scholarly journals A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position, Movement, and Function of Woronin Bodies in Aspergillus oryzae

2014 ◽  
Vol 13 (7) ◽  
pp. 866-877 ◽  
Author(s):  
Pei Han ◽  
Feng Jie Jin ◽  
Jun-ichi Maruyama ◽  
Katsuhiko Kitamoto
Keyword(s):  
Aspergillus Oryzae ◽  
Fluorescent Protein ◽  
Terminal Region ◽  
Middle Region ◽  
Content Type ◽  
Fusion Construct ◽  
Egfp Fusion Protein ◽  
Woronin Body ◽  
Green Fluorescent ◽  
And Function

ABSTRACT The Woronin body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin body tethering protein that contains highly conserved N- and C-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies.

scholarly journals Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

2014 ◽  
Vol 14 (2) ◽  
pp. 182-193 ◽  
Author(s):  
Areti Gkourtsa ◽  
Janny van den Burg ◽  
Karin Strijbis ◽  
Teja Avula ◽  
Sietske Bijvoets ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Genetic Interaction ◽  
Phenotypic Analysis ◽  
Content Type ◽  
Associated Functions ◽  
Increased Sensitivity ◽  
Green Fluorescent ◽  
And Function

ABSTRACT Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro . A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3 Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167 Δ/Δ and rvs161 Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans : the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.

scholarly journals The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation

2012 ◽  
Vol 11 (3) ◽  
pp. 311-323 ◽  
Author(s):  
Yainitza Hernández-Rodríguez ◽  
Susan Hastings ◽  
Michelle Momany
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Germ Tube ◽  
Fluorescent Protein ◽  
Early Growth ◽  
Content Type ◽  
Green Fluorescent ◽  
And Function ◽  
Cytoskeletal Elements ◽  
Germ Tubes

ABSTRACTIn yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungusAspergillus nidulansthere are five septin genes,aspA(an ortholog ofSaccharomyces cerevisiae CDC11),aspB(an ortholog ofS. cerevisiae CDC3),aspC(an ortholog ofS. cerevisiae CDC12),aspD(an ortholog ofS. cerevisiae CDC10), andaspE(found only in filamentous fungi). TheaspBgene was previously reported to be the most highly expressedAspergillus nidulansseptin and to be essential. Using improved gene targeting techniques, we found that deletion ofaspBis not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained inΔaspDand lost inΔaspAandΔaspCstrains. Interestingly, in theΔaspEmutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Nicolas J. Delalez ◽  
Richard M. Berry ◽  
Judith P. Armitage
Keyword(s):  
Copy Number ◽  
Motor Proteins ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Content Type ◽  
Rotation Direction ◽  
Cell Measurement ◽  
Biological Complexes ◽  
And Function

ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, inEscherichia coli. Our results confirm that,in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.IMPORTANCEThe flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

scholarly journals Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

2010 ◽  
Vol 9 (9) ◽  
pp. 1398-1402 ◽  
Author(s):  
Guillermo Aguilar-Osorio ◽  
Patricia A. vanKuyk ◽  
Bernhard Seiboth ◽  
Dirk Blom ◽  
Peter S. Solomon ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Fluorescent Protein ◽  
Northern Analysis ◽  
Expression Data ◽  
Mannitol Dehydrogenase ◽  
Content Type ◽  
Green Fluorescent ◽  
Developmental Differentiation ◽  
Encoding Genes ◽  
Mannitol Cycle

ABSTRACT The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.

scholarly journals Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS256Tempering of WalKR Expression

2013 ◽  
Vol 57 (7) ◽  
pp. 3240-3249 ◽  
Author(s):  
Christopher R. E. McEvoy ◽  
Brian Tsuji ◽  
Wei Gao ◽  
Torsten Seemann ◽  
Jessica L. Porter ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Infection Model ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Phenotype Analysis ◽  
Dosing Regimens ◽  
Mrsa Strains ◽  
Green Fluorescent

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains often arise by mutations in the essential two-component regulatorwalKR; however their impact onwalKRfunction has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus(VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256insertions in thewalKR5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKRgene expression in the IS256mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256insertions had replaced two SigA-likewalKRpromoters with weaker, hybrid promoters. Removal of IS256reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaAbut no change in expression ofsakandsceDin both IS256mutants. Whole-genome sequencing of the two mutants revealed an additional IS256insertion withinagrCfor one mutant, and we confirmed that this mutation abolishedagrfunction. These data provide the first substantial analysis ofwalKRpromoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction inwalKRexpression; however, the mechanisms by which this occurs remain to be determined.

scholarly journals Asc Modulates the Function of NLRC4 in Response to Infection of Macrophages by Legionella pneumophila

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Christopher L. Case ◽  
Craig R. Roy
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Microbial Infection ◽  
Content Type ◽  
Caspase 1 ◽  
Cell Death Pathway ◽  
Dependent Cell ◽  
Green Fluorescent ◽  
In Cells

ABSTRACTNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 byLegionella pneumophilaoccurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages byL. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta followingL. pneumophilainfection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.IMPORTANCECaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacteriumLegionella pneumophilainduces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

scholarly journals Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Shireen M. Kotay ◽  
Rodney M. Donlan ◽  
Christine Ganim ◽  
Katie Barry ◽  
Bryan E. Christensen ◽  
...  
Keyword(s):  
Patient Care ◽  
Sampling Methods ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Air Sampling ◽  
Multidrug Resistant ◽  
Hand Washing ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACT An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods. IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

Sign in / Sign up

Export Citation Format

Share Document