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Proteomics has been widely used to characterize milk protein content in livestock species and unravel protein biological functions. The aim of this work was to characterize the Girgentana goat breed whole milk and milk fraction proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. Bulk milk samples from two farms locate in Palermo and Agrigento (Sicily) areas were collected during the early stage of lactation. Initially, 2D-PAGE and protein detection protocols were set-up. In particular, 2D-PAGE analyses were carried out on whole milk using 18-cm IPG strips ranging from pH 3-10 and 4-7 and 12.5% polyacrylamide gel for first and the second dimension separation, respectively. Then, staining methods (silver-, colloidal Coomassie Blue G-250- and fluorescent dye-based method) were tested to reveal protein spots in 2D-gels. In silico 2D-gel image analysis, performed using Image Master 2D Platinum software, revealed that silver and fluorescent (based on SyproTM Ruby) staining methods showed similar results in terms of sensitivity/resolution. Then, centrifuge-based protocols were set-up to obtain milk protein fractions. The protein profiles of whole milk, caseins, whey and milk fat globule protein fractions were qualitatively compared in silico. This analysis showed that casein content decreased in both whey and fat globule protein fraction respect to caseins fraction which, instead, missed specific protein spots revealing in whey and/or fat globule fraction. Finally, a global protein differential expression analysis of whole milk samples from Palermo and Agrigento, respectively, was performed by 2D-Differential Gel Electrophoresis (2D-DIGE) using pH range 3-10. In silico analysis of the 2D-DIGE gel images (at least three per sample) showed a total of 143 differentially abundant spots with more than 1.5 fold change in volume and a statistically significant ANOVA value (p < 0.05) with 58 up-regulated and 84 down-regulated in Palermo farm milk respect to the Agrigento one. In addition, differential expression analysis will be also performed on milk fractions from Palermo and Agrigento, respectively, and the differentially abundant proteins and others showing invariant abundance will be identified by mass spectrometry to generate reference milk proteomic maps of Girgentana goat breed which could be useful to identify specific molecular marker assessing milk quality.

Actinomycetes are an important source of biologically active compounds, like antibiotics, antitumor agents, and immunosuppressors. Genome sequencing is revealing that this class of microorganisms has larger genomes relative to other bacteria and uses a considerable fraction of its coding capacity (5–10%) for the production of mostly cryptic secondary metabolites. To access actinomycetes biosynthetic capabilities or to improve the pharmacokinetic properties and production yields of these chemically complex compounds, genetic manipulation of the producer strains can be performed. Heterologous expression in amenable hosts can be useful to exploit and to explore the genetic potential of actinomycetes and not cultivable but interesting bacteria. Artificial chromosomes that can be stably integrated into the Streptomyces genome were constructed and demonstrated to be effective for transferring entire biosynthetic gene clusters from intractable actinomycetes into more suitable hosts. In this paper, the construction of several shuttle Escherichia coli-Streptomyces artificial chromosomes is discussed together with old and new strategies applied to improve heterologous production of secondary metabolites.

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.

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.

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.

8701-BC cells were shown to release “membrane vesicles” playing a role in tumor progression mechanisms. On the other hand, production of “exosomes”, smaller vesicles known to be involved in immune response activation, had not been revealed. The first goal of this study was to separate different vesicle populations from 8701-BC cell conditioned medium. To this aim, the medium was differentially centrifuged. Western analysis revealed that the 15,000xg pelletted fraction contains β1-integrin, which had been shown to be clustered in membrane vesicles shed by 8701-BC cells, but not Hsc70, a protein found in exosomes. On the contrary, Hsc70 is detectable while β1-integrin is not present in the fraction obtained by a further centrifugation at 100,000xg from 15,000xg supernatant. Moreover, the absence of Cytochrom C in both fractions excludes the contamination with apoptotic vesicles.These results suggested that 8701-BC cells release both membrane vesicles and exosomes and that their separation can be achieved by differential centrifugation.Then, to analyze the whole protein content of the vesicle preparations, a proteomic approach was chosen. Protein 2D-PAGE analysis was performed and the gel images were analyzed in silico, using ImageMaster 2D Platinum software. The preliminary comparative proteomic analysis revealed a set of protein spots differently abundant in the vesicle fractions. These data strongly encourage for further investigation using 2D-PAGE coupled with MS-MALDI-TOF analysis which could highlight physiological roles of the two different kinds of vesicles.