DOCSLIB.ORG

Production of Omega-3 Fatty Acids in Rhodococcus Opacus PD630

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Production of Omega-3 Fatty Acids in Rhodococcus Opacus PD630 Production of omega-3 fatty acids in Rhodococcus opacus PD630 Borom Blakie A thesis submitted for the degree of Master of Science at the University of Otago, Dunedin, New Zealand. April 2015 Abstract Omega-3 fatty acids (ω-3 FA) are essential to human health. However, the primary source of ω-3 FA are declining fish stocks. A more sustainable alternative is the cultivation of marine bacteria, however, the yields to date are less than favourable compared with those of fish. Oleaginous bacteria, when genetically engineered with the 16-27 kb pfaA-E gene cluster that encodes ω-3 FA synthesis, may potentially overcome this yield deficiency. One such species is Rhodococcus opacus PD630, which is able to accumulate up to 87% of its cell dry weight as fatty acids. This fatty acid accumulation makes R. opacus PD630 highly desirable as a biotechnological producer strain. However, before ω-3 FA may be produced by genetically engineered R. opacus PD630, work must be undertaken to develop expression vectors carrying the pfaA-E gene cluster. New transformation protocols, capable of handling an expression vector of 30-35 kb, twice the size of the largest reported to have been transformed into a Rhodococcus spp. to date, must also be developed. This thesis describes the development of new protocols, based on electroporation and sonoporation, for the transformation of R. opacus PD630. An electroporation based protocol was successfully developed, with an efficiency of transformation of 5.317 x104 R. opacus PD630 transformants per µg of DNA using the E. coli -Rhodococcus spp. shuttle vector pBS305, compared with 81.15 for the most efficient method previously reported, and 7.158 x106 for Escherichia coli DH5α. A novel method for the generation and recovery of R. opacus PD630 protoplasts for transformation was successfully developed. Protoplast electroporation was found to be ineffective, though protoplasts were able to be used for the extraction of plasmid DNA in the identification of putative transformants. Sonoporation was found to be an ineffective means of transformation, both in E. coli DH5α and R. opacus PD630. ii The development of rhodococcal vectors carrying pfa genes was attempted with LT- PCR from Shewanella baltica chromosomal DNA, and both restriction cloning and PCR of the transgenic expression vector pDHA4. LT-PCR was found to be ineffective at amplifying the 16-20 kb pfa gene cluster of S. baltica, even after optimisation to minimise non-target binding. Restriction cloning of pDHA4 was successful in obtaining the pfa gene cluster, though at a yield insufficient for further use in cloning. PCR amplification of the gene cluster proved to be successful, however, an absence of selective pressure for the gene cluster when inserted into transgenic vectors, and a failure to recover any transformants in E. coli DH5α, meant that this approach was abandoned. PCR amplification of the Rhodococcus spp. gene expression and plasmid maintenance genes from the E. coli -Rhodococcus spp. shuttle vector pTip QT1 was successful. These genes were able to be inserted into pDHA4, although no transformants of E. coli DH5α carrying the construct were able to be recovered. De novo DNA synthesis is a possibility for the construction of the desired transgenic vector. To determine if pDHA4, a purportedly E. coli-only plasmid, could be maintained by R. opacus PD630, a derivative lacking the pfa gene cluster, pDHA4VO, was constructed. Expression of the chloramphenicol resistance gene of pDHA4VO in a confirmed R. opacus PD630 transformant, suggested that R. opacus PD630 was capable of the heterologous expression of DNA from pDHA4. This is the first reported incidence of heterologous expression in R. opacus PD630 without the use of an E. coli -Rhodococcus spp. shuttle vector. Transformation was attempted by electroporation with pDHA4, although no confirmed R. opacus PD630 transformants were recovered. Differences in the GC content between pDHA4 and pDHA4VO