Metatranscriptomic analysis of community structure and metabolism of the rhizosphere microbiome.

Plant-microbe interactions in the rhizosphere, the region of soil influenced by plant roots, are integral to biogeochemical cycling, and maintenance of plant health and productivity. Interactions between model plants and microbes are well understood, but relatively little is about the plant microbiome. Here, comparative metatranscriptomics was used to determine taxonomic compositions and metabolic responses of microbes in soil and the rhizospheres of wheat, oat and pea. Additionally a wild-type oat was compared to a mutant (sad1) deficient in production of antifungal avenacins. Analyses of taxonomic compositions and functions based on rRNA and protein coding genes agreed that rhizosphere microbiomes differed from soil and between plant species. Pea had a stronger effect than wheat and oat, suggesting distinct cereal and legume microbiomes. Proportions of eukaryotic rRNA in the oat and pea rhizospheres were more than fivefold higher than in the wheat rhizosphere or soil. Nematodes and bacterivorous protozoa were enriched in all rhizospheres, while the pea rhizosphere was highly enriched for fungi. Only the eukaryotic community was distinct from wild-type oat in the sad1 mutant, suggesting avenacins have a broader role than protecting from fungal pathogens. The addition of an internal RNA standard allowed quantitative determination of global transcriptional activity in each environment. This was generally higher in the rhizospheres, particularly pea, than in soil. Taxa known to possess metabolic traits potentially important for rhizosphere colonisation, plant growth promotion and pathogenesis were selected by plants. Such traits included cellulose and other plant polymer degradation, nitrogen fixation, hydrogen oxidation, methylotrophy and antibiotic production. These functions were also more highly expressed in rhizospheres than soil. Microbes also induced functions involved in chemotaxis, motility, attachment, pathogenesis, responses to oxidative stress, cycling of nitrogen and sulphur, acquisition of phosphorous, iron and other metals, as well as metabolism of a variety of sugars, aromatics, organic and amino acids, many plant species specific. Profiling microbial communities with metatranscriptomics allowed comparison of relative and quantitative abundance of microbes and their metabolism, from multiple samples, across all domains of life, without PCR bias. This revealed profound differences in the taxonomic composition and metabolic functions of rhizosphere microbiomes between crop plants and soil

Selección de bioinoculantes agrícolas mediante la caracterización molecular y funcional de bacterias asociadas a Zea mays y su análisis práctico en la mejora del rendimiento de cultivos de maíz, canónigos, albahaca, berros y rúcula

[ES]En la actualidad, la población mundial está en aumento (Soby 2013), por lo que surge la necesidad de incrementar también la producción de alimentos sin afectar por ello al medio ambiente (Sayer and Cassman 2013). Por estos motivos, actualmente se buscan alternativas como los biofertilizantes basados en microorganismos beneficiosos, ya que a través de diferentes mecanismos, son capaces de promover el crecimiento vegetal, como las denominadas PGPB (Plant Growth Promoting Bacteria) (García-Fraile et al. 2015). En este sentido, se aislaron bacterias endófitas de maíz que son capaces de habitar en el interior de los tejidos vegetales. Todos los aislados fueron caracterizados de manera fenotípica y genotípica. Además, se evaluaron su capacidades PGPB in vitro con el fin de seleccionar cepas con capacidades promotoras del crecimiento vegetal. Por lo que finalmente se seleccionaron las cepas de Rhizobium zeae CRZM18RT, nueva especie descrita en este trabajo y Rhizobium leucaenae CRZM52R, con las que realizamos ensayos de producción en los cultivos de maíz, rúcula, albahaca, canónigos y berros, obteniendo prometedores incrementos de estos cultivos con la inoculación de las bacterias citadas. Por otra parte, se seleccionó una cepa de Streptomyces con capacidad para inhibir tanto in vitro como in planta diferentes hongos fitopatógenos. Se secuenció y analizó el genoma de las cepas de interés con el fin de complementar la información de los génes implicados en la capacidad promotora del crecimiento vegetal

