Phylogenomic analysis expands the known repertoire of single-stranded DNA viruses in benthic zones of the South Indian Ocean.

Single-stranded (ss) DNA viruses are ubiquitous and constitute some of the most diverse entities on Earth. Most studies have focused on ssDNA viruses from terrestrial environments resulting in a significant deficit in benthic ecosystems including aphotic zones of the South Indian Ocean (SIO). Here, we assess the diversity and phylogeny of ssDNA in deep waters of the SIO using a combination of established viral taxonomy tools and a Hidden Markov Model based approach. Replication initiator protein-associated (Rep) phylogenetic reconstruction and sequence similarity networks were used to show that the SIO hosts divergent and as yet unknown circular Rep-encoding ssDNA viruses. Several sequences appear to represent entirely novel families, expanding the repertoire of known ssDNA viruses. Results suggest that a small proportion of these viruses may be circular genetic elements, which may strongly influence the diversity of both eukaryotes and prokaryotes in the SIO. Taken together, our data show that the SIO harbours a diverse assortment of previously unknown ssDNA viruses. Due to their potential to infect a variety of hosts, these viruses may be crucial for marine nutrient recycling through their influence of the biological carbon pump.

Bacteria and Archaea Regulate Particulate Organic Matter Export in Suspended and Sinking Marine Particle Fractions.

The biological carbon pump (BCP) in the Southern Ocean is driven by phytoplankton productivity and is a significant organic matter sink. However, the role of particle-attached (PA) and free-living (FL) prokaryotes (bacteria and archaea) and their diversity in influencing the efficiency of the BCP is still unclear. To investigate this, we analyzed the metagenomes linked to suspended and sinking marine particles from the Sub-Antarctic Southern Ocean Time Series (SOTS) by deploying a Marine Snow Catcher (MSC), obtaining suspended and sinking particulate material, determining organic carbon and nitrogen flux, and constructing metagenome-assembled genomes (MAGs). The suspended and sinking particle-pools were dominated by bacteria with the potential to degrade organic carbon. Bacterial communities associated with the sinking fraction had more genes related to the degradation of complex organic carbon than those in the suspended fraction. Archaea had the potential to drive nitrogen metabolism via nitrite and ammonia oxidation, altering organic nitrogen concentration. The data revealed several pathways for chemoautotrophy and the secretion of recalcitrant dissolved organic carbon (RDOC) from CO2, with bacteria and archaea potentially sequestering particulate organic matter (POM) via the production of RDOC. These findings provide insights into the diversity and function of prokaryotes in suspended and sinking particles and their role in organic carbon/nitrogen export in the Southern Ocean. IMPORTANCE The biological carbon pump is crucial for the export of particulate organic matter in the ocean. Recent studies on marine microbes have shown the profound influence of bacteria and archaea as regulators of particulate organic matter export. Yet, despite the importance of the Southern Ocean as a carbon sink, we lack comparable insights regarding microbial contributions. This study provides the first insights regarding prokaryotic contributions to particulate organic matter export in the Southern Ocean. We reveal evidence that prokaryotic communities in suspended and sinking particle fractions harbor widespread genomic potential for mediating particulate organic matter export. The results substantially enhance our understanding of the role played by microorganisms in regulating particulate organic matter export in suspended and sinking marine fractions in the Southern Ocean.

CrAssphage May Be Viable Markers of Contamination in Pristine and Contaminated River Water.

