Pilin Gene Research Articles

Proteomic characterization of a pilR regulatory mutant of Azoarcus sp. strain BH72 with the aid of gel‐based and gel‐free approaches

The proteome of the grass endophyte Azoarcus sp. strain BH72 was analyzed by a combination of gel-based methods by means of 2-DE and MS and a gel-free approach via LC-MS/MS. Among the identified 785 proteins, synthesis of around 100 conserved hypothetical proteins could be confirmed. Membrane proteins were detected at a higher rate in the gel-free than in the gel-based approach. The abundance of proteins in the constructed proteome reference map was analyzed and the 30 most abundant proteins were determined. The reference map was then used as a starting point to characterize the regulon under control of the response regulator PilR. PilR is part of the two-component regulatory system PilSR controlling type IV pilin gene (pilAB) expression in strain BH72, which was strongly decreased (19.7-fold) in the pilR-mutant. Changes of protein composition in the wild type and the regulatory mutant were compared by the gel-based and gel-free analyses. Proteins responsible for amino acid and energy metabolism, chaperones as well as proteins that are involved in iron metabolism and iron storage were present in a pilR-mutant at different levels than in the wild-type strain. Levels of the transcriptional regulator Flp were also dependent on PilR, indicating that PilR might be part of a hierarchical regulatory cascade.

Mucosal Adhesion Properties of the ProbioticLactobacillus rhamnosusGG SpaCBA and SpaFED Pilin Subunits

Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.

Structural and functional specificity of Dichelobacter nodosus Pili: Synthesis of pilin protein D. nodosus in various recombinant bacterial strains

Sheep hoof rot is a serious infectious disease specific to this type of animal; its pathogenic agent is anaerobic gram-negative bacillus Dichelobacter nodosus. Very strict requirements for cultivation conditions and slow rate of accumulation of bacterial mass make the vaccine and diagnistcs production laborious and expensive. Fimbriae (pili) are one of the main factors of D. nodosus virulence, since they have both antigenic and immunogenic properties that allow them to be added to vaccines to intensify immunogenicity. After it was determined that the fimA gene, which encodes the fimbrial subunit, attempts were made to obtain recombinant pili. The production of genetically engineered fimbriae serves to simplify and lower the cost of the process of vaccine production, as well as to improve immune-protective qualities of the obtained preparations. The first system of bacterial expression was selected for E. coli, however, the assembly of D. nodosus pili on its surface failed due to significant differences in the biogenesis and fimbria structure of these microorganisms. The morphogenesis of D. nodosus pili succeeded in Pseudomonas aeruginosa, which is characterized by the most similar structure of pilin subunits. Sheep’s immunity after injection of recombinant fimbria, native fimbria, and whole cells of D. nodosus was almost analogous in intensity. The article presents a review of data from publications on the production of recombinant cultures of P. aeruginosa with D. nodosus pili and expression of pilin genes in various bacterial systems.

Functional analyses of pilin-like proteins from Francisella tularensis: complementation of type IV pilus phenotypes in Neisseria gonorrhoeae

Accumulating evidence from a number of studies strongly suggests that proteins orthologous to those involved in type IV pili (Tfp) assembly and function are required for Francisella pathogenicity. However, the molecular mechanisms by which the components exert their influence on virulence remain poorly understood. Owing to the conservation and promiscuity of Tfp biogenesis machineries, expression of Tfp pilins in heterologous species has been used successfully to analyse organelle structure-function relationships. In this study we expressed a number of Francisella pilin genes in the Tfp-expressing pathogen Neisseria gonorrhoeae lacking its endogenous pilin subunit. Two gene products, the orthologous PilA proteins from Francisella tularensis subspecies tularensis and novicida, were capable of restoring the expression of Tfp-like appendages that were shown to be dependent upon the neisserial Tfp biogenesis machinery for surface localization. Expression of Francisella PilA pilins also partially restored competence for natural transformation in N. gonorrhoeae. This phenotype was not complemented by expression of the PulG and XcpT proteins, which are equivalent components of the related type II protein secretion system. Taken together, these findings provide compelling, although indirect, evidence of the potential for Francisella PilA proteins to express functional Tfp.

