Deciphering the functional and structural complexity of the Solar Lake flat mat microbial benthic communities.

The Solar Lake in Taba, Egypt, encompasses one of the few modern-day microbial mats' systems metabolically analogous to Precambrian stromatolites. Solar Lake benthic communities and their adaptation to the Lake's unique limnological cycle have not been described for over two decades. In this study, we revisit the flat mat and describe the summer's shallow water versus exposed microbial community; the latter occurs in response to the seasonal partial receding of water. We employed metagenomic NovaSeq-6000 shotgun sequencing and 16S rRNA, mcrA, and dsrB quantitative PCR. A total of 292 medium-to-high-quality metagenome-assembled genomes (MAGs) were reconstructed. At the structural level, Candidatus Aenigmatarchaeota, Micrarchaeota, and Omnitrophota MAGs were exclusively detected in the shallow-water mats, whereas Halobacteria and Myxococcota MAGs were specific to the exposed microbial mat. Functionally, genes involved in reactive oxygen species (ROS) detoxification and osmotic pressure were more abundant in the exposed than in the shallow-water microbial mats, whereas genes involved in sulfate reduction/oxidation and nitrogen fixation were ubiquitously detected. Genes involved in the utilization of methylated amines for methane production were predominant when compared with genes associated with alternative methanogenesis pathways. Solar Lake methanogen MAGs belonged to Methanosarcinia, Bathyarchaeia, Candidatus Methanofastidiosales, and Archaeoglobales. The latter had the genetic capacity for anaerobic methane oxidation. Moreover, Coleofasciculus chthonoplastes, previously reported to dominate the winter shallow-water flat mat, had a substantial presence in the summer. These findings reveal the taxonomic and biochemical microbial zonation of the exposed and shallow-water Solar Lake flat mat benthic community and their capacity to ecologically adapt to the summer water recession. IMPORTANCE: Fifty-five years ago, the extremophilic "Solar Lake" was discovered on the Red Sea shores, garnering microbiologists' interest worldwide from the 1970s to 1990s. Nevertheless, research on the lake paused at the turn of the millennium. In our study, we revisited the Solar Lake benthic community using a genome-centric approach and described the distinct microbial communities in the exposed versus shallow-water mat unveiling microbial zonation in the benthic communities surrounding the Solar Lake. Our findings highlighted the unique structural and functional adaptations employed by these microbial mat communities. Moreover, we report new methanogens and phototrophs, including an intriguing methanogen from the Archaeoglobales family. We describe how the Solar Lake's flat mat microbial community adapts to stressors like oxygen intrusion and drought due to summer water level changes, which provides insights into the genomic strategies of microbial communities to cope with altered and extreme environmental conditions.

PLM-ARG: antibiotic resistance gene identification using a pretrained protein language model.

MOTIVATION: Antibiotic resistance presents a formidable global challenge to public health and the environment. While considerable endeavors have been dedicated to identify antibiotic resistance genes (ARGs) for assessing the threat of antibiotic resistance, recent extensive investigations using metagenomic and metatranscriptomic approaches have unveiled a noteworthy concern. A significant fraction of proteins defies annotation through conventional sequence similarity-based methods, an issue that extends to ARGs, potentially leading to their under-recognition due to dissimilarities at the sequence level. RESULTS: Herein, we proposed an Artificial Intelligence-powered ARG identification framework using a pretrained large protein language model, enabling ARG identification and resistance category classification simultaneously. The proposed PLM-ARG was developed based on the most comprehensive ARG and related resistance category information (>28K ARGs and associated 29 resistance categories), yielding Matthew's correlation coefficients (MCCs) of 0.983 ± 0.001 by using a 5-fold cross-validation strategy. Furthermore, the PLM-ARG model was verified using an independent validation set and achieved an MCC of 0.838, outperforming other publicly available ARG prediction tools with an improvement range of 51.8%-107.9%. Moreover, the utility of the proposed PLM-ARG model was demonstrated by annotating resistance in the UniProt database and evaluating the impact of ARGs on the Earth's environmental microbiota. AVAILABILITY AND IMPLEMENTATION: PLM-ARG is available for academic purposes at https://github.com/Junwu302/PLM-ARG, and a user-friendly webserver (http://www.unimd.org/PLM-ARG) is also provided.

Optimization design of interdigitated microelectrodes with an insulation layer on the connection tracks to enhance efficiency of assessment of the cell viability.

BACKGROUND: Microelectrical Impedance Spectroscopy (µEIS) is a tiny device that utilizes fluid as a working medium in combination with biological cells to extract various electrical parameters. Dielectric parameters of biological cells are essential parameters that can be extracted using µEIS. µEIS has many advantages, such as portability, disposable sensors, and high-precision results. RESULTS: The paper compares different configurations of interdigitated microelectrodes with and without a passivation layer on the cell contact tracks. The influence of the number of electrodes on the enhancement of the extracted impedance for different types of cells was provided and discussed. Different types of cells are experimentally tested, such as viable and non-viable MCF7, along with different buffer solutions. This study confirms the importance of µEIS for in vivo and in vitro applications. An essential application of µEIS is to differentiate between the cells' sizes based on the measured capacitance, which is indirectly related to the cells' size. The extracted statistical values reveal the capability and sensitivity of the system to distinguish between two clusters of cells based on viability and size. CONCLUSION: A completely portable and easy-to-use system, including different sensor configurations, was designed, fabricated, and experimentally tested. The system was used to extract the dielectric parameters of the Microbeads and MCF7 cells immersed in different buffer solutions. The high sensitivity of the readout circuit, which enables it to extract the difference between the viable and non-viable cells, was provided and discussed. The proposed system can extract and differentiate between different types of cells based on cells' sizes; two other polystyrene microbeads with different sizes are tested. Contamination that may happen was avoided using a Microfluidic chamber. The study shows a good match between the experiment and simulation results. The study also shows the optimum number of interdigitated electrodes that can be used to extract the variation in the dielectric parameters of the cells without leakage current or parasitic capacitance.

Assessing and Reassessing the Association of Comorbidities and Coinfections in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) has posed an enormous global health and economic burden. To date, 324 million confirmed cases and over 5.5 million deaths have been reported. Several studies have reported comorbidities and coinfections associated with complicated and serious COVID-19 infections. Data from retrospective, prospective, case series, and case reports from various geographical locations were assessed, which included ~ 2300 COVID-19 patients with varying comorbidities and coinfection. We report that Enterobacterales with Staphylococcus aureus was the most while Mycoplasma pneumoniae was the least prevalent coinfection in COVID-19 patients with a comorbidity. In this order, hypertension, diabetes, cardiovascular disease, and pulmonary disease were the prevalent comorbidities observed in COVID-19 patients. There was a statistically significant difference in the prevalent comorbidities observed in patients coinfected with Staphylococcus aureus and COVID-19 and a statistically non-significant difference in the prevalent comorbidities in patients coinfected with Mycoplasma pneumoniae and COVID-19 as compared to similar infections in non-COVID-19 coinfection. We report a significant difference in the prevalent comorbidities recorded in COVID-19 patients with varying coinfections and varying geographic study regions. Our study provides informative data on the prevalence of comorbidities and coinfections in COVID-19 patients to aid in evidence-based patient management and care.

Metagenomic profiling of antibiotic resistance genes in Red Sea brine pools.

Antibiotic resistance (AR) is an alarming global health concern, causing an annual death rate of more than 35,000 deaths in the US. AR is a natural phenomenon, reported in several pristine environments. In this study, we report AR in pristine Red Sea deep brine pools. Antimicrobial resistance genes (ARGs) were detected for several drug classes with tetracycline and macrolide resistance being the most abundant. As expected, ARGs abundance increased in accordance with the level of human impact with pristine Red Sea samples having the lowest mean ARG level followed by estuary samples, while activated sludge samples showed a significantly higher ARG level. ARG hierarchical clustering grouped drug classes for which resistance was detected in Atlantis II Deep brine pool independent of the rest of the samples. ARG abundance was significantly lower in the Discovery Deep brine pool. A correlation between integrons and ARGs abundance in brine pristine samples could be detected, while insertion sequences and plasmids showed a correlation with ARGs abundance in human-impacted samples not seen in brine pristine samples. This suggests different roles of distinct mobile genetic elements (MGEs) in ARG distribution in pristine versus human-impacted sites. Additionally, we showed the presence of mobile antibiotic resistance genes in the Atlantis II brine pool as evidenced by the co-existence of integrases and plasmid replication proteins on the same contigs harboring predicted multidrug-resistant efflux pumps. This study addresses the role of non-pathogenic environmental bacteria as a silent reservoir for ARGs, and the possible horizontal gene transfer mechanism mediating ARG acquisition.

Impact of innovative nanoadditives on biodigesters microbiome.

Nanoparticles (NPs) supplementation to biodigesters improves the digestibility of biowaste and the generation of biogas. This study investigates the impact of innovative nanoadditives on the microbiome of biodigesters. Fresh cow manure was anaerobically incubated in a water bath under mesophilic conditions for 30 days. Three different NPs (zinc ferrite, zinc ferrite with 10% carbon nanotubes and zinc ferrite with 10% C76 fullerene) were separately supplemented to the biodigesters at the beginning of the incubation period. Methane and hydrogen production were monitored daily. Manure samples were collected from the digesters at different time points and the microbial communities inside the biodigesters were investigated via real-time PCR and 16 S rRNA gene amplicon-sequencing. The results indicate that zinc ferrite NPs enhanced biogas production the most. The microbial community was significantly affected by NPs addition in terms of archaeal and bacterial 16 S rRNAgene copy numbers. The three ZF formulations NPs augmented the abundance of members within the hydrogenotrophic methanogenic phyla Methanobacteriaceae. While Methanomassiliicoccacaea were enriched in ZF/C76 supplemented biodigester due to a significant increase in hydrogen partial pressure, probably caused by the enrichment of Spirochaetaceae (genus Treponema). Overall, NPs supplementation significantly enriched acetate-producing members within Hungateiclostridiaceae in ZF/CNTs, Dysgonomonadaceae in ZF and Spirochaetaceae ZF/C76 biodigesters.

Bioprospecting the microbiome of Red Sea Atlantis II brine pool for peptidases and biosynthetic genes with promising antibacterial activity.

BACKGROUND: The search for novel antimicrobial agents is crucial as antibiotic-resistant pathogens continue to emerge, rendering the available antibiotics no longer effective. Likewise, new anti-cancer drugs are needed to combat the emergence of multi-drug resistant tumors. Marine environments are wealthy sources for natural products. Additionally, extreme marine environments are interesting niches to search for bioactive natural compounds. In the current study, a fosmid library of metagenomic DNA isolated from Atlantis II Deep Lower Convective Layer (ATII LCL), was functionally screened for antibacterial activity as well as anticancer effects. RESULTS: Two clones exhibited antibacterial effects against the marine Bacillus Cc6 strain, namely clones 102-5A and 88-1G and they were further tested against eleven other challenging strains, including six safe relatives of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter  spp.), a safe relative to Mycobacterium tuberculosis and four resistant clinical isolates. Clone 88-1G resulted in clear zones of inhibition against eight bacterial strains, while clone 102-5A resulted in zones of inhibition against five bacterial strains. The whole cell lysates of clone 88-1G showed 15% inhibition of Mtb ClpP protease -Mycobacterium tuberculosis drug target-, while whole cell lysates of clone 102-5A showed 19% inhibition of Mtb ClpP protease. Whole cell lysates from the selected clones exhibited anticancer effects against MCF-7 breast cancer cells (cell viability at 50% v/v was 46.2% ± 9.9 for 88-1G clone and 38% ± 7 for 102-5A clone), U2OS osteosarcoma cells (cell viability at 50% v/v was 64.6% ± 12.3 for 88-1G clone and 28.3% ± 1.7 for 102-5A clone) and 1BR hTERT human fibroblast cells (cell viability at 50% v/v was 74.4% ± 5.6 for 88-1G clone and 57.6% ± 8.9 for 102-5A clone). Sequencing of 102-5A and 88-1G clones, and further annotation detected putative proteases and putative biosynthetic genes in clones 102-5A and 88-1G, respectively. CONCLUSIONS: The ATII LCL metagenome hosts putative peptidases and biosynthetic genes that confer antibiotic and anti-cancer effects. The tested clones exhibited promising antibacterial activities against safe relative strains to ESKAPE pathogens and Mycobacterium tuberculosis. Thus, searching the microbial dark matter of extreme environments is a promising approach to identify new molecules with pharmaceutical potential use.

