Biological activity of silver nanoparticles synthesized using viticultural waste.

This research paper presents a novel approach to the green synthesis of silver nanoparticles (AgNPs) using viticultural waste, allowing to obtain NP dispersions with distinct properties and morphologies (monodisperse and polydisperse AgNPs, referred to as mAgNPs and pAgNPs) and to compare their biological activities. Our synthesis method utilized the ethanolic extract of Vitis vinifera pruning residues, resulting in the production of mAgNPs and pAgNPs with average sizes of 12 ± 5 nm and 19 ± 14 nm, respectively. Both these AgNPs preparations demonstrated an exceptional stability in terms of size distribution, which was maintained for one year. Antimicrobial testing revealed that both types of AgNPs inhibited either the growth of planktonic cells or the metabolic activity of biofilm sessile cells in Gram-negative bacteria and yeasts. No comparable activity was found towards Gram-positives. Overall, pAgNPs exhibited a higher antimicrobial efficacy compared to their monodisperse counterparts, suggesting that their size and shape may provide a broader spectrum of interactions with target cells. Both AgNP preparations showed no cytotoxicity towards a human keratinocyte cell line. Furthermore, in vivo tests using a silkworm animal model indicated the biocompatibility of the phytosynthesized AgNPs, as they had no adverse effects on insect larvae viability. These findings emphasize the potential of targeted AgNPs synthesized from viticultural waste as environmentally friendly antimicrobial agents with minimal impact on higher organisms.

Actinoplanes oblitus sp. nov., producing the glycopeptide antibiotic A477.

In 1973, Eli Lilly and Company described the filamentous actinomycete producing the glycopeptide antibiotic A477 as an Actinoplanes species on the basis of its morphological and physiological features and deposited it as NRRL 3884T. In this paper, we report that the phylogenetic analysis based on the 16S rRNA gene sequence and the whole genome phylogenomic study indicate that NRRL 3884T forms a distinct monophyletic line within the genus Actinoplanes, being most closely related to Actinoplanes octamycinicus NBRC 14524T [99.6 % 16S rRNA gene similarity, 89.4 % average nucleotide identity (ANI), 46.0 % digital DNA-DNA hybridization (dDDH)] and Actinoplanes ianthinogenes NBRC 13996T (98.8 % 16S rRNA gene similarity, 89.0 % ANI, 47.0 % dDDH). NRRL 3884T forms an extensively branched, non-fragmented vegetative mycelium; either sterile aerial hyphae or regular subglobose sporangia are formed depending on cultivation conditions. The cell wall contains meso-2,6-diaminopimelic acid and 2,6-diamino-3-hydroxypimelic acid and the diagnostic sugars are glucose, mannose and ribose with a minor amount of rhamnose. The predominant menaquinone (MK) is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H8). Mycolic acids are absent. The diagnostic phospholipids are diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids are anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0, with moderate amounts of anteiso-C15 : 0 and iso-C17 : 0. The genomic G+C content is 71.5 mol%. Significant differences in the genomic, morphological, chemotaxonomic and biochemical data between NRRL 3884T and the two most closely related Actinoplanes type strains clearly demonstrate that NRRL 3884T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes oblitus sp. nov. is proposed. The type strain is NRRL 3884T (=DSM 116196T).

Biocatalyzed Synthesis of Glycostructures with Anti-infective Activity.

