Reinhold-Hurek, Barbara
Loading...
Preferred name
Reinhold-Hurek, Barbara
Official Name
Reinhold-Hurek, Barbara
9 results
Now showing 1 - 9 of 9
- Some of the metrics are blocked by yourconsent settings
Item type:Publication, Molecular characterization of a novel quorum sensing system of the diazotrophic grass endophyte Azoarcus sp. strain BH72(2010-07-01); ; ; Quorum sensing (QS) is a cell density dependent regulatory mechanism which is involved in regulation of several processes in bacteria. Azoarcus sp. BH72 communicates via QS and conditioned culture supernatant, obtained from a culture grown until the stationary growth phase, shows inducing ability on QS target gene expression.The presented study aimed to investigate the novel QS system of Azoarcus sp. BH72 by applying several methods: Comparative proteomic studies with Azoarcus wild type grown under QS conditions based on two-dimensional gel electrophoresis and mass spectrometry with an additional genome wide microarray approach were performed to detect QS regulated genes and proteins. Beside that, QS experiments were carried out with reporter gene studies by means of determination of beta-glucuronidase activity encoded by the gene uidA. While the QS dependent regulation of the pilAB operon, involved in the formation of type IV pili, could be validated by reporter gene studies, it could be shown for the first time that expression of azo3874, encoding a conserved hypothetical secreted protein, was as well induced under QS conditions. Additionally, the proteome experiments revealed that 18 % of the detected proteins were differentially synthesized under QS conditions. Furthermore, transcriptome studies showed that the expression of around 10 % of all Azoarcus sp. BH72 genes was under QS control. The computational analyses of regulated genes and proteins showed that several cellular processes such as type IV pili formation/regulation, oxidative stress response, transport pathways (iron, nickel, and phosphate) and energy metabolism were altered under QS conditions. The expression of several genes, encoding regulatory proteins were under QS control indicating that quorum sensing regulation is organized in a complex hierarchy in the studied beta-proteobacterium. Comparative proteomic studies with gel-based and gel-free methods with a pilR-mutant revealed that the responser regulator PilR is besides regulation of type IV pili biogenesis involved in activation as well as repression of a number of processes.QS experiments with different bacterial species could clearly show that conditioned culture supernatants from Azoarcus communis SWub3 or Azospira oryzae 6a3 induced the Azoarcus pilAB as well as azo3874 gene expression 2-fold. This observation indicates that Azoarcus sp. BH72 is capable of interspecies communication and that the novel quorum sensing system might be widespread in bacteria.Concluding, the presented study demonstrates that density dependent regulation is important for this grass endophyte.doctoral thesis250 169 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Untersuchungen zu Genregulationskaskaden in Azoarcus sp. BH72 unter besonderer Berücksichtigung interaktionsrelevanter GeneThe aim of this study is to elucidate signal transduction pathways in the grass endophyte Azoarcus sp. BH72. The work comprises two main parts. The first part includes computer based analysis of the Azoarcus sp. BH72 genome and the endophytic metagenome from rice with respect to genes involved in signal transduction and transcription. The second part comprises the experimental investigation of two-component-systems and their role for interaction of Azoarcus sp. BH72 with kallar grass, rice and fungi.doctoral thesis299 208 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Design and application of an oligonucleotide microarray (nifH-phylochip) for nifH gene-based detection of nitrogen-fixing prokaryotes(2005-01-11); ; ; Biological nitrogen fixation, the enzymatic reduction of N:sub:2:/sub: to ammonium, is anexclusively prokaryotic process which is crucial to balance the global nitrogen cycle.The key enzyme of this process -- nitrogenase -- has been highly conserved throughevolution. Transcription of one of the nitrogenase structural genes, nifH, provides apractical genetic marker for nitrogen fixing conditions and diazotrophic activities, as itis not constitutively expressed and is regulated in response to factors that controlnitrogen fixation. Our study focused on developing an oligonucleotide microarray(nifH-phylochip) for the nifH gene-based detection of nitrogen-fixing prokaryotes andapplying it to assess the functional diversity of diazotrophic prokaryotes within thecontext of complex environmental samples.doctoral thesis227 128 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, STUDIES ON THE REGULATION OF GENES RELATED TO NITROGEN FIXATION AND N-ASSIMILATION IN Azoarcus sp. strain BH72:THE ROLE OF NtrBCThe aim of this work is to study the regulation of genes related to nitrogen fixation and N-assimilation in Azoarcus sp. strain BH72 and elucidate the role of NtrBC in this regulatory process. Characterization of the transcripts for nitrogenase and its transcription activator, revealed unusual as well as common features in strain BH72. Success has been made to clone and characterize ntrBC of strain BH72, which act as an important two-component system in sensing and initialising the N-regulatory cascade. An ntrBC deletion mutant although nif in phenotype, was affected in N2 fixation. Nitrate had a severe effect on the ntrBC mutant and even derepression of its nitrogenase. Unusually the 'switching motility' was found to be upregulated in BntrBsp. Interestingly, for the first time, GlnY was found to be expressed (albeit at low level) in the ntrBC mutant along with down regulation of the other two PII proteins. Analyses of the expression of some nitrogen assimilatory genes revealed that their transcription was also affected by nitrogen.Thus it appears that NtrBC of Azoarcus strain BH72 has a significant contribution on the N-metabolism.doctoral thesis343 247 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Determination of factors involved in establishment of Azoarcus sp. strain BH72-rice interaction(2013-01-23); ; ; Endophytic bacteria are known to have many beneficial effects on plants and hence plant endophytic interactions are extensively investigated. To understand and further manipulate endophytic interactions for enhancement of plant growth and productivity, it is necessary to determine factors which are responsible for establishment of such an interaction. Azoarcus sp. strain BH72, our model organism, colonizes roots of rice in high numbers and is also capable of endophytic nitrogen fixation inside the roots. Very few factors involved in establishment of Azoarcus-rice interactions have been investigated till date. In this work, attempts were made to further identify factors which might be responsible for establishment of this interaction. The recently discovered type VI secretion system (T6SS) found in many plant associated bacteria is known to be involved in host interaction in many of these organisms. Two gene clusters (sci and imp) encoding the putative T6SS have also been detected in Azoarcus sp. strain BH72 and are speculated to play an important role in host interaction. Recent, RT-PCR based studies revealed expression of the imp cluster was nitrogen regulated. Experiments were performed to determine transcriptional regulators involved in expression of the imp cluster. This would lead to a better understanding of the molecular basis of plant-endophyte interaction. Compounds secreted by the plant as root exudates are also known to influence behavior of the interacting microbe by alteration of specific gene expression patterns in the microbe. To investigate if Azoarcus sp. strain BH72 is also influenced by root exudates of rice variety Nipponbare, oligonucleotide transcriptome microarrays were applied. The analysis revealed that expression of 4.4% of Azoarcus transcriptome was significantly affected in response to exudates. For few of the differentially expressed genes, functional analysis was performed to determine the rhizosphere competence and colonization capacity of the mutant, where the respective gene was inactivated, in comparