Genomic and Gene-Expression Comparisons among Phage-Resistant Type-IV Pilus Mutants of Pseudomonas syringae pathovar phaseolicola

AbstractPseudomonas syringaepv.phaseolicola(Pph) is a significant bacterial pathogen of agricultural crops, and phage ϕ6 and other members of the dsRNA virus familyCystoviridaeundergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage ϕ6 as a model system in evolutionary biology, Pph resistance to phage ϕ6 remains poorly characterized. To investigate differences between phage ϕ6 resistant Pph strains, we examined genomic and gene expression variation among three bacterial genotypes that differ in the number of type IV pili expressed per cell: ordinary (wild-type), non-piliated, and super-piliated. Genome sequencing of non-piliated and super-piliated Pph identified few mutations that separate these genotypes from wild type Pph – and none present in genes known to be directly involved in type IV pilus expression. Expression analysis revealed that 81.1% of GO terms up-regulated in the non-piliated strain were down-regulated in the super-piliated strain. This differential expression is particularly prevalent in genes associated with respiration — specifically genes in the tricarboxylic acid cycle (TCA) cycle, aerobic respiration, and acetyl-CoA metabolism. The expression patterns of the TCA pathway appear to be generally up and down-regulated, in non-piliated and super-piliated Pph respectively. As pilus retraction is mediated by an ATP motor, loss of retraction ability might lead to a lower energy draw on the bacterial cell, leading to a different energy balance than wild type. The lower metabolic rate of the super-piliated strain is potentially a result of its loss of ability to retract.

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Characterization of Type IV Pilus Genes in Pseudomonas syringae pv. tomato DC3000

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.11.1048 ◽

1998 ◽

Vol 11 (11) ◽

pp. 1048-1056 ◽

Cited By ~ 70

Author(s):

Elina Roine ◽

Deanna M. Raineri ◽

Martin Romantschuk ◽

Mark Wilson ◽

David N. Nunn

Keyword(s):

Pseudomonas Syringae ◽

Lytic Bacteriophage ◽

Type Iv Pilus ◽

Tomato Dc3000 ◽

Plant Leaf ◽

Type Iv ◽

Pilus Biogenesis ◽

Population Sizes ◽

Pila Gene ◽

Prepilin Peptidase

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage φ6. 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 φ6 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 φ6. 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.

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Faculty Opinions recommendation of Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1013554.192053 ◽

2003 ◽

Author(s):

Virginia L Miller

Keyword(s):

Gene Cluster ◽

Citrobacter Rodentium ◽

Type Iv Pilus ◽

Type Iv ◽

Gastrointestinal Colonization

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Faculty Opinions recommendation of The Che4 pathway of Myxococcus xanthus regulates type IV pilus-mediated motility.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019833.226437 ◽

2004 ◽

Author(s):

George Ordal

Keyword(s):

Myxococcus Xanthus ◽

Type Iv Pilus ◽

Type Iv

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Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)92810-3 ◽

1991 ◽

Vol 266 (21) ◽

pp. 14064-14071

Author(s):

C.M. Overall ◽

J.L. Wrana ◽

J. Sodek

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Human Fibroblasts ◽

Type Iv Collagenase ◽

Type Iv ◽

Growth Factor Beta ◽

Post Transcriptional Regulation

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Analysis of Arabidopsis JAZ gene expression during Pseudomonas syringae pathogenesis

Molecular Plant Pathology ◽

10.1111/j.1364-3703.2011.00727.x ◽

2011 ◽

Vol 13 (1) ◽

pp. 46-57 ◽

Cited By ~ 68

Author(s):

AGNES J. DEMIANSKI ◽

KWI MI CHUNG ◽

BARBARA N. KUNKEL

Keyword(s):

Gene Expression ◽

Pseudomonas Syringae

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Type IV Pilus Assembly Proficiency and Dynamics Influence Pilin Subunit Phospho-Form Macro- and Microheterogeneity in Neisseria gonorrhoeae

PLoS ONE ◽

10.1371/journal.pone.0096419 ◽

2014 ◽

Vol 9 (5) ◽

pp. e96419 ◽

Cited By ~ 1

Author(s):

Åshild Vik ◽

Jan Haug Anonsen ◽

Finn Erik Aas ◽

Finn Terje Hegge ◽

Norbert Roos ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Type Iv Pilus ◽

Type Iv ◽

Pilin Subunit ◽

Pilus Assembly

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Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

Microbiology ◽

10.1099/mic.0.001080 ◽

2021 ◽

Vol 167 (9) ◽

Author(s):

Marie Zachary ◽

Susanne Bauer ◽

Maximilian Klepsch ◽

Katharina Wagler ◽

Bruno Hüttel ◽

...

Keyword(s):

Target Genes ◽

Target Prediction ◽

Type Iv Pilus ◽

Content Type ◽

Type Iv ◽

Gene Expression Control ◽

Initial Characterization ◽

Pilus Biogenesis ◽

Regulatory Rnas

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae , TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

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Purification and Three-Dimensional Electron Microscopy Structure of the Neisseria meningitidis Type IV Pilus Biogenesis Protein PilG

Journal of Bacteriology ◽

10.1128/jb.00648-07 ◽

2007 ◽

Vol 189 (17) ◽

pp. 6389-6396 ◽

Cited By ~ 25

Author(s):

Richard F. Collins ◽

Muhammad Saleem ◽

Jeremy P. Derrick

Keyword(s):

Electron Microscopy ◽

Neisseria Meningitidis ◽

Metal Ion ◽

Polyacrylamide Gel Electrophoresis ◽

Three Dimensional ◽

Type Ii Secretion ◽

Type Ii ◽

Type Iv Pilus ◽

Type Iv ◽

Pilus Biogenesis

ABSTRACT Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 Å in length and 80 Å in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower “waist” region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.

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