Molecules containing carbohydrate moieties play essential roles in fighting a variety of bacterial and viral infections. Consequently, the design of new carbohydrate-containing drugs or vaccines has attracted great attention in recent years as means to target several infectious diseases.Conventional methods to produce these compounds face numerous challenges because their current production technology is based on chemical synthesis, which often requires several steps and uses environmentally unfriendly reactants, contaminant solvents, and inefficient protocols. The search for sustainable processes such as the use of biocatalysts and eco-friendly solvents is of vital importance. Therefore, their use in a variety of reactions leading to the production of pharmaceuticals has increased exponentially in the last years, fueled by recent advances in protein engineering, enzyme directed evolution, combinatorial biosynthesis, immobilization techniques, and flow biocatalysis. In glycochemistry and glycobiology, enzymes belonging to the families of glycosidases, glycosyltransferases (Gtfs), lipases, and, in the case of nucleoside and nucleotide analogues, also nucleoside phosphorylases (NPs) are the preferred choices as catalysts.In this Account, on the basis of our expertise, we will discuss the recent biocatalytic and sustainable approaches that have been employed to synthesize carbohydrate-based drugs, ranging from antiviral nucleosides and nucleotides to antibiotics with antibacterial activity and glycoconjugates such as neoglycoproteins (glycovaccines, GCVs) and glycodendrimers that are considered as very promising tools against viral and bacterial infections.In the first section, we will report the use of NPs and N-deoxyribosyltransferases for the development of transglycosylation processes aimed at the synthesis of nucleoside analogues with antiviral activity. The use of deoxyribonucleoside kinases and hydrolases for the modification of the sugar moiety of nucleosides has been widely investigated.Next, we will describe the results obtained using enzymes for the chemoenzymatic synthesis of glycoconjugates such as GCVs and glycodendrimers with antibacterial and antiviral activity. In this context, the search for efficient enzymatic syntheses represents an excellent strategy to produce structure-defined antigenic or immunogenic oligosaccharide analogues with high purity. Lipases, glycosidases, and Gtfs have been used for their preparation.Interestingly, many authors have proposed the use Gtfs originating from the biosynthesis of natural glycosylated antibiotics such as glycopeptides, macrolides, and aminoglycosides. These have been used in the chemoenzymatic semisynthesis of novel antibiotic derivatives by modification of the sugar moiety linked to their complex scaffold. These contributions will be described in the last section of this review because of their relevance in the fight against the spreading phenomenon of antibiotic resistance. In this context, the pioneering in vivo synthesis of novel derivatives obtained by genetic manipulation of producer strains (combinatorial biosynthesis) will be shortly described as well.All of these strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can contribute to the sustainable development of complex glycan-based therapies and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and selectivity. The use of enzymatic synthesis is becoming a reality in the pharmaceutical industry and in drug discovery to rapidly afford collections of new antibacterial or antiviral molecules with improved specificity and better metabolic stability.

Heterologous Expression Reveals Ancient Properties of Tei3­A VanS Ortholog from the Teicoplanin Producer Actinoplanes teichomyceticus

Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus­the producer of the last-resort-drug teicoplanin­has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.

Laboratory scale cultivation of Salinispora tropica in shake flasks and mechanically stirred bioreactors.

OBJECTIVE: Marine actinomycetes from the genus Salinispora have an unexploited biotechnological potential. To accurately estimate their application potential however, data on their cultivation, including biomass growth kinetics, are needed but only incomplete information is currently available. RESULTS: This work provides some insight into the effect of temperature, salinity, nitrogen source, glucose concentration and oxygen supply on growth rate, biomass productivity and yield of Salinispora tropica CBN-440T. The experiments were carried out in unbaffled shake flasks and agitated laboratory-scale bioreactors. The results show that the optimum growth temperature lies within the range 28-30 °C, salinity is close to sea water and the initial glucose concentration is around 10 g/L. Among tested nitrogen sources, yeast extract and soy peptone proved to be the most suitable. The change from unbaffled to baffled flasks increased the volumetric oxygen transfer coefficient (kLa) as did the use of agitated bioreactors. The highest specific growth rate (0.0986 h-1) and biomass productivity (1.11 g/L/day) were obtained at kLa = 28.3 h-1. A further increase in kLa was achieved by increasing stirrer speed, but this led to a deterioration in kinetic parameters. CONCLUSIONS: Improvement of S. tropica biomass growth kinetics of was achieved mainly by identifying the most suitable nitrogen sources and optimizing kLa in baffled flasks and agitated bioreactors.

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System.

RESEARCH BACKGROUND: In recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil. EXPERIMENTAL APPROACH: We used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates. RESULTS AND CONCLUSIONS: Profile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol. NOVELTY AND SCIENTIFIC CONTRIBUTION: Our results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes.

