The negative impact of the massive use of synthetic pesticides on the environment and on human health has stimulated the search for environment-friendly practices for controlling plant diseases and ...pests. Among them, biocontrol, which relies on using beneficial organisms or their products (bioactive molecules and/or hydrolytic enzymes), holds the greatest promise and is considered a pillar of integrated pest management. Chitinases are particularly attractive to this purpose since they have fungicidal, insecticidal, and nematicidal activities. Here, current knowledge on the biopesticidal action of microbial and viral chitinases is reviewed, together with a critical analysis of their future development as biopesticides.
•Chitinases are promising candidates for integrated pest management strategies.•Chitinases have fungicidal, insecticidal, and nematicidal activities.•Chitinases are harmless for plants and vertebrates that do not contain chitin.•Uncovering novel chitinases from untapped microbial diversity potentiates biocontrol.•Metagenomics is a powerful tool for discovering novel chitinases.
Glycopeptide antibiotics (GPAs) are considered drugs of “last resort” for the treatment of life-threatening infections caused by relevant Gram-positive pathogens (enterococci, staphylococci, and ...clostridia). Driven by the issue of the never-stopping evolution of bacterial antibiotic resistance, research on GPA biosynthesis and resistance is developing fast in modern “post-genomic” era. It is today widely accepted that resistance mechanisms emerging in pathogens have been acquired from the soil-dwelling antibiotic-producing actinomycetes, which use them to avoid suicide during production, rather than being orchestrated
de novo
by pathogen bacteria upon continued treatment. Actually, more and more genomes of GPA producers are being unraveled, carrying a broad collection of differently arranged GPA resistance (named
van
) genes. In the producer actinomycetes,
van
genes are generally associated with the antibiotic biosynthetic gene clusters (BGCs) deputed to GPA biosynthesis, being probably transferred/arranged together, favoring a possible co-regulation between antibiotic production and self-resistance. GPA BGC-associated
van
genes have been also found mining public databases of bacterial genomic and metagenomic sequences. Interestingly, some BGCs for antibiotics, seemingly unrelated to GPAs (e.g., feglymycin), carry
van
gene homologues. Herein, we would like to cover the recent advances on the distribution of GPA resistance genes in genomic and metagenomics datasets related to GPA potential/proved producer microorganisms. A thorough understanding of GPA resistance in the producing microorganisms may prove useful in the future surveillance of emerging mechanisms of resistance to this clinically relevant antibiotic class.
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.
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.
Enzymes are increasingly applied as biocatalysts for fulfilling industrial needs in a variety of applications and there is a bursting of interest for novel therapeutic proteins. Consequently, ...developing appropriate expression platforms for efficiently producing such recombinant proteins represents a crucial challenge. It is nowadays widely accepted that an ideal ‘universal microbial host’ for heterologous protein expression does not exist. Indeed, the first-choice microbes, as
Escherichia coli
or yeasts, possess known intrinsic limitations that inevitably restrict their applications. In this scenario, bacteria belonging to the
Streptomyces
genus need to be considered with more attention as promising, alternative, and versatile platforms for recombinant protein production. This is due to their peculiar features, first-of-all their natural attitude to secrete proteins in the extracellular milieu. Additionally, streptomycetes are considered robust and scalable industrial strains and a wide range of tools for their genetic manipulation is nowadays available. This mini-review includes an overview of recombinant protein production in streptomycetes, covering nearly 100 cases of heterologous proteins expressed in these Gram-positives from the 1980s to December 2019. We investigated homologous sources, heterologous hosts, and molecular tools (promoters/vectors/signal peptides) used for the expression of these recombinant proteins. We reported on their final cellular localization and yield. Thus, this analysis might represent a useful source of information, showing pros and cons of using streptomycetes as platform for recombinant protein production and paving the way for their more extensive use in future as alternative heterologous hosts.
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 teichomyceticus—the producer of the last-resort-drug teicoplanin—has 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.
