scholarly journals Association Between Loss of Type IV Pilus Synthesis Ability and Phenotypic Variation in the Cucurbit Pathogenic Bacterium Acidovorax citrulli

2018 ◽  
Vol 31 (5) ◽  
pp. 548-559 ◽  
Author(s):  
Tally Rosenberg ◽  
Bolaji Babajide Salam ◽  
Saul Burdman
Keyword(s):  
Phenotypic Variation ◽  
Seed Transmission ◽  
Colony Morphology ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv ◽  
Acidovorax Citrulli ◽  
Transmission Electron ◽  
Transposon Mutants ◽  
Transposon Insertions

Acidovorax citrulli is the causal agent of bacterial fruit blotch of cucurbits. We have shown that functional type IV pili (T4P) are required for full virulence of this bacterium. To identify A. citrulli genes required for T4P activity, we screened a library of about 10,000 transposon mutants of A. citrulli M6 for altered T4P-mediated twitching motility. This screen led to the identification of 50 mutants impaired in twitching ability due to transposon insertions into 20 different genes. Representative mutants with disruptions in these genes were further characterized. All mutants were compromised in their virulence in seed transmission and stem inoculation assays and had reduced biofilm formation ability relative to wild-type M6. When grown on nutrient agar, most mutants produced colonies with a translucent and fuzzy appearance, in contrast to the opaque and smooth appearance of wild-type colonies. The colony morphology of these mutants was identical to that of previously reported phenotypic variants of strain M6. The exceptions were M6 mutants disrupted in genes tonB, pilT, pilW, and pilX that exhibited typical wild-type colony morphology, although lacking twitching haloes surrounding the colony. Transmission electron microscopy revealed that most mutants lacked the ability to produce T4P. The exceptions were mutants with disruptions in tonB, pilT, pilW, and pilX genes that were shown to produce these appendages. These findings support the idea that colony phenotypic variation in A. citrulli is determined by the lack of ability to synthesize T4P but not by lack of T4P functionality.

scholarly journals Type IV Pili Are Required for Virulence, Twitching Motility, and Biofilm Formation of Acidovorax avenae subsp. citrulli

2009 ◽  
Vol 22 (8) ◽  
pp. 909-920 ◽  
Author(s):  
Ofir Bahar ◽  
Tal Goffer ◽  
Saul Burdman
Keyword(s):  
Biofilm Formation ◽  
Host Plants ◽  
Seed Transmission ◽  
Type Iv Pili ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv ◽  
Group I ◽  
Tn5 Mutant

Acidovorax avenae subsp. citrulli is the causal agent of bacterial fruit blotch (BFB), a threatening disease of watermelon, melon, and other cucurbits. Despite the economic importance of BFB, relatively little is known about basic aspects of the pathogen's biology and the molecular basis of its interaction with host plants. To identify A. avenae subsp. citrulli genes associated with pathogenicity, we generated a transposon (Tn5) mutant library on the background of strain M6, a group I strain of A. avenae subsp. citrulli, and screened it for reduced virulence by seed-transmission assays with melon. Here, we report the identification of a Tn5 mutant with reduced virulence that is impaired in pilM, which encodes a protein involved in assembly of type IV pili (TFP). Further characterization of this mutant revealed that A. avenae subsp. citrulli requires TFP for twitching motility and wild-type levels of biofilm formation. Significant reductions in virulence and biofilm formation as well as abolishment of twitching were also observed in insertional mutants affected in other TFP genes. We also provide the first evidence that group I strains of A. avenae subsp. citrulli can colonize and move through host xylem vessels.

scholarly journals Drosophilaas a Model Host forPseudomonas aeruginosaInfection

2001 ◽  
Vol 183 (4) ◽  
pp. 1466-1471 ◽  
Author(s):  
David A. D'Argenio ◽  
Larry A. Gallagher ◽  
Celeste A. Berg ◽  
Colin Manoil
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Drosophila Melanogaster ◽  
Gene Cluster ◽  
Virulence Factors ◽  
Fruit Fly ◽  
Colony Morphology ◽  
Twitching Motility ◽  
Wild Type ◽  
Signal Transduction System

ABSTRACT Using the fruit fly Drosophila melanogaster as model host, we have identified mutants of the bacterium Pseudomonas aeruginosa with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in pilGHIJKL chpABCDE, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The pil chp genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.

scholarly journals Differential Regulation of Twitching Motility and Elastase Production by Vfr in Pseudomonas aeruginosa

2002 ◽  
Vol 184 (13) ◽  
pp. 3605-3613 ◽  
Author(s):  
Scott A. Beatson ◽  
Cynthia B. Whitchurch ◽  
Jennifer L. Sargent ◽  
Roger C. Levesque ◽  
John S. Mattick
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv ◽  
Allelic Deletion ◽  
Pyocyanin Production ◽  
Null Mutants

ABSTRACT Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

scholarly journals A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Prem P. Kandel ◽  
Hongyu Chen ◽  
Leonardo De La Fuente
Keyword(s):  
Natural Transformation ◽  
Gene Knockout ◽  
Xylella Fastidiosa ◽  
Twitching Motility ◽  
Wild Type ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Type Iv ◽  
Overlap Extension ◽  
Pilin Subunit

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

scholarly journals FimX, a Multidomain Protein Connecting EnvironmentalSignals to Twitching Motility in Pseudomonasaeruginosa

2003 ◽  
Vol 185 (24) ◽  
pp. 7068-7076 ◽  
Author(s):  
Bixing Huang ◽  
Cynthia B. Whitchurch ◽  
John S. Mattick
Keyword(s):  
Fluorescent Protein ◽  
Response Regulator ◽  
Type Iv Pili ◽  
Environmental Cues ◽  
Twitching Motility ◽  
Wild Type ◽  
Protein Fusion ◽  
Type Iv ◽  
Low Levels ◽  
Signal Transduction Proteins