Viruses are the most biologically abundant entities and may be ideal indicators of fecal pollutants in water. Anthropogenic activities have triggered drastic ecosystem changes in rivers, leading to substantial shifts in chemical and biological attributes. Here, we evaluate the viability of using the presence of crAssphage as indicators of fecal contamination in South African rivers. Shotgun analysis revealed diverse crAssphage viruses in these rivers, which are impacted by chemical and biological pollution. Overall, the diversity and relative abundances of these viruses was higher in contaminated sites compared to pristine locations. In contrast to fecal coliform counts, crAssphage sequences were detected in pristine rivers, supporting the assertion that the afore mentioned marker may be a more accurate indicator of fecal contamination. Our data demonstrate the presence of diverse putative hosts which includes members of the phyla Bacteroidota, Pseudomonadota, Verrucomicrobiota, and Bacillota. Phylogenetic analysis revealed novel subfamilies, suggesting that rivers potentially harbor distinct and uncharacterized clades of crAssphage. These data provide the first insights regarding the diversity, distribution, and functional roles of crAssphage in rivers. Taken together, the results support the potential application of crAssphage as viable markers for water quality monitoring. IMPORTANCE Rivers support substantial populations and provide important ecosystem services. Despite the application of fecal coliform tests and other markers, we lack rapid and reproducible approaches for determining fecal contamination in rivers. Waterborne viral outbreaks have been reported even after fecal indicator bacteria (FIB) were suggested to be absent or below regulated levels of coliforms. This indicates a need to develop and apply improved indicators of pollutants in aquatic ecosystems. Here, we evaluate the viability of crAssphage as indicators of fecal contamination in two South African rivers. We assess the abundance, distribution, and diversity of these viruses in sites that had been predicted pristine or contaminated by FIB analysis. We show that crAssphage are ideal and sensitive markers for fecal contamination and describe novel clades of crAss-like phages. Known crAss-like subfamilies were unrepresented in our data, suggesting that the diversity of these viruses may reflect geographic locality and dependence.

Metagenome-Assembled Genomes of Four Southern Ocean Archaea Harbor Multiple Genes Linked to Polyethylene Terephthalate and Polyhydroxybutyrate Plastic Degradation.

Here, we present four archaeal metagenome-assembled genomes (MAGs) (three Thaumarchaeota MAGs and one Thermoplasmatota MAG) from a polar upwelling zone in the Southern Ocean. These archaea harbor putative genes encoding enzymes such as polyethylene terephthalate (PET) hydrolases (PETases) and polyhydroxybutyrate (PHB) depolymerases, which are associated with microbial degradation of PET and PHB plastics.

Microbial ecology of the Southern Ocean.

The Southern Ocean (SO) distributes climate signals and nutrients worldwide, playing a pivotal role in global carbon sequestration. Microbial communities are essential mediators of primary productivity and carbon sequestration, yet we lack a comprehensive understanding of microbial diversity and functionality in the SO. Here, we examine contemporary studies in this unique polar system, focusing on prokaryotic communities and their relationships with other trophic levels (i.e. phytoplankton and viruses). Strong seasonal variations and the characteristic features of this ocean are directly linked to community composition and ecosystem functions. Specifically, we discuss characteristics of SO microbial communities and emphasise differences from the Arctic Ocean microbiome. We highlight the importance of abundant bacteria in recycling photosynthetically derived organic matter. These heterotrophs appear to control carbon flux to higher trophic levels when light and iron availability favour primary production in spring and summer. Conversely, during winter, evidence suggests that chemolithoautotrophs contribute to prokaryotic production in Antarctic waters. We conclude by reviewing the effects of climate change on marine microbiota in the SO.

Four Lentisphaerae Family Metagenome-Assembled Genomes from the South Atlantic Ocean.

We present four Lentisphaerae metagenome-assembled genomes (MAGs) from the South Atlantic Ocean. The medium-quality genomes, affiliated with the family of Lentisphaeraceae, ranged from 4.86 to 5.46 Mbp and harbored the genetic capacity to produce secondary metabolites. This resource provides a basis for investigating the functional attributes of this phylum.

Zetaproteobacteria Pan-Genome Reveals Candidate Gene Cluster for Twisted Stalk Biosynthesis and Export.

