Uncultured Species Research Articles

Quinones are growth factors for the human gut microbiota

BackgroundThe human gut microbiome has been linked to numerous components of health and disease. However, approximately 25% of the bacterial species in the gut remain uncultured, which limits our ability to properly understand, and exploit, the human microbiome. Previously, we found that growing environmental bacteria in situ in a diffusion chamber enables growth of uncultured species, suggesting the existence of growth factors in the natural environment not found in traditional cultivation media. One source of growth factors proved to be neighboring bacteria, and by using co-culture, we isolated previously uncultured organisms from the marine environment and identified siderophores as a major class of bacterial growth factors. Here, we employ similar co-culture techniques to grow bacteria from the human gut microbiome and identify novel growth factors.ResultsBy testing dependence of slow-growing colonies on faster-growing neighboring bacteria in a co-culture assay, eight taxonomically diverse pairs of bacteria were identified, in which an “induced” isolate formed a gradient of growth around a cultivatable “helper.” This set included two novel species Faecalibacterium sp. KLE1255—belonging to the anti-inflammatory Faecalibacterium genus—and Sutterella sp. KLE1607. While multiple helper strains were identified, Escherichia coli was also capable of promoting growth of all induced isolates. Screening a knockout library of E. coli showed that a menaquinone biosynthesis pathway was required for growth induction of Faecalibacterium sp. KLE1255 and other induced isolates. Purified menaquinones induced growth of 7/8 of the isolated strains, quinone specificity profiles for individual bacteria were identified, and genome analysis suggests an incomplete menaquinone biosynthetic capability yet the presence of anaerobic terminal reductases in the induced strains, indicating an ability to respire anaerobically.ConclusionsOur data show that menaquinones are a major class of growth factors for bacteria from the human gut microbiome. These organisms are taxonomically diverse, including members of the genus Faecalibacterium, Bacteroides, Bilophila, Gordonibacter, and Sutterella. This suggests that loss of quinone biosynthesis happened independently in many lineages of the human microbiota. Quinones can be used to improve existing bacterial growth media or modulate the human gut microbiota by encouraging the growth of important symbionts, such as Faecalibacterium species.

English

Bacteria regulate their social activities and physiological processes through a quorum sensing (QS) mechanism. Many bacteria inhabit the mesohyl matrix of sponges. This symbiotic relationship is believed to contribute to the health and nutrition of sponges by production of protective antibiotics, the acquisition of limiting nutrients and nitrogen fixation. Marine isolates were analysed by bioassays using two different acylated homoserine lactone (AHL) reporter bacteria Chromobacterium violaceum 026 (CV026) and Agrobacterium tumefaciens KYC55. Thirty four marine isolates activated the A. tumefaciens and produce blue color. Using degenerate LuxS primers, partial LuxS gene sequences were detected in both Gram-negative and Gram-positive species including Bacillus species, Proteus species, uncultured Bacterium species, Micrococcus species and Bacterium NLAE-zl-H221. Using degenerate LuxR primers, partial LuxR gene sequences were detected in Proteus spp., Providencia species and Bacterium NLAE-zl-H221. Partial LuxS gene sequences were detected in both Gram-negative and Gram-positive. The results thus assure the fact that LuxS gene has been proposed to be a universal interspecies signaling molecule. This study showed that, Proteus mirabilis strain SZH18, Proteus vulgaris and Bacterium NLAE-zl-H221 have both luxS and luxR genes, thus it was claimed that these isolates have the potential to use two different QS systems, for intraspecies communication and communication with other species. Key words: Quorum sensing, autoinducers, biofilm, double layer method, quorum quenching, N-acyl homoserine lactones.

Novel mastreviruses identified in Australian wild rice

Most known mastreviruses (family Geminiviridae) infect members of the grass family, Poaceae. Although the greatest number of grass-infecting mastrevirus species have been discovered in Africa, it is apparent that the ten grass-infecting mastrevirus species that have so far only been discovered in south-east Queensland have a degree of diversity that rivals that observed in Africa. In this study, we have used a deep sequencing approach to identify two new mastrevirus species, tentatively named rice latent virus 1 and 2 (RLV 1 and 2), from two, undescribed wild rice species (Oryza AA genome group) in Cape York Peninsula, Queensland. The sequences of these new viruses had less than 70% identity with any previously identified mastrevirus, and therefore their discovery vastly expands the known diversity of monocot-infecting mastreviruses in Australia. This study also highlights the potential risks of novel crop pathogens emerging from uncultivated grass species, as the wild rice hosts are very closely related to domesticated rice.

Molecular diversity, geographic distribution and host range of monocot-infecting mastreviruses in Africa and surrounding islands

Maize streak virus (MSV), an important pathogen of maize in Africa, is the most extensively studied member of the Mastrevirus genus in the family Geminiviridae. Comparatively little is known about other monocot-infecting African mastreviruses, most of which infect uncultivated grasses. Here we determine the complete sequences of 134 full African mastrevirus genomes from predominantly uncultivated Poaceae species. Based on established taxonomic guidelines for the genus Mastrevirus, these genomes could be classified as belonging to the species Maize streak virus, Eragrostis minor streak virus, Maize streak Reunion virus, Panicum streak virus, Sugarcane streak Reunion virus and Sugarcane streak virus. Together with all other publicly available African monocot-infecting mastreviruses, the 134 new isolates extend the known geographical distributions of many of these species, including MSV which we found infecting Digitaria sp. on the island of Grand Canaria: the first definitive discovery of any African monocot-infecting mastreviruses north-west of the Saharan desert. These new isolates also extend the known host ranges of both African mastrevirus species and the strains within these. Most notable was the discovery of MSV-C isolates infecting maize which suggests that this MSV strain, which had previously only ever been found infecting uncultivated species, may be in the process of becoming adapted to this important staple crop.

Observation of Wild Seaweed Species in Labuhanbua Waters, Indonesia: a preliminary assessment for aquaculture development

Seaweed industry has been growing up and is supplied by either wild or cultivated seaweed crops. This study was aimed to present relevant information regarding ecological availability of wild seaweed in Labuhanbua coastal waters, Sumbawa Regency, West Nusa Tenggara and potential use of important species as candidate species for aquaculture. 46 sampling stations were determined along line transects perpendicular to coastal line; and seaweeds sampling were conducted during low tide by using 1 x 1 m2 quadrat transect. Field data consist of in-situ parameter including number of seaweed species and coverage area of each species; and ex-situ parameters consist of carbohydrate, protein, total C, total N, and total P content of seaweeds. The results showed that 33 species were found and three species has the most widely distribu tion, i.e. Padina sp., Dictyota dichotoma, and Gracilaria salicornia. Turbinaria, Dictyota, Padina, Stoechospermum, Hydroclathrus, Halimeda, and Chaetomorpha might be some important species that could be develop as aquaculture species candidates among other uncultivated species that were found along this study location. They have potencies as human food, livestock feed, neutraceuicals, cosmetics, pulp, textile, biofuel and any other industries; but conversely, they were found in lower density at Labuhanbua coastal waters. These species should be develop through aquaculture technology, involve genetic improvement and possibly genetic engineering. Commercial scale cultivation of those important seaweed species will contribute to industrial needs and prevent decreasing of wild seaweed availability in natural ecosystem.

Is the role of human female reproductive tract microbiota underestimated?

An issue that is currently undergoing extensive study is the influence of human vaginal microbiota (VMB) on the health status of women and their neonates. Healthy women are mainly colonised with lactobacilli such as Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus iners; however, other bacteria may be elements of the VMB, particularly in women with bacterial vaginosis. The implementation of culture-independent molecular methods in VMB characterisation, especially next-generation sequencing, have provided new information regarding bacterial diversity in the vagina, revealing a large number of novel, fastidious, and/or uncultivated bacterial species. These molecular studies have contributed new insights regarding the role of bacterial community composition. In this study, we discuss recent findings regarding the reproductive tract microbiome. Not only bacteria but also viruses and fungi constitute important components of the reproductive tract microbiome. We focus on aspects related to the impact of the maternal microbiome on foetal development, as well as the establishment of the neonatal microbiomes, including the placenta microbiome, and the haematogenous source of intrauterine infection. We also discuss whether the role of the vaginal microbiome is currently understood and appreciated.