ABSTRACT Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.

scholarly journals Twitching Motility Is Essential for Virulence in Dichelobacter nodosus

2008 ◽  
Vol 190 (9) ◽  
pp. 3323-3335 ◽  
Author(s):  
Xiaoyan Han ◽  
Ruth M. Kennan ◽  
John K. Davies ◽  
Leslie A. Reddacliff ◽  
Om P. Dhungyel ◽  
...  
Keyword(s):  
Cell Movement ◽  
Foot Rot ◽  
Twitching Motility ◽  
Wild Type ◽  
Cell Adherence ◽  
Secretion Pathway ◽  
Dichelobacter Nodosus ◽  
Fimbrial Subunit ◽  
Type Iv ◽  
Protease Secretion

ABSTRACTType IV fimbriae are essential virulence factors ofDichelobacter nodosus, the principal causative agent of ovine foot rot. ThefimAfimbrial subunit gene is required for virulence, butfimAmutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either thepilTorpilUgene eliminated the ability to carry out twitching motility. However, thepilTmutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas thepilUmutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway inD. nodosus. It was postulated that sufficient fimbrial retraction must occur in thepilUmutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in apilUmutant ofD. nodosus, aberrant motion was detected in an equivalent mutant ofPseudomonas aeruginosa. These observations explain how inD. nodosusprotease secretion can occur in apilUmutant but not in apilTmutant. In addition, virulence studies with sheep showed that both thepilTandpilUmutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.

scholarly journals Loss of Virulence of the Phytopathogen Ralstonia solanacearum Through Infection by φRSM Filamentous Phages

2012 ◽  
Vol 102 (5) ◽  
pp. 469-477 ◽  
Author(s):  
Hardian S. Addy ◽  
Ahmed Askora ◽  
Takeru Kawasaki ◽  
Makoto Fujie ◽  
Takashi Yamada
Keyword(s):  
Ralstonia Solanacearum ◽  
Virulence Genes ◽  
Host Cells ◽  
Extracellular Polysaccharides ◽  
Twitching Motility ◽  
Wild Type ◽  
Tomato Plants ◽  
Type Iv ◽  
Infected Cells ◽  
Filamentous Phages

φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

scholarly journals A LysR-Type Transcriptional Regulator in Burkholderia cenocepacia Influences Colony Morphology and Virulence

2007 ◽  
Vol 76 (1) ◽  
pp. 38-47 ◽  
Author(s):  
Steve P. Bernier ◽  
David T. Nguyen ◽  
Pamela A. Sokol
Keyword(s):  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Burkholderia Cenocepacia ◽  
Colony Morphology ◽  
Infection Model ◽  
Bacterial Cells ◽  
Wild Type ◽  
Transmission Electron ◽  
Lung Histopathology ◽  
Seedling Infection

ABSTRACT Burkholderia cenocepacia strain K56-2 typically has rough colony morphology on agar medium; however, shiny colony variants (shv) can appear spontaneously. These shv all had a minimum of 50% reduction in biomass formation and were generally avirulent in an alfalfa seedling infection model. Three shv—K56-2 S15, K56-2 S76, and K56-2 S86—were analyzed for virulence in a chronic agar bead model of respiratory infection and, although all shv were able to establish chronic infection, they produced significantly less lung histopathology than the rough K56-2. Transmission electron microscopy revealed that an extracellular matrix surrounding bacterial cells was absent or reduced in the shv compared to the rough wild type. Transposon mutagenesis was performed on the rough wild-type strain and a mutant with an insertion upstream of ORF BCAS0225, coding for a putative LysR-type regulator, exhibited shiny colony morphology, reduced biofilm production, increased N-acyl homoserine lactone production, and avirulence in alfalfa. The rough parental colony morphotype, biofilm formation, and virulence in alfalfa were restored by providing BCAS0225 in trans in the BCAS0225::pGSVTp-luxCDABF mutant. Introduction of BCAS0225 restored the rough morphotype in several shv which were determined to have spontaneous mutations in this gene. In the present study, we show that the conversion from rough wild type to shv in B. cenocepacia correlates with reduced biofilm formation and virulence, and we determined that BCAS0225 is one gene involved in the regulation of these phenotypes.

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 Pseudomonas aeruginosa Exhibits Directed Twitching Motility Up Phosphatidylethanolamine Gradients

2001 ◽  
Vol 183 (2) ◽  
pp. 763-767 ◽  
Author(s):  
Daniel B. Kearns ◽  
Jayne Robinson ◽  
Lawrence J. Shimkets
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Solid Surfaces ◽  
Twitching Motility ◽  
Wild Type ◽  
Directed Movement ◽  
Endogenous Factors ◽  
Type Iv ◽  
Cell Velocity ◽  
Dependent Form

ABSTRACT Pseudomonas aeruginosa translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from wild-type cells a lipid that caused directed movement. Wild-type P. aeruginosa, but not a pilJ pilus-deficient mutant, showed biased movement up gradients of phosphatidylethanolamine (PE) established in agar. Activity was related to the fatty acid composition of the lipid, as two synthetic PE species, dilauroyl and dioleoyl PE, were capable of directing P. aeruginosa motility while many other species were inactive. P. aeruginosa PE did not contain either laurate or oleate, implying that the native attractant species contains different fatty acids. Uniform concentrations of PE increased cell velocity, suggesting that chemokinesis may be at least partly responsible for directed movement. We speculate that PE-directed twitching motility may be involved in biofilm formation and pathogenesis.

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