The ecophysiology of nitrite-oxidizing bacteria in the genus Nitrospira

Ein Kernprozess des biogeochemischen Stickstoffzyklus in natürlichen Lebensräumen und technischen Anlagen (z. B. Kläranlagen) ist die Nitrifikation. Dieser aerobe Prozess untergliedert sich in zwei Stufen, der oxidativen Umwandlung von Ammoniak zu Nitrit bzw. von Nitrit zu Nitrat. Zwei verschiedene funktionelle Gruppen chemolithotropher Mikroorganismen sind dabei katalytisch an der Stoffumsetzung beteiligt: die Ammoniak oxidierenden Bakterien (AOB) bzw. Archaeen (AOA) und die Nitrit oxidierenden Bakterien (NOB). Ein Hauptaugenmerk in dieser Doktorarbeit wurde auf NOB der Gattung Nitrospira gelegt. Diese Gruppe von Bakterien ist maßgeblich an der Stickstoffeleminierung in Kläranlagen beteiligt und ist darüber hinaus weit verbreitet in den unterschiedlichsten aquatischen und terrestrischen Lebensräumen. Bisher ist wenig bekannt über die Ökophysiologie dieser meist unkultivierbaren NOB. In dieser Arbeit konnte zum ersten Mal auf Metagenomsequenzen dieser Bakteriengruppe zurückgegriffen werden und zusammen mit dem Einsatz verschiedener molekularbiologischer Techniken erschlossen sich bis dato unbekannte ökophysiologische Charakteristika dieser NOB. Der gekoppelte Einsatz von Fluoreszenz in situ Hybridisierung (FISH) und quantitativer Bildanalyse ergab Hinweise für eine Nischendifferenzierung zweier Nitrospira Populationen in einem Biofilm aufgrund ihrer Präferenz für verschiedene Nitritkonzentrationen. Weitere physiologische Adaptationen dieser NOB ergaben sich aus Metagenomanalysen. Nitrospira besitzt das detoxifizierende Enzym Chloritdismutase. Es ist gut möglich, dass Nitrospira den Abbau von Chlor-Komponenten mit der Nitrifizierung an kontaminierten Standorten koppelt. Die Genomdaten lieferten darüber hinaus erste Einblicke in das bisher unbekannte Nitrit-oxidierende System dieser NOB. Diese Doktorarbeit erweitert unser bisheriges Verständnis über die Ökophysiologie Nitrit-oxidierender Nitrospiren. Der gekoppelte Einsatz neuer molekularer Methoden mit ersten Metagenomdaten ermöglichte eine detaillierte Beschreibung dieser langsam wachsenden, meist unkultivierbaren NOB.Nitrification is a key process of the biogeochemical nitrogen-cycle in natural and engineered habitats. The two steps of aerobic nitrification, the oxidation of ammonia to nitrite and subsequently from nitrite to nitrate, are catalysed by two functional groups of chemolithotrophic prokaryotes: the ammonia-oxidizing bacteria (AOB) and archaea (AOA), and the nitrite-oxidizing bacteria (NOB). This thesis focused on the genus Nitrospira, one of the important but yet less intensively studied groups of NOB. Nitrospira-like bacteria are the dominant NOB in wastewater treatment plants and the most diverse known NOB in nature, being widespread in various aquatic and terrestrial habitats. However, ecophysiological and genomic data of these mostly uncultured NOB are either scarce or lacking. Molecular and, for the first time metagenomic data retrieved in the course of this thesis brought forward novel ecophysiological traits of these NOB. Fluorescence in situ hybridization and quantitative image analysis provided indication of a different spatial arrangement of members of two Nitrospira sub-populations in a biofilm suggesting a niche differentiation of the coexisting NOB with respect to their preferred concentrations of nitrite. Metagenome analyses revealed further ecophysiological adaptations of these NOB. Nitrospira harbours a gene encoding the detoxifying enzyme chlorite dismutase. Therefore, Nitrospira might link the bioremediation of chloroxo compounds with nitrogen turnover at contaminated sites. Moreover, the metagenomic data uncovered a unique nitrite-oxidizing system in this NOB. This thesis extends our knowledge of the ecophysiology of nitrite-oxidizing Nitrospira-like bacteria. Combining novel molecular methods with metagenomic data allowed in-depth characterization of this slow-growing, mostly uncultured NOB

Structural studies of three enzymes: telomerase, the methyltransferase CobJ and pectate lyase

PhDThis thesis investigates the structure and function of three enzymes of biotechnological and biomedical interest: telomerase from Caenorhabtidis elegans, pectate lyase from Bacillus subtilis and the methyltransferase CobJ from Rhodobacter capsulatus. Telomerase is a ribonucleoprotein found in all eukaryotes and its function is to maintain telomere length, sustain chromosome integrity and circumvent the end-replication problem. The protein requires two subunits to function, telomerase reverse transcriptase (TERT), the catalytic component, and an intrinsic RNA template (TR). The TR makes telomerase a unique reverse transcriptase using the template in the synthesis of short iterative sequences which cap the ends of telomeres. This work reports the successful cloning of a small and therefore potentially crystallisable TERT from C. elegans and expression trials of this catalytic component. Cobalamin (vitamin B12) is an intricate small molecule belonging to a group of compounds called cyclic tetrapyrroles. Its biosynthesis is achieved through a complex pathway encompassing over thirty different enzyme-mediated reactions. Within this pathway there are seven methyltransferases which add eight S-adenosyl-methionine (SAM) derived methyl groups to the macrocycle. CobJ catalyses the methylation of C17 and ring contraction at C20, this reaction which exudes C20 from the tetrapyrrole ring is unprecedented in nature. In this thesis I report the crystallisation of native CobJ and refinement and validation of a high resolution structure along side co-crystallisation and soaking experiments aimed at capturing an enzyme-tetrapyrrole complex. Pectate lyase (BsPel) is an enzyme secreted from the bacterium B. subtilis, it is one of many enzymes secreted by plant pathogens that is responsible for soft rot disease in plants and vegetables. The lyase utilises anti β-elimination chemistry to cleave an α-1,4- glycosidic link present in polygalacturonate the major component of the plant cell wall. 3 The structure of BsPel in complex with hexagalacturonate and a cobalt metal has been solved confirming the position and role of the putative catalytic base Arg 279 in the abstraction of a proton from C5 in galacturonate.Biotechnology and Biological Science Research Council for Studentship

Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised

A METE mutant of Chlamydomonas reinhardtii provides new perspectives on the evolution of vitamin B12 auxotrophy

Vitamin B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Roughly half of all algae require vitamin B12, and the phylogenetic distribution of this trait suggests that it has evolved on multiple occasions. Previous work using artificial evolution generated a metE mutant of Chlamydomonas reinhardtii (hereafter metE7) that requires B12 for growth. Here, I use metE7 to investigate how a newly-evolved B12 auxotroph might cope with B12 limitation and interact with B12-producing bacteria. Compared to the closely related natural B12 auxotroph Lobomonas rostrata, metE7 has a higher requirement and lower binding affinity for B12. B12 deprivation of metE7 caused an increase in cell diameter, indicative of a decreased rate of cell division relative to growth. Other responses included an accumulation of starch and triacylglycerides at the expense of polar lipids and free fatty acids, and a decrease in photosynthetic pigments, proteins and free amino acids. This is reminiscent of nitrogen deprivation, but closer investigation revealed that B12 deprivation caused a substantial increase in reactive oxygen species, which preceded a rapid decline in cell viability. This might be explained by the observation that there was no induction of non-photochemical quenching, unlike under nitrogen deprivation. The metabolite S-adenosyl homocysteine, a potent inhibitor of methylation, increased substantially during B12 deprivation, as did the transcripts for several enzymes of one-carbon metabolism. The rhizobial bacterium Mesorhizobium loti formed a commensal relationship with wild-type C. reinhardtii, benefiting from the alga’s photosynthate. When co-cultured with metE7 this interaction should be considered a mutualism as the alga was dependent on bacterial B12. Adding B12 or glycerol to the coculture increased the cell density of metE7 and M. loti respectively, revealing that the rate of nutrient transfer between species was the factor limiting growth. It was unsurprising therefore that when grown in triculture, the wild type soon outcompeted metE7, even when M. loti growth and B12 production were fuelled by added glycerol. B12 release was also shown to be critical for mutualism: a mutant of E. coli that released more B12 into the media was, in contrast to its parental strain, able to support metE7. The mutual support of metE7 and M. loti over 6 months during an artificial evolution experiment illustrates that newly-evolved B12 auxotrophs could survive in the presence of B12producers, but it was also demonstrated that M. loti could not produce sufficient B12 to favour the evolution of B12 dependence in C. reinhardtii. In summary, this work has provided an insight into the challenges of evolving B12 auxotrophy making it all the more remarkable that it is such a common trait.Biotechnology and Biological Sciences Research Council (BBSRC

Molecular Aspects of Plant Salinity Stress and Tolerance

This book presents the advances in plant salinity stress and tolerance, including mechanistic insights revealed using powerful molecular tools and multi-omics and gene functions studied by genetic engineering and advanced biotechnological methods. Additionally, the use of plant growth-promoting rhizobacteria in the improvement of plant salinity tolerance and the underlying mechanisms and progress in breeding for salinity-tolerant rice are comprehensively discussed. Clearly, the published data have contributed to the significant progress in expanding our knowledge in the field of plant salinity stress and the results are valuable in developing salinity-stress-tolerant crops; in benefiting their quality and productivity; and eventually, in supporting the sustainability of the world food supply

