VgrG proteins and that these interactions not only lead to effector stabilisation, but also to their secretion. In summary, this work will give sound evidence that an effector delivered by the T6SS requires a chaperone module for stabilisation and an adaptor module for its connection to the T6SS spike.
Results from this thesis highlight the various modes that are at P. aeruginosa’ s disposal to deliver its many T6SS effectors, which confer a significant advantage during bacterial competition. Overall, this work advances our understanding on the delivery mechanisms of T6SS effectors and thus, in general, of this fantastic nanomachine.Open Acces
Insights into the life styles of Pseudomonas stutzeri
No full textRamos, Juan Luis; Filloux, Alain (eds.). Pseudomonas. Volume 6: Molecular Microbiology, Infection and Biodiversity. Springer, 2010, p.177-198. e-ISBN 978-90-481-3909-5. ISBN 978-90-481-3908-8. DOI 10.1007/978-90-481-3909-5_6Peer Reviewe
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