Gene cloning and characterization of an alkane hydroxylase system in the long chain n-alkane- degrader Gordonia sp. SoCg

Degradazione di pesticidi organofosforici in matrici ambientali

Matrici quali acqua, suolo ed aria sono costantemente soggette a forti pressioni antropiche. I pesticidi pur avendo apportato grandi benefici alle attività agricole influiscono negativamente sugli equilibri degli ecosistemi. Circa 85-90% dei principi attivi applicati in agricoltura non raggiunge gli organismi a cui sono destinati, ma viene disperso nell’ambiente. In tale contesto l’individuazione di microrganismi deputati alla loro degradazione diventa un importante mezzo per la salvaguardia ambientale e della salute umana. Lo scopo del presente lavoro è stato quello di isolare batteri del suolo capaci di degradare composti organofosforici, i quali rivestono una notevole importanza nelle pr…

Dbv4 is DNA-binding protein acting as a positive regulator of the glycopeptide antibiotic A40926 biosynthesis

ISWI ATP-dependent remodeling of nucleoplasmic ω-speckles in the brain of Drosophila melanogaster.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) belong to the RNA-binding proteins family. They are involved in processing heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs. These proteins participate in every step of mRNA cycle, such as mRNA export, localization, translation, stability and alternative splicing. At least 14 major hnRNPs, which have structural and functional homologues in mammals, are expressed in Drosophila melanogaster. Until now, six of these hnRNPs are known to be nucleus-localized and associated with the long non-coding RNA (lncRNA) heat shock responsive ω (hsrω) in the omega speckle compartments (ω-speckles). The chromatin remodeler ISWI is the catalytic subunit …

Biodegradazione di miscele di gasolio in suoli contaminati

Il biorisanamento mediante l’utilizzo di microrganismi ha un ruolo fondamentale nelle pratiche di trasformazione di molecole potenzialmente tossiche quali gli idrocarburi, che quotidianamente vengono riversati negli ecosistemi provocando fenomeni di antropizzazione in equilibri naturali particolarmente sensibili. Numerosi ceppi batterici sono abili degradatori e mineralizzatori di sostanze inquinanti xenobiotiche di sintesi e recalcitranti non esistenti in natura, quali i derivati di idrocarburi aromatici e policlorobifenili (PCB). Mediante colture su mezzo minerale Bushnell-Haas agarizzato (BH) in presenza di una miscela di idrocarburi come unica fonte di carbonio sono stati isolati 5 cepp…

Exploring long chain n-alkane metabolism in Gordonia sp. strain SoCg

Many microorganisms are able to degrade aliphatic hydrocarbons and a relationship between n-alkane utilization and storage compound synthesis has been described in bacteria. The Gram positive GC-rich n-alkane degrader Gordonia sp. strain SoCg, isolated from a long-term accidentally contaminated beach in Sicily, is able to grow on long n-alkanes up to. It carries a single copy of the alkane hydroxylase gene alkB on its chromosome and its alk cluster revealed a genomic organization similar to other alk clusters of alkane-degrading Gram positive bacteria. The alk gene expression, analysed by Real-time RT-PCR, is induced by n-hexadecane and n-triacontane and coupled to alkane consumption. Inter…

GENE CLONING AND CHRACTERIZATION OF AN ALKANE HYDROXYLASE SYSTEM IN A LONG CHAIN N.ALKANE. DEGRADER GORDONIA SP.