Actinoplanes teichomyceticus NRRL B-16726 is the only known producer of the clinically important glycopeptide antibiotic teicoplanin. The producing strain is highly self-resistant to teicoplanin. ...Although the biosynthesis of teicoplanin has been investigated, much of our understanding of self-resistance in the producing strain is based on the extrapolation of existing data about glycopeptide resistance (mediated by the expression of vanRS-vanHAX genes) in other actinomycetes and cocci. The organization of the operons carrying putative van orthologues in A. teichomyceticus differs from known precedents, further adding up to the uncertainty about teicoplanin self-resistance mechanisms. Here, we determined operon structure of the teicoplanin resistance genes in A. teichomyceticus. Although Tei15* is necessary to activate teicoplanin biosynthetic genes, the expression of van orthologues was shown to be independent of Tei15*. We further showed that tei7 promoter driving the expression of vanHAX orthologues is dependent on Tei2 (VanR). Finally, we demonstrate the utility of the tei2 promoter as a new tool to achieve strong constitutive expression in A. teichomyceticus.
Microbial chitinases are gaining interest as promising candidates for controlling plant pests. These enzymes can be used directly as biocontrol agents as well as in combination with chemical ...pesticides or other biopesticides, reducing their environmental impact and/or enhancing their efficacy. Chitinolytic enzymes can target two different structures in insects: the cuticle and the peritrophic matrix (PM). PM, formed by chitin fibrils connected to glycoproteins and proteoglycans, represents a physical barrier that plays an essential role in midgut physiology and insect digestion, and protects the absorptive midgut epithelium from food abrasion or pathogen infections. In this paper, we investigate how two recently discovered metagenome-sourced chitinases (Chi18H8 and 53D1) affect,
and
, the PM integrity of
, a model system among Lepidoptera. The two chitinases were produced in
or, alternatively, in the unconventional - but more environmentally acceptable -
. Although both the proteins dramatically altered the structure of
PM
, when administered orally only 53D1 caused adverse and marked effects on larval growth and development, inducing mortality and reducing pupal weight. These
results demonstrate that 53D1 is a promising candidate as insecticide protein.
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.
Glycopeptides are considered antibiotics of last resort for the treatment of life-threatening infections caused by relevant Gram-positive human pathogens, such as Staphylococcus aureus, Enterococcus ...spp. and Clostridium difficile. The emergence of glycopeptide-resistant clinical isolates, first among enterococci and then in staphylococci, has prompted research for second generation glycopeptides and a flurry of activity aimed at understanding resistance mechanisms and their evolution. Glycopeptides are glycosylated non-ribosomal peptides produced by a diverse group of soil actinomycetes. They target Gram-positive bacteria by binding to the acyl-D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the growing peptidoglycan on the outer surface of the cytoplasmatic membrane. Glycopeptide-resistant organisms avoid such a fate by replacing the D-Ala-D-Ala terminus with D-alanyl-D-lactate (D-Ala-D-Lac) or D-alanyl-D-serine (D-Ala-D-Ser), thus markedly reducing antibiotic affinity for the cellular target. Resistance has manifested itself in enterococci and staphylococci largely through the expression of genes (named van) encoding proteins that reprogram cell wall biosynthesis and, thus, evade the action of the antibiotic. These resistance mechanisms were most likely co-opted from the glycopeptide producing actinomycetes, which use them to avoid suicide during antibiotic production, rather than being orchestrated by pathogen bacteria upon continued treatment. van-like gene clusters, similar to those described in enterococci, were in fact identified in many glycopeptide-producing actinomycetes, such as Actinoplanes teichomyceticus, which produces teicoplanin, and Streptomyces toyocaensis, which produces the A47934 glycopeptide. In this paper, we describe the natural and semi-synthetic glycopeptide antibiotics currently used as last resort drugs for Gram-positive infections and compare the van gene-based strategies of glycopeptide resistance among the pathogens and the producing actinomycetes. Particular attention is given to the strategy of immunity recently described in Nonomuraea sp. ATCC 39727. Nonomuraea sp. ATCC 39727 is the producer of A40926, which is the natural precursor of the second generation semi-synthetic glycopeptide dalbavancin, very recently approved for acute bacterial skin and skin structure infections. A thorough understanding of glycopeptide immunity in this producing microorganism may be particularly relevant to predict and eventually control the evolution of resistance that might arise following introduction of dalbavancin and other second generation glycopeptides into clinics.
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