Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors.
An environmental isolate Comamonas testosteroni RF2 has been previously described to cometabolize trichloroethene (TCE), 1,2-cis-dichloroethene (cDCE), 1,2-trans-dichloroethene (tDCE), and 1,1-dichloroethene (1,1DCE) when grown on phenol and lactate sodium. In this study, three vinyl chloride (VC) degrading strains, Mycobacterium aurum L1, Pseudomonas putida PS, and Rhodococcus ruber Sm-1 were used to form consortia with the strain RF2 in terms to achieve the removal of VC along with above-mentioned chloroethenes. Degradation assays were performed for a binary mixture of cDCE and VC as well as for a mixture of TCE, all DCEs and VC. The consortium composed of C. testosteroni RF2 and M. aurum L1 showed to be the most efficient towards the removal of cDCE (6.01 mg L-1) in the binary mixture with VC (10 mg L-1) and was capable of efficiently removing chloroethenes in the mixture sample at the initial concentrations of 116 μg L-1 for TCE, 662 μg L-1 for cDCE, 42 μg L-1 for tDCE, 16 μg L-1 for 1,1DCE, and 7 mg L-1 for VC with a removal efficiency of nearly 100% for all of the compounds. Although complete removal of VC took a significantly longer time than the removal of other chloroethenes, the consortium composed of C. testosteroni RF2 and M. aurum L1 displayed strong bioremediation potential for aquifers with downstream contamination characterized by the presence of less chlorinated ethenes.
Rhodococcus spp. strains are widespread in diverse natural and anthropized environments thanks to their high metabolic versatility, biodegradation activities, and unique adaptation capacities to several stress conditions such as the presence of toxic compounds and environmental fluctuations. Additionally, the capability of Rhodococcus spp. strains to produce high value-added products has received considerable attention, mostly in relation to lipid accumulation. In relation with this, several works carried out omic studies and genome comparative analyses to investigate the genetic and genomic basis of these anabolic capacities, frequently in association with the bioconversion of renewable resources and low-cost substrates into triacylglycerols. This review is focused on these omic analyses and the genetic and metabolic approaches used to improve the biosynthetic and bioconversion performance of Rhodococcus. In particular, this review summarizes the works that applied heterologous expression of specific genes and adaptive laboratory evolution approaches to manipulate anabolic performance. Furthermore, recent molecular toolkits for targeted genome editing as well as genome-based metabolic models are described here as novel and promising strategies for genome-scaled rational design of Rhodococcus cells for efficient biosynthetic processes application.
Rhodococci are bacteria which can survive under various extreme conditions, in the presence of toxic compounds, and in other hostile habitats. Their tolerance of unfavorable conditions is associated with the structure of their cell wall and their large array of enzymes, which degrade or detoxify harmful compounds. Their physiological and biotechnological properties, together with tools for their genetic manipulation, enable us to apply them in biotransformations, biodegradation and bioremediation. Many such biotechnological applications cause stresses that positively or negatively affect their efficiency. Whereas numerous reviews on rhodococci described their enzyme activities, the optimization of degradation or production processes, and corresponding technological solutions, only a few reviews discussed some specific effects of stresses on the physiology of rhodococci and biotechnological processes. This review aims to comprehensively describe individual stress responses in Rhodococcus strains, the interconnection of different types of stresses and their consequences for cell physiology. We examine here the responses to (1) environmental stresses (desiccation, heat, cold, osmotic and pH stress), (2) the presence of stress-inducing compounds (metals, organic compounds and antibiotics) in the environment (3) starvation and (4) stresses encountered during biotechnological applications. Adaptations of the cell envelope, the formation of multicellular structures and stresses induced by the interactions of hosts with pathogenic rhodococci are also included. The roles of sigma factors of RNA polymerase in the global regulation of stress responses in rhodococci are described as well. Although the review covers a large number of stressful conditions, our intention was to provide an overview of the selected stress responses and their possible connection to biotechnological processes, not an exhaustive survey of the scientific literature. The findings on stress responses summarized in this review and the demonstration of gaps in current knowledge may motivate researchers working to fill these gaps.
