Search

Estuaries provide many ecosystem services and host a majority of the world’s population. Here, the response of microbial communities after a record-breaking flood event in a highly urbanized estuary was followed. Hurricane Harvey (hereafter Harvey) was a category 4 hurricane that made landfall on the Texas coast in 2017 and lashed the Houston area with 1.4–1.7 × 1010 m3 of rainfall, disrupting the natural gradients of nutrients and salinity. Here, we utilized metagenomics to analyze how Harvey altered the microbial community of Galveston Bay over five weeks following the storm. We hypothesized that the community would shift from a marine dominated community to that of a terrestrial and freshwater origin. We found that following the storm there were changes in the distribution of species with specific metabolic capacities, such as Cyanobacteria, enriched in oxygenic photosynthesis and nitrogen fixation genes, as well as Verrucomicrobia and Betaproteobacteria, with high prevalence of the SOX complex and anoxygenic photosynthesis genes. On the other hand, dominant members of the community with more diverse metabolic capabilities showed less fluctuations in their distribution. Our results highlight how massive precipitation disturbances can alter microbial communities and how the coalescence of diverse microorganisms creates a resilient community able to maintain ecosystem services even when the system is in an altered state.

Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared >99.2% average nucleotide identity, low frequency of SNP’s, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.

Hurricane Harvey was the wettest hurricane in US history bringing record rainfall and widespread flooding in Houston, TX. The resulting storm- and floodwaters largely emptied into the Galveston Bay. Surface water was collected from 10 stations during five cruises to investigate the concentrations and sources of 16 priority polycyclic aromatic hydrocarbons (PAHs), and relative abundances of PAH-degrading bacteria. Highest PAH levels (102-167 ng/L) were detected during the first sampling event, decreasing to 36-69 ng/L within a week. Four sites had elevated concentrations of carcinogenic benzo[a]pyrene that exceeded the Texas Standard for Surface Water threshold. The highest relative abundances of known PAH-degrading bacteria Burkholderiaceae, Comamonadaceae, and Sphingomonadales were detected during the first and second sampling events. PAH origins were about 60% pyrogenic, 2% petrogenic, and the remainder of mixed sources. This study improves our understanding on the fate, source, and distributions of PAHs in Galveston Bay after an extreme flooding event.

Prokaryotes constitute the majority of sedimentary biomass, where they cycle organic carbon and regulate organic matter transformation. The microbes inhabiting sediment are diverse and the factors controlling microbial community composition are not fully understood. Here, we characterized the prokaryotic community using 16S rRNA gene sequencing in 24 stratigraphic layers within a 89 cm (dated to ~1900 years old) sediment core from an anchialine sinkhole in the Bahamas with a stratified water column and anoxic bottom water. The microbial community was dominated by members of the Alphaproteobacteria, Dehalococcoidia, Gammaproteobacteria, Bathyarchaeota, and Campylobacter classes. Most interestingly, subsurface microbial community structure could be correlated to previous evidence for timewise changes in the main source of organic matter that was supplied to the sediment accumulating during the last 2000 years, which itself was caused by regional terrestrial vegetation changes. The C:N ratio was correlated to the relative abundance of the microbial classes, and the microbial communities followed three previously determined time periods based on the source of organic matter, which suggests that the carbon source at time of deposition influences the resultant subsurface microbial community composition. These results show that carbon source is a driver of the microbial community composition inhabiting anoxic sediment, which could have implications for improving understanding of carbon cycling in coastal sedimentary basins.

A simple, pervasive biological entity in the ocean sheds light on evolution

Microbial interactions influence nearly one-half of the global biogeochemical flux of major elements of the marine ecosystem. Despite their ecological importance, microbial interactions remain poorly understood and even less is known regarding the effects of anthropogenic perturbations on these microbial interactions. The Deepwater Horizon oil spill exposed the Gulf of Mexico to ∼4.9 million barrels of crude oil over 87 days. We determined the effects of oil exposure on microbial interactions using short- and long-term microcosm experiments with and without Macondo surrogate oil. Microbial activity determined using radiotracers revealed that oil exposure negatively affected substrate uptake by prokaryotes within 8 h and by eukaryotes over 72 h. Eukaryotic uptake of heterotrophic exopolymeric substances (EPS) was more severely affected than prokaryotic uptake of phototrophic EPS. In addition, our long-term exposure study showed severe effects on photosynthetic activity. Lastly, changes in microbial relative abundances and fewer co-occurrences among microbial species were mostly driven by photosynthetic activity, treatment (control vs. oil), and prokaryotic heterotrophic metabolism. Overall, oil exposure affected microbial co-occurrence and/or interactions possibly by direct reduction in abundance of one of the interacting community members and/or indirect by reduction in metabolism (substrate uptake or photosynthesis) of interacting members.

Extreme weather events such as tropical storms and hurricanes deliver large amounts of freshwater (stormwater and river discharge) and associated dissolved organic carbon (DOC) to estuaries and the coastal ocean, affecting water quality, and carbon budgets. Hurricane Harvey produced an unprecedented 1000-year flood event in 2017 that inundated the heavily urbanized and industrialized Houston/Galveston region (TX, United States). Within a week, storm-associated floodwater delivered 87 +/- 18 Gg of terrigenous dissolved organic carbon (tDOC) to Galveston Bay and the Gulf of Mexico continental shelves. In situ decay constants of 8.75-28.33 year(-1) resulted in the biomineralization of similar to 70% of tDOC within 1 month of discharge from the flood plain. The high removal efficiency of tDOC was linked to a diverse microbial community capable of degrading a wide repertoire of dissolved organic matter (DOM), and suggested hurricane-induced flood events affect net CO2 exchange and nutrient budgets in estuarine watersheds and coastal seas.

Hurricane Harvey (category four storm) made landfall along the coast of Texas (United States) and then stalled out over Texas and Louisiana, releasing 1.29 × 1011 m3 of precipitation over 5 days. This caused extensive flooding that elevated freshwater river discharge and land runoff into Galveston Bay and eventually into the Gulf of Mexico. The floodwaters delivered a significant influx of dissolved organic carbon (DOC), organic pollutants and nutrients along with terrestrial and freshwater associated microbes. Over the 24 days following the flooding event, samples were collected on five cruises across Galveston Bay from the mouth of the San Jacinto River (Houston, TX, United States) to the Gulf of Mexico. Parameters measured for this study include: water quality (temperature, salinity, pH, and dissolved oxygen), nutrients (NO3–, NO2–, NH4+, Pi, and HSiO3–), polycyclic aromatic hydrocarbons (PAHs), pharmaceuticals (cotinine, carbamazepine, carbamazepine-epoxide, and prednisone), biocide (imidacloprid), DOC, bacteria, and the eukaryotic community composition (16S and 18S rRNA genes). In the week after the flood event, bay-wide salinities decreased to 0–5 compared to the higher pre-Harvey salinities of 20–30 (recorded 5 days before the flood). Water treatment facilities and petrochemical plants were compromised due to the heavy flooding in the region. Increased concentrations of DOC, nutrients, PAHs, pharmaceuticals, and biocides were observed across Galveston Bay immediately following the storm. During the 4 weeks after Harvey, concentrations of DOC, nutrients, and organic pollutants began to decrease coinciding with rising salinities as the freshwater was flushed into the Gulf of Mexico and seawater began moving back into the Bay. Successive blooms of Chlorophyta, diatoms (Bacillariophyta), and dinoflagellates (Dinophyta) occurred similar to post-storm communities from past hurricanes that have impacted estuarine systems along the Gulf of Mexico. The bacterial community showed an increase in the abundance of bacteria associated with terrestrial soils and freshwater at the initial time point and then decreased over time and were replaced by their estuarine and marine relatives within the month following the hurricane. The eukaryotic community changed substantially following Harvey and did not recover to pre-Harvey conditions during our study period, suggesting a longer recovery time compared to the prokaryotes. Although the water quality parameters and prokaryotic community showed signs of returning to pre-Harvey conditions within the month following the flood event, long-term impacts need to be measured in the years following the flood.

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere., Nature Biotechnology, 37 (1), ISSN:1546-1696, ISSN:1087-0156