are thought to be a contributing factor, and may be indicative of the range of GC contents of DNA which R. opacus PD630 is capable of expressing heterologously. iii Acknowledgements First and foremost, my thanks to my supervisor, Dr. Robin Simmonds, for providing me with the opportunity to undertake this research. His patience and guidance during the course of my study have been invaluable to my development as a scientist. A special thanks to Dr. Felicity McLeod, whose patience and assistance as a post-doctoral fellow were very welcome whilst I settled into research. Thanks also to the other members of the lab: Katie, Chris N. and Chris R., for their advice and commiseration on working with Rhodococcus. I am grateful for the friendships I’ve made in the write-up room, and the support and encouragement that they have fostered. Anthony, Rowan, Maggie, Jonny, Susan, Matt, Manmeet, Sainur, Hoan and Rachel, thanks for the company and the adventures. I extend the same gratitude to the others who have come and gone in my time, and the wider department, listing the names of whom would most probably occupy a document similar in length to this thesis. Thanks to my friends from school, especially Matt, Kurt and Sehan, for encouraging me to undertake an MSc in the first instance, and for supporting me during the course of it. However, I’ll reserve judgment on whether or not to thank the three of you for encouraging me to look into undertaking a PhD. Thanks also to the teachers at Otago Boys’ High School, who taught me the value discipline and hard work, and fostered my burgeoning interest in the biological sciences. Finally, thanks to my parents for their support and assistance in getting this far, and to my siblings, their spouses and children, for all the encouragement and kindness they have shown me. iv Table of Contents Abstract ii Acknowledgements iv List of Abbreviations ix List of Figures xi List of Tables xiii 1. Introduction 1 __1.1. Human health and omega-3 fatty acids 1 __1.2. Sources of ω-3 FA 1 __1.3. Genetic engineering using pfa genes 6 __1.4. Oleaginous bacteria 8 __1.5. PUFA production in R. opacus 9 _____ 1.5.1. Principle 9 _____ 1.5.2. Transformability 10 __1.6. Wider effects of sourcing ω-3 FA from R. opacus 12 __1.7. Hypothesis and study objectives 13 2. Materials and methods 14 __2.1. Stock solutions 14 _____ 2.1.1. General reagents 15 _____ 2.1.2. Antibiotic stock solutions 16 _____ 2.1.3. Media 16 _____ _____2.1.3.1. Growth media 16 _____ __________ _2.1.3.1.1. Commercially sourced media 17 _____ __________ _2.1.3.1.2. Manually prepared media 17 _____ _____2.1.3.2. Transformation media 17 _____ _____2.1.3.3. Freezer stock medium 18 _____ 2.1.4. Protoplasting solutions 18 _____ 2.1.5. Microscope stains 19 _____ 2.1.6. Alkaline lysis reagents 20 _____ 2.1.7. Plasmid preparation buffers 20 _____ 2.1.8. Gel electrophoresis solutions 21 _____ _____2.1.8.1. Gel electrophoresis buffers 21 _____ _____2.1.8.2. Agarose electrophoresis gels 21 _____ _____2.1.8.3. Loading dye 22 __2.2. Bacterial strains, culture conditions and freezer stocks 22 _____ 2.2.1. Bacterial strains and culture conditions 22 _____ 2.2.2. Freezer stocks 25 __2.3. Plasmids 25 __2.4. Microscopy 28 _____ 2.4.1. Light microscopy 28 _____ 2.4.2. Electron microscopy 30 _____ _____2.4.2.1. Transmission electron microscopy 30 _____ _____2.4.2.2. Cryo-transmission electron microscopy 31 v __2.5. Serial dilutions and plating 31 __2.6. Growth and inhibition 32 _____ 2.6.1. Growth curves 32 _____ 2.6.2. R. opacus PD630 morphology during growth 33 _____ 2.6.3. Growth inhibition and function of selective antibiotics 33 _____ _____2.6.3.1. Growth inhibition 33 _____ _____2.6.3.2. Susceptibility to selective antibiotics 35 _____ 2.6.4. R. opacus protoplasts, their viability, and their morphology 35 _____ _____2.6.4.1. Protoplasting 35 _____ ___________ 2.6.4.1.1. Assaf and Dick method 35 _____ ___________ 2.6.4.1.2. Study-generated method 37 _____ _____2.6.4.2. Protoplast viability and morphology 38 __2.7. DNA extractions 38 _____ 2.7.1. E. coli 39 _____ _____2.7.1.1. Commercial DNA extraction kits 39 _____ _____2.7.1.2. Untergasser’s alkaline lysis method 39 _____ 2.7.2. R. opacus PD630 41 _____ _____2.7.2.1. DNeasy pre-treatment for gram positive bacteria 41 _____ _____2.7.2.2. Sambrook and Russell alkaline lysis miniprep 41 _____ 2.7.3. S. baltica 42 _____ 2.7.4. Gel extraction of DNA 43 __2.8. Transformation 43 _____ 2.8.1. E. coli 45 _____ _____2.8.1.1. Preparation of electrocompetent cells and electroporation 45 _________________conditions _____ _____2.8.1.2. Sonoporation 45 _____ 2.8.2. R. opacus 46 _____ _____2.8.2.1. Electrocompetent cell preparation and electroporation - 46 _________________general methods _____ ___________ 2.8.2.1.1. Arenskötter method 46 _____ ___________ 2.8.2.1.2. Desomer method 47 _____ ___________ 2.8.2.1.3. Kalscheuer method 47 _____ ___________ 2.8.2.1.4. Study-generated methods - vegetative cells 47 _____ ___________ ________2.8.2.1.4.1 Optimisation of vegetative cell _____ 48 ___________ ______________________ electroporation _____ ___________ 2.8.2.1.5. Study-generated methods - protoplasts 48 _____ _____2.8.2.2. Sonoporation 48 _____ ___________ 2.8.2.2.1. Optimisation of sonoporation 49 _____ 2.8.3. Calculation of killing rates and efficiency of transformation 49 __2.9.
Load more

Recommended publications
  • Responses to Ecopollutants and Pathogenization Risks of Saprotrophic Rhodococcus Species
    pathogens Review Responses to Ecopollutants and Pathogenization Risks of Saprotrophic Rhodococcus Species Irina B. Ivshina 1,2,*, Maria S. Kuyukina 1,2 , Anastasiia V. Krivoruchko 1,2 and Elena A. Tyumina 1,2 1 Perm Federal Research Center UB RAS, Institute of Ecology and Genetics of Microorganisms UB RAS, 13 Golev Str., 614081 Perm, Russia; [email protected] (M.S.K.); [email protected] (A.V.K.); [email protected] (E.A.T.) 2 Department of Microbiology and Immunology, Perm State University, 15 Bukirev Str., 614990 Perm, Russia * Correspondence: [email protected]; Tel.: +7-342-280-8114 Abstract: Under conditions of increasing environmental pollution, true saprophytes are capable of changing their survival strategies and demonstrating certain pathogenicity factors. Actinobacteria of the genus Rhodococcus, typical soil and aquatic biotope inhabitants, are characterized by high ecological plasticity and a wide range of oxidized organic substrates, including hydrocarbons and their derivatives. Their cell adaptations, such as the ability of adhering and colonizing surfaces, a complex life cycle, formation of resting cells and capsule-like structures, diauxotrophy, and a rigid cell wall, developed against the negative effects of anthropogenic pollutants are discussed and the risks of possible pathogenization of free-living saprotrophic Rhodococcus species are proposed. Due to universal adaptation features, Rhodococcus species are among the candidates, if further anthropogenic pressure increases, to move into the group of potentially pathogenic organisms with “unprofessional” parasitism, and to join an expanding list of infectious agents as facultative or occasional parasites. Citation: Ivshina, I.B.; Kuyukina, Keywords: actinobacteria; Rhodococcus; pathogenicity factors; adhesion; autoaggregation; colonization; M.S.; Krivoruchko, A.V.; Tyumina, defense against phagocytosis; adaptive strategies E.A.
    [Show full text]
  • The Rhodococcus Equi Virulence Protein Vapa Disrupts Endolysosome Function and Stimulates Lysosome Biogenesis
    This is a repository copy of The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/104956/ Version: Accepted Version Article: Rofe, Adam P, Davis, Luther J, Whittingham, Jean L et al. (3 more authors) (2016) The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis. Microbiology Open. e00416. ISSN 2045-8827 https://doi.org/10.1002/mbo3.416 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis. Adam P. Rofe1, Luther J. Davis2, Jean L. Whittingham3, Elizabeth C. Latimer- Bowman2, Anthony J. Wilkinson3 and Paul R. Pryor1,4* 1 Department of Biology, Wentworth Way, University of York, York. YO10 5DD. United Kingdom; 2 Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Hills Rd, Cambridge, CB2 0XY; 3 Structural Biology Laboratory, Department of Chemistry, University of York York.
