pilD Gene Research Articles

Determining the role of the pilD gene in Francisella tularensis susceptibility to resazomycins.

Antimicrobial resistant strains (AMRs) of bacteria have become such a severe worldwide issue that the United Nations General assembly met in 2016 to discuss the topic of AMRs. Due to the increase in the number of antibiotic-resistant strains, new therapeutics against AMRs must be developed. Resazomycins are resazurin-based compounds that exhibit antimicrobial activity against F. tularensis as well as other Gram-negative bacteria including N. gonorrheae. The mode of action of these antibiotics is not understood. Approximately half of forty-eight resazurin-resistant F. tularensis isolates had a mutation in the genes FTL_0959 (pilD) and FTL_1306 (dipA). The objective of this project is to determine how pilD affects F. tularensis susceptibility to resazomycins. In F. tularensis, pilD encodes for the cytoplasmic membrane peptidase responsible for processing the prepilin subunits in type IV pilin assembly proteins. We are currently working to generate a pilD null deletion mutant using standard molecular genetic techniques. We will first confirm the sensitivity of the pilD deletion mutant to resazomycins. Understanding the role of pilD in resazomycin susceptibility would facilitate further development of these compounds as potential treatments for tularemia and gonorrhea.

Two forms of Vibrio vulnificus metalloprotease VvpE are secreted via the type II general secretion system

Vibrio vulnificus has been known to secrete one form of metalloprotease VvpE (45 kDa) that is cleaved to 34 kDa-VvpE and 11 kDa-C-terminal propeptide via extracellular autoproteolysis. However, we found that extracellular secretion of both the 34 and 45 kDa forms of VvpE began in the early growth phase; moreover, 34 kDa-VvpE existed as the major form in V. vulnificus cell lysates and culture supernatants. In addition, extracellular secretion of both 34 and 45 kDa-VvpE was blocked by mutation of the pilD gene, which encodes for the type IV leader peptidase/N-methyltransferase of the type II general secretion system, and the blocked VvpE secretion was recovered by in trans-complementation of the wild-type pilD gene. These results indicate that 34 kDa-VvpE is the major form secreted along with 45 kDa-VvpE from the early growth phase via the PilD-mediated type II general secretion system.

Oxygen‐dependent autoaggregation in Shewanella oneidensis MR‐1

In aerobic chemostat cultures maintained at 50% dissolved O(2) tension (3.5 mg l(-1) dissolved O(2)), Shewanella oneidensis strain MR-1 rapidly aggregated upon addition of 0.68 mM CaCl(2) and retained this multicellular phenotype at high dilution rates. Confocal microscopy analysis of the extracellular matrix material contributing to the stability of the aggregate structures revealed the presence of extracellular DNA, protein and glycoconjugates. Upon onset of O(2)-limited growth (dissolved O(2) below detection) however, the Ca(2+)-supplemented chemostat cultures of strain MR-1 rapidly disaggregated and grew as motile dispersed cells. Global transcriptome analysis comparing aerobic aggregated to O(2)-limited unaggregated cells identified genes encoding cell-to-cell and cell-to-surface adhesion factors whose transcription increased upon exposure to increased O(2) concentrations. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologues of metal reduction genes, including mtrD (SO1782), mtrE (SO1781) and mtrF (SO1780). Our data indicate that mechanisms involved in autoaggregation of MR-1 are dependent on the function of pilD gene which encodes a putative prepilin peptidase. Mutants of S. oneidensis strain MR-1 deficient in PilD and associated pathways, including type IV and Msh pili biogenesis, displayed a moderate increase in sensitivity to H(2)O(2). Taken together, our evidence indicates that aggregate formation in S. oneidensis MR-1 may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

Production of Vibrio vulnificus metalloprotease VvpE begins during the early growth phase: Usefulness of gelatin-zymography

