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The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. We found that MazF-Da exhibited the highest activity at around 60°C. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. The recognition sequence of the N36A mutant differed from that of the wild type. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties.

Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus Thiothrix. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus Thiothrix with the proposed names Thiothrix subterranea sp. nov. Ku-5T, Thiothrix litoralis sp. nov. AST, and “Candidatus Thiothrix anitrata” sp. nov. A52. The complete genome sequences of Thiothrix fructosivorans QT and Thiothrix unzii A1T were determined. Complete genomes of seven Thiothrix isolates, as well as two MAGs, were used for pangenome analysis. The Thiothrix core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (SoxAXBYZ), direct (soeABC) and indirect (aprAB, sat) pathways of sulfite oxidation, sulfur oxidation complex Dsr (dsrABEFHCEMKLJONR), sulfide oxidation systems SQR (sqrA, sqrF), and FCSD (fccAB) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.

UCYN-A is one of the most widespread and important marine diazotrophs. Its unusual distribution in both cold/warm and coastal/oceanic waters challenges current understanding about what drives the biogeography of diazotrophs. This study assessed the community assembly processes of the nitrogen-fixing cyanobacterium UCYN-A, developing a framework of assembly processes underpinning the microbial biogeography and diversity. High-throughput sequencing and a qPCR approach targeting the nifH gene were used to investigate three tropical seas: the Bay of Bengal, the Western Pacific Ocean, and the South China Sea. Based on the neutral community model and two types of null models calculating the β-nearest taxon index and the normalized stochasticity ratio, we found that stochastic assembly processes could explain 66–92% of the community assembly; thus, they exert overwhelming influence on UCYN-A biogeography and diversity. Among the deterministic processes, temperature and coastal/oceanic position appeared to be the principal environmental factors driving UCYN-A diversity. In addition, a close linkage between assembly processes and UCYN-A abundance/diversity/drivers can provide clues for the unusual global distribution of UCYN-A.

During the 1960s, small quantities of radioactive materials were co-disposed with chemical waste at the Little Forest Legacy Site (LFLS, Sydney, Australia). The microbial function and population dynamics in a waste trench during a rainfall event have been previously investigated revealing a broad abundance of candidate and potentially undescribed taxa in this iron-rich, radionuclide-contaminated environment. Applying genome-based metagenomic methods, we recovered 37 refined archaeal MAGs, mainly from undescribed DPANN Archaea lineages without standing in nomenclature and ‘Candidatus Methanoperedenaceae’ (ANME-2D). Within the undescribed DPANN, the newly proposed orders ‘Ca. Gugararchaeales’, ‘Ca. Burarchaeales’ and ‘Ca. Anstonellales’, constitute distinct lineages with a more comprehensive central metabolism and anabolic capabilities within the ‘Ca. Micrarchaeota’ phylum compared to most other DPANN. The analysis of new and extant ‘Ca. Methanoperedens spp.’ MAGs suggests metal ions as the ancestral electron acceptors during the anaerobic oxidation of methane while the respiration of nitrate/nitrite via molybdopterin oxidoreductases would have been a secondary acquisition. The presence of genes for the biosynthesis of polyhydroxyalkanoates in most ‘Ca. Methanoperedens’ also appears to be a widespread characteristic of the genus for carbon accumulation. This work expands our knowledge about the roles of the Archaea at the LFLS, especially, DPANN Archaea and ‘Ca. Methanoperedens’, while exploring their diversity, uniqueness, potential role in elemental cycling, and evolutionary history.

“Candidatus Liberibacter asiaticus” (CLas) is an unculturable phloem-limited α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). HLB is currently threatening citrus production worldwide. Understanding the CLas biology is critical for HLB management. In this study, a novel single-stranded DNA (ssDNA) phage, CLasMV1, was identified in a CLas strain GDHZ11 from Guangdong Province of China through a metagenomic analysis. The CLasMV1 phage had a circular genome of 8,869 bp with eight open reading frames (ORFs). While six ORFs remain uncharacterized, ORF6 encoded a replication initiation protein (RIP), and ORF8 encoded a major capsid protein (MCP). Based on BLASTp search against GenBank database, amino acid sequences of both MCP and RIP shared similarities (coverage > 50% and identity > 25%) to those of phages in Microviridae, an ssDNA phage family. Phylogenetic analysis revealed that CLasMV1 MCP and RIP sequences were clustered with genes from CLas and “Ca. L. solanacearum” (CLso) genomes and formed a unique phylogenetic lineage, designated as a new subfamily Libervirinae, distinct to other members in Microviridae family. No complete integration form but partial sequence (∼1.9 kb) of CLasMV1 was found in the chromosome of strain GDHZ11. Read-mapping analyses on additional 15 HiSeq data sets of CLas strains showed that eight strains harbored complete CLasMV1 sequence with variations in single-nucleotide polymorphisms (SNPs) and small sequence insertions/deletions (In/Dels). PCR tests using CLasMV1-specific primer sets detected CLasMV1 in 577 out of 1,006 CLas strains (57%) from southern China. This is the first report of Microviridae phage associated with CLas, which expands our understanding of phage diversity in CLas and facilitates current research in HLB.

The potential metabolism and ecological roles of many microbial taxa remain unknown because insufficient genomic data are available to assess their functional potential. Two such microbial “dark matter” taxa are the Candidatus bacterial phyla Cloacimonadota and Omnitrophota, both of which have been identified in global anoxic environments, including (but not limited to) organic-carbon-rich lakes. Using 24 metagenome-assembled genomes (MAGs) obtained from an Antarctic lake (Ace Lake, Vestfold Hills), novel lineages and novel metabolic traits were identified for both phyla. The Cloacimonadota MAGs exhibited a capacity for carbon fixation using the reverse tricarboxylic acid cycle driven by oxidation of hydrogen and sulfur. Certain Cloacimonadota MAGs encoded proteins that possess dockerin and cohesin domains, which is consistent with the assembly of extracellular cellulosome-like structures that are used for degradation of polypeptides and polysaccharides. The Omnitrophota MAGs represented phylogenetically diverse taxa that were predicted to possess a strong biosynthetic capacity for amino acids, nucleosides, fatty acids, and essential cofactors. All of the Omnitrophota were inferred to be obligate fermentative heterotrophs that utilize a relatively narrow range of organic compounds, have an incomplete tricarboxylic acid cycle, and possess a single hydrogenase gene important for achieving redox balance in the cell. We reason that both Cloacimonadota and Omnitrophota form metabolic interactions with hydrogen-consuming partners (methanogens and Desulfobacterota, respectively) and, therefore, occupy specific niches in Ace Lake.

Sap-feeding insects in the order Hemiptera associate with obligate endosymbionts that are required for survival and facultative endosymbionts that can potentially modify resistance to stress, enemies, development, and reproduction. In the superfamily Psylloidea, the jumping plant lice (psyllids), less is known about the diversity and prevalence of their endosymbionts compared to other sap-feeding pests such as aphids (Aphididae). To address this knowledge gap, using 16S rRNA sequencing we identify symbionts across divergent psyllid host lineages from around the world. Taking advantage of a new comprehensive phylogenomic analyses of Psylloidea, we included psyllid samples from 44 species of 35 genera of five families, collected from 11 international locations for this study. Across psyllid lineages, a total of 91 OTUs were recovered, predominantly of the Enterobacteriaceae (68%). The diversity of endosymbionts harbored by each psyllid species was low with an average of approximately 3 OTUs. Two clades of endosymbionts (clade 1 and 2), belonging to Enterobacteriaceae, were identified that appear to be long term endosymbionts of the psyllid families Triozidae and Psyllidae, respectively. We also conducted high throughput metagenomic sequencing on three Ca. Liberibacter infected psyllid species (Russelliana capsici, Trichochermes walkeri, and Macrohomotoma gladiata), initially identified from 16S rRNA sequencing, to obtain more genomic information on these putative Liberibacter plant pathogens. The phylogenomic analyses from these data identified a new Ca. Liberibacter species, Candidatus Liberibacter capsica, that is a potential pathogen of solanaceous crops. This new species shares a distant ancestor with Ca. L. americanus, which occurs in the same range as R. capsici in South America. We also detected the first association between a psyllid specializing on woody hosts and the Liberibacter species Ca. L. psyllaurous, which is a globally distributed pathogen of herbaceous crop hosts in the Solanaceae. Finally, we detected a potential association between a psyllid pest of figs (M. gladiata) and a Ca. Liberibacter related to Ca. L. asiaticus, which causes severe disease in citrus. Our findings reveal a wider diversity of associations between facultative symbionts and psyllids than previously reported and suggest numerous avenues for future work to clarify novel associations of ecological, evolutionary, and pathogenic interest.

