Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine

The organisms thriving under extreme conditions better than any other organism living on Earth, fascinate by their hostile growing parameters, physiological features, and their production of valuable bioactive metabolites. This is the case of microorganisms (bacteria, archaea, and fungi) that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As along as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions on which the compounds are produced in the microbial community without more stress than sharing the same substratum with their peers, the salt. In this review are reported the molecules described and produced by halophilic microorganisms with a spectrum of action in vitro: antimicrobial and anticancer. The action mechanisms of these molecules, the urgent need to introduce alternative lead compounds and the current aspects on the exploitation and its limitations are discussed.España, MINECO CGL2017-83385-

The role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by <em>Serratia marcescens </em>Db10

Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Serratia marcescens Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Ser. marcescens Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against Staphylococcus aureus. Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of Ser. marcescens to inhibit competitor bacteria by the production of antimicrobial secondary metabolites

Diverse secondary metabolites are expressed in particle-associated and free-living microorganisms of the permanently anoxic Cariaco Basin

Secondary metabolites play essential roles in ecological interactions and nutrient acquisition, and are of interest for their potential uses in medicine and biotechnology. Genome mining for biosynthetic gene clusters (BGCs) can be used for the discovery of new compounds. Here, we use metagenomics and metatranscriptomics to analyze BGCs in free-living and particle-associated microbial communities through the stratified water column of the Cariaco Basin, Venezuela. We recovered 565 bacterial and archaeal metagenome-assembled genomes (MAGs) and identified 1154 diverse BGCs. We show that differences in water redox potential and microbial lifestyle (particle-associated vs. free-living) are associated with variations in the predicted composition and production of secondary metabolites. Our results indicate that microbes, including understudied clades such as Planctomycetota, potentially produce a wide range of secondary metabolites in these anoxic/euxinic waters

Microbial communities and bioactive compounds in marine sponges of the family Irciniidae-a review

Marine sponges harbour complex microbial communities of ecological and biotechnological importance. Here, we propose the application of the widespread sponge family Irciniidae as an appropriate model in microbiology and biochemistry research. Half a gram of one Irciniidae specimen hosts hundreds of bacterial species-the vast majority of which are difficult to cultivate-and dozens of fungal and archaeal species. The structure of these symbiont assemblages is shaped by the sponge host and is highly stable over space and time. Two types of quorum-sensing molecules have been detected in these animals, hinting at microbe-microbe and host-microbe signalling being important processes governing the dynamics of the Irciniidae holobiont. Irciniids are vulnerable to disease outbreaks, and concerns have emerged about their conservation in a changing climate. They are nevertheless amenable to mariculture and laboratory maintenance, being attractive targets for metabolite harvesting and experimental biology endeavours. Several bioactive terpenoids and polyketides have been retrieved from Irciniidae sponges, but the actual producer (host or symbiont) of these compounds has rarely been clarified. To tackle this, and further pertinent questions concerning the functioning, resilience and physiology of these organisms, truly multi-layered approaches integrating cutting-edge microbiology, biochemistry, genetics and zoology research are needed.Portuguese Foundation [PTDC/MAR/101431/2008, PTDC/BIA-MIC/3865/2012]; European Regional Development Fund (ERDF) through the Operational Competitiveness Programme (COMPETE); national funds through FCT (Foundation for Science and Technology) [PEst-C/MAR/LA0015/2011]; FCT [SFRH/BD/60873/2009]info:eu-repo/semantics/publishedVersio

A large scale prediction of bacteriocin gene blocks suggests a wide functional spectrum for bacteriocins

Bacteriocins are peptide-derived molecules produced by bacteria, whose recently-discovered functions include virulence factors and signalling molecules as well as their better known roles as antibiotics. To date, close to five hundred bacteriocins have been identified and classified. Recent discoveries have shown that bacteriocins are highly diverse and widely distributed among bacterial species. Given the heterogeneity of bacteriocin compounds, many tools struggle with identifying novel bacteriocins due to their vast sequence and structural diversity. Many bacteriocins undergo post-translational processing or modifications necessary for the biosynthesis of the final mature form. Enzymatic modification of bacteriocins as well as their export is achieved by proteins whose genes are often located in a discrete gene cluster proximal to the bacteriocin precursor gene, referred to as \textit{context genes} in this study. Although bacteriocins themselves are structurally diverse, context genes have been shown to be largely conserved across unrelated species. Using this knowledge, we set out to identify new candidates for context genes which may clarify how bacteriocins are synthesized, and identify new candidates for bacteriocins that bear no sequence similarity to known toxins. To achieve these goals, we have developed a software tool, Bacteriocin Operon and gene block Associator (BOA) that can identify homologous bacteriocin associated gene clusters and predict novel ones. We discover that several phyla have a strong preference for bactericon genes, suggesting distinct functions for this group of molecules. Availability: https://github.com/idoerg/BOAComment: Accepted for publication in BMC Bioinformatic

