Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

Frye, Stephan A.; Assalkhou, Reza; Collins, Richard F.; Ford, Robert C.; Petersson, Christoffer; Derrick, Jeremy P.; Tønjum, Tone

doi:10.1099/mic.0.2006/000315-0

Cited by 32 publications

(35 citation statements)

References 56 publications

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“…The presence of intact pili on M1M2 suggests that enough disulphidebonded PilQ is formed to produce the secretin without DsbA-catalysed folding, but it may be that the function of the pore or a separable PilQ activity in DNA binding and uptake is affected more profoundly. These findings are also in agreement with the recent discovery that the PilQ complex binds DNA in the core of its central channel (Assalkhou et al, 2007;Frye et al, 2006), explaining why DNA binding and uptake are reduced in the DsbA1/2 mutant. Assalkou et al (2007) showed that PilQ bound DNA irrespective of the presence of DUS, which was confirmed in our assays, as binding of plasmid DNA with and without DUS was comparably reduced in M1M2.…”

Section: Discussionsupporting

confidence: 81%

Reduced DNA binding and uptake in the absence of DsbA1 and DsbA2 of Neisseria meningitidis due to inefficient folding of the outer-membrane secretin PilQ

Sinha¹,

Ambur

Langford³

et al. 2008

Microbiology

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DsbA ensures the correct folding of many exported bacterial proteins by forming intramolecular disulphide bonds in the bacterial periplasm. The pathogen Neisseria meningitidis is unusual in its possession of three different dsbA genes (dsbA1, dsbA2 and dsbA3), encoding two membrane-anchored (DsbA1 and DsbA2) and one periplasmic (DsbA3) thiol-disulphide oxidoreductase enzymes. In this study, the involvement of DsbA1 and DsbA2 in natural competence was confirmed and attributed to events in the early stages of the transformation process. Strains lacking both DsbA1 and DsbA2 were reduced in competence as a result of decreased DNA binding and uptake. Overexpression of DsbA3 could not overcome this defect, suggesting differences in substrate specificity and protein-folding abilities between the DsbA homologues. Competence in Neisseria is dependent on the expression of type IV pili, which are extruded and retracted through the outer-membrane secretin PilQ. Both DsbA1 and DsbA2 were able to specifically bind PilQ in solid-phase overlay assays. Consistent with this, deletion of both dsbA1 and dsbA2 resulted in reduced levels of PilQ, confirming inefficient folding of PilQ, while pilus expression was apparently unaffected. The secretin PilQ is involved in DNA binding and transport as well as pilus biogenesis, and the defect in PilQ folding resulting from the absence of DsbA1 and DsbA2 is revealed in the observed decreased DNA binding and uptake.

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Section: Discussionsupporting

confidence: 81%

Reduced DNA binding and uptake in the absence of DsbA1 and DsbA2 of Neisseria meningitidis due to inefficient folding of the outer-membrane secretin PilQ

Sinha¹,

Ambur

Langford³

et al. 2008

Microbiology

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“…Within the complex is a long tapered cavity that is 90 Å in height, and 87 Å in diameter at its broadest point. The topology of the PilQ complex was investigated using insertion epitopes and immunogold labelling [40]. These studies showed that both the N-terminal and C-terminal regions of PilQ localize to the periplasm, including an insert at residue 205, which maps to the arms of the complex.…”

