Bioprospecting natural products in marine bacteria from fjord environments are attractive due to their unique geographical features. Although Actinobacteria are well known for producing a myriad of bioactive compounds, investigations regarding fjord-derived marine Actinobacteria are scarce. In this study, the diversity and biotechnological potential of Actinobacteria isolated from marine sediments within the Comau fjord, in Northern Chilean Patagonia, were assessed by culture-based approaches. The 16S rRNA gene sequences revealed that members phylogenetically related to the Micrococcaceae, Dermabacteraceae, Brevibacteriaceae, Corynebacteriaceae, Microbacteriaceae, Dietziaceae, Nocardiaceae and Streptomycetaceae families were present at the Comau fjord. A high diversity of cultivable Actinobacteria (10 genera) was retrieved by using only five different isolation media. Four isolates belonging to Arthrobacter, Brevibacterium, Corynebacterium and Kocuria genera showed 16S rRNA gene identity <98.7% suggesting that they are novel species. Physiological features such as salt tolerance, artificial sea water requirement, growth temperature, pigmentation and antimicrobial activity were evaluated. Arthrobacter, Brachybacterium, Curtobacterium, Rhodococcus and Streptomyces isolates showed strong inhibition against both Gram-negative Pseudomonas aeruginosa, Escherichia coli and Salmonella enterica and Gram-positive Staphylococcus aureus, Listeria monocytogenes. Antimicrobial activities in Brachybacterium, Curtobacterium and Rhodococcus have been scarcely reported, suggesting that non-mycelial strains are a suitable source of bioactive compounds. In addition, all strains bear at least one of the biosynthetic genes coding for NRPS (91%), PKS I (18%) and PKS II (73%).Our results indicate that the Comau fjord is a promising source of novel Actinobacteria with biotechnological potential for producing biologically active compounds.

The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions −5 and −36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37°C, a nutrient-rich environment, high osmolarity, and the presence of Mn ²⁺ . Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans . The EDL933Δ sepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans . These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.

Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase—named Chi18H8 and belonging to family 18 glycosyl hydrolases—was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 μg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential. We report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate. Expression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies.

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d -alanyl- d -alanine of lipid II with d -alanyl- d -lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide A40926, which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward A40926 during vegetative growth was 4 μg/ml, but this increased to ca. 20 μg/ml during A40926 production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for A40926 production contains a gene, vanY (ORF7), potentially encoding an enzyme capable of removing the terminal d -Ala residue of pentapeptide peptidoglycan precursors. Analysis of UDP-linked peptidoglycan precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide UDP-MurNAc- l -Ala- d -Glu- meso -Dap- d -Ala and only traces of the pentapeptide UDP-MurNAc- l -Ala- d -Glu- meso -Dap- d -Ala- d -Ala. This suggested a novel mechanism of glycopeptide resistance in Nonomuraea sp. ATCC 39727 that was based on the d , d -carboxypeptidase activity of vanY . Consistent with this, a vanY -null mutant of Nonomuraea sp. ATCC 39727 demonstrated a reduced level of glycopeptide resistance, without affecting A40926 productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae , resulted in higher levels of glycopeptide resistance.

Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10⁷–10⁹ protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments.