Search

Echinacea purpurea is a plant cultivated worldwide for its pharmaceutical properties, mainly related to the stimulation of the immune system in the treatment of respiratory infections. The cypselas (fruits) of E. purpurea were examined in order to investigate the presence, localization and potential function(s) of endophytic microorganisms. Electron and confocal microscopy observations showed that three different components of microorganisms were associated to cypselas of E. purpurea: (i) one endocellular bacterial component in the cotyledons, enclosed within the host membrane; (ii) another more generic bacterial component adhering to the external side of the perianth; and (iii) a fungal component inside the porous layer of the perianth, the woody and porous modified residual of the flower, in the form of numerous hyphae able to cross the wall between adjacent cells. Isolated bacteria were affiliated to the genera Paenibacillus, Pantoea, and Sanguibacter. Plate tests showed a general resistance to six different antibiotics and also to an antimicrobial-producing Rheinheimera sp. test strain. Finally, microbiome-deprived E. purpurea seeds showed a reduced ability to germinate, suggesting an active role of the microbiome in the plant vitality. Our results suggest that the endophytic bacterial community of E. purpurea, previously found in roots and stem/leaves, might be already carried at the seed stage, hosted by the cotyledons. A further microbial fungal component is transported together with the seed in the perianth of the cypsela, whose remarkable structure may be considered as an adaptation for fungal transportation, and could influence the capability of the seed to germinate in the soil.Key Points• The fruit of Echinacea purpurea contains fungi not causing any damage to the plant.• The seed cotyledons contain endocellular bacteria.• Seed/fruit deprived of the microbiome showed a reduced ability to germinate.

In the present work, culture-based and culture-independent investigations were performed to determine the microbiota structure of the coelomic fluid of Mediterranean sea urchin Paracentrotus lividus individuals collected from two distinct geographical sites neighboring a high-density population bay and a nature reserve, respectively. Next Generation Sequencing analysis of 16S rRNA gene (rDNA) showed that members of the Proteobacteria, Bacteroidetes and Fusobacteria phyla, which have been previously reported to be commonly retrieved from marine invertebrates, dominate the overall population of microorganisms colonizing this liquid tissue, with minority bacterial genera exhibiting remarkable differences among individuals. Our results showed that there is a correlation between microbiota structure and geographical location of the echinoderm collection site, highlighting over-representation of metagenomic functions related to amino acid and bioactive peptides metabolism in specimens inhabiting the nature reserve. Finally, we also described the developmental delay and aberrations exhibited by sea urchin embryos exposed to distinct bacterial isolates, and showed that these defects rely upon hydrophilic compound(s) synthesized by the bacterial strains assayed. Altogether, our findings lay the groundwork to decipher the relationships of bacteria with sea urchins in their aquatic environment, also providing an additional layer of information to understand the biological roles of the coelomic fluid.

TIn this work, the bacterial community putatively involved in BNR events of a UCT-MBMBR pilot plant was elucidated by both culture-dependent and metagenomics DNA analyses. The presence of bacterial isolates belonging to Bacillus (in the anoxic compartment) and to Acinetobacter, Stenotrophomonas, Rhodococcus, Escherichia and Aeromonas (in the aerobic compartment) is in agreement with the nitrification/denitrification processes observed in the plant. Moreover, the study of bacterial community structure by NGS revealed a microbial diversity suggesting a biochemical complexity which can be further explored and exploited to improve UCT-MBMBR plant performance.

The chemical synthesis of novel chemotherapeutical leads is evolving thanks to possibility to design molecules with desired physical-chemical and, thus, biological properties. The imidazolium salts, recently proven effective to inhibit bacterial and/or cancer cell growth, posses an amphiphilic nature that is conferred by the imidazolium cation having a polar head generally coupled with aliphatic side chains. Thus, biological properties of imidazolium salts can be tuned through modifications involving the cation structure and/or the anion nature. By covalently linking two imidazolium rings, di-imidazolium salts were obtainedobtain differing in: i) kind of anions; ii) geometric isomerization of di-imidazolium anions; iii) length of the imidazolium alkyl side chains. Preliminarly, eleven di-imidazolium salts, differing for their anionic counterparts, were assayed for: i) antibacterial property, quantified as the minimal concentration inhibiting at least the 90% of bacterial growth (MIC90) using Escherichia coli and Micrococcus luteus as Gram-negative and Gram-positive tester strains; ii) antitumoral activity measured as the concentration inhibiting the 50% of cell growth (IC50) using SKBR-3 breast cancer cell line. All the assayed salts possesses biological activity showing i) 0.1-0.5 and 25-50 µg/ml as MIC90 values against of M. luteus, and E. coli; respectively, and ii) 30-55 µg/ml as IC50 values. Among the tested di-imidazolium salts, three were chosen to further investigate the relationship between biological efficacy and either length of alkyl side chains or imidazolium ring configurations. Geometric isomerization revealed few or no effect while a positive correlation between alkyl chain length and cell-growth inhibitory efficacy was shown. Although further studies have to be performed to elucidate the molecular mechanisms leading to cell growth arrest, this study provide insights on the attractive possibility of di-imidazolium salt exploitation as chemotherapeutical compounds whose activity can be tuned by modifying structural characteristics.

HspR (heat shock protein regulator) acts as a negative regulator of different genes in many bacteria. In Streptomyces coelicolor hspR gene is part and the transcriptional repressor of the dnaK operon which encodes the DnaK, GrpE, DnaJ chaperone machines and HspR itself. Our experiments led us to the discovery of a second promoter, internal to dnaK operon, located upstream hspR gene. Transcription from this promoter was detected at 30 degrees C indicating that hspR could play a key physiological role.

Klebsiella oxytoca DSM 29614 (KO) is a strain that produces, under anaerobic conditions, bacterial exopolysaccharides (EPSs), made of four rhamnose (Rha), two glucuronic acids (GlcA) and one galactose (Gal) bound by α and β glycosidic bonds1,2, showing metal-binding properties3. In particular, KO in the presence of AgNO3 is able to synthesize silver nanoparticles (AgNPs) incorporated within the EPS (AgNPs-EPS). The AgNPs-EPS, may contain Ag+1 when KO growing in the presence of oxygen and Ag° under anaerobic conditions, giving a different biological activity4. In the present study were evaluated the cytotoxic effects of AgNPs-EPS, produced under aerobic and anaerobic conditions, on breast cancer cell line SK-BR3. Since cancer cells have an altered metabolism in the glycolytic direction (Warburg effect), and utilize glucose as an energy font even in the presence of O2, they are able to sense the presence of the sugar present in the EPS and to pick it with greater efficiency compared to normal cells. Currently, moreover, most of the silver nanoparticles are used as antimicrobial, antifungal, antioxidant and anti-infiammatory agents in biotechnology and bioengineering, in textile engineering and in water treatment, and compared to available cytotoxic chemotherapy, they could afford to developing a biocompatible and cost-effective method of treatment for cancer. The AgNPs-EPS treatments (5 μg/ml) caused a dose dependent behavior resulting in a conspicuous inhibition of cell proliferation rate, dramatic morphological changes with apoptotic features and general proteomic modulation and the most important effects were obtained by treatment with AgNPs-EPS biosynthesised aerobically, which has been demonstrated with voltammetric analysis which issues a greater presence of Ag+1. Proteomic analysis showed modulation of several proteins related to oxidative stress and apoptotic and mitochondrial pathways. Taken together, these results offer new important elements in support of the potential antitumoral activity of AgNPs-EPS.

A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividansColon, two colonsNmESAC50 and S. lividansColon, two colonsNmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT-PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividansColon, two colonsNmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.

Background NAI-107, produced by the actinomycete Microbispora sp. ATCC-PTA-5024, is a promising lantibiotic active against Gram-positive bacteria and currently in late preclinical-phase. Lantibiotics (lanthionine-containing antibiotics) are ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded by structural genes as precursor peptides. The biosynthesis of biologically active compounds is developmentally controlled and it depends upon a variety of environmental stimuli and conditions. Inorganic phosphate (Pi) usually negatively regulates biologically-active molecule production in Actinomycetes, while it has been reported to have a positive control on lantibiotic production in Firmicutes strains. So far, no information is available concerning the Pi effect on lantibiotic biosynthesis in Actinomycetes. Results After having developed a suitable defined medium, Pi-limiting conditions were established and confirmed by quantitative analysis of polyphosphate accumulation and of expression of selected Pho regulon genes, involved in the Pi-limitation stress response. Then, the effect of Pi on Microbispora growth and NAI-107 biosynthesis was investigated in a defined medium containing increasing Pi amounts. Altogether, our analyses revealed that phosphate is necessary for growth and positively influences both growth and NAI-107 production up to a concentration of 5 mM. Higher Pi concentrations were not found to further stimulate Microbispora growth and NAI-107 production. Conclusion These results, on one hand, enlarge the knowledge on Microbispora physiology, and, on the other one, could be helpful to develop a robust and economically feasible production process of NAI-107 as a drug for human use. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0133-0) contains supplementary material, which is available to authorized users.

Bacterial infections on a sutured wound represent a critical problem, and the preparation of suture threads possessing antimicrobial properties is valuable. In this work, poly(caprolactone) (PCL) monofilaments were compounded at the concentration of 1, 2 and 4 % (w/w), respectively, to the antiseptic chlorhexidine diacetate (CHX). The incorporation was carried out in the melt by a single-step methodology, i.e. “online” approach. Mechanical tests revealed that the incorporation of CHX does not significantly change tensile properties of PCL fibres as the thermal profile adopted to prepare the compounded fibres does not compromise the antibacterial activity of CHX. In fact, CHX confers to compounded PCL fibres’ antimicrobial property even at the lowest CHX concentration as revealed by microbiological assays performed on Escherichia coli, Micrococcus luteus and Bacillus subtilis strains. The scanning electron microscope micrographs and energy-dispersive X-ray analysis of compounded threads revealed that CHX is uniformly distributed on fibre surface and that the overall amount of superficial CHX increases by increasing compounded CHX concentration. This distribution determines a biphasic CHX release kinetics characterized by an initial rapid solubilisation of superficial CHX micro-crystals, followed by a slow and gradual release of CHX incorporated in the bulk. Interestingly, the compounded threads did not show any toxic effect compromising cell viability of human fibroblasts in vitro, differently from that observed using an equal amount of pure CHX. Thus, this study originally demonstrated the effectiveness of an “online” approach to confer antimicrobial properties to an organic thermoplastic polymeric material commonly used for medical devices.

Arid lands and deserts cover one fifth of the earth’s land surface and they are currently increasing due to the global warming. Besides the natural desert settings, human activities lead to the desertification of certain areas: municipal landfills and caves are the most common examples of such anthropogenic deserts. Massive pollution and lack of natural soil profiles create harsh conditions and hinder the spontaneous rehabilitation of such areas. Phytoremediation is often used for renaturalization of such degraded soils. Two case studies are presented aiming at demonstrating that the plant-microbe interactions are essential for improving the soil quality and allowing the formation of crops. In the case study of the closed municipall landfill of “Bellolampo” (Sicily, Italy) the performances of newly planted legume shrubs inoculated with selected rhizobial and mychorrizal symbionts, were compared with uninoculated plants. Results showed that microbial symbionts significantly enhanced plant survival and growth. This study demonstrates the importance of the selection procedure aimed to the identification of the best performing strains. Selection of high-performing strains should start with the isolation of microbes from the same region where they will be applied, aiming at exploiting the endemicity, an important feature to increase the chances of soil quality promotion. Moreover, microbial symbionts often show unique metabolic properties due to the co-evolution with the specific host. In natural deserts and arid lands, where few vascular plant species can live, primary colonizers of the barren mineral substrates and suitable hosts for strain selection are lichens. These symbiotic holobionts harbor specific bacterial communities as dense as up to more than 1010 bacterial cells per gram of biomass (dw). These communities are metabolically active and contribute to the survival of the lichen symbiosis in extreme habitats. The wide microbial diversity of the symbioses associated to desert lichens could be a valuable source of new microbial strains particularly suitable for biotechnological applications in arid lands.