Investigation of the role of gene clusters in terpene biosynthesis in Sorghum bicolor

The staple crop Sorghum bicolor shows potential as a source of secondary metabolite-based biofuels due to its diverse phenotype and chemical profile. S. bicolor produces a variety of high-energy metabolites, including terpenes which are a potential renewable source of fuel additives. Information on the biosynthetic and genetic pathways by which S. bicolor terpenes are produced is limited and these pathways must be better understood before they can be engineered for human applications. Recent work on plant biosynthetic pathways has shown that terpenes can be modified by the products of clustered genes. Identification of biosynthetic gene clusters may accelerate the elucidation of complete pathways, but few have been characterized in S. bicolor. The aims of this thesis were to identify a putative terpene biosynthetic gene cluster in S. bicolor, characterize the terpene synthase in the cluster, and express the terpene synthase alongside clustered enzymes to determine if they modify the terpene skeleton structure. The terpene synthase Sobic.001G339000 was found to produce a novel sesquiterpene product. Mass spectra analysis suggested that the novel product was similar to guaiol and β-eudesmol and possibly shared a mass (222.2 Da) and chemical formula (C15H26O) with these compounds. Transient expression of the putative gene cluster in N. benthamiana produced a metabolite of a significantly higher mass than anticipated based on the hypothesized mass of the unknown terpene. Elucidation of a structure by NMR spectroscopy will be required to characterize the unknown terpene product. Once the structure of the terpene is known, analysis of the metabolic profile of transfected N. benthamiana will be simplified and the effect of clustered enzymes on the terpene product can be better explored

Production of Secondary Metabolites in Extreme Environments: Food- and Airborne Wallemia spp. Produce Toxic Metabolites at Hypersaline Conditions

<div><p>The food- and airborne fungal genus <i>Wallemia</i> comprises seven xerophilic and halophilic species: <i>W</i>. <i>sebi</i>, <i>W</i>. <i>mellicola</i>, <i>W</i>. <i>canadensis</i>, <i>W</i>. <i>tropicalis</i>, <i>W</i>. <i>muriae</i>, <i>W</i>. <i>hederae</i> and <i>W</i>. <i>ichthyophaga</i>. All listed species are adapted to low water activity and can contaminate food preserved with high amounts of salt or sugar. In relation to food safety, the effect of high salt and sugar concentrations on the production of secondary metabolites by this toxigenic fungus was investigated. The secondary metabolite profiles of 30 strains of the listed species were examined using general growth media, known to support the production of secondary metabolites, supplemented with different concentrations of NaCl, glucose and MgCl₂. In more than two hundred extracts approximately one hundred different compounds were detected using high-performance liquid chromatography-diode array detection (HPLC-DAD). Although the genome data analysis of <i>W</i>. <i>mellicola</i> (previously <i>W</i>. <i>sebi sensu lato</i>) and <i>W</i>. <i>ichthyophaga</i> revealed a low number of secondary metabolites clusters, a substantial number of secondary metabolites were detected at different conditions. Machine learning analysis of the obtained dataset showed that NaCl has higher influence on the production of secondary metabolites than other tested solutes. Mass spectrometric analysis of selected extracts revealed that NaCl in the medium affects the production of some compounds with substantial biological activities (wallimidione, walleminol, walleminone, UCA 1064-A and UCA 1064-B). In particular an increase in NaCl concentration from 5% to 15% in the growth media increased the production of the toxic metabolites wallimidione, walleminol and walleminone.</p></div

Genomic Insights Into New Species of the Genus Halomicroarcula Reveals Potential for New Osmoadaptative Strategies in Halophilic Archaea

