Biochemical analysis and environmental applications of petroleum degrading bacteria

A bacterial stain, Spet, was isolated from a Greek petroleum polluted soil, near Athens, using petroleum as sole carbon and energy source and it was taxonomically identified as closest to Pseudomonas aeruginosa based on 16S rDNA sequence, G+C content, fatty acids analysis and several biochemical assays. Strain Spyr1 isolated from a Greek creosote polluted soil using pyrene as sole carbon and energy source was found to belong to Mycobacterium sp. based on 16S rDNA gene sequence analysis. Spyr1, exhibited a very broad substrate profile, being able to utilize anthracene, phenanthrene, fluorine, fluoranthene and pyrene as sole carbon and energy source. For soil biodegradation studies four different types of soil were used: a non-polluted spiked soil, a heavily polluted soil, an aged polluted soil and a non-polluted spiked soil that was left to age for six months. Spyr1 and Spet cells acclimated to grow in petroleum and pyrene as the sole carbon and energy source were immobilized in three different materials: Alumina pellets, Macroporous sinter glass SiranR, and an alginate/starch mixture. Alumina was disintegrated during biodegradation studies in liquid cultures. Both freely suspended and immobilized Spyr1 cells on porous glass beads showed the same degradation ability in liquid suspensions whereas only about 58% was removed in soil microcosms using immobilized cells. Alginate/starch entrapped cells have given good yields in the removal of pyrene as well as mixture of PAHs. Pyrene degradation by entrapped cells in liquid cultures and in soil was complete within 12 days and 17 days, while pyrene degradation by free cells occurred within 12 and 14 days, respectively. Phenathrene was degraded completely after 14 days of incubation while 4% pyrene and 2% fluoranthene persisted after 23 days of incubation with free cells. After 23 days of incubation with entrapped cells, 2% phenathrene, 10% pyrene and 8% fluoranthene remained in the soil. The biodegradation of petroleum in liquid cultures and soil was monitored using free and immobilized Spet cells. Petroleum degradation by free and alginate/starch entrapped cells in liquid cultures and in soil after 21 days resulted to 60% degradation, respectively. 60% and 31% petroleum was degraded in liquid cultures and in soil using Siran immobilized Spet cells, respectively.Για τη μελέτη της βιοεξυγίανσης ρυπασμένων από πετρελαιοειδή εδαφών, απομονώθηκε με την μέθοδο του εμπλουτισμού ένα βακτηριακό στέλεχος, Spet, το οποίο χρησιμοποιεί πετρέλαιο ως μοναδική πηγή άνθρακα και ενέργειας. Κύτταρα Spet αποδομούν επίσης πετρέλαιο κίνησης και πίσσα, αναπτύσσονται σε ευρύ φάσμα n-αλκανίων C5-C28 με ιδιαίτερη προτίμηση σε αλκάνια μεσαίας αλυσίδας. Η ταυτοποίηση του στελέχους έγινε με μελέτη των μορφολογικών χαρακτηριστικών του, μικροβιολογικό και βιοχημικό χαρακτηρισμό, λιπιδιακή ανάλυση και ανάλυση των ισοπρενοειδών κινονών καθώς και μελέτη του γονιδιώματός του με προσδιορισμό της περιεκτικότητας % G+C και ανάλυση της αλληλουχίας 16S rRNA. Το στέλεχος Spet ανήκει στο γένος Pseudomonas sp. με συγγενέστερο είδος το Pseudomonas aeruginosa. Το στέλεχος Spet εμφανίζει μεγάλη ανθεκτικότητα σε ευρύ φάσμα αντιβιοτικών, εμφανίζει κίνηση, σχηματίζει βιοφιλμ και παράγει ραμνολιπίδια. Στο στέλεχος Spet ανιχνεύθηκαν γονίδια που κωδικεύουν υδροξυλάσες που συμμετέχουν στην αποδόμηση των αλκανίων. Η ανίχνευση έγινε ύστερα από την ενίσχυση με PCR των αλληλουχιών των γονιδίων των υδροξυλασών alkB1 και alkB2 χρησιμοποιώντας τους κατάλληλους εκκινητές. Ακολούθησαν πειράματα αποδόμησης PAHs από τους μικροοργανισμούς Spyr1, Spyr2, Spyr3, Sphe3, Sflu4, Sflu5 και Ophe1 και πετρελαίου από το στέλεχος Spet α) σε τεχνητά ρυπασμένο χώμα, β) σε παλαιά ρυπασμένο χώμα, γ) σε πιο πρόσφατα και ιδιαίτερα ρυπασμένο χώμα και δ) σε τεχνητά ρυπασμένο χώμα το οποίο είχε αφεθεί να παλαιώσει για 6 μήνες στο εξωτερικό περιβάλλον. Διαπιστώθηκε ότι (i) μίγματα μικροοργανισμών και μίγματα υποστρωμάτων δρουν συνεργιστικά, (ii) ο χρόνος επαφής χώματος-υποστρώματος επηρεάζει αρνητικά την αποδόμηση και (iii) η προσθήκη μικροοργανισμών (βιοενίσχυση) με την ταυτόχρονη προσθήκη θρεπτικών συστατικών (βιοδιέγερση) ήταν πιο αποτελεσματική στην αποδόμηση των ρυπογόνων ενώσεων. Το στέλεχος Spet απομονώθηκε και χαρακτηρίστηκε στην παρούσα εργασία. Τα στελέχη Spyr1, Spyr2, Spyr3, Sphe3, Sflu4 και Sflu5 απομονώθηκαν με την μέθοδο του εμπλουτισμού από έδαφος ρυπασμένο με κρεοζωτέλαιο που προέρχεται από τις εγκαταστάσεις του εργοστάσιου κατεργασίας ξύλου ΕΛΒΙΕΞ στην περιοχή Ιωαννίνων - Κόνιτσας χρησιμοποιώντας αντίστοιχα πυρένιο για τα τρία πρώτα, φαινανθρένιο, και φλουορανθένιο για τα δύο τελευταία ως μοναδική πηγή άνθρακα και ενέργειας (Καλλιμάνης Αριστείδης, διδακτορική διατριβή 2004). Το βακτηριακ

High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate, or oxalate (sulphite could not be tested in uptake assays due to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.Publisher PDFPeer reviewe

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and II termed NS (nitrate signature) I and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn(168) in NS1 and Asn(459) in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg(368) and Are respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn(168)/Arg(368) and Asn(459)/Arg(87) residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn(168)/Are(368) and Asn(459)/Arg(87) pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.</p

Complete genome sequence of Mycobacterium sp. strain (Spyr1) and reclassification to Mycobacterium gilvum Spyr1

Abstract Mycobacterium sp.Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated

Complete genome sequence of Mycobacterium sp strain (Spyr1) and reclassification to Mycobacterium gilvum Spyr1

Mycobacterium sp. Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BackgroundThe fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.ResultsWe have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli.ConclusionsMany aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi