Gradients of bacteria in the oceanic water column reveal finely-resolved vertical distributions.

Bacterial communities directly influence ecological processes in the ocean, and depth has a major influence due to the changeover in primary energy sources between the sunlit photic zone and dark ocean. Here, we examine the abundance and diversity of bacteria in Monterey Bay depth profiles collected from the surface to just above the sediments (e.g., 2000 m). Bacterial abundance in these Pacific Ocean samples decreased by >1 order of magnitude, from 1.22 ±0.69 ×106 cells ml-1 in the variable photic zone to 1.44 ± 0.25 ×105 and 6.71 ± 1.23 ×104 cells ml-1 in the mesopelagic and bathypelagic, respectively. V1-V2 16S rRNA gene profiling showed diversity increased sharply between the photic and mesopelagic zones. Weighted Gene Correlation Network Analysis clustered co-occurring bacterial amplicon sequence variants (ASVs) into seven subnetwork modules, of which five strongly correlated with depth-related factors. Within surface-associated modules there was a clear distinction between a 'copiotrophic' module, correlating with chlorophyll and dominated by e.g., Flavobacteriales and Rhodobacteraceae, and an 'oligotrophic' module dominated by diverse Oceanospirillales (such as uncultured JL-ETNP-Y6, SAR86) and Pelagibacterales. Phylogenetic reconstructions of Pelagibacterales and SAR324 using full-length 16S rRNA gene data revealed several additional subclades, expanding known microdiversity within these abundant lineages, including new Pelagibacterales subclades Ia.B, Id, and IIc, which comprised 4-10% of amplicons depending on the subclade and depth zone. SAR324 and Oceanospirillales dominated in the mesopelagic, with SAR324 clade II exhibiting its highest relative abundances (17±4%) in the lower mesopelagic (300-750 m). The two newly-identified SAR324 clades showed highest relative abundances in the photic zone (clade III), while clade IV was extremely low in relative abundance, but present across dark ocean depths. Hierarchical clustering placed microbial communities from 900 m samples with those from the bathypelagic, where Marinimicrobia was distinctively relatively abundant. The patterns resolved herein, through high resolution and statistical replication, establish baselines for marine bacterial abundance and taxonomic distributions across the Monterey Bay water column, against which future change can be assessed.

Novel Alphaproteobacteria transcribe genes for nitric oxide transformation at high levels in a marine oxygen-deficient zone.

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

Assessing the impacts of oil contamination on microbial communities in a Niger Delta soil.

Oil spills are a global challenge, contaminating the environment with organics and metals known to elicit toxic effects. Ecosystems within Nigeria's Niger Delta have suffered from prolonged severe spills for many decades but the level of impact on the soil microbial community structure and the potential for contaminant bioremediation remains unclear. Here, we assessed the extent/impact of an oil spill in this area 6 months after the accident on both the soil microbial community/diversity and the distribution of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHDGNα) genes, responsible for encoding enzymes involved in the degradation of PAHs, across the impacted area. Analyses confirmed the presence of oil contamination, including metals such as Cr and Ni, across the whole impacted area and at depth. The contamination impacted on the microbial community composition, resulting in a lower diversity in all contaminated soils. Gamma-, Delta-, Alpha- proteobacteria and Acidobacteriia dominated 16S rRNA gene sequences across the contaminated area, while Ktedonobacteria dominated the non-contaminated soils. The PAH-RHDαGN genes were only detected in the contaminated area, highlighting a clear relationship with the oil contamination/hydrocarbon metabolism. Correlation analysis indicated significant positive relationships between the oil contaminants (organics, Cr and Ni), PAH-RHDαGN gene, and the presence of bacteria/archaea such as Anaerolinea, Spirochaetia Bacteroidia Thermoplasmata, Methanomicrobia, and Methanobacteria indicating that the oil contamination not only impacted the microbial community/diversity present, but that the microbes across the impacted area and at depth were potentially playing an important role in degrading the oil contamination present. These findings provide new insights on the level of oil contamination remaining 6 months after an oil spill, its impacts on indigenous soil microbial communities and their potential for in situ bioremediation within a Niger Delta's ecosystem. It highlights the strength of using a cross-disciplinary approach to assess the extent of oil pollution in a single study.

Effects of color variation and physiological state on ascidian microbiomes.

Ascidians, known for their color variation, host species-specific microbial symbiont communities. Some ascidians can also transition into a nonfiltering (resting) physiological state. Recent studies suggest that the microbial symbiont communities may vary across different physiological states and color morphs of the host. The colonial ascidian, Polyclinum constellatum, which exhibits several color morphs in the Caribbean Sea, periodically ceases its filtering activity. To investigate if color variation in P. constellatum is indicative of sibling speciation, we sequenced fragments of the ribosomal 18S rRNA and the mitochondrial cytochrome oxidase subunit I genes. Additionally, we sequenced a fragment of the 16S rRNA gene to characterize the microbial communities of two common color morphs (red and green) in colonies that were either actively filtering (active) or nonfiltering (resting). Phylogenetic analyses of both ascidian genes resulted in well-supported monophyletic clades encompassing all color variants of P. constellatum. Interestingly, no significant differences were observed among the microbial communities of the green and red morphs, suggesting that color variation in this species is a result of intraspecific variation. However, the host's physiological state significantly influenced the microbial community structure. Nonfiltering (resting) colonies hosted higher relative abundances of Kiloniella (Alphaproteobacteria) and Fangia (Gammaproteobacteria), while filtering colonies hosted more Reugeria (Alphaproteobacteria) and Endozoicomonas (Gammaproteobacteria). This study demonstrates that microbial symbiont communities serve as reliable indicators of the taxonomic state of their host and are strongly influenced by the host's feeding condition.

Host association and intracellularity evolved multiple times independently in the Rickettsiales.

The order Rickettsiales (Alphaproteobacteria) encompasses multiple diverse lineages of host-associated bacteria, including pathogens, reproductive manipulators, and mutualists. Here, in order to understand how intracellularity and host association originated in this order, and whether they are ancestral or convergently evolved characteristics, we built a large and phylogenetically-balanced dataset that includes de novo sequenced genomes and a selection of published genomic and metagenomic assemblies. We perform detailed functional reconstructions that clearly indicates "late" and parallel evolution of obligate host-association in different Rickettsiales lineages. According to the depicted scenario, multiple independent horizontal acquisitions of transporters led to the progressive loss of biosynthesis of nucleotides, amino acids and other metabolites, producing distinct conditions of host-dependence. Each clade experienced a different pattern of evolution of the ancestral arsenal of interaction apparatuses, including development of specialised effectors involved in the lineage-specific mechanisms of host cell adhesion and/or invasion.

Co-evolution of gene transfer agents and their alphaproteobacterial hosts.

Gene transfer agents (GTAs) are enigmatic elements that resemble small viruses and are known to be produced during nutritional stress by some bacteria and archaea. The production of GTAs is regulated by quorum sensing, under which a small fraction of the population acts as GTA producers, while the rest becomes GTA recipients. In contrast to canonical viruses, GTAs cannot propagate themselves because they package pieces of the producing cell's genome. In alphaproteobacteria, GTAs are mostly vertically inherited and reside in their hosts' genomes for hundreds of millions of years. While GTAs' ability to transfer genetic material within a population and their long-term preservation suggest an increased fitness of GTA-producing microbes, the associated benefits and type of selection that maintains GTAs are poorly understood. By comparing rates of evolutionary change in GTA genes to the rates in gene families abundantly present across 293 alphaproteobacterial genomes, we detected 59 gene families that likely co-evolve with GTA genes. These gene families are predominantly involved in stress response, DNA repair, and biofilm formation. We hypothesize that biofilm formation enables the physical proximity of GTA-producing cells, limiting GTA-derived benefits only to a group of closely related cells. We further conjecture that the population structure of biofilm-forming sub-populations ensures that the trait of GTA production is maintained despite the inevitable rise of "cheating" genotypes. Because release of GTA particles kills the producing cell, maintenance of GTAs is an exciting example of social evolution in a microbial population.IMPORTANCEGene transfer agents (GTAs) are viruses domesticated by some archaea and bacteria as vehicles for carrying pieces of the host genome. Produced under certain environmental conditions, GTA particles can deliver DNA to neighboring, closely related cells. The function of GTAs remains uncertain. While making GTAs is suicidal for a cell, GTA-encoding genes are widespread in genomes of alphaproteobacteria. Such GTA persistence implies functional benefits but raises questions about how selection maintains this lethal trait. By showing that GTA genes co-evolve with genes involved in stress response, DNA repair, and biofilm formation, we provide support for the hypothesis that GTAs facilitate DNA exchange during the stress conditions and present a model for how GTAs persist in biofilm-forming bacterial populations despite being lethal.

Novel evolutionary insights on the interactions of the Holosporales (Alphaproteobacteria) with eukaryotic hosts from comparative genomics.

Holosporales are an alphaproteobacterial order engaging in obligate and complex associations with eukaryotes, in particular protists. The functional and evolutionary features of those interactions are still largely undisclosed. Here, we sequenced the genomes of two members of the species Bealeia paramacronuclearis (Holosporales, Holosporaceae) intracellularly associated with the ciliate protist Paramecium, which resulted in high correspondence. Consistent with the short-branched early-divergent phylogenetic position, Bealeia presents a larger functional repertoire than other Holosporaceae, comparable to those of other Holosporales families, particularly for energy metabolism and motility. Our analyses indicate that different Holosporales likely experienced at least partly autonomous genome reduction and adaptation to host interactions, for example regarding dependence on host biotin driven by multiple independent horizontal acquisitions of transporters. Among Alphaproteobacteria, this is reminiscent of the convergently evolved Rickettsiales, which however appear more diverse, possibly due to a probably more ancient origin. We identified in Bealeia and other Holosporales the plasmid-encoded putative genetic determinants of R-bodies, which may be involved in a killer trait towards symbiont-free hosts. While it is not clear whether these genes are ancestral or recently horizontally acquired, an intriguing and peculiar role of R-bodies is suggested in the evolution of the interactions of multiple Holosporales with their hosts.

Phosphate-related genomic islands as drivers of environmental adaptation in the streamlined marine alphaproteobacterial HIMB59.

IMPORTANCE: These results shed light on the evolutionary strategies of microbes with streamlined genomes to adapt and survive in the oligotrophic conditions that dominate the surface waters of the global ocean. At the individual level, these microbes have been subjected to evolutionary constraints that have led to a more efficient use of nutrients, removing non-essential genes named as "streamlining theory." However, at the population level, they conserve a highly diverse gene pool in flexible genomic islands resulting in polyclonal populations on the same genomic background as an evolutionary response to environmental pressures. Localization of these islands at equivalent positions in the genome facilitates horizontal transfer between clonal lineages. This high level of environmental genomic heterogeneity could explain their cosmopolitan distribution. In the case of the order HIMB59 within the class Alphaproteobacteria, two factors exert evolutionary pressure and determine this intraspecific diversity: phages and the concentration of P in the environment.

Potential of psychrotolerant rhizobacteria for the growth promotion of wheat (Triticum aestivum L.).

Wheat is the second most important staple crop grown and consumed worldwide. Temperature fluctuations especially the cold stress during the winter season reduces wheat growth and grain yield. Psychrotolerant plant growth-promoting rhizobacteria (PGPR) may improve plant stress-tolerance in addition to serve as biofertilizer. The present study aimed to isolate and identify PGPR, with the potential to tolerate cold stress for subsequent use in supporting wheat growth under cold stress. Ten psychrotolerant bacteria were isolated from the wheat rhizosphere at 4 °C and tested for their ability to grow at wide range of temperature ranging from -8 °C to 36 °C and multiple plant beneficial traits. All bacteria were able to grow at 4 °C to 32 °C temperature range and solubilized phosphorus except WR23 at 4 °C, whereas all the bacteria solubilized phosphorus at 28 °C. Seven bacteria produced indole-3-acetic acid at 4 °C, whereas all produced indole-3-acetic acid at 28 °C. Seven bacteria showed the ability to fix nitrogen at 4 °C, while all the bacteria fixed nitrogen at 28 °C. Only one bacterium showed the potential to produce cellulase at 4 °C, whereas four bacteria showed the potential to produce cellulase at 28 °C. Seven bacteria produced pectinase at 4 °C, while one bacterium produced pectinase at 28 °C. Only one bacterium solubilized the zinc at 4 °C, whereas six bacteria solubilized the zinc at 28 °C using ZnO as the primary zinc source. Five bacteria solubilized the zinc at 4 °C, while seven bacteria solubilized the zinc at 28 °C using ZnCO3 as the primary zinc source. All the bacteria produced biofilm at 4 °C and 28 °C. In general, we noticed behavior of higher production of plant growth-promoting substances at 28 °C, except pectinase assay. Overall, in vitro testing confirms that microbes perform their inherent properties efficiently at optimum temperatures rather than the low temperatures due to high metabolic rate. Five potential rhizobacteria were selected based on the in vitro testing and evaluated for plant growth-promoting potential on wheat under controlled conditions. WR22 and WR24 significantly improved wheat growth, specifically increasing plant dry weight by 42% and 58%, respectively. 16S rRNA sequence analysis of WR22 showed 99.78% similarity with Cupriavidus campinensis and WR24 showed 99.9% similarity with Enterobacter ludwigii. This is the first report highlighting the association of C. campinensis and E. ludwigii with wheat rhizosphere. These bacteria can serve as potential candidates for biofertilizer to mitigate the chilling effect and improve wheat production after field-testing.

The Improvement of Fluorescence In Situ Hybridization Technique Based on Explorations of Symbionts in Cicadas.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is widely used for the identification of microbes in complex samples, but it suffers from some limitations resulting in the weak or even absence of fluorescence signals of microbe(s), which may lead to the underestimation or misunderstanding of a microbial community. Herein, we explored symbionts in the bacteriomes and fat bodies of cicadas using modified FISH, aiming to improve this technique. We initially revealed that the probes of Candidatus Sulcia muelleri (Sulcia) and the yeast-like fungal symbiont (YLS) are suitable for detection of these symbionts in all cicadas and some other species of Auchenorrhyncha, whereas the probe of Candidatus Hodgkinia cicadicola (Hodgkinia) is only suitable for detection of Hodgkinia in a few cicada species. The fluorescence signal of Sulcia, Hodgkinia and YLS exhibited weak intensity without the addition of unlabeled oligonucleotides (helpers) and heat shock in some cicadas; however, it can be significantly improved by the addition of both helpers and heat shock. Results of this study suggest that heat shock denaturing rRNA and proteins of related microbe(s) together with helpers binding to the adjacent region of the probe's target sites prevent the re-establishment of the native secondary structure of rRNA; therefore, suitable probe(s) can more easily access to the probe's target sites of rRNA. Our results provide new information for the significant improvement of hybridization signal intensities of microbes in the FISH experiment, making it possible to achieve a more precise understanding of the microbial distribution, community and density in complex samples.

Complete genome analysis of type strain of a novel bacterial family Temperatibacteraceae fam. nov., isolated from surface seawater.

Novel bacterial resources are valuable for studying bacterial taxonomy, bacterial evolution, and genome mining of novel antibiotics, antitumor agents, and immune modulators. In this study, we de novo sequenced the type strain of a novel bacterial family, Temperatibacteraceae fam. Nov., belonging to class Alphaproteobacteria of phylum Pseudomonadota. The type strain, Temperatibacter marinus NBRC 110045T, is mesophilic and was isolated from surface seawater around Muroto city of Japan at a depth of 0.5 m. Here, the sequenced complete genome of strain NBRC 110045T is composed of a circular chromosome of 3,184,799 bp with a mean G + C content of 43.71%. Genome analysis was applied to reveal the genetic basis of its cellular activities. Cellular regulation and signaling was analyzed to infer the regulatory mechanism of its limited growth temperature range. Genomic features of the novel family Temperatibacteraceae may expand our knowledge on environmental adaptation, genetic evolution and natural product discovery of marine bacteria.

Design-Build-Test of Synthetic Promoters for Inducible Gene Regulation in Alphaproteobacteria.

Inducible gene expression is useful for biotechnological applications and for studying gene regulation and function in bacteria. Many inducible systems that perform in model organisms such as the Gammaproteobacterium Escherichia coli do not perform well in other bacteria that are of biotechnological interest. Typical problems include weak or leaky expression. Here, we describe an invention named ACIT (Alphaproteobacteria chromosomally integrating transcription-control cassette) that is carried on a suicide plasmid to enable insertion into the chromosome of the host. ACIT consists of multiple DNA fragments specifically arranged in a cassette that allows tight transcription control over any gene or gene cluster of interest following homologous recombination. At the heart of the invention is the ability to modify or exchange parts, e.g., promoters, to suit particular bacteria and growth conditions, allowing for customized gene expression control. Furthermore, ACIT provides a basis for a design-build-test approach for controlling gene expression in less studied bacteria. We describe examples of its control over pigment and exopolysaccharide production, growth, cell form, and social behavior in various Alphaproteobacteria.

Rhizobacterial Isolates from Prosopis limensis Promote the Growth of Raphanus sativus L. Under Salt Stress.

Microbial biotechnology employes techniques that rely based on the natural interactions that occur in ecosystems. Bacteria, including rhizobacteria, play an important role in plant growth, providing agricultural crops with an alternative that can mitigate the negative effects of abiotic stress, such as those caused by saline environments. In this study, bacterial isolates were obtained from soil and roots of Prosopis limensis Bentham from the department of Lambayeque, Peru. This region has high salinity levels, therefore, the collected samples were used to isolate plant growth-promoting rhizobacteria (PGPR), which were identified through morphological, and physical-biochemical characteristics. These salt tolerant bacteria were screened phosphate solubilization, indole acetic acid, deaminase activity and molecular characterization by 16S rDNA sequencing. Eighteen samples from saline soils of the Prosopis limensis plants in the northern coastal desert of San Jose district, Lambayeque, Peru. The bacterial isolates were screened for salt tolerance ranging from 2 to 10%, a total of 78 isolates were found. Isolates 03, 13 and 31 showed maximum salt tolerance at 10%, in vitro ACC production, phosphate solubilization and IAA production. The three isolates were identified by sequencing the amplified 16S rRNA gene and were found to be Pseudomonas sp. 03 (MW604823), Pseudomonas sp. 13 (MW604824) and Bordetella sp. 31 (MW604826). These microorganisms promoted the germination of radish plants and increased the germination rates for treatments T2, T3 and T4 by 129, 124 and 118% respectively. The beneficial effects of salt tolerant PGPR isolates isolated from saline environments can be new species, used to overcome the detrimental effects of salt stress on plants. The biochemical response and inoculation of the three isolates prove the potential of using these strains as a source of products that can be employed for the development of new compounds proving their potential as biofertilizers for saline environments.

No Transcriptional Compensation for Extreme Gene Dosage Imbalance in Fragmented Bacterial Endosymbionts of Cicadas.

Bacteria that form long-term intracellular associations with host cells lose many genes, a process that often results in tiny, gene-dense, and stable genomes. Paradoxically, the some of the same evolutionary processes that drive genome reduction and simplification may also cause genome expansion and complexification. A bacterial endosymbiont of cicadas, Hodgkinia cicadicola, exemplifies this paradox. In many cicada species, a single Hodgkinia lineage with a tiny, gene-dense genome has split into several interdependent cell and genome lineages. Each new Hodgkinia lineage encodes a unique subset of the ancestral unsplit genome in a complementary way, such that the collective gene contents of all lineages match the total found in the ancestral single genome. This splitting creates genetically distinct Hodgkinia cells that must function together to carry out basic cellular processes. It also creates a gene dosage problem where some genes are encoded by only a small fraction of cells while others are much more abundant. Here, by sequencing DNA and RNA of Hodgkinia from different cicada species with different amounts of splitting-along with its structurally stable, unsplit partner endosymbiont Sulcia muelleri-we show that Hodgkinia does not transcriptionally compensate to rescue the wildly unbalanced gene and genome ratios that result from lineage splitting. We also find that Hodgkinia has a reduced capacity for basic transcriptional control independent of the splitting process. Our findings reveal another layer of degeneration further pushing the limits of canonical molecular and cell biology in Hodgkinia and may partially explain its propensity to go extinct through symbiont replacement.

Ancylobacter radicis sp. nov., a novel aerobic methylotrophic bacteria associated with plants.

The two novel bacterial strains, designated as VTT and ML, were isolated from roots of cinquefoil (Potentilla sp.) and leaves of meadow-grass (Poa sp.) on the flooded bank of lake, respectively. These isolates were Gram-negative, non-spore-forming, non-motile, rod-shaped cells, utilized methanol, methylamine, and polycarbon compounds as carbon and energy sources. In the whole-cell fatty acid pattern of strains prevailed C18:1ω7c and C19:0cyc. Based on the phylogenetic analysis of 16S rRNA gene sequences, strains VTT and ML were closely related to the representatives of the genus Ancylobacter (98.3-98.5%). The assembled genome of strain VTT has a total length of 4.22 Mbp, and a G + C content is 67.3%. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strain VTT and closely related type strains of genus Ancylobacter were 78.0-80.6%, 73.8-78.3% and 22.1-24.0%, respectively, that clearly lower than proposed thresholds for species. On the basis of the phylogenetic, phenotypic, and chemotaxonomic analysis, isolates VTT and ML represent a novel species of the genus Ancylobacter, for which the name Ancylobacter radicis sp. nov. is proposed. The type strain is VTT (= VKM B-3255T = CCUG 72400T). In addition, novel strains were able to dissolve insoluble phosphates, to produce siderophores and plant hormones (auxin biosynthesis). According to genome analysis genes involved in the biosynthesis of siderophores, polyhydroxybutyrate, exopolysaccharides and phosphorus metabolism, as well as the genes involved in the assimilation of C1-compounds (natural products of plant metabolism) were found in the genome of type strain VTT.

A Metagenomic and Amplicon Sequencing Combined Approach Reveals the Best Primers to Study Marine Aerobic Anoxygenic Phototrophs.

Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of aerobic anoxygenic phototrophic (AAP) bacteria, the pufM gene has been established as the genetic marker for this particular functional group, although available primers are known to have amplification biases. We review here the existing primers for pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from contrasting marine environments to evaluate their performance. By comparing the taxonomic composition of communities retrieved with metagenomics and with different amplicon approaches, we show that the commonly used PCR primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach, as well as the use of other combinations of the existing and newly designed primers, show that these groups are in fact less abundant than previously observed, and that a great proportion of pufM sequences are affiliated to uncultured representatives, particularly in the open ocean. Altogether, the framework developed here becomes a better alternative for future studies based on the pufM gene and, additionally, serves as a reference for primer evaluation of other functional genes.

A novel bacteriobiont of the Arctic lichen Flavocetraria nivalis, Lichenifustis flavocetrariae gen. nov, sp. nov. demonstrating hydrolytic properties and containing a full set of the Calvin-Benson-Bassham cycle genes.

A Gram-negative, strictly aerobic, chemoorganotrophic, bacteriochlorophyll a-containing, slow-growing bacterium was isolated from the lichen Flavocetraria nivalis and designated strain BP6-180914 T. Cells of this strain were large nonmotile rods, which reproduced by binary fission. Cells grew under oxic conditions and were able to utilize sugars and several polysaccharides, including starch and pectin. Strain BP6-180914 T was psychrotolerant and moderately acidophilic growing at 4-35 °C (optimum 20-28 °C) and between pH 4.0 and 7.5 (optimum 4.5-5.5). The major fatty acids were C18:1ω7c, C19:0 cyclo, C16:0 and C18:0. The polar lipids were diphosphatidylglycerols, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylcholines, unidentified aminolipids, and a number of glycolipids, the major one being an unidentified glycolipid. The quinone was Q-10. The DNA G + C content was 63.65%. Comparative 16S rRNA gene sequence analysis revealed that strain BP6-180914 T was a member of the order Hyphomicrobiales and belonged to the family Lichenihabitantaceae defined by the lichen-dwelling facultative aerobic chemo-organotroph Lichenihabitans psoromatis (92.7% sequence similarity). The results of phylogenomic and genomic relatedness analyses showed that strain BP6-180914 T could clearly be distinguished from other species in the order Hyphomicrobiales with average nucleotide identity values of < 74.05% and genome-to-genome distance values of < 21.1%. The AAI value of 65.9% between strain BP6-180914 T and L. psoromatis allowed us to assign this strain to the novel genus of the family Lichenihabitantaceae. Therefore, it is proposed that strain BP6-180914 T represents a novel species in a new genus, Lichenifustis flavocetrariae gen. nov., sp. nov.; strain BP6-180914 T (= KCTC 92872 T = VKM B-3641 T = UQM 41506 T) is the type strain.

Intracytoplasmic-membrane development in alphaproteobacteria involves the homolog of the mitochondrial crista-developing protein Mic60.

Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 in R. sphaeroides. We show that the three α helices of mitochondrial Mic60's mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression led to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs and have therefore improved our understanding of the nature of the mitochondrial ancestor.

Ecophysiology and genomics of the brackish water adapted SAR11 subclade IIIa.

The Order Pelagibacterales (SAR11) is the most abundant group of heterotrophic bacterioplankton in global oceans and comprises multiple subclades with unique spatiotemporal distributions. Subclade IIIa is the primary SAR11 group in brackish waters and shares a common ancestor with the dominant freshwater IIIb (LD12) subclade. Despite its dominance in brackish environments, subclade IIIa lacks systematic genomic or ecological studies. Here, we combine closed genomes from new IIIa isolates, new IIIa MAGS from San Francisco Bay (SFB), and 460 highly complete publicly available SAR11 genomes for the most comprehensive pangenomic study of subclade IIIa to date. Subclade IIIa represents a taxonomic family containing three genera (denoted as subgroups IIIa.1, IIIa.2, and IIIa.3) that had distinct ecological distributions related to salinity. The expansion of taxon selection within subclade IIIa also established previously noted metabolic differentiation in subclade IIIa compared to other SAR11 subclades such as glycine/serine prototrophy, mosaic glyoxylate shunt presence, and polyhydroxyalkanoate synthesis potential. Our analysis further shows metabolic flexibility among subgroups within IIIa. Additionally, we find that subclade IIIa.3 bridges the marine and freshwater clades based on its potential for compatible solute transport, iron utilization, and bicarbonate management potential. Pure culture experimentation validated differential salinity ranges in IIIa.1 and IIIa.3 and provided detailed IIIa cell size and volume data. This study is an important step forward for understanding the genomic, ecological, and physiological differentiation of subclade IIIa and the overall evolutionary history of SAR11.

On the origin of mitochondria: a multilayer network approach.

Backgound: The endosymbiotic theory is widely accepted to explain the origin of mitochondria from a bacterial ancestor. While ample evidence supports the intimate connection of Alphaproteobacteria to the mitochondrial ancestor, pinpointing its closest relative within sampled Alphaproteobacteria is still an open evolutionary debate. Many different phylogenetic methods and approaches have been used to answer this challenging question, further compounded by the heterogeneity of sampled taxa, varying evolutionary rates of mitochondrial proteins, and the inherent biases in each method, all factors that can produce phylogenetic artifacts. By harnessing the simplicity and interpretability of protein similarity networks, herein we re-evaluated the origin of mitochondria within an enhanced multilayer framework, which is an extension and improvement of a previously developed method. Methods: We used a dataset of eight proteins found in mitochondria (N = 6 organisms) and bacteria (N = 80 organisms). The sequences were aligned and resulting identity matrices were combined to generate an eight-layer multiplex network. Each layer corresponded to a protein network, where nodes represented organisms and edges were placed following mutual sequence identity. The Multi-Newman-Girvan algorithm was applied to evaluate community structure, and bifurcation events linked to network partition allowed to trace patterns of divergence between studied taxa. Results: In our network-based analysis, we first examined the topology of the 8-layer multiplex when mitochondrial sequences disconnected from the main alphaproteobacterial cluster. The resulting topology lent firm support toward an Alphaproteobacteria-sister placement for mitochondria, reinforcing the hypothesis that mitochondria diverged from the common ancestor of all Alphaproteobacteria. Additionally, we observed that the divergence of Rickettsiales was an early event in the evolutionary history of alphaproteobacterial clades. Conclusion: By leveraging complex networks methods to the challenging question of circumscribing mitochondrial origin, we suggest that the entire Alphaproteobacteria clade is the closest relative to mitochondria (Alphaproteobacterial-sister hypothesis), echoing recent findings based on different datasets and methodologies.