RecQ DNA helicase HRDC domains are critical determinants in Neisseria gonorrhoeae pilin antigenic variation and DNA repair

Neisseria gonorrhoeae (Gc), an obligate human bacterial pathogen, utilizes pilin antigenic variation to evade host immune defences. Antigenic variation is driven by recombination between expressed (pilE) and silent (pilS) copies of the pilin gene, which encodes the major structural component of the type IV pilus. We have investigated the role of the GcRecQ DNA helicase (GcRecQ) in this process. Whereas the vast majority of bacterial RecQ proteins encode a single 'Helicase and RNase D C-terminal' (HRDC) domain, GcRecQ encodes three tandem HRDC domains at its C-terminus. Gc mutants encoding versions of GcRecQ with either two or all three C-terminal HRDC domains removed are deficient in pilin variation and sensitized to UV light-induced DNA damage. Biochemical analysis of a GcRecQ protein variant lacking two HRDC domains, GcRecQDeltaHRDC2,3, shows it has decreased affinity for single-stranded and partial-duplex DNA and reduced unwinding activity on a synthetic Holliday junction substrate relative to full-length GcRecQ in the presence of Gc single-stranded DNA-binding protein (GcSSB). Our results demonstrate that the multiple HRDC domain architecture in GcRecQ is critical for structure-specific DNA binding and unwinding, and suggest that these features are central to GcRecQ's roles in Gc antigenic variation and DNA repair.

Expression of two distinct types of pili by a hospital-acquired Enterococcus faecium isolate

Surface filamentous structures designated pili, and implicated in virulence, have been found on the surfaces of several Gram-positive pathogens. This work describes the conditional expression of two phenotypically distinct pilus-like structures, designated PilA and PilB, on the surface of a hospital-adapted Enterococcus faecium bloodstream isolate. E. faecium is an emerging Gram-positive opportunistic pathogen that can cause severe disease, particularly in immunocompromised patients. Expression of PilA- and PilB-type pili was analysed during different phases of growth in broth culture. During growth, PilA and PilB pilin subunits were expressed around the cross-wall in early-exponential-phase cells. Polymerization and migration of short PilB-type pili towards the poles occurred in cells from the exponential phase and long polymerized pili were expressed at the poles of cells grown to stationary phase. In contrast, PilA-type pili were not expressed in broth culture, but only when cells were grown on solid media. Furthermore, surface expression of the PilA- and PilB-type pili was regulated in a temperature-dependent manner, as polymerization of two distinct types of pili at the surface only occurred when cells were grown at 37 degrees C; no pili were observed on cells grown at 21 degrees C. Hospital-aquired E. faecium isolates were specifically enriched in pilin gene clusters, suggesting that conditional expression of pili may contribute to E. faecium pathogenesis.

Novel Proteins That Modulate Type IV Pilus Retraction Dynamics in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses type IV pili to colonize various materials and for surface-associated twitching motility. We previously identified five phylogenetically distinct alleles of pilA in P. aeruginosa, four of which occur in genetic cassettes with specific accessory genes (J. V. Kus, E. Tullis, D. G. Cvitkovitch, and L. L. Burrows, Microbiology 150:1315-1326, 2004). Each of the five pilin alleles, with and without its associated pilin accessory gene, was used to complement a group II PAO1 pilA mutant. Expression of group I or IV pilA genes restored twitching motility to the same extent as the PAO1 group II pilin. In contrast, poor twitching resulted from complementation with group III or group V pilA genes but increased significantly when the cognate tfpY or tfpZ accessory genes were cointroduced. The enhanced motility was linked to an increase in recoverable surface pili and not to alterations in total pilin pools. Expression of the group III or V pilins in a PAO1 pilA-pilT double mutant yielded large amounts of surface pili, regardless of the presence of the accessory genes. Therefore, poor piliation in the absence of the TfpY and TfpZ accessory proteins results from a net increase in PilT-mediated retraction. Similar phenotypes were observed for tfpY single and tfpY-pilT double knockout mutants of group III strain PA14. A PilAV-TfpY chimera produced few surface pili, showing that the accessory proteins are specific for their cognate pilin. The genetic linkage between specific pilin and accessory genes may be evolutionarily conserved because the accessory proteins increase pilus expression on the cell surface, thereby enhancing function.

