scholarly journals Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae

2016 ◽  
Vol 198 (17) ◽  
pp. 2352-2359 ◽  
Author(s):  
Yoshito Kawakita ◽  
Miki Kinoshita ◽  
Yukio Furukawa ◽  
Isil Tulum ◽  
Yuhei O. Tahara ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Coiled Coil ◽  
Core Structure ◽  
Gliding Motility ◽  
Pathogenic Bacterium ◽  
Predicted Amino Acid Sequence ◽  
Content Type ◽  
Gliding Mechanism

ABSTRACTMycoplasma pneumoniaeis a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed.IMPORTANCEHuman mycoplasma pneumonia is caused by a pathogenic bacterium,Mycoplasma pneumoniae. This tiny, 2-μm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma “gliding motility” is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism.

scholarly journals Proteins P24 and P41 Function in the Regulation of Terminal-Organelle Development and Gliding Motility in Mycoplasma pneumoniae

2007 ◽  
Vol 189 (20) ◽  
pp. 7442-7449 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Duncan C. Krause
Keyword(s):  
Cell Division ◽  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Gliding Motility ◽  
Atypical Pneumonia ◽  
Reading Frame ◽  
Spatial Positioning ◽  
Time Lapse Imaging ◽  
Gliding Mechanism

ABSTRACT Mycoplasma pneumoniae is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal-organelle development during cell division. Disruption of M. pneumoniae open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminal-organelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a P28− mutant established that the MPN311 mutant phenotype was not a function of loss of P28.

scholarly journals Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism ofMycoplasma pneumoniae

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Akihiro Kawamoto ◽  
Lisa Matsuo ◽  
Takayuki Kato ◽  
Hiroki Yamamoto ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Conformational Changes ◽  
Hexagonal Lattice ◽  
Gliding Motility ◽  
Influenza Viruses ◽  
Pathogenic Bacterium ◽  
Dimensional Structure ◽  
Internal Core ◽  
Electron Cryotomography ◽  
Gliding Mechanism

ABSTRACTMycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding.IMPORTANCEHuman mycoplasma pneumonia, epidemic all over the world in recent years, is caused by a pathogenic bacterium,Mycoplasma pneumoniae. This tiny bacterium, about 2 µm in cell body length, glides on the surface of the human trachea to infect the host by binding to sialylated oligosaccharides, which are also the binding targets of influenza viruses. The mechanism of mycoplasmal gliding motility is not related to any other well-studied motility systems, such as bacterial flagella and cytoplasmic motor proteins. Here, we visualized the attachment organelle, a cellular architecture for gliding, three dimensionally by using electron cryotomography and other conventional methods. A possible gliding mechanism has been suggested based on the architectural images.

scholarly journals Mutant Analysis Reveals a Specific Requirement for Protein P30 in Mycoplasma pneumoniae Gliding Motility

2005 ◽  
Vol 187 (18) ◽  
pp. 6281-6289 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Jarrat L. Jordan ◽  
Duncan C. Krause
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Gliding Motility ◽  
Host Cells ◽  
Cellular Polarity ◽  
Wild Type ◽  
Developmental Defects ◽  
C Terminus ◽  
Quantitative Examination

ABSTRACT The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.

scholarly journals Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Celina Frank ◽  
Dieter Jendrossek
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Eukaryotic Cells ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Polyphosphate Granules

ABSTRACT Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971–29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. IMPORTANCE The uptake of alphaproteobacterium-like cells by ancestors of eukaryotic cells and subsequent conversion of these alphaproteobacterium-like cells to mitochondria are thought to be key steps in the evolution of the first eukaryotic cells. The identification of acidocalcisomes in two alphaproteobacterial species some years ago and the presence of homologs of the vacuolar proton-translocating pyrophosphatase HppA, a marker protein of the acidocalcisome membrane in eukaryotes, in virtually all species within the alphaproteobacteria suggest that eukaryotic acidocalcisomes might also originate from related structures in ancestors of alphaproteobacterial species. Accordingly, alphaproteobacterial acidocalcisomes and eukaryotic acidocalcisomes should have similar features. Since hardly any information is available on bacterial acidocalcisomes, this study aimed at the characterization of organelle-like structures in alphaproteobacterial cells, with A. tumefaciens as an example.

scholarly journals Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression inClostridium perfringens

2010 ◽  
Vol 77 (2) ◽  
pp. 471-478 ◽  
Author(s):  
Andrea H. Hartman ◽  
Hualan Liu ◽  
Stephen B. Melville
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Yellow Fluorescent Protein ◽  
Inducible Promoter ◽  
Gliding Motility ◽  
Glucuronidase Activity ◽  
Atpase Gene ◽  
Regulated Gene Expression ◽  
Promoter System

