Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility.

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

Analysis of Microbiomes from Ultra-Low Biomass Surfaces Using Novel Surface Sampling and Nanopore Sequencing.

The rapid assessment of microbiomes from ultra-low biomass environments such as cleanrooms or hospital operating rooms has a number of applications for human health and spacecraft manufacturing. Current techniques often employ lengthy protocols using short-read DNA sequencing technology to analyze amplified DNA and have the disadvantage of a longer analysis time and lack of portability. Here, we demonstrate a rapid (~24 hours) on-site nanopore-based sequencing approach to characterize the microbiome of a NASA Class 100K cleanroom where spacecraft components are assembled. This approach employs a modified protocol of Oxford Nanopore's Rapid PCR Barcoding Kit in combination with the recently developed Squeegee-Aspirator for Large Sampling Area (SALSA) surface sampling device. Results for these ultra-low biomass samples revealed DNA amplification ~1 to 2 orders of magnitude above process control samples and were dominated primarily by Paracoccus and Acinetobacter species. Negative control samples were collected to provide critical data on background contamination, including Cutibacerium acnes, which most likely originated from the sampling reagents-associated microbiome (kitome). Overall, these results provide data on a novel approach for rapid low-biomass DNA profiling using the SALSA sampler combined with modified nanopore sequencing. These data highlight the critical need for employing multiple negative controls, along with using DNA-free reagents and techniques, to enable a proper assessment of ultra-low biomass samples.

Draft genome sequencing of Naganishia species isolated from the polar environments.

The draft genomes of five Naganishia strains were sequenced using MinION and annotated using Funannotate pipeline. Phylogenetic and genomic analyses were performed to provide their genetic relationships, diversity, and potential functional capabilities. This approach will aid in understanding their potential to survive under microgravity and their resilience to extreme environments.

Genomic characterization and radiation tolerance of Naganishia kalamii sp. nov. and Cystobasidium onofrii sp. nov. from Mars 2020 mission assembly facilities.

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

Microbial Technologies in Waste Management, Energy Generation and Climate Change: Implications on Earth and Space.

Microbes are important decomposers of organic waste. By decomposing organic waste and using it for their growth, microbes play an important role in maintaining ecosystem's carbon and nitrogen cycles. An ecosystem's microbial shift may disturb it's carbon/nitrogen cycle as a result of any climate change or humanitarian factors, but heat produced by various instruments and greenhouse gases contribute significantly to global warming which in turn may be related to microbial shift of ecosystems. To reduce greenhouse gas emissions and global warming, innovative clean energy production methods must be employed to develop fuels with minimal greenhouse effect. Biofuels, such as bioethanol, provide clean energy with less carbon dioxide emissions. For the production of bioethanol, it is always recommended to use microbes that are capable of decomposing complex organic matter (cellulose, lignin, hemicellulose). Some microbes can efficiently decompose complex organic matter due to the presence of genetic machinery that produces cellulases and ß-glucosidase. The membrane transporters are also important for microbes in uptake of simple sugars for metabolism and ethanol production. Microbial technologies are addressing the future needs for not only organic waste management but also clean energy/bioethanol production. However, the role of these technologies on space missions and extraterrestrial settings needs to be explored to improve long term space missions.

Draft Genome Sequences of Fungi Isolated from Mars 2020 Spacecraft Assembly Facilities.

During the Mars 2020 mission, several fungal strains were isolated from surfaces where spacecraft components were assembled. Draft genome sequencing and characterization will help identify the genes responsible for radiation resistance, supporting the development of countermeasures to prevent fungal contamination of extraterrestrial environments.

Isomaltooligosaccharides utilization and genomic characterization of human infant anti-inflammatory Bifidobacterium longum and Bifidobacterium breve strains.

This study was carried out to understand the probiotic features, ability to utilize non-digestible carbohydrates and comparative genomics of anti-inflammatory Bifidobacterium strains isolated from human infant stool samples. Bacterial strains were isolated from the stool samples using serial dilution on MRS agar plates supplemented with 0.05% l-cysteine hydrochloride and mupirocin. Molecular characterization of the strains was carried out by 16S rRNA gene sequencing. Anti-inflammatory activity was determined using TNF-α and lipopolysaccharide (LPS) induced inflammation in Caco2 cells. Probiotic attributes were determined as per the established protocols. Isomaltooligosaccharides (IMOS) utilization was determined in the broth cultures. Whole genome sequencing and analysis was carried out for three strains. Four obligate anaerobic, Gram positive Bifidobacterium strains were isolated from the infant stool samples. Strains were identified as Bifidobacterium longum Bif10, B. breve Bif11, B. longum Bif12 and B. longum Bif16. The strains were able to prevent inflammation in the Caco2 cells through lowering of IL8 production that was caused by TNF-α and LPS treatment. The strains exhibited desirable probiotic attributes such as acid and bile tolerance, mucin binding, antimicrobial activity, bile salt hydrolase activity, cholesterol lowering ability and could ferment non-digestible carbohydrates such as isomaltooligosaccharides and raffinose. Furthermore, Isomaltooligosaccharides supported the optimum growth of the strains in vitro, which was comparable to that on glucose. Strains could metabolize IMOS through cell associated α-glucosidase activity. Genomic features revealed the presence of genes responsible for the utilization of IMOS and for the probiotic attributes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03141-2.

