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Small genes (40,000 small-gene families in ~2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.

A weekly conference series paired with lectures entitled "Microbiome-MX: exploring the Microbiota and Microbiome Research in Mexico" was organized to provide a multidisciplinary overview of the most recent research done in Mexico using high-throughput sequencing. Scientists and postgraduate students from several disciplines such as microbiology, bioinformatics, virology, immunology, nutrition, and medical genomics gathered to discuss state of the art in each of their respective subjects of expertise, as well as advances, applications and new opportunities on microbiota/microbiome research. In particular, high-throughput sequencing is a crucial tool to understand the challenges of a megadiverse developing country as Mexico, and moreover to know the scientific capital and capabilities available for collaboration. The conference series addressed three main topics important for Mexico: i) the complex role of microbiota in health and prevalent diseases such as obesity, diabetes, inflammatory bowel disease, tuberculosis, HIV, autoimmune diseases and gastric cancer; ii) the use of local, traditional and prehispanic products as pre/probiotics to modulate the microbiota and improve human health; and iii) the impact of the microbiota in shaping the biodiversity of economically important terrestrial and marine ecosystems. Herein, we summarize the contributions that Mexican microbiota/microbiome research is making to the global trends, describing the highlights of the conferences and lectures, rather than a review of the state-of-the-art of this research. This meeting report also presents the efforts of a multidisciplinary group of scientist to encourage collaborations and bringing this research field closer for younger generations.

Fungi represent a group of eukaryotic microorganisms that are an important part of the plant microbiome. They produce a vast array of metabolites, including fungal volatile organic compounds (fVOCs). However, the diversity and biological activities of fVOCs emitted by the mycobiota of plants native to arid and semi-arid environments remain under-explored. We characterized the chemical diversity of fVOCs produced by 22 representative members of the microbiome of agaves and cacti using SPME-GC-MS. We further tested the effects of pure compounds on the growth and development of Arabidopsis thaliana and host plants. Members of the Sordariomycetes (nine strains), Eurotiomycetes (three), Dothideomycetes (eight), Saccharomycetes (one) and Mucoromycetes (one) were included in our study. We identified 94 fungal organic volatiles classified into nine chemical classes. Terpenes showed the greatest chemical diversity, followed by alcohols and aliphatic compounds. We discovered that camphene and benzyl benzoate, together with the widely distributed and already tested benzyl alcohol, 2-phenylethyl alcohol and 3-methyl-1-butanol, improved plant growth and development of A. thaliana, Agave tequilana and Agave salmiana. Our studies on the fungal VOCs from desert plants underscore an untapped chemical diversity with promising biotechnological applications.

Aims The effects of aridity on soil and water-use efficient (WUE) crop species are relatively well known. However, the understanding of its impacts on the dynamics of below-ground microorganisms associated with plant roots is less well understood. Methods To investigate the influence of increasing aridity on the dynamics of the fungal communities, samples from the root endosphere and rhizosphere associated with the prickly pear cactus trees (Opuntia ficus-indica) growing along the aridity gradient were collected and the internal transcribed spacer (ITS) were sequenced. The diversity and network analyses of fungal taxa were determined along with standard measurements of soil parameters. Results We found that (i) the fungal community exhibited similar alpha diversity and shared a set of core taxa within the rhizosphere and endosphere, but there was significant beta diversity differences; (ii) the relative abundance of major phyla was higher in the rhizosphere than in the endosphere; (iii) arbuscular endomycorrhizal colonization was highest in the humid climate and decreased under lower-arid, and was negatively correlated with increased concentration of Ca2+ in the soil; (iv) increased aridity correlated with increased connectivity of the soil microbial-root fungal networks in the arid soils, producing a highly cohesive network in the upper-arid area; and (v) distinct fungal hubs sculpt the fungal microbiome network structure in the rhizosphere and endosphere within each bioclimatic zone. Conclusions Our findings highlight the importance of gradient analysis-based correlation network as a powerful approach to understand changes in the diversity, the dynamics, and the structure of fungal communities associated with the rhizosphere-endosphere interaction and led to the identification of microbes at each bioclimatic zone that are potentially involved in promoting the survival, protection, and growth of Opuntia trees. The variability of fungal hubs composition depending on plant compartment and bioclimatic zone will give key implications for the application of rhizospheric fungi and endophytes as microbial inoculants in agriculture, as well as in the conservation and restoration of cacti plants in arid and semi-arid lands against the backdrop of climate change. Overall, this study will enhance our understanding of the microbiomes'dynamic of CAM plants in nature.

The plant microbiota can affect host fitness via the emission of microbial volatile organic compounds (mVOCs) that influence growth and development. However, evidence of these molecules and their effects in plants from arid ecosystems is limited. We screened the mVOCs produced by 40 core and representative members of the microbiome of agaves and cacti in their interaction with Arabidopsis thaliana and Nicotiana benthamiana. We used SPME-GC-MS to characterize the chemical diversity of mVOCs and tested the effects of selected compounds on growth and development of model and host plants. Our study revealed that approximately 90% of the bacterial strains promoted plant growth both in A. thaliana and N. benthamiana. Bacterial VOCs were mainly composed of esters, alcohols, and S-containing compounds with 25% of them not previously characterized. Remarkably, ethyl isovalerate, isoamyl acetate, 3-methyl-1-butanol, benzyl alcohol, 2-phenylethyl alcohol, and 3-(methylthio)-1-propanol, and some of their mixtures, displayed beneficial effects in A. thaliana and also improved growth and development of Agave tequilana and Agave salmiana in just 60 days. Volatiles produced by bacteria isolated from agaves and cacti are promising molecules for the sustainable production of crops in arid and semi-arid regions.

Phaseolus lunatus (lima bean) has been a model organism to understand direct and indirect defenses against herbivores. We investigated the identity and diversity of endophytic fungi associated with young, mature and old leaves of lima bean in its natural environment. We recovered 758 isolates that were identified and classified in 142 fungal species from 66 genera, with Curvularia , Fusarium , Phomopsis , Alternaria and Colletotrichum being the most abundant and diverse Ascomycota genera. Our data revealed that species richness and diversity increased with leaf age, this factor being also important for the structure of the endophytic community. The occurrence patterns of fungal species was highly heterogeneous among leaves, and only fourteen species were recovered from all three foliar ages investigated. In vitro dual assays between endophytes and the plant pathogen Colletotrichum lindemuthianum revealed that most endophytes tested can inhibit the growth of this pathogen, suggesting their potential as biocontrol agents.

The evaluation of metabolomics data sets involves multiples steps of data processing, statistical analysis and interpretation. XCMS is a commonly employed program for analyzing metabolomics data. It exists in two distinct variants: XCMS/R and XCMS Online. XCMS/R is a package for the statistics and graphics language R which requires manual coding, allowing for highly customized workflows. In contrast, XCMS Online provides a user-friendly web interface with modules for statistics and metabolic pathway integration. We demonstrate the step-by-step use of both platforms through the analysis of a liquid-chromatography/high-resolution mass spectrometry dataset of flowers of Arabidopsis thaliana (accessions Col and Ws). Both workflows identify common features in the twelve samples (2 accessions × 6 biological replicates). However, the general purpose of the programs is different. XCMS/R is more focused on optimizing the parameter settings and statistical testing/visualization. In contrast, XCMS Online performs most of the tasks automatically and supports the mapping of results on biochemical pathways.

Symptoms of Tar Spot Complex (TSC) in corn appear in the form of black spots surrounded by a chlorotic halo known as fish-eye. The crop with these symptoms was found at San Pablo Autopan, Estado de Mexico. The stromal region of foliar lesions was cut longitudinally, showing perithecia, asci and ascospores typical of Phyllachora maydis. The results were confirmed by PCR-ITS with ITS 1 and ITS4 initiators, cloned by pJET, before being sequenced and compared against NCBI’s data base. Phyllachora identity was confirmed. This is the first report where TSC pathogen has been detected under conditions that had not been previously reported, for instance, at 2600 meters above the sea level.

Summary Desert plants are hypothesized to survive the environmental stress inherent to these regions in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood.Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing.Phylogenetic profiling revealed that the composition of prokaryotic communities was primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana exhibited lower levels of prokaryotic diversity compared with native agaves, although no differences in microbial diversity were found in the endosphere.Agaves shared core prokaryotic and fungal taxa known to promote plant growth and confer tolerance to abiotic stress, which suggests common principles underpinning Agave–microbe interactions., See also the Commentary by Hacquard

Phylogenetic profiling and metagenomics of crassulacean acid metabolism (CAM) plants from North America have revealed diverse and structured microbial communities with core microbial taxa in each plant compartment. In silico functional analyses suggest that microorganisms in the rhizosphere and phyllosphere of CAM plants differentially benefit their host plants to succeed in drylands.

Agaves are major biotic resources in arid and semi-arid ecosystems. Despite their ecological, economical and cultural relevance, many aspects of the microbial communities associated with agaves are still unknown. Here, we investigated the bacterial communities associated with two Agave species by 16S rRNA- Denaturing gradient gel electrophoresis fingerprinting and sequencing. We also evaluated the effects of biotic and abiotic factors in the structure of the bacterial communities. In parallel, we isolated and characterized diazotrophic bacteria associated with agaves, as Agave soils are characterized by their low nitrogen content. Our results demonstrate that in Agave, the structure of prokaryotic assemblages was mostly influenced by the community group, where the soil, episphere, and endosphere were clearly distinct. Proteobacteria (γ and α), Actinobacteria, and Acidobacteria were the dominant phyla. Bacterial communities in the episphere of agaves were mainly influenced by the host species, whereas in the endosphere were affected by the season. Fifteen bacterial taxa were common and abundant in the endosphere of both Agave species during the dry season. Notably, some of the confirmed diazotrophic strains belonged to this group, suggesting a possible beneficial role in planta.

The assembly of fungal communities on stone materials is mainly influenced by the differential bioreceptivity of such materials and environmental conditions. However, little is known about the role of fungal interactions in the colonization and establishment of fungal species. We analyzed the effects of intra- and interspecific interactions between 11 species of fungi in oligotrophic and copiotrophic media and on limestone coupons. In a previous study, these species were the most frequently isolated in the epilithic biofilms of limestone walls exposed to a subtropical climate. In the culture media, we found a greater frequency of intra- and interspecific inhibitory effects in the oligotrophic medium than in the copiotrophic medium. On the limestone coupons, all fungi were able to establish; however, the colonization success rate varied significantly. Cladosporium cladosporioides had a less extensive colonization in isolation (control) than in dual interactions (coexistence) with other species. Phoma eupyrena exhibited the highest colonization success rate and competitive dominance among all tested species. X-ray diffraction (XRD) and scanning electron microscope (SEM) analyses revealed that Pestalotiopsis maculans and Paraconiothyrium sp. produced calcium oxalate crystals during their growth on coupon surfaces, both in isolation and in dual interactions. Our results demonstrate that interactions between abundant fungal species influence the fungal colonization on substrates, the biomineralization and the fungal community assemblage growing in limestone biofilms.

© 2016 Fonseca-García, Coleman-Derr, Garrido, Visel, Tringe and Partida-Martínez. Cactaceae represents one of the most species-rich families of succulent plants native to arid and semi-arid ecosystems, yet the associations Cacti establish with microorganisms and the rules governing microbial community assembly remain poorly understood. We analyzed the composition, diversity, and factors influencing above- and below-ground bacterial, archaeal, and fungal communities associated with two native and sympatric Cacti species: Myrtillocactus geometrizans and Opuntia robusta. Phylogenetic profiling showed that the composition and assembly of microbial communities associated with Cacti were primarily influenced by the plant compartment; plant species, site, and season played only a minor role. Remarkably, bacterial, and archaeal diversity was higher in the phyllosphere than in the rhizosphere of Cacti, while the opposite was true for fungi. Semi-arid soils exhibited the highest levels of microbial diversity whereas the stem endosphere the lowest. Despite their taxonomic distance, M. geometrizans and O. robusta shared most microbial taxa in all analyzed compartments. Influence of the plant host did only play a larger role in the fungal communities of the stem endosphere. These results suggest that fungi establish specific interactions with their host plant inside the stem, whereas microbial communities in the other plant compartments may play similar functional roles in these two species. Biochemical and molecular characterization of seed-borne bacteria of Cacti supports the idea that these microbial symbionts may be vertically inherited and could promote plant growth and drought tolerance for the fitness of the Cacti holobiont. We envision this knowledge will help improve and sustain agriculture in arid and semi-arid regions of the world.

Toxinogenic endobacteria were isolated from a collection of Rhizopus spp. representing highly diverse geographic origins and ecological niches. All endosymbionts belonged to the Burkholderia rhizoxinica complex according to matrix-assisted laser desorption ionization-time of flight biotyping and multilocus sequence typing, suggesting a common ancestor. Comparison of host and symbiont phylogenies provides insights into possible cospeciation and horizontal-transmission events.

Infections by Rhizopus spp. account for about 90% of zygomycoses, many of which are lethal in immunocompromised patients. We recently noted that several strains of Rhizopus microsporus harbour rare bacterial endosymbionts (Burkholderia sp.) for the production of 'mycotoxins', which might play a role as virulence factors in human Rhizopus infections. In this study eight clinical Rhizopus spp. isolates have been investigated for the presence of toxin-producing bacterial endosymbionts. By metabolomic data, PCR targeting bacterial 16S rDNA and microscopic investigations with fluorescence dyes we provide three lines of evidence showing that the fungal strains are not associated with endofungal bacteria. Consequently, toxin-producing bacteria are not essential for Rhizopus infections and the development of zygomycoses in humans.

Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic cyclopeptide rhizonin. R. microsporus and isolated endobacteria were selected for freeze–fracture electron microscopy, which allowed visualization of bacterial cells within the fungal cytosol by their two parallel-running envelope membranes and by the fine structure of the lipopolysaccharide layer of the outer membrane. Two representatives of bacterial endosymbionts were chosen for phylogenetic analyses on the basis of full 16S rRNA gene sequences, which revealed that the novel fungal endosymbionts formed a monophyletic group within the genus Burkholderia. Inter-sequence similarities ranged from 98.94 to 100 %, and sequence similarities to members of the Burkholderia pseudomallei group, the closest neighbours, were 96.74–97.38 %. In addition, the bacterial strains were distinguished from their phylogenetic neighbours by their fatty acid profiles and other biochemical characteristics. The phylogenetic studies based on 16S rRNA gene sequence data, together with conclusive DNA–DNA reassociation experiments, strongly support the proposal that these strains represent two novel species within the genus Burkholderia, for which the names Burkholderia rhizoxinica sp. nov. (type strain, HKI 454T=DSM 19002T=CIP 109453T) and Burkholderia endofungorum sp. nov. (type strain, HKI 456T=DSM 19003T=CIP 109454T) are proposed.

The antitumour agent rhizoxin is a fungal metabolite produced by Rhizopus microsporus, the pathogen that causes one of the most destructive diseases of rice crops, rice seedling blight. Or so we thought. Now it has been discovered that Rhizopus is not the true producer of rhizoxin. In fact it is synthesized by a bacterium of the genus Burkholderia, living in the fungus as an endosymbiont. Rhizoxin causes cell-cycle arrest in the plant cells, and the fungal pathogen and its symbiont both benefit from the decaying plant matter produced. A number of plant pathogenic fungi belonging to the genus Rhizopus are infamous for causing rice seedling blight. This plant disease is typically initiated by an abnormal swelling of the seedling roots without any sign of infection by the pathogen1,2,3,4. This characteristic symptom is in fact caused by the macrocyclic polyketide metabolite rhizoxin that has been isolated from cultures of Rhizopus sp.5,6. The phytotoxin exerts its destructive effect by binding to rice β-tubulin, which results in inhibition of mitosis and cell cycle arrest7,8. Owing to its remarkably strong antimitotic activity in most eukaryotic cells, including various human cancer cell lines, rhizoxin has attracted considerable interest as a potential antitumour drug9,10. Here we show that rhizoxin is not biosynthesized by the fungus itself, but by endosymbiotic, that is, intracellular living, bacteria of the genus Burkholderia. Our unexpected findings unveil a remarkably complex symbiotic-pathogenic relationship that extends the fungus–plant interaction to a third, bacterial, key-player, and opens new perspectives for pest control.

Microorganisms of subaerial biofilms, which develop on the surface of stone monuments, have been widely studied due to their impact on Cultural Heritage deterioration. Nevertheless, these extremophile microorganisms are poorly characterised. In this study, a cyanobacterial strain isolated from an archaeological zone in north-central Mexico was characterised through a polyphasic study based on ecological, morphological, and molecular data. Phylogenetic analyses were done using sequences of the 16S rRNA gene and the 16S-23S ITS region. Based on the polyphasic data, a new species corresponding to the genus Nodosilinea is described. This alkaliphilic species has optimum growth at pH of 9. Its fatty acid profile indicates that C18:1, 9E and C16:0 are its most abundant fatty acids. Extracellular polymeric substance production was verified on this strain. Only a few species of the genus Nodosilinea have been documented and described, and this is the first report of their presence in central Mexico. Hence the name Nodosilinea chupicuarensis sp. nov. is proposed.

As revealed by genome sequencing, the biology of prokaryotes with reduced genomes is strikingly diverse. These include free-living prokaryotes with ∼800 genes as well as endosymbiotic bacteria with as few as ∼140 genes. Comparative genomics is revealing the evolutionary mechanisms that led to these small genomes. In the case of free-living prokaryotes, natural selection directly favored genome reduction, while in the case of endosymbiotic prokaryotes neutral processes played a more prominent role. However, new experimental data suggest that selective processes may be at operation as well for endosymbiotic prokaryotes at least during the first stages of genome reduction. Endosymbiotic prokaryotes have evolved diverse strategies for living with reduced gene sets inside a host-defined medium. These include utilization of host-encoded functions (some of them coded by genes acquired by gene transfer from the endosymbiont and/or other bacteria); metabolic complementation between co-symbionts; and forming consortiums with other bacteria within the host. Recent genome sequencing projects of intracellular mutualistic bacteria showed that previously believed universal evolutionary trends like reduced G+C content and conservation of genome synteny are not always present in highly reduced genomes. Finally, the simplified molecular machinery of some of these organisms with small genomes may be used to aid in the design of artificial minimal cells. Here we review recent genomic discoveries of the biology of prokaryotes endowed with small gene sets and discuss the evolutionary mechanisms that have been proposed to explain their peculiar nature.

Targeted gene inactivation and metabolic profiling revealed that the cryptic PKS-NRPS gene cluster in the genome of the plant commensal Pseudomonas fluorescens Pf-5 codes for the biosynthesis of antiproliferative and antifungal rhizoxin derivatives.

Plant–microbe interactions are ubiquitous. Plants are threatened by pathogens, but they are even more commonly engaged in neutral or mutualistic interactions with microbes: belowground microbial plant associates are mycorrhizal fungi, Rhizobia, and plant-growth promoting rhizosphere bacteria, aboveground plant parts are colonized by internally living bacteria and fungi (endophytes) and by microbes in the phyllosphere (epiphytes). We emphasize here that a completely microbe-free plant is an exotic exception rather than the biologically relevant rule. The complex interplay of such microbial communities with the host–plant affects multiple vital parameters such as plant nutrition, growth rate, resistance to biotic and abiotic stressors, and plant survival and distribution. The mechanisms involved reach from direct ones such as nutrient acquisition, the production of plant hormones, or direct antibiosis, to indirect ones that are mediated by effects on host resistance genes or via interactions at higher trophic levels. Plant-associated microbes are heterotrophic and cause costs to their host plant, whereas the benefits depend on the current environment. Thus, the outcome of the interaction for the plant host is highly context dependent. We argue that considering the microbe-free plant as the “normal” or control stage significantly impairs research into important phenomena such as (1) phenotypic and epigenetic plasticity, (2) the “normal” ecological outcome of a given interaction, and (3) the evolution of plants. For the future, we suggest cultivation-independent screening methods using direct PCR from plant tissue of more than one fungal and bacterial gene to collect data on the true microbial diversity in wild plants. The patterns found could be correlated to host species and environmental conditions, in order to formulate testable hypotheses on the biological roles of plant endophytes in nature. Experimental approaches should compare different host–endophyte combinations under various relevant environmental conditions and study at the genetic, epigenetic, transcriptional, and physiological level the parameters that cause the interaction to shift along the mutualism–parasitism continuum.

Background Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic zygomycete Rhizopus microsporus, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on the evolution and genetic potential of this model organism, we analysed the whole genome of B. rhizoxinica HKI 0454 - a type strain of endofungal Burkholderia species. Results The genome consists of a structurally conserved chromosome and two plasmids. Compared to free-living Burkholderia species, the genome is smaller in size and harbors less transcriptional regulator genes. Instead, we observed accumulation of transposons over the genome. Prediction of primary metabolic pathways and transporters suggests that endosymbionts consume host metabolites like citrate, but might deliver some amino acids and cofactors to the host. The rhizoxin biosynthesis gene cluster shows evolutionary traces of horizontal gene transfer. Furthermore, we analysed gene clusters coding for nonribosomal peptide synthetases (NRPS). Notably, B. rhizoxinica lacks common genes which are dedicated to quorum sensing systems, but is equipped with a large number of virulence-related factors and putative type III effectors. Conclusions B. rhizoxinica is the first endofungal bacterium, whose genome has been sequenced. Here, we present models of evolution, metabolism and tools for host-symbiont interaction of the endofungal bacterium deduced from whole genome analyses. Genome size and structure suggest that B. rhizoxinica is in an early phase of adaptation to the intracellular lifestyle (genome in transition). By analysis of tranporters and metabolic pathways we predict how metabolites might be exchanged between the symbiont and its host. Gene clusters for biosynthesis of secondary metabolites represent novel targets for genomic mining of cryptic natural products. In silico analyses of virulence-associated genes, secreted proteins and effectors might inspire future studies on molecular mechanisms underlying bacterial-fungal interaction.

Mould fungi are not only well known for food spoilage through toxin formation but also for the production of fermented foods. In Asian countries, the fermentation of soy beans and tofu for tempe and sufu production with various Rhizopus strains is widespread. Here we report the finding of toxinogenic bacteria in a starter culture used for sufu production. By means of metabolic profiling of the fungus under standard conditions for tempe and sufu production, we found that toxins of the rhizoxin complex are produced in critical amounts. Considering that rhizoxins are severe toxins with strong antimitotic activity it is important to notice that our findings uncover a health-threatening symbiosis in food processing. A simple PCR method for detecting toxinogenic endofungal bacteria in starter cultures is proposed.

Mycotoxins are compounds of fungal origin that can adversely affect human, animal and plant health through food spoilage or infection, even to the point of epidemics such as turkey X disease and ergotism. The biosynthetic pathways of various mycotoxins (such as aflatoxin and fumonisins) are generally well understood. However, two examples have recently been described where a mycotoxin is not synthesized by the fungus itself but by bacteria residing within the fungal cytosol. These discoveries have implications in various fields, such as ecology, medicine and food processing.

The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of beta-tubulin sequences in the kingdom Fungi. A total of 49 new beta-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism--mutualism shift in insensitive fungi.

A novel prototype polymer-coated adsorbent (PCA) has been developed for the effective expanded bed recovery of protein products from particulate feedstocks. The adsorbents were manufactured using the three-phase emulsification process by which the selected core phases (anion- and cation-exchangers and a custom-assembled pseudo-affinity adsorbent) were coated by an agarose gel. This new non-stick exterior coating acts as a sieve reducing the non-specific binding of cell and cell debris without diminution of selective capture of target protein from complex feedstocks such as whole microbial broths and cell disruptates. The new coated adsorbents were subjected to physical and hydrodynamical comparison with the performance of their uncoated adsorbents. Hydrodynamic characteristics (e.g. axial dispersion coefficient (Daxl) and Bodenstein number (Bo)) of PCA demonstrated a marked robustness in the face of biomass loading disrupted yeast cells. In addition, each adsorbent was compared with its uncoated native form during the expanded bed adsorption of one of two intracellular proteins (i.e. glyceraldehyde 3-phosphate dehydrogenase and cytochrome c) from a 20% (ww/v) yeast disruptate. The performance parameters of efficiency of washing, purification factor, turbidity of the eluted product and protein recovery in all analysed cases were favourable to the coated materials. In particular, exploiting PCA reduced significantly undesirable adsorption of cells without significant loss of binding capacity for the target product. The generic application of such adsorbents and their potential for the recovery of target products from complex feedstock is discussed, whilst other application such as the subtractive purification of nanoparticles were detailed in our previous publication.

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this “first mycotoxin from lower fungi” is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia . By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.

The potent antimitotic polyketide macrolide rhizoxin, the causal agent of rice seedling blight, is not produced by the fungus Rhizopus microsporus, as has been believed for over two decades, but by endosymbiotic bacteria that reside within the fungal mycelium. Here we report the successful isolation and large-scale fermentation of the bacterial endosymbiont ("Burkholderia rhizoxina") in pure culture, which resulted in a significantly elevated (10x higher) production of antimitotics. In addition to several known rhizoxin derivatives, numerous novel natural and semisynthetic variants were isolated, and their structures were fully elucidated. Cell-based assays as well as tubulin binding experiments revealed that methylated seco-rhizoxin derivatives are 1000-10000 times more active than rhizoxin and thus rank among the most potent antiproliferative agents known to date. Furthermore, more stable didesepoxy rhizoxin analogues were obtained by efficiently inhibiting a putative P-450 monooxygenase involved in macrolide tailoring.

Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic fungus Rhizopus microsporus . The vertically transmitted endosymbiont not only delivers the antimitotic macrolide rhizoxin to its host but is also essential for vegetative spore formation of the fungus. To shed light on the genetic equipment of this model organism, we sequenced the whole genome of B. rhizoxinica HKI 0454, thus providing the first genomic insight into an intracellular mutualist of a fungal species. The 3.75-Mb genome consists of a chromosome and two strain-specific plasmids. The primary metabolism appears to be specialized for the uptake of fungal metabolites. Besides the rhizoxin biosynthesis gene cluster, there are 14 loci coding for nonribosomal peptide synthetase (NRPS) assembly lines, which represent novel targets for genomic mining of cryptic natural products. Furthermore, the endosymbionts are equipped with a repertoire of virulence-related factors, which can now be studied to elucidate molecular mechanisms underlying bacterial-fungal interaction.

SummaryBacterial endosymbionts play essential roles for many organisms, and thus specialized mechanisms have evolved during evolution that guarantee the persistence of the symbiosis during or after host reproduction [1, 2]. The rice seedling blight fungus Rhizopus microsporus represents a unique example of a mutualistic life form in which a fungus harbors endobacteria (Burkholderia sp.) for the production of a phytotoxin [3]. Here we report the unexpected observation that in the absence of endosymbionts, the host is not capable of vegetative reproduction. Formation of sporangia and spores is restored only upon reintroduction of endobacteria. To monitor this process, we succeeded in GFP labeling cultured endosymbionts. We also established a laserbeam transformation technique for the first controlled introduction of bacteria into fungi to observe their migration to the tips of the aseptate hyphae. The persistence of this fungal-bacterial mutualism through symbiont-dependent sporulation is intriguing from an evolutionary point of view and implies that the symbiont produces factors that are essential for the fungal life cycle. Reproduction of the host has become totally dependent on endofungal bacteria, which in return provide a highly potent toxin for defending the habitat and accessing nutrients from decaying plants. This scenario clearly highlights the significance for a controlled maintenance of this fungal-bacterial symbiotic relationship.