    [Show full text]
  • Impact of Diesel and Biodiesel Contamination on Soil Microbial Community Activity and Structure Eduardo K
    www.nature.com/scientificreports OPEN Impact of diesel and biodiesel contamination on soil microbial community activity and structure Eduardo K. Mitter1*, James J. Germida2 & J. Renato de Freitas2 Soil contamination as a result of oil spills is a serious issue due to the global demand for diesel fuel. As an alternative to diesel, biodiesel has been introduced based on its high degradability rates and potential for reducing of greenhouse gases emissions. This study assessed the impacts diesel and biodiesel contamination on soil microbial community activity and structure. Our results suggest higher microbial activity in biodiesel contaminated soils and analysis of PLFA profles confrmed shifts in microbial community structure in response to contamination. High-throughput 16S rRNA amplicon sequencing also revealed a lower bacterial richness and diversity in contaminated soils when compared to control samples, supporting evidence of the detrimental efects of hydrocarbons on soil microbiota. Control samples comprised mostly of Actinobacteria, whereas Proteobacteria were predominantly observed in diesel and biodiesel contaminated soils. At genus level, diesel and biodiesel amendments highly selected for Rhodococcus and Pseudomonas spp., respectively. Moreover, predicted functional profles based on hydrocarbon-degrading enzymes revealed signifcant diferences between contaminated soils mostly due to the chemical composition of diesel and biodiesel fuel. Here, we also identifed that Burkholderiaceae, Novosphingobium, Anaeromyxobacter, Pseudomonas and Rhodococcus were the main bacterial taxa contributing to these enzymes. Together, this study supports the evidence of diesel/biodiesel adverse efects in soil microbial community structure and highlights microbial taxa that could be further investigated for their biodegradation potential. Petroleum derived diesel fuel is a major source of energy throughout the world and one of the most widespread soil contaminants1,2.
    [Show full text]
  • Evolutionary Transitions Between Beneficial and Phytopathogenic
    RESEARCH ARTICLE Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management Elizabeth A Savory1†, Skylar L Fuller1,2†, Alexandra J Weisberg1†, William J Thomas1, Michael I Gordon1, Danielle M Stevens1, Allison L Creason1,2‡, Michael S Belcher1, Maryna Serdani1, Michele S Wiseman1, Niklaus J Gru¨ nwald3, Melodie L Putnam1, Jeff H Chang1,2,4* 1Department of Botany and Plant Pathology, Oregon State University, Corvallis, United States; 2Molecular and Cellular Biology Program, Oregon State University, Corvallis, United States; 3Horticultural Crops Research Laboratory, United States Department of Agriculture and Agricultural Research Service, Corvallis, United States; 4Center for Genome Research, Oregon State University, Corvallis, United States Abstract Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize *For correspondence: the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids [email protected] in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple †These authors contributed lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent equally to this work introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence
    [Show full text]
  • Detection of Rhodococcus Fascians, the Causative Agent of Lily Fasciation in South Korea
    pathogens Article Detection of Rhodococcus fascians, the Causative Agent of Lily Fasciation in South Korea Joon Moh Park 1,†, Jachoon Koo 2,†, Se Won Kang 3, Sung Hee Jo 4 and Jeong Mee Park 4,* 1 Forest Resource Research Division, Jeollabuk-do Forest Environment Research Institute, Jinan 55454, Korea; [email protected] 2 Division of Science Education and Institute of Fusion Science, Jeonbuk National University, Jeonju 54896, Korea; [email protected] 3 Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeonguep 56212, Korea; [email protected] 4 Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-42-860-4346 † These authors contributed equally. Abstract: Rhodococcus fascians is an important pathogen that infects various herbaceous perennials and reduces their economic value. In this study, we examined R. fascians isolates carrying a virulence gene from symptomatic lily plants grown in South Korea. Phylogenetic analysis using the nucleotide sequences of 16S rRNA, vicA, and fasD led to the classification of the isolates into four different strains of R. fascians. Inoculation of Nicotiana benthamiana with these isolates slowed root growth and resulted in symptoms of leafy gall. These findings elucidate the diversification of domestic pathogenic R. fascians and may lead to an accurate causal diagnosis to help reduce economic losses in the bulb market. Citation: Park, J.M.; Koo, J.; Kang, S.W.; Jo, S.H.; Park, J.M. Detection of Keywords: fasciation; fasD; lily; Rhodococcus fascians; vicA Rhodococcus fascians, the Causative Agent of Lily Fasciation in South Korea.
