Biosynthetic specificity of the rhamnosyltransferase gene of Mycobacterium avium serovar 2 as determined by allelic exchange mutagenesis

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3193-3202 ◽  
Author(s):  
Joel N. Maslow ◽  
Vida R. Irani ◽  
Sun-Hwa Lee ◽  
Torsten M. Eckstein ◽  
Julia M. Inamine ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Multicopy Plasmid ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Counter Selection ◽  
Alditol Acetate ◽  
In Trans ◽  
Green Fluorescent

In prior studies, through recombinant expression in Mycobacterium smegmatis, the rtfA gene of Mycobacterium avium was shown to encode a rhamnosyltransferase that catalyses the addition of rhamnose (Rha) to the 6-deoxytalose of serovar 2-specific glycopeptidolipid (GPL). Whether RtfA also catalyses the transfer of Rha to the alaninol of the lipopeptide core is unknown. An isogenic rtfA mutant of M. avium serovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained the katG gene of Mycobacterium bovis and the gene encoding green fluorescent protein (gfp). Overexpression of KatG in M. avium resulted in increased susceptibility to isoniazid, thus providing counter-selection by enriching for clones that had lost plasmid DNA. Plasmid loss was confirmed by screening for gfp-negative clones to select putative allelic exchange mutants. Two exchange mutants were created, confirmed by Southern hybridization, and demonstrated loss of serovar 2-specific GPL by thin-layer chromatography (TLC). Gas chromatography of alditol acetate derivatives revealed the loss of Rha and the terminal 2,3-O-Me-fucose and preservation of 3-O-Me-Rha and 3,4-O-Me-Rha substituents at the terminal alaninol of the lipopeptide core. Complementation of rtfA in trans through an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724. This result shows that rtfA encodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange for M. avium as a tool for future genetic studies involving this species.

scholarly journals Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis, and Generation of Mutants with Deletions at the pknG, relA, and lsr2 Loci

2008 ◽  
Vol 74 (6) ◽  
pp. 1687-1695 ◽  
Author(s):  
Kun Taek Park ◽  
John L. Dahl ◽  
John P. Bannantine ◽  
Raúl G. Barletta ◽  
Jongsam Ahn ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Fluorescent Protein ◽  
Virulent Strain ◽  
Green Fluorescent Protein Gene ◽  
Effective Vaccine ◽  
Allelic Exchange ◽  
Functional Studies ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Green Fluorescent ◽  
Transduction Frequency

ABSTRACT Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 × 10−7 to 2.9 × 10−7. Three genes were selected for mutagenesis: pknG and relA, which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis, and lsr2, a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis (M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.

Recruitment of the LIM protein hic-5 to focal contacts of human platelets

1998 ◽  
Vol 111 (15) ◽  
pp. 2181-2188 ◽  
Author(s):  
J. Hagmann ◽  
M. Grob ◽  
A. Welman ◽  
G. van Willigen ◽  
M.M. Burger
Keyword(s):  
Fluorescent Protein ◽  
Human Platelets ◽  
Gene Encoding ◽  
Chain Reactions ◽  
Amino Terminal ◽  
Culture Cells ◽  
Lim Domains ◽  
Focal Contacts ◽  
Green Fluorescent ◽  
Carboxy Terminal

Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxy-terminal LIM domains and LD motifs in the amino-terminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.

scholarly journals Feature of Adhesins Produced by Human Clinical Isolates of Mycobacterium intracellulare, Mycobacterium intracellulare subsp. chimaera and Closely Related Species

2020 ◽  
Vol 8 (8) ◽  
pp. 1154
Author(s):  
Louise H. Lefrancois ◽  
Thierry Cochard ◽  
Maxime Branger ◽  
Olivia Peuchant ◽  
Cyril Conde ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Related Species ◽  
Fluorescent Protein ◽  
Severe Disease ◽  
A549 Cells ◽  
Clinical Isolates ◽  
Heparin Binding ◽  
Closely Related Species ◽  
Mycobacterium Intracellulare ◽  
Green Fluorescent

The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.