Recent studies have demonstrated that expression of the vvpE gene begins during the early growth phase albeit at low levels. However, we found that the traditional protease assay method that is used to measure caseinolytic activity in culture supernatants is not suitable for the measurement of extracellular VvpE that is produced at low levels during the early growth phase. By using gelatin-zymography in place of the protease assay, we could specifically detect only VvpE of several proteases produced by Vibrio vulnificus. Moreover, we could sensitively measure VvpE produced at low levels during the early growth phase, which was consistent with transcription of the vvpE gene. The extracellular production of VvpE was reduced or delayed by mutation of the pilD gene which encodes for the type IV leader peptidase/ N-methyltransferase associated with the type II general secretion system; the delayed production of VvpE was recovered by in trans complementation of the wild-type pilD gene. These results indicate that VvpE begins to be produced during the early growth phase via the PilD-mediated type II general secretion system, and that the use of gelatin-zymography is recommended as a simple method for the sensitive and specific detection of VvpE production.

Microevolution through DNA exchange among strains of Neisseria meningitidis isolated during an outbreak in the Czech Republic

Neisseria meningitidis is a highly variable bacterium. Indeed, N. meningitidis is naturally competent for transformation, and horizontal DNA exchange between strains may lead to mosaic genetic loci in N. meningitidis. We studied such an exchange in nature during an epidemic provoked by N. meningitidis. This epidemic started in the Czech Republic in 1993 and the original epidemic clone was shown to have the antigenic formula (serogroup:serotype:serosubtype) C:2a:P1.2,5. We analysed 145 meningococcal strains isolated in the Czech Republic between 1993 and 1997 using serological and genetic typing methods (multilocus enzyme electrophoresis and polymorphism of pilA and pilD genes). This analysis showed that genetic exchange between epidemic and endemic strains had occurred. Exchanges involved mostly surface- exposed structures such as the capsule, giving rise to new meningococcal variants. The expansion of these variants should be kept under close surveillance.

Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000.

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.

Amino Acid Substitutions in PilD, a Bifunctional Enzyme ofPseudomonas aeruginosa: EFFECT ON LEADER PEPTIDASE AND N-METHYLTRANSFERASE ACTIVITIES IN VITRO AND IN VIVO

Subunits of type IV pili and a subset of proteins of the type II extracellular protein secretion apparatus undergo two consecutive post-translational modifications: leader peptide cleavage, followed by methylation of the newly created N-terminal amino acid. These two reactions are carried out by a single bifunctional enzyme encoded in Pseudomonas aeruginosa by the pilD gene. Properties of PilD mutants at positions Gly95 and/or Lys96 which were differentially affected in leader peptidase and N-methyltransferase function were characterized. None of the single amino acid substitutions showed a significant alteration in their ability to cleave the prepilin leader peptide; however, two double mutants did exhibit a modest reduction in the efficiency of cleavage. In contrast, a significant decrease of N-methyltransferase activity was detected in PilD having substitutions at Gly95. Mutants with substitutions at position Lys96 showed a variable effect on N-methyltransferase activity with an apparent requirement for any charged amino acid at this position. Absence of N-methyltransferase activity did not appear to interfere with the ability of P. aeruginosa to assemble functional pili. Moreover, pilin monomers isolated from P. aeruginosa expressing PilD with Gly95 substitutions were not methylated. Although complete methylation does not appear to be absolutely required for pilus assembly in P. aeruginosa, this modification may be important for pilus function in its natural habitat.

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion.

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.

Identification and characterization of pilG, a highly conserved pilus‐assembly gene in pathogenic Neisseria

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus-assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus-assembly protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of M(r) 38 kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group B polysaccharide-producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Mc are extremely conserved. Hybridization studies strongly suggest that pilG-related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Mc chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.

Characterization of the pilF—pilD pilus‐assembly locus of Neisseria gonorrhoeae

Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre-pilin peptidase, were unable to process the pre-pilin subunit into pilin and thus were non-piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S-pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide-binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.

Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W.