The Succession of Bacterial Community Structure in Groundwater from a 250-m Gallery in the Horonobe Underground Research Laboratory

ABSTRACTWe investigated the change in bacterial community structure after drilling boreholes, 09-V250-M02 and 09-V250-M03, in the 250-m deep research gallery of the Horonobe Underground Research Laboratory. In the 09-V250-M02 borehole, ϵ-Proteobacteria were predominantly detected in the clone library analyses of the groundwater samples conducted immediately after drilling. All the ϵ-Proteobacteria clones were closely related to Arcobacter spp., which are known to be sulfide-oxidizing chemoautotrophic bacteria. After 4 years, the microbial structure drastically changed, and most detected operational taxonomic units were uncultured species such as candidate division OP9 and Chloroflexi relatives, which are frequently detected in deep sea sediments. The results indicated that the microbial community structure was drastically affected by borehole drilling and was concomitant with oxidation perturbation. However, these disturbed microbial communities changed within a few years to a microbial community composed of uncultivated species such as OP9 and Chloroflexi.

A Clostridium Group IV Species Dominates and Suppresses a Mixed Culture Fermentation by Tolerance to Medium Chain Fatty Acids Products.

A microbial community is engaged in a complex economy of cooperation and competition for carbon and energy. In engineered systems such as anaerobic digestion and fermentation, these relationships are exploited for conversion of a broad range of substrates into products, such as biogas, ethanol, and carboxylic acids. Medium chain fatty acids (MCFAs), for example, hexanoic acid, are valuable, energy dense microbial fermentation products, however, MCFA tend to exhibit microbial toxicity to a broad range of microorganisms at low concentrations. Here, we operated continuous mixed population MCFA fermentations on biorefinery thin stillage to investigate the community response associated with the production and toxicity of MCFA. In this study, an uncultured species from the Clostridium group IV (related to Clostridium sp. BS-1) became enriched in two independent reactors that produced hexanoic acid (up to 8.1 g L−1), octanoic acid (up to 3.2 g L−1), and trace concentrations of decanoic acid. Decanoic acid is reported here for the first time as a possible product of a Clostridium group IV species. Other significant species in the community, Lactobacillus spp. and Acetobacterium sp., generate intermediates in MCFA production, and their collapse in relative abundance resulted in an overall production decrease. A strong correlation was present between the community composition and both the hexanoic acid concentration (p = 0.026) and total volatile fatty acid concentration (p = 0.003). MCFA suppressed species related to Clostridium sp. CPB-6 and Lactobacillus spp. to a greater extent than others. The proportion of the species related to Clostridium sp. BS-1 over Clostridium sp. CPB-6 had a strong correlation with the concentration of octanoic acid (p = 0.003). The dominance of this species and the increase in MCFA resulted in an overall toxic effect on the mixed community, most significantly on the Lactobacillus spp., which resulted in a decrease in total hexanoic acid concentration to 32 ± 2% below the steady-state average. As opposed to the current view of MCFA toxicity broadly leading to production collapse, this study demonstrates that varied tolerance to MCFA within the community can lead to the dominance of some species and the suppression of others, which can result in a decreased productivity of the fermentation.

A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea.

ABSTRACTHeterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO2-reducing methanogenic anaerobes (methanogens) from the domain Archaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F420 (F420H2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F420H2 and reduction of ferredoxin with the exergonic oxidation of F420H2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting.

PCR-activated cell sorting as a general, cultivation-free method for high-throughput identification and enrichment of virus hosts

Characterizing virus-host relationships is critical for understanding the impact of a virus on an ecosystem, but is challenging with existing techniques, particularly for uncultivable species. We present a general, cultivation-free approach for identifying phage-associated bacterial cells. Using PCR-activated cell sorting, we interrogate millions of individual bacteria for the presence of specific phage nucleic acids. If the nucleic acids are present, the bacteria are recovered via sorting and their genomes analyzed. This allows targeted recovery of all possible host species in a diverse population associated with a specific phage, and can be easily targeted to identify the hosts of different phages by modifying the PCR primers used for detection. Moreover, this technique allows quantification of free phage particles, as benchmarked against the “gold standard” of virus enumeration, the plaque assay.

