scholarly journals 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

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 815-821 ◽  
Author(s):  
Hiroyuki Azakami ◽  
Hiromi Akimichi ◽  
Yuichiro Noiri ◽  
Shigeyuki Ebisu ◽  
Akio Kato
Keyword(s):  
Gene Locus ◽  
Solid Medium ◽  
Shuttle Vector ◽  
Phase Variation ◽  
Homologous Gene ◽  
Eikenella Corrodens ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Pilin Gene ◽  
Genomic Recombination

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.

scholarly journals Eikenella corrodens Phase Variation Involves a Posttranslational Event in Pilus Formation

1999 ◽  
Vol 181 (14) ◽  
pp. 4154-4160 ◽  
Author(s):  
Maria T. Villar ◽  
Jennifer T. Helber ◽  
Becky Hood ◽  
Michael R. Schaefer ◽  
Rona L. Hirschberg
Keyword(s):  
Sequence Analysis ◽  
Phase Variation ◽  
S Phase ◽  
Open Reading Frames ◽  
Major Type ◽  
Eikenella Corrodens ◽  
Dichelobacter Nodosus ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Phase Variants

ABSTRACT The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate E. corrodens VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designatedpilA1, pilA2, pilB, andhagA. Both pilA1 and pilA2 predict a type IV pilin. The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein. The protein predicted by hagAresembles a hemagglutinin. The region containing these four ORFs was designated the pilA locus. DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundantpilA1 transcript initiating upstream of pilA1and terminating at a predicted hairpin structure betweenpilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassingpilA1, pilA2, and pilB. Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E. corrodens.

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

2006 ◽  
Vol 59 (6) ◽  
pp. 1818-1830 ◽  
Author(s):  
Anna-Lena Forslund ◽  
Kerstin Kuoppa ◽  
Kerstin Svensson ◽  
Emelie Salomonsson ◽  
Anders Johansson ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Direct Repeat ◽  
Type Iv ◽  
Virulence Attenuation ◽  
Type Iv Pilin ◽  
Pilin Gene

scholarly journals Expression of Multiple Pili by Legionella pneumophila: Identification and Characterization of a Type IV Pilin Gene and Its Role in Adherence to Mammalian and Protozoan Cells

1998 ◽  
Vol 66 (4) ◽  
pp. 1768-1775 ◽  
Author(s):  
Barbara J. Stone ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Insertion Mutation ◽  
Wild Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Wild Type Phenotype ◽  
Type Iv Pilin ◽  
Legionnaires Disease ◽  
Pilin Gene

ABSTRACT Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 μm) or short (0.1 to 0.6 μm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL ). An insertion mutation was constructed in pilEL and introduced into theL. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that thepilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires’ disease and invasion of amoebas in the environment.

scholarly journals PpdD Type IV Pilin of Escherichia coliK-12 Can Be Assembled into Pili in Pseudomonas aeruginosa

2000 ◽  
Vol 182 (3) ◽  
pp. 848-854 ◽  
Author(s):  
Nathalie Sauvonnet ◽  
Pierre Gounon ◽  
Anthony P. Pugsley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Low Levels ◽  
Fusion Analysis ◽  
K 12 ◽  
Pilin Gene

ABSTRACT Escherichia coli K-12 possesses at least 16 chromosomal genes related to genes involved in the formation of type IV pili in other gram-negative bacteria. However, E. coli K-12 does not produce type IV pili when grown under standard laboratory conditions. The results of reverse transcription-PCR, operon fusion analysis, and immunoblotting demonstrated that several of the putativeE. coli piliation genes are expressed at very low levels. Increasing the level of expression of the major pilin gene (ppdD) and the linked assembly genes hofB andhofC (homologues of the Pseudomonas aeruginosatype IV pilus assembly genes pilB and pilC) did not lead to pilus production. However, expression of theppdD gene in P. aeruginosa led to assembly of PpdD into pili that were recognized by antibodies directed against the PpdD protein. Assembly of PpdD into pili in P. aeruginosawas dependent on the expression of the pilB andpilC genes and independent of expression of the P. aeruginosa pilin structural gene pilA.

scholarly journals Role of the Eikenella corrodens pilALocus in Pilus Function and Phase Variation

2001 ◽  
Vol 183 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Maria T. Villar ◽  
Rona L. Hirschberg ◽  
Michael R. Schaefer
Keyword(s):  
Phase Variation ◽  
S Phase ◽  
Related Phenomenon ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Eikenella Corrodens ◽  
Type Iv ◽  
And Function ◽  
Phase Variants

ABSTRACT The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolateE. corrodens VA1. Earlier work defined the pilAlocus which includes pilA1, pilA2,pilB, and hagA. Both pilA1 andpilA2 predict a type IV pilin, whereas pilBpredicts a putative pilus assembly protein. The role ofhagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilAmutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated forpilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation ofhagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.

scholarly journals Protein Nanowires: the Electrification of the Microbial World and Maybe Our Own

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Derek R. Lovley ◽  
Dawn E. Holmes
Keyword(s):  
Electron Transport ◽  
Long Range ◽  
Electronic Devices ◽  
Electricity Production ◽  
Microbial World ◽  
Electrically Conductive ◽  
Anaerobic Digesters ◽  
Content Type ◽  
Type Iv ◽  
Type Iv Pilin

ABSTRACT Electrically conductive protein nanowires appear to be widespread in the microbial world and are a revolutionary “green” material for the fabrication of electronic devices. Electrically conductive pili (e-pili) assembled from type IV pilin monomers have independently evolved multiple times in microbial history as have electrically conductive archaella (e-archaella) assembled from homologous archaellin monomers. A role for e-pili in long-range (micrometer) extracellular electron transport has been demonstrated in some microbes. The surprising finding of e-pili in syntrophic bacteria and the role of e-pili as conduits for direct interspecies electron transfer have necessitated a reassessment of routes for electron flux in important methanogenic environments, such as anaerobic digesters and terrestrial wetlands. Pilin monomers similar to those found in e-pili may also be a major building block of the conductive “cables” that transport electrons over centimeter distances through continuous filaments of cable bacteria consisting of a thousand cells or more. Protein nanowires harvested from microbes have many functional and sustainability advantages over traditional nanowire materials and have already yielded novel electronic devices for sustainable electricity production, neuromorphic memory, and sensing. e-pili can be mass produced with an Escherichia coli chassis, providing a ready source of material for electronics as well as for studies on the basic mechanisms for long-range electron transport along protein nanowires. Continued exploration is required to better understand the electrification of microbial communities with microbial nanowires and to expand the “green toolbox” of sustainable materials for wiring and powering the emerging “Internet of things.”

scholarly journals Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility

2009 ◽  
Vol 191 (21) ◽  
pp. 6513-6524 ◽  
Author(s):  
Hanjeong Harvey ◽  
Marc Habash ◽  
Francisca Aidoo ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antigenic Variation ◽  
Intact Protein ◽  
Twitching Motility ◽  
Beta Turn ◽  
Type Iv ◽  
Pilin Subunit ◽  
Type Iv Pilin ◽  
Intersubunit Interactions ◽  
Pilus Assembly

ABSTRACT PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.

scholarly journals Acinetobacterstrains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated toO-glycosylation of multiple proteins

2015 ◽  
Vol 96 (5) ◽  
pp. 1023-1041 ◽  
Author(s):  
Christian M. Harding ◽  
Mohamed A. Nasr ◽  
Rachel L. Kinsella ◽  
Nichollas E. Scott ◽  
Leonard J. Foster ◽  
...  
Keyword(s):  
The Other ◽  
Type Iv ◽  
Type Iv Pilin

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

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

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

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