Twisted stalks are morphologically unique bacterial extracellular organo-metallic structures containing Fe(III) oxyhydroxides that are produced by microaerophilic Fe(II)-oxidizers belonging to the Betaproteobacteria and Zetaproteobacteria. Understanding the underlying genetic and physiological mechanisms of stalk formation is of great interest based on their potential as novel biogenic nanomaterials and their relevance as putative biomarkers for microbial Fe(II) oxidation on ancient Earth. Despite the recognition of these special biominerals for over 150 years, the genetic foundation for the stalk phenotype has remained unresolved. Here we present a candidate gene cluster for the biosynthesis and secretion of the stalk organic matrix that we identified with a trait-based analyses of a pan-genome comprising 16 Zetaproteobacteria isolate genomes. The "stalk formation in Zetaproteobacteria" (sfz) cluster comprises six genes (sfz1-sfz6), of which sfz1 and sfz2 were predicted with functions in exopolysaccharide synthesis, regulation, and export, sfz4 and sfz6 with functions in cell wall synthesis manipulation and carbohydrate hydrolysis, and sfz3 and sfz5 with unknown functions. The stalk-forming Betaproteobacteria Ferriphaselus R-1 and OYT-1, as well as dread-forming Zetaproteobacteria Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8 contain distant sfz gene homologs, whereas stalk-less Zetaproteobacteria and Betaproteobacteria lack the entire gene cluster. Our pan-genome analysis further revealed a significant enrichment of clusters of orthologous groups (COGs) across all Zetaproteobacteria isolate genomes that are associated with the regulation of a switch between sessile and motile growth controlled by the intracellular signaling molecule c-di-GMP. Potential interactions between stalk-former unique transcription factor genes, sfz genes, and c-di-GMP point toward a c-di-GMP regulated surface attachment function of stalks during sessile growth.

Evolutionary stasis of a deep subsurface microbial lineage.

Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared >99.2% average nucleotide identity, low frequency of SNP's, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.

Ancestral Absence of Electron Transport Chains in Patescibacteria and DPANN.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.

Phages Actively Challenge Niche Communities in Antarctic Soils.

By modulating the structure, diversity, and trophic outputs of microbial communities, phages play crucial roles in many biomes. In oligotrophic polar deserts, the effects of katabatic winds, constrained nutrients, and low water availability are known to limit microbial activity. Although phages may substantially govern trophic interactions in cold deserts, relatively little is known regarding the precise ecological mechanisms. Here, we provide the first evidence of widespread antiphage innate immunity in Antarctic environments using metagenomic sequence data from hypolith communities as model systems. In particular, immunity systems such as DISARM and BREX are shown to be dominant systems in these communities. Additionally, we show a direct correlation between the CRISPR-Cas adaptive immunity and the metavirome of hypolith communities, suggesting the existence of dynamic host-phage interactions. In addition to providing the first exploration of immune systems in cold deserts, our results suggest that phages actively challenge niche communities in Antarctic polar deserts. We provide evidence suggesting that the regulatory role played by phages in this system is an important determinant of bacterial host interactions in this environment.IMPORTANCE In Antarctic environments, the combination of both abiotic and biotic stressors results in simple trophic levels dominated by microbiomes. Although the past two decades have revealed substantial insights regarding the diversity and structure of microbiomes, we lack mechanistic insights regarding community interactions and how phages may affect these. By providing the first evidence of widespread antiphage innate immunity, we shed light on phage-host dynamics in Antarctic niche communities. Our analyses reveal several antiphage defense systems, including DISARM and BREX, which appear to dominate in cold desert niche communities. In contrast, our analyses revealed that genes which encode antiphage adaptive immunity were underrepresented in these communities, suggesting lower infection frequencies in cold edaphic environments. We propose that by actively challenging niche communities, phages play crucial roles in the diversification of Antarctic communities.

Charting the Complexity of the Marine Microbiome through Single-Cell Genomics.

Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.

Active Fungal Communities in Asymptomatic Eucalyptus grandis Stems Differ between a Susceptible and Resistant Clone.

Fungi represent a common and diverse part of the microbial communities that associate with plants. They also commonly colonise various plant parts asymptomatically. The molecular mechanisms of these interactions are, however, poorly understood. In this study we use transcriptomic data from Eucalyptus grandis, to demonstrate that RNA-seq data are a neglected source of information to study fungal-host interactions, by exploring the fungal transcripts they inevitably contain. We identified fungal transcripts from E. grandis data based on their sequence dissimilarity to the E. grandis genome and predicted biological functions. Taxonomic classifications identified, amongst other fungi, many well-known pathogenic fungal taxa in the asymptomatic tissue of E. grandis. The comparison of a clone of E. grandis resistant to Chrysoporthe austroafricana with a susceptible clone revealed a significant difference in the number of fungal transcripts, while the number of fungal taxa was not substantially affected. Classifications of transcripts based on their respective biological functions showed that the fungal communities of the two E. grandis clones associate with fundamental biological processes, with some notable differences. To shield the greater host defence machinery in the resistant E. grandis clone, fungi produce more secondary metabolites, whereas the environment for fungi associated with the susceptible E. grandis clone is more conducive for building fungal cellular structures and biomass growth. Secreted proteins included carbohydrate active enzymes that potentially are involved in fungal-plant and fungal-microbe interactions. While plant transcriptome datasets cannot replace the need for designed experiments to probe plant-microbe interactions at a molecular level, they clearly hold potential to add to the understanding of the diversity of plant-microbe interactions.

Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa.

BACKGROUND: Bacillus endophyticus is a soil plant-endophytic bacterium, while B. anthracis is the causative agent of anthrax. The virulence factors of B. anthracis are the plasmid encoded tripartite toxins (pXO1) and poly-γ-glutamic acid (PGA) capsule (pXO2). B. endophyticus isolated alongside B. anthracis from animals that died of anthrax in Northern Cape Province (NCP), South Africa, harbored polyglutamate genes. The study compared the characteristics of B. anthracis and B. endophyticus with other Bacillus species with a focus on the presence of the PGA capsule or/and unbound PGA. The morphology and whole genome sequence analysis of B. endophyticus strains and B. anthracis were compared. RESULTS: In conventional microbiology, B. endophyticus showed gram-positive round-shaped rods in single/short chains, which were endospore-forming, non-motile, non-haemolytic with white and dry colonies, and γ-phage resistant. B. anthracis was differentiated from B. endophyticus based on the latter's box-shaped rods in pairs/long chains, white-grey and slimy colonies, encapsulated and γ-phage susceptible. The study identified a PGA polyglutamate synthase operon that consisted of pgsBCA, γ-glutamyltranspeptidase (ggt) and pgsE in B. endophyticus genomes. CONCLUSIONS: PGA regions of B. anthracis contain capBCADE genes located in the pXO2 required for capsulation formation, while B. endophyticus contain the pgsBCAE genes in the chromosome. Whole genome and microbiology analysis identified B. endophyticus, as a non-capsuled endospore-forming bacterium that consists of PGA required for biosynthesis. B. endophyticus strains do not synthesize surface associated PGA, therefore capsule visualization of B. anthracis is a key diagnostic characteristic. The study highlights the significance of using whole genome shotgun sequencing to identify virulence and other important genes that might be present amongst unknown samples from natural outbreaks. None of the B. anthracis related plasmids or virulence genes were found in the B. endophyticus genomes.

A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa.

Species within the Neoromicia bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, Neoromicia capensis have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the Circoviridae family, and also identified sequence data from the Adenoviridae, Coronaviridae, Herpesviridae, Parvoviridae, Papillomaviridae, Phenuiviridae, and Picornaviridae families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the Adenoviridae, Coronaviridae, and Herpesviridae families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous Neoromicia-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread throughout Neoromicia populations in South Africa. Furthermore, similar adenovirus sequences were detected within these populations throughout several years. With the exception of the coronaviruses, the study represents the first report of sequence data from several viral families within a Southern African insectivorous bat genus; highlighting the need for continued investigations in this regard.

A reservoir of 'historical' antibiotic resistance genes in remote pristine Antarctic soils.

BACKGROUND: Soil bacteria naturally produce antibiotics as a competitive mechanism, with a concomitant evolution, and exchange by horizontal gene transfer, of a range of antibiotic resistance mechanisms. Surveys of bacterial resistance elements in edaphic systems have originated primarily from human-impacted environments, with relatively little information from remote and pristine environments, where the resistome may comprise the ancestral gene diversity. METHODS: We used shotgun metagenomics to assess antibiotic resistance gene (ARG) distribution in 17 pristine and remote Antarctic surface soils within the undisturbed Mackay Glacier region. We also interrogated the phylogenetic placement of ARGs compared to environmental ARG sequences and tested for the presence of horizontal gene transfer elements flanking ARGs. RESULTS: In total, 177 naturally occurring ARGs were identified, most of which encoded single or multi-drug efflux pumps. Resistance mechanisms for the inactivation of aminoglycosides, chloramphenicol and ß-lactam antibiotics were also common. Gram-negative bacteria harboured most ARGs (71%), with fewer genes from Gram-positive Actinobacteria and Bacilli (Firmicutes) (9%), reflecting the taxonomic composition of the soils. Strikingly, the abundance of ARGs per sample had a strong, negative correlation with species richness (r = - 0.49, P < 0.05). This result, coupled with a lack of mobile genetic elements flanking ARGs, suggests that these genes are ancient acquisitions of horizontal transfer events. CONCLUSIONS: ARGs in these remote and uncontaminated soils most likely represent functional efficient historical genes that have since been vertically inherited over generations. The historical ARGs in these pristine environments carry a strong phylogenetic signal and form a monophyletic group relative to ARGs from other similar environments.

The draft genome sequence of Hymenobacter sp. CRA2 isolated from Nama Karoo shrub land soils from South Africa.

Here we report the draft genome sequence of Hymenobacter sp. CRA2 isolated from the Nama Karoo shrub land soils of the Northern Cape, South Africa. This genome is approximately 5.88 Mb long and the assembly comprised 45 contigs. The draft genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession number NZ_MVBC00000000 and is available for download at: https://www.ncbi.nlm.nih.gov/nuccore/NZ_MVBC00000000.1.

Draft genome sequence of Massilia sp. KIM isolated from South African grassland biome soils.

Massilia sp. are aerobic, Gram-negative, rod-shaped bacteria that are found in air, water, and soils. Here we describe the draft genome sequence of Massilia sp. KIM, isolated from the South African grassland soils. The total length of the genome was estimated at 5.73 Mb, comprised of 17 contigs. The draft genome has been deposited in the DDBJ/EMBL/GenBank under the accession MVAD10000000 and is available for download at: https://www.ncbi.nlm.nih.gov/nuccore/MVAD00000000. Additionally, the raw short reads are available in the NCBI SRA database under the accession number: SRR5469241.

Draft genome sequence of Thermoactinomyces sp. strain AS95 isolated from a Sebkha in Thamelaht, Algeria.

The members of the genus Thermoactinomyces are known for their protein degradative capacities. Thermoactinomyces sp. strain AS95 is a Gram-positive filamentous bacterium, isolated from moderately saline water in the Thamelaht region of Algeria. This isolate is a thermophilic aerobic bacterium with the capacity to produce extracellular proteolytic enzymes. This strain exhibits up to 99 % similarity with members of the genus Thermoactinomyces, based on 16S rRNA gene sequence similarity. Here we report on the phenotypic features of Thermoactinomyces sp. strain AS95 together with the draft genome sequence and its annotation. The genome of this strain is 2,558,690 bp in length (one chromosome, but no plasmid) with an average G + C content of 47.95 %, and contains 2550 protein-coding and 60 RNA genes together with 64 ORFs annotated as proteases.

The Geobacillus Pan-Genome: Implications for the Evolution of the Genus.

The genus Geobacillus is comprised of a diverse group of spore-forming Gram-positive thermophilic bacterial species and is well known for both its ecological diversity and as a source of novel thermostable enzymes. Although the mechanisms underlying the thermophilicity of the organism and the thermostability of its macromolecules are reasonably well understood, relatively little is known of the evolutionary mechanisms, which underlie the structural and functional properties of members of this genus. In this study, we have compared 29 Geobacillus genomes, with a specific focus on the elements, which comprise the conserved core and flexible genomes. Based on comparisons of conserved core and flexible genomes, we present evidence of habitat delineation with specific Geobacillus genomes linked to specific niches. Our analysis revealed that Geobacillus and Anoxybacillus share a high proportion of genes. Moreover, the results strongly suggest that horizontal gene transfer is a major factor deriving the evolution of Geobacillus from Bacillus, with genetic contributions from other phylogenetically distant taxa.