Search

During evolution, chloroplasts, which originated by endosymbiosis of a prokaryotic ancestor of today’s cyanobacteria with a eukaryotic host cell, were established as the site for photosynthesis. Therefore, chloroplast organelles are loaded with transition metals including iron, copper, and manganese, which are essential for photosynthetic electron transport due to their redox capacity. Although transport, storage, and cofactor-assembly of metal ions in chloroplasts are tightly controlled and crucial throughout plant growth and development, knowledge on the molecular nature of chloroplast metal-transport proteins is still fragmentary. Here, we characterized the soluble, ATP-binding ABC-transporter subunits ABCI10 and ABCI11 in Arabidopsis thaliana, which show similarities to components of prokaryotic, multisubunit ABC transporters. Both ABCI10 and ABCI11 proteins appear to be strongly attached to chloroplast-intrinsic membranes, most likely inner envelopes for ABCI10 and possibly plastoglobuli for ABCI11. Loss of ABCI10 and ABCI11 gene products in Arabidopsis leads to extremely dwarfed, albino plants showing impaired chloroplast biogenesis and deregulated metal homeostasis. Further, we identified the membrane-intrinsic protein ABCI12 as potential interaction partner for ABCI10 in the inner envelope. Our results suggest that ABCI12 inserts into the chloroplast inner envelope membrane most likely with five predicted α-helical transmembrane domains and represents the membrane-intrinsic subunit of a prokaryotic-type, energy-coupling factor (ECF) ABC-transporter complex. In bacteria, these multisubunit ECF importers are widely distributed for the uptake of nickel and cobalt metal ions as well as for import of vitamins and several other metabolites. Therefore, we propose that ABCI10 (as the ATPase A-subunit) and ABCI12 (as the membrane-intrinsic, energy-coupling T-subunit) are part of a novel, chloroplast envelope-localized, AAT energy-coupling module of a prokaryotic-type ECF transporter, most likely involved in metal ion uptake.

Heterotrophic nanoflagellates (HNANs) play a pivotal role as consumers of picoplankton, remineralizers and carbon vectors, yet knowledge on how prey quantity and quality affect HNAN physiology remains limited. In a series of grazing experiments using an uncharacterized member of the HNAN assemblage, we found that growth (μ) and ingestion rate (IR) varied when offering heterotrophic bacteria (HB), Synechococcus spp. (Syn), Ostreococcus lucimarinus (Ost) or a combination of all 3 prey types. Highest average μ rates (1.8 d-1) were detected on HB at densities of ~106 cells ml-1 and maximum IR on Syn (485 pg C d-1) at ~106 cells ml-1. Independent of prey type, flagellate μ increased with IR up to ~50 pg C d-1. A relatively low P-content in Ost was linked to shifts in C:N:P ratios of the HNAN in the single-prey experiment and when Ost was offered as part of the mixed assemblage. Presented with a mixed diet, the highest contribution to daily C intake came from Ost with 50%, followed by HB with 46% and Syn with only 4%. C-based gross growth efficiencies (GGEs) were higher when feeding on HB and mixed prey, compared to both picophototrophs, while N- and P-based GGEs in mixed prey treatments markedly exceeded those when feeding on any single prey. The findings in this study corroborate the importance of investigating the biogeochemical role of HNANs in relation to prey availability and quality to refine estimates of energy transfer within the microbial loop.

Microbial communities in cave ecosystems have specific survival strategies, which is far from being well explicated. Here, we reported the genetic and functional diversity of bacteria and archaea in typical limestone (Kashmir Cave) and silicate-containing (Tiser Cave) caves. X-ray diffraction (XRD) and Fourier transform infrared spectroscopic (FTIR) analyses revealed the different geochemical and mineral compositions of the two caves. Amplicon barcode sequencing revealed the dominancy of Actinobacteria and Proteobacteria in Kashmir and Tiser Caves. Bacteroidetes and Firmicutes were the dominant phyla in Tiser Cave, and the abundance is relatively small in Kashmir Cave. Archaea was also abundant prokaryotes in Kashmir Cave, but it only accounted for 0.723% of the total prokaryote sequences in Tiser Cave. Functional analysis based on metagenomic sequencing data revealed that a large number of functional potential genes involved in nutrient metabolism and biosynthesis of bioactive compounds in Tiser and Kashmir Cave samples could significantly influence the biogeochemical cycle and secondary metabolite production in cave habitats. In addition, the two caves were also found to be rich in biosynthetic genes, encoding bioactive compounds, such as monobactam and prodigiosin, indicating that these caves could be potential habitats for the isolation of antibiotics. This study provides a comprehensive insight into the diversity of bacteria and archaea in cave ecosystems and helps to better understand the special survival strategies of microorganisms in cave ecosystems.Key points• Geochemically distinct caves possess unique microbial community structure.• Cavernicoles could be important candidates for antibiotic production.• Cavernicoles are important for biogeochemical cycling.

Background Characterizing phage–host interactions is critical to understanding the ecological role of both partners and effective isolation of phage therapeuticals. Unfortunately, experimental methods for studying these interactions are markedly slow, low-throughput, and unsuitable for phages or hosts difficult to maintain in laboratory conditions. Therefore, a number of in silico methods emerged to predict prokaryotic hosts based on viral sequences. One of the leading approaches is the application of the BLAST tool that searches for local similarities between viral and microbial genomes. However, this prediction method has three major limitations: (i) top-scoring sequences do not always point to the actual host; (ii) mosaic virus genomes may match to many, typically related, bacteria; and (iii) viral and host sequences may diverge beyond the point where their relationship can be detected by a BLAST alignment. Results We created an extension to BLAST, named Phirbo, that improves host prediction quality beyond what is obtainable from standard BLAST searches. The tool harnesses information concerning sequence similarity and bacteria relatedness to predict phage–host interactions. Phirbo was evaluated on three benchmark sets of known virus–host pairs, and it improved precision and recall by 11–40 percentage points over currently available, state-of-the-art, alignment-based, alignment-free, and machine-learning host prediction tools. Moreover, the discriminatory power of Phirbo for the recognition of virus–host relationships surpassed the results of other tools by at least 10 percentage points (area under the curve = 0.95), yielding a mean host prediction accuracy of 57% and 68% at the genus and family levels, respectively, and drops by 12 percentage points when using only a fraction of viral genome sequences (3 kb). Finally, we provide insights into a repertoire of protein and ncRNA genes that are shared between phages and hosts and may be prone to horizontal transfer during infection. Conclusions Our results suggest that Phirbo is a simple and effective tool for predicting phage–host relationships.

The emergence of multicellularity is a key event in the evolution of life and is an attractive challenge among researchers, including those investigating the artificial design of cellular behavior1. Multicellular organisms are widely distributed on Earth, and retracing the specific conditions conducive for the initial transition from unicellularity to multicellularity is difficult. However, by examining organisms that inhabit unique (e.g., isolated) environmental niches, we may be able to get a glimpse into primitive multicellularity in the context of a given environment. Here we report the discovery of a new bacterium that displayed multicellular-like characteristics and behavior. The bacterium, which was isolated adjacent to an underground stream in a limestone cave, is to be named Jeongeupia sacculi sp. nov. HS-3. On a solid surface, HS-3 self-organizes its filamentous cells to form an appearance similar to the nematic phase of a liquid crystal2. Mature colonies produce and accommodate clusters of coccobacillus progeny, and release them upon contact with water. HS-3 demonstrated novel, spatiotemporally regulated multicellularity that can resolve the so-called ‘competition-dispersal trade-off’ problem3. This study illustrates a hypothetical missing link on the emergence of multicellularity.