Description and Genome Characterization of Three Novel Fungal Strains Isolated from Mars 2020 Mission-Associated Spacecraft Assembly Facility Surfaces-Recommendations for Two New Genera and One Species.

National Aeronautics and Space Administration's (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains.

Draft Genome Sequence of Bifidobacterium pseudocatenulatum Bif4, Isolated from Healthy Infant Feces.

We report the 2.24-Mb draft genome sequence of Bifidobacterium pseudocatenulatum Bif4, isolated from a fecal sample from a healthy infant. The specific annotations revealed genes predictive of its probiotic attributes.

Genomic analysis of Indian strains of Salmonella enterica subsp. enterica serovar Typhi indicates novel genetic repertoire for pathogenicity and adaptations.

In the era of emerging antibiotic resistance, Salmonella enterica subsp. enterica serovar Typhi the causative agent of typhoid, is a threat for healthcare systems in developing countries especially India, where the disease is highly endemic. Genetic diversity among different strains may be the cause of variable severity of disease in different regions of the world. To explore this genetic diversity, genome annotation by rapid annotation using subsystem technology (RAST) was carried out for genomes of four Salmonella Typhi strains from two distinct areas available in the public domain. Two clinical strains were from India (P-stx-12 and E02-1180) and the other two strains considered as reference strains were from the endemic regions of Papua New Guinea (UJ308A and UJ816A). We report that Indian clinical strains possess several similar genes responsible for virulence and pathogenicity as those present in the reference strains. Interestingly, Indian clinical strains also possess 34 additional potential virulence genes that are absent in the reference strains, suggesting the more dreadful nature of Indian clinical strains as compared to those causing endemic typhoid. Indian strains contained genes coding for; Colicin V and bacteriocin production; multidrug resistance efflux pumps; ABC transporters; Type III and Type VI secretion systems, siderophore aerobactin, pathogenicity islands and Vi polysaccharide biosynthesis and transport. These unique genes are also reported in the genomes of other six clinical strains of India analyzed through RAST and IslandViewer 4 for validation purpose. This study highlights the presence of potential genes as molecular targets to overcome the future endemic outbreaks in India.

A genomic analysis of Mycobacterium immunogenum strain CD11_6 and its potential role in the activation of T cells against Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis (Mtb) is an etiological agent of tuberculosis (TB). Tuberculosis is a mounting problem worldwide. The only available vaccine BCG protects the childhood but not adulthood form of TB. Therefore, efforts are made continuously to improve the efficacy of BCG by supplementing it with other therapies. Consequently, we explored the possibility of employing Mycobacterium immunogenum (Mi) to improve BCG potential to protect against Mtb. RESULTS: We report here the genome mining, comparative genomics, immunological and protection studies employing strain CD11_6 of Mi. Mycobacterium immunogenum was isolated from duodenal mucosa of a celiac disease patient. The strain was whole genome sequenced and annotated for identification of virulent genes and other traits that may make it suitable as a potential vaccine candidate. Virulence profile of Mi was mapped and compared with two other reference genomes i.e. virulent Mtb strain H37Rv and vaccine strain Mycobacterium bovis (Mb) AFF2122/97. This comparative analysis revealed that Mi is less virulent, as compared to Mb and Mtb, and contains comparable number of genes encoding for the antigenic proteins that predict it as a probable vaccine candidate. Interestingly, the animals vaccinated with Mi showed significant augmentation in the generation of memory T cells and reduction in the Mtb burden. CONCLUSION: The study signifies that Mi has a potential to protect against Mtb and therefore can be a future vaccine candidate against TB.

Cross-Talk Between Gluten, Intestinal Microbiota and Intestinal Mucosa in Celiac Disease: Recent Advances and Basis of Autoimmunity.

Celiac disease (CD) is an autoimmune disorder of the small intestine, caused by gluten induced inflammation in some individuals susceptible to genetic and environmental influences. To date, pathophysiology of CD in relation to intestinal microbiota is not known well. This review relies on contribution of intestinal microbiome and oral microbiome in pathogenesis of CD based on their interactions with gluten, thereby highlighting the role of upper gastrointestinal microbiota. It has been hypothesized that CD might be triggered by additive effects of immunotoxic gluten peptides and intestinal dysbiosis (microbial imbalance) in the people with or without genetic susceptibilities, where antibiotics may be deriving dysbiotic agents. In contrast to the intestinal dysbiosis, genetic factors even seem secondary in disease outcome thus suggesting the importance of interaction between microbes and dietary factors in immune regulation at intestinal mucosa. Moreover, association of imbalanced counts of some commensal microbes in intestine of CD patients suggests the scope for probiotic therapies. Lactobacilli and specific intestinal and oral bacteria are potent source of gluten degrading enzymes (glutenases) that may contribute to commercialization of a novel glutenase therapy. In this review, we shall discuss advantages and disadvantages of food based therapies along with probiotic therapies where probiotic therapies are expected to emerge as the safest biotherapies among other in-process therapies. In addition, this review emphasizes on differential targets of probiotics that make them suitable to manage CD as along with glutenase activity, they also exhibit immunomodulatory and intestinal microbiome modulatory properties.