ABSTRACTClostridium perfringensis a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working withC. perfringens, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is anEscherichia coli-C. perfringensshuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of genebgaL(bgaR-PbgaL). To measure transcription at thebgaLpromoter in pKRAH1, theE. colireporter genegusA, encoding β-glucuronidase, was placed downstream of the PbgaLpromoter to make plasmid pAH2. When transformed into three strains ofC. perfringens, pAH2 exhibited lactose-inducible expression.C. perfringensstrain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated genebglR, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express theyfp-pilBgene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility inC. perfringens. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in apilCmutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

scholarly journals Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

2013 ◽  
Vol 79 (7) ◽  
pp. 2218-2224 ◽  
Author(s):  
Jeffrey L. Bose ◽  
Paul D. Fey ◽  
Kenneth W. Bayles
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Exchange System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genetic Tools ◽  
Allelic Exchange ◽  
Green Fluorescent

ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

scholarly journals Fluorescence-Based Bacterial Bioreporter for Specific Detection of Methyl Halide Emissions in the Environment

2013 ◽  
Vol 79 (21) ◽  
pp. 6561-6567 ◽  
Author(s):  
Muhammad Farhan Ul Haque ◽  
Thierry Nadalig ◽  
Françoise Bringel ◽  
Hubert Schaller ◽  
Stéphane Vuilleumier
Keyword(s):  
Fluorescent Protein ◽  
Stratospheric Ozone ◽  
Yellow Fluorescent Protein ◽  
Attractive Alternative ◽  
Detailed Knowledge ◽  
Methyl Halides ◽  
Natural Sources ◽  
Content Type ◽  
Degrading Bacteria ◽  
Methyl Halide

ABSTRACTMethyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH3Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics ofMethylobacterium extorquensCM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase genecmuAfused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced intoM. extorquensCM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plantArabidopsis thalianain particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.

scholarly journals MoTea4-Mediated Polarized Growth Is Essential for Proper Asexual Development and Pathogenesis in Magnaporthe oryzae

2010 ◽  
Vol 9 (7) ◽  
pp. 1029-1038 ◽  
Author(s):  
Rajesh N. Patkar ◽  
Angayarkanni Suresh ◽  
Naweed I. Naqvi
Keyword(s):  
Actin Cytoskeleton ◽  
Magnaporthe Oryzae ◽  
Fluorescent Protein ◽  
Function Analysis ◽  
Coiled Coil ◽  
Polarized Growth ◽  
Wild Type ◽  
Cortical Actin ◽  
Content Type ◽  
Aerial Hyphae

ABSTRACT Polarized growth is essential for cellular development and function and requires coordinated organization of the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly and bipolar growth in fission yeast and directional mycelial growth in Aspergillus. Here, we characterize Tea4 in the rice blast fungus Magnaporthe oryzae (MoTea4). Similar to its orthologs, MoTea4-green fluorescent protein (MoTea4-GFP) showed punctate distribution confined to growth zones, particularly in the mycelial tips, aerial hyphae, conidiophores, conidia, and infection structures (appressoria) in Magnaporthe. MoTea4 was dispensable for vegetative growth in Magnaporthe. However, loss of MoTea4 led to a zigzag morphology in the aerial hyphae and a huge reduction in conidiation. The majority of the tea4Δ conidia were two celled, as opposed to the tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coil domains of MoTea4 are necessary for proper conidiation in Magnaporthe. The tea4Δ conidia failed to produce proper appressoria and consequently failed to infect the host plants. The tea4Δ conidia and germ tubes showed disorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to the wild-type conidia, the tea4Δ conidia showed aberrant germination, poor cytoplasmic streaming, and persistent accumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment of germinating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorial development in Magnaporthe. We show that MoTea4 plays an important role in organizing the F-actin cytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and pathogenic development in Magnaporthe.

scholarly journals Use of Degradation Tags To Control Protein Levels in the Cyanobacterium Synechocystis sp. Strain PCC 6803

2013 ◽  
Vol 79 (8) ◽  
pp. 2833-2835 ◽  
Author(s):  
Brian P. Landry ◽  
Jana Stöckel ◽  
Himadri B. Pakrasi
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Terminal Protein ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Protein Levels ◽  
Steady State Fluorescence ◽  
Tunable Range ◽  
Control Protein ◽  
Synechocystis Sp

ABSTRACTWe generated a collection ofssrA-based C-terminal protein degradation tags with different degradation strengths. The steady-state fluorescence levels of different enhanced yellow fluorescent protein (eYFP) tag variants in aSynechocystissp. indicated a tunable range from 1% to 50% of untagged eYFP.

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