Nezara viridula(L.) in Central Texas: I. New Host Plant Associations and Reproductive Status of Adults Encountered Within

Southern green stink bug, Nezara viridula (L.), and other phytophagous stink bug species continue to plague producers of row crops (e.g., cotton [Gossypium hirsutum L.] and soybean [Glycine max (L.) Merrill]) in Texas and the remainder of the Cotton Belt in the southeastern United States. Developing effective management tactics for N. viridula requires a thorough understanding of factors contributing to continued production of field pest populations. Uncultivated plant species in the absence of cultivated crops are key factors known to support N. viridula. This report identifies 17 new host plant associations (from nine plant families) for N. viridula in Central Texas; 12 of these associations were previously unidentified altogether in Central Texas while five plant species associations were identified in other regions of North America and the world. Also, reproductive status data of adults in the new associations aid in identifying new plant species capable of harboring reproductive adults. The new plant species associated with N. viridula should be monitored for developing populations that might eventually, or simultaneously, infest cultivated crops.

Combining culture‐dependent and culture‐independent molecular methods for the isolation and purification of a potentially novel anaerobic species from pit mud in a Chinese liquor distillery

Strain S12–27–1-3-5 (a potentially novel anaerobic species) with a 16S rRNA sequence homology of <97% was isolated and purified from pit mud by combining culture-dependent and culture-independent molecular methods, such as cloning of 16S rRNA, amplified rRNA restriction analysis, and denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the 16S rRNA gene indicated that strain S12–27–1-3-5 was related to Aminobacterium mobile strain ILE-3 DSM 12262T and Aminobacterium colombiense strain DSM 12261T (95 and 96% similarity value, respectively). The results verified that cloning of the 16S rRNA was efficient to identify whether a potentially new bacterial taxon existed in impure isolates and that the DGGE method was a powerful tool for screening the target bacteria and for identifying duplicate strains. Therefore, the application of the culture-independent molecular methods for the isolation and purification of a potentially novel species was effective. Strain S12–27–1-3-5 (= DSM 27871 = JCM 19605 = CICC 10731T) was an anaerobic amino acid-degrading bacterium. The results of fermentation experiments demonstrated that strain S12–27–1-3-5 produced volatile fatty acids (VFAs) and the presence of Methanosarcina barkeri enhanced the generation of VFAs, which contribute to the aroma composition of Chinese liquor. This work could enrich the species resources and promote the development and utilization of an uncultured species. Copyright © 2016 The Institute of Brewing & Distilling

Natural Anaplasmataceae infection in Rhipicephalus bursa ticks collected from sheep in the French Basque Country

Rhipicephalus bursa is one of 79 species of the genus Rhipicephalus in the family of Ixodidae. In this study, we investigated Anaplasmataceae bacteria associated with R. bursa collected after an epizootic outbreak of ovine anaplasmosis. 76 adult ticks, (60 male and 16 female ticks), were removed from sheep in two farms and all identified as R. bursa, all females were partially engorged. We found that 50% of the ticks were positive in the initial Anaplasmataceae qPCR screening. Bacterial species was identified by analyzing the sequences of amplicons of 23S rRNA, groEL and rpoB genes. 22.4% of ticks contained DNA of Anaplasma phagocytophilum and 7.9% the DNA of Anaplasma ovis. Based on 23S rRNA and groEL genes analysis, we found that 19.7% of ticks contained a potentially new species of Ehrlichia. We propose the status of Candidatus for this uncultured species and we provisionally name it Candidatus Ehrlichia urmitei. No Wolbachia were identified. These results show that R. bursa can be a carrier of Anaplasmataceae bacteria.

Functional characterization of a gene locus from an uncultured gut Bacteroides conferring xylo-oligosaccharides utilization to Escherichia coli.

In prominent gut Bacteroides strains, sophisticated strategies have been evolved to achieve the complete degradation of dietary polysaccharides such as xylan, which is one of the major components of the plant cell wall. Polysaccharide Utilization Loci (PULs) consist of gene clusters encoding different proteins with a vast arsenal of functions, including carbohydrate binding, transport and hydrolysis. Transport is often attributed to TonB-dependent transporters, although major facilitator superfamily (MFS) transporters have also been identified in some PULs. However, until now, few of these transporters have been biochemically characterized. Here, we targeted a PUL-like system from an uncultivated Bacteroides species that is highly prevalent in the human gut metagenome. It encodes three glycoside-hydrolases specific for xylo-oligosaccharides, a SusC/SusD tandem homolog and a MFS transporter. We combined PUL rational engineering, metabolic and transcriptional analysis in Escherichia coli to functionally characterize this genomic locus. We demonstrated that the SusC and the MFS transporters are specific for internalization of linear xylo-oligosaccharides of polymerization degree up to 3 and 4 respectively. These results were strengthened by the study of growth dynamics and transcriptional analyses in response to XOS induction of the PUL in the native strain, Bacteroides vulgatus.

Assemble CRISPRs from metagenomic sequencing data.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) allows more specific and efficient gene editing than all previous genetic engineering systems. These exciting discoveries stem from the finding of the CRISPR system being an adaptive immune system that protects the prokaryotes against exogenous genetic elements such as phages. Despite the exciting discoveries, almost all knowledge about CRISPRs is based only on microorganisms that can be isolated, cultured and sequenced in labs. However, about 95% of bacterial species cannot be cultured in labs. The fast accumulation of metagenomic data, which contains DNA sequences of microbial species from natural samples, provides a unique opportunity for CRISPR annotation in uncultivable microbial species. However, the large amount of data, heterogeneous coverage and shared leader sequences of some CRISPRs pose challenges for identifying CRISPRs efficiently in metagenomic data. In this study, we developed a CRISPR finding tool for metagenomic data without relying on generic assembly, which is error-prone and computationally expensive for complex data. Our tool can run on commonly available machines in small labs. It employs properties of CRISPRs to decompose generic assembly into local assembly. We tested it on both mock and real metagenomic data and benchmarked the performance with state-of-the-art tools. The source code and the documentation of metaCRISPR is available at https://github.com/hangelwen/metaCRISPR CONTACT: yannisun@msu.edu.

A catalogue of 136 microbial draft genomes from Red Sea metagenomes.

Earth is expected to continue warming and the Red Sea is a model environment for understanding the effects of global warming on ocean microbiomes due to its unusually high temperature, salinity and solar irradiance. However, most microbial diversity analyses of the Red Sea have been limited to cultured representatives and single marker gene analyses, hence neglecting the substantial uncultured majority. Here, we report 136 microbial genomes (completion minus contamination is ≥50%) assembled from 45 metagenomes from eight stations spanning the Red Sea and taken from multiple depths between 10 to 500 m. Phylogenomic analysis showed that most of the retrieved genomes belong to seven different phyla of known marine microbes, but more than half representing currently uncultured species. The open-access data presented here is the largest number of Red Sea representative microbial genomes reported in a single study and will help facilitate future studies in understanding the physiology of these microorganisms and how they have adapted to the relatively harsh conditions of the Red Sea.

Linking the Physicochemical Properties with the Abundance and Diversity of Rhizospheric Bacterial Population Inhabiting Paddy Soil Based on a Concerted Multivariate Analysis of PCR-DGGE and RISA

ABSTRACTTo unravel the existence of dominant bacterial population in the paddy fields of Eastern Uttar Pradesh, India and their relation to the prevailing soil physicochemistry using multivariate statistical analyses, a cumulative culture-independent 16S rRNA based Polymerase chain reaction-Denaturing gradient gel electrophoresis (PCR-DGGE) and a 16S-23S ribosomal intergenic spacer analysis (RISA) have been performed. Detrended correspondence analysis (DCA) and principal component analysis (PCA) biplot analyses were used to assess the relation between soil bacterial population and its physicochemistry. DCA analysis exhibited a strong dependence of bacterial existence on the soil physicochemical variables, such as organic matter, total nitrogen, inorganic nutrients, temperatures, and moisture status. Soil dehydrogenase activity (DHA) was assessed to check the metabolic activity of all soil samples which showed a range of 0.012–0.050 nmol TPF g−1 min−1 with significant variation (p < 0.01). Out of 96 bands excised, 45 different phylotypes were obtained using both techniques which elucidated the abundance of Cyanobacteria over other soil bacterial population. Scytonema sp., Leptolyngbya sp. and different uncultured cyanobacterial species were the major genera found. Profiling data obtained through PCR-DGGE and RISA were used in alpha diversity and rarefaction curve analysis suggested site 6 (Chandauli) as the most diversity rich site. Thus extensive dataset of weighted and unweighted variables generated through DGGE and RISA coupled with metabolic functioning of soil and multivariate analyses provided an excellent opportunity to map the soil microbial structure in paddy fields and their regulation with existing soil environment.