scholarly journals Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Catherine P. Chia ◽  
Noriko Inoguchi ◽  
Kyle C. Varon ◽  
Bradley M. Bartholomai ◽  
Hideaki Moriyama
Keyword(s):  
Dictyostelium Discoideum ◽  
Active Sites ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Unicellular Eukaryote ◽  
Gene Encoding ◽  
Conserved Motifs ◽  
N Terminus ◽  
Key Enzyme ◽  
Green Fluorescent

Abstract Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

scholarly journals The hetF Gene Product Is Essential to Heterocyst Differentiation and Affects HetR Function in the Cyanobacterium Nostoc punctiforme

2001 ◽  
Vol 183 (8) ◽  
pp. 2654-2661 ◽  
Author(s):  
Francis C. Y. Wong ◽  
John C. Meeks
Keyword(s):  
Fluorescent Protein ◽  
Filamentous Cyanobacterium ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Nostoc Punctiforme ◽  
Combined Nitrogen ◽  
In The Wild ◽  
Step Down ◽  
Green Fluorescent ◽  
Heterocyst Development

ABSTRACT A novel gene, hetF, was identified as essential for heterocyst development in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133. In the absence of combined nitrogen, hetF mutants were unable to differentiate heterocysts, whereas extra copies of hetF intrans induced the formation of clusters of heterocysts. Sequences hybridizing to a hetF probe were detected only in heterocyst-forming cyanobacteria. The inactivation and multicopy effects of hetF were similar to those of hetR, which encodes a self-degrading serine protease thought to be a central regulator of heterocyst development. Increased transcription ofhetR begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in heterocysts. In the hetFmutant, this increase in hetR transcription was delayed, and a hetR promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of hetR occurred in all cells rather than only in developing heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the hetF mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of hetR and localization of the HetR protein in differentiating heterocysts.

scholarly journals Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

2006 ◽  
Vol 72 (9) ◽  
pp. 6088-6093 ◽  
Author(s):  
Helen Rawsthorne ◽  
Kevin N. Turner ◽  
David A. Mills
Keyword(s):  
Fluorescent Protein ◽  
Group Ii Intron ◽  
Multicopy Plasmid ◽  
Bacterial Genomes ◽  
Gfp Expression ◽  
High Frequencies ◽  
Multicopy Integration ◽  
Group Ii ◽  
Green Fluorescent ◽  
High Level

ABSTRACT Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

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.

Comparative production of human interleukin-2 fused with green fluorescent protein in several recombinant expression systems

2005 ◽  
Vol 24 (3) ◽  
pp. 225-233 ◽  
Author(s):  
Hyung Joon Cha ◽  
Hwa Sung Shin ◽  
Hye Jung Lim ◽  
Hye Sook Cho ◽  
Nimish N. Dalal ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Interleukin 2 ◽  
Expression Systems ◽  
Green Fluorescent

scholarly journals Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E

2007 ◽  
Vol 74 (3) ◽  
pp. 653-659 ◽  
Author(s):  
Harry B. Hines ◽  
Alexander D. Kim ◽  
Robert G. Stafford ◽  
Shirin S. Badie ◽  
Ernst E. Brueggeman ◽  
...  
Keyword(s):  
Natural Product ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Sulfhydryl Group ◽  
Cleavage Sites ◽  
Gene Encoding ◽  
Fluorescent Substrate ◽  
C Terminus ◽  
Protease Cleavage Sites ◽  
Green Fluorescent

ABSTRACT The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

scholarly journals Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence

2003 ◽  
Vol 69 (7) ◽  
pp. 4214-4218 ◽  
Author(s):  
J. Reunanen ◽  
P. E. J. Saris
Keyword(s):  
Green Fluorescent Protein ◽  
Lactococcus Lactis ◽  
Culture Supernatant ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Regulatory Proteins ◽  
Protein Fluorescence ◽  
Gene Encoding ◽  
Two Component ◽  
Green Fluorescent

ABSTRACT A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 μg of nisin in cheese, and 1 μg of nisin per ml in salad dressings.

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