Kingella kingae Expresses Type IV Pili That Mediate Adherence to Respiratory Epithelial and Synovial Cells

Kingella kingae is a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency of K. kingae disease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed that K. kingae expresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by a pilA homolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of the K. kingae genome, we identified two genes in physically separate locations on the chromosome that encode homologs of the Neisseria PilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of the K. kingae PilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediating K. kingae adherence.

The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative Escherichia coli Mediates Binding to Extracellular Matrix Proteins

Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is mediated by aggregative adherence fimbriae (AAF); however, the receptors involved in EAEC adherence remain uncharacterized. Adhesion to extracellular matrix proteins is commonly observed among enteric pathogens, so we addressed the hypothesis that EAEC may bind to extracellular matrix proteins commonly found in the intestine. We found that EAEC prototype strain 042 adhered more abundantly to surfaces that were precoated with the extracellular matrix proteins fibronectin, laminin, and type IV collagen. Differences in fibronectin binding of almost 2 orders of magnitude were observed between EAEC 042 and a mutant in the AAF/II major pilin gene, aafA. Purified AafA, refolded as a donor strand complementation construct, bound fibronectin in a dose-dependent manner. Addition of fibronectin to the apical surfaces of polarized T84 cell monolayers augmented EAEC 042 adherence, and this effect required expression of aafA. Finally, increased bacterial adherence was observed when apical secretion of fibronectin was induced by adenosine in polarized T84 cells. Binding to fibronectin may contribute to colonization of the gastrointestinal tract by EAEC.

Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans : a Termination Cascade

The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis.

PilR, a Transcriptional Regulator for Pilin and Other Genes Required for Fe(III) Reduction in Geobacter sulfurreducens

Growth using Fe(III) as a terminal electron acceptor is a critical physiological process in Geobacter sulfurreducens. However, the mechanisms of electron transfer during Fe(III) reduction are only now being understood. It has been demonstrated that the pili in G. sulfurreducens function as microbial nanowires conducting electrons onto Fe(III) oxides. A number of c-type cytochromes have also been shown to play important roles in Fe(III) reduction. However, the regulatory networks controlling the expression of the genes involved in such processes are not well known. Here we report that the expression of pilA, which encodes the pilistructural protein, is directly regulated by a two-component regulatory system in which PilR functions as an RpoN-dependent enhancer binding protein. Surprisingly, a deletion of the pilR gene affected not only insoluble Fe(III) reduction, which requires pili, but also soluble Fe(III) reduction, which, in contrast, does not require pili. Gene expression profiling using whole-genome DNA microarray and quantitative RT-PCR analyses obtained with a PilR-deficient mutant revealed that the expression of pilA and other pilin-related genes are downregulated, while many c-type cytochromes involved in Fe(III) reduction were differentially regulated. This is the first instance of an enhancer binding protein implicated in regulating genes involved in Fe(III) respiratory functions.

Caratterizzazione di isolati di Pseudomonas aeruginosa provenienti da pazienti affetti da fibrosi cistica

Pseudomonas aeruginosa is the major pulmonary pathogen that causes morbidity and mortality in burned, immunocompromised and cystic fibrosis patients. Among the various virulence factors, type IV-pili, play a major role in mediating bacteria-host cells interactions, in formation of biofilm and for twiching motility.These pili are composed of pilin, 15000-16000 molecular weight monomeric subunit, synthesized from pilA gene. The N-terminal region of pilin protein is strong conserved and is important for the oligomerization.The C-terminal region is less conserved and contains a disulfide-bonded loop (DSL) structure that is thought to interact with the eukaryotic glycolipid receptor “asialo GM1”. Analysis of pilin allele distribution among isolates from various sources revealed the presence of six groups of pilin allels characterized by different DSL sequence and different associated accessory genes in pilA chromosomal locus. 81 P. aeruginosa isolates were recovered from cystic fibrosis patients during a 3 years period. 30 of these strains were grown and their genomic DNA was prepared using a rapid method for gram-negative bacteria. PCR primers were used for amplification of pilA and adjacent sequences revealing the presence of three different amplification products. One of these is highly homologous with pilA gene of PA14 strain, the others are identical to PA103 and PAK pilA genes. Our study revealed in the prevalence of isolates with group II pilin genes from Cystic Fibrosis compared with other groups that are predominant in previous studies.

