Proteomic analysis of 2,4,6-trinitrotoluene degrading yeast Yarrowia lipolytica

© 2017 Khilyas, Lochnit and Ilinskaya. 2,4,6-trinitrotoluene (TNT) is a common component of many explosives. The overproduction and extensive usage of TNT significantly contaminates the environment. TNT accumulates in soils and aquatic ecosystems and can primarily be destroyed by microorganisms. Current work is devoted to investigation of Yarrowia lipolytica proteins responsible for TNT transformation through the pathway leading to protonated Meisenheimer complexes and nitrite release. Here, we identified a unique set of upregulated membrane and cytosolic proteins of Y. lipolytica, which biosynthesis increased during TNT transformation through TNT-monohydride-Meisenheimer complexes in the first step of TNT degradation, through TNT-dihydride-Meisenheimer complexes in the second step, and the aromatic ring denitration and degradation in the last step. We established that the production of oxidoreductases, namely, NADH flavin oxidoreductases and NAD(P)+-dependent aldehyde dehydrogenases, as well as transferases was enhanced at all stages of the TNT transformation by Y. lipolytica. The up-regulation of several stress response proteins (superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase) was also detected. The involvement of intracellular nitric oxide dioxygenase in NO formation during nitrite oxidation was shown. Our results present at the first time the full proteome analysis of Y. lipolytica yeast, destructor of TNT

Assessment of a biogas-generating microbial community in a pilot-scale anaerobic reactor

In this work bacteria and methanogenic archaea utilizing agricultural wastes in a pilot-scale biogas reactor were examined using sequencing and terminal restriction fragment length polymorphism analysis. Based on the analyses of 16S rRNA genes, Clostridia represented the most diverse group in the digester. Of the Clostridia, unclassified Clostridiales and the members of the genera Anaerotruncus and Tissierella were detected at high abundances. The representatives of the bacterial phyla Bacteroidetes and Proteobacteria were also defined, but in minor proportions, and were assigned to non-dominant communities. Within the phylum Euryarchaeota, the members of the orders Methanosarcinales and Methanomicrobiales were found at high levels. Methanogenic archaea were analyzed using both 16S rRNA and mcrA genes. Actually good results were received using both approaches; however, the rRNA gene method missed the non-dominant order Methanobacteriales. © 2013 The Society for Biotechnology, Japan

Effect of ferrihydrite on 2,4,6-trinitrotoluene biotransformation by an aerobic yeast

This study investigated the impact of ferrihydrite on the pathway and rate of 2,4,6-trinitrotoluene (TNT) transformation by Yarrowia lipolytica AN-L15. The presence of ferrihydrite in the culture medium decreased the rate of TNT biotransformation but resulted in the accumulation of the same TNT metabolites as in the absence of ferrihydrite, albeit at slightly different concentrations. Transformation products observed included aromatic ring reduction products, such as hydride-Meisenheimer complexes, and nitro group reduction products, such as hydroxylamino- and amino-dinitrotoluenes. Independently of the presence of ferrihydrite the subsequent degradation of the hydride complex(es) resulted in the release of nitrite followed by its conversion to nitrate and nitric oxide at the low pH values observed during yeast cultivation. Nitric oxide generation was ascertained by electron spin resonance spectroscopy. In addition, increased Fe3+-reduction was observed in the presence of TNT and Y. lipolytica. This study demonstrates that in the presence of yeast cells, TNT-hydride complexes were formed at approximately the same level as in the presence of ferrihydrite, opening up the possibility of aromatic ring cleavage, instead of promoting the production of potentially toxic nitro group reduction products in the presence of iron minerals. © 2012 Springer Science+Business Media Dordrecht

Production of Siderophores by Serratia marcescens and the Role of MacAB Efflux Pump in Siderophores Secretion

© 2016, Springer Science+Business Media New York.Human opportunistic pathogen Serratia marcescens secrete siderophores to enable growth under iron-limiting conditions. Iron acquisition genes are among a few known virulence factors of S. marcescens. Siderophore export systems in Gram-negative bacteria are not fully understood. There is some evidence for involvement of efflux pumps in the export of synthesized enterobactin-like molecules. The goal of this study was to characterize siderophore production by two different strains of S. marcescens, SM6 and SR41-8000, and to evaluate the role of efflux pump MacAB in siderophore secretion by these strains. We showed that both strains produced siderophores in CAS agar assay. We further showed that both strains were able to secrete catecholate siderophores in response to iron starvation. MacAB efflux pump played a role in siderophore secretion of S. marcescens SR41-8000 strain but was dispensable for accumulation of these molecules in the culture supernatant of S. marcescens strain SM6