    [Show full text]
  • Rhodococcus As a Versatile Biocatalyst in Organic Synthesis
    International Journal of Molecular Sciences Review Rhodococcus as a Versatile Biocatalyst in Organic Synthesis Hanna Busch, Peter-Leon Hagedoorn and Ulf Hanefeld * Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands; [email protected] (H.B.); [email protected] (P.L.H.) * Correspondence: [email protected] Received: 9 September 2019; Accepted: 24 September 2019; Published: 26 September 2019 Abstract: The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research. Keywords: Rhodococcus; biocatalysis; organic synthesis; enzyme; drugs 1. Introduction The genus Rhodococcus belongs to the phylum Actinobacteria and its members are aerobic, Gram-positive, non-motile bacteria showing high GC-contents [1]. Numerous members have been isolated from copious sources such as soil, groundwater, marine sediments, internal organs of insects, diseased and healthy animals, or plants-to name a few [2].
    [Show full text]
  • Fatty Acids As a Tool to Understand Microbial Diversity and Their Role in Food Webs of Mediterranean Temporary Ponds
    Molecules 2014, 19, 5570-5598; doi:10.3390/molecules19055570 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Fatty Acids as a Tool to Understand Microbial Diversity and Their Role in Food Webs of Mediterranean Temporary Ponds Carla C.C.R. de Carvalho 1,* and Maria-José Caramujo 2,† 1 IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, Lisbon 1049-001, Portugal 2 Centre for Environmental Biology, Faculty of Sciences, Universidade de Lisboa, Campo Grande C2, Lisbon 1749-016, Portugal; E-Mail: [email protected] † Current address: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, Roma 00161, Italy. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +351-218-419-594; Fax: +351-218-419-062. Received: 6 February 2014; in revised form: 27 April 2014 / Accepted: 28 April 2014 / Published: 30 April 2014 Abstract: Temporary Mediterranean ponds are complex ecosystems which support a high diversity of organisms that include heterotrophic microorganisms, algae, crustaceans, amphibians and higher plants, and have the potential to supply food and a resting place to migratory birds. The role of heterotrophs at the base of the food web in providing energy to the higher trophic levels was studied in temporary ponds in Central and Southern Portugal. The relative quantification of the hetero and autotrophic biomass at the base of the food web in each pond was derived from the polar fatty acid (PLFA) composition of seston through the application of the matrix factorization program CHEMTAX that used specific PLFA and their relative proportion as markers for e.g., classes of bacteria, algae and fungi.
    [Show full text]
  • Adaptive Response of Rhodococcus Opacus PWD4 to Salt and Phenolic Stress on the Level of Mycolic Acids Carla C
    de Carvalho et al. AMB Expr (2016) 6:66 DOI 10.1186/s13568-016-0241-9 ORIGINAL ARTICLE Open Access Adaptive response of Rhodococcus opacus PWD4 to salt and phenolic stress on the level of mycolic acids Carla C. C. R. de Carvalho1, Martin A. Fischer2, Sandra Kirsten2, Birgit Würz3, Lukas Y. Wick3 and Hermann J. Heipieper2* Abstract Mycolata form a group of Gram-positive bacteria with unique cell envelope structures that are known for their high tolerance against antibiotics and both aromatic and aliphatic hydrocarbons. An important part of the unique surface structure of the mycolata is the presence of long chain α-alkyl-β-hydroxy fatty acids, the mycolic acids. In order to investigate the adaptive changes in the mycolic acid composition, we investigated the composition of mycolic acids during the response both to osmotic stress caused by NaCl and to 4-chlorophenol in Rhodococcus opacus PWD4. This bacterium was chosen as it is known to adapt to different kinds of stresses. In addition, it is a potential biocatalyst in bioremediation as well as for biotechnological applications. In the present study, cells of R. opacus PWD4, grown in liquid cultures, responded to toxic concentrations of NaCl by increasing the ratio between mycolic acids and mem- brane phospholipid fatty acids (MA/PLFA-ratio). Cells reacted to both NaCl and 4-chlorophenol by decreasing both the average chain length and the unsaturation index of their mycolic acids. These changes in mycolic acid composi- tion correlated with increases in cell surface hydrophobicity and saturation of membrane fatty acids, demonstrating the relation between mycolic acid and phospholipid synthesis and their contribution to cell surface properties of R.