Type II Secretion and Type IV Pili of Francisella

Francisella tularensis genomes encode homologues of type IV pili. Though several F. tularensis genes required for Tfp expression are homologous to genes required for type II secretion (T2S), these gene clusters mainly bear structural signatures that are typical of Tfp. There is preliminary evidence that different F. tularensis subspecies express Tfp-like surface structures, but there are also some interesting differences between the subspecies. One difference between the nonpathogenic subspecies novicida (F. novicida) and the highly pathogenic type A strains is in sequence of one of the predicted pilin genes, pilA. In contrast, type B strains show several differences compared to type A strains, two predicted pilin genes and the pilT gene are pseudogenes, while pilA is identical to pilA that is encoded by the type A strains. This is likely significant as PilA contributes to virulence of type B strains while PilT is essential for Tfp retraction in other bacterial pathogens. Tfp-mediated protein secretion is only evident in in vitro grown F. novicida. Surprisingly, secretion of several F. novicida proteins was dependent on pilA and other genes with postulated roles in Tfp expression. F. novicida secretion mutants were more virulent in the mouse infection model. Thus, in F. novicida, Tfp gene clusters serve both T2S and Tfp assembly and it is tempting to speculate that evolution of the pathogenic subspecies involved loss of functional T2S via structural changes of PilA and additional genes, including those that encode some of the Tfp-secreted proteins.

Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracellular macromolecular surface appendages. Likewise, S. solfataricus binding proteins have been suggested to assemble in higher ordered surface structures as well, tentatively termed the bindosome. Here we show that S. solfataricus contains a specific system that is needed for the functional surface localization of sugar binding proteins. This system, encoded by the bas (bindosome assembly system) operon, is composed of five proteins: basABC, three homologues of so-called bacterial (pseudo)pilins; BasE, a cytoplasmic ATPase; and BasF, an integral membrane protein. Deletion of either the three (pseudo)pilin genes or the basEF genes resulted in a severe defect of the cells to grow on substrates which are transported by sugar binding proteins containing class III signal peptides, while growth on glucose and maltose was restored when the corresponding genes were reintroduced in these cells. Concomitantly, DeltabasABC and DeltabasEF cells were severely impaired in glucose uptake even though the sugar binding proteins were normally secreted across the cytoplasmic membrane. These data underline the hypothesis that the bas operon is involved in the functional localization of sugar binding proteins at the cell surface of S. solfataricus. In contrast to surface structure assembly systems of Gram-negative bacteria, the bas operon seems to resemble an ancestral simplified form of these machineries.

