scholarly journals Mycobacterium avium Genes Associated with the Ability To Form a Biofilm

2006 ◽  
Vol 72 (1) ◽  
pp. 819-825 ◽  
Author(s):  
Yoshitaka Yamazaki ◽  
Lia Danelishvili ◽  
Martin Wu ◽  
Molly MacNab ◽  
Luiz E. Bermudez
Keyword(s):  
Mycobacterium Avium ◽  
Fatty Acid Biosynthesis ◽  
Fluorescent Protein ◽  
Tca Cycle ◽  
Promoter Library ◽  
The Tca Cycle ◽  
Important Pathway ◽  
Mutant Bank ◽  
Green Fluorescent ◽  
Mycobacterial Strain

ABSTRACT Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression ≥2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm.

pckA-deficient Mycobacterium bovis BCG shows attenuated virulence in mice and in macrophages

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1829-1835 ◽  
Author(s):  
Keyi Liu ◽  
Jinzhi Yu ◽  
David G. Russell
Keyword(s):  
Green Fluorescent Protein ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Green Fluorescent Protein Expression ◽  
Fluorescent Protein ◽  
Bacterial Load ◽  
Tca Cycle ◽  
Wild Type ◽  
The Tca Cycle ◽  
Encoding Gene ◽  
Green Fluorescent

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation and phosphorylation of oxaloacetate (OAA) to form phosphoenolpyruvate (PEP). In this study, the regulation of the PEPCK-encoding gene pckA was examined through the evaluation of green fluorescent protein expression driven by the pckA promoter. The results showed that pckA was upregulated by acetate or palmitate but downregulated by glucose. Deletion of the pckA gene of Mycobacterium bovis BCG led to a reduction in the capacity of the bacteria to infect and survive in macrophages. Moreover, mice infected with ΔpckA BCG were able to reduce the bacterial load much more effectively than mice infected with the parental wild-type bacteria. This attenuated virulence was reflected in the degree of pathology, where granuloma formation was diminished both in numbers and degree. The data indicate that PEPCK activity is important during establishment of infection. Whether its role is in the gluconeogenic pathway for carbohydrate formation or in the conversion of PEP to OAA to maintain the TCA cycle remains to be determined.

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 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.

scholarly journals Systematic identification of a panel of strong promoter regions from Listeria monocytogenes for fine-tuning gene expression

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Qianyu Ji ◽  
Junfei Ma ◽  
Shuying Wang ◽  
Qing Liu
Keyword(s):  
Listeria Monocytogenes ◽  
Fluorescent Protein ◽  
Fine Tuning ◽  
Egfp Expression ◽  
Promoter Regions ◽  
Strong Promoter ◽  
Yield Increase ◽  
Promoter Library ◽  
Green Fluorescent ◽  
Systematic Identification

Abstract Background Attenuated Listeria monocytogenes (Lm) has been widely used as a vaccine vector in the prevention and treatment of pathogen infection and tumor diseases. In addition, previous studies have proved that the attenuated Lm can protect zebrafish from Vibrio infections, indicating that the attenuated Lm has a good application prospect in the field of aquatic vaccines. However, the limitation mainly lies in the lack of a set of well-characterized natural promoters for the expression of target antigens in attenuated Lm. Results In our study, candidate strong promoters were identified through RNA-seq analysis, and characterized in Lm through enhanced green fluorescent protein (EGFP). Nine native promoters that showed stronger activities than that of the known strong promoter P36 under two tested temperatures (28 and 37 °C) were selected from the set, and P29 with the highest activity was 24-fold greater than P36. Furthermore, we demonstrated that P29 could initiate EGFP expression in ZF4 cells and zebrafish embryos. Conclusions This well-characterized promoter library can be used to fine-tune the expression of different proteins in Lm. The availability of a well-characterized promoter toolbox of Lm is essential for the analysis of yield increase for biotechnology applications.

scholarly journals Genomic Approach to Identifying the Putative Target of and Mechanisms of Resistance to Mefloquine in Mycobacteria

2005 ◽  
Vol 49 (9) ◽  
pp. 3707-3714 ◽  
Author(s):  
Lia Danelishvili ◽  
Martin Wu ◽  
Lowell S. Young ◽  
Luiz E. Bermudez
Keyword(s):  
Fluorescent Protein ◽  
Genomic Library ◽  
Antimycobacterial Activity ◽  
Acid Synthesis ◽  
Function Evaluation ◽  
Promoter Library ◽  
Cellular Replication ◽  
Green Fluorescent