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.

Background A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant. Results Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production. Conclusion Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.

The actinomycete Nonomuraea produces the glycopeptide A40926, precursor of dalbavancin. Dalbavancin is a novel lipoglycopeptide agent against emerging resistant Gram-positive cocci with superior pharmacodynamics properties compared to vancomycin. Chemically, a glycopeptide antibiotic consists of a heptapeptide core constituted by proteinogenic and nonproteinogenic amino acids such as 3,5-dihydroxyphenylglycine (DPG) and 4-hydroxyphenylglycine (HPG). The heptapeptide is assembled by a nonribosomal peptide synthetase and modified by oxidative cross-linking of the electron-rich aromatic side chains, halogenation, sulfation, methylation, acylation, and glycosylation. 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. The purpose of this study is to investigate the role of these regulators in A40926 biosynthesis. Mutants in each regulator were generated. Bioassay and HPLC analysis revealed that Dbv3 and Dbv4 are necessary for antibiotic production. Dbv4, a StrR-like protein, was found to positively regulate the transcription of 13 genes, encoding DPG synthesizing enzymes and modification enzymes. Dbv3 is a putative LuxR-like regulator and the search for Dbv3-targeted genes is ongoing by transcriptional and biochemical analysis. Surprisingly, the response regulator Dbv6 does not seem to influence A40926 production. Chemical and biochemical analysis are in progress to elucidate its role. These studies will provide new insights in gene expression of gram-positive bacteria, in particular they will elucidate the complex regulatory network governing antibiotic production. Moreover, these analyses will offer a platform for the rational manipulation of Nonomuraea for increased production of A40926.

Both industry and academia have shown a growing interest in materials with antimicrobial properties suitable for food packaging applications. In this study, we prepared and characterized thin films of ethylene-co-vinyl acetate (EVA) copolymer with antimicrobial properties. The films were prepared with a film blowing process by incorporating a nisin preparation as an antimicrobial agent in the melt. Two grades of EVA containing 14 and 28% (wt/wt) vinyl acetate (EVA 14 and EVA 28, respectively) and two commercial formulations of nisin with different nominal activities were used. The effect of the nisin concentration also was evaluated. The films with the highest antimicrobial activity were those formulated with nisin at the highest activity and EVA with the highest content of vinyl acetate. The use of the commercial formulation of nisin with high activity in the EVA films allowed reduction in the amount of nisin needed to provide antimicrobial properties. Consequently, the mechanical properties of these films were only slightly inferior to those of the pure polymers. In contrast, films prepared by incorporating more of the nisin with lower activity had poor mechanical properties. The effect of different processing temperatures used in the preparation of the films on the antimicrobial properties of the films also was evaluated. The materials displayed antimicrobial properties even when they were prepared at temperatures as high as 160 °C, probably because of the very short processing time (60 to 90 s) required for preparation.

