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.
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.
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.
Clinically relevant glycopeptide antibiotics remain among the most successful classes of natural antibacterials. This success, however, is endangered by the spread of glycopeptide resistance genes, ...also known as van genes. Thus, it is important to trace and comprehend possible routes of van gene dissemination. In the current work, we present a comprehensive bioinformatic analysis aimed at mapping the occurrence of van genes beyond the Actinobacteria phylum—the most likely natural reservoir of van genes. We show that two additional classes of Gram-positive bacteria, Erysipelotrichia and Ktedonobacteria, as well as one class of Gram-negative bacteria, Anaerolineae, carry van genes. Additionally, we demonstrate that various new genera belonging to the classes Clostridia and Bacilli also carry van genes. The majority of discovered van loci are co-localized with MGE-related genes of various types. Finally, we propose a phylogeny-based scenario for the spread of van genes, unraveling a network of consequential horizontal gene transfer events linking the phylum Actinobacteria with the five other bacterial classes carrying van genes.
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-440
T
. 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 (k
L
a) 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 k
L
a = 28.3 h
−1
. A further increase in k
L
a 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 k
L
a in baffled flasks and agitated bioreactors.
Genome sequencing has revealed that
spp. represent a still largely unexplored source of specialized metabolites.
ATCC 39727 is the most studied representative species since it produces the ...glycopeptide antibiotic (GPA) A40926 - the precursor of the clinically relevant antibiotic dalbavancin, approved by the FDA in 2014 for the treatment of acute skin infections caused by multi-drug resistant Gram-positive pathogens. The clinical relevance of dalbavancin has prompted increased attention on A40926 biosynthesis and its regulation. In this paper, we investigated how to enhance the genetic toolkit for members of the
genus, which have proved quite recalcitrant to genetic manipulation. By constructing promoter-probe vectors, we tested the activity of 11 promoters (heterologous and native) using the GusA reporter system in
and in
; this latter species is phylogenetically distant from
and also possesses the genetic potential to produce A40926 or a very similar GPA. Finally, the strongest constitutive promoter analyzed in this study,
, was used to overexpress the cluster-situated regulatory genes controlling A40926 biosynthesis (
and
from
and
from
) in
.
, and the growth and productivity of the best performing strains were assessed at bioreactor scale using an industrial production medium. Overexpression of positive pathway-specific regulatory genes resulted in a significant increase in the level of A40926 production in
, providing a new knowledge-based approach to strain improvement for this valuable glycopeptide antibiotic.
Microbial communities inhabiting the Antarctic Ocean show psychrophilic and halophilic adaptations conferring interesting properties to the enzymes they produce, which could be exploited in ...biotechnology and bioremediation processes. Use of cold- and salt-tolerant enzymes allows to limit costs, reduce contaminations, and minimize pretreatment steps. Here, we report on the screening of 186 morphologically diverse microorganisms isolated from marine biofilms and water samples collected in Terra Nova Bay (Ross Sea, Antarctica) for the identification of new laccase activities. After primary screening, 13.4 and 10.8% of the isolates were identified for the ability to oxidize 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the dye azure B, respectively. Amongst them, the marine
sp. strain M68 showed the highest activity. Production of its laccase-like activity increased six-fold when copper was added to culture medium. Enzymatic activity-guided separation coupled with mass spectrometry identified this intracellular laccase-like protein (named Ant laccase) as belonging to the copper resistance system multicopper oxidase family. Ant laccase oxidized ABTS and 2,6-dimethoxy phenol, working better at acidic pHs The enzyme showed a good thermostability, with optimal temperature in the 40-50°C range and maintaining more than 40% of its maximal activity even at 10°C. Furthermore, Ant laccase was salt- and organic solvent-tolerant, paving the way for its use in harsh conditions. To our knowledge, this is the first report concerning the characterization of a thermo- and halo-tolerant laccase isolated from a marine Antarctic bacterium.
En 1982, Gilbert Simondon écrit une lettre, jamais envoyée, à Jacques Derrida. Dans ces pages émerge le concept fondamental de techno-esthétique. La réflexion esthétique que Simondon y esquisse ...consiste en une reprise et une radicalisation de ce qu’il avait déjà théorisé dans Du mode d’existence des objets techniques, à propos du rapport très strict que l’on peut envisager entre technique et esthétique : la sensibilité humaine a toujours été connectée avec des prolongements techniques par lesquels cette sensibilité de l’individu se réorganise, tout en réorganisant ce qui l’entoure. Cet article vise à montrer que ce type de conception techno-esthétique est également présent dans certains textes de John Dewey. La convergence entre les deux auteurs s’exprime également dans leur façon de considérer le rapport, que l’on peut qualifier de co-constitution, entre l’organisme et son milieu ; tout comme pour Simondon, qui développe la notion de « milieu associé », il n’est possible, pour Dewey, de concevoir un individu qu’à partir de sa relation à un milieu. Par là, c’est le parallélisme entre les notions de « transduction » et de « transaction » qui sera à explorer.
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
ATCC 33076 (Ramo5) and the chersinamycin BGC in
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 (
and
) were heterologously expressed in
NRRL B-16726 and
ATCC 39727, which produce the clinically-relevant GPAs teicoplanin and A40926, respectively. Recombinant strains of NRRL B-16726 and ATCC 39727 expressing
exhibited improved antibiotic production, although the expression of
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.
Food quality is also related to safety and prevention of spoilage. Biological antimicrobial agents represent suitable alternatives to clinical preservatives in food industry to increase both safety ...and stability of aliments. Here, we focused on the enzyme D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, a well-studied protein for biotechnological use based on its stability, high activity, and easy recombinant production. DAAO catalyzes the O2-dependent oxidative deamination of D-enantiomer of amino acids generating α-keto acids, ammonia, and hydrogen peroxide. DAAO shows antibacterial activity on both Gram-positive and Gram-negative bacteria in the presence of D-alanine when tested on plates and reduced by half their growth when tested on liquid cultures. Control experiments performed with alternative amino acid-specific flavoenzymes (able or not to generate H2O2 acting on amino acids), a DAAO inactive variant, catalase (H2O2 scavenger), and L-amino acids instead of D-alanine identified H2O2 as the antibacterial agent. DAAO showed a good ability to decrease the bacterial growth on various food stuffs: e.g., 10-fold less colonies were formed on grated cheese incubated for 16 h at 37°C when a tiny amount (0.01 mg corresponding to 1.2 units) of DAAO was added. No exogenous D-amino acids were added since DAAO used the ones naturally occurring or the ones generated during ripening. Notably, simultaneously to H2O2 generation, DAAO also acts as O2-scavenger thus further hampering food deterioration.
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