Evaluation of a Thermophilic, Psychrostable, and Heavy Metal-Resistant Red Sea Brine Pool Esterase.

Lipolytic enzymes catalyze the hydrolysis and synthesis of ester compounds. They are valuable in the pulp, food, and textile industries. This study aims to comprehensively evaluate the extreme properties of a hormone-sensitive lipase (EstATII-TM) isolated from the Red Sea Atlantis II brine pool. EstATII-TM was cloned, expressed, and its biochemical activities were assessed under different conditions. EstATII-TM catalytic properties and resistance to different metal ions were compared to commercial thermophilic esterases under different temperatures. Phylogenetically, EstATII-TM was assigned to the GDSAG motif subfamily of hormone-sensitive lipase. The optimal enzyme activity was evident at a temperature of 30 °C and pH 7-8. The enzyme retained 84.9% of its activity at 0.5 M NaCl. EstATII-TM maintained 93% to 97% activity at -40 and -20 °C, respectively. EstATII-TM activity was significantly enhanced, up to 10-fold, at temperatures ranging from 45 to 65 °C in the presence of 1 mM Cu2+, Cd2+, Ba2+, Mn2+, and Zn2+. EstATII-TM showed superior catalytic activity and resistance-to/enhancement-by metal ions compared to two commercial thermophilic esterases. The Red Sea Atlantis II brine EstATII-TM is characterized by tolerance to high temperatures, stability to hot and cold conditions, as well as toxic heavy metal contamination, making it an ideal candidate for industrial processes.

Abundance of integrons in halophilic bacteria.

Integrons are genetic platforms used for expressing open reading frames (ORFs) arranged in gene cassettes. Excision and integration of gene cassettes are controlled by their associated integron integrase (IntI). Using IntegronFinder software, we analyzed all complete halophilic genomes available in the HaloDom database, along with selected partial halophilic genomes. We identified 18 new complete bacterial integrons and 46 clusters of attC sites lacking a neighboring integron integrase (CALINs). Different classes of insertion sequences (ISs) were also identified within and near integrons and CALINs, with an abundance of IS1182 elements and different ISs that can presumably mobilize adjacent genomic structures. Different promoters of intI genes (PintI) showed nearby binding sites for arginine repressor (ArgR), raising the possibility that IntI expression and recombination activity are regulated by these proteins. Our findings revealed the existence of new integrons in halophilic bacteria with possible adaptive roles.

Antimicrobial and Wound-Healing Activities of Graphene-Reinforced Electrospun Chitosan/Gelatin Nanofibrous Nanocomposite Scaffolds.