DBV4 IS A DNA BINDING PROTEIN ACTING AS A POSITIVE REGULATOR OF THE GLYCOPEPTIDE ANTIBIOTIC A40926 BIOSYNTHESIS

Degradation of long-chain n-alkanes in soil microcosms by two actinobacteria

The ability of two recently isolated actinobacteria, that degrade medium and long chain n-alkanes in laboratory water medium, was investigated in soil microcosms using different standard soils that were artificially contaminated with n-alkanes of different length (C(12)- C(20)- C(24)- C(30)). The two strains, identified as Nocardia sp. SoB and Gordonia sp. SoCp, revealed a similar high HC degradation efficiency with an average of 75% alkane degraded after 28 days incubation. A selectivity of bacteria towards n-alkanes of different length was detected as well as a consistent effect of soil texture and other soil physical chemical characteristics on degradation. It was demonstrated the specif…

Differential proteomic analysis of an engineered Streptomyces coelicolor strain reveals metabolic pathways supporting growth on n-hexadecane

The alkB gene, encoding an alkane monooxygenase in the actinomycete Gordonia sp. SoCg, was expressed in the non-alkane-degrading actinomycete Streptomyces coelicolor M145. The resulting engineered strain, M145-AH, can grow on n-hexadecane as sole carbon source. To unravel proteins associated with growth on n-alkanes, proteome of M145-AH after 6, 24, and 48 h of incubation in the Bushnell-Haas (BH) mineral medium containing n-hexadecane as sole carbon source (H condition) and in BH without any carbon source (0 condition) were compared using 2D-differential gel electrophoresis. Proteome analysis revealed significant changes only at 48 h, showing 48 differentially abundant proteins identified …

Gordonia sp. SoCg alkB gene confers the ability to degrade and use n-alkanes as carbon source in Gram positive bacteria

Gordonia sp. SoCg, a Gram positive strain able to grow on long chain n-alkanes1, possess a single copy of alkB2 gene, whose product is required for n-alkane hydroxylation3. An analysis of alkB flanking regions revealed five ORFs which were designed as orf1, rubA3, rubA4, rubB and alkU, according to the sequence 14 homology with that of known alk clusters3. In G. sp. SoCg the transcription of these genes was induced by long-chain and solid n-alkanes as revealed by quantitative RT-PCR, and the essential role of alkB in nalkane degradation was demonstrated by the construction of an alkB disruption mutant strain3. The SoCg alkB gene was successfully expressed in Streptomyces coelicolor M145 (M1…

Isolation Of Organophosphorus Pesticides Bacteria Degraders From Agricultural Sicilian Soils.

Pesticides are generally categorized according to their great or low persistence in the environment. Although new biological, chemical, and management technologies are continually being developed to provide more sustainable production alternatives, it is expected that the use of pesticides will continue to be an essential tool in the integrated pest management. Pesticides are biologically active compounds designed to interfere with metabolic processes. Organophosphorus pesticides (OP) pesticides are less persistent than Organochlorine pesticides (OC), they are not without environmental risks, so this justifies the social concern about their level in different kind of matrices like soil, wat…

Cloning of an alkane hydroxylase system in a long chain n-alkane- degrader Gordonia sp.

Five Gram-positive GC rich n-alkane degraders were isolated from a long-term accidentally contaminated beach in Sicily and identified as one Nocardia, two Rhodococcus and two Gordonia strains (Quatrini et al., 2008 J. Appl. Microbiol. 104:251-9). All the isolates were able to grow on long and very long chain n-alkanes up to C36. Diverging alkane-hydroxylase encoding genes (alkB) were detected by PCR using degenerated primers in all the strains. Multiple sequences were obtained from the Nocardia strain while only one alkB gene was detected in Rhodococcus and Gordonia. The aim of this work is to genetically characterize the alk cluster in one of the two Gordonia strains called SoCg. Pulsed Fi…

Isolation and characterization of Gordonia SoCg n-alkane degradation cluster

Gordonia strain SoCg is a Gram-positive GC rich n-alkane degrader, isolated from a long-term contaminated beach in Sicily for her ability to degrade long (dodecane C12) and very long chain n-alkanes up to hexatriacontane (C36) (P.Quatrini et al., J. Appl. Microbiol., 2007). PCR analysis, using degenerated primers, reveled that it carries one alkane-hydroxylase gene (alkB); PFGE and Southern analysis showed that this gene is localized on the chromosome. In order to isolate the alk cluster of Gordonia strain SoCg, an enriched gene bank was constructed in E.coli DH10B by isolating restriction fragments of the desired size from a preparative gel. One clone, containing a DNA insert of about 7 kb…

