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

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.

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A new twist on bacterial motility – two distinct type IV pili revealed by cryoEM

10.1101/720938 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alexander Neuhaus ◽

Muniyandi Selvaraj ◽

Ralf Salzer ◽

Julian D. Langer ◽

Kerstin Kruse ◽

...

Keyword(s):

Natural Transformation ◽

Thermus Thermophilus ◽

Distinct Type ◽

Type Iv Pili ◽

Dna Uptake ◽

Molecular Architecture ◽

Surface Adhesion ◽

Cellular Functions ◽

Type Iv ◽

New Type

SummaryMany bacteria express flexible protein filaments on their surface that enable a variety of important cellular functions. Type IV pili are examples of such filaments and are comprised of a helical assembly of repeating pilin subunits. Type IV pili are involved in motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). They are therefore powerful structures that enable bacterial proliferation and genetic adaptation, potentially leading to the development of pathogenicity and antibiotic resistance. They are also targets for drug development.By a complement of experimental approaches, we show that the bacterium Thermus thermophilus produces two different forms of type IV pilus. We have determined the structures of both and built atomic models. The structures answer key unresolved questions regarding the molecular architecture of type IV pili and identify a new type of pilin. We also delineate the roles of the two filaments in promoting twitching and natural transformation.

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Pilin-Like Proteins in the Extremely Thermophilic Bacterium Thermus thermophilus HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.3695-3700.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 3695-3700 ◽

Cited By ~ 42

Author(s):

Alexandra Friedrich ◽

Judit Rumszauer ◽

Anke Henne ◽

Beate Averhoff

Keyword(s):

Natural Transformation ◽

Thermus Thermophilus ◽

Thermophilic Bacterium ◽

Type Iv Pili ◽

Dna Uptake ◽

Significant Similarity ◽

High Frequencies ◽

Type Iv ◽

Genes Encoding ◽

Pilus Biogenesis

ABSTRACT The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Δkat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.

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Structure of a type IV pilus machinery in the open and closed state

eLife ◽

10.7554/elife.07380 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 60

Author(s):

Vicki AM Gold ◽

Ralf Salzer ◽

Beate Averhoff ◽

Werner Kühlbrandt

Keyword(s):

Thermus Thermophilus ◽

Type Iv Pili ◽

Gram Negative Bacteria ◽

Dna Uptake ◽

Type Ii ◽

Secretion Systems ◽

Macromolecular Complexes ◽

Closed State ◽

Type Iv

Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.

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The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation

Microbiology ◽

10.1099/mic.0.29189-0 ◽

2006 ◽

Vol 152 (12) ◽

pp. 3623-3631 ◽

Cited By ~ 29

Author(s):

Kenlee Nakasugi ◽

Charles J. Svenson ◽

Brett A. Neilan

Keyword(s):

Natural Transformation ◽

Heterotrophic Bacteria ◽

Fold Increase ◽

Hypothetical Protein ◽

Type Iv Pili ◽

Spectrometric Analysis ◽

Dna Uptake ◽

Wild Type ◽

Type Iv ◽

Pcc 6803

The gene slr0388 was previously annotated to encode a hypothetical protein in Synechocystis sp. strain PCC 6803. When a positively phototactic strain of this cyanobacterium was insertionally inactivated at slr0388, the mutants were not transformable, and appeared to aggregate as a result of increased bundling of type IV pili. Also, these mutants were rendered non-phototactic compared to the wild-type. Quantitative real-time PCR revealed a 3.5-fold increase in pilA1 transcript levels in the mutant over wild-type cells, while there were no changes in the level of pilT1 and comA transcripts. Supernatant from mutant liquid culture contained more PilA1 protein, confirmed by mass spectrometric analysis, compared to the wild-type cells, which corresponded to the increase in pilA1 transcripts. The increase in PilA1 subunits may contribute to the bundling morphology of pili that was observed, which in turn may act to retard DNA uptake by hindering the retraction of pili. This gene is therefore proposed to be designated comF, as it possesses a phosphoribosyltransferase domain, a distinguishing feature of other ComF proteins of naturally transformable heterotrophic bacteria. This report is the second of a competence-related gene from Synechocystis sp. strain PCC 6803, the product of which does not show homology to other well-studied type IV pili proteins.

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

Applied and Environmental Microbiology ◽

10.1128/aem.03218-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 644-652 ◽

Cited By ~ 18

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.

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Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1322889111 ◽

2014 ◽

Vol 111 (10) ◽

pp. E953-E961 ◽

Cited By ~ 45

Author(s):

Katja Siewering ◽

Samta Jain ◽

Carmen Friedrich ◽

Mariam T. Webber-Birungi ◽

Dmitry A. Semchonok ◽

...

Keyword(s):

Type Iv Pili ◽

Host Cells ◽

Gram Negative Bacteria ◽

Dependent Manner ◽

Dna Uptake ◽

Oligomeric State ◽

Gram Negative ◽

Type Iv ◽

Surface Assembly ◽

Peripheral Structure

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the β-proteobacteriumNeisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. InN. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacteriumMyxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.

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DNA Uptake Sequence-Mediated Enhancement of Transformation in Neisseria gonorrhoeae Is Strain Dependent

Journal of Bacteriology ◽

10.1128/jb.00442-10 ◽

2010 ◽

Vol 192 (17) ◽

pp. 4436-4444 ◽

Cited By ~ 30

Author(s):

Paul M. Duffin ◽

H. Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Plasmid Dna ◽

Natural Transformation ◽

Dna Transformation ◽

Dna Uptake ◽

Efficient Transformation ◽

Type Iv ◽

Different Strains ◽

Strain Dependent

ABSTRACT Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing the nonpalindromic 10- or 12-nucleotide sequence 5′- AT GCCGTCTGAA-3′ (additional semiconserved nucleotides are underlined), termed the DNA uptake sequence (DUS10 or DUS12). Here we investigated the effects of the DUS on transformation and DNA uptake for several laboratory strains of N. gonorrhoeae. We found that all strains showed efficient transformation of DUS containing DNA (DUS10 and DUS12) but that the level of transformation with DNA lacking a DUS (DUS0) was variable in different strains. The DUS-enhanced transformation was 20-fold in two strains, FA1090 and FA19, but was approximately 150-fold in strains MS11 and 1291. All strains tested provide some level of DUS0 transformation, and DUS0 transformation was type IV pilus dependent. Competition with plasmid DNA revealed that transformation of MS11 was enhanced by the addition of excess plasmid DNA containing a DUS while FA1090 transformation was competitively inhibited. Although FA1090 was able to mediate much more efficient transformation of DNA lacking a DUS than was MS11, DNA uptake experiments showed similar levels of uptake of DNA containing and lacking a DUS in FA1090 and MS11. Finally, DNA uptake was competitively inhibited in both FA1090 and MS11. Taken together, our data indicate that the role of the DUS during DNA transformation is variable between strains of N. gonorrhoeae and may influence multiple steps during transformation.

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MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

International Journal of Molecular Sciences ◽

10.3390/ijms22105328 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5328

Author(s):

Miao Ma ◽

Margaux Lustig ◽

Michèle Salem ◽

Dominique Mengin-Lecreulx ◽

Gilles Phan ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Cell Division ◽

Membrane Proteins ◽

Outer Membrane ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

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