Biology subjects

Haro-Moreno, J. M.

Publications and source records attributed to Haro-Moreno, J. M..

15 recordsLinked to original sources

bioRxiv2026

Population dynamics of Pelagibacterales clonal lineages: ecological-consortia and frequency modulation

Pelagibacterales gMED is the dominant epipelagic genomospecies in the western Mediterranean Sea. We used the O-chain biosynthesis gene clusters, OBCs, as clonal barcodes to analyse strain-level population structure. In total, 385 OBC-defined clonal lineages were tracked across Mediterranean metagenomes spanning 14 years and depths from 5 to 90 m within the photic zone, with between 128 and 336 detected per metagenome. The relative conservation of dominant OBC types across years, seasons, and geographic locations indicated a persistently high and stable clonal diversity. Abundance distributions were log-normal in all metagenomes -- the canonical signature of bet-hedging portfolio dynamics. Co-varying consortia of clonal lineages were detected, identifying ecologically coherent guilds whose relative abundances fluctuate in concert across environmental gradients, particularly depth during the stratified season. Consortia membership showed no correlation with core-genome phylogeny, indicating convergent ecological adaptation independently of ancestry. Our work reveals another layer of complexity below the community level, which together with its modulation, provides the gMED population with a robust and versatile genetic repertoire for the degradation of dissolved organic matter under the fluctuating conditions of the epipelagic ocean. We have named this mechanistic model frequency-modulation (FM).

microbiology↗

bioRxiv2025

Type IV pili increase phage encounter in marine cyanobacteria

Type IV pili are extracellular bacterial appendages with diverse functions, including biofilm formation, motility, conjugation, transformation, pathogenicity, and secretion. In planktonic marine cyanobacteria, they also enhance buoyancy and protect against grazing, yet their consequences for the oceanic carbon pump and microbial loop remain poorly understood. Pili production in dilute marine ecosystems entails ecological trade-offs, including high nutrient investment and, as we show here, increased vulnerability to phage infection. Using high-resolution electron microscopy, we observed cyanophages attaching along the entire length of extracellular pili, indicating that these filaments act as structural amplifiers of viral encounter in diffusion-limited environments. While piliated and non-piliated Synechococcus exhibited similar mortality in isolation, competition assays revealed a strong selective disadvantage for piliated cells, which were more rapidly depleted in mixed populations. This effect was most pronounced for cyanophage S-CAM7, which displayed higher pili-binding affinity and drove competitive exclusion of piliated hosts. To extend these findings to natural settings, we analysed single-cell amplified genomes (SAGs) observing pili genes encoded by 14.7% of total cyanobacterial SAGs. This proportion increased to 28.1% in virally infected SAGs (virocells; representing 3.8% of the total cyanobacterial SAGs), consistent with encounter-driven susceptibility of piliated cells in the environment. Together, our results identify type IV pili as double-edged structures: while conferring ecological advantages, they expose cells to viral attack by increasing encounter probability when pilus-attached phage are transport to the cell surface during pili retraction. This fitness cost may help explain the patchy distribution of pili in natural populations and the extreme sequence variability of pilins, reflecting an ongoing evolutionary arms race with phage.

microbiology↗

bioRxiv2025

Structural basis for no retinal binding in flotillin-associated rhodopsins

Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins were reported to lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins) were identified and suggested to lack the typical retinal binding pocket despite preserving the lysine residue in many members of the group. Here we present cryo-EM structures of paralog FArhodopsin and proteorhodopsin from marine bacteria Pseudothioglobus. The structures revealed pentameric assemblies of both proteins similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Thus, our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of the FArhodopsin clade.

biophysics↗

bioRxiv2025

The Type IV Secretion System of Patescibacteria is homologous to the bacterial monoderm conjugation machinery

The Candidate Phyla Radiation, also known as Patescibacteria, represents a vast and diverse division of bacteria that has come to light via culture-independent "omics" technologies. Their limited biosynthetic capacity, along with evidence of their growth as obligate epibionts on other bacteria, suggests a broad reliance on host organisms for their survival. Nevertheless, our understanding of the molecular mechanisms governing their metabolism and lifestyle remains limited. The Type IV Secretion System (T4SS) represents a superfamily of translocation systems with a wide range of functional roles. T4SS genes have been identified in the CPR group Saccharibacteria as essential for their epibiotic growth. In this study, we used a comprehensive bioinformatics approach to investigate the diversity and distribution of T4SS within the Patescibacteria lineage. The phylogenetic analysis of the T4SS signature protein VirB4 suggests that most of these proteins cluster into a distinct monophyletic group with a shared ancestry to the MPFFATA class of T4SS. This class is found in the conjugative elements of Firmicutes, Actinobacteria, Tenericutes, and Archaea, indicating a possible horizontal gene transfer from these monoderm microorganisms to CPR bacteria. We identified additional T4SS components near virB4, particularly those associated with the MPFFATA class, as well as homologs of other T4SS classes, such as VirB2-like pilins, and observed their varied arrangements across different CPR phyla. The absence of a relaxase in most of these T4SS clusters suggests that the system has been co-opted for other functions in CPR bacteria. The proximity of T4SS components to the origin of replication (gene dnaA) in some CPR suggests a potential mechanism for increased expression. The broad ubiquity of a phylogenetically distinct T4SS in CPR, combined with its chromosomal location, underscores the significance of T4SS in the biology of Patescibacteria. Impact statementThe Candidate Phyla Radiation (CPR), or Patescibacteria, represents a highly diverse bacterial group that constitutes a significant fraction of the microbial dark matter. Known for their minimal biosynthetic capabilities and dependence on host bacteria, CPR bacteria exemplify fascinating yet poorly understood lifestyles. Our research sheds light on the molecular strategies underlying their survival by focusing on the Type IV Secretion System (T4SS), a versatile bacterial machinery typically involved in DNA transfer and effector molecule delivery. We revealed that T4SS is not only widespread in CPR but also forms a distinct evolutionary lineage, likely acquired via horizontal gene transfer from Gram-positive bacteria, and we illustrated its gene organization across different CPR phyla. Unlike canonical systems, CPR T4SS lacks key components for conjugation, hinting at a novel functional adaptation. Remarkably, T4SS genes in CPR are often located near the replication origin, suggesting a regulatory role linked to their expression. This research suggests the repurposing of T4SS in CPR to thrive under extreme reliance on other organisms. By unraveling the genomic and evolutionary peculiarities of T4SS in CPR, our work provides valuable insights into their unique biology, broadens our understanding of microbial diversity and innovation, and highlights this system as a strong candidate to sustain their parasitic episymbiotic lifestyle. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2024

Microdiversity in marine pelagic ammonia-oxidizing archaeal populations

The knowledge of the different population-level processes operating within a species, and the genetic variability of the individual prokaryotic genomes, is key to understanding the adaptability of microbial populations. Here, we characterized the flexible genome of ammonia-oxidizing archaeal (AOA) populations using a metagenomic recruitment approach and long-read (PacBio HiFi) metagenomic sequencing. In the lower photic zone of the western Mediterranean Sea, the genomes Nitrosopelagicus brevis CN25 and Nitrosopumilus catalinensis SPOT1 had the highest recruitment values among AOA. These two complete genomes were used to analyze the diversity of flexible genes (variable from strain to strain) by analyzing the long-reads located within the flexible genomic islands (fGIs) identified through their under-recruitment. Both AOA genomes had a large fGI involved in the glycosylation of exposed structures, highly variable and rich in glycosyltransferases. N. brevis had two fGIs related to the transport of phosphorus and ammonium respectively. N. catalinensis had fGIs involved in phosphorus transportation and metal uptake. A flexible genomic island (fGI5) previously reported as "unassigned function" in N. brevis could be associated with defense. These findings demonstrate that the microdiversity of marine microbe populations, including AOA, can be effectively characterized using an approach that incorporates new third-generation sequencing metagenomics.

genomics↗

bioRxiv2024

Diversity of the O-chain biosynthetic cluster in the Pelagibacterales

We have used single-amplified genomes (SAGs) and long-read metagenomics to examine the diversity of the O-chain polysaccharide biosynthesis cluster (OBC) in marine bacteria of the Pelagibacterales order. OBCs are notorious for their diversity and have been used to type strains in pathogens and saprophytes, but their patterns of variation in free-living bacteria are little known. We found that, for these marine heterotrophic bacteria, the diversity is comparable to that of saprophytes, such as Enterobacteriales i.e. nearly each strain carries a different OBC. However, although OBC inheritance was largely vertical, the existence of some shared clusters allowed a comparative analysis. The OBCs diverge along with the genome, which is taken as indicative of old horizontal gene transfer (HGT) events. Only 14 cases of recent HGT were detected and they happened independently of taxonomy or location. Thus, although the O-chain is a major phage receptor in Gram-negative bacteria, the exchange of the complete cluster seems to play a minor role in the phage-bacterium arms-race. By long-read metagenomics, we could detect 380 different OBCs in a single sampling site in the Mediterranean. A single population (single species and sample) of the endemic Ia.3/VII genomospecies had a set of 158 OBCs of which 130 were different. This large diversity in clonal lineages might reflect the large amount of metabolic pathways required to deal with the enormous chemical diversity of dissolved organic matter in the ocean.