Section: The Outer Membrane Secretinmentioning

confidence: 99%

Section: The Outer Membrane Secretinmentioning

confidence: 99%

Type IV pili: paradoxes in form and function

Craig

2008

Current Opinion in Structural Biology

249

263

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Type IV pili are filaments on the surfaces of many Gram-negative bacteria that mediate an extraordinary array of functions, including adhesion, motility, microcolony formation and secretion of proteases and colonization factors. Their prominent display on the surfaces of many bacterial pathogens, their vital role in virulence, and their ability to elicit an immune response make Type IV pilus structures particularly relevant for study as targets for component vaccines and therapies. Structural studies of the pili and components of the pilus assembly apparatus have proven extremely challenging, but new approaches and methods have produced important breakthroughs that are advancing our understanding of pilus functions and their complex assembly mechanism. These structures provide insights into the biology of Type IV pili as well as that of the related bacterial secretion and archaeal flagellar systems. This review will summarize the most recent structural advances on Type IV pili and their assembly components and highlight their significance.Type IV pili are homopolymers of a 15-20 kDa pilin subunit that emanate from the surfaces of many Gram-negative bacteria and at least one Gram-positive organism. These filaments, which appear smooth and featureless by electron microscopy (EM), are 6-9 nm in diameter and several microns in length (Fig. 1). Beneath their plain façade lies an exquisite helical architecture that provides for strength, flexibility and a multitude of functions, including twitching and gliding motility, adhesion, immune escape, DNA uptake, biofilm formation, microcolony formation, secretion, phage transduction and signal transduction. Unlike other bacterial pili, which use as few as two proteins for assembly [1,2], Type IV pilus biogenesis requires a dozen or more proteins, many of which share sequence conservation among divergent species. Pili are assembled, and in some cases disassembled, rapidly using powerful molecular motors that hydrolyze ATP. The proteins involved in pilus biogenesis form a dynamic but poorly-defined complex that spans both bacterial membranes and the intervening periplasm. Our knowledge of Type IV pili presents several paradoxes: What type of molecular architecture yields such thin flexible filaments that can withstand stresses greater than 100 pN? How does a single filament design provide for such functional diversity? How does the assembly apparatus allow for rapid polymerization and depolymerization at a rate of more than 1000 subunits/second? This review will focus on the latest structural findings, which help to explain these paradoxes and advance our understanding of this remarkable biological machine.Type IV pilus assembly involves 12 or more proteins that in many cases are encoded within the same operon. Several key components are utilized in all Type IV pilus systems and are also found in Type II secretion and archaeal flagellar systems [3][4][5]. These are: the pilin subunit; an inner membrane prepilin peptidase that cleaves the N-terminal leader peptide; an ...

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“…2A). As has been done to investigate various types of OM proteins, including other iron transporters (22,23,50,89,106,122,127,130), we introduced a His 6 -tagged version of LbtU into 130b and then used anti-His antibodies and immunoblot analysis to track the location of LbtU in cellular fractions. LbtU-His 6 did not diminish the growth of the wild type and complemented an iron-related growth defect of an lbtU mutant (see below, Fig.…”

Section: Vol 193 2011mentioning

confidence: 99%

Legionella pneumophila LbtU Acts as a Novel, TonB-Independent Receptor for the Legiobactin Siderophore

et al. 2011

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Gram-negative Legionella pneumophila produces a siderophore (legiobactin) that promotes lung infection. We previously determined that lbtA and lbtB are required for the synthesis and secretion of legiobactin. DNA sequence and reverse transcription-PCR (RT-PCR) analyses now reveal the presence of an iron-repressed gene (lbtU) directly upstream of the lbtAB-containing operon. In silico analysis predicted that LbtU is an outer membrane protein consisting of a 16-stranded transmembrane ␤-barrel, multiple extracellular domains, and short periplasmic tails. Immunoblot analysis of cell fractions confirmed an outer membrane location for LbtU. Although replicating normally in standard media, lbtU mutants, like lbtA mutants, were impaired for growth on iron-depleted agar media. While producing typical levels of legiobactin, lbtU mutants were unable to use supplied legiobactin to stimulate growth on iron-depleted media and displayed an inability to take up iron. Complemented lbtU mutants behaved as the wild type did. The lbtU mutants were also impaired for infection in a legiobactin-dependent manner. Together, these data indicate that LbtU is involved in the uptake of legiobactin and, based upon its location, is most likely the Legionella siderophore receptor. The sequence and predicted two-dimensional (2D) and 3D structures of LbtU were distinct from those of all known siderophore receptors, which generally contain a 22-stranded ␤-barrel and an extended N terminus that binds TonB in order to transduce energy from the inner membrane. This observation coupled with the fact that L. pneumophila does not encode TonB suggests that LbtU is a new type of receptor that participates in a form of iron uptake that is mechanistically distinct from the existing paradigm.

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Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

Cited by 32 publications

References 56 publications

Reduced DNA binding and uptake in the absence of DsbA1 and DsbA2 of Neisseria meningitidis due to inefficient folding of the outer-membrane secretin PilQ

Reduced DNA binding and uptake in the absence of DsbA1 and DsbA2 of Neisseria meningitidis due to inefficient folding of the outer-membrane secretin PilQ

Type IV pili: paradoxes in form and function

Legionella pneumophila LbtU Acts as a Novel, TonB-Independent Receptor for the Legiobactin Siderophore

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