Comparative genomics for studying the proteomes of mucosal microorganisms

A tremendous number of microorganisms are known to interact with their animal hosts. The outcome of the interactions between microbes and their animal hosts range from modulating the maintenance of homeostasis to the establishment of processes leading to pathogenesis. Of the numerous species known to inhabit humans, the great majority live on mucosal surfaces which are highly defended. Despite their importance in human health, little is known about the molecular and cellular basis of most host-microbe interactions across the tremendous diversity of mucosal-adapted microorganisms. The ever-increasing availability of genome sequence data allows systematic comparative genomics studies to identify proteins with potential important molecular functions at the host-microbe interface. In this study, a genome-wide analysis was performed on 3,021,490 protein sequences derived from 867 complete microbial genome sequences across the three domains of cellular life. The ability of microbes to thrive successfully in a mucosal environment was examined in relation to functional genomics data from a range of publicly available databases. Particular emphasis was placed on the extracytoplasmic proteins of microorganisms that thrive on human mucosal surfaces. These proteins form the interface between the complex host-microbe and microbe-microbe interactions. The large amounts of data involved, combined with the numerous analytical techniques that need to be performed makes the study intractable with conventional bioinformatics. The lack of habitat annotations for microorganisms further compounds the problem of identifying the microbial extracytoplasmic proteins playing important roles in the mucosal environments. In order to address these problems, a distributed high throughput computational workflow was developed, and a system for mining biomedical literature was trained to automatically identify microorganisms’ habitats. The workflow integrated existing bioinformatics tools to identify and characterise protein-targeting signals, cell surface-anchoring features, protein domains and protein families. This study successfully demonstrated a large-scale comparative genomics approach utilising a system called Microbase to harness Grid and Cloud computing technologies. A number of conserved protein domains and families that are significantly associated with a speiii iv cific set of mucosa-inhabiting microorganisms were identified. These conserved protein regions of which their functions were either characterised or unknown, were quite narrow in their coverage of taxa distribution, with only a few protein domains more widely distributed, suggesting that mucosal microorganisms evolved different solutions in their strategies and mechanisms for their survival in the host mucosal environments. Metabolic and biological processes common to many mucosal microorganisms included: carbohydrate and amino acid metabolisms, signal transduction, adhesion to host tissues or contents in mucosal environments (e.g. food remnants, mucins), and resistance to host defence mechanisms. Invasive or virulence factors were also identified in pathogenic strains. Several extracytoplasmic protein families were shared among prominent bacterial members of gut microbiota and microbial eukaryotes known to thrive in the same environment, suggesting that the ability of microbes to adapt to particular niches can be influenced by lateral gene transfer. A large number of conserved regions or protein families that potentially play important roles in the mucosa-microbe interactions were revealed by this study. Several of these candidates were proteins of unknown function. The identified candidates were subjected to more detailed computational analysis providing hypothesis for their function that will be tested experimentally in order to contribute to our understanding of the complex host-microbe interactions. Among the candidates of unknown function, a novel M60-like domain was identified. The domain was deposited in the Pfam database with accession number PF13402. The M60-like domain is shared amongst a broad range of mucosal microorganisms as well as their vertebrate hosts. Bioinformatics analyses of the M60-like domain suggested a potential catalytic function of the conserved motif as gluzincins metalloproteases. Targeting signals were detected across microbial M60-likecontaining proteins. Mucosa-related carbohydrate-binding modules (CBMs), CBM32 was also identified on several proteins containing M60-like domains encoded by known mucosal commensals and pathogens. The co-occurrence of the CBMs and M60-like domain, as well as annotated potential peptidase function unveiled a new functional context for the CBM, which is typically connected with carbohydrate processing enzymes but not proteases. The CBM domains linked with members of different protease families are likely to enable these proteases to bind to specific glycoproteins from host animals further highlighting the importance of proteases and CBMs (CBM32 and CBM5_12) in host-microbe interactions.EThOS - Electronic Theses Online ServiceMedical School, Newcastle UniversityGBUnited Kingdo

Senescence

The book "Senescence" is aimed to describe all the phenomena related to aging and senescence of all forms of life on Earth, i.e. plants, animals and the human beings. The book contains 36 carefully reviewed chapters written by different authors, aiming to describe the aging and senescent changes of living creatures, i.e. plants and animals