Description of the bacterial RNA polymerase inhibitor GE23077-producer Actinomadura sp. NRRL B-65521T as Actinomadura lepetitiana sp. nov.

The filamentous actinomycete that produces the antibiotic GE23077 was isolated by the Lepetit Research Group from a soil sample collected in Thailand, and it was classified as a member of the genus Actinomadura on the basis of its morphology and cell-wall composition. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain formed a distinct monophyletic line within the genus Actinomadura, and it was most closely related to Actinomadura bangladeshensis DSM 45347T (99.31 % similarity) and Actinomadura mexicana DSM 44485T (98.94 %). The GE23077-producing strain formed an extensively branched, non-fragmented vegetative mycelium; no pseudosporangia were formed and the arthrospores were organized in slightly twisted chains. The cell wall contained meso-2,6-diaminopimelic acid and the diagnostic sugar was madurose. The predominant menaquinone was MK-9(H6), with minor amounts of MK-9(H8) and MK-9(H4). The diagnostic phospholipids were phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were C16 : 0 and tuberculostearic acid (10-methyloctadecanoic acid), followed by minor amounts of C18:1ω9c, C16:1ω7c and 10-methylheptadecanoic acid. The genomic DNA G+C content was 71.77 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, and the low DNA-DNA relatedness between the GE23077-producing strain and closely related type strains clearly demonstrate that it represents a novel species of the genus Actinomadura, for which the name Actinomadura lepetitiana sp. nov. is proposed. The type strain is NRRL B-65521T(=LMG 31258T=DSM 109019T).

Teicoplanin biosynthesis: unraveling the interplay of structural, regulatory, and resistance genes.

Teicoplanin (Tcp) is a clinically relevant glycopeptide antibiotic (GPA) that is produced by the actinobacterium Actinoplanes teichomyceticus. Tcp is a front-line therapy for treating severe infections caused by multidrug-resistant Gram-positive pathogens in adults and infants. In this review, we provide a detailed overview of how Tcp is produced by A. teichomyceticus by describing Tcp biosynthesis, regulation, and resistance. We summarize the knowledge gained from in vivo and in vitro studies to provide an integrated model of teicoplanin biosynthesis. Then, we discuss genetic and nutritional factors that contribute to the regulation of teicoplanin biosynthesis, focusing on those that have been successfully applied for improving teicoplanin production. A current view on teicoplanin self-resistance mechanisms in A. teichomyceticus is given, and we compare the Tcp biosynthetic gene cluster with other glycopeptide gene clusters from actinoplanetes and from unidentified isolates/metagenomics samples. Finally, we provide an outlook for further directions in studying Tcp biosynthesis and regulation.

Regulation of teicoplanin biosynthesis: refining the roles of tei cluster-situated regulatory genes.

Teicoplanin is a frontline glycopeptide antibiotic produced by Actinoplanes teichomyceticus. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the tei biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in A. teichomyceticus. We demonstrate that the tei cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of tei biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in A. teichomyceticus is not co-regulated with antibiotic production. The gene tei31*, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.

Classification of Actinoplanes sp. ATCC 33076, an actinomycete that produces the glycolipodepsipeptide antibiotic ramoplanin, as Actinoplanes ramoplaninifer sp. nov.

Strain ATCC 33076, which produces the antibiotic ramoplanin, was isolated from a soil sample collected in India, and it was classified as a member of the genus Actinoplanes on the basis of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain forms a distinct clade within the genus Actinoplanes, and it is most closely related to Actinoplanes deccanensis IFO 13994T (98.71 % similarity) and Actinoplanes atraurantiacus Y16T (98.33 %). The strain forms an extensively branched substrate mycelium; the sporangia are formed very scantily and are globose with irregular surface. Spores are oval and motile. The cell wall contains meso-diaminopimelic acid and the diagnostic sugars are xylose and arabinose. The predominant menaquinone is MK-9(H6), with minor amounts of MK-9(H4) and MK-9(H2). Mycolic acids are absent. The diagnostic phospholipids are phosphatidylethanolamine, hydroxyphosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids are anteiso-C17 : 0 and iso-C16 : 0, followed by iso-C15 : 0 and moderate amounts of anteiso-C15 : 0, iso-C17 : 0 and C18 : 1ω9c. The genomic DNA G+C content is 71.4 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 33076 and closely related type strains, clearly demonstrated that strain ATCC 33076 represents a novel species of the genus Actinoplanes, for which the name Actinoplanes ramoplaninifer sp. nov. is proposed. The type strain is ATCC 33076T (=DSM 105064T=NRRL B-65484T).

Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The first acidobacterial laccase-like multicopper oxidase revealed by metagenomics shows high salt and thermo-tolerance.

Metagenomics is a powerful tool that allows identifying enzymes with novel properties from the unculturable component of microbiomes. However, thus far only a limited number of laccase or laccase -like enzymes identified through metagenomics has been subsequently biochemically characterized. This work describes the successful bio-mining of bacterial laccase-like enzymes in an acidic bog soil metagenome and the characterization of the first acidobacterial laccase-like multicopper oxidase (LMCO). LMCOs have hitherto been mostly studied in fungi and some have already found applications in diverse industries. However, improved LMCOs are in high demand. Using molecular screening of a small metagenomic library (13,500 clones), a gene encoding a three-domain LMCO (LacM) was detected, showing the highest similarity to putative copper oxidases of Candidatus Solibacter (Acidobacteria). The encoded protein was expressed in Escherichia coli, purified by affinity chromatography and biochemically characterized. LacM oxidized a variety of phenolic substrates, including two standard laccase substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), k cat/k M  = 8.45 s-1 mM-1; 2,6-dimethoxyphenol (2,6-DMP), k cat/k M  = 6.42 s-1 mM-1), next to L-3,4-dihydroxyphenylalanine (L-DOPA), vanillic acid, syringaldazine, pyrogallol, and pyrocatechol. With respect to the latter two lignin building blocks, LacM showed the highest catalytic activity (k cat/k M  = 173.6 s-1 mM-1) for pyrogallol, with ca. 20% activity preserved even at pH 8.0. The enzyme was thermostable and heat-activated in the interval 40-60 °C, with an optimal activity on ABTS at 50 °C. It was rather stable at high salt concentration (e.g., 34% activity preserved at 500 mM NaCl) and in the presence of organic solvents. Remarkably, LacM decolored azo and triphenylmethane dyes, also in the absence of redox mediators.

Classification of Nonomuraea sp. ATCC 39727, an actinomycete that produces the glycopeptide antibiotic A40926, as Nonomuraea gerenzanensis sp. nov.

Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic dalbavancin), was isolated from a soil sample collected in India, and it was originally classified as a member of the genus Actinomadura on the base of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicates that the strain forms a distinct clade within the genus Nonomuraea, and it is most closely related to Nonomuraea angiospora DSM 43173T (98.72 % similarity) and Nonomuraea jabiensis A4036T (98.69 %). The strain forms an extensively branched substrate mycelium and aerial hyphae that form spiral chains of spores with ridged surfaces. The cell wall contains meso-diaminopimelic acid and the whole-cell sugars are glucose, ribose, galactose, mannose and madurose (madurose as the diagnostic sugar). The N-acyl type of muramic acid is acetyl. The predominant menaquinone is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H0). The polar-lipid profile includes diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylmethylethanolamine, phosphatidylinositol and a series of uncharacterized phospholipids, glycolipids and phosphoglycolipids. The major cellular fatty acids are iso-C16 : 0 and 10-methyl C17 : 0. The genomic DNA G+C content is 71.2 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 39727 and closely related type strains, clearly demonstrated that strain ATCC 39727 represents a novel species of the genus Nonomuraea, for which the name Nonomuraea gerenzanensis sp. nov. is proposed. The type strain is ATCC 39727T ( = DSM 100948T).

A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil.

Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of sizes of 21 to 40 kb, yielding a total of approximately 5.8 GB of cloned soil DNA. Using genetic screenings by repeated PCR cycles aimed to detect gene sequences of the bacterial chitinase A-class (hereby named chi A genes), we identified and characterized five fosmids carrying candidate genes for chitinolytic enzymes. The analysis thus allowed access to the genomic (fosmid-borne) context of these genes. Using the chiA-targeted PCR, which is based on degenerate primers, the five fosmids all produced amplicons, of which the sequences were related to predicted chitinolytic enzyme-encoding genes of four different host organisms, including Stenotrophomonas maltophilia. Sequencing and de novo annotation of the fosmid inserts confirmed that each one of these carried one or more open reading frames that were predicted to encode enzymes active on chitin, including one for a chitin deacetylase. Moreover, the genetic contexts in which the putative chitinolytic enzyme-encoding genes were located were unique per fosmid. Specifically, inserts from organisms related to Burkholderia sp., Acidobacterium sp., Aeromonas veronii, and the chloroflexi Nitrolancetus hollandicus and/or Ktedonobacter racemifer were obtained. Remarkably, the S. maltophilia chiA-like gene was found to occur in two different genetic contexts (related to N. hollandicus/K. racemifer), indicating the historical occurrence of genetic reshufflings in this part of the soil microbiota. One fosmid containing the insert composed of DNA from the N. hollandicus-like organism (denoted 53D1) was selected for further work. Using subcloning procedures, its putative gene for a chitinolytic enzyme was successfully brought to expression in an E. coli host. On the basis of purified protein preparations, the produced protein was characterized as a chitobiosidase of 43.6 kDa, with a pI of 4.83. Given its activity spectrum, it can be typified as a halotolerant chitobiosidase.

Relationship between glycopeptide production and resistance in the actinomycete Nonomuraea sp. ATCC 39727.

Glycopeptides and ß-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the ß-lactam-sensitive D,D-peptidase/D,D-carboxypeptidase encoded by vanYn, the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanYn gene dosage and expressed vanHatAatXat from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanYn, complementing a vanYn mutant, or duplicating vanYn had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYn activity. The heterologous expression of vanHatAatXat increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanYn-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.

New insights into glycopeptide antibiotic binding to cell wall precursors using SPR and NMR spectroscopy.

Glycopeptide antibiotics, such as vancomycin and teicoplanin, are used to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. They inhibit bacterial cell wall biosynthesis by binding to the D-Ala-D-Ala C-terminus of peptidoglycan precursors. Vancomycin-resistant bacteria replace the dipeptide with the D-Ala-D-Lac depsipeptide, thus reducing the binding affinity of the antibiotics with their molecular targets. Herein, studies of the interaction of teicoplanin, teicoplanin-like A40926, and of their semisynthetic derivatives (mideplanin, MDL63,246, dalbavancin) with peptide analogues of cell-wall precursors by NMR spectroscopy and surface plasmon resonance (SPR) are reported. NMR spectroscopy revealed the existence of two different complexes in solution, when the different glycopeptides interact with Ac2KdAlaDAlaOH. Despite the NMR experimental conditions, which are different from those employed for the SPR measurements, the NMR spectroscopy results parallel those deduced in the chip with respect to the drastic binding difference existing between the D-Ala and the D-Lac terminating analogues, confirming that all these antibiotics share the same primary molecular mechanism of action and resistance. Kinetic analysis of the interaction between the glycopeptide antibiotics and immobilized AcKdAlaDAlaOH by SPR suggest a dimerization process that was not observed by NMR spectroscopy in DMSO solution. Moreover, in SPR, all glycopeptides with a hydrophobic acyl chain present stronger binding with a hydrophobic surface than vancomycin, indicating that additional interactions through the employed surface are involved. In conclusion, SPR provides a tool to differentiate between vancomycin and other glycopeptides, and the calculated binding affinities at the surface seem to be more relevant to in vitro antimicrobial activity than the estimations from NMR spectroscopy analysis.

Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics.

Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.

The pathway-specific regulatory genes, tei15* and tei16*, are the master switches of teicoplanin production in Actinoplanes teichomyceticus.