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multipleturnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been characterized – but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

AaRSs (aminoacyl-tRNA synthetases) group into two ten-member classes throughout evolution, with unique active site architectures defining each class. Most are monomers or homodimers but, for no apparent reason, many bacterial GlyRSs are heterotetramers consisting of two catalytic α-subunits and two tRNA-binding β-subunits. The heterotetrameric GlyRS from Escherichia coli (EcGlyRS) was historically tested whether its α- and β-polypeptides, which are encoded by a single mRNA with a gap of three in-frame codons, are replaceable by a single chain. Here, an unprecedented X-shaped structure of EcGlyRS shows wide separation of the abutting chain termini seen in the coding sequences, suggesting strong pressure to avoid a single polypeptide format. The structure of the five-domain β-subunit is unique across all aaRSs in current databases, and structural analyses suggest these domains play different functions on α-subunit binding, ATP coordination and tRNA recognition. Moreover, the X-shaped architecture of EcGlyRS largely fits with a model for how two classes of tRNA synthetases arose, according to whether enzymes from opposite classes can simultaneously co-dock onto separate faces of the same tRNA acceptor stem. While heterotetrameric GlyRS remains the last structurally uncharacterized member of aaRSs, our study contributes to a better understanding of this ancient and essential enzyme family.

In this study, fluorescence in situ hybridization (FISH) and PCR-amplified fragments of the 16SrDNA gene were used to determine prokaryotes diversity in Urmia Salt Lake. Prokaryote cell population in Urmia lake range from 3.1 ± 0.3 × 106, 2 ± 0.2 × 108, 4 ± 0.3 × 108, and 1.8 ± 0.2 × 108 cells ml−1 for water, soil, sediment, and salt samples by DAPI (4, 6-diamidino-2-phenylindole) direct count, respectively. The proportion of bacteria and archaea in the samples determinable by FISH ranged between 36.1 and 55% and 48.5 and 55.5%, respectively. According to the DGGE method, some bands were selected and separated from the gel, then amplified and sequenced. The results of sequences were related to two phyla Proteobacteria (16.6%) and Bacteroidetes (83.3%), which belonged to four genera Salinibacter, Mangroviflexus, Pseudomonas, and Cesiribacter, and the archaeal sequences were related to Euryarchaeota phyla and three genera Halonotius, Haloquadratum, and Halorubrum. According to our results, it seems that prokaryotic populations in this hypersaline environment are more diverse than expected, and bacteria are so abundant and diverse and form the metabolically active part of the microbial population inhabiting this extreme environment. Molecular dependent and independent approaches revealed a different aspect of this environment microbiota.

Metagenomic and traditional paleolimnological approaches are suitable to infer past biological and environmental changes, however, they are often applied independently, especially in tropical regions. We combined both approaches to investigate Holocene Prokaryote and Eukaryote diversity and microbial metabolic pathways in ancient Lake Chalco, Mexico. Here, we report on diversity among a large number of lineages (36,722 OTUs) and functional diversity (27,636,243 non-clustered predicted proteins, and 6,144 annotated protein-family genes). The most abundant domain is Bacteria (81%), followed by Archaea (15%) and Eukarya (3%). We also determined the diversity of protein families and their relationship to metabolic pathways. The early Holocene (> 11,000 cal years BP) lake was characterized by cool, freshwater conditions, which later became warmer and hyposaline (11,000–6,000 cal years BP). We found high abundances of cyanobacteria, and fungi groups associated with mature forests in these sediments. Bacteria and Archaea include mainly anaerobes and extremophiles that are involved in the sulfur, nitrogen, and carbon cycles. We found evidence for early human impacts, including landscape modifications and lake eutrophication, which began ~ 6,000 cal years BP. Subsaline, temperate conditions were inferred for the past 5,000 years. Finally, we found nitrogen-fixing bacteria and protein-family genes that are linked to contaminated environments, as well as several fungal pathogens of crops in near-surface sediments.

Prokaryote genomics started in earnest in 1995, with the complete sequences of two small bacterial genomes, those of Haemophilus influenzae and Mycoplasma genitalium. During the next quarter century, the prokaryote genome database has been growing exponentially, with no saturation in sight. For most of these 25 years, genome sequencing remained limited to cultivable microbes. Together with next-generation sequencing methods, advances in metagenomics and single-cell genomics have lifted this limitation, providing for an increasingly unbiased characterization of the global prokaryote diversity. Advances in computational genomics followed the progress of genome sequencing, even if occasionally lagging behind. Several major new branches of bacteria and archaea were discovered, including Asgard archaea, the apparent closest relatives of eukaryotes and expansive groups of bacteria and archaea with small genomes thought to be symbionts of other prokaryotes. Comparative analysis of numerous prokaryote genomes spanning a wide range of evolutionary distances changed the conceptual foundations of microbiology, supplanting the notion of species genomes with fixed gene sets with that of dynamic pangenomes and the notion of a single Tree of Life (ToL) with a statistical tree-like trend among individual gene trees. Strides were also made towards a theory and quantitative laws of prokaryote genome evolution.

Background Menaquinones are constituents of prokaryote cell membranes where they play important functions during electron transport. Menaquinone profiles are strongly recommended for species classification when proposing a new Actinomycetes taxon. Presently, the most widely used methods to determine menaquinones are based on freeze-dried cells. Taxonomic research in our lab has revealed that menaquinone concentrations are low for some species of the genus Microbacterium, leading to difficulties in identifying menaquinones. Results Menaquinones extracted using the novel lysozyme-chloroform-methanol (LCM) method were comparable in quality to those obtained using the Collins method, the most widely used method. All tested strains extracted via the LCM method showed higher concentrations of menaquinones than those extracted via the Collins method. For some Microbacterium strains, the LCM method exhibited higher sensitivity than the Collins method, and more trace menaquinones were detected with the LCM method than the Collins method. In addition, LCM method is faster than the Collins method because it uses wet cells. Conclusion The LCM method is a simple, rapid and efficient technique for the extraction and identification of menaquinones from Actinomycetes.

Universal primers for SSU rRNA genes allow profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show relative abundance for all rRNA genes, universal primers are rarely used, due to various concerns including amplicon length variation and its effect on bioinformatic pipelines. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this approach. Using these mocks, we show that universal primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed versus expected abundance correlations (slope = 0.88 vs. 0.67–0.79), and mock community members with single mismatches to the primer were strongly underestimated (threefold to eightfold). Using field samples, both primers yielded similar 18S beta-diversity patterns (Mantel test, p < 0.001) but differences in relative proportions of many rarer taxa. To test for length biases, we mixed mock communities (16S + 18S) before PCR and found a twofold underestimation of 18S sequences due to sequencing bias. Correcting for the twofold underestimation, we estimate that, in Southern California field samples (1.2–80 μm), there were averages of 35% 18S, 28% chloroplast 16S, and 37% prokaryote 16S rRNA genes. These data demonstrate the potential for universal primers to generate comprehensive microbiome profiles.

A pangenome is the complete set of genes (core and accessory) present in a phylogenetic clade. We hypothesize that a pangenome's accessory gene content is structured and maintained by selection. To test this hypothesis, we interrogated the genomes of 40 Pseudomonas species for statistically significant coincident (i.e. co-occurring/avoiding) gene patterns. We found that 86.7% of common accessory genes are involved in ≥1 coincident relationship. Further, genes that co-occur and/or avoid each other - but are not vertically inherited - are more likely to share functional categories, are more likely to be simultaneously transcribed, and are more likely to produce interacting proteins, than would be expected by chance. These results are not due to coincident genes being adjacent to one another on the chromosome. Together, these findings suggest that the accessory genome is structured into sets of genes that function together within a given strain. Given the similarity of the Pseudomonas pangenome with open pangenomes of other prokaryotic species, we speculate that these results are generalizable. Keyword: pangenome, microbial genomics, selection.

Deep-sea hypersaline anoxic basins are polyextreme environments in the ocean’s interior characterized by the high density of brines that prevents mixing with the overlaying seawater, generating sharp chemoclines and redoxclines up to tens of meters thick that host a high concentration of microbial communities. Yet, a fundamental understanding of how such pycnoclines shape microbial life and the associated biogeochemical processes at a fine scale, remains elusive. Here, we applied high-precision sampling of the brine–seawater transition interface in the Suakin Deep, located at 2770 m in the central Red Sea, to reveal previously undocumented fine-scale community structuring and succession of metabolic groups along a salinity gradient only 1 m thick. Metagenomic profiling at a 10-cm-scale resolution highlighted spatial organization of key metabolic pathways and corresponding microbial functional units, emphasizing the prominent role and significance of salinity and oxygen in shaping their ecology. Nitrogen cycling processes are especially affected by the redoxcline with ammonia oxidation processes being taxa and layers specific, highlighting also the presence of novel microorganisms, such as novel Thaumarchaeota and anammox, adapted to the changing conditions of the chemocline. The findings render the transition zone as a critical niche for nitrogen cycling, with complementary metabolic networks, in turn underscoring the biogeochemical complexity of deep-sea brines., This research was supported by King Abdullah University of Science and Technology through baseline funding and Red Sea Research Center competitive fund to DD. We thank the crew of R/V Thuwal and CMOR for their support in sampling the brines, especially Brian Hession and Thomas Hoover. We also thank personal of the KAUST Bioscience Core Lab for their assistance with sequencing.

In an alluvial aquifer in the River Fulda Valley (Germany) the influence of agricultural inputs on the subterranean physical, chemical and biological relationships was examined. A 40-year-old (1977–1981) comprehensive data set on the groundwater microbiome plus metazoa was now analysed for the first time in full (measurements for up to 4 years: hydrological, chemical, physical, prokaryote, and metazoa characteristics). Four hydrogeochemically different groundwater zones were identified across the floodplain. In addition, the prokaryote (Archaea and Bacteria) and metazoan communities differed among the four zones. The hydraulic exchange between the alluvial aquifer and the River Fulda influenced the sites closest to the river, leading to the highest prokaryote and metazoan biomasses at these locations. An organic carbon plume zone of anthropogenic origin exhibited high prokaryote abundances and production, which were higher than in the surrounding mixing zone. This mixing zone represented a transition area to the river-influenced sites as well as to the fourth zone, which was characterized by high nutrient levels from intense agriculture and which exhibited low prokaryote abundance and activity and intermediate metazoan abundance. Despite high prokaryote productivity, metazoa did not favor the organic carbon plume, due probably to low oxygen concentrations. At the sites, where metazoa occurred, their biomass corresponded mostly to about one hundredth of the prokaryote biomass. The main implication from this new analysis of an old data set is that even on a coarse taxonomical resolution, patterns emerge that show – in a geologically homogeneous area – an unprecedented complexity among different groundwater zones resulting from different external influences of natural as well as anthropogenic origin. Future studies need to ascertain an adequate temporal and spatial resolution.

Background The genomes of bacteria and archaea evolve by extensive loss and gain of genes which, for any group of related prokaryotic genomes, result in the formation of a pangenome with the universal, asymmetrical U-shaped distribution of gene commonality. However, the evolutionary factors that define the specific shape of this distribution are not thoroughly understood. Results We investigate the fit of simple models of genome evolution to the empirically observed gene commonality distributions and genome intersections for 33 groups of closely related bacterial genomes. A model with an infinite external gene pool available for gene acquisition and constant genome size (IGP-CGS model), and two gene turnover rates, one for slow- and the other one for fast-evolving genes, allows two approaches to estimate the parameters for gene content dynamics. One is by fitting the model prediction to the distribution of the number of genes shared by precisely k genomes (gene commonality distribution) and another by analyzing the distribution of the number of genes common for k genome sets (k-cores). Both approaches produce a comparable overall quality of fit, although the former significantly overestimates the number of the universally conserved genes, while the latter overestimates the number of singletons. We further explore the effect of dropping each of the assumptions of the IGP-CGS model on the fit to the gene commonality distributions and show that models with either a finite gene pool or unequal rates of gene loss and gain (greater gene loss rate) eliminate the overestimate of the number of singletons or the core genome size. Conclusions We examine the assumptions that are usually adopted for modeling the evolution of the U-shaped gene commonality distributions in prokaryote genomes, namely, those of infinitely many genes and constant genome size. The combined analysis of genome intersections and gene commonality suggests that at least one of these assumptions is invalid. The violation of both these assumptions reflects the limited ability of prokaryotes to gain new genes. This limitation seems to stem, at least partly, from the horizontal gene transfer barrier, i.e., the cost of accommodation of foreign genes by prokaryotes. Further development of models taking into account the complexity of microbial evolution is necessary for an improved understanding of the evolution of prokaryotes.

Kongsfjorden is a typical glacier fjord in the European Arctic. In order to know the distribution and diversity of planktonic prokaryotes in the fjord subjected to strong physical gradient related with local glacier dynamics, prokaryote communities along a transect extending from the outer to the inner fjord was investigated in the summer of 2015 using 454 pyrosequencing of archaeal and bacterial 16S rRNA genes. Higher diversity and species richness were detected in Bacteria than in Archaea in each sample. Archaeal 16S rRNA gene sequences were not detected in the outer area of the fjord. Nitrososphaeria within the phylum Crenarchaeota and Poseidoniia within the Thermoplasmatota were two dominant archaeal groups in the fjord. At the genus level, Nitrosopumilus, Nitrosopelagicus, MGIIa-L1, and MGIIb–O2 dominated seawater archaeal communities. Higher abundances of Poseidoniale-related archaea were observed in the deep water than in the surface water. The Proteobacteria, Bacteroidota, and Actinobacteriota dominated the planktonic bacterial communities, with Verrucomicrobiota (represented by Verrucomicrobiae) dominating the middle area of the fjord. At the class level, Bacteroidia, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria dominated the bacterial communities within Kongsfjorden. Sequences affiliated with genera Polaribacter and Yoonia were frequently detected in the fjord. Results suggest that planktonic archaeal and bacterial community compositions in Kongsfjorden can be driven by different major environmental factors.

Only a few studies have reported on viral dynamics in estuarine environments, and the factors regulating viral ecology are not well known. We herein aimed to address the factors controlling virus–host relationships in tropical estuaries. To accomplish this, we combine a historical data series (2011–2017), containing virus and prokaryote abundances, with environmental data from Guanabara Bay, Brazil. Water quality gradients and tides were found to regulate the structure of prokaryotic communities, and seasonal patterns added complexity to the dynamics of viral populations. Prediction models showed a balance between expected and recorded virus–host relationships. However, changes in virus-microbial ratio (VMR) were found in areas with microorganism increases and regions showing critical environmental changes. Our results demonstrated that VMR changes are multifactorial and cannot be associated with any specific factor. Neither Kill-the-Winner nor Piggyback-the-Winner models can predict VMR in a highly dynamic environment strongly influenced by eutrophication. In sum, it can be concluded that special care is necessary when analyzing models for predicting virus and host abundances, as ecological factors control host distribution and play a significant role in the virus–host relationship.

A halophilic archaeon named strain LR21T was isolated from a salt mine in Yunnan Province, PR China. Cells were spherical, Gram-stain-negative and motile. Strain LR21T grew at 20–50 °C (optimum, 42 °C), with 8–30 % (w/v) NaCl (optimum, 23 %) and at pH 5.5–9.5 (optimum, pH 7.5–8.5). Mg2+ was not required for growth. The major polar lipid profile comprised phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Strain LR21T had two dissimilar 16S rRNA genes (rrnA and rrnB) and they were closely related to Halomicroarcula limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T with sequence similarities of 95.3–99.0, 93.0–96.2 and 93.2–95.9 %, respectively, and much lower values to other members. The rpoB′ gene sequence similarities between strain LR21T and Hma. limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T were 95.2, 91.2 and 91.2 % respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain LR21T and Hma. limicola YGHS32T, were 89.0 and 90.1 %, respectively. DNA relatedness between strains LR21T and Hma. limicola YGHS32T determined by in silico DNA–DNA hybridization was 36.8 %. Values of ANI and AAI between strain LR21T and other members in the genus Halomicroarcula were far below 95 % and the DNA–DNA relatedness values between strain LR21T and its close relatives were much lower than 70 %, which is far below the boundary for delineation of a new species prokaryote. The DNA G+C content of strain LR21T was 62.0 mol% (genome). The results suggested that strain LR21T represents a novel species of the genus Halomicroarcula , for which the name Halomicroarcula amylolytica sp. nov. is proposed. The type strain is LR21T (=CGMCC 1.13611T=NBRC 113588T).

Prochlorococcus is the most abundant photosynthetic prokaryote on our planet. The extensive ecological literature on the Prochlorococcus collective (PC) is based on the assumption that it comprises one single genus comprising the species Prochlorococcus marinus, containing itself a collective of ecotypes. Ecologists adopt the distributed genome hypothesis of an open pan-genome to explain the observed genomic diversity and evolution patterns of the ecotypes within PC. Novel genomic data for the PC prompted us to revisit this group, applying the current methods used in genomic taxonomy. As a result, we were able to distinguish the five genera: Prochlorococcus, Eurycolium, Prolificoccus, Thaumococcus, and Riococcus. The novel genera have distinct genomic and ecological attributes.

We experimentally evaluated the temporal (interday and interseason) and spatial variability in microbial plankton responses to vitamin B12 and/or B1 supply (solely or in combination with inorganic nutrients) in coastal and oceanic waters of the northeast Atlantic Ocean. Phytoplankton and, to a lesser extent, prokaryotes were strongly limited by inorganic nutrients. Interday variability in microbial plankton responses to B vitamins was limited compared to interseason variability, suggesting that B-vitamin availability might be partially controlled by factors operating at seasonal scale. Chlorophyll a (Chl a) concentration and prokaryote biomass (PB) significantly increased after B-vitamin amendments in 13 % and 21 %, respectively, of the 216 cases (36 experiments × 6 treatments). Most of these positive responses were produced by treatments containing either B12 solely or B12 combined with B1 in oceanic waters, which was consistent with the significantly lower average vitamin-B12 ambient concentrations compared to that in the coastal station. Negative responses, implying a decrease in Chl a or PB, represented 21 % for phytoplankton and 26 % for prokaryotes. Growth stimulation by B1 addition was more frequent on prokaryotes than in phytoplankton, suggesting that B1 auxotrophy in the sampling area could be more widespread in prokaryotes than in phytoplankton. Negative responses to B vitamins were generalized in coastal surface waters in summer and were associated with a high contribution of Flavobacteriales to the prokaryote community. This observation suggests that the external supply of B12 and/or B1 may promote negative interactions between microbial components when B-vitamin auxotrophs are abundant. The microbial response patterns to B12 and/or B1 amendments were significantly correlated with changes in the prokaryotic community composition, highlighting the pivotal role of prokaryotes in B-vitamin cycling in marine ecosystems.

Here, we report on the biochemical characterization of a new glycosylated bacteriocin (glycocin), ASM1, produced by Lactobacillus plantarum A-1 and analysis of the A-1 bacteriocinogenic genes. ASM1 is 43 amino acids in length with Ser18-O- and Cys43-S-linked N-acetylglucosamine moieties that are essential for its inhibitory activity. Its only close homologue, glycocin F (GccF), has five amino acid substitutions all residing in the flexible C-terminal 'tail' and a lower IC50 (0.9 nm) compared to that of ASM1 (1.5 nm). Asm/gcc genes share the same organization (asmH← →asmABCDE→F), and the asm genes reside on an 11 905-bp plasmid dedicated to ASM1 production. The A-1 genome also harbors a gene encoding a 'rare' bactofencin-type bacteriocin. As more examples of prokaryote S-GlcNAcylation are discovered, the functions of this modification may be understood.

Viruses are some of the most abundant biological entities on Earth, and prokaryote virus are the dominant members of the viral community. Because of the diversity of prokaryote virus, functional annotation cannot be performed on a large number of genes from newly discovered prokaryote virus by searching the current database; therefore, the development of an alignment-free algorithm for functional annotation of prokaryote virus proteins is important to understand the viral community. The identification of prokaryote virus virion proteins (PVVPs) is a critical step for many viral analyses, such as species classification, phylogenetic analysis and the exploration of how prokaryote virus interact with their hosts. Although a series of PVVP prediction tools have been developed, the performance of these tools is still not satisfactory. Moreover, viral metagenomic data contains fragmented sequences, leading to the existence of some incomplete genes. Therefore, a tool that can identify partial PVVPs is also needed. In this work, we present a novel algorithm, called VirionFinder, to identify the complete and partial PVVPs from non-prokaryote virus virion proteins (non-PVVPs). VirionFinder uses the sequence and biochemical properties of 20 amino acids as the mathematical model to encode the protein sequences and uses a deep learning technique to identify whether a given protein is a PVVP. Compared with the state-of-the-art tools using artificial benchmark datasets, the results show that under the same specificity (Sp), the sensitivity (Sn) of VirionFinder is approximately 10–34% much higher than the Sn of these tools on both complete and partial proteins. When evaluating related tools using real virome data, the recognition rate of PVVP-like sequences of VirionFinder is also much higher than that of the other tools. We expect that VirionFinder will be a powerful tool for identifying novel virion proteins from both complete prokaryote virus genomes and viral metagenomic data. VirionFinder is freely available at https://github.com/zhenchengfang/VirionFinder.

Relationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.

Sirtuins are deacetylase enzymes widely distributed in all domains of life. Although for decades they have been related only to histones deacetylation in eukaryotic organisms, today they are considered global regulators in both prokaryotes and eukaryotes. Despite the important role of sirtuins in humans, the knowledge about bacterial sirtuins is still limited. Several proteomics studies have shown that bacterial sirtuins deacetylate a large number of lysines in vivo, although the effect that this deacetylation causes in most of them remains unknown. To date, only the regulation of a few bacterial sirtuin substrates has been characterized, being their metabolic roles widely distributed: carbon and nitrogen metabolism, DNA transcription, protein translation, or virulence. One of the most current topics on acetylation and deacetylation focuses on studying stoichiometry using quantitative LC-MS/MS. The results suggest that prokaryotic sirtuins deacetylate at low stoichiometry sites, although more studies are needed to know if it is a common characteristic of bacterial sirtuins and its biological significance. Unlike eukaryotic organisms, bacteria usually have one or few sirtuins, which have been reported to have closer phylogenetic similarity with the human Sirt5 than with any other human sirtuin. In this work, in addition to carrying out an in-depth review of the role of bacterial sirtuins in their physiology, a phylogenetic study has been performed that reveals the evolutionary differences between sirtuins of different bacterial species and even between homologous sirtuins.

Little is known regarding how community assembly and species association vary with habitat and depth. Here, we examined the assembly and association of protistan and bacterial communities across a coast-shelf-slope-basin gradient of the South China Sea using high-throughput sequencing of the V3 and V4 regions of the rRNA gene transcript. Our study revealed that homogenizing dispersal and drift exerted an influence on protistan communities comparable to that on bacterial communities. In contrast, selection and dispersal limitation exerted contrasting effects on the two microbial communities. Community assembly was governed to a greater degree by selection than by dispersal limitation in the bacterial community, and this was much lower in the protistan community. Moreover, this organismal assembly pattern was robust with habitat and depth. However, the relative importance of selection to dispersal limitation varied with habitat and depth in both communities, where horizontally it was higher offshore than nearshore and vertically it was lower in the bottom or deep chlorophyll maximum (DCM) than on the surface. The offshore possessed more microbial network complexity and more associations among microbial taxa than the nearshore, and vertically, the bottom possessed more complexity than the surface and the DCM. Moreover, temperature is strongly associated with the composition and co-occurrence of microbial communities, implying that temperature plays a dominant role in the selection of the protistan-bacterial microbiome across a coast-to-basin continuum. This study contributes to our understanding of the assembly mechanism and species association of protistan-bacterial microbiota across multiple habitats and depths. IMPORTANCE Microbial organisms play a crucial role in global nutrient cycling. Few studies have attempted to simultaneously investigate the community assembly of microeukaryotes and prokaryotes and their association patterns in oceanic waters. This is especially true regarding how they vary with habitats and depths despite the fact that they are essential for developing a more holistic understanding of marine ecosystems. This study revealed the differential actions of selection and dispersal limitation and species association across a coast-to-basin continuum on the marine protistan-bacterial microbiome. Moreover, temperature was identified as a crucial factor driving the structure and co-occurrence of protistan and bacterial communities. The results emphasize that the differences in community assembly and association patterns between nearshore and offshore of the main constituents of the ocean microbiota should be considered to understand their current and future configurations. This is especially crucial in the context of climate change, as the response of ocean microbiota to nearshore and offshore temperature changes remains unknown.

Prokaryotic whole cell biosensors are effective yet inexpensive, renewable and environmentally substitutes for many contemporary sensors and diagnostic devices. Unfortunately, many previously constructed prokaryotic biosensors are plagued by poor performances, which prohibit their use in real life applications. Biosafety concerns, promiscuous analyte detection, analyte insensitivity, low output dynamic range and high background expression are common issues. Engineering principles and strategies from the emerging field of synthetic biology offer unprecedented solutions. They accelerate new biosensor developments and improve biosensor behaviors. Addition of novel devices and modules from synthetic biology further augments functions beyond sensing and render them safer. Thanks to this, prokaryotic whole cell biosensors have enjoyed a renaissance in recent years, and they hold promises to address the increasing demand for marketable biosensors.

The origin of eukaryotes remains unclear1–4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as ‘Asgard’ archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon—‘Candidatus Prometheoarchaeum syntrophicum’ strain MK-D1—is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle–engulf–endogenize (also known as E3) model., Isolation and characterization of an archaeon that is most closely related to eukaryotes reveals insights into how eukaryotes may have evolved from prokaryotes.

It is believed that eukaryotes arise from prokaryotes, which means that organelles can form de novo in prokaryotes. Such events, however, had not been observed previously. Here, we report the biogenesis of organelles in the endosymbiotic cyanobacterium TDX16 (prokaryote) that was released from its senescent/necrotic host cell of green alga Haematococcus pluvialis (eukaryote). Microscopic observations showed that organelle biogenesis in TDX16 initiated with cytoplasm compartmentalization, followed by de-compartmentalization, DNA allocation, and re-compartmentalization, as such two composite organelles-the primitive chloroplast and primitive nucleus sequestering minor and major fractions of cellular DNA respectively were formed. Thereafter, the eukaryotic cytoplasmic matrix was built up from the matrix extruded from the primitive nucleus; mitochondria were assembled in and segregated from the primitive chloroplast, whereby the primitive nucleus and primitive chloroplast matured into the nucleus and chloroplast respectively. While mitochondria subsequently turned into double-membraned vacuoles after matrix degradation. Results of pigment analyses, 16S rRNA and genome sequencing revealed that TDX16 is a phycocyanin-containing cyanobacterium resembling Chroococcidiopsis thermalis, which had acquired 9,017,401 bp DNAs with 10,301 genes from its host. Accordingly, we conclude that organelle biogenesis in TDX16 is achieved by hybridizing the acquired eukaryotic DNAs with its own one and expressing the hybrid genome. The formation of organelles in cyanobacterium TDX16 is the first case of organelle biogenesis in prokaryotes observed so far, which sheds an unprecedented light on eukaryotes and their connections with prokaryotes, and thus has broad implications on biology.

The European flat oyster (Ostrea edulis L.) represents an economically important oyster production in Southern Italy, widespread in natural beds along the coast. The practice to be eaten raw is an everlasting concern for possible health risk with a need to stringently monitor the health of aquatic environment. A screening survey using histopathological examination was undertaken by harvesting O. edulis from different sites along the Apulian coast of Italy. Tissue samples of the digestive gland, kidney, gonad, and gill were provided for morphologic study in light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) analysis. The LM observations revealed spherical cytoplasmic inclusions as basophilic prokaryote colonies in 13/250 oysters. The TEM and SEM confirmed the presence of intracytoplasmic inclusions of Rickettsia-like organisms (RLOs), merely in the epithelial cells of the digestive gland tubule tissues in the 13 oysters. Within intracytoplasmic vacuoles, RLOs exhibited a prokaryotic characteristic ultrastructure with transverse binary fission, a DNA zone full of chromatin fibers and a granular periplasmic ribosome zone. O. edulis were found positive for RLOs in wild oysters from Manfredonia, while the other sites were found free of pathological inclusions. Thus, we present the first report of a Rickettsia-like infection in the Apulian wild oyster (O. edulis) from Italy, including an ultrastructural description and pathological characterization.

In Archaea and Bacteria, the arrays called CRISPRs for ‘clustered regularly interspaced short palindromic repeats’ and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR–Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR–Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.

Southern rice black-streaked dwarf virus (SRBSDV) causes disease in crops, which reduces the quality and yield. Several commercial antiviral agents are available to control the SRBSDV induced disease. However, the mechanism of antiviral agents controlling SRBSDV is largely unknown. Identifying targets in SRBSDV is a key step of antiviral agent discovery. Here, we investigated the potential protein target of the antiviral agent dufulin. We cloned and expressed a soluble viroplasmic P6 protein in the prokaryote Escherichia coli and the eukaryote Spodoptera frugiperda 9. The dissociation constants of dufulin with the purified P6 protein from E. coli and S. frugiperda 9 expression systems were 4.49 and 4.95 μM, respectively, indicating a strong binding affinity between dufulin and P6 protein. In vivo, dufulin significantly inhibited the expression of both P6 protein and P6 gene in the SRBSDV-infected rice leaves. This inhibition on P6 protein expression was also observed in transformed Nicotiana benthamiana where the P6 was overexpressed. Our data also showed that dufulin inhibited the duplication of SRBSDV in a dose-dependent manner in infected rice leaves with a half maximum effective concentration of 3.32 mM. It is therefore concluded that dufulin targets the viroplasmic protein P6 to inhibit the virulence of SRBSDV.

Microbial communities are important regulators of many processes in all ecosystems. Understanding of ecosystem processes requires at least an overview of the involved microorganisms. While in-depth identification of microbial species in environmental samples can be achieved by next generation sequencing, profiling of whole microbial communities can be accomplished via less labour-intensive approaches. Especially automated ribosomal intergenic spacer analysis (ARISA) are of interest as they are highly specific even at fine scales and widely applicable for environmental samples. Yet, established protocols lack the possibility to compare prokaryotic and eukaryotic communities as different primer sets are necessary. However, shifts in the eukaryote to prokaryote ratio can be a useful indicator for ecosystem processes like decomposition or nutrient cycling. We propose a protocol to analyse prokaryotic and eukaryotic communities using a single primer pair based reaction based on a region with variable length (V4, which is about 180 bp shorter in prokaryotes compared to eukaryotes) in the small ribosomal subunit flanked by two highly conservative regions. Shifts in the prokaryotic and eukaryotic ratio between samples can be reliably detected by fragment length polymorphism analysis as well as sequencing of this region. Together with established approaches such as ARISA or 16S and ITS rDNA sequencing, this can provide a more complex insight into microbial community shifts and ecosystem processes.

High-throughput screening identified a new class of antibacterial compounds based on N-pyridyl-substituted p- and m-carboxypiperidine amides. Constructs with the two reporter genes RFP and Katushka2S allowed the detection of compounds that inhibit protein translation against DNA biosynthesis. Some agents of this class demonstrated antibacterial activity by inhibiting translation. The most promising compound had an MIC of 12 μg/mL. Thus, this class could become a valuable platform for developing new compounds with higher antibacterial activity and selectivity.

The Paracoccus sp. is one of the best-characterized prokaryote that has served as a model organism to study the dentrification and sulphur oxidation processes. However, its ability of dentrification and sulphur oxidation in coastal shrimp ponds is not reported much. In the present work, the Gram-negative, neutrophilic, facultatively lithoautotrophic bacterium Paracoccus saliphilus strain SPUM isolated from coastal shrimp ponds of Urran, Maharashtra, was studied for its efficiency of simultaneous heterotrophic nitrification–aerobic denitrification and sulphur oxidation processes. The maximum removal rate of nitrite and nitrate was 11.22 ± 0.31 and 14.17 ± 0.31 mg of NO3-N/l respectively after 24 h of incubation, while the sulphate-sulphur production observed was 190 ± 4.3 mg l−1 with a change in pH from 8.0 to 7.4 ± 0.08 after 12 days of incubation. The strain was characterized using universal 16S rRNA gene primers revealing high similarity (> 99%) with Paracoccus saliphilus belonging to α-proteobacteria. The isolate could express sulphate thiolesterase/sulphate thiohydrolase, soxB gene which is essential for sulphur oxidation. From all the results, it has been found that the strain SPUM could play a major role in simultaneous aerobic nitrification/denitrification and sulphur oxidation processes to overcome the toxicity of nitrogenous and sulphur-reducing compounds respectively in coastal aquaculture and wastewater systems.

Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthe...

Anthropogenic activities, such as mining and agriculture, have resulted in many freshwater systems having elevated concentrations of copper. Despite the prevalence of this contamination, and the vital ecological function of prokaryotes, just three studies have investigated prokaryote community responses to copper concentration in freshwater sediments. To address this, the current study investigated these communities in outdoor mesocosms spiked with varying copper concentrations. We profiled the prokaryotic communities at the taxonomic level, using next-generation high-throughput sequencing techniques, as well as their function, using baiting with leaf analogues, and Biolog Ecoplates for community-level physiological profiling. Sediments containing just 46 mg kg−1 of copper, had distinctly different microbial communities compared with controls, as determined by both DNA and RNA 16S ribosomal RNA gene (rRNA) profiling. In addition to this, sediment communities displayed a greatly reduced utilisation of carbon substrates under elevated copper, while the communities recruited onto leaf analogues were also disparate from those of control ponds. Given the vital role of prokaryotes in ecosystem processes, including carbon cycling, these changes are potentially of great ecological relevance, and are seen to occur well below the ‘low risk’ sediment quality guideline values (SQGV) used by regulatory bodies internationally.

Archaea and Bacteria have evolved different defence strategies that target virtually all steps of the viral life cycle. The diversified virion morphotypes and genome contents of archaeal viruses result in a highly complex array of archaea-virus interactions. However, our understanding of archaeal antiviral activities lags far behind our knowledges of those in bacteria. Here we report a new archaeal defence system that involves DndCDEA-specific DNA phosphorothioate (PT) modification and the PbeABCD-mediated halt of virus propagation via inhibition of DNA replication. In contrast to the breakage of invasive DNA by DndFGH in bacteria, DndCDEA-PbeABCD does not degrade or cleave viral DNA. The PbeABCD-mediated PT defence system is widespread and exhibits extensive interdomain and intradomain gene transfer events. Our results suggest that DndCDEA-PbeABCD is a new type of PT-based virus resistance system, expanding the known arsenal of defence systems as well as our understanding of host-virus interactions., The prokaryote defence system Dnd relies on phosphorothioation (PT) of DNA backbone to distinguish between self and non-self DNA. Here, Xiong et al. describe a previously uncharacterized PT-based antiviral system that is independent of the canonical Dnd and is widespread in Archaea and Bacteria.

The pangenome contains all genes encoded by a species, with the core genome present in all strains and the accessory genome in only a subset. Coincident gene relationships are expected within the accessory genome, where the presence or absence of one gene is influenced by the presence or absence of another. Here, we analysed the accessory genome of an Escherichia coli pangenome consisting of 400 genomes from 20 sequence types to identify genes that display significant co-occurrence or avoidance patterns with one another. We present a complex network of genes that are either found together or that avoid one another more often than would be expected by chance, and show that these relationships vary by lineage. We demonstrate that genes co-occur by function, and that several highly connected gene relationships are linked to mobile genetic elements. We find that genes are more likely to co-occur with, rather than avoid, another gene, suggesting that cooperation is more common than conflict in the accessory genome. This work furthers our understanding of the dynamic nature of prokaryote pangenomes and implicates both function and mobility as drivers of gene relationships.Data summaryAll Supplementary Data files and the Python scripts used in the analyses are available at doi.org/10.17639/nott.7103.Impact statementThe pangenome of a species encompasses the core genes encoded by all genomes, as well as the accessory genes found in only a subset. Much remains to be understood about the relationships and interactions between accessory genes; in particular, what drives pairs of genes to appear together in the same genome, or what prevents them from being in the same genome together, more often than expected by chance. How these co-occurrence and avoidance relationships develop, and what effect they have on the dynamics and evolution of the pangenome as a whole, is largely unknown. Here, we present a springboard for understanding prokaryote pangenome evolution by uncovering significant gene relationships in a model Escherichia coli pangenome. We identify mobile genetic elements and the sharing of common function as possible driving forces behind the co-occurrence of accessory genes. Furthermore, this work offers an extensive dataset from which gene relationships could be identified for any gene of interest in this E. coli accessory genome, providing a rich resource for the community.

Aquaglyceroporins (AQPs) are membrane proteins that function in osmoregulation and the uptake of low molecular weight solutes, in particular glycerol and urea. The AQP family is highly conserved, with two major subfamilies having arisen very early in prokaryote evolution and retained by eukaryotes. A complex evolutionary history indicates multiple lineage-specific expansions, losses and not uncommonly a complete loss. Consequently, the AQP family is highly evolvable and has been associated with significant events in life on Earth. In the African trypanosomes, a role for the AQP2 paralogue, in sensitivity to two chemotherapeutic agents, pentamidine and melarsoprol, is well established, albeit with the mechanisms for cell entry and resistance unclear until very recently. Here, we discuss AQP evolution, structure and mechanisms by which AQPs impact drug sensitivity, suggesting that AQP2 stability is highly sensitive to mutation while serving as the major uptake pathway for pentamidine.

Population-level comparisons of prokaryotic genomes must take into account the substantial differences in gene content, resulting from frequent horizontal gene transfer, gene duplication and gene loss. However, the automated annotation of prokaryotic genomes is imperfect, and errors due to fragmented assemblies, contamination, diverse gene families and mis-assemblies accumulate over the population, leading to profound consequences when analysing the set of all genes found in a species. Here we introduce Panaroo, a graph based pangenome clustering tool that is able to account for many of the sources of error introduced during the annotation of prokaryotic genome assemblies. We verified our approach through extensive simulations of de novo assemblies using the infinitely many genes model and by analysing a number of publicly available large bacterial genome datasets. Using a highly clonal Mycobacterium tuberculosis dataset as a negative control case, we show that failing to account for annotation errors can lead to pangenome estimates that are dominated by error. We additionally demonstrate the utility of the improved graphical output provided by Panaroo by performing a pan-genome wide association study in Neisseria gonorrhoeae and by analysing gene gain and loss rates across 51 of the major global pneumococcal sequence clusters. Panaroo is freely available under an open source MIT licence at https://github.com/gtonkinhill/panaroo.

The increase in global temperatures predicted by climate change models presents a serious problem for agriculture because high temperatures reduce crop yields. Protein biochemistry is at the core of plant heat stress response, and understanding the interactions between protein biochemistry and temperature will be key to developing heat-tolerant crop varieties. Current experimental studies of proteome-wide plant thermostability are limited by the complexity of plant proteomes: evaluating function for thousands of proteins across a variety of temperatures is simply not feasible with existing technologies. In this paper, we use homologous prokaryote sequences to predict plant Pfam temperature adaptation and gain insights into how thermostability varies across the proteome for three species: maize, Arabidopsis, and poplar. We find that patterns of Pfam domain adaptation across organelles are consistent and highly significant between species, with cytosolic proteins having the largest range of predicted Pfam stabilities and a long tail of highly-stable ribosomal proteins. Pfam adaptation in leaf and root organs varies between species, and maize root proteins have more low-temperature Pfam domains than do Arabidopsis or poplar root proteins. Both poplar and maize populations have an excess of low-temperature mutations in Pfam domains, but only the mutations identified in poplar accessions have a negative effect on Pfam temperature adaptation overall. These Pfam domain adaptation profiles provide insight into how different plant structures adapt to their surrounding environment and can help inform breeding or protein editing strategies to produce heat-tolerant crops.

Protein thermostability is important for fitness but difficult to measure across the proteome. Fortunately, protein thermostability is correlated with prokaryote optimal growth temperatures (OGTs), which can be predicted from genome features. Models that can predict temperature sensitivity across the prokaryote-eukaryote divide would help inform how eukaryotes adapt to elevated temperatures, such as those predicted by climate change models. In this study we test whether prediction models can cross the prokaryote-eukaryote divide to predict protein stability in both prokaryotes and eukaryotes. We compare models built using a) the whole proteome, b) Pfam domains, and c) individual amino acid residues. Proteome-wide models accurately predict prokaryote optimal growth temperatures (r2 up to 0.93), while site-specific models demonstrate that nearly half of the proteome is associated with optimal growth temperature in both Archaea and Bacteria. Comparisons with the small number of eukaryotes with temperature sensitivity data suggest that site-specific models are the most transferable across the prokaryote-eukaryote divide. Using the site-specific models, we evaluated temperature sensitivity for 323,850 amino acid residues in 2,088 Pfam domain clusters in Archaea and Bacteria species separately. 59.0% of tested residues are significantly associated with OGT in Archaea and 75.2% of tested residues are significantly associated with OGT in Bacteria species at a 5% false discovery rate. These models make it possible to identify which Pfam domains and amino acid residues are involved in temperature adaptation and facilitate future research questions about how species will fare in the face of increasing environmental temperatures.

Early colonization and succession of soil microbial communities are essential for soil development and nutrient accumulation. Herein we focused on the changes in pioneer prokaryotic communities in rhizosphere and bulk soils along the high-elevation glacier retreat chronosequence, the northern Himalayas, Tibetan Plateau. Rhizosphere soils showed substantially higher levels of total organic carbon, total nitrogen, ammonium, and nitrate than bulk soils. The dominant prokaryotes were Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Crenarchaeota, Bacteroidetes, and Planctomycetes, which totally accounted for more than 75% in relative abundance. The dominant genus Candidatus Nitrososphaera occurred at each stage of the microbial succession. The richness and evenness of soil prokaryotes displayed mild succession along chronosequene. Linear discriminant analysis effect size (LEfSe) analysis demonstrated that Proteobacteria (especially Alphaproteobacteria) and Actinobacteria were significantly enriched in rhizosphere soils compared with bulk soils. Actinobacteria, SHA_109, and Thermoleophilia; Betaproteobacteria and OP1.MSBL6; and Planctomycetia and Verrucomicrobia were separately enriched at each of the three sample sites. The compositions of prokaryotic communities were substantially changed with bulk and rhizosphere soils and sampling sites, indicating that the communities were dominantly driven by plants and habitat-specific effects in the deglaciated soils. Additionally, the distance to the glacier terminus also played a significant role in driving the change of prokaryotic communities in both bulk and rhizosphere soils. Soil C/N ratio exhibited a greater effect on prokaryotic communities in bulk soils than rhizosphere soils. These results indicate that plants, habitat, and glacier retreat chronosequence collectively control prokaryotic community composition and succession.

The ultimate role of prokaryote (bacteria and archaea) as the decomposer of the ecosystem, easily interrelates its community structure with the surrounding environment. In the present study, we compared prokaryote community structure and identified their abode preference as an indicator of wetland ecosystem in three distict wetland ecosystem using three universal prokaryote primers for the firdt time. Our results showed that Alpha diversity indices and phylogenetic differential abundance pattern did not differ significantly with primers, however those were significantly different across wetlands. Microbial community composition revealed distinct pattern in each wetland with each primer. Overall comparison of bacterial communities in three different wetland sediment revealed highest bacterial richness and diversity in Bhomra (freshwater wetland) followed by Malencho (brackish water bheri) and EKW (sewage-fed wetland). Indicator genus analysis identified 21, 4, and 29 unique genera as the diagnostic genus having abode preference for Bhomra, EKW and Malencho, respectively. Prediction of potential roles of these microbes based on close OTU similarity revealed more abode preference of sulphate reducing microbes in Malencho and methanogens in Bhomra. Distinct phylogenetic differential abundance pattern alongwith microbial abode preference and their potential functional role predicts ecosystem variables shapes microbial diversity. The variation in community composition of prokaryotes in response to ecosystem variables, can serve as the most sensitive bioindicator of ecosystem assessment and management.

Special issue Polar Microbes.-- 22 pages, 4 figures, 5 tables, supplementary material https://doi.org/10.3390/microorganisms9020317.-- Data Availability Statement: All data is reported in the present article.-- This research is part of POLARCSIC activities, The ocean surface microlayer (SML), with physicochemical characteristics different from those of subsurface waters (SSW), results in dense and active viral and microbial communities that may favor virus–host interactions. Conversely, wind speed and/or UV radiation could adversely affect virus infection. Furthermore, in polar regions, organic and inorganic nutrient inputs from melting ice may increase microbial activity in the SML. Since the role of viruses in the microbial food web of the SML is poorly understood in polar oceans, we aimed to study the impact of viruses on prokaryotic communities in the SML and in the SSW in Arctic and Antarctic waters. We hypothesized that a higher viral activity in the SML than in the SSW in both polar systems would be observed. We measured viral and prokaryote abundances, virus-mediated mortality on prokaryotes, heterotrophic and phototrophic nanoflagellate abundance, and environmental factors. In both polar zones, we found small differences in environmental factors between the SML and the SSW. In contrast, despite the adverse effect of wind, viral and prokaryote abundances and virus-mediated mortality on prokaryotes were higher in the SML than in the SSW. As a consequence, the higher carbon flux released by lysed cells in the SML than in the SSW would increase the pool of dissolved organic carbon (DOC) and be rapidly used by other prokaryotes to grow (the viral shunt). Thus, our results suggest that viral activity greatly contributes to the functioning of the microbial food web in the SML, which could influence the biogeochemical cycles of the water column, This research was part of the ATOS project, funded as part of the Spanish contribution to the International Polar Year (IPY) by the Spanish Ministry of Science and Innovation (POL200600550/CTM). J.A.B.’s work was supported by a PhD fellowship from the Spanish Ministerio de Ciencia e Innovación (FPU grant), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote Escherichia coli K-12, but the identification of whole sets of their direct targets is impossible with use of in vivo approaches. For this end, the most direct and quick approach is to identify the TF-binding sites in vitro on the genome. We then developed and utilized the gSELEX screening system in vitro for identification of more than 150 E. coli TF-binding sites along the E. coli genome. Based on the number of predicted regulatory targets, we classified E. coli K-12 TFs into four groups, altogether forming a hierarchy ranging from a single-target TF (ST-TF) to local TFs, global TFs, and nucleoid-associated TFs controlling as many as 1,000 targets. Using the collection of purified TFs and a library of genome DNA segments from a single and the same E. coli K-12, we identified here a total of 11 novel ST-TFs, CsqR, CusR, HprR, NorR, PepA, PutA, QseA, RspR, UvrY, ZraR, and YqhC. The regulation of single-target promoters was analyzed in details for the hitherto uncharacterized QseA and RspR. In most cases, the ST-TF gene and its regulatory target genes are adjacently located on the E. coli K-12 genome, implying their simultaneous transfer in the course of genome evolution. The newly identified 11 ST-TFs and the total of 13 hitherto identified altogether constitute the minority group of TFs in E. coli K-12.

Streptomyces is a model filamentous prokaryote to study multicellular differentiation and a rich reservoir for antibiotics discovery. In their natural conditions, Streptomyces grows at the interface of porous soil, air, and water. The morphological development of Streptomyces is traditionally performed on agar plates and mostly studied at the population levels. However, the detailed lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools which can mimic their natural conditions in the soil. Here, we developed a simple assembled microfluidic device for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level. The microfluidic device composed of a microchannel for loading samples and supplying nutrients, microwell arrays for seeding and growth of single spores, and air-filled chambers aside of the microwells that facilitate growth of aerial hyphae and spores. A unique feature of this device is that each microwell is surrounded by a 1.5 µm gap connected to an air-filled chamber which provide stabilized water-air interface. We used this device to observe the development of single Streptomyces spores and found that unlike those in bulk liquid culture, Streptomyces can differentiate at water-air interfaces in microscale liquid culture. Finally, we demonstrated that phenotypic A-Factor assay can be performed at defined time point of its lifecycle. This microfluidic device could become a robust tool for studying Streptomyces multi-cellular differentiation and interaction at single cell level.IMPORTANCEWe describe a microfluidic device that mimics the natural porous environment for the growth and development of Streptomyces, the model system for bacterial multicellularity. The microfluidic device is used for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level, including growth of aerial filaments. The aerial hyphae development of Streptomyces at the water-air interface was observed at real time in the microfluidic device. The early growth, opportunistic transformation (in the gap), and merging of aerial hyphae of Streptomyces in the microfluidic device were observed for the first time. It will play an important role in finding single-cell heterogeneity to study secondary metabolites related to the complex lifecycle of Streptomyces.