- MeSH
- bakteriální léková rezistence MeSH
- diskové difúzní antimikrobiální testy metody MeSH
- Klebsiella oxytoca izolace a purifikace MeSH
- mikrobiální testy citlivosti metody MeSH
- Rhodococcus equi izolace a purifikace MeSH
- Shigella sonnei izolace a purifikace MeSH
- Streptococcus pneumoniae izolace a purifikace MeSH
- testování odbornosti laboratoří * MeSH
Nanofiber scaffolds provide numerous advantages over common carriers engineered for microorganisms. The most important advantage is an increased speed of primary surface colonization (up to four times faster), which shortens the time required for the areal biofilm formation and optimum performance of attached microorganisms (higher efficiency of biological activity of up to twice as fast). Image analysis predicts early formation of biofilm even in beginning stages; analysis of biofilm reveals the different structures of bacterial colonies on both scaffolds (higher porosity, size, and number of bacterial colonies on nanofiber's surface). The image analysis correlates well with determinations of dry matter (linear correlation of 0.96) and proteins (linear correlation of 0.89).
- MeSH
- biofilmy * MeSH
- kultivační média * MeSH
- nanovlákna * MeSH
- polyurethany MeSH
- poréznost MeSH
- Rhodococcus MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Extended soil contamination by polychlorinated biphenyls (PCBs) represents a global environmental issue that can hardly be addressed with the conventional remediation treatments. Rhizoremediation is a sustainable alternative, exploiting plants to stimulate in situ the degradative bacterial communities naturally occurring in historically polluted areas. This approach can be enhanced by the use of bacterial strains that combine PCB degradation potential with the ability to promote plant and root development. With this aim, we established a collection of aerobic bacteria isolated from the soil of the highly PCB-polluted site "SIN Brescia-Caffaro" (Italy) biostimulated by the plant Phalaris arundinacea. The strains, selected on biphenyl and plant secondary metabolites provided as unique carbon source, were largely dominated by Actinobacteria and a significant number showed traits of interest for remediation, harbouring genes homologous to bphA, involved in the PCB oxidation pathway, and displaying 2,3-catechol dioxygenase activity and emulsification properties. Several strains also showed the potential to alleviate plant stress through 1-aminocyclopropane-1-carboxylate deaminase activity. In particular, we identified three Rhodococcus strains able to degrade in vitro several PCB congeners and to promote lateral root emergence in the model plant Arabidopsis thaliana in vivo. In addition, these strains showed the capacity to colonize the root system and to increase the plant biomass in PCB contaminated soil, making them ideal candidates to sustain microbial-assisted PCB rhizoremediation through a bioaugmentation approach.
- MeSH
- Arabidopsis růst a vývoj mikrobiologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biodegradace MeSH
- exprese genu MeSH
- katechol-2,3-dioxygenasa genetika metabolismus MeSH
- kořeny rostlin růst a vývoj mikrobiologie MeSH
- látky znečišťující půdu metabolismus MeSH
- lyasy štěpící vazby C-C genetika metabolismus MeSH
- oxidace-redukce MeSH
- Phalaris růst a vývoj mikrobiologie MeSH
- polychlorované bifenyly metabolismus MeSH
- půda chemie MeSH
- půdní mikrobiologie MeSH
- Rhodococcus enzymologie genetika MeSH
- sekundární metabolismus genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.
Úvod: 13 – 20 miliónov ľudí vo veku 15 až 64 rokov užíva kokaín. Podľa Výboru OSN pre drogy a kriminalitu (UNODC) dosahuje globálna ročná prevalencia porúch súvisiacich s užívaním kokaínu 0,3 až 0,4 %. Nízkomolekulové preparáty sú len okrajovo efektívne v liečbe látkových závislostí. Neexistuje žiadna schválená medikácia na liečbu závislosti od kokaínu. V teoretických možnostiach liečby závislosti od kokaínu je základom buď vylúčiť alebo spomaliť vstup látky do centrálneho nervového systému. Metodika: Publikovaná literatúra a prezentované prednášky o biologickej liečbe kokaínovej závislosti. Výsledky: Perspektívou biologickej liečby sú: 1. vakcíny, ktoré stimulujú imunitný systém na tvorbu protilátok proti kokaínu; 2. pasívna imunizácia podávaním monoklonálnych protilátok priamo namierených proti kokaínu; 3. geneticky modifikované esterázy, ktoré katalyzujú hydrolýzu kokaínu rýchlejšie než prirodzený enzým. Podanie zmutovanej butyrylcholínesterázy viazanej na albumín séra výrazne redukuje toxikologický aj behaviorálny efekt kokaínu u nehumánnych primátov. Súvisí to s rapídnou degradáciou kokaínu v plazme a nárastu hydrolýzou metabolizovaného produktu kokaínu – ekgonínu metylesteru. Diskusia: Imunoterapia je pomocou prevencie recidív u motivovaných pacientov s cieľom udržania ich abstinencie. Pravdepodobne bude menej účinná u ťažko kokaínovo závislých s cieľom indukovať u nich abstinenciu. Môže byť úspešná v spojení s behaviorálnymi a inými farmakologickými procesmi. Záver: Pre klinickú prax by bolo prínosné úspešné ukončenie klinických skúšaní biologík a ich schválenie príslušnými (nad-)národnými liekovými kontrolnými inštitúciami. Biologiká na liečbu kokaínovej závislosti by sa tak mohli stať súčasťou liečebných postupov tejto závažnej poruchy.