Metagenomic studies on prokaryotic diversity of hypersaline soils from the Odiel saltmarshes, South-west Spain, revealed a high proportion of genomic sequences not related to previously cultivated taxa, that might be related to haloarchaea with a high environmental and nutritional flexibility. In this study, we used a culturomics approach in order to isolate new haloarchaeal microorganisms from these hypersaline soils. Four haloarchaeal strains, designated strains F24AT, F28, F27T, and F13T, phylogenetically related to the genus Halomicroarcula, were isolated and characterized in detail. The phylogenomic tree based on the 100 orthologous single-copy genes present in the genomes of these four strains as well as those of the type strains of the species Halomicroarcula pellucida CECT 7537T, Halomicroarcula salina JCM 18369T and Halomicroarcula limicola JCM 18640T, that were determined in this study, revealed that these four new isolates clustered on three groups, with strains F24AT and F28 within a single cluster, and altogether with the species of Halomicroarcula. Additionally, Orthologous Average Nucleotide Identity (OrthoANI), digital DNA-DNA hybridization (dDDH) and Average Amino-acid Identity (AAI) values, likewise phenotypic characteristics, including their polar lipids profiles, permitted to determine that they represent three new species, for which we propose the names Halomicroarcula rubra sp. nov. (type strain F13T), Halomicroarcula nitratireducens sp. nov. (type strain F27T) and Halomicroarcula salinisoli sp. nov. (type strain F24AT). An in deep comparative genomic analysis of species of the genus Halomicroarcula, including their metabolism, their capability to biosynthesize secondary metabolites and their osmoregulatory adaptation mechanisms was carried out. Although they use a salt-in strategy, the identification of the complete pathways for the biosynthesis of the compatible solutes trehalose and glycine betaine, not identified before in any other haloarchaea, might suggest alternative osmoadaptation strategies for this group. This alternative osmoregulatory mechanism would allow this group of haloarchaea to be versatile and eco-physiologically successful in hypersaline environments and would justify the capability of the species of this genus to grow not only on environments with high salt concentrations [up to 30% (w/v) salts], but also under intermediate to low salinities.España Junta de Andalucía (grants US-1263771 [US/JUNTA/FEDER/UE], P20_01066 and BIO-213, which included FEDER funds)Spanish Ministry of Science and Innovation-State Research Agency /FEDER (projects CGL2017-83385-P and PID2020-118136GB-I00

Prospecting Novel Microbiomes for Antibiotic Compounds using Metagenomics and Genome Mining

There has been a void in the discovery and development of new antibiotic classes over the past four decades due, in part, to the traditional bioprospecting pipeline becoming inefficient from high compound rediscovery rates and high costs. The need for new antibiotic classes is urgent as antimicrobial drug resistant infections are now a major public health concern. Strategies such as exploring novel environments, use of next-generation sequencing, and metagenomics may reduce rediscovery rates and costs which could help accelerate lead discovery and encourage greater participation in bioprospecting. Whole genome-sequencing and analysis was used to characterise four bacterial strains (Y1-4) isolated from raw honey that were shown to have antibiotic activity. The isolates were identified as Bacillus and were closely related but distinctive strains with variations amongst their secondary metabolite profiles. All isolates contained a gene cluster homologous to AS- 48, a circular bacteriocin produced by Enterococcus faecalis, which has broad-spectrum antibiotic activity. To date, no example of this bacteriocin has been reported in Bacillus. This work demonstrated the value of whole-microbial genome sequencing for dereplication. A pipeline for the low-cost sequencing and assembly of bacterial genomes using Oxford Nanopore MinION was developed in order to produce contiguous and accurate genome assemblies for taxonomic and bioprospecting analysis. The pipeline developed used a combination of Nanopore draft assembly by Canu and polishing with RACON and Nanopolish, with final polishing with Illumina reads using Pilon. The Nanopore-only assembly of Streptomyces coelicolor A3(2) produced was contiguous and covered 98.9 % of reference. AntiSMASH analysis identified the full secondary metabolite profile of the genome through homology searches. However, indel rates were high (66.82 per 100 kbp) causing fragmented gene annotations which limited secondary metabolite structure prediction. Illumina read polishing reduced indels (2.03 per 100 kbp) and enabled accurate structure prediction from the identified biosynthetic pathways. This demonstrates that Nanopore sequencing can provide a viable dereplication strategy by detection of known biosynthetic pathways. Additionally, supplementation with Illumina sequencing can allow for structure prediction of biosynthetic pathways which could inform chemical extraction strategies for novel pathways. Nanopore sequencing was further utilised to characterise an antibiotic producing isolate (KB16) active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus from the hot spring of the Roman Baths, UK. Genomic analysis showed KB16 to be highly related to Streptomyces canus and to contain 26 putative secondary metabolite gene clusters - some of which were potentially novel. One of the gene clusters was identified as encoding the antibiotic albaflavenone. Attempts to chemically identify the antibiotic produced by KB16 showed that it may produce multiple antimicrobial compounds. These findings demonstrate the value in prospecting underexplored environments such as the Roman Baths for microbially-derived antimicrobial leads. A PCR screen was used to amplify NRPS and PKS gene fragments from a human oral metagenome. Analysis of the fragments suggested that some are from uncharacterised gene clusters. Nanopore shotgun metagenomic sequencing was used to profile the water of the Roman Baths which revealed a diverse microbiome of species with reported metabolic characteristics that are in keeping with the known geochemistry of the waters and aligned with 16S rRNA analysis. Further analysis also identified putative heavy metal resistance genes which can be a co-marker for their metabolism and aligned with the chemical properties of the water. These findings demonstrate the potential value in these sites for bioprospecting whilst also giving insight that can inform bioprospecting strategies. The investigations also highlight the utility of Nanopore sequencing for taxonomic and functional gene profiling of environmental microbiomes. In combination these findings have all contributed information on novel environments, potential isolate leads, and cost-efficient methodologies to accelerate the discovery of microbially-derived antibiotics