A soluble form of the pilus protein FimA targets the VDAC-hexokinase complex at mitochondria to suppress host cell apoptosis.

Inhibition of apoptotic response of host cells during an early phase of infection is a strategy used by many enteroinvasive bacterial pathogens to enhance their survival. Here, we report the identification of a soluble form of the pilus protein FimA from the culture supernatants of E. coli K1, Salmonella, and Shigella that can potently inhibit Bax-mediated release of cytochrome c from isolated mitochondria. Similar to the infected cells, HCT116 cells stably expressing FimA display a delay in the integration of Bax into outer mitochondrial membrane induced by apoptotic stimuli. FimA targets to mitochondria through binding to VDAC1, which is a prerequisite step for E. coli K1 to render the short-term blockade of apoptotic death in the host cells. Interestingly, FimA strengthens the VDAC1-hexokinase interaction and prevents dissociation of hexokinase from VDAC1 triggered by apoptotic stimuli. Together, these data thus reveal a paradigm of antiapoptosis mechanism undertaken by the enteroinvasive bacteria.

Initiation of phage infection by partial unfolding and prolyl isomerization.

Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time.

Role of plasmids in the formation of E. coli polycellular forms.

The formation of polycellular forms by E. coli strain K-12 cells containing F-like plasmids pAP22-2 and pAP42 was studied by the method of small-angle laser scattering. The efficiency and patterns of the resultant cell associations depend on genetic characteristics of the studied plasmids.

Bacterial motility: secretory secrets of gliding bacteria.

Many bacteria glide over surfaces without the aid of flagella. Gliding is still somewhat mysterious, but recent studies show that it involves specialized secretory systems that assemble membrane-associated filaments, and the recognition of extracellular components that trigger movement via transmembrane transducers.

Tying rings for sex.

The primary component of the sex pilus encoded by IncP (RP4) and Ti plasmids has been identified as a circular pilin protein with a peptide bond between the amino and carboxyl terminus. Here, we review the key experiments that led to this discovery, and the present mechanistic model for pilin-precursor processing and the cyclization reaction. In addition, we discuss the implications for horizontal gene transfer in bacterial conjugation.

Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H(2)-based ithoautotrophy and anaerobiosis.

The self-transmissible megaplasmid pHG1 carries essential genetic information for the facultatively lithoautotrophic and facultatively anaerobic lifestyles of its host, the Gram-negative soil bacterium Ralstonia eutropha H16. We have determined the complete nucleotide sequence of pHG1. This megaplasmid is 452,156 bp in size and carries 429 potential genes. Groups of functionally related genes form loose clusters flanked by mobile elements. The largest functional group consists of lithoautotrophy-related genes. These include a set of 41 genes for the biosynthesis of the three previously identified hydrogenases and of a fourth, novel hydrogenase. Another large cluster carries the genetic information for denitrification. In addition to a dissimilatory nitrate reductase, both specific and global regulators were identified. Also located in the denitrification region is a set of genes for cytochrome c biosynthesis. Determinants for several enzymes involved in the mineralization of aromatic compounds were found. The genes for conjugative plasmid transfer predict that R.eutropha forms two types of pili. One of them is related to the type IV pili of pathogenic enterobacteria. pHG1 also carries an extensive "junkyard" region encompassing 17 remnants of mobile elements and 22 partial or intact genes for phage-type integrase. Among the mobile elements is a novel member of the IS5 family, in which the transposase gene is interrupted by a group II intron.

Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F(+) cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.

Protein circlets as sex pilus subunits.

The largest circular protein structures discovered define a class of transfer proteins acting in bacterial conjugation and type IV secretion. Proteins ranging from 73 to 78 residues with head-to-tail peptide bonds constitute the major subunit of conjugative pili of some type IV secretion systems. Their plasmid-encoded precursors are enzymatically processed and cyclized before being assembled into pili. These extra-cellular surface filaments mediate physical contact between donor and recipient cell or pathogen and host cell. Pili are essential prerequisites for DNA and protein transfer. A membrane-bound signal peptidase-like enzyme is responsible for the circularization reaction. Site-directed mutagenesis and mass spectrometry has been used extensively to unravel the mechanism of the enzyme-substrate interaction of the pilin maturation process.

The pathogenic neisseriae contain an inactive rpoN gene and do not utilize the pilE sigma54 promoter.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

Type IV pilus assembly proficiency and dynamics influence pilin subunit phospho-form macro- and microheterogeneity in Neisseria gonorrhoeae.

The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp) colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE). In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC). Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A) required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae.

The pvc operon regulates the expression of the Pseudomonas aeruginosa fimbrial chaperone/usher pathway (cup) genes.

The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators.

Virulence regulons of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli is frequently associated with travelers' diarrhea and is a leading cause of infant and childhood mortality in developing countries. Disease is dependent upon the orchestrated expression of enterotoxins, flexible adhesive pili, and other virulence factors. Both the heat-labile (LT) and heat-stable (ST-H) enterotoxins are regulated at the level of transcription by cAMP-receptor protein which represses the expression of LT while activating expression of ST-H. The expression of many different serotypes of adhesive pili is regulated by Rns, a member of the AraC family that represents a subgroup of conserved virulence regulators from several enteric pathogens. These Rns-like regulators recognize similar DNA binding sites, and a compiled sequence logo suggests they may bind DNA through both major and minor groove interactions. These regulators are also tempting targets for novel therapeutics because they play pivotal roles during infection. To that end, high-throughput screens have begun to identify compounds that inhibit the activity of these regulators, predominately by interfering with DNA binding.

Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

pilQ Missense mutations have diverse effects on PilQ multimer formation, piliation, and pilus function in Neisseria gonorrhoeae.

Type IV pili are required for virulence in Neisseria gonorrhoeae, as they are involved in adherence to host epithelium, twitching motility, and DNA transformation. The outer membrane secretin PilQ forms a homododecameric ring through which the pilus is proposed to be secreted. pilQ null mutants are nonpiliated, and thus, all pilus-dependent functions are eliminated. Mutagenesis was performed on the middle one-third of pilQ, and mutants with colony morphologies consistent with the colony morphology of nonpiliated or underpiliated bacteria were selected. Nineteen mutants, each with a single amino acid substitution, were isolated and displayed diverse phenotypes in terms of PilQ multimer stability, pilus expression, transformation efficiency, and host cell adherence. The 19 mutants were grouped into five phenotypic classes based on functionality. Four of the five mutant classes fit the current model of pilus functionality, which proposes that a functional pilus assembly apparatus, not necessarily full-length pili, is required for transformation, while high levels of displayed pili are required for adherence. One class, despite having an underpiliated colony morphology, expressed high levels of pili yet adhered poorly, demonstrating that pilus expression is necessary but not sufficient for adherence and indicating that PilQ may be directly involved in host cell adherence. The collection of phenotypes expressed by these mutants suggests that PilQ has an active role in pilus expression and function.

Expression and assembly of a functional type IV secretion system elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli.

Conditions perturbing protein homeostasis are known to induce cellular stress responses in prokaryotes and eukaryotes. Here we show for the first time that expression and assembly of a functional type IV secretion (T4S) machinery elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli. After induction of T4S genes by a nutritional upshift and assembly of functional DNA transporters encoded by plasmid R1-16, host cells activated the CpxAR envelope stress signaling system, as revealed by induction or repression of downstream targets of the CpxR response regulator. Furthermore, we observed elevated transcript levels of cytoplasmic stress genes, such as groESL, with a concomitant increase of sigma(32) protein levels in cells expressing T4S genes. A traA null mutant of plasmid R1-16, which lacks the functional gene encoding the major pilus protein pilin, showed distinctly reduced stress responses. These results corroborated our conclusion that the activation of bacterial stress networks was dependent on the presence of functional T4S machinery. Additionally, we detected increased transcription from the rpoHp(1) promoter in the presence of an active T4S system. Stimulation of rpoHp(1) was dependent on the presence of CpxR, suggesting a hitherto undocumented link between CpxAR and sigma(32)-regulated stress networks.

I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells.

Two pilus receptors are identified for the pathogenic Neisseria, CD46 and complement receptor 3. An intimate association between the asialoglycoprotein receptor and gonococcal lipooligosaccharide mediates invasion of primary, male urethral epithelial cells (UECs); however, studies to identify pilus receptors on these cells have not been performed. Based on our previous studies we reasoned that the I-domain-containing (IDC), alpha(1)- and alpha(2)-integrins might serve as pilus receptors on UECs and on urethral tissue. Confocal microscopy revealed colocalization of pilus with alpha(1) and alpha(2) integrins on UECs and tissue. We found that recombinant I-domain and antibodies directed against the alpha(1)- and alpha(2)-integrins inhibited gonococcal association with UECs and with immortal cell lines of variable origin. Gonococcus-integrin colocalization occurred at early time points post infection, but this interaction dissociated with extended infection. Similarly, Western Blot analyses revealed that gonococcal pilin coimmunoprecipitates with alpha(1)- and alpha(2)-integrins. However, studies performed in parallel and that were designed to capture CD46-pilus immune complexes indicated that a CD46-pilus interaction did not occur. Collectively, these data suggest that while CD46 might be able to bind gonococcal pilus, IDC integrins are preferentially used as the initial docking site for gonococci on UECs, on urethral tissue and on some immortal cell lines.

Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy.

The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.

Interaction of the globular domains of pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.

Gene 3 protein (pIII), a minor coat protein at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. Infection is thought to start with the adsorption of the D2 domain of pIII to the tip of the pilus, retraction of the pilus, and penetration of the E. coli cell membrane mediated by an interaction between the D1 domain of pIII and the Tol protein complex in the membrane. A subgene encoding the pIII-D1D2 di-domain was created, and the subgene was successfully overexpressed in E. coli cells. Domains D1 and D2 were separated after limited proteolysis of a modified pIII-D1D2 (designated pIII-D1D2.trp) into which two tryptic cleavage sites were introduced at appropriate points. The purified pIII-D1D2 di-domain and pIII-D2 domain were able to bind to the F-pilus, competing with the wild-type pIII and delaying infection by the intact filamentous phage. The pIII-D1 domain was unable to bind to the F-pilus by this criterion. This provides conclusive evidence that the pIII-D2 domain is responsible for the adsorption to the tip of the F-pilus and can achieve this in the absence of domain D1, opening the way to identifying the molecular basis of the interaction of pIII-D2 with the pilus.

The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus.

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus. We report the discovery of four additional pil genes: pilD, a homologue of type IV prepilin leader peptidases; and pilG, pilH and pilI, which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH. Null mutants of pilG, pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.

Contact stimulation of Tgl and type IV pili in Myxococcus xanthus.

Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.