    [Show full text]
  • Phenotypic Characterization of Rhodococcus Equi Biofilm Grown In
    pathogens Article Phenotypic Characterization of Rhodococcus equi Biofilm Grown In Vitro and Inhibiting and Dissolving Activity of Azithromycin/Rifampicin Treatment Elisa Rampacci 1,* , Maria Luisa Marenzoni 1, Stefano Giovagnoli 2 , Fabrizio Passamonti 1 , Mauro Coletti 1 and Donatella Pietrella 3 1 Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy; [email protected] (M.L.M.); [email protected] (F.P.); [email protected] (M.C.) 2 Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy; [email protected] 3 Department of Pharmaceutical Sciences, Biochemical Sciences and Health Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy; [email protected] * Correspondence: [email protected] Received: 8 November 2019; Accepted: 3 December 2019; Published: 4 December 2019 Abstract: Microbial biofilm has been implicated in a wide range of chronic infections. In spite of the fact that Rhodococcus equi is a recognized cause of chronic disease in animals and humans, few studies have focused on the sessile phenotype of R. equi. The aim of this research was to phenotypically characterize the biofilm development of R. equi and its answerability for hypo-responsiveness to macrolides and rifampicin. Biofilm formation is initiated by bacterial adhesion to the surface. In this work, the ability of R. equi to adhere to the surface of human lung epithelial cells was detected by a fluorometric adhesion test performed on 40 clinical isolates. Subsequently, the capability of R. equi to produce biofilm was investigated by colorimetric, fluorescence and scanning electron microscopy analysis, revealing a general slow growth of rhodococcal biofilm and different sessile phenotypes among field isolates, some also including filamented bacteria.
    [Show full text]
  • Rhodococcus Opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass
    Washington University in St. Louis Washington University Open Scholarship Engineering and Applied Science Theses & Dissertations McKelvey School of Engineering Winter 12-15-2019 Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass Drew Michael Delorenzo Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/eng_etds Part of the Biomedical Engineering and Bioengineering Commons, and the Microbiology Commons Recommended Citation Delorenzo, Drew Michael, "Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass" (2019). Engineering and Applied Science Theses & Dissertations. 492. https://openscholarship.wustl.edu/eng_etds/492 This Dissertation is brought to you for free and open access by the McKelvey School of Engineering at Washington University Open Scholarship. It has been accepted for inclusion in Engineering and Applied Science Theses & Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS McKelvey School of Engineering Department of Energy, Environmental, and Chemical Engineering Dissertation Examination Committee: Dr. Tae Seok Moon (chair) Dr. Gautam Dantas Dr. Marcus Foston Dr. Himadri Pakrasi Dr. Fuzhong Zhang Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass by Drew Michael DeLorenzo A dissertation presented to The Graduate School of Washington University
    [Show full text]
  • Rhodococcus As Biofactories for Microbial Oil Production