Genome sequence and comparative genomic analysis of a clinically important strain CD11-4 of Janibacter melonis isolated from celiac disease patient.

BACKGROUND: Janibacter melonis and other member of this genus are known to cause bacteremia and serious clinical comorbidities, but there is no study reporting about pathogenicity attributes of J. melonis. Janibacter terrae is known to cause lethal infection. Reporting the genome of J. melonis CD11-4 and comparative genomics with other members of genus has provided some novel insights that can enable us to understand the mechanisms responsible for its pathogenicity in humans. RESULTS: Comparative genomic analysis by Rapid Annotation Server and Technology revealed the presence of similar virulence determinant genes in both J. terrae NBRC 107853T and J. melonis CD11-4. Like J. terrae NBRC 107853T, J. melonis CD11-4 contained two genes responsible for resistance against ß-lactam class of antibiotics and two genes for resistance against fluoroquinolones. Interestingly, J. melonis CD11-4 contained a unique gene coding for multidrug resistance efflux pumps unlike all other members of this genus. It also contained two genes involved in Toxin-antitoxin Systems that were absent in J. terrae NBRC 107853T but were present in some other members of genus. CONCLUSIONS: Genome annotations of J. melonis CD11-4 revealed that it contained similar or more virulence repertoire like J. terrae NBRC 107853T. Like other gut pathogens, J. melonis possesses key virulence determinant genes for antibiotic resistance, invasion, adhesion, biofilm formation, iron acquisition and to cope with stress response, thereby indicating that strain J. melonis CD11-4 could be a gut pathogen.

Genome Sequence of Kocuria polaris Strain CD08_4, an Isolate from the Duodenal Mucosa of a Celiac Disease Patient.

We report here the 3.8-Mb genome sequence of Kocuria polaris strain CD08_4, an isolate from the duodenal mucosa of a celiac disease patient. The genome consists of specific virulence determinant genes, antibiotic resistance genes, genes for coping with oxidative stress, and genes responsible for iron acquisition and metabolism, suggestive of its pathogenic attributes.

Genome Insight and Comparative Pathogenomic Analysis of Nesterenkonia jeotgali Strain CD08_7 Isolated from Duodenal Mucosa of Celiac Disease Patient.

Species of the genus Nesterenkonia have been isolated from different ecological niches, especially from saline habitats and reported as weak human pathogens causing asymptomatic bacteraemia. Here, for the first time we are reporting the genome sequence and pathogenomic analysis of a strain designated as CD08_7 isolated from the duodenal mucosa of a celiac disease patient, identified as Nesterenkonia jeotgali. To date, only five strains of the genus Nesterenkonia (N. massiliensis strain NP1T, Nesterenkonia sp. strain JCM 19054, Nesterenkonia sp. strain F and Nesterenkonia sp. strain AN1) have been whole genome sequenced and annotated. In the present study we have mapped and compared the virulence profile of N. jeotgali strain CD08_7 along with other reference genomes which showed some characteristic features that could contribute to pathogenicity. The RAST (Rapid Annotation using Subsystem Technology) based genome mining revealed more genes responsible for pathogenicity in strain CD08_7 when compared with the other four sequenced strains. The studied categories were resistance to antibiotic and toxic compounds, invasion and intracellular resistance, membrane transport, stress response, osmotic stress, oxidative stress, phages and prophages and iron acquisition. A total of 1431 protein-encoding genes were identified in the genome of strain CD08_7 among which 163 were predicted to contribute for pathogenicity. Out of 163 genes only 59 were common to other genome, which shows the higher levels of genetic richness in strain CD08_7 that may contribute to its functional versatility. This study provides a comprehensive analysis on genome of N. jeotgali strain CD08_7 and possibly indicates its importance as a clinical pathogen.

Genome Sequence of Kocuria palustris Strain CD07_3 Isolated from the Duodenal Mucosa of a Celiac Disease Patient.

We report here the 2.8-Mb genome of Kocuria palustris strain CD07_3 isolated from the duodenal mucosa of a celiac disease (CD) patient. The genome of the bacterium consists of specific virulence factor genes and antibiotic resistance genes that depict its pathogenic potential.

Genome Sequencing of Serinicoccus chungangensis Strain CD08_5 Isolated from Duodenal Mucosa of a Celiac Disease Patient.

For the first time, we report here the 3.5-Mb genome of Serinicoccus chungangensis strain CD08_5, isolated from duodenal mucosa from a celiac disease (CD) patient. The specific annotations obtained revealed genes associated with virulence, disease, and defense, which predict its probable role in the pathogenesis of CD.