microbiology↗

bioRxiv2023

Flotillin-Associated rhodopsin (FArhodopsin), a widespread paralog of proteorhodopsin in aquatic bacteria with streamlined genomes

Microbial rhodopsins are often found more than once in a single genome (paralogs) that often have different functions. We screened a large dataset of open ocean single-amplified genomes (SAGs) for co-occurrences of multiple rhodopsin genes. Many such cases were found among Pelagibacterales (SAR11), HIMB59 and the Gammaproteobacteria Pseudothioglobus SAGs. These genomes always had a bona fide proteorhodopsin and a separate cluster of genes containing a second rhodopsin associated with a predicted flotillin coding gene and have thus been named flotillin-associated rhodopsins (FArhodopsins). They are quite divergent from the other proteorhodopsin paralog and contain either DTT, DTL or DNI motives in their key functional amino acids. FArhodopsins are mainly associated with the lower layers of the epipelagic zone. All marine FArhodopsins had the retinal binding lysine, but we found their relatives in freshwater metagenomes that lack this key amino acid. Alfa-fold predictions of marine FArhodopsins indicate that their retinal pocket might be very reduced or absent, hinting that they are retinal-less (blind). Freshwater FArhodopsins were more diverse than marine FArhodopsins, but we could not determine if they are present as paralogs of other rhodopsins, due to the lack of SAGs or isolates. Although the function of FArhodopsins could not be established, their conserved genomic context indicated involvement in the formation of membrane microdomains. The conservation of FArhodopsins in diverse and globally abundant microorganisms suggests that they may be important in the adaptation to the twilight zone of aquatic environments. IMPORTANCERhodopsins have been shown to play a key role in the ecology of aquatic microbes. Here we describe a group of widespread rhodopsins in aquatic microbes associated with dim light conditions. Their characteristic genomic context found in both marine and freshwater environments indicates a novel potential involvement in membrane microstructure that could be important for the function of the co-existing proteorhodopsin proton pumps. The absence or reduction of the retinal binding pocket points to drastically different physiology. In addition to their ecological importance, novel rhodopsins have biotechnological potential in the nascent field of optogenetics.

microbiology↗

bioRxiv2022

Cluster-driven evolution and modularity uncover paths of cholera emergence

Cholera, an acute secretory diarrhea, is caused by strains from a phylogenetically confined group within the Vibrio cholerae species, the pandemic cholera group (PCG). To date, the molecular and evolutionary factors that enable the isolated emergence of toxigenic V. cholerae from environmental populations remain mostly enigmatic. Comprehensive analyses of over 1,100 V. cholerae genomes, including novel environmental isolates from this study, reveal that the species consists of four major clades and several minor ones. PCG belongs to a large clade located within a lineage shared with environmental strains, the pandemic cholera lineage. This hierarchical classification provided us with a framework to unravel the eco-evolutionary dynamics of the genetic determinants associated with the emergence of toxigenic V. cholerae. Our analyses indicate that this phenomenon is largely dependent on the acquisition of unique modular gene clusters and allelic variations that confer a competitive advantage during intestinal colonization. We determined that certain PCG-associated alleles are essential for successful colonization whereas others provide a non-linear competitive advantage, acting as a critical bottleneck that elucidates the isolated emergence of PCG. For instance, toxigenic strains encoding non-PCG alleles of a) tcpF or b) a sextuple allelic exchange mutant for genes tcpA, toxT, VC0176, VC1791, rfbT and ompU, lose their ability to colonize the intestine. Interestingly, these alleles do not play a role in the colonization of model environmental reservoirs. Our study uncovers the evolutionary roots of toxigenic V. cholerae and offers a tractable approach for investigating the emergence of pathogenic clones within an environmental population. SIGNIFICANCEThe underlying factors that lead to specific strains within a species to emerge as human pathogens remain mostly enigmatic. Toxigenic clones of the cholera agent, Vibrio cholerae, are encompassed within one phylogenomic clade, the pandemic cholera group (PCG). Here, we investigate the molecular and evolutionary factors that explain the confined nature of this group. Our analyses determined that the emergence of PCG is largely dependent on the acquisition of unique modular gene clusters and allelic variations that confer a competitive advantage during intestinal colonization. These allelic variations act as a critical bottleneck that elucidates the isolated emergence of PCG and provides a tractable blueprint for the study of the emergence of pathogenic clones within an environmental population.