Genomic Insights into (Extreme) Life at the Bottom of the Sea

If you lived some 2,500 meters below the ocean’s surface in waters oscillating between 2 ° and 40 °C, what sorts of genes would you need? In a new study, Kathleen Scott, Stefan Sievert, and their colleagues shed light on the special adaptations required for such extreme living by sequencing and analyzing the complete genome of the extremophilic bacterium Thiomicrospira crunogena XCL-2. First isolated in 1985 from deep-sea hydrothermal vents along the East Pacific Rise in the South Pacific, T. crunogena has since been found in both Atlantic and Pacific Ocean vents, revealing its critical role in these ecosystems. It belongs to the diverse group of bacteria called gammaproteobacteria, which includes the human pathogens Escherichia coli and Salmonella. T. crunogena is what’s known as an obligate chemolithoautotroph—it can grow using carbon dioxide as its sole carbon source and (in this case) sulfur as an energy source. Much like photosynthetic bacteria and plants use the sun’s energy to produce food, T. crunogena uses the oxidation of reduced sulfur compounds as an energy source for carbon fixation (synthesizing carbon-based sugars and other molecules) and cellular maintenance. And like their photosynthetic counterparts, these chemolithoautotrophs function as primary producers at the base of their vent community. Hydrothermal vents release geothermally heated seawater through fissures along the volcanically active mid-ocean ridge. These carbon dioxide- and sulfide-rich plumes periodically mix with cold, oxygenated bottom water, creating eddies and forcing hydrothermal vent communities to contend with dramatic fluctuations in environmental conditions. One way T. crunogena copes with these oscillations is by using carbon-concentrating mechanisms that allow growth to continue when carbon dioxide levels drop. Scott et al. studied the content and structure of the microbe’s genome for evidence of other adaptations required to thrive in its extreme environment. The T. crunogena genome is confined to a single chromosome that is densely packed with genes involved in electron transport (used to gain energy from sulfur compounds), energy and carbon metabolism, along with those required for nucleotide and amino acid synthesis and other cellular processes. The authors found only one genetic system for energy generation (and a possible alternate), which is perhaps not surprising for a microbe with limited energy options. They found all the components of the Sox system, a sulfur-oxidizing pathway typically found in microbes with more flexible strategies for carbon and energy metabolism. Together, these Sox genes completely oxidize, or strip electrons, from a variety of reduced sulfur-related compounds (producing sulfate). The microbe also harbors an enzyme that stops short of complete oxidation to sulfate (producing elemental sulfur instead). Because complete oxidation generates enough acid to inhibit growth, the authors suspect that this alternate pathway may curtail further acidification when acid levels rise. In any case, T. crunogena appears to be the first obligate chemolithoautotrophic sulfur-oxidizing bacteria to rely primarily on the Sox system for oxidizing sulfur compounds, raising the possibility that either the Sox system evolved in T. crunogena’s obligate autotrophic ancestors or the microbe lost its flexibility but retained its Sox pathway. Interestingly, however, this obligate chemotroph, which spends its life affixed to a hydrothermal vent, has proportionally more regulatory and signaling molecules than a free-living planktonic species. This enhanced repertoire likely reflects the different demands of life in extreme, volatile conditions—which requires rapid, flexible cellular responses—compared with the relatively stable existence of plankton floating on the open ocean. T. crunogena also has more flexibility in acquiring nutrients, based on the number of genes encoding transporters involved in the uptake of inorganic nitrogen and phosphate. And the microbe has a number of chemotaxis genes, which likely direct it to favorable microhabitats along the vent, as well as pilin genes, which allow it to set up shop in the best locations. With the first complete genome of a cosmopolitan autotrophic hydrothermal vent bacterium, researchers can further explore the genetic and physiological mechanisms that allow life to thrive in hostile environments at the bottom of the sea. And by comparing the T. crunogena genome to the genomes of autotrophic bacteria living in hot springs and other extreme environments around the world, they can begin to piece together the evolutionary history of primary production at the boundaries of life.

Two type IV pili ofVibrio parahaemolyticusplay different roles in biofilm formation

Vibrio parahaemolyticus RIMD2210633 has two sets of type IV-A pilus genes. One set is similar to that found in other Gram-negative bacteria, such as Pseudomonas aeruginosa, Vibrio cholerae (chitin-regulated pilus; ChiRP), and Vibrio vulnificus. The other is homologous to the genes for the mannose-sensitive hemagglutinin (MSHA) pilus. In this study, we analyzed the effects of the deletions in the pilin genes for each type IV pilus (the ChiRP and the MSHA pilus) on biofilm formation. Although the MSHA pilin mutant formed aggregates, the number of bacteria that attached directly to the coverslip was reduced, suggesting that this pilus contributes to the bacterial attachment to the surface of the coverslip. In contrast, the ChiRP mutant attached to the surface of the coverslip, but did not form aggregates, suggesting that ChiRP plays a role in bacterial agglutination during biofilm formation. These results suggest that the two type IV pili of V. parahaemolyticus contribute to biofilm formation in different ways. Both mutants showed a lower fitness for adsorption onto chitin particles than that of the wild type. Collectively, these data suggest that the use of two type IV pili is a refined strategy of V. parahaemolyticus for survival in natural environments.

