Four additional natural 7-deazaguanine derivatives in phages and how to make them.

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

Nutrient driven transcriptional changes during phage infection in an aquatic Gammaproteobacterium.

Phages modulate bacterial metabolism during infection by regulating gene expression, which influences aquatic nutrient cycling. However, the effects of shifting nutrient regimes are less understood. Here, we analyzed transcriptomes of an ecologically relevant Gammaproteobacterium and its lytic phage in high (HNM) and low (LNM) nutrient medium. Despite different infection characteristics, including reduced burst size and longer latent period in LNM, the phage had a fixed expression profile. Bacterial transcription was instead different depending on nutrient regime, with HNM bacteria focusing on growth while LNM bacteria focused on motility and membrane transport. Additionally, phage infection had a larger effect on bacterial gene expression in LNM compared to HNM, e.g. suppressing increased iron uptake and altering expression of phosphorus uptake genes. Overall, phage infection influenced host metabolism more in LNM, which was more similar to natural conditions, emphasizing the importance of considering natural conditions to understand phage and host ecology.

Dynamics of Baltic Sea phages driven by environmental changes.

Phage predation constitutes a major mortality factor for bacteria in aquatic ecosystems, and thus, directly impacts nutrient cycling and microbial community dynamics. Yet, the population dynamics of specific phages across time scales from days to months remain largely unexplored, which limits our understanding of their influence on microbial succession. To investigate temporal changes in diversity and abundance of phages infecting particular host strains, we isolated 121 phage strains that infected three bacterial hosts during a Baltic Sea mesocosm experiment. Genome analysis revealed a novel Flavobacterium phage genus harboring gene sets putatively coding for synthesis of modified nucleotides and glycosylation of bacterial cell surface components. Another novel phage genus revealed a microdiversity of phage species that was largely maintained during the experiment and across mesocosms amended with different nutrients. In contrast to the newly described Flavobacterium phages, phages isolated from a Rheinheimera strain were highly similar to previously isolated genotypes, pointing to genomic consistency in this population. In the mesocosm experiment, the investigated phages were mainly detected after a phytoplankton bloom peak. This concurred with recurrent detection of the phages in the Baltic Proper during summer months, suggesting an influence on the succession of heterotrophic bacteria associated with phytoplankton blooms.

Polynucleobacter paneuropaeus sp. nov., characterized by six strains isolated from freshwater lakes located along a 3000 km north-south cross-section across Europe.

Six Polynucleobacter (Burkholderiaceae, Betaproteobacteria) strains isolated from different freshwater lakes located across Europe were taxonomically investigated. Phylogenetic analyses based on 16S rRNA gene sequences assigns all six strains to the cryptic species complex PnecC within the genus Polynucleobacter. Analyses of partial glutamine synthetase (glnA) genes suggests that all six strains belong to the species-like taxon designated F15 in previous papers. Comparative genome analyses reveal that the six strains form a genomically coherent group characterized by whole-genome average nucleotide identity (gANI) values of >98 % but separated by gANI values of <88 % from the type strains and representatives of the 16 previously described Polynucleobacter species. In phylogenetic analyses based on nucleotide sequences of 319 orthologous genes, the six strains represent a monophyletic cluster that is clearly separated from all other described species. Genome sizes of the six strains range from 1.61 to 1.83 Mbp, which is smaller than genome sizes of the majority of type strains representing previously described Polynucleobacter species. By contrast, the G+C content of the DNA of the strains is well in the range of 44.8-46.6 mol% previously found for other type strains of species affiliated with the subgroup PnecC. Variation among the six strains representing the new species is evident in a number of traits. These include gene content differences, for instance regarding a gene cluster encoding anoxygenic photosynthesis, as well as phenotypic traits. We propose to name the new species represented by the six strains Polynucleobacter paneuropaeus sp. nov. and designate strain MG-25-Pas1-D2T (=DSM 103454T =CIP 111323T) as the type strain.

Genomic divergence and cohesion in a species of pelagic freshwater bacteria.

BACKGROUND: In many prokaryotic genera a clustered phylogeny is observed, akin to the occurrence of species in sexually reproducing organisms. For some taxa, homologous recombination has been invoked as the underlying mechanism providing genomic cohesion among conspecific individuals. Whether this mechanism is applicable to prokaryotes in freshwaters with low habitat connectivity - i.e. elevated geographic barriers to gene flow - is unclear. To investigate further we studied genomic trends within the globally abundant PnecC cluster (genus Polynucleobacter, Betaproteobacteria) and analyzed homologous recombination within the affiliated species P. asymbioticus. RESULTS: Comparisons among 20 PnecC genomes revealed a clearly discontinuous distribution of nucleotide sequence similarities. Among the nine conspecific individuals (P. asymbioticus) all average nucleotide identity (ANI) values were greater than 97%, whereas all other comparisons exhibited ANI values lower than 85%. The reconstruction of recombination and mutation events for the P. asymbioticus core genomes yielded an r/m ratio of 7.4, which is clearly above estimated thresholds for recombination to act as a cohesive force. Hotspots of recombination were found to be located in the flanking regions of genomic islands. Even between geographically separated habitats a high flux of recombination was evident. While a biogeographic population structure was suggested from MLST data targeting rather conserved loci, such a structure was barely visible when whole genome data was considered. However, both MLST and whole genome data showed evidence of differentiation between two lineages of P. asymbioticus. The ratios of non-synonymous to synonymous substitution rates as well as growth rates in transplantation experiments suggested that this divergence was not selectively neutral. CONCLUSIONS: The high extent of homologous recombination among P. asymbioticus bacteria can act as a cohesive force that effectively counteracts genetic divergence. At least on a regional scale, homologous recombination can act across geographically separated ecosystems and therefore plays an important role in the evolution and consistency of bacterial freshwater species. A species model akin to the biological species concept may be applicable for P. asymbioticus. Nonetheless, two genetically distinct lineages have emerged and further research may clarify if their divergence has been initiated by reinforced geographical barriers or has been evolving in sympatry.

Microdiversification of a Pelagic Polynucleobacter Species Is Mainly Driven by Acquisition of Genomic Islands from a Partially Interspecific Gene Pool.

Microdiversification of a planktonic freshwater bacterium was studied by comparing 37 Polynucleobacter asymbioticus strains obtained from three geographically separated sites in the Austrian Alps. Genome comparison of nine strains revealed a core genome of 1.8 Mb, representing 81% of the average genome size. Seventy-five percent of the remaining flexible genome is clustered in genomic islands (GIs). Twenty-four genomic positions could be identified where GIs are potentially located. These positions are occupied strain specifically from a set of 28 GI variants, classified according to similarities in their gene content. One variant, present in 62% of the isolates, encodes a pathway for the degradation of aromatic compounds, and another, found in 78% of the strains, contains an operon for nitrate assimilation. Both variants were shown in ecophysiological tests to be functional, thus providing the potential for microniche partitioning. In addition, detected interspecific horizontal exchange of GIs indicates a large gene pool accessible to Polynucleobacter species. In contrast to core genes, GIs are spread more successfully across spatially separated freshwater habitats. The mobility and functional diversity of GIs allow for rapid evolution, which may be a key aspect for the ubiquitous occurrence of Polynucleobacter bacteria. IMPORTANCE: Assessing the ecological relevance of bacterial diversity is a key challenge for current microbial ecology. The polyphasic approach which was applied in this study, including targeted isolation of strains, genome analysis, and ecophysiological tests, is crucial for the linkage of genetic and ecological knowledge. Particularly great importance is attached to the high number of closely related strains which were investigated, represented by genome-wide average nucleotide identities (ANI) larger than 97%. The extent of functional diversification found on this narrow phylogenetic scale is compelling. Moreover, the transfer of metabolically relevant genomic islands between more distant members of the Polynucleobacter community provides important insights toward a better understanding of the evolution of these globally abundant freshwater bacteria.

Polynucleobacter wuianus sp. nov., a free-living freshwater bacterium affiliated with the cryptic species complex PnecC.

Strain QLW-P1FAT50C-4T, isolated from a shallow, acidic freshwater pond located in the Austrian Alps at an altitude of 1300 m, was characterized by investigation of phenotypic, chemotaxonomic and genomic traits. As shown previously, phylogenetic analyses based on 16S rRNA gene sequences placed the strain in the cryptic species complex PnecC within the genus Polynucleobacter. The major fatty acids of the strain were C16 : 1ω7c and C18 : 1ω7c. The strain has a genome of 2.23 Mbp with a DNA G+C content of 44.9 mol%. The strain encodes a seemingly complete gene cluster for anoxygenic photosynthesis but lacks typical genes for CO2 assimilation. In order to resolve the phylogenetic position of the strain within the species complex PnecC, concatenated partial sequences of eight housekeeping genes were analysed. The phylogenetic reconstruction obtained did not place strain QLW-P1FAT50C-4T close to any of the five previously described species within subcluster PnecC. Pairwise average nucleotide identity (ANI) comparisons of whole-genome sequences suggested that strain QLW-P1FAT50C-4T (=DSM 24008T=CIP 111100T) represents a novel species, for which we propose the name Polynucleobacter wuianus sp. nov.

Geographic population structure and distinct intra-population dynamics of globally abundant freshwater bacteria.

Implications of geographic separation and temporal dynamics on the evolution of free-living bacterial species are widely unclear. However, the vast amount of metagenome sequencing data generated during the last decades from various habitats around the world provides an unprecedented opportunity for such investigations. Here, we exploited publicly available and new freshwater metagenomes in combination with the genomes of abundant freshwater bacteria to reveal geographic and temporal population structure. We focused on species that were detected across broad geographic ranges at high enough sequence coverage for meaningful population genomic analyses, associated with the predominant freshwater taxa acI, LD12, Polynucleobacter, and Candidatus Methylopumilus. Despite the broad geographic ranges, each species appeared as a sequence-discrete cluster, in contrast to abundant marine taxa, for which continuous diversity structures were reported on a global scale. Population differentiation increased significantly with spatial distance in all species, but notable dispersal barriers (e.g. oceanic) were not apparent. Yet, the different species showed contrasting rates of geographic divergence and strikingly different intra-population dynamics in time series within individual habitats. The change in an LD12 population over 7 years was minor (FST = 0.04) compared to differentiation between lakes, whereas a Polynucleobacter population displayed strong changes within merely 2 months (FST up to 0.54), similar in scale to differentiation between populations separated by thousands of kilometers. The slowly and steadily evolving LD12 population showed high strain diversity, whereas the dynamic Polynucleobacter population exhibited alternating clonal expansions of mostly two strains only. Based on the contrasting population structures, we propose distinct models of speciation.

Exploring environmental intra-species diversity through non-redundant pangenome assemblies.

At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different environmental niches and can have a profound influence on microbial community dynamics. As a consequence, any individual genome or population will contain merely a fraction of the total genetic diversity of any operationally defined "species", whose ecological potential can thus be only fully understood by studying all of their genomes and the genes therein. This concept, known as the pangenome, is valuable for studying microbial ecology and evolution, as it partitions genomes into core (present in all the genomes from a species, and responsible for housekeeping and species-level niche adaptation among others) and accessory regions (present only in some, and responsible for intra-species differentiation). Here we present SuperPang, an algorithm producing pangenome assemblies from a set of input genomes of varying quality, including metagenome-assembled genomes (MAGs). SuperPang runs in linear time and its results are complete, non-redundant, preserve gene ordering and contain both coding and non-coding regions. Our approach provides a modular view of the pangenome, identifying operons and genomic islands, and allowing to track their prevalence in different populations. We illustrate this by analysing intra-species diversity in Polynucleobacter, a bacterial genus ubiquitous in freshwater ecosystems, characterized by their streamlined genomes and their ecological versatility. We show how SuperPang facilitates the simultaneous analysis of allelic and gene content variation under different environmental pressures, allowing us to study the drivers of microbial diversification at unprecedented resolution.

Ecogenomics and cultivation reveal distinctive viral-bacterial communities in the surface microlayer of a Baltic Sea slick.

Visible surface films, termed slicks, can extensively cover freshwater and marine ecosystems, with coastal regions being particularly susceptible to their presence. The sea-surface microlayer (SML), the upper 1-mm at the air-water interface in slicks (herein slick SML) harbors a distinctive bacterial community, but generally little is known about SML viruses. Using flow cytometry, metagenomics, and cultivation, we characterized viruses and bacteria in a brackish slick SML in comparison to non-slick SML as well as seawater below slick and non-slick areas (subsurface water = SSW). Size-fractionated filtration of all samples distinguished viral attachment to hosts and particles. The slick SML contained higher abundances of virus-like particles, prokaryotic cells, and dissolved organic carbon compared to non-slick SML and SSW. The community of 428 viral operational taxonomic units (vOTUs), 426 predicted as lytic, distinctly differed across all size fractions in the slick SML compared to non-slick SML and SSW. Specific metabolic profiles of bacterial metagenome-assembled genomes and isolates in the slick SML included a prevalence of genes encoding motility and carbohydrate-active enzymes (CAZymes). Several vOTUs were enriched in slick SML, and many virus variants were associated with particles. Nine vOTUs were only found in slick SML, six of them being targeted by slick SML-specific clustered-regularly interspaced short palindromic repeats (CRISPR) spacers likely originating from Gammaproteobacteria. Moreover, isolation of three previously unknown lytic phages for Alishewanella sp. and Pseudoalteromonas tunicata, abundant and actively replicating slick SML bacteria, suggests that viral activity in slicks contributes to biogeochemical cycling in coastal ecosystems.

Lipocalin Q83 reveals a dual ligand binding mode with potential implications for the functions of siderocalins.

Siderocalins are particular lipocalins that participate in the innate immune response by interfering with bacterial siderophore-mediated iron uptake. Additionally, siderocalins are involved in several physiological and pathological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. Here we show that siderocalin Q83 displays an unexpected dual ligand binding mode as it can bind enterobactin and unsaturated fatty acids simultaneously. The solution structure of the siderocalin Q83 in complex with arachidonic acid and enterobactin reveals molecular details of this novel dual binding mode and the determinants of fatty acid binding specificity. Our results suggest that Q83 is a metabolic hub linking iron and fatty acid pathways. This unexpected coupling might contribute to the pleiotropic functions of siderocalins.

Siderocalin Q83 exhibits differential slow dynamics upon ligand binding.

Siderocalin Q83 is a small soluble protein that has the ability to bind two different ligands (enterobactin and arachidonic acid) simultaneously in two distinct binding sites. Here we report that Q83 exhibits an intriguing dynamic behavior. In its free form, the protein undergoes significant micro-to-millisecond dynamics. When binding arachidonic acid, the motions of the arachidonic acid binding site are quenched while the dynamics at the enterobactin binding site increases. Reciprocally, enterobactin binding to Q83 quenches the motions at the enterobactin binding site and increases the slow dynamics at the arachidonic acid binding site. Additionally, in the enterobactin-bound state, the excited state of the arachidonic acid binding site resembles the arachidonic acid-bound state. These observations strongly suggest an allosteric regulation where binding of one ligand enhances the affinity of Q83 for the other one. Additionally, our data strengthen the emerging view of proteins as dynamic ensembles interconverting between different sub-states with distinct functionalities.

Opening a next-generation black box: Ecological trends for hundreds of species-like taxa uncovered within a single bacterial >99% 16S rRNA operational taxonomic unit.

Current knowledge on environmental distribution and taxon richness of free-living bacteria is mainly based on cultivation-independent investigations employing 16S rRNA gene sequencing methods. Yet, 16S rRNA genes are evolutionarily rather conserved, resulting in limited taxonomic and ecological resolutions provided by this marker. The faster evolving protein-encoding gene priB was used to reveal ecological patterns hidden within a single operational taxonomic unit (OTU) defined by >99% 16S rRNA sequence similarity. The studied subcluster PnecC of the genus Polynucleobacter represents a ubiquitous group of abundant freshwater bacteria with cosmopolitan distribution, which is very frequently detected by diversity surveys of freshwater systems. Based on genome taxonomy and a large set of genome sequences, a sequence similarity threshold for delineation of species-like taxa could be established. In total, 600 species-like taxa were detected in 99 freshwater habitats scattered across three regions representing a latitudinal range of 3,400 km (42°N to 71°N) and a pH gradient of 4.2 to 8.6. In addition to the unexpectedly high richness, the increased taxonomic resolution revealed structuring of Polynucleobacter communities by a couple of macroecological trends, which was previously only demonstrated for phylogenetically much broader groups of bacteria. An unexpected pattern was the almost complete compositional separation of Polynucleobacter communities of Ca2+ -rich and Ca2+ -poor habitats. This compositional pattern strongly resembled the vicariance of plant species on silicate and limestone soils. The new cultivation-independent approach presented opened a window to an incredible, previously unseen diversity, and enables investigations aiming on deeper understanding of how environmental conditions shape bacterial communities and drive evolution of free-living bacteria.

Continental-Scale Gene Flow Prevents Allopatric Divergence of Pelagic Freshwater Bacteria.

Allopatric divergence is one of the principal mechanisms for speciation of macro-organisms. Microbes by comparison are assumed to disperse more freely and to be less limited by dispersal barriers. However, thermophilic prokaryotes restricted to geothermal springs have shown clear signals of geographic isolation, but robust studies on this topic for microbes with less strict habitat requirements are scarce. Furthermore, it has only recently been recognized that homologous recombination among conspecific individuals provides species coherence in a wide range of prokaryotes. Recombination barriers thus may define prokaryotic species boundaries, yet, the extent to which geographic distance between populations gives rise to such barriers is an open question. Here, we investigated gene flow and population structure in a widespread species of pelagic freshwater bacteria, Polynucleobacter paneuropaeus. Through comparative genomics of 113 conspecific strains isolated from freshwater lakes and ponds located across a North-South range of more than 3,000 km, we were able to reconstruct past gene flow events. The species turned out to be highly recombinogenic as indicated by significant signs of gene transfer and extensive genome mosaicism. Although genomic differences increased with spatial distance on a regional scale (<170 km), such correlations were mostly absent on larger scales up to 3,400 km. We conclude that allopatric divergence in European P. paneuropaeus is minor, and that effective gene flow across the sampled geographic range in combination with a high recombination efficacy maintains species coherence.

Ecological Differentiation in Two Major Freshwater Bacterial Taxa Along Environmental Gradients.

Polynucleobacter (Burkholderiaceae, Betaproteobacteria) and Limnohabitans (Comamonadaceae, Betaproteobacteria) are abundant freshwater bacteria comprising large genetic and taxonomic diversities, with species adapted to physico-chemically distinct types of freshwater systems. The relative importance of environmental drivers, i.e., physico-chemistry, presence of microeukaryotes and geographic position for the diversity and prevalence has not been investigated for both taxa before. Here, we present the first pan-European study on this topic, comprising 255 freshwater lakes. We investigated Limnohabitans and Polynucleobacter using an amplicon sequencing approach of partial 16S rRNA genes along environmental gradients. We show that physico-chemical factors had the greatest impact on both genera. Analyses on environmental gradients revealed an exceptionally broad ecological spectrum of operational taxonomic units (OTUs). Despite the coarse resolution of the genetic marker, we found OTUs with contrasting environmental preferences within Polynucleobacter and Limnohabitans subclusters. Such an ecological differentiation has been characterized for PnecC and LimC before but was so far unknown for less well studied subclusters such as PnecA and PnecB. Richness and abundance of OTUs are geographically clustered, suggesting that geographic diversity patterns are attributable to region-specific physico-chemical characteristics (e.g., pH and temperature) rather than latitudinal gradients or lake sizes.

Combined Methylome, Transcriptome and Proteome Analyses Document Rapid Acclimatization of a Bacterium to Environmental Changes.

Polynucleobacter asymbioticus strain QLW-P1DMWA-1T represents a group of highly successful heterotrophic ultramicrobacteria that is frequently very abundant (up to 70% of total bacterioplankton) in freshwater habitats across all seven continents. This strain was originally isolated from a shallow Alpine pond characterized by rapid changes in water temperature and elevated UV radiation due to its location at an altitude of 1300 m. To elucidate the strain's adjustment to fluctuating environmental conditions, we recorded changes occurring in its transcriptomic and proteomic profiles under contrasting experimental conditions by simulating thermal conditions in winter and summer as well as high UV irradiation. To analyze the potential connection between gene expression and regulation via methyl group modification of the genome, we also analyzed its methylome. The methylation pattern differed between the three treatments, pointing to its potential role in differential gene expression. An adaptive process due to evolutionary pressure in the genus was deduced by calculating the ratios of non-synonymous to synonymous substitution rates for 20 Polynucleobacter spp. genomes obtained from geographically diverse isolates. The results indicate purifying selection.