ABSTRACT The emergence of mycobacterial resistance to multiple antimicrobials emphasizes the need for new compounds. The antimycobacterial activity of mefloquine has been recently described. Mycobacterium avium, Mycobacterium smegmatis, and Mycobacterium tuberculosis are susceptible to mefloquine in vitro, and activity was evidenced in vivo against M. avium. Attempts to obtain resistant mutants by both in vitro and in vivo selection have failed. To identify mycobacterial genes regulated in response to mefloquine, we employed DNA microarray and green fluorescent protein (GFP) promoter library techniques. Following mefloquine treatment, RNA was harvested from M. tuberculosis H37Rv, labeled with 32P, and hybridized against a DNA array. Exposure to 4× MIC resulted in a significant stress response, while exposure to a subinhibitory concentration of mefloquine triggered the expression of genes coding for enzymes involved in fatty acid synthesis, the metabolic pathway, and transport across the membrane and other proteins of unknown function. Evaluation of gene expression using an M. avium GFP promoter library exposed to subinhibitory concentrations of mefloquine revealed more than threefold upregulation of 24 genes. To complement the microarray results, we constructed an M. avium genomic library under the control of a strong sigma-70 (G13) promoter in M. smegmatis. Resistant clones were selected in 32 μg/ml of mefloquine (wild-type M. avium, M. tuberculosis, and M. smegmatis are inhibited by 8 μg/ml), and the M. avium genes associated with M. smegmatis resistant to mefloquine were sequenced. Two groups of genes were identified: one affecting membrane transport and one gene that apparently is involved in regulation of cellular replication.

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 Phenotypes of Fission Yeast Defective in Ubiquinone Production Due to Disruption of the Gene for p-Hydroxybenzoate Polyprenyl Diphosphate Transferase

2000 ◽  
Vol 182 (24) ◽  
pp. 6933-6939 ◽  
Author(s):  
Naonori Uchida ◽  
Kengo Suzuki ◽  
Ryoichi Saiki ◽  
Tomohiro Kainou ◽  
Katsunori Tanaka ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Minimal Medium ◽  
Fluorescent Protein ◽  
Sulfur Metabolism ◽  
Transfer System ◽  
Protein Fusion ◽  
Electron Transfer System ◽  
Important Pathway ◽  
Green Fluorescent ◽  
Ubiquinone 10

ABSTRACT Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps.p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not grow on minimal medium supplemented with glucose. Expression ofCOQ2 from Saccharomyces cerevisiae in the defective S. pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 andppt1 are functional homologs. Theppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and α-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that theppt1-deficient strain is sensitive to H2O2 and Cu2+. Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H2S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S. pombe. Thus, analysis of the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.

Liquid-liquid extraction of succinate using a dicopper cryptate

2020 ◽  
Author(s):  
Riccardo Mobili ◽  
Sonia La Cognata ◽  
Francesca Merlo ◽  
Andrea Speltini ◽  
Massimo Boiocchi ◽  
...  
Keyword(s):  
Aqueous Phase ◽  
Visible Spectrum ◽  
Tca Cycle ◽  
Residual Concentration ◽  
Waste Products ◽  
Liquid Liquid Extraction ◽  
The Tca Cycle ◽  
Quantitative Extraction ◽  
Uv Visible

<div> <p>The extraction of the succinate dianion from a neutral aqueous solution into dichloromethane is obtained using a lipophilic cage-like dicopper(II) complex as the extractant. The quantitative extraction exploits the high affinity of the succinate anion for the cavity of the azacryptate. The anion is effectively transferred from the aqueous phase, buffered at pH 7 with HEPES, into dichloromethane. A 1:1 extractant:anion adduct is obtained. Extraction can be easily monitored by following changes in the UV-visible spectrum of the dicopper complex in dichloromethane, and by measuring the residual concentration of succinate in the aqueous phase by HPLC−UV. Considering i) the relevance of polycarboxylates in biochemistry, as e.g. normal intermediates of the TCA cycle, ii) the relevance of dicarboxylates in the environmental field, as e.g. waste products of industrial processes, and iii) the recently discovered role of succinate and other dicarboxylates in pathophysiological processes including cancer, our results open new perspectives for research in all contexts where selective recognition, trapping and extraction of polycarboxylates is required. </p> </div>

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

Sign in / Sign up

Export Citation Format

Share Document