microbiology↗

bioRxiv2021

Phylogenomics of SAR116 clade reveals two subclades with different evolutionary trajectories and important role in the ocean sulfur cycle

The SAR116 clade within the class Alphaproteobacteria represents one of the most abundant groups of heterotrophic bacteria inhabiting the surface of the ocean. The small number of cultured representatives of SAR116 (only two to date) is a major bottleneck that has prevented an in-depth study at the genomic level to understand the relationship between genome diversity and its role in the marine environment. In this study, we use all publicly available genomes to provide a genomic overview of the phylogeny, metabolism and biogeography within the SAR116 clade. This increased genomic diversity revealed has led to the discovery of two subclades of SAR116 that, despite having similar genome size (ca. 2.4 Mb) and coexist in the same environment, display different properties in their genomic make up. One represents a novel subclade for which no pure cultures have been isolated and is composed mainly of single-amplified genomes (SAGs). Genomes within this subclade showed convergent evolutionary trajectories with more streamlining features, such as low GC content (ca. 30%), short intergenic spacers (<22 bp) and strong purifying selection (low dN/dS). Besides, they were more abundant in metagenomic databases recruiting also at the deep chlorophyll maximum. Less abundant and restricted to the upper photic layers of the global ocean, the other subclade of SAR116, enriched in MAGs, accommodated the only two pure cultures. Genomic analysis suggested that both clades have a significant role in the sulfur cycle with differences in the way in which both clades can metabolize the dimethylsulfoniopropionate (DMSP). IMPORTANCESAR116 clade of Alphaproteobacteria is an ubiquitous group of heterotrophic bacteria inhabiting the surface of the ocean, but the information about their ecology and population genomic diversity is scarce due to the difficulty of getting pure culture isolates. The combination of single-cell genomics and metagenomics has become an alternative approach to study this kind of microbes. Our results expand the understanding of the genomic diversity, distribution, and lifestyles within this clade and provide evidence of different evolutionary trajectories in the genome make-up of the two subclades that could serve to understand how evolutionary pressure can drive different adaptations to the same environment. Therefore, the SAR116 clade represents an ideal model organism for the study of the evolutionary streamlining of genomes in microbes that have relatively close relatedness to each other.

microbiology↗

bioRxiv2020

Nasopharyngeal microbial communities of patients infected with SARS-COV-2 that developed COVID-19.

BackgroundSARS-CoV-2 is an RNA virus causing COVID-19. The clinical characteristics and epidemiology of COVID-19 have been extensively investigated, however studies focused on the patients microbiota are still lacking. In this study, we investigated the nasopharyngeal microbiome composition of patients who developed different severity levels of COVID-19. We performed Rdna-SSU (16S) sequencing from nasopharyngeal swab samples obtained from SARS-CoV-2 positive (56) and negative (18) patients in the province of Alicante (Spain) in their first visit to the hospital. Positive SARS-CoV-2 patients were observed and later categorized in mild (symptomatic without hospitalization), moderate (hospitalization) and severe (admission to ICU). We compared the microbiome diversity and OTU composition among severity groups using Similarity Percentage (SIMPER) analysis and Maaslin2. We also built bacterial co-abundance networks for each group using Fastpar. ResultsStatistical analysis indicated differences in the nasopharyngeal microbiome of COVID19 patients. 62 OTUs were found exclusively in SARS-CoV-2 positive patients, mostly classified as members of the phylum Bacteroidetes (18) and Firmicutes (25). OTUs classified as Prevotella were found to be significantly more abundant in patients that developed more severe COVID-19. Furthemore, co-abundance analysis indicated a loss of network complexity among samples from patients that later developed more severe symptoms. ConclusionsOur preliminary study shows that the nasopharyngeal microbiome of COVID-19 patients showed differences in the composition of specific OTUs and complexity of co-abundance networks. These microbes with differential abundances among groups could serve as biomarkers for COVID-19 severity. Nevertheless, further studies with larger sample sizes should be conducted to validate these results. IMPORTANCEThis work has studied the microbiota of the nasopharyngeal tract in COVID19 patients using advanced techniques of molecular microbiology. Diverse microorganisms, most of which are harmless or even beneficial to the host, colonize the nasopharyngeal tract. These microorganisms are the microbiota, and they are present in every people. However, changes in this microbiota could be related to different diseases as cancer, gastrointestinal pathologies or even COVID19. This study has been performed to investigate the microbiota from patients with COVID19, in order to determinate its implication in the pathology severity. The results obtained showed that it is possible that several specific microorganisms are present only in patients with severe COVID19. These data, could be used as a prognostic biomarker to early detect whose patients will develop a severe COVID19 and improve their clinical management.

microbiology↗

bioRxiv2020

Long read metagenomics, the next step?

BackgroundThird-generation sequencing has penetrated little in metagenomics due to the high error rate and dependence for assembly on short-read designed bioinformatics. However, 2nd generation sequencing metagenomics (mostly Illumina) suffers from limitations, particularly in allowing assembly of microbes with high microdiversity or retrieving the flexible (adaptive) compartment of prokaryotic genomes. ResultsHere we have used different 3rd generation techniques to study the metagenome of a well-known marine sample from the mixed epipelagic water column of the winter Mediterranean. We have compared Oxford Nanopore and PacBio last generation technologies with the classical approach using Illumina short reads followed by assembly. PacBio Sequel II CCS appears particularly suitable for cellular metagenomics due to its low error rate. Long reads allow efficient direct retrieval of complete genes (473M/Tb) and operons before assembly, facilitating annotation and compensates the limitations of short reads or short-read assemblies. MetaSPAdes was the most appropriate assembly program when used in combination with short reads. The assemblies of the long reads allow also the reconstruction of much more complete metagenome-assembled genomes, even from microbes with high microdiversity. The flexible genome of reconstructed MAGs is much more complete and allows rescuing more adaptive genes. ConclusionsFor most applications of metagenomics, from community structure analysis to ecosystem functioning, long-reads should be applied whenever possible. Particularly for in-silico screening of biotechnologically useful genes, or population genomics, long-read metagenomics appears presently as a very fruitful approach and can be used from raw reads, before a computing-demanding (and potentially artefactual) assembly step.

microbiology↗

bioRxiv2020

Microbiome of the Black Sea water column analyzed by genome centric metagenomics

BackgroundThe Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former albeit with lower salinity and (mostly) temperature. In spite of its well-known hydrology and physico chemistry, this enormous water mass remains poorly studied at the microbial genomics level. ResultsWe have sampled its different water masses and analyzed the microbiome by classic and genome-resolved metagenomics generating a large number of metagenome-assembled genomes (MAGs) from them. The oxic zone presents many similarities to the global ocean while the euxinic water mass has similarities to other similar aquatic environments of marine or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environments and includes Cyanobacteria (Synechococcus), photoheterotrophs (largely with marine relatives), facultative/microaerophilic microbes again largely marine, chemolithotrophs (N and S oxidizers) and a large number of anaerobes, mostly sulfate reducers but also a few methanogens and a large number of "dark matter" streamlined genomes of largely unpredictable ecology. ConclusionsThe Black Sea presents a mixture of similarities to other water bodies. The photic zone has many microbes in common with that of the Mediterranean with the relevant exception of the absence of Prochlorococcus. The chemocline already presents very different characteristics with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. Finally the euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy generating metabolism and a small but detectable methanogenesis.We are adding critical information about this unique and important ecosystem and its microbiome.

microbiology↗

bioRxiv2020

Staphylococcus sciuri C2865 from a distinct subspecies cluster as reservoir of the novel transferable trimethoprim resistance gene, dfrE, and adaptation driving mobile elements

Four methicillin-resistant Staphylococcus sciuri (MRSS) strains isolated from stranded dogs showed trimethoprim (TMP) resistance, while all staphylococcal TMP resistant dihydrofolate reductase genes (dfr) were negative. An in-depth whole-genome-sequencing approach on strain C2865 was followed for resistome and mobilome profiling, and for comparative genomics with S. sciuri group available genomes. Lack of species host tropism was observed, with MRSS C2865 placed at a separate sub-branch within S. sciuri species, close to the average nucleotide identity to be considered a different species (95-96%). S. sciuri proved a pronounced accessory genome (73% of genes), while MRSS C2865 distinctively harboured the highest total gene number and highest number of unique genes, with 75% associated to the recognised mobilome. A novel multidrug resistance mosaic plasmid (pUR2865-34) with several adaptive, mobilization (oriT mimic) and segregational stability (Type Ib par system) traits and two small single resistance plasmids were identified. Plasmid pUR2865-34 enclosed a novel staphylococcal TMP resistance gene, named dfrE, which shared highest identity with dfr of soil-related Paenibacillus anaericanus (68%). DfrE conferred high-level TMP resistance in S. aureus and Escherichia coli. Database searches revealed that dfrE was formerly denoted (dfr_like) in an Exiguobacterium spp. from a fish-farm sediment and that was present but unnoticed in several staphylococcal and onemacrococcal genomes with different epidemiological backgrounds. Novel chromosomal site-specific mobile islands with resourceful traits were identified, including a multidrug-resistant SCCmec cassette lacking cassette chromosome recombinase (Ccr) genes, a staphylococcal pathogenicity island of the SaPI4 family, and three unrelated siphoviridae prophages, two of which enclosed recombinases with the conserved Ccr-motif. We reveal a novel staphylococcal TMP resistance dfrE gene already present in diverse bacterial backgrounds. We confirm the ubiquity, high genome plasticity and low host tropism of S. sciuri highlighting its role as a resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability. Author summaryStaphylococcus spp. are ubiquitous bacteria present in diverse ecological niches, including humans, animals and the environment. They are clinically relevant opportunistic pathogens and are notorious for their ability to acquire antimicrobial resistance (AMR) and virulence properties, resulting in a significant impact for Public Health. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information across bacterial species. Staphylococcus sciuri represents one of the most ancestral species in the genus and has been suggested a reservoir for AMR genes. Here, following a refined whole genome sequencing approach we determined the entire genome of an animal and environment-associated multidrug resistant (MDR) S. sciuri strain uncovering a novel acquired staphylococcal TMP resistance gene already spread among different bacterial species from different epidemiological backgrounds. We also reveal several additional MGEs, including a novel MDR mobilizable plasmid that encloses several adaptive and stabilization features, and novel mobilizable chromosomal islands with resourceful traits, including three unrelated prophages. Together with comparative genomics, we confirm the ubiquity, high intraspecies heterogenicity, genome plasticity and low host tropism of this species, highlighting its role as resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability.

microbiology↗

bioRxiv2019

Uncovering a hidden diversity: optimized protocols for the extraction of bacteriophages from soil

BackgroundBacteriophages are the most numerous biological entities on earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil samples will shed light not only on the yet largely unknown phage diversity, but also may result in novel insights into phage biology and functioning. Unfortunately, the study of soil viromes lags far behind any other ecological model system, due to the heterogeneous soil matrix that rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is therefore a fundamental prerequisite for replicable results and comparative virome studies.\n\nResultsWe here report the optimization of protocols for extraction of bacteriophage DNA from soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with a viral community consisting of phages from different families (106 PFU/g soil): Listeria phage {Phi}A511 (Myovirus), Staphylococcus phage {Phi}2638A{Delta}LCR (Siphovirus), and Escherichia phage {Phi}T7 (Podovirus). The efficacy of bacteriophage (i) elution, (ii) filtration, (iii) concentration, and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for non-amplification dependent sequencing and low 16S rRNA gene contamination levels ([≤] 0.2 {per thousand}). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65 %. In addition, 468 novel circularized soil phage genomes in size up to 235 kb were obtained from over 29,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity.\n\nConclusionWe have shown a dramatically enhanced extraction of the soil phage community by protocol optimization that has proven robustness in both a culture-depended as well as through metaviromic analysis. Our huge data set of manually curated soil viral contigs roughly doubles the amount of currently available soil virome data, and provide insights into the yet largely undescribed soil viral sequence space.

microbiology↗

bioRxiv2017

Fine Stratification Of Microbial Communities Through A Metagenomic Profile Of The Photic Zone

Most marine metagenomic studies of the marine photic zone analyze only samples taken at one or two depths. However, when the water column is stratified, physicochemical parameters change dramatically over relatively short depth intervals. We sampled the photic water column every 15m depth at a single point of an off-shore Mediterranean site during a period of strong stratification (early autumn) to evaluate the effects of small depth increases on the microbiome. Using genomic assembly and metagenomic read recruitment, we found major shifts in the community structure over small variations of depth, with most microbes showing a distribution limited to layers approximately 30 meters thick (stenobathic). Only some representatives of the SAR11 clade and the Sphingomonadaceae appeared to be eurybathic, spanning a greater range of depths. These results were confirmed by studying a single gene (rhodopsin) for which we also found narrow depth distributions. Our results highlight the importance of considering vertical distribution as a major element when analyzing the presence of marine clades and species or comparing the microbiome present at different locations.

microbiology↗