    molecules Review Rhodococcus as Biofactories for Microbial Oil Production Héctor M. Alvarez * , Martín A. Hernández, Mariana P. Lanfranconi, Roxana A. Silva and María S. Villalba INBIOP (Instituto de Biociencias de la Patagonia), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Naturales y Ciencias de la Salud, Universidad Nacional de la Patagonia San Juan Bosco, Ruta Provincial N◦ 1, Km 4-Ciudad Universitaria, Comodoro Rivadavia 9000, Chubut, Argentina; [email protected] (M.A.H.); [email protected] (M.P.L.); [email protected] (R.A.S.); [email protected] (M.S.V.) * Correspondence: [email protected]; Tel.: +54-297-488-6168 Abstract: Bacteria belonging to the Rhodococcus genus are frequent components of microbial commu- nities in diverse natural environments. Some rhodococcal species exhibit the outstanding ability to produce significant amounts of triacylglycerols (TAG) (>20% of cellular dry weight) in the presence of an excess of the carbon source and limitation of the nitrogen source. For this reason, they can be considered as oleaginous microorganisms. As occurs as well in eukaryotic single-cell oil (SCO) producers, these bacteria possess specific physiological properties and molecular mechanisms that differentiate them from other microorganisms unable to synthesize TAG. In this review, we summa- rized several of the well-characterized molecular mechanisms that enable oleaginous rhodococci to produce significant amounts of SCO. Furthermore, we highlighted the ability of these microorganisms to degrade a wide range of carbon sources coupled to lipogenesis. The qualitative and quantitative oil production by rhodococci from diverse industrial wastes has also been included. Finally, we summarized the genetic and metabolic approaches applied to oleaginous rhodococci to improve SCO production.
    [Show full text]
  • Rhizosphere Bacterium Rhodococcus Sp. P1Y Metabolizes Abscisic Acid to Form Dehydrovomifoliol
    biomolecules Article Rhizosphere Bacterium Rhodococcus sp. P1Y Metabolizes Abscisic Acid to Form Dehydrovomifoliol Oleg S. Yuzikhin 1,2 , Natalia E. Gogoleva 3,4, Alexander I. Shaposhnikov 1 , Tatyana A. Konnova 5, Elena V. Osipova 3, Darya S. Syrova 1, Elena A. Ermakova 3, Valerii P. Shevchenko 6, Igor Yu. Nagaev 6, Konstantin V. Shevchenko 6, Nikolay F. Myasoedov 6, Vera I. Safronova 1, Alexey L. Shavarda 7, Anton A. Nizhnikov 1,7 , Andrey A. Belimov 1 and Yuri V. Gogolev 3,4,* 1 All-Russia Research Institute for Agricultural Microbiology, Podbelskogo sh. 3, Pushkin, 196608 Saint-Petersburg, Russia; [email protected] (O.S.Y.); [email protected] (A.I.S.); [email protected] (D.S.S.); [email protected] (V.I.S.); [email protected] (A.A.N.); [email protected] (A.A.B.) 2 All-Russian Research Institute of Plant Protection, Podbelskogo sh. 3, Pushkin, 196608 Saint-Petersburg, Russia 3 Kazan Institute of Biochemistry and Biophysics, Federal Research Center “Kazan Scientific Center of the RAS”, Lobachevsky Street, 2/31, 420111 Kazan, Tatarstan Republic, Russia; [email protected] (N.E.G.); [email protected] (E.V.O.); [email protected] (E.A.E.) 4 Kazan Federal University Institute of Fundamental Medicine and Biology, K.Marx, 76, 420012 Kazan, Russia 5 Laboratory of Plant Infectious Diseases, FRC Kazan Scientific Center of RAS, 420111 Kazan, Tatarstan Republic, Russia; [email protected] 6 Institute of Molecular Genetics, Russian Academy of Sciences, Akademika Kurchatova Square, 2, 123182 Moscow, Russia; [email protected] (V.P.S.); [email protected] (I.Y.N.); [email protected] (K.V.S.); Citation: Yuzikhin, O.S.; Gogoleva, [email protected] (N.F.M.) 7 Department of Genetics and Biotechnology, Saint-Petersburg State University, University Embankment, N.E.; Shaposhnikov, A.I.; Konnova, 199034 Saint-Petersburg, Russia; [email protected] T.A.; Osipova, E.V.; Syrova, D.S.; * Correspondence: [email protected]; Tel.: +7-917-916-8381 Ermakova, E.A.; Shevchenko, V.P.; Nagaev, I.Y..; Shevchenko, K.V.; et al.
    [Show full text]