Four components of the apparatus of extracellular protein secretion of Pseudomonas aeruginosa, Xcpt, -U, -V, and -W (XcpT-W), are synthesized as precursors with short N-terminal leader peptides that share sequence similarity with the pilin subunit of this organism. A specialized leader peptidase/methylase, product of the pilD gene, has been shown to cleave the leader peptide from prepilin and to methylate the N-terminal phenylalanine of the mature pilin. Antibodies were prepared against XcpT-W and used to purify each of these proteins. Sequence analysis of XcpT-W has shown that these proteins, like mature pilin, contain N-methylphenylalanine as the N-terminal amino acid. Analysis of cellular fractions from wild-type and pilD mutant strains of P. aeruginosa showed that the precursor forms of XcpT-W are located predominantly in the bacterial inner membrane, and their localization is not altered after PilD-mediated removal of the leader sequence. These studies demonstrate that the biogenesis of the apparatus of extracellular protein secretion and that of type IV pili share a requirement for PilD. This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W and subsequently methylates the amino group of the N-terminal phenylalanine of each of its substrates.

A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family.

Precursors of the type IV pilins of a number of bacterial pathogens, as well as related proteins involved in extracellular protein export and DNA uptake, are synthesized with short basic leader sequences. Maturation of these proteins involves two consecutive posttranslational modifications. The leader sequence is first proteolytically removed by specialized endopeptidases, of which the prototype is encoded by the pilD gene of Pseudomonas aeruginosa. Subsequently, the amino termini of these proteins are methylated. Here we demonstrate that PilD, in addition to cleaving the amino-terminal leader sequences of prepilin, also catalyzes N-methylation of the amino-terminal phenylalanine of the mature pilin, using S-adenosyl-L-methionine as a methyl donor. Thus, to our knowledge, PilD is the first characterized bacterial N-methyltransferase. Complete inhibition of N-methylation, but not peptide cleavage, by structural analogues of S-adenosyl-L-methionine suggests that PilD is a bifunctional enzyme with proteolytic and methylation activities carried out within two distinct active sites.

Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase.

In the Gram-negative pathogen Pseudomonas aeruginosa, mutants in the gene for the prepilin peptidase (pilD) are pleiotropic, as they not only fail to process pilin but also accumulate in the periplasm, in their mature form, several toxins and hydrolytic enzymes that are normally exported to the external medium (excreted). We have suggested that this excretion defect is due to the lack of PilD-dependent processing of proteins that share sequences in common with the prepilin subunit and that are components of a protein-excretion machinery. In this paper we report the isolation and characterization of transposon-induced excretion mutants with phenotypes similar to that of a pilD gene mutant. Using oligonucleotide probes designed to recognize sequences encoding the cleavage site of the type IV prepilins, we have isolated four linked genes with the predicted putative PilD-dependent cleavage site. Site-specific mutations within these genes have shown that they are required for protein excretion, and PilD-dependent processing of at least one of the four encoded proteins was demonstrated. Evidence suggests that similar components play a role in protein excretion in a wide variety of Gram-negative bacteria.

Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase.

The related type IV pilins produced by Pseudomonas aeruginosa, Neisseria gonorrhoeae, Bacteroides nodosus, and Moraxella bovis are synthesized as precursors with short, six- or seven-amino acid N-terminal leader peptides. We have previously observed that P. aeruginosa mutations in pilD, a gene required for pilus biogenesis, result in the accumulation of unprocessed prepilin in the membrane and a general defect in the excretion of a number of extracellular enzymes. An endopeptidase activity has been detected in detergent-solubilized inner membrane of P. aeruginosa and shown to correctly cleave the prepilin of P. aeruginosa and N. gonorrhoeae. It is absent from pilD mutants, increased by pilD overexpression, and conferred on Escherichia coli by the introduction of the pilD gene. The pilD gene product, purified by immunoaffinity chromatography with antibody to a PilD-derived synthetic peptide, was identified with the endopeptidase. PilD appears to be a prototype of a class of enzymes that process not only type IV pilin precursors but also components of a protein-excretion apparatus of Gram-negative bacteria.