Nitrogen cycling microbes, including nitrite-oxidizing bacteria (NOB), perform critical ecosystem functions that help mitigate anthropogenic stresses and maintain ecosystem health. Activity of these beneficial nitrogen cycling microbes is dictated in part by the microorganisms’ response to physicochemical conditions, such as temperature, pH, and nutrient availability. NOB from the newly described Candidatus Nitrotoga genus have been detected in a wide range of habitats across the globe, yet only a few organisms within the genus have been physiologically characterized. For freshwater systems where NOB are critical for supporting aquatic life, Ca. Nitrotoga have been previously detected but little is known about the physiological potential of these organisms or their response to changing environmental conditions. Here, we determined functional response to environmental change for a representative freshwater species of Ca. Nitrotoga (Ca. Nitrotoga sp. CP45, enriched from a Colorado river). The physiological findings demonstrated that CP45 maintained nitrite oxidation at pH levels of 5–8, at temperatures from 4 to 28°C, and when incubated in the dark. Light exposure and elevated temperature (30°C) completely halted nitrite oxidation. Ca. Nitrotoga sp. CP45 maintained nitrite oxidation upon exposure to four different antibiotics, and potential rates of nitrite oxidation by river sediment communities were also resilient to antibiotic stress. We explored the Ca. Nitrotoga sp. CP45 genome to make predictions about adaptations to enable survival under specific conditions. Overall, these results contribute to our understanding of the versatility of a representative freshwater Ca. Nitrotoga sp. Identifying the specific environmental conditions that maximize NOB metabolic rates may ultimately direct future management decisions aimed at restoring impacted systems.

Boreal lakes and ponds produce two-thirds of the total natural methane emissions above the latitude of 50° North. These lake emissions are regulated by methanotrophs which can oxidize up to 99% of the methane produced in the sediments and the water column. Despite their importance, the diversity and distribution of the methanotrophs in lakes are still poorly understood. Here, we used shotgun metagenomic data to explore the diversity and distribution of methanotrophs in 40 oxygen-stratified water bodies in boreal and subarctic areas in Europe and North America. In our data, gammaproteobacterial methanotrophs (order Methylococcales) generally dominated the methanotrophic communities throughout the water columns. A recently discovered lineage of Methylococcales, Candidatus Methylumidiphilus, was present in all the studied water bodies and dominated the methanotrophic community in lakes with a high relative abundance of methanotrophs. Alphaproteobacterial methanotrophs were the second most abundant group of methanotrophs. In the top layer of the lakes, characterized by low CH4 concentration, their abundance could surpass that of the gammaproteobacterial methanotrophs. These results support the theory that the alphaproteobacterial methanotrophs have a high affinity for CH4 and can be considered stress-tolerant strategists. In contrast, the gammaproteobacterial methanotrophs are competitive strategists. In addition, relative abundances of anaerobic methanotrophs, Candidatus Methanoperedenaceae and Candidatus Methylomirabilis, were strongly correlated, suggesting possible co-metabolism. Our data also suggest that these anaerobic methanotrophs could be active even in the oxic layers. In non-metric multidimensional scaling, alpha- and gammaproteobacterial methanotrophs formed separate clusters based on their abundances in the samples, except for the gammaproteobacterial Candidatus Methylumidiphilus, which was separated from these two clusters. This may reflect similarities in the niche and environmental requirements of the different genera within alpha- and gammaproteobacterial methanotrophs. Our study confirms the importance of O2 and CH4 in shaping the methanotrophic communities and suggests that one variable cannot explain the diversity and distribution of the methanotrophs across lakes. Instead, we suggest that the diversity and distribution of freshwater methanotrophs are regulated by lake-specific factors.