The More the Merrier: Multiple Genomes in a Parasitic Prokaryote

The notion that bacteria and other single-celled “prokaryotes” carry only one genome copy per cell is as ingrained as the notion that they lack a distinct nucleus. (Organisms with one genome copy are “haploid”; organisms with two, including humans, are “diploid”; and organisms with more than two, such as plants, are “polyploid.”) This assumption is based on decades of research on Escherichia coli and a select group of other bacterial species. But researchers have been finding indications that some bacteria, under certain conditions, have more genetic material in a single cell than would be expected for a single genome. And now, using a combination of cell sorting, fluorescent microscopy, microarray, and quantitative PCR techniques, Deborah Tobiason and Hank Seifert present strong evidence that the bacterium that causes gonorrhea, (Neisseria gonorrhoeae), is indeed polyploid. N. gonorrhoeae successfully infects at-risk individuals by reshuffling the genetic code for proteins expressed on its hairlike “pili” protrusions through a process called gene conversion. During gene conversion, one gene incorporates DNA from a variant copy that carries slightly different information. The high frequency of gene conversion events enhances pili protein diversity, which increases the likelihood that the human immune system won't detect the pathogen. It is conceivable that N. gonorrhoeae might briefly express two copies of the pilin gene in one cell after DNA replication, before cell division. But Tobiason and Seifert thought that the high frequency of gene conversion required for antigenic diversity might instead be supported by the presence of extra genome copies. To explore this possibility, the researchers had to distinguish the amount of DNA associated with genome duplication prior to cell division from that found in a nondividing cell. They labeled the DNA of N. gonorrhoeae with fluorescent markers and, as a source of comparison, did the same for E. coli, which has provided a wellspring of knowledge on chromosomal DNA replication. Cells were either left untreated, allowing exponential growth, or were treated with an antibiotic that inhibits new rounds of replication but allows any DNA replication already in progress to finish. Cells in “stationary phase”—during which cell division stops and no new DNA replication begins, but active replication forks complete their task—were also labeled. Both antibiotic treated cells and stationary cells produce fully replicated chromosomes. Using a standard cell sorting technique called flow cytometry, the researchers separated cells based on their DNA content. Treated and stationary phase E. coli cells produced individual cells with the equivalent of two, four, or eight genomes per cell. Since multiple initiation events on the chromosome can occur per round of cell division, the number of replicated genomes reflects the number of replication forks active when cell division stops. Untreated E. coli cells, however, revealed a wide range of DNA content per cell, likely reflecting the presence of multiple replication forks at various stages of replication and cell division. The DNA content of untreated gonococcal cells also varied, with most cells having between four and six genome equivalents. Gonococcus exists as single (monococcal) and double spheres (diplococcal)—it is these two forms, Tobiason and Seifert show with fluorescence microscopy, that explains the range of DNA content in different cell populations found with flow cytometry. Monococci had, on average, about three to six genome equivalents, while diplococci averaged about four to ten per cell. To explore the reason for the expanded DNA content, the researchers treated gonococcal cells with antibiotics to generate fully replicated chromosomes. These experiments suggested that actively dividing cells have two or four pairs of active replication forks. They used independent measures to confirm that both monococci and diplococci contain fully replicated chromosomes with a single pair of active replication forks on each chromosome—which means that the DNA content they saw in gonococcal cells is not the result of multiple replication initiation events on a single chromosome, as occurs with E. coli, but represents multiple, fully replicated chromosomes. Future studies will have to determine whether polyploidy underlies pilin antigenic variation and the bacterium's success as a human pathogen. But this study leaves little doubt that it's time to retire the notion that all prokaryotes are haploid.

Atypical EnteropathogenicEscherichia coli: Typical Pathogens?