Introduction: The United Nations Office on Drugs and Crime (UNODC) estimates the annual prevalence of cocaine use between 0.3 – 0.4 %, affecting 13 – 20 million individuals between 15 and 64 years of age. Small molecule approaches have only been marginally successful in developing therapies for substance use disorders (SUDs). There are no approved medications to treat cocaine addiction. Biological approaches are all grounded on a common mechanism: either exclude or retard the entry of cocaine from the central nervous system. Methods: Published literature and conference lectures about biological therapies of cocaine addiction. Results: There are currently three means of achieving this objective: 1. vaccines that stimulate the immune system to produce antibodies directed against cocaine specific; 2. passive immunization by administering monoclonal antibodies directed against cocaine; 3. genetically engineered esterases that catalyze the hydrolysis of cocaine in a much greater way than the wild-type enzyme. Administration of mutated butyrylcholinesterase linked to serum albumin dramatically reduces both the toxicological and behavioral effects of cocaine in nonhuman primates. These effects are attributable to a rapid degradation of cocaine in plasma and subsequent greater hydrolysis of ecgonine methylester. Discussion: Immunotherapies may help to prevent relapse in addicted patients who are motivated to remain abstinent, however they are less likely to be successful for inducing abstinence in heavily cocaine addicted individuals. To enhance chances for effectiveness, they should be combined with other behavioral and pharmacological therapies. Conclusion: Hopefully, those challenges will be resolved and biologics for SUDs will be approved by the FDA or related authorities, to become part of the therapeutic armamentarium to curtail this serious disorder.
- Klíčová slova
- antikokainové vakcíny,
- MeSH
- biologická terapie * MeSH
- butyrylcholinesterasa farmakologie metabolismus MeSH
- experimenty na zvířatech MeSH
- klinické zkoušky jako téma MeSH
- kokain * farmakologie metabolismus škodlivé účinky MeSH
- lidé MeSH
- monoklonální protilátky farmakologie terapeutické užití MeSH
- myši MeSH
- poruchy spojené s užíváním psychoaktivních látek farmakoterapie MeSH
- Rhodococcus izolace a purifikace MeSH
- vakcíny aplikace a dávkování terapeutické užití MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- Publikační typ
- přehledy MeSH
The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution ( Pavlova, M. et al. Nat. Chem. Biol. 2009 , 5 , 727 - 733 ), which is 32 times more active than the wild-type enzyme and is currently the most active variant known against that substrate. Recent evidence has shown that the structural basis responsible for the higher activity of DhaA31 was poorly understood. Here we have undertaken a comprehensive computational study of the main steps involved in the biocatalytic hydrolysis of 1,2,3-trichloropropane to decipher the structural basis for such enhancements. Using molecular dynamics and quantum mechanics approaches we have surveyed (i) the substrate binding, (ii) the formation of the reactive complex, (iii) the chemical step, and (iv) the release of the products. We showed that the binding of the substrate and its transport through the molecular tunnel to the active site is a relatively fast process. The cleavage of the carbon-halogen bond was previously identified as the rate-limiting step in the wild-type. Here we demonstrate that this step was enhanced in DhaA31 due to a significantly higher number of reactive configurations of the substrate and a decrease of the energy barrier to the SN2 reaction. C176Y and V245F were identified as the key mutations responsible for most of those improvements. The release of the alcohol product was found to be the rate-limiting step in DhaA31 primarily due to the C176Y mutation. Mutational dissection of DhaA31 and kinetic analysis of the intermediate mutants confirmed the theoretical observations. Overall, our comprehensive computational approach has unveiled mechanistic details of the catalytic cycle which will enable a balanced design of more efficient enzymes. This approach is applicable to deepen the biochemical knowledge of a large number of other systems and may contribute to robust strategies in the development of new biocatalysts.
- MeSH
- biokatalýza * MeSH
- hydrolasy chemie genetika metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- mutace MeSH
- počítačová simulace * MeSH
- Rhodococcus enzymologie MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
