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 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 Imaging OmpR Binding to Native Chromosomal Loci in Escherichia coli

2010 ◽  
Vol 192 (15) ◽  
pp. 4045-4053 ◽  
Author(s):  
Elizabeth A. Libby ◽  
Seda Ekici ◽  
Mark Goulian
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Chromosomal Location ◽  
Response Regulator ◽  
Yellow Fluorescent Protein ◽  
Growth Conditions ◽  
Inhomogeneous Distribution ◽  
Wild Type ◽  
Protein Fusion ◽  
Chromosomal Loci

ABSTRACT Previously, an unexplained subcellular localization was reported for a functional fluorescent protein fusion to the response regulator OmpR in Escherichia coli. The pronounced regions of increased fluorescence, or foci, are dependent on OmpR phosphorylation and do not occupy fixed, easily identifiable positions, such as the poles or mid-cell. Here we show that the foci are due to OmpR-YFP (yellow fluorescent protein) fusion binding to specific sites in the chromosome. To identify positions of foci and quantify their fluorescence intensity, we used a simple system to tag virtually any chromosomal location with arrays of lacO or tetO. The brightest foci colocalize with the OmpR-regulated gene ompF, which is strongly expressed under our growth conditions. When we increased OmpR-YFP phosphorylation by stimulating the EnvZ/OmpR system with procaine, we observed a small increase in OmpR-YFP fluorescence at ompF and a significant increase at the OmpR-regulated gene ompC. This supports a model of hierarchical binding of OmpR to the ompF and ompC promoters. Our results explain the inhomogeneous distribution of OmpR-YFP fluorescence in cells and further demonstrate that for a transcription factor expressed at wild-type levels, binding to native sites in the chromosome can be imaged and quantified by fluorescence microscopy.

scholarly journals Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Vincent Nieto ◽  
Abby R. Kroken ◽  
Melinda R. Grosser ◽  
Benjamin E. Smith ◽  
Matteo M. E. Metruccio ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Host Cell ◽  
Type Iv Pili ◽  
Fluorescent Imaging ◽  
Bacterial Motility ◽  
Twitching Motility ◽  
Wild Type ◽  
Content Type ◽  
Type Iv

ABSTRACT Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s−1 while dividing intracellularly. Similar results were obtained with flagellin (fliC) and flagellum assembly (flhA) mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, pilA mutants (lacking T4P) and pilT mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules. IMPORTANCE Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa. Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo, and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.

scholarly journals tonB3 Is Required for Normal Twitching Motility and Extracellular Assembly of Type IV Pili

2004 ◽  
Vol 186 (13) ◽  
pp. 4387-4389 ◽  
Author(s):  
Bixing Huang ◽  
Kelin Ru ◽  
Zheng Yuan ◽  
Cynthia B. Whitchurch ◽  
John S. Mattick
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv

ABSTRACT Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.

scholarly journals Mutations in Type I and Type IV Pilus Biosynthetic Genes Affect Twitching Motility Rates in Xylella fastidiosa

2007 ◽  
Vol 189 (20) ◽  
pp. 7507-7510 ◽  
Author(s):  
Leonardo De La Fuente ◽  
Thomas J. Burr ◽  
Harvey C. Hoch
Keyword(s):  
Standard Error ◽  
Flow Direction ◽  
Xylella Fastidiosa ◽  
Type Iv Pili ◽  
Type I ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Rate Of Movement

ABSTRACT Xylella fastidiosa possesses both type I and type IV pili at the same cell pole. By use of a microfluidic device, the speed of twitching movement by wild-type cells on a glass surface against the flow direction of media was measured as 0.86 (standard error [SE], 0.04) μm min−1. A type I pilus mutant (fimA) moved six times faster (4.85 [SE, 0.27] μm min−1) and a pilY1 mutant moved three times slower (0.28 [SE, 0.03] μm min−1) than wild-type cells. Type I pili slow the rate of movement, while the putative type IV pilus protein PilY1 is likely important for attachment to surfaces.

scholarly journals Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants

2003 ◽  
Vol 48 (6) ◽  
pp. 1511-1524 ◽  
Author(s):  
Mikkel Klausen ◽  
Arne Heydorn ◽  
Paula Ragas ◽  
Lotte Lambertsen ◽  
Anders Aaes-Jørgensen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Type Iv Pili ◽  
Wild Type ◽  
Type Iv

scholarly journals Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

2005 ◽  
Vol 187 (3) ◽  
pp. 829-839 ◽  
Author(s):  
Poney Chiang ◽  
Marc Habash ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Distribution Patterns ◽  
Opportunistic Pathogen ◽  
Twitching Motility ◽  
Polar Localization ◽  
Type Iv ◽  
And Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

scholarly journals Spermine analogue-regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

2009 ◽  
Vol 422 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Anne Uimari ◽  
Tuomo A. Keinänen ◽  
Anne Karppinen ◽  
Patrick Woster ◽  
Pekka Uimari ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fusion Gene ◽  
Wild Type Mouse ◽  
Egfp Expression ◽  
Wild Type ◽  
Protein Fusion ◽  
Reverse Transcription Pcr ◽  
Fetal Fibroblasts ◽  
Spermine Analogues ◽  
Ssat Activity

SSAT (Spermidine/spermine N1-acetyltransferase, also known as SAT1), the key enzyme in the catabolism of polyamines, is turned over rapidly and there is only a low amount present in the cell. In the present study, the regulation of SSAT by spermine analogues, the inducers of the enzyme, was studied in wild-type mouse fetal fibroblasts, expressing endogenous SSAT, and in the SSAT-deficient mouse fetal fibroblasts transiently expressing an SSAT–EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N1,N11-diethylnorspermine), CPENSpm (N1-ethyl-N11-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N1-ethyl-N11-[(cycloheptyl)methy]-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity respectively. The level of activity detected correlated with the presence of SSAT and SSAT–EGFP proteins, the latter localizing both in the cytoplasm and nucleus. RT–PCR (reverse transcription–PCR) results suggested that the analogue-affected regulation of SSAT–EGFP expression occurred, mainly, after transcription. In wild-type cells, DENSpm increased the amount of SSAT mRNA, and both DENSpm and CHENSpm affected splicing of the SSAT pre-mRNA. Depleted intracellular spermidine and spermine levels inversely correlated with detected SSAT activity. Interestingly, the analogues also reduced polyamine levels in the SSAT-deficient cells expressing the EGFP control. The results from the present study show that the distinct SSAT regulation by different analogues involves regulatory actions at multiple levels, and that the spermine analogues, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.

scholarly journals Motility and adhesion through type IV pili in Gram-positive bacteria

2016 ◽  
Vol 44 (6) ◽  
pp. 1659-1666 ◽  
Author(s):  
Kurt H. Piepenbrink ◽  
Eric J. Sundberg
Keyword(s):  
Bacterial Species ◽  
Cellular Adhesion ◽  
Type Iv Pili ◽  
Twitching Motility ◽  
Gram Positive ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
Type Iv ◽  
Current State ◽  
Highly Hydrophobic

Type IV pili are hair-like bacterial surface appendages that play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, and horizontal gene transfer. These extracellular fibers are composed exclusively or primarily of many copies of one or more pilin proteins, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. Type IV pili have been characterized extensively in Gram-negative bacteria, and recent advances in high-throughput genomic sequencing have revealed that they are also widespread in Gram-positive bacteria. Here, we review the current state of knowledge of type IV pilus systems in Gram-positive bacterial species and discuss them in the broader context of eubacterial type IV pili.

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