scholarly journals The type IVa pilus machinery is pre-installed during cell division

2016 ◽  
Author(s):  
Tyson Carter ◽  
Ryan N.C. Buensuceso ◽  
Stephanie Tammam ◽  
Ryan P. Lamers ◽  
Hanjeong Harvey ◽  
...  
Keyword(s):  
Cell Division ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Cell Recruitment ◽  
Type Iv ◽  
Membrane Invagination ◽  
Dynamic Assembly ◽  
Polar Organelle

ABSTRACTType IV pili (T4aP) are ubiquitous microbial appendages used for adherence, twitching motility, DNA uptake, and electron transfer. Many of these functions depend on dynamic assembly and disassembly of the pilus by a megadalton-sized, cell envelope-spanning protein complex located at the poles of rod-shaped bacteria. How the T4aP assembly complex becomes integrated into the cell envelope in the absence of dedicated peptidoglycan (PG) hydrolases is unknown. After ruling out potential involvement of housekeeping PG hydrolases in installation of the T4aP machinery in P. aeruginosa, we discovered that key components of inner (PilMNOP) and outer (PilQ) membrane subcomplexes are recruited to future sites of cell division. Mid-cell recruitment of a fluorescently tagged alignment subcomplex component, mCherry-PilO, depended on PilQ secretin monomers – specifically, their N-terminal PG-binding AMIN domains. PilP, which connects PilO to PilQ, was required for recruitment, while PilM, which is structurally similar to divisome component FtsA, was not. Recruitment preceded secretin oligomerization in the outer membrane, as loss of the PilQ pilotin, PilF, had no effect on localization. These results were confirmed in cells chemically blocked for cell division prior to outer membrane invagination. The hub protein FimV and a component of the Polar Organelle Coordinator complex – PocA – were independently required for mid-cell recruitment of PilO and PilQ. Together, these data reveal an integrated, energy-efficient strategy for the targeting and pre-installation – rather than retrofit – of the T4aP system into nascent poles, without the need for dedicated PG-remodelling enzymes.

scholarly journals Phylogenomics Reveals a Diverse Rickettsiales Type IV Secretion System

2010 ◽  
Vol 78 (5) ◽  
pp. 1809-1823 ◽  
Author(s):  
Joseph J. Gillespie ◽  
Kelly A. Brayton ◽  
Kelly P. Williams ◽  
Marco A. Quevedo Diaz ◽  
Wendy C. Brown ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Cell Envelope ◽  
Secretion System ◽  
Current Data ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Dna Uptake ◽  
Type Iv ◽  
Conserved Core

ABSTRACT With an obligate intracellular lifestyle, Alphaproteobacteria of the order Rickettsiales have inextricably coevolved with their various eukaryotic hosts, resulting in small, reductive genomes and strict dependency on host resources. Unsurprisingly, large portions of Rickettsiales genomes encode proteins involved in transport and secretion. One particular transporter that has garnered recent attention from researchers is the type IV secretion system (T4SS). Homologous to the well-studied archetypal vir T4SS of Agrobacterium tumefaciens, the R ickettsiales v ir homolog (rvh) T4SS is characterized primarily by duplication of several of its genes and scattered genomic distribution of all components in several conserved islets. Phylogeny estimation suggests a single event of ancestral acquirement of the rvh T4SS, likely from a nonalphaproteobacterial origin. Bioinformatics analysis of over 30 Rickettsiales genome sequences illustrates a conserved core rvh scaffold (lacking only a virB5 homolog), with lineage-specific diversification of several components (rvhB1, rvhB2, and rvhB9b), likely a result of modifications to cell envelope structure. This coevolution of the rvh T4SS and cell envelope morphology is probably driven by adaptations to various host cells, identifying the transporter as an important target for vaccine development. Despite the genetic intractability of Rickettsiales, recent advancements have been made in the characterization of several components of the rvh T4SS, as well as its putative regulators and substrates. While current data favor a role in effector translocation, functions in DNA uptake and release and/or conjugation cannot at present be ruled out, especially considering that a mechanism for plasmid transfer in Rickettsia spp. has yet to be proposed.

scholarly journals Requirement of Novel Competence Genes pilT andpilU of Pseudomonas stutzeri for Natural Transformation and Suppression of pilT Deficiency by a Hexahistidine Tag on the Type IV Pilus Protein PilAI

2001 ◽  
Vol 183 (16) ◽  
pp. 4694-4701 ◽  
Author(s):  
Stefan Graupner ◽  
Nicole Weger ◽  
Monika Sohni ◽  
Wilfried Wackernagel
Keyword(s):  
Amino Acid ◽  
Natural Transformation ◽  
Pseudomonas Stutzeri ◽  
Amino Acid Identity ◽  
Binding Motif ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Insertional Inactivation

ABSTRACT The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termedpilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [3H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termedpilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When thepilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gmr cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT + in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

scholarly journals Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Courtney K. Ellison ◽  
Triana N. Dalia ◽  
Catherine A. Klancher ◽  
Joshua W. Shaevitz ◽  
Zemer Gitai ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Type Iv Pili ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Protein Inhibitor ◽  
Acinetobacter Baylyi ◽  
Type Iv ◽  
Surface Sensing ◽  
Virulence Protein ◽  
Labeling Method

AbstractBacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis.

scholarly journals Type IV Pilus Biogenesis, Twitching Motility, and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF, PilT1, and PilT2

2013 ◽  
Vol 80 (2) ◽  
pp. 644-652 ◽  
Author(s):  
Ralf Salzer ◽  
Friederike Joos ◽  
Beate Averhoff
Keyword(s):  
Natural Transformation ◽  
Thermophilic Bacteria ◽  
Bacterial Species ◽  
Thermus Thermophilus ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Content Type ◽  
Ecological Diversification ◽  
Type Iv

ABSTRACTNatural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species.Thermus thermophilusHB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whetherT. thermophilushas any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that inT. thermophilus, T4P and natural transformation are linked but distinct systems.

scholarly journals The Caulobacter Tol-Pal Complex Is Essential for Outer Membrane Integrity and the Positioning of a Polar Localization Factor

2010 ◽  
Vol 192 (19) ◽  
pp. 4847-4858 ◽  
Author(s):  
Yi-Chun Yeh ◽  
Luis R. Comolli ◽  
Kenneth H. Downing ◽  
Lucy Shapiro ◽  
Harley H. McAdams
Keyword(s):  
Cell Division ◽  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Protein Localization ◽  
Membrane Integrity ◽  
Division Plane ◽  
Localization Factor ◽  
Complete Cell ◽  
And Function

ABSTRACTCell division inCaulobacter crescentusinvolves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast toEscherichia coli, theCaulobacterTol-Pal complex is essential. Cryo-electron microscopy images of theCaulobactercell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild-type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Coimmunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of thetrans-envelope Tol-Pal complex releases TipN from its subcellular position. TheCaulobacterTol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor.

scholarly journals Cryo-EM structure of the bifunctional secretin complex of Thermus thermophilus

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Edoardo D'Imprima ◽  
Ralf Salzer ◽  
Ramachandra M Bhaskara ◽  
Ricardo Sánchez ◽  
Ilona Rose ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Natural Transformation ◽  
Thermus Thermophilus ◽  
Homology Model ◽  
Type Iv Pili ◽  
Gram Negative Bacteria ◽  
Dna Uptake ◽  
Type Iv ◽  
Periplasmic Domain

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

scholarly journals Novel mechanisms of type IV pilus regulation in Acinetobacter baylyi

2020 ◽  
Author(s):  
Courtney K. Ellison ◽  
Triana N. Dalia ◽  
Catherine A. Klancher ◽  
Joshua W. Shaevitz ◽  
Zemer Gitai ◽  
...  
Keyword(s):  
Model Organism ◽  
Type Iv Pili ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Acinetobacter Baylyi ◽  
Model Species ◽  
Type Iv ◽  
Surface Sensing ◽  
Chemosensory Pathway ◽  
Virulence Protein

AbstractBacteria employ extracellular appendages called type IV pili (T4P) to interact with their environment. T4P are essential for diverse microbial behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility (1). While T4P have been studied extensively, our understanding of these nanomachines largely comes from work on a few model species. Here, we develop Acinetobacter baylyi as a new model organism to study T4P and uncover several unreported mechanisms of T4P regulation. First, using recently-developed T4P-labeling methods (2, 3), we demonstrate that A. baylyi T4P are synthesized on one side of the cell body along the long axis of the cell, and we uncover that this pattern is dependent on components of a conserved chemosensory pathway. Second, we overturn the current dogma that T4P extension occurs through the action of a single, highly conserved ATP-hydrolyzing motor (ATPase) called PilB by showing that T4P synthesis in A. baylyi is dependent on two partially redundant and phylogenetically distinct motors, PilB and PilB2. Third, we uncover a small protein inhibitor of T4P synthesis that specifically inhibits PilB but not PilB2 activity. Together, these results demonstrate novel mechanisms of T4P regulation, which have broad implications for the unexplored diversity of T4P biology in microbial species.

scholarly journals Outer membrane lipoprotein DolP interacts with the BAM complex and promotes fitness during envelope stress response

2020 ◽  
Author(s):  
David Ranava ◽  
Yiying Yang ◽  
Luis Orenday-Tapia ◽  
François Rousset ◽  
Catherine Turlan ◽  
...  
Keyword(s):  
Cell Division ◽  
Outer Membrane ◽  
Detrimental Effect ◽  
Critical Determinant ◽  
Cell Recruitment ◽  
Bam Complex ◽  
Outer Membrane Lipoprotein ◽  
Envelope Stress ◽  
Late Step ◽  
Membrane Lipoprotein

AbstractIn Gram-negative bacteria, coordinated remodelling of the outer membrane (OM) and the peptidoglycan is crucial for envelope integrity. Envelope stress caused by unfolded OM proteins (OMPs) activates sigmaE (σE) in Enterobacteria. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyzes OMP-folding. Elevated σE activity, however, can be detrimental for OM integrity. Here we report that DolP (YraP), a σE-upregulated OM lipoprotein important for envelope integrity, is a novel interactor of BAM and we demonstrate that OM-assembled BamA is a critical determinant of the BAM-DolP complex. Mid-cell recruitment of DolP had been previously associated to activation of septal peptidoglycan remodelling during cell division, but its role during envelope stress was unknown. We now show that DolP promotes cell fitness upon stress-induced activation of σE and opposes a detrimental effect caused by the overaccumulation of BAM in the OM. During envelope stress, DolP loses its association with the mid-cell, thus suggesting a possible link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

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