Escherichia coli is both the most abundant facultative commensal of the human gastrointestinal tract and the most common bacterial cause of human diarrhea (1). However, precise recognition of E. coli pathotypes remains problematic. Enteropathogenic E. coli (EPEC), classically associated with outbreaks of infant diarrhea, harbors distinctive chromosomal (the locus of enterocyte effacement, or LEE island) and plasmidborne (residing on the EPEC adherence factor, or EAF, plasmid) virulence factors, which are linked by common gene regulators (1). At the Second International Conference on held in Sao Paulo, Brazil, in 1995, the foremost authorities in the field proclaimed the global importance of such typical EPEC (tEPEC) but pondered the clinical relevance of strains carrying only the LEE island (dubbed at that conference atypical EPEC, or aEPEC) (2). Had aEPEC lost the EAF plasmid? Had it incidentally acquired only fragments or incomplete packages of virulence-associated genes? Or were some aEPEC true pathogens of humans or animals? In this issue of Emerging Infectious Diseases, Nguyen et al. propose a distinct role for aEPEC in human infection (3). Previously, these investigators reported a high prevalence of aEPEC among pediatric diarrhea patients in Melbourne, including both infants and older children (in contrast to the strong tendency for infants to be infected with tEPEC) (4). Now these authors show that, in contrast to patients infected with other pathogens, patients infected with aEPEC are far more likely to experience diarrhea past 14 days, the point long recognized as a clinical watershed that heralds increased risk for illness and death. aEPEC's prevalence among diarrhea patients, the pathogen's strong association with diarrheal symptoms, and the infection's distinctively persistent nature argue for a high disease burden in Melbourne. Although the authors define aEPEC strictly on the basis of positivity for the LEE eae gene and failure to amplify a bfpA pilin gene (not assessing additional plasmid loci), the absence of tEPEC serotypes and the occurrence of disease in children older than infants suggest that these are indeed aEPEC. This communication also illustrates 2 principles that should be recognized more generally with regard to diarrheogenic E. coli: 1) one can authoritatively implicate a particular strain as a pathogen by (only) outbreak implication or by volunteer studies, but one cannot definitively prove by any data that a putative pathotype is not a human pathogen; and 2) the implication of pathogenicity for 1 strain is not sufficient to implicate any similar strain as a pathogen. Thus, once definitive evidence is presented that a particular strain is pathogenic, the challenge is to determine the genotypic or phenotypic fingerprints that permit extrapolation to other strains. This obstacle has long proved daunting for the enteroaggregative pathotype (shown to be pathogenic in both volunteer studies and outbreaks), and it may prove similarly frustrating for aEPEC. Diarrheal epidemiology studies today are able to identify a likely etiologic agent for most patients, in contrast to studies of 20 years ago, because of improved diagnostic tests and the identification of new pathogens, but the task is not finished. Additional studies are needed in many places to refine our understanding of putative and emerging pathogens and to determine their full epidemiologic roles.

Plasmid-mediated genomic recombination at the pilin gene locus enhances the N-acetyl-d-galactosamine-specific haemagglutination activity and the growth rate of Eikenella corrodens

Eikenella corrodens belongs to a group of periodontopathogenic bacteria and forms unique corroding colonies on solid medium due to twitching motility. It is believed that an N-acetyl-D-galactosamine (GalNAc)-specific lectin on the cell surface contributes significantly to its pathogenicity and can be estimated by its haemagglutination (HA) activity. Recently, a plasmid, pMU1, from strain 1073 has been found; this plasmid affects pilus formation and colony morphology. To identify the gene involved in these phenomena, ORF 4 and ORFs 5-6 on pMU1 were separately subcloned into a shuttle vector, and the resultant plasmids were introduced into E. corrodens 23834. Transformants with the ORF 4 gene, which is identified to be a homologous gene of the type IV pilin gene-specific recombinase, lost their pilus structure and formed non-corroding colonies on a solid medium, whereas transformants with ORFs 5-6 exhibited the same phenotype as the host strain 23834. Southern analysis showed that the introduction of the ORF 4 gene into strain 23834 resulted in genomic recombination at the type IV pilin gene locus. The hybridization pattern of these transformants was similar to that of strain 1073. These results suggest that ORF 4 on pMU1 encodes a site-specific recombinase and causes genomic recombination of the type IV pilin gene locus. Furthermore, the introduction of ORF 4 into strain 23834 increased GalNAc-specific HA activity to a level equivalent to that of strain 1073. Although the morphological colony changes and loss of pilus structure are also observed in phase variation, genomic recombination of the type IV pilin gene locus did not occur in these variants. Moreover, an increase was not observed in the GalNAc-specific HA activity of these variants. These results suggested that the loss of pilus structure, the morphological change in colonies and the increase in HA activity due to plasmid pMU1 might be caused by a mechanism that differs from phase variation, such as a genomic recombination of the type IV pilin gene locus.

Direct repeat‐mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis

Francisella tularensis, the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella. In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat-mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage-like cell lines. Importantly, the pilin-negative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.