Introduction: Alkanes are biodegraded to generate the corresponding primary alcohol trough alkane hydroxylases (AHs) consisting on an integral membrane alkane monooxygenase (AlkB) and two soluble proteins, rubredoxin and rubredoxin reductase. Recently, an alkB gene was reported to be involved in degradation of long chain n-alkanes in the actinobacterium Gordonia sp. SoCg. This gene was expressed in Streptomyces coelicolor M145 which is unable to degrade n-alkanes. Results: The engineered strain, M145-AH, can biotransform n-hexadecane into the corresponding 1-hexadecanol and it is able to grow on n-hexadecane as sole carbon source. Changes in global protein expression associated with n-hexadecane metabolism in M145-AH were studied using a differential proteomic approach. M145-AH was incubated in three different conditions using a mineral medium supplemented with hexadecane or glucose as the sole carbon source or without any carbon source, respectively. Total proteins, extracted from samples collected after 6, 24 and 48 h of incubation in the three conditions, were analyzed by 2D-Differential Gel Electrophoresis (2D-DIGE). Differentially abundant protein spots were identified by mass spectrometry. The expression profile of proteins involved in central carbon metabolism, amino acid and protein biosynthesis, fatty acid metabolism and respiration revealed a gradual metabolic adaptation to n-hexadecane utilization, which is similar to that of specialized alkane-degraders. Conclusion: Thus, the addition of the alkB gene confers to Streptomyces coelicolor the ability to use an insoluble recalcitrant contaminant as an usual carbon source. These data, expanding the knowledge on n-alkane bioconversion mechanisms in Gram-positive bacteria, provide new technological platforms for bioremediation studies and strategies.

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 (rubredoxin), and RubB (rubredoxin reductase) were induced by both n -hexadecane and n -triacontane, which were chosen as representative long-chain liquid and solid n -alkane molecules, respectively. Biotransformation of n -hexadecane into the corresponding 1-hexadecanol was detected by solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME/GC-MS) analysis. The Gordonia SoCg alkB was heterologously expressed in Escherichia coli BL21 and in Streptomyces coelicolor M145, and both hosts acquired the ability to transform n -hexadecane into 1-hexadecanol, but the corresponding long-chain alcohol was never detected on n -triacontane. However, the recombinant S. coelicolor M145-AH, expressing the Gordonia alkB gene, was able to grow on n -triacontane as the sole C source. A SoCg alkB disruption mutant that is completely unable to grow on n -triacontane was obtained, demonstrating the role of an AlkB-type AH system in degradation of solid n -alkanes.

Background A bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics. Results One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These orfs encode two proteins with high similarity to a putative membrane protein of Streptomyces coelicolor and to the nogalamycin resistance protein SnorO of Streptomyces nogalater, respectively. The role of these two Orfs is unknown in Planobispora. Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker. Conclusion Two Planobispora rosea orfs are responsible for increasing intracellular amounts and storage of thiostrepton in Streptomyces lividans.

A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially and constitutively expressed protein isoforms, which were associated to 203 ORFs in the A. balhimycina genome. These data, providing insights on the major metabolic pathways/molecular processes operating in this organism, were used to compile 2-DE reference maps covering 3-10, 4-7 and 4.5-5.5 pH gradients available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal-proteome-maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis, energetic and redox balance, sugar/amino sugar metabolism, balhimycin biosynthesis and transcriptional regulation or with hypothetical and/or unknown function. Interestingly, proteins involved in the biosynthesis of balhymycin precursors, such as amino acids, amino sugars and central carbon metabolism intermediates, were up-regulated during antibiotic production. qRT-PCR analysis revealed that 8 out of 14 up-regulated genes showed a positive correlation between changes at translational and transcriptional expression level. Furthermore, proteomic analysis of two non-producing mutants, restricted to a sub-set of differentially expressed proteins, showed that most proteins required for the biosynthesis of balhimycin precursors are down-regulated in both mutants. These findings suggest that primary metabolic pathways support anabolic routes leading to balhimycin biosynthesis and the differentially expressed genes are interesting targets for the construction of high-yielding producer strains by rational genetic engineering.

