The ability of most bacterial flagellar motors to reverse the direction of rotation is crucial for efficient chemotaxis. In Escherichia coli, motor reversals are mediated by binding of phosphorylated chemotaxis protein CheY to components of the flagellar rotor, FliM and FliN, which induces a conformational switch of the flagellar C-ring. Here, we show that for Shewanella putrefaciens, Vibrio parahaemolyticus and likely a number of other species an additional transmembrane protein, ZomB, is critically required for motor reversals as mutants lacking ZomB exclusively exhibit straightforward swimming also upon full phosphorylation or overproduction of CheY. ZomB is recruited to the cell poles by and is destabilized in the absence of the polar landmark protein HubP. ZomB also co-localizes to and may thus interact with the flagellar motor. The ΔzomB phenotype was suppressed by mutations in the very C-terminal region of FliM. We propose that the flagellar motors of Shewanella, Vibrio and numerous other species harboring orthologs to ZomB are locked in counterclockwise rotation and may require interaction with ZomB to enable the conformational switch required for motor reversals. Regulation of ZomB activity or abundance may provide these species with an additional means to modulate chemotaxis efficiency.
Many pathogenic bacteria use the type III secretion system (T3SS) injectisome to manipulate host cells by injecting virulence-promoting effector proteins into the host cytosol. The T3SS is activated upon host cell contact, and its activation is accompanied by an arrest of cell division; hence, many species maintain a T3SS-inactive sibling population to propagate efficiently within the host. The enteric pathogen Yersinia enterocolitica utilizes the T3SS to prevent phagocytosis and inhibit inflammatory responses. Unlike other species, almost all Y. enterocolitica are T3SS-positive at 37°C, which raises the question, how these bacteria are able to propagate within the host, that is, when and how they stop secretion and restart cell division after a burst of secretion. Using a fast and quantitative in vitro secretion assay, we have examined the initiation and termination of type III secretion. We found that effector secretion begins immediately once the activating signal is present, and instantly stops when this signal is removed. Following effector secretion, the bacteria resume division within minutes after being introduced to a non-secreting environment, and the same bacteria are able to re-initiate effector secretion at later time points. Our results indicate that Y. enterocolitica use their type III secretion system to promote their individual survival when necessary, and are able to quickly switch their behavior toward replication afterwards, possibly gaining an advantage during infection.
The type III secretion system is the common core of two bacterial molecular machines: the flagellum and the injectisome. The flagellum is the most widely distributed prokaryotic locomotion device, whereas the injectisome is a syringe‐like apparatus for inter‐kingdom protein translocation, which is essential for virulence in important human pathogens. The successful concept of the type III secretion system has been modified for different bacterial needs. It can be adapted to changing conditions, and was found to be a dynamic complex constantly exchanging components. In this review, we highlight the flexibility, adaptivity, and dynamic nature of the type III secretion system.
Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. We show that in Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.
2Many gastrointestinal pathogens use a type III secretion system (T3SS) to manipulate host cells. 3 Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has 4 been unclear how premature effector secretion is prevented during infection. We found that at 5 low external pH, such as in the stomach, the components at the proximal interface of the 6 injectisome are temporarily released to the bacterial cytosol, preventing protein secretion. Low 7 external pH is sensed in the periplasm and leads to a partial dissociation of the inner membrane 8 injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS 9 components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of 10
The original version of this Article contained an error in the Methods, section 'Single-particle tracking photoactivated localization microscopy (sptPALM)', where an equation incorrectly readThis has been corrected in both the PDF and HTML versions of the Article.
In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics and live-cell microscopy, we determined the contribution of the pilotin to assembly, function and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In the absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. In line, the majority of export apparatus subcomplexes is mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, the export of late T3SS substrates including the virulence effectors is hardly affected in these bacteria, whereas early T3SS substrates, such as the needle subunits, are only exported to a very low degree. We found that pilotins transiently interact with the secretin and the large export apparatus component SctV, but mostly localize throughout the bacterial membrane, where they form transient mobile clusters, which do not colocalize with assembled injectisomes. In addition, pilotins interact with non-T3SS components, including sugar transporters. Pilotins therefore have additional functions downstream injectisome assembly, which include the regulation of type III secretion and a potential new link to the cellular energy metabolism.
The Type III Secretion System (T3SS) is a complex transmembrane apparatus used by many Gram‐negative pathogens for protein translocation and host invasion. An essential T3SS component is the needle, a hollow substructure extending from the bacterial membrane, spanning the space from the bacterium to the host cytosol. The needle is formed by helical polymerization of a single small protein, which is secreted by the T3SS and assembled outside the bacterium. Visualization of the needle previously depended on immunofluorescence, which requires cell fixation. We hereby describe a simple methodology for T3SS needle fluorescent tagging and visualization, which enables live‐cell microscopy and investigation of needle kinetics and dynamics.Support or Funding InformationThis study was supported in part by the DAAD RISE Scholarship, Reed College Biology Undergraduate Student Travel Award, Reed College Summer Opportunity Fellowship, Reed College Undergraduate Research Opportunity Grant and Galakatos funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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