Phosphate-controlled regulator for the biosynthesis of the dalbavancin precursor A40926

ABSTRACT The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene ( dbv ) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14 - dbv8 and the dbv30 - dbv35 operons, as well as dbv4 , were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-bind…

Proteomic analysis of Amycolatopsis balhimycina to reveal primary metabolism proteins involved in balhimycin production

Dbv4 is a DNA-binding protein acting as a positive regulator of the glycopeptide antibiotic A40926 biosynthesis

Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila’s Whole Larvae and Tissues

The identification and study of protein’s function in several model organisms is carried out using both nuclear and cytoplasmic extracts. For a long time, Drosophila’s embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood. Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila’s larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation.

Effect of osmotic and heat shock on the biosynthesis of glycopeptide antibiotics in actinomycetes

Involvement of an Alkane Hydroxylase System of Gordonia sp. Strain SoCg in Degradation of Solid n-Alkanes▿

ABSTRACT Enzymes involved in oxidation of long-chain n -alkanes are still not well known, especially those in Gram-positive bacteria. This work describes the alkane degradation system of the n -alkane degrader actinobacterium Gordonia sp. strain SoCg, which is able to grow on n -alkanes from dodecane (C 12 ) to hexatriacontane (C 36 ) as the sole C source. SoCg harbors in its chromosome a single alk locus carrying six open reading frames (ORFs), which shows 78 to 79% identity with the alkane hydroxylase (AH)-encoding systems of other alkane-degrading actinobacteria. Quantitative reverse transcription-PCR showed that the genes encoding AlkB (alkane 1-monooxygenase), RubA3 (rubredoxin), RubA4…

Biodiversity of organophosphorus-degrading bacteria isolated from agricultural soils

Organophosphorus (OP) insecticides are used all over the world for crop protection. Although OPs are less persistent than organochlorine pesticides, they still constitute an environmental risk thus increasing the social concern about their levels in soils, surface and ground waters. Biodegradation by microorganisms is primarily responsible for elimination of the OP insecticides released to the environment. In the present study the OP degrading potential of agricultural soils with different agronomic history was investigated. Using enrichment cultures, with parathion or dimethoate as the sole C and energy sources, 47 bacterial isolates were obtained from OPs contaminated and pristine agricul…

Loss of ISWI Function in Drosophila Nuclear Bodies Drives Cytoplasmic Redistribution of Drosophila TDP-43

Over the past decade, evidence has identified a link between protein aggregation, RNA biology, and a subset of degenerative diseases. An important feature of these disorders is the cytoplasmic or nuclear aggregation of RNA-binding proteins (RBPs). Redistribution of RBPs, such as the human TAR DNA-binding 43 protein (TDP-43) from the nucleus to cytoplasmic inclusions is a pathological feature of several diseases. Indeed, sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration share as hallmarks ubiquitin-positive inclusions. Recently, the wide spectrum of neurodegenerative diseases characterized by RBPs functions’ alteration and loss was coll…

Exploring long chain n-alkane metabolism in Gordonia sp. strain SoCg

Biodegradation of organophosphorus pesticides by soil bacteria.

A number of studies in the 1980s and 1990s showed that crop-protection products, applied to drained fields, could move downwards through the soil profile and to the groundwater. Organophosphorus insecticides (OPs) are used all over the world for crop protection, for other agricultural practices such as sheep dipping and, in aquaculture, for the control of sea lice. Ops besides showing a specific neurotoxicity and have also been related to various modern diseases, including Creutzfeldt–Jakob (CJD) and the Gulf War syndrome. Although OPs are less persistent than Organoclorine pesticides (OCs), they still constitute an environmental risks thus increasing the social concern about their levels i…

Chromosomal advanced technologies to study the regulation of erythromycin biosynthesis