Major Pilin Research Articles

Off‐pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly.

Structure of the Neisseria meningitidis Type IV pilus.

Neisseria meningitidis use Type IV pili (T4P) to adhere to endothelial cells and breach the blood brain barrier, causing cause fatal meningitis. T4P are multifunctional polymers of the major pilin protein, which share a conserved hydrophobic N terminus that is a curved extended α-helix, α1, in X-ray crystal structures. Here we report a 1.44 Å crystal structure of the N. meningitidis major pilin PilE and a ∼6 Å cryo-electron microscopy reconstruction of the intact pilus, from which we built an atomic model for the filament. This structure reveals the molecular arrangement of the N-terminal α-helices in the filament core, including a melted central portion of α1 and a bridge of electron density consistent with a predicted salt bridge necessary for pilus assembly. This structure has important implications for understanding pilus biology.

Structural Diversity in the Type IV Pili of Multidrug-resistant Acinetobacter

Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating that A. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species.

Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform.

Type II secretion systems (T2SSs) promote secretion of folded proteins playing important roles in nutrient acquisition, adaptation and virulence of Gram-negative bacteria. Protein secretion is associated with the assembly of type 4 pilus (T4P)-like fibres called pseudopili. Initially membrane embedded, pseudopilin and T4 pilin subunits share conserved transmembrane segments containing an invariant Glu residue at the fifth position, E5. Mutations of E5 in major T4 pilins and in PulG, the major pseudopilin of the Klebsiella T2SS abolish fibre assembly and function. Among the four minor pseudopilins, only PulH required E5 for secretion of pullulanase, the substrate of the Pul T2SS. Mass-spectrometry analysis of pili resulting from the co-assembly of PulG(E5A) variant and PulG(WT) ruled out an E5 role in pilin processing and N-methylation. A bacterial two-hybrid analysis revealed interactions of the full-length pseudopilins PulG and PulH with the PulJ-PulI-PulK priming complex and with the assembly factors PulM and PulF. Remarkably, PulG(E5A) and PulH(E5A) variants were defective in interaction with PulM but not with PulF, and co-purification experiments confirmed the E5-dependent interaction between native PulM and PulG. These results reveal the role of E5 in a recruitment step critical for assembly of the functional T2SS, likely relevant to T4P assembly systems.

Type IV pili promote early biofilm formation by Clostridium difficile.

Increasing morbidity and mortality from Clostridium difficile infection (CDI) present an enormous challenge to healthcare systems. Clostridium difficile express type IV pili (T4P), but their function remains unclear. Many chronic and recurrent bacterial infections result from biofilms, surface-associated bacterial communities embedded in an extracellular matrix. CDI may be biofilm mediated; T4P are important for biofilm formation in a number of organisms. We evaluate the role of T4P in C. difficile biofilm formation using RNA sequencing, mutagenesis and complementation of the gene encoding the major pilin pilA1, and microscopy. RNA sequencing demonstrates that, in comparison to other growth phenotypes, C. difficile growing in a biofilm has a distinct RNA expression profile, with significant differences in T4P gene expression. Microscopy of T4P-expressing and T4P-deficient strains suggests that T4P play an important role in early biofilm formation. A non-piliated pilA1 mutant forms an initial biofilm of significantly reduced mass and thickness in comparison to the wild type. Complementation of the pilA1 mutant strain leads to formation of a biofilm which resembles the wild-type biofilm. These findings suggest that T4P play an important role in early biofilm formation. Novel strategies for confronting biofilm infections are emerging; our data suggest that similar strategies should be investigated in CDI.

Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

Type IV pili are important virulence factors for many pathogens, including Pseudomonas aeruginosa Transcription of the major pilin gene-pilA-is controlled by the PilS-PilR two-component system in response to unknown signals. The absence of a periplasmic sensing domain suggested that PilS may sense an intramembrane signal, possibly PilA. We suggest that direct interactions between PilA and PilS in the inner membrane reduce pilA transcription when PilA levels are high. Overexpression in trans of PilA proteins with diverse and/or truncated C termini decreased native pilA transcription, suggesting that the highly conserved N terminus of PilA was the regulatory signal. Point mutations in PilA or PilS that disrupted their interaction prevented autoregulation of pilA transcription. A subset of PilA point mutants retained the ability to interact with PilS but could no longer decrease pilA transcription, suggesting that interaction between the pilin and sensor kinase is necessary but not sufficient for pilA autoregulation. Furthermore, PilS's phosphatase motif was required for the autoregulation of pilA transcription, suggesting that under conditions where PilA is abundant, the PilA-PilS interaction promotes PilR dephosphorylation and thus down-regulation of further pilA transcription. These data reveal a clever bacterial inventory control strategy in which the major subunit of an important P. aeruginosa virulence factor controls its own expression.

Diversity and Evolution of Type IV pili Systems in Archaea.

Many surface structures in archaea including various types of pili and the archaellum (archaeal flagellum) are homologous to bacterial type IV pili systems (T4P). The T4P consist of multiple proteins, often with poorly conserved sequences, complicating their identification in sequenced genomes. Here we report a comprehensive census of T4P encoded in archaeal genomes using sensitive methods for protein sequence comparison. This analysis confidently identifies as T4P components about 5000 archaeal gene products, 56% of which are currently annotated as hypothetical in public databases. Combining results of this analysis with a comprehensive comparison of genomic neighborhoods of the T4P, we present models of organization of 10 most abundant variants of archaeal T4P. In addition to the differentiation between major and minor pilins, these models include extra components, such as S-layer proteins, adhesins and other membrane and intracellular proteins. For most of these systems, dedicated major pilin families are identified including numerous stand alone major pilin genes of the PilA family. Evidence is presented that secretion ATPases of the T4P and cognate TadC proteins can interact with different pilin sets. Modular evolution of T4P results in combinatorial variability of these systems. Potential regulatory or modulating proteins for the T4P are identified including KaiC family ATPases, vWA domain-containing proteins and the associated MoxR/GvpN ATPase, TFIIB homologs and multiple unrelated transcription regulators some of which are associated specific T4P. Phylogenomic analysis suggests that at least one T4P system was present in the last common ancestor of the extant archaea. Multiple cases of horizontal transfer and lineage-specific duplication of T4P loci were detected. Generally, the T4P of the archaeal TACK superphylum are more diverse and evolve notably faster than those of euryarchaea. The abundance and enormous diversity of T4P in hyperthermophilic archaea present a major enigma. Apparently, fundamental aspects of the biology of hyperthermophiles remain to be elucidated.

Differentiation of Crohn's Disease-Associated Isolates from Other Pathogenic Escherichia coli by Fimbrial Adhesion under Shear Force.

Shear force exerted on uropathogenic Escherichia coli adhering to surfaces makes type-1 fimbriae stretch out like springs to catch on to mannosidic receptors. This mechanism is initiated by a disruption of the quaternary interactions between the lectin and the pilin of the two-domain FimH adhesin and transduces allosterically to the mannose-binding pocket of FimH to increase its affinity. Mannose-specific adhesion of 14 E. coli pathovars was measured under flow, using surface plasmon resonance detection on functionalized graphene-coated gold interfaces. Increasing the shear had important differential consequences on bacterial adhesion. Adherent-invasive E. coli, isolated from the feces and biopsies of Crohn’s disease patients, consistently changed their adhesion behavior less under shear and displayed lower SPR signals, compared to E. coli opportunistically infecting the urinary tract, intestines or loci of knee and hip prostheses. We exemplified this further with the extreme behaviors of the reference strains UTI89 and LF82. Whereas their FimA major pilins have identical sequences, FimH of LF82 E. coli is marked by the Thr158Pro mutation. Positioned in the inter-domain region known to carry hot spots of mutations in E. coli pathotypes, residue 158 is indicated to play a structural role in the allosteric regulation of type-1 fimbriae-mediated bacterial adhesion.

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin.

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.

Homo-trimeric Structure of the Type IVb Minor Pilin CofB Suggests Mechanism of CFA/III Pilus Assembly in Human Enterotoxigenic Escherichia coli

In gram-negative bacteria, the assembly of type IV pilus (T4P) and the evolutionally related pseudopilus of type II secretion system involves specialized structural proteins called pilins and pseudopilins, respectively, and is dynamically regulated to promote bacterial pathogenesis. Previous studies have suggested that a structural “tip”-like hetero-complex formed through the interaction of at least three minor (pseudo) pilins plays an important role in this process, while some members of the pathogenic type IVb subfamily are known to have only one such minor pilin subunit whose function is still unknown. Here, we determined the crystal structure of the type IVb minor pilin CofB of colonization factor antigen/III from human enterotoxigenic Escherichia coli at 1.88-Å resolution. The crystal structure, in conjunction with physicochemical analysis in solution, reveals a symmetrical homo-trimeric arrangement distinct from the hetero-complexes of minor (pseudo) pilins observed in other T4P and type II secretion systems. Each CofB monomer adopts a unique three-domain architecture, in which the C-terminal β-sheet-rich lectin domain can effectively initiate trimer association of its pilin-like N-terminal domain through extensive hydrophobic interactions followed by domain swapping at the central hinge-like domain. Deletion of cofB produces a phenotype with no detectable pili formation on the cell surface, while molecular modeling indicates that the characteristic homo-trimeric structure of CofB is well situated at the pilus tip of colonization factor antigen/III formed by the major pilin CofA, suggesting a role for the minor pilin in the efficient initiation of T4P assembly.

Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

The involvement of the PilQ secretin of type IV pili in phage infection in Ralstonia solanacearum

PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum.

Characterization of a Unique Tetrasaccharide and Distinct Glycoproteome in the O-Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica.

Broad-spectrum O-linked protein glycosylation is well characterized in the major Neisseria species of importance to human health and disease. Within strains of Neisseria gonorrhoeae, N. meningitidis, and N. lactamica, protein glycosylation (pgl) gene content and the corresponding oligosaccharide structure are fairly well conserved, although intra- and interstrain variability occurs. The status of such systems in distantly related commensal species, however, remains largely unexplored. Using a strain of deeply branching Neisseria elongata subsp. glycolytica, a heretofore unrecognized tetrasaccharide glycoform consisting of di-N-acetylbacillosamine-glucose-di-N-acetyl hexuronic acid-N-acetylhexosamine (diNAcBac-Glc-diNAcHexA-HexNAc) was identified. Directed mutagenesis, mass spectrometric analysis, and glycan serotyping confirmed that the oligosaccharide is an extended version of the diNAcBac-Glc-based structure seen in N. gonorrhoeae and N. meningitidis generated by the successive actions of PglB, PglC, and PglD and glucosyltransferase PglH orthologues. In addition, a null mutation in the orthologue of the broadly conserved but enigmatic pglG gene precluded expression of the extended glycoform, providing the first evidence that its product is a functional glycosyltransferase. Despite clear evidence for a substantial number of glycoprotein substrates, the major pilin subunit of the endogenous type IV pilus was not glycosylated. The latter finding raises obvious questions as to the relative distribution of pilin glycosylation within the genus, how protein glycosylation substrates are selected, and the overall structure-function relationships of broad-spectrum protein glycosylation. Together, the results of this study provide a foundation upon which to assess neisserial O-linked protein glycosylation diversity at the genus level. Broad-spectrum protein glycosylation systems are well characterized in the pathogenic Neisseria species N. gonorrhoeae and N. meningitidis. A number of lines of evidence indicate that the glycan components in these systems are subject to diversifying selection and suggest that glycan variation may be driven in the context of glycosylation of the abundant and surface-localized pilin protein PilE, the major subunit of type IV pili. Here, we examined protein glycosylation in a distantly related, nonpathogenic neisserial species, Neisseria elongata subsp. glycolytica. This system has clear similarities to the systems found in pathogenic species but makes novel glycoforms utilizing a glycosyltransferase that is widely conserved at the genus level but whose function until now remained unknown. Remarkably, PilE pilin is not glycosylated in this species, a finding that raises important questions about the evolutionary trajectories and overall structure-function relationships of broad-spectrum protein glycosylation systems in bacteria.

Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin, PilE

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilEΔ1-28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilXNm and PilVNm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilXNm and PilVNm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.

Crystal Structure of the Minor Pilin CofB, the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.

Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus.

ABSTRACTThe nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus.IMPORTANCE We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of the pilA gene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa

Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.

Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation.

ABSTRACTExpression of type four pili (Tfp) is essential for virulence in Neisseria meningitidis. Pili mediate adhesion, bacterial aggregation, and DNA uptake. In N. meningitidis, the major pilin subunit is encoded by the pilE gene. In some strains, PilE is subject to phase and antigenic variation, which can alter Tfp properties and together offer a possible mechanism of immune escape. Pilin expression and antigenic variation can be modulated in response to environmental cues; however, the precise mechanisms of such regulation remain unclear. We identified a promoter in the pilE locus, 3′ of the pilE coding sequence, on the antisense (AS) strand which is conserved in meningococci. We show that this promoter directs transcription of an AS RNA that is expressed during specific growth phases and in response to salt stress. Furthermore, we demonstrate that the transcript encompasses sequences complementary to the entire pilE coding sequence and 5′ untranslated region. AS RNAs can regulate the gene on the sense strand by altering transcript stability or translation. However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes in pilE transcript or protein level. Instead, our data indicate that the AS RNA influences pilin antigenic variation. This work provides further insights into the complex regulation of pilin expression and variation in pathogenic Neisseria. IMPORTANCE Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. Some strains of N. meningitidis are able to vary the sequence of the major subunit (PilE) of the Tfp. The mechanisms underlying this variation are not fully defined, but the process requires several noncoding elements that are found adjacent to the pilE gene. In this work, we identified a cis-encoded RNA antisense to pilE in N. meningitidis. By using Northern blotting and RT-PCR analysis, we found that the RNA is expressed in stationary phase or following salt stress. Our work also indicates that this RNA does not significantly affect pilE or pilin expression levels but instead appears to modulate pilin variation.

Type IV Pilus: One Architectural Problem, Many Structural Solutions

Type IV pili are long appendages found at the surface of many bacteria, composed of an oligomerized pilinprotein and involved in processes such as adherence, motility and DNA transfer. In this issue of Structure, Piepenbrink and colleagues report the first structure a major pilin from a Gram-positive bacterium, revealing an unprecedented stabilization mechanism that may have implications for pilus evolution.

Uptake of extracellular DNA: competence induced pili in natural transformation of Streptococcus pneumoniae.

Transport of DNA across bacterial membranes involves complex DNA uptake systems. In Gram-positive bacteria, the DNA uptake machinery shares fundamental similarities with type IV pili and type II secretion systems. Although dedicated pilus structures, such as type IV pili in Gram-negative bacteria, are necessary for efficient DNA uptake, the role of similar structures in Gram-positive bacteria is just beginning to emerge. Recently two essentially very different pilus structures composed of the same major pilin protein ComGC were proposed to be involved in transformation of the Gram-positive bacterium Streptococcus pneumoniae – one is a long, thin, type IV pilus-like fiber with DNA binding capacity and the other one is a pilus structure that was thicker, much shorter and not able to bind DNA. Here we discuss how competence induced pili, either by pilus retraction or by a transient pilus-related opening in the cell wall, may mediate DNA uptake in S. pneumoniae.