12 pages, 4 figures, 1 table, supplementary material https://www.frontiersin.org/articles/10.3389/fmicb.2020.524828/full#supplementary-material.-- The datasets generated for this study can be found in the OSF https://osf.io/7pm3u/, doi: 10.17605/OSF.IO/7PM3U. Protist SAG assemblies are available through NCBI Bioproject PRJNA655200, The predominant model of the role of viruses in the marine trophic web is that of the “viral shunt,” where viral infection funnels a substantial fraction of the microbial primary and secondary production back to the pool of dissolved organic matter. Here, we analyzed the composition of non-eukaryotic DNA associated with individual cells of small, planktonic protists in the Gulf of Maine (GoM) and the Mediterranean Sea. We found viral DNA associated with a substantial fraction cells from the GoM (51%) and the Mediterranean Sea (35%). While Mediterranean SAGs contained a larger proportion of cells containing bacterial sequences (49%), a smaller fraction of cells contained bacterial sequences in the GoM (19%). In GoM cells, nearly identical bacteriophage and ssDNA virus sequences where found across diverse lineages of protists, suggesting many of these viruses are non-infective. The fraction of cells containing viral DNA varied among protistan lineages and reached 100% in Picozoa and Choanozoa. These two groups also contained significantly higher numbers of viral sequences than other identified taxa. We consider mechanisms that may explain the presence of viral DNA in protistan cells and conclude that protistan predation on free viral particles contributed to the observed patterns. These findings confirm prior experiments with protistan isolates and indicate that the viral shunt is complemented by a viral link in the marine microbial food web. This link may constitute a sink of viral particles in the ocean and has implications for the flow of carbon through the microbial food web, This work was supported by the NSF awards OCE-0623288, OCE-1335810, and OIA-1826734 to RS as well as by the projects INTERACTOMICS (CTM2015-69936-P, MINECO/FEDER, Spain) and MicroEcoSystems (Research Council of Norway 240904, Norway) to RL, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Hurricane Harvey was the wettest hurricane in US history bringing record rainfall and widespread flooding in Houston, TX. The resulting storm- and floodwaters largely emptied into the Galveston Bay. Surface water was collected from 10 stations during five cruises to investigate the concentrations and sources of 16 priority polycyclic aromatic hydrocarbons (PAHs), and relative abundances of PAH-degrading bacteria. Highest PAH levels (102-167 ng/L) were detected during the first sampling event, decreasing to 36-69 ng/L within a week. Four sites had elevated concentrations of carcinogenic benzo[a]pyrene that exceeded the Texas Standard for Surface Water threshold. The highest relative abundances of known PAH-degrading bacteria Burkholderiaceae, Comamonadaceae, and Sphingomonadales were detected during the first and second sampling events. PAH origins were about 60% pyrogenic, 2% petrogenic, and the remainder of mixed sources. This study improves our understanding on the fate, source, and distributions of PAHs in Galveston Bay after an extreme flooding event.

Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds.

Volcanic arcs are the surface expression of magmatic systems that result from subduction of mostly oceanic lithosphere at convergent plate boundaries. Arcs with a submarine component include intraoceanic arcs and island arcs that span almost 22,000 km on Earth’s surface, and the vast majority of them are located in the Pacific region. Hydrothermal systems hosted by submarine arc volcanoes commonly contain a large component of magmatic fluid. This magmatic-hydrothermal signature, coupled with the shallow water depths of arc volcanoes and their high volatile contents, strongly influences the chemistry of the fluids and resulting mineralization and likely has important consequences for the biota associated with these systems. The high metal content and very acidic fluids in these hydrothermal systems are thought to be important analogs to numerous porphyry copper and epithermal gold deposits mined today on land. During International Ocean Discovery Program (IODP) Expedition 376 (5 May–5 July 2018), a series of five sites was drilled on Brothers volcano in the Kermadec arc. The expedition was designed to provide the missing link (i.e., the third dimension) in our understanding of hydrothermal activity and mineral deposit formation at submarine arc volcanoes and the relationship between the discharge of magmatic fluids and the deep biosphere. Brothers volcano hosts two active and distinct hydrothermal systems: one is seawater influenced and the other is affected by magmatic fluids (largely gases). In total, 222.4 m of volcaniclastics and lavas were recovered from the five sites drilled, which include Sites U1527 and U1530 in the Northwest (NW) Caldera seawater-influenced hydrothermal field; Sites U1528 and U1531 in the magmatic fluid-influenced hydrothermal fields of the Upper and Lower Cones, respectively; and Site U1529, located within an area of low crustal magnetization that marks the West (W) Caldera upflow zone on the caldera floor. Downhole logging and borehole fluid sampling were completed at two sites, and two tests of a prototype turbine-driven coring system (designed by the Center for Deep Earth Exploration [CDEX] at Japan Agency for Marine-Earth Science and Technology [JAMSTEC]) for drilling and coring hard rocks were conducted. Core recovered from all five sites consists of dacitic volcaniclastics and lava flows with only limited chemical variability relative to the overall range in composition of dacites in the Kermadec arc. Pervasive alteration with complex and variable mineral assemblages attest to a highly dynamic hydrothermal system. The upper parts of several drill holes at the NW Caldera hydrothermal field are characterized by secondary mineral assemblages of goethite + opal + zeolites that result from low-temperature (

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus-host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus-host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage-host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host-virus interactions in complex microbial communities.