Proteomic analysis of 2,4,6-trinitrotoluene degrading yeast Yarrowia lipolytica

© 2017 Khilyas, Lochnit and Ilinskaya. 2,4,6-trinitrotoluene (TNT) is a common component of many explosives. The overproduction and extensive usage of TNT significantly contaminates the environment. TNT accumulates in soils and aquatic ecosystems and can primarily be destroyed by microorganisms. Current work is devoted to investigation of Yarrowia lipolytica proteins responsible for TNT transformation through the pathway leading to protonated Meisenheimer complexes and nitrite release. Here, we identified a unique set of upregulated membrane and cytosolic proteins of Y. lipolytica, which biosynthesis increased during TNT transformation through TNT-monohydride-Meisenheimer complexes in the first step of TNT degradation, through TNT-dihydride-Meisenheimer complexes in the second step, and the aromatic ring denitration and degradation in the last step. We established that the production of oxidoreductases, namely, NADH flavin oxidoreductases and NAD(P)+-dependent aldehyde dehydrogenases, as well as transferases was enhanced at all stages of the TNT transformation by Y. lipolytica. The up-regulation of several stress response proteins (superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase) was also detected. The involvement of intracellular nitric oxide dioxygenase in NO formation during nitrite oxidation was shown. Our results present at the first time the full proteome analysis of Y. lipolytica yeast, destructor of TNT

Virulence of Pigmented Serratia marcescens Strain SM6 and its Nalidixic Acid-Resistant Derivative in White Outbred Mice

© 2016, Springer Science+Business Media New York.Human opportunistic pathogen Serratia marcescens is a bacterium with broad host range representing a growing problem for public health. Little is known about virulence factors used by S. marcescens to colonize the host. We used white outbred mice to study virulence of S. marcescens wild type strain SM6 and its nalidixic acid-resistant derivative during intraperitoneal infection. We showed that both strains were lethal for mice at 107–108 CFU and death occurred within 24–48 h post infection. No mortality was noticed at 106 CFU. Bacteria were isolated from hearts, lungs, kidneys, livers, spleens, intestines, and blood of infected animals. Resistance to nalidixic acid did not affect virulence of S. marcescens. Thus, this strain can be used in the future virulence studies to simplify tracking and recovery of S. marcescens from infected tissue

Assessment of a biogas-generating microbial community in a pilot-scale anaerobic reactor

In this work bacteria and methanogenic archaea utilizing agricultural wastes in a pilot-scale biogas reactor were examined using sequencing and terminal restriction fragment length polymorphism analysis. Based on the analyses of 16S rRNA genes, Clostridia represented the most diverse group in the digester. Of the Clostridia, unclassified Clostridiales and the members of the genera Anaerotruncus and Tissierella were detected at high abundances. The representatives of the bacterial phyla Bacteroidetes and Proteobacteria were also defined, but in minor proportions, and were assigned to non-dominant communities. Within the phylum Euryarchaeota, the members of the orders Methanosarcinales and Methanomicrobiales were found at high levels. Methanogenic archaea were analyzed using both 16S rRNA and mcrA genes. Actually good results were received using both approaches; however, the rRNA gene method missed the non-dominant order Methanobacteriales. © 2013 The Society for Biotechnology, Japan

Assessment of a biogas-generating microbial community in a pilot-scale anaerobic reactor

In this work bacteria and methanogenic archaea utilizing agricultural wastes in a pilot-scale biogas reactor were examined using sequencing and terminal restriction fragment length polymorphism analysis. Based on the analyses of 16S rRNA genes, Clostridia represented the most diverse group in the digester. Of the Clostridia, unclassified Clostridiales and the members of the genera Anaerotruncus and Tissierella were detected at high abundances. The representatives of the bacterial phyla Bacteroidetes and Proteobacteria were also defined, but in minor proportions, and were assigned to non-dominant communities. Within the phylum Euryarchaeota, the members of the orders Methanosarcinales and Methanomicrobiales were found at high levels. Methanogenic archaea were analyzed using both 16S rRNA and mcrA genes. Actually good results were received using both approaches; however, the rRNA gene method missed the non-dominant order Methanobacteriales. © 2013 The Society for Biotechnology, Japan