Shotgun metagenomic analysis of kombucha mutualistic community exposed to Mars-like environment outside the International Space Station.

Kombucha is a multispecies microbial ecosystem mainly composed of acetic acid bacteria and osmophilic acid-tolerant yeasts, which is used to produce a probiotic drink. Furthermore, Kombucha Mutualistic Community (KMC) has been recently proposed to be used during long space missions as both a living functional fermented product to improve astronauts' health and an efficient source of bacterial nanocellulose. In this study, we compared KMC structure and functions before and after samples were exposed to the space/Mars-like environment outside the International Space Station in order to investigate the changes related to their re-adaptation to Earth-like conditions by shotgun metagenomics, using both diversity and functional analyses of Community Ecology and Complex Networks approach. Our study revealed that the long-term exposure to space/Mars-like conditions on low Earth orbit may disorganize the KMC to such extent that it will not restore the initial community structure; however, KMC core microorganisms of the community were maintained. Nonetheless, there were no significant differences in the community functions, meaning that the KMC communities are ecologically resilient. Therefore, despite the extremely harsh conditions, key KMC species revived and provided the community with the genetic background needed to survive long periods of time under extraterrestrial conditions.

Resistance Sniffer: An online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using next generation sequencing data.

The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the problems that are associated with current phenotypic drug susceptibility testing. However, the application of WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up.ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial genome datasets in different file formats including raw Illumina fastq read files. The program was validated on sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.

Complete genome sequence and epigenetic profile of Bacillus velezensis UCMB5140 used for plant and crop protection in comparison with other plant-associated Bacillus strains.

The application of biocontrol biopesticides based on plant growth-promoting rhizobacteria (PGPR), particularly members of the genus Bacillus, is considered a promising perspective to make agricultural practices sustainable and ecologically safe. Recent advances in genome sequencing by third-generation sequencing technologies, e.g., Pacific Biosciences' Single Molecule Real-Time (PacBio SMRT) platform, have allowed researchers to gain deeper insights into the molecular and genetic mechanisms of PGPR activities, and to compare whole genome sequences and global patterns of epigenetic modifications. In the current work, this approach was used to sequence and compare four Bacillus strains that exhibited various PGPR activities including the strain UCMB5140, which is used in the commercial biopesticide Phytosubtil. Whole genome comparison and phylogenomic inference assigned the strain UCMB5140 to the species Bacillus velezensis. Strong biocontrol activities of this strain were confirmed in several bioassays. Several factors that affect the evolution of active PGPR B. velezensis strains were identified: (1) horizontal acquisition of novel non-ribosomal peptide synthetases (NRPS) and adhesion genes; (2) rearrangements of functional modules of NRPS genes leading to strain specific combinations of their encoded products; (3) gain and loss of methyltransferases that can cause global alterations in DNA methylation patterns, which eventually may affect gene expression and regulate transcription. Notably, we identified a horizontally transferred NRPS operon encoding an uncharacterized polypeptide antibiotic in B. velezensis UCMB5140. Other horizontally acquired genes comprised a possible adhesin and a methyltransferase, which may explain the strain-specific methylation pattern of the chromosomal DNA of UCMB5140. KEY POINTS: • Whole genome sequence of the active PGPR Bacillus velezensis UCMB5140. • Identification of genetic determinants responsible for PGPR activities. • Role of methyltransferases and epigenetic mechanisms in evolution of bacteria.

Selection of marker genes for genetic barcoding of microorganisms and binning of metagenomic reads by Barcoder software tools.

BACKGROUND: Metagenomic approaches have revealed the complexity of environmental microbiomes with the advancement in whole genome sequencing displaying a significant level of genetic heterogeneity on the species level. It has become apparent that patterns of superior bioactivity of bacteria applicable in biotechnology as well as the enhanced virulence of pathogens often requires distinguishing between closely related species or sub-species. Current methods for binning of metagenomic reads usually do not allow for identification below the genus level and generally stops at the family level. RESULTS: In this work, an attempt was made to improve metagenomic binning resolution by creating genome specific barcodes based on the core and accessory genomes. This protocol was implemented in novel software tools available for use and download from http://bargene.bi.up.ac.za /. The most abundant barcode genes from the core genomes were found to encode for ribosomal proteins, certain central metabolic genes and ABC transporters. Performance of metabarcode sequences created by this package was evaluated using artificially generated and publically available metagenomic datasets. Furthermore, a program (Barcoding 2.0) was developed to align reads against barcode sequences and thereafter calculate various parameters to score the alignments and the individual barcodes. Taxonomic units were identified in metagenomic samples by comparison of the calculated barcode scores to set cut-off values. In this study, it was found that varying sample sizes, i.e. number of reads in a metagenome and metabarcode lengths, had no significant effect on the sensitivity and specificity of the algorithm. Receiver operating characteristics (ROC) curves were calculated for different taxonomic groups based on the results of identification of the corresponding genomes in artificial metagenomic datasets. The reliability of distinguishing between species of the same genus or family by the program was nearly perfect. CONCLUSIONS: The results showed that the novel online tool BarcodeGenerator ( http://bargene.bi.up.ac.za /) is an efficient approach for generating barcode sequences from a set of complete genomes provided by users. Another program, Barcoder 2.0 is available from the same resource to enable an efficient and practical use of metabarcodes for visualization of the distribution of organisms of interest in environmental and clinical samples.

EuGI: a novel resource for studying genomic islands to facilitate horizontal gene transfer detection in eukaryotes.

BACKGROUND: Genomic islands (GIs) are inserts of foreign DNA that have potentially arisen through horizontal gene transfer (HGT). There are evidences that GIs can contribute significantly to the evolution of prokaryotes. The acquisition of GIs through HGT in eukaryotes has, however, been largely unexplored. In this study, the previously developed GI prediction tool, SeqWord Gene Island Sniffer (SWGIS), is modified to predict GIs in eukaryotic chromosomes. Artificial simulations are used to estimate ratios of predicting false positive and false negative GIs by inserting GIs into different test chromosomes and performing the SWGIS v2.0 algorithm. Using SWGIS v2.0, GIs are then identified in 36 fungal, 22 protozoan and 8 invertebrate genomes. RESULTS: SWGIS v2.0 predicts GIs in large eukaryotic chromosomes based on the atypical nucleotide composition of these regions. Averages for predicting false negative and false positive GIs were 20.1% and 11.01% respectively. A total of 10,550 GIs were identified in 66 eukaryotic species with 5299 of these GIs coding for at least one functional protein. The EuGI web-resource, freely accessible at http://eugi.bi.up.ac.za , was developed that allows browsing the database created from identified GIs and genes within GIs through an interactive and visual interface. CONCLUSIONS: SWGIS v2.0 along with the EuGI database, which houses GIs identified in 66 different eukaryotic species, and the EuGI web-resource, provide the first comprehensive resource for studying HGT in eukaryotes.

Assembling metagenomes, one community at a time.

BACKGROUND: Metagenomics allows unprecedented access to uncultured environmental microorganisms. The analysis of metagenomic sequences facilitates gene prediction and annotation, and enables the assembly of draft genomes, including uncultured members of a community. However, while several platforms have been developed for this critical step, there is currently no clear framework for the assembly of metagenomic sequence data. RESULTS: To assist with selection of an appropriate metagenome assembler we evaluated the capabilities of nine prominent assembly tools on nine publicly-available environmental metagenomes, as well as three simulated datasets. Overall, we found that SPAdes provided the largest contigs and highest N50 values across 6 of the 9 environmental datasets, followed by MEGAHIT and metaSPAdes. MEGAHIT emerged as a computationally inexpensive alternative to SPAdes, assembling the most complex dataset using less than 500 GB of RAM and within 10 hours. CONCLUSIONS: We found that assembler choice ultimately depends on the scientific question, the available resources and the bioinformatic competence of the researcher. We provide a concise workflow for the selection of the best assembly tool.

Role of the horizontal gene exchange in evolution of pathogenic Mycobacteria.

BACKGROUND: Mycobacterium tuberculosis is one of the most dangerous human pathogens, the causative agent of tuberculosis. While this pathogen is considered as extremely clonal and resistant to horizontal gene exchange, there are many facts supporting the hypothesis that on the early stages of evolution the development of pathogenicity of ancestral Mtb has started with a horizontal acquisition of virulence factors. Episodes of infections caused by non-tuberculosis Mycobacteria reported worldwide may suggest a potential for new pathogens to appear. If so, what is the role of horizontal gene transfer in this process? RESULTS: Availing of accessibility of complete genomes sequences of multiple pathogenic, conditionally pathogenic and saprophytic Mycobacteria, a genome comparative study was performed to investigate the distribution of genomic islands among bacteria and identify ontological links between these mobile elements. It was shown that the ancient genomic islands from M. tuberculosis still may be rooted to the pool of mobile genetic vectors distributed among Mycobacteria. A frequent exchange of genes was observed between M. marinum and several saprophytic and conditionally pathogenic species. Among them M. avium was the most promiscuous species acquiring genetic materials from diverse origins. CONCLUSIONS: Recent activation of genetic vectors circulating among Mycobacteria potentially may lead to emergence of new pathogens from environmental and conditionally pathogenic Mycobacteria. The species which require monitoring are M. marinum and M. avium as they eagerly acquire genes from different sources and may become donors of virulence gene cassettes to other micro-organisms.

Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats.

The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5°C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65°C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16°C, was shown to contain halopiezophilic-like enzymes that are most active at 70°C and with denaturing temperatures of 71.4°C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.

Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp Rimicaris exoculata by using functional metagenomics.

The shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (≤52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (≤356 U mg(-1)) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities.

Temperate bacteriophages collected by outer membrane vesicles in Komagataeibacter intermedius.

The acetic acid bacteria have mainly relevance for bacterial cellulose production and fermented bio-products manufacture. The purpose of this study was to identify temperate bacteriophages in a cellulose-producing bacterial strain Komagataeibacter intermedius IMBG180. Prophages from K. intermedius IMBG180 were induced with mitomycin C and nalidixic acid. Transmission electron microscopy analysis exhibited tailed bacteriophages belonging to Myoviridae. A PCR assay targeting the capsid gene of the myoviruses proved phylogenetic position of induced phages. Nalidixic acid was poor inducer of prophages, however, it induced the OMV-like particles release. Size of OMVs depended on an antibiotic applied for phage induction and varied in the range of 30-80 and 120-200 nm. Inside some of them, tails of phages have been visible. Under conditions, inducing prophages, OMVs acted as the collectors of formed phage particles, using outer membrane receptors for phage detection (in this case, outer membrane siderophore receptor), and fulfilled therefore "a cleaning," as well as defensive functions, preventing bacteriophage spread outside population. This is the first description of myoviruses affiliated to K. intermedius, as well as outer membrane vesicles interaction with phages within this host.

Analysis of genomic rearrangements, horizontal gene transfer and role of plasmids in the evolution of industrial important Thermus species.

BACKGROUND: Bacteria of genus Thermus inhabit both man-made and natural thermal environments. Several Thermus species have shown biotechnological potential such as reduction of heavy metals which is essential for eradication of heavy metal pollution; removing of organic contaminants in water; opening clogged pipes, controlling global warming among many others. Enzymes from thermophilic bacteria have exhibited higher activity and stability than synthetic or enzymes from mesophilic organisms. RESULTS: Using Meiothermus silvanus DSM 9946 as a reference genome, high level of coordinated rearrangements has been observed in extremely thermophilic Thermus that may imply existence of yet unknown evolutionary forces controlling adaptive re-organization of whole genomes of thermo-extremophiles. However, no remarkable differences were observed across species on distribution of functionally related genes on the chromosome suggesting constraints imposed by metabolic networks. The metabolic network exhibit evolutionary pressures similar to levels of rearrangements as measured by the cross-clustering index. Using stratigraphic analysis of donor-recipient, intensive gene exchanges were observed from Meiothermus species and some unknown sources to Thermus species confirming a well established DNA uptake mechanism as previously proposed. CONCLUSION: Global genome rearrangements were found to play an important role in the evolution of Thermus bacteria at both genomic and metabolic network levels. Relatively higher level of rearrangements was observed in extremely thermophilic Thermus strains in comparison to the thermo-tolerant Thermus scotoductus. Rearrangements did not significantly disrupt operons and functionally related genes. Thermus species appeared to have a developed capability for acquiring DNA through horizontal gene transfer as shown by the donor-recipient stratigraphic analysis.

Staphylococcus aureus associated with surgical site infections in Western Kenya reveals genomic hotspots for pathogen evolution.

Objectives. Staphylococcus aureus is one of the most common pathogens attributed to hospital infections. Although S. aureus infections have been well studied in developed countries, far less is known about the biology of the pathogen in sub-Saharan Africa. Methods. Here, we report on the isolation, antibiotic resistance profiling, whole genome sequencing, and genome comparison of six multi-drug resistant isolates of S. aureus obtained from a referral hospital in Kakamega, Western Kenya. Results. Five of the six isolates contained a 20.7 kb circular plasmid carrying blaZ (associated with resistance to ß-lactam antibiotics). These five strains all belonged to the same sequence type, ST152. Despite the similarity of the plasmid in these isolates, whole genome sequencing revealed that the strains differed, depending on whether they were associated with hospital-acquired or community-acquired infections. Conclusion. The intriguing finding is that the hospital-acquired and the community-acquired isolates of S. aureus belonging to the same genotype, ST152, formed two separate sub-clusters in the phylogenetic tree and differed by the repertoire of accessory virulence genes. These data suggest ongoing adaptive evolution and significant genomic plasticity.

Whole genome sequencing and genotyping Klebsiella pneumoniae multi-drug resistant hospital isolates from Western Kenya.

Objectives: Klebsiella pneumoniae are a frequent cause of nosocomial infections worldwide. Sequence type 147 (ST147) has been reported as a major circulating high-risk lineage in many countries, and appears to be a formidable platform for the dissemination of antimicrobial resistance (AMR) determinants. However, the distribution of this pathogen in Western African hospitals has been scarcely studied. The main objective of this work was to perform whole genome sequencing of K. pneumoniae isolates from a referral hospital in Kakamega (Kenya) for genotyping and identification of AMR and virulence determinants. Methods: In total, 15 K. pneumoniae isolates showing a broad spectrum antimicrobial resistance were selected for whole genome sequencing by Illumina HiSeq 2500 platform. Results: ST147 was the dominant lineage among the highly-resistant K. pneumoniae isolates that we sequenced. ST147 was associated with both community- and the hospital-acquired infections, and with different infection sites, whereas other STs were predominantly uropathogens. Multiple antibiotic resistance and virulence determinants were detected in the genomes including extended-spectrum ß-lactamases (ESBL) and carbapenemases. Many of these genes were plasmid-borne. Conclusions: Our data suggest that the evolutionary success of ST147 may be linked with the acquisition of broad host-range plasmids, and their propensity to accrue AMR and virulence determinants. Although ST147 is a dominant lineage in many countries worldwide, it has not been previously reported as prevalent in Africa. Our data suggest an influx of new nosocomial pathogens with new virulence genes into African hospitals from other continents.

Interplay of intracellular and trans-cellular DNA methylation in natural archaeal consortia.

DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano-sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous cTAG methylation, typical of Haloferax, was accompanied in this strain by methylation at four other motifs, including GDGcHC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGAaG methylation increased in highly expressed genes, while CcTTG and GTCGaGG methylation could be linked to restriction-modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self-restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo-ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host.

Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach.

The esterases and lipases from the α/ß hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46'N, 2°59'W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated.

Analysis of Whole-Genome Sequences of Pathogenic Gram-Positive and Gram-Negative Isolates from the Same Hospital Environment to Investigate Common Evolutionary Trends Associated with Horizontal Gene Exchange, Mutations and DNA Methylation Patterning.

Hospital-acquired infections are a generally recognized problem for healthcare professionals. Clinical variants of Gram-negative and Gram-positive pathogens are characterized with enhanced antibiotic resistance and virulence due to mutations and the horizontal acquisition of respective genetic determinants. In this study, two Escherichia coli, two Klebsiella pneumoniae, three Pseudomonas aeruginosa, two Staphylococcus aureus, one Staphylococcus epidermidis and one Streptococcus pneumoniae showing broad spectra of antibiotic resistance were isolated from patients suffering from nosocomial infections in a local hospital in Almaty, Kazakhstan. The aim of the study was to compare general and species-specific pathways of the development of virulence and antibiotic resistance through opportunistic pathogens causing hospital-acquired infections. The whole-genome PacBio sequencing of the isolates allowed for the genotyping and identification of antibiotic resistance and virulence genetic determinants located in the chromosomes, plasmids and genomic islands. It was concluded that long-read sequencing is a useful tool for monitoring the epidemiological situation in hospitals. Marker antibiotic resistance mutations common for different microorganisms were identified, which were acquired due to antibiotic-selective pressure in the same clinical environment. The genotyping and identification of strain-specific DNA methylation motifs were found to be promising in estimating the risks associated with hospital infection outbreaks and monitoring the distribution and evolution of nosocomial pathogens.

The Effect of Three Complexes of Iodine with Amino Acids on Gene Expression of Model Antibiotic Resistant Microorganisms Escherichia coli ATCC BAA-196 and Staphylococcus aureus ATCC BAA-39.

1. BACKGROUND: Iodine is a broad-spectrum antimicrobial disinfectant for topical application. Recent studies have shown promising results on the applicability of an iodine-containing complex, FS-1, against antibiotic-resistant pathogens. It was hypothesized that the antimicrobial activity of iodine-containing complexes may be modulated by the organic moiety of the complex, i.e., amino acids. 2. METHODS: Gene regulation and metabolic alterations were studied in two model multidrug-resistant microorganisms, Staphylococcus aureus ATCC BAA-39, and Escherichia coli ATCC BAA-196, treated with three complexes containing iodine and three different amino acids: glycine, L-alanine, and L-isoleucine. The bacterial cultures were exposed to sub-lethal concentrations of the complexes in the lagging and logarithmic growth phases. Gene regulation was studied by total RNA sequencing and differential gene expression analysis. 3. RESULTS: The central metabolism of the treated bacteria was affected. An analysis of the regulation of genes involved in stress responses suggested the disruption of cell wall integrity, DNA damage, and oxidative stress in the treated bacteria. 4. CONCLUSIONS: Previous studies showed that the application of iodine-containing complexes, such as FS-1, serves as a supplement to common antibiotics and can be a promising way to combat antibiotic-resistant pathogens. Current results shed light on possible mechanisms of this action by disrupting the cell wall barriers and imposing oxidative stress. It was also found that the effect of the complexes on metabolic pathways varied in the tested microorganisms depending on the organic moiety of the complexes and the growth phase when the complexes had been applied.

Functional diversity of nanohaloarchaea within xylan-degrading consortia.

Extremely halophilic representatives of the phylum Candidatus Nanohaloarchaeota (members of the DPANN superphyla) are obligately associated with extremely halophilic archaea of the phylum Halobacteriota (according to the GTDB taxonomy). Using culture-independent molecular techniques, their presence in various hypersaline ecosystems around the world has been confirmed over the past decade. However, the vast majority of nanohaloarchaea remain uncultivated, and thus their metabolic capabilities and ecophysiology are currently poorly understood. Using the (meta)genomic, transcriptomic, and DNA methylome platforms, the metabolism and functional prediction of the ecophysiology of two novel extremely halophilic symbiotic nanohaloarchaea (Ca. Nanohalococcus occultus and Ca. Nanohalovita haloferacivicina) stably cultivated in the laboratory as members of a xylose-degrading binary culture with a haloarchaeal host, Haloferax lucentense, was determined. Like all known DPANN superphylum nanoorganisms, these new sugar-fermenting nanohaloarchaea lack many fundamental biosynthetic repertoires, making them exclusively dependent on their respective host for survival. In addition, given the cultivability of the new nanohaloarchaea, we managed to discover many unique features in these new organisms that have never been observed in nano-sized archaea both within the phylum Ca. Nanohaloarchaeota and the entire superphylum DPANN. This includes the analysis of the expression of organism-specific non-coding regulatory (nc)RNAs (with an elucidation of their 2D-secondary structures) as well as profiling of DNA methylation. While some ncRNA molecules have been predicted with high confidence as RNAs of an archaeal signal recognition particle involved in delaying protein translation, others resemble the structure of ribosome-associated ncRNAs, although none belong to any known family. Moreover, the new nanohaloarchaea have very complex cellular defense mechanisms. In addition to the defense mechanism provided by the type II restriction-modification system, consisting of Dcm-like DNA methyltransferase and Mrr restriction endonuclease, Ca. Nanohalococcus encodes an active type I-D CRISPR/Cas system, containing 77 spacers divided into two loci. Despite their diminutive genomes and as part of their host interaction mechanism, the genomes of new nanohaloarchaea do encode giant surface proteins, and one of them (9,409 amino acids long) is the largest protein of any sequenced nanohaloarchaea and the largest protein ever discovered in cultivated archaea.

Nanohaloarchaea as beneficiaries of xylan degradation by haloarchaea.

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt-induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon 'Candidatus Nanohalobium constans'. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea-mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome-inferred trophic relations in two extremely halophilic xylan-degrading three-member consortia. We succeeded in genome assembly and closure for all members of both xylan-degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan-degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan-hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea-host associations using microscopy, multi-omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano-sized archaea can be readily isolated in binary co-cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals.

Mupirocin Promotes Wound Healing by Stimulating Growth Factor Production and Proliferation of Human Keratinocytes.

Mupirocin has been reported for its role in the treatment of infected wounds through its antibacterial activity, however the role of mupirocin in promoting wound healing via alternative mechanisms has not been extensively evaluated. This study aimed to evaluate the potential effect of mupirocin to promote wound healing, not only through its antibacterial activity but by increasing human keratinocyte proliferation and growth factor production. In the scratch assay, using human keratinocytes (HaCat), mupirocin (at 0.1 and 0.2 mM) significantly increased wound closure compared to the vehicle control. Cell viability, measured from the scratch assay, verified the increase in wound closure, where mupirocin at both concentrations showed higher cell viability compared to the vehicle control. In addition, mupirocin at 0.1 mM significantly stimulated the production of hepatocyte growth factor and M-CSF in HaCat cells, whereas at 0.2 mM, PDGF-AA and EPO were increased. The findings of this study suggest that mupirocin, which is commonly used as an antibacterial agent for the treatment of wounds, also facilitates the wound healing process by stimulating the proliferation of human keratinocytes and enhancing the production of several growth factors involved in wound healing. This is the first report on the effect of mupirocin on growth factors expressed by human keratinocytes as well as the stimulation of keratinocyte proliferation.