The original version of this Article contained errors in the units of concentration of three reagents listed in the Methods. These errors have all been corrected in both the PDF and HTML versions of the Article.

Microbial communities living in deeply buried sediment may be adapted to long-term energy limitation as they are removed from new detrital energy inputs for thousands to millions of years. However, sediment layers near the underlying oceanic crust may receive inputs from below that influence microbial community structure and/or activity. As part of the Census of Deep Life, we used 16S rRNA gene tag pyrosequencing on DNA extracted from a spectrum of deep sediment-basement interface samples from the subsurface of the Juan de Fuca Ridge flank (collected on IODP Expedition 327) to examine this possible basement influence on deep sediment communities. This area experiences rapid sedimentation, with an underlying basaltic crust that hosts a dynamic flux of hydrothermal fluids that diffuse into the sediment. Chloroflexi sequences dominated tag libraries in all sediment samples, with variation in the abundance of other bacterial groups (e.g., Actinobacteria, Aerophobetes, Atribacteria, Planctomycetes, and Nitrospirae). These variations occur in relation to the type of sediment (clays versus carbonate-rich) and the depth of sample origin, and show no clear connection to the distance from the discharge outcrop or to basement fluid microbial communities. Actinobacteria-related sequences dominated the basalt libraries, but these should be viewed cautiously due to possibilities for imprinting from contamination. Our results indicate that proximity to basement or areas of seawater recharge is not a primary driver of microbial community composition in basal sediment, even though fluids diffusing from basement into sediment may stimulate microbial activity.

Sinking marine oil snow was found to be a major mechanism in the transport of spilled oil from the surface to the deep sea following the Deepwater Horizon (DwH) oil spill. Marine snow formation is primarily facilitated by extracellular polymeric substances (EPS), which are mainly composed of proteins and carbohydrates secreted by microorganisms. While numerous bacteria have been identified to degrade oil, there is a paucity of knowledge on bacteria that produce EPS in response to oil and Corexit exposure in the northern Gulf of Mexico (nGoM). In this study, we isolated bacteria from surface water of the nGoM that grow on oil or Corexit dispersant. Among the 100 strains isolated, nine were identified to produce remarkable amounts of EPS. 16S rRNA gene analysis revealed that six isolates (strains C1, C5, W10, W11, W14, W20) belong to the genus Alteromonas; the others were related to Thalassospira (C8), Aestuariibacter (C12), and Escherichia (W13a). The isolates preferably degraded alkanes (17-77%), over polycyclic aromatic hydrocarbons (0.90-23%). The EPS production was determined in the presence of a water accommodated fraction (WAF) of oil, a chemical enhanced WAF (CEWAF), Corexit, and control. The highest production of visible aggregates was found in Corexit followed by CEWAF, WAF, and control; indicating that Corexit generally enhanced EPS production. The addition of WAF and Corexit did not affect the carbohydrate content, but significantly increased the protein content of the EPS. On the average, WAF and CEWAF treatments had nine to ten times more proteins, and Corexit had five times higher than the control. Our results reveal that Alteromonas and Thalassospira, among the commonly reported bacteria following the DwH spill, produce protein rich EPS that could have crucial roles in oil degradation and marine snow formation. This study highlights the link between EPS production and bacterial oil-degrading capacity that should not be overlooked during spilled oil clearance.

The distribution of viral genotypes in the ocean and their evolutionary relatedness remain poorly constrained. This paper presents data on the genetic diversity and evolutionary relationships of 1.2-kb DNA polymerase ( pol ) gene fragments from podoviruses. A newly designed set of PCR primers was used to amplify DNA directly from coastal sediment and water samples collected from inlets adjacent to the Strait of Georgia, British Columbia, Canada, and from the northeastern Gulf of Mexico. Restriction fragment length polymorphism analysis of 160 cloned PCR products revealed 29 distinct operational taxonomic units (OTUs), with OTUs within a site typically being more similar than those among sites. Phylogenetic analysis of the DNA pol gene fragments demonstrated high similarity between some environmental sequences and sequences from the marine podoviruses roseophage SIO1 and cyanophage P60, while others were not closely related to sequences from cultured phages. Interrogation of the CAMERA database for sequences from metagenomics data demonstrated that the amplified sequences were representative of the diversity of podovirus pol sequences found in marine samples. Our results indicate high genetic diversity within marine podovirus communities within a small geographic region and demonstrate that the diversity of environmental polymerase gene sequences for podoviruses is far more extensive than previously recognized.

Metagenomic studies have revealed that ssDNA phages from the family Microviridae subfamily Gokushovirinae are widespread in aquatic ecosystems. It is hypothesized that gokushoviruses occupy specialized niches, resulting in differences among genotypes traversing water column gradients. Here, we use degenerate primers that amplify a fragment of the gene encoding the major capsid protein to examine the diversity of gokushoviruses in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia. Amplicon sequencing of samples from the mixed oxic surface (10 m) and deeper anoxic (200 m) layers indicated a diverse assemblage of gokushoviruses, with greater richness at 10 m than 200 m. A comparison of amplicon sequences with sequences selected on the basis of RFLP patterns from eight surface samples collected over a one-year period revealed that gokushovirus diversity was higher in spring and summer during stratification, and lower in fall and winter after deep-water renewal, consistent with seasonal variability within gokushovirus populations. Phylogenetic analysis of clustered amplicons revealed at least five new phylogenetic clades of previously unknown sequences, with the most abundant group associated with viruses that infect SUP05, a ubiquitous and abundant member of marine oxygen minimum zones. Our results provide persuasive evidence that, while specific gokushovirus genotypes may have a narrow host range, hosts for gokushoviruses in Saanich Inlet consist of a wide range of bacterial taxa, including SUP05, a taxonomic clade of gamma proteobacterial sulfur oxidizers. Members of SUP05 are abundant in Saanich Inlet and involved in carbon, nitrogen, and sulfur cycling along the redoxline; thus, gokushoviruses are likely important mortality agents of these bacteria and have consequent influences on biogeochemical cycling in this system.

A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a three km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32 % of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

The Streptococcus thermophilus virulent pac -type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos - and pac -type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization—time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac - and cos -type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos -type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed.

Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus . First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (dif SL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine dif SL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when dif SL sites are located on chromosome dimers. Moreover, the XerS/dif SL recombination requires the streptococcal protein FtsKSL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs., Author Summary In bacteria, genetic information is mainly carried by a single circular chromosome. The replication of this circular molecule sometimes leads to the formation of a chromosome dimer unable to segregate in the daughter cells during the division process. In the bacterial model E. coli, chromosome dimers are resolved in monomers by site-specific recombination: two recombinases (XerC and XerD) cooperatively catalyze the recombination at a chromosomal site (dif), located at the junction of the two replication arms. This recombination is intimately coupled to cell division by the control of the septal protein FtsK. Xer recombination machinery as described in E. coli appears highly conserved among bacterial species. We show by comparative genomics and genetic studies that Streptococci use an alternative Xer recombination system, renamed XerS/dif SL, which is composed of a single recombinase phylogenetically unrelated to XerCD proteins and a noncanonical dif site. We also demonstrate that the streptococcal FtsK protein localizes at the division septum and operates the XerS/dif SL recombination. This is the first identification of an alternative Xer recombination system in prokaryotes to out knowledge, which might indicate that other unusual chromosome dimer resolution systems could exist in bacterial phyla where a canonical dif site is not detected.

Bacterial antagonists of Helminthosporium solani were submitted to different tests in order to determine the role of antibiosis in their antagonistic interaction. Among the bacterial strains tested, seven (Alcaligenes piechaudii, Aquaspirillum autotrophicum, Cellulomonas fimi, Pseudomonas chlororaphis, Pseudomonas putida (strains 94-19 and E-30) and Streptomyces griseus) were shown to produce agar diffusible metabolites inhibiting H. solani mycelial growth and/or conidial germination. Differential activity was revealed when diffusible metabolites were extracted from either pure cultures of these antagonists or dual culture in the presence of H. solani. The results presented indicate that the methodology employed could be a decisive factor in whether or not antibiosis can be identified as a mode of action of biocontrol agents., Des antagonistes bactériens envers Helminthosporium solani ont été soumis à différents essais en vue de déterminer le rôle de l'antibiose dans l'antagonisme observé. Parmi les souches bactériennes évaluées, sept (Alcaligenes piechaudii, Aquaspirillum autotrophicum, Cellulomonas fimi, Pseudomonas chlororaphis, Pseudomonas putida (souches 94-19 et E-30) et Streptomyces griseus) ont produit dans le milieu gélosé des métabolites inhibant la croissance mycélienne et/ou la germination des conidies de H. solani. Les métabolites produits en culture pure et en culture mixte ont présenté une activité différente sur la croissance mycélienne de H. solani. Les résultats présentés indiquent que la méthodologie employée peut être un facteur déterminant dans l'identification de l'antibiose comme mode d'action d'un agent antagoniste.