Pathogenic antibiotic-resistant bacteria are an unprecedented threat to health care worldwide. The range of antibiotics active against these bacteria is narrow; it includes teicoplanin, a "last resort" drug, which is produced by the filamentous actinomycete Actinoplanes teichomyceticus. In this report, we determine the functions of tei15* and tei16*, pathway-specific regulatory genes that code for StrR- and LuxR-type transcriptional factors, respectively. The products of these genes are master switches of teicoplanin biosynthesis, since their inactivation completely abolished antibiotic production. We show that Tei15* positively regulates the transcription of at least 17 genes in the cluster, whereas the targets of Tei16* still remain unknown. Integration of tei15* or tei16* under the control of the aminoglycoside resistance gene aac(3)IV promoter into attBϕC31 site of the A. teichomyceticus chromosome increased teicoplanin productivity to nearly 1 g/L in TM1 industrial medium. The expression of these genes from the moderate copy number episomal vector pKC1139 led to 3-4 g/L teicoplanin, while under the same conditions, wild type produced approximately 100 mg/L. This shows that a significant increase in teicoplanin production can be achieved by a single step of genetic manipulation of the wild-type strain by increasing the expression of the tei regulatory genes. This confirms that natural product yields can be increased using rational engineering once suitable genetic tools have been developed. We propose that this new technology for teicoplanin overproduction might now be transferred to industrial mutants of A. teichomyceticus.

The use of bones as tools in Late Lower Paleolithic of Central Italy.

The Latium area in Italy has yielded rich evidence of Lower Paleolithic sites with both faunal remains, artefacts, and human fossil remains, such as the Ceprano human skull. Many are the sites where lithic industry has been found in association with bone industry. Medium and large animals were a key resource because they provided an enormous amount of meat and fat. However, they were extensively exploited for their bones, rich in marrow, and as raw material for tool production. Bone tools are so far few documented for early period of time and especially for the Middle Pleistocene in Western Europe. We report here evidence of bone tools and their efficiency of use for hominin groups living in the Frosinone-Ceprano basin during the MIS 11/10, a key period which records behavioral innovations and onset of the Neanderthal behaviors. In three sites, Isoletta, Colle Avarone and Selvotta, several bone tools and bone flakes have been discovered (MIS 11/10). They were associated to stone artefacts part of the hominins tool-kit. Technological and use-wear analyses conducted on these bone industries, dated between 410 and 430 ka, yield relevant results to understand the effectiveness of the bones tools found associated with lithic series, including handaxes.

The Impact of Heterologous Regulatory Genes from Lipodepsipeptide Biosynthetic Gene Clusters on the Production of Teicoplanin and A40926.

StrR-like pathway-specific transcriptional regulators (PSRs) function as activators in the biosynthesis of various antibiotics, including glycopeptides (GPAs), aminoglycosides, aminocoumarins, and ramoplanin-like lipodepsipeptides (LDPs). In particular, the roles of StrR-like PSRs have been previously investigated in the biosynthesis of streptomycin, novobiocin, GPAs like balhimycin, teicoplanin, and A40926, as well as LDP enduracidin. In the current study, we focused on StrR-like PSRs from the ramoplanin biosynthetic gene cluster (BGC) in Actinoplanes ramoplaninifer ATCC 33076 (Ramo5) and the chersinamycin BGC in Micromonospora chersina DSM 44151 (Chers28). Through the analysis of the amino acid sequences of Ramo5 and Chers28, we discovered that these proteins are phylogenetically distant from other experimentally investigated StrR PSRs, although all StrR-like PSRs found in BGCs for different antibiotics share a conserved secondary structure. To investigate whether Ramo5 and Chers28, given their phylogenetic positions, might influence the biosynthesis of other antibiotic pathways governed by StrR-like PSRs, the corresponding genes (ramo5 and chers28) were heterologously expressed in Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727, which produce the clinically-relevant GPAs teicoplanin and A40926, respectively. Recombinant strains of NRRL B-16726 and ATCC 39727 expressing chers28 exhibited improved antibiotic production, although the expression of ramo5 did not yield the same effect. These results demonstrate that some StrR-like PSRs can "cross-talk" between distant biosynthetic pathways and might be utilized as tools for the activation of silent BGCs regulated by StrR-like PSRs.