Capturing One of the Human Gut Microbiome's Most Wanted: Reconstructing the Genome of a Novel Butyrate-Producing, Clostridial Scavenger from Metagenomic Sequence Data.

The role of the microbiome in health and disease is attracting great attention, yet we still know little about some of the most prevalent microorganisms inside our bodies. Several years ago, Human Microbiome Project (HMP) researchers generated a list of “most wanted” taxa: bacteria both prevalent among healthy volunteers and distantly related to any sequenced organisms. Unfortunately, the challenge of assembling high-quality genomes from a tangle of metagenomic reads has slowed progress in learning about these uncultured bacteria. Here, we describe how recent advances in sequencing and analysis allowed us to assemble “most wanted” genomes from metagenomic data collected from four stool samples. Using a combination of both de novo and guided assembly methods, we assembled and binned over 100 genomes from an initial data set of over 1,300 Gbp. One of these genome bins, which met HMP’s criteria for a “most wanted” taxa, contained three essentially complete genomes belonging to a previously uncultivated species. This species is most closely related to Eubacterium desmolans and the clostridial cluster IV/Clostridium leptum subgroup species Butyricicoccus pullicaecorum (71–76% average nucleotide identity). Gene function analysis indicates that the species is an obligate anaerobe, forms spores, and produces the anti-inflammatory short-chain fatty acids acetate and butyrate. It also appears to take up metabolically costly molecules such as cobalamin, methionine, and branch-chained amino acids from the environment, and to lack virulence genes. Thus, the evidence is consistent with a secondary degrader that occupies a host-dependent, nutrient-scavenging niche within the gut; its ability to produce butyrate, which is thought to play an anti-inflammatory role, makes it intriguing for the study of diseases such as colon cancer and inflammatory bowel disease. In conclusion, we have assembled essentially complete genomes from stool metagenomic data, yielding valuable information about uncultured organisms’ metabolic and ecologic niches, factors that may be required to successfully culture these bacteria, and their role in maintaining health and causing disease.

Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular β-proteobacteria.

New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

Molecular cytogenetic characterization of Dasypyrum breviaristatum chromosomes in wheat background revealing the genomic divergence between Dasypyrum species.

BackgroundThe uncultivated species Dasypyrum breviaristatum carries novel diseases resistance and agronomically important genes of potential use for wheat improvement. The development of new wheat-D. breviaristatum derivatives lines with disease resistance provides an opportunity for the identification and localization of resistance genes on specific Dasypyrum chromosomes. The comparison of wheat-D. breviaristatum derivatives to the wheat-D. villosum derivatives enables to reveal the genomic divergence between D. breviaristatum and D. villosum.ResultsThe mitotic metaphase of the wheat- D. breviaristatum partial amphiploid TDH-2 and durum wheat -D. villosum amphiploid TDV-1 were studied using multicolor fluorescent in situ hybridization (FISH). We found that the distribution of FISH signals of telomeric, subtelomeric and centromeric regions on the D. breviaristatum chromosomes was different from those of D. villosum chromosomes by the probes of Oligo-pSc119.2, Oligo-pTa535, Oligo-(GAA)7 and Oligo-pHv62-1. A wheat line D2139, selected from a cross between wheat lines MY11 and TDH-2, was characterized by FISH and PCR-based molecular markers. FISH analysis demonstrated that D2139 contained 44 chromosomes including a pair of D. breviaristatum chromosomes which had originated from the partial amphiploid TDH-2. Molecular markers confirmed that the introduced D. breviaristatum chromosomes belonged to homoeologous group 7, indicating that D2139 was a 7Vb disomic addition line. The D2139 displayed high resistance to wheat stripe rust races at adult stage plant, which may be inherited from, D. breviaristatum chromosome 7Vb.ConclusionThe study present here revealed that the large divergence between D. breviaristatum and D. villosum with respected to the organization of different repetitive sequences. The identified wheat- D. breviaristatum chromosome addition line D2139 will be used to produce agronomically desirable germplasm for wheat breeding.