This study aims at preparing electrospun chitosan/gelatin nanofiber scaffolds reinforced with different amounts of graphene nanosheets to be used as antibacterial and wound-healing scaffolds. Full characterization was carried out for the different fabricated scaffolds before being assessed for their antimicrobial activity against Escherichia coli and Staphylococcus aureus, cytotoxicity, and cell migration capacity. Raman and transmission electron microscopies confirmed the successful reinforcement of nanofibers with graphene nanosheets. Scanning electron microscopy and porosity revealed that nanofibers reinforced with 0.15% graphene nanosheets produced the least diameter (106 ± 30 nm) and the highest porosity (90%), in addition to their good biodegradability and swellability. However, the excessive increase in graphene nanosheet amount produced beaded nanofibers with decreased porosity, swellability, and biodegradability. Interestingly, nanofibers reinforced with 0.15% graphene nanosheets showed E. coli and S. aureus growth inhibition percents of 50 and 80%, respectively. The cell viability assay showed no cytotoxicity on human fibroblasts when cultured with either unreinforced or reinforced nanofibers. The cell migration was higher in the case of reinforced nanofibers when compared to the unreinforced nanofibers after 24 and 48 h, which is substantially associated with the great effect of the graphene nanosheets on the cell migration capability. Unreinforced and reinforced nanofibers showed cell migration results up to 93.69 and 97%, respectively, after 48 h.

The association of group IIB intron with integrons in hypersaline environments.

BACKGROUND: Group II introns are mobile genetic elements used as efficient gene targeting tools. They function as both ribozymes and retroelements. Group IIC introns are the only class reported so far to be associated with integrons. In order to identify group II introns linked with integrons and CALINS (cluster of attC sites lacking a neighboring integron integrase) within halophiles, we mined for integrons in 28 assembled metagenomes from hypersaline environments and publically available 104 halophilic genomes using Integron Finder followed by blast search for group II intron reverse transcriptases (RT)s. RESULTS: We report the presence of different group II introns associated with integrons and integron-related sequences denoted by UHB.F1, UHB.I2, H.ha.F1 and H.ha.F2. The first two were identified within putative integrons in the metagenome of Tanatar-5 hypersaline soda lake, belonging to IIC and IIB intron classes, respectively at which the first was a truncated intron. Other truncated introns H.ha.F1 and H.ha.F2 were also detected in a CALIN within the extreme halophile Halorhodospira halochloris, both belonging to group IIB introns. The intron-encoded proteins (IEP) s identified within group IIB introns belonged to different classes: CL1 class in UHB.I2 and bacterial class E in H.ha.Fa1 and H.ha.F2. A newly identified insertion sequence (ISHahl1) of IS200/605 superfamily was also identified adjacent to H. halochloris CALIN. Finally, an abundance of toxin-antitoxin (TA) systems was observed within the identified integrons. CONCLUSION: So far, this is the first investigation of group II introns within integrons in halophilic genomes and metagenomes from hypersaline environments. We report the presence of group IIB introns associated with integrons or CALINs. This study provides the basis for understanding the role of group IIB introns in the evolution of halophiles and their potential biotechnological role.

Novel Inulin Electrospun Composite Nanofibers: Prebiotic and Antibacterial Activities.

Inspired by the rampant digestive disorders and the vast bacterial infections, this study aimed at fabricating nanofibers made of inulin/polyvinyl alcohol (PVA) composite nanofibers (CNFs) using the electrospinning technique and testing their prebiotic and antibacterial activities. The inulin/PVA CNFs were tested for prebiotic activity with Lactobacillus species while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used to assess the antibacterial potentiality. During the fabrication of the CNFs, different electrospinning parameters have been carefully controlled, in order to produce nanofibers with relatively uniform diameter, fewer beads, and high integrity. The different parameters included variable solution concentration (material ratio varied from 14 to 20 wt %), applied voltage (varied from 15 to 25 kV), and solution flow (ranged between 0.005 and 0.5 mL/min). The chemical characteristics, thermal stability, and morphology of the formed CNFs were comprehensively characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. Selected CNFs, showing the best diameter uniformity and integrity, were tested for the prebiotic and antimicrobial activity. A 38% increase in prebiotic activity of CNFs, compared to their bulk solution, was observed. The antibacterial activity of the selected CNFs was enhanced, from ∼40% (pure inulin) to 70% (inulin/PVA CNFs) against E. coli and 45% against S. aureus. This study investigates the prebiotic and antibacterial activities of PVA/inulin CNFs and provides the foundation for inulin/PVA CNF use in the healthcare sector, as in disinfectants and/or digestive disorders.

Fabrication of Poly(vinyl alcohol)/Chitosan/Bidens pilosa Composite Electrospun Nanofibers with Enhanced Antibacterial Activities.

Due to the current challenges faced by the increasing rate of drug-resistant bacteria, attention is gradually shifting from synthetic antimicrobial chemical compounds to natural products that are ecofriendly with a wide spectrum of properties. The aim of this research was to successfully fabricate electrospun nanofibers from poly(vinyl alcohol) (PVA), PVA blended with Bidens pilosa and chitosan composite blends and investigate their potential antibacterial activities against Escherichia coli and Staphylococcus aureus. Fabrication of nanofibers was performed by the electrospinning technique, which applies high voltage on the polymer, forcing it to spin off as a jet onto a plate collector. Characterization of the nanofibers was successfully performed by scanning electron microscopy and Fourier transform infrared spectroscopy. Antibacterial assessment was carried out by colony forming unit enumeration. The results obtained revealed a 12% increase in growth inhibition of bacteria in composite nanofibers as compared with their parental forms, which were >91 and 79%, respectively. Chitosan nanofibers have been extensively researched, and their antibacterial properties have been studied. However B. pilosa antibacterial properties in a nanofiber form have not been previously reported. These composite nanofibers open new avenues toward using natural materials as potent antibacterial agents.

Insights into Red Sea Brine Pool Specialized Metabolism Gene Clusters Encoding Potential Metabolites for Biotechnological Applications and Extremophile Survival.

The recent rise in antibiotic and chemotherapeutic resistance necessitates the search for novel drugs. Potential therapeutics can be produced by specialized metabolism gene clusters (SMGCs). We mined for SMGCs in metagenomic samples from Atlantis II Deep, Discovery Deep and Kebrit Deep Red Sea brine pools. Shotgun sequence assembly and secondary metabolite analysis shell (antiSMASH) screening unraveled 2751 Red Sea brine SMGCs, pertaining to 28 classes. Predicted categorization of the SMGC products included those (1) commonly abundant in microbes (saccharides, fatty acids, aryl polyenes, acyl-homoserine lactones), (2) with antibacterial and/or anticancer effects (terpenes, ribosomal peptides, non-ribosomal peptides, polyketides, phosphonates) and (3) with miscellaneous roles conferring adaptation to the environment/special structure/unknown function (polyunsaturated fatty acids, ectoine, ladderane, others). Saccharide (80.49%) and putative (7.46%) SMGCs were the most abundant. Selected Red Sea brine pool sites had distinct SMGC profiles, e.g., for bacteriocins and ectoine. Top promising candidates, SMs with pharmaceutical applications, were addressed. Prolific SM-producing phyla (Proteobacteria, Actinobacteria, Cyanobacteria), were ubiquitously detected. Sites harboring the largest numbers of bacterial and archaeal phyla, had the most SMGCs. Our results suggest that the Red Sea brine niche constitutes a rich biological mine, with the predicted SMs aiding extremophile survival and adaptation.

Antibacterial and anticancer activities of orphan biosynthetic gene clusters from Atlantis II Red Sea brine pool.