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 (M145-AH), and the production of 1-hexadecanol from n-hexadecane oxidation was observed3. A differential study of global gene expression of M145-AH cultures was performed, where n-hexadecane (C16) glucose (GLU) and none (NC) were provided as only carbon source, respectively. Proteomic analysis, based on 2D-DIGE and MS procedures, revealed a gradual metabolic adaptation to n-hexadecane utilization, not dissimilar from that one revealed in specialized alkane-degraders4. In addition, expression profiles of central carbon metabolism enzymes revealed that the addition of a single gene confers the ability to use recalcitrant pollutants as simple sugars in Streptomyces. Altogether these data, expanding the knowledge on n-alkane bioconversion mechanisms in Gram positive bacteria, could provide new technological platforms for bioremediation studies and strategies. Quatrini, P., Scaglione, G., De Pasquale, C., Riela, S., & Puglia, A. M. (2008). Isolation of gram-positive n-alkane degraders from a hydrocarbon- contaminated mediterranean shoreline. Journal of Applied Microbiology, 104(1), 251-259. Lo Piccolo, L., De Pasquale, C., Fodale, R., Puglia, A. M. & Quatrini, P. . An alkane hydroxylase system of Gordonia sp. Strain SoCg is involved in degradation of solid n-alkanes. Applied and Environmental Microbiology. In revision. van Beilen, J. B., & Funhoff, E. G. (2007). Alkane hydroxylases involved in microbial alkane degradation. Applied Microbiology and Biotechnology, 74(1), 13-21. Sabirova, J. S., Ferrer, M., Regenhardt, D., Timmis, K. N., & Golyshin, P. N. (2006). Proteomic insights into metabolic adaptations in Alcanivorax borkumensis induced by alkane utilization. Journal of Bacteriology, 188(11), 3763-3773.

Susceptibility to desertification in southern Europe is increasing and rehabilitation of desertification-threatened Mediterranean soils is a challenge due to the inhospitality of the environment. In particular, recovery of anthropogenic soils (mainly human-derived artefacts from housing construction and other inert materials or topsoil of terminal phase municipal landfills) cannot rely on spontaneous processes and low-cost/low-impact strategies are needed to prevent desertification. Mediterranean wild legume shrubs have great potential for soil recovery and conservation against desertification, thanks to drought resistance, and their symbiosis with N2-fixing rhizobia and arbuscular mycorrhizal fungi. In this study double inoculated autochthonous wild legume shrubs (the genistea Spartium junceum L. and the thermopsidea Anagyris foetida L.) were used in a long-term trial to recover an anthropogenic hill in a semi-arid site of southern Italy, mainly composed of inert and human-derived artefacts. Microbial inoculants strongly enhanced plant establishment and growth on the anthropogenic soil in the greenhouse and in the field up to two years. Automated Ribosomal Intergenic Spacer Analysis (ARISA) and bacterial cultivation revealed a dramatic effect of the tripartite symbiosis on the structure of soil bacterial communities that largely overcomes plant species effect and suggests synergism of AMF with the bacterial community of the mycorhizosphere. Our results demonstrate that microsymbiont inoculation on wild legume shrubs is a promising strategy to rehabilitate anthropogenic soils in Mediterranean semi-arid regions.

Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protocol to purify, fractionate, blot, and hybridize Streptomyces RNA. It is based on grinding by a high-performance laboratory ball mill, followed by prompt lysis with acid phenol-guanidinium, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.

The tryptophan is a precursor of the lipopeptide calcium dependent antibiotic (CDA), produced from Streptomyces coelicolor and closely related to important antibiotics such as daptomycin. We have focused our attention on the correlation between CDA production and tryptophan metabolism in order to identify new strategies aimed at increasing the production of peptide antibiotics.

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. Interestingly, SPME GC-MS analysis revealed extracellular production of hexadecyl-hexadecanoic acid (wax ester), during growth on n-triacontane. Degenerated oligonucleotides were used to PCR amplify the Wax Esther Synthase/Acyl-CoA:Diacylglycerol Acyltransferase encoding gene (atfa), responsible for intracellular wax esther synthesis by estherification between palmitoyl-CoA and 1-haxadecanol in the n-alkane degrader Acinetobacter sp. ADP1. The amplicon showed 75% similarity sequence to a putative acyl-CoA transferase gene of Nocardia farcinica. Only one copy of atfa-like gene was detected in Gordonia SoCg chromosome. qRT-PCR analysis showed an up-regulation of atfa gene in the presence of long chain n-alkanes, correlating, for the first time, long chain n-alkane metabolism and wax esthers extracellular production.