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

2019 ◽  
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.

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

2003 ◽  
Vol 69 (7) ◽  
pp. 3695-3700 ◽  
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.

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

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3623-3631 ◽  
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.

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

eLife ◽  
2015 ◽  
Vol 4 ◽  
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.

scholarly journals The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold

2020 ◽  
Vol 295 (19) ◽  
pp. 6594-6604 ◽  
Author(s):  
Devon Sheppard ◽  
Jamie-Lee Berry ◽  
Rémi Denise ◽  
Eduardo P. C. Rocha ◽  
Steve Matthews ◽  
...  
Keyword(s):  
Natural Transformation ◽  
Dna Uptake ◽  
Human Pathogens ◽  
Unusual Structure ◽  
Widespread Distribution ◽  
Solution Structures ◽  
Type Iv ◽  
Filament Assembly ◽  
Pilin Subunit ◽  
Type Iv Pilin

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

scholarly journals DNA Binding: a Novel Function of Pseudomonas aeruginosa Type IV Pili

2005 ◽  
Vol 187 (4) ◽  
pp. 1455-1464 ◽  
Author(s):  
Erin J. van Schaik ◽  
Carmen L. Giltner ◽  
Gerald F. Audette ◽  
David W. Keizer ◽  
Daisy L. Bautista ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Binding ◽  
Biofilm Formation ◽  
Quaternary Structure ◽  
Natural Transformation ◽  
Ex Vivo ◽  
Specific Binding ◽  
Type Iv Pili ◽  
Type Iv ◽  
Bacteriophage Infection

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.

scholarly journals Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

2010 ◽  
Vol 192 (12) ◽  
pp. 3093-3102 ◽  
Author(s):  
Manuela Tripepi ◽  
Saheed Imam ◽  
Mechthild Pohlschröder
Keyword(s):  
Critical Role ◽  
Type Iv Pili ◽  
Haloferax Volcanii ◽  
Surface Adhesion ◽  
Functional Roles ◽  
Bacterial Flagella ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Flagellar Assembly ◽  
Bacterial Type

ABSTRACT Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.

scholarly journals Natural transformation in Gram-negative bacteria thriving in extreme environments: from genes and genomes to proteins, structures and regulation

Extremophiles ◽  
2021 ◽  
Author(s):  
Beate Averhoff ◽  
Lennart Kirchner ◽  
Katharina Pfefferle ◽  
Deniz Yaman
Keyword(s):  
Genetic Information ◽  
Natural Transformation ◽  
Thermus Thermophilus ◽  
Extreme Environments ◽  
Opportunistic Pathogen ◽  
Dna Uptake ◽  
Acinetobacter Baylyi ◽  
And Function ◽  
Transformation Systems ◽  
Evolutionary Lineages

AbstractExtremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.

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