BACKGROUND: Cancer and infectious diseases are problematic because of continuous emergence of drug resistance. One way to address this enormous global health threat is bioprospecting the unlikeliest environments, such as extreme marine niches, which have tremendous biodiversity that is barely explored. One such environment is the Red Sea brine pool, Atlantis II Deep (ATII). Here, we functionally screened a fosmid library of metagenomic DNA isolated from the ATII lower convective layer (LCL) for antibacterial and anticancer activities. RESULTS: Selected clones, 14-7E and 10-2G, displayed antibacterial effects on the marine strain Bacillus sp. Cc6. Moreover, whole cell lysates from 14-7E and 10-2G exhibited decreased cell viability against MCF-7 (39.1% ± 6.6, 42% ± 8.1 at 50% v/v) and U2OS cells (35.7% ± 1.9, 79.9% ± 5.9 at 50% v/v), respectively. By sequencing the insert DNA from 14-7E and 10-2G, we identified two putative orphan biosynthetic gene clusters. Both clusters harbored putative ATP-binding cassette (ABC) transporter permeases and S-adenosylmethionine-related genes. Interestingly, the biosynthetic gene cluster identified on 14-7E is of archaeal origin and harbors a putative transcription factor. Several identified genes may be responsible for the observed antibacterial and anticancer activities. The 14-7E biosynthetic gene cluster may be encoding enzymes producing a specialized metabolite (effect of detected genes involved in C-C bond formation and glycosylation). The bioactivity may also be due to predicted subtilases encoded by this cluster. The 10-2G cluster harbored putative glycosyltransferase and non-ribosomal peptide synthase genes; thus the observed activity of this clone could be caused by a bioactive peptide. CONCLUSIONS: The ATII LCL prokaryotic metagenome hosts putative orphan biosynthetic gene clusters that confer antibiotic and anticancer effects. Further biochemical studies should characterize the detected bioactive components, and the potential use of 14-7E metabolite for antibiosis and 10-2G metabolite as a selective anti-breast cancer drug.

Distinct domains of Escherichia coli IgaA connect envelope stress sensing and down-regulation of the Rcs phosphorelay across subcellular compartments.

In enterobacteria, the Rcs system (Regulator of capsule synthesis) monitors envelope integrity and induces a stress response when damages occur in the outer membrane or in the peptidoglycan layer. Built around a two-component system, Rcs controls gene expression via a cascade of phosphoryl transfer reactions. Being particularly complex, Rcs also involves the outer membrane lipoprotein RcsF and the inner membrane essential protein IgaA (Intracellular growth attenuator). RcsF and IgaA, which are located upstream of the phosphorelay, are required for normal Rcs functioning. Here, we establish the stress-dependent formation of a complex between RcsF and the periplasmic domain of IgaA as the molecular signal triggering Rcs. Moreover, molecular dissection of IgaA reveals that its negative regulatory role on Rcs is mostly carried by its first N-terminal cytoplasmic domain. Altogether, our results support a model in which IgaA regulates Rcs activation by playing a direct role in the transfer of signals from the cell envelope to the cytoplasm. This remarkable feature further distinguishes Rcs from other envelope stress response systems.

Novel thermostable antibiotic resistance enzymes from the Atlantis II Deep Red Sea brine pool.

The advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence-based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3'-aminoglycoside phosphotransferase [APH(3')] and a class A beta-lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3') was active in vivo. Interestingly, APH(3') proved to be thermostable (Tm  = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a Tm  = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.

Insertion sequences enrichment in extreme Red sea brine pool vent.

Mobile genetic elements are major agents of genome diversification and evolution. Limited studies addressed their characteristics, including abundance, and role in extreme habitats. One of the rare natural habitats exposed to multiple-extreme conditions, including high temperature, salinity and concentration of heavy metals, are the Red Sea brine pools. We assessed the abundance and distribution of different mobile genetic elements in four Red Sea brine pools including the world's largest known multiple-extreme deep-sea environment, the Red Sea Atlantis II Deep. We report a gradient in the abundance of mobile genetic elements, dramatically increasing in the harshest environment of the pool. Additionally, we identified a strong association between the abundance of insertion sequences and extreme conditions, being highest in the harshest and deepest layer of the Red Sea Atlantis II Deep. Our comparative analyses of mobile genetic elements in secluded, extreme and relatively non-extreme environments, suggest that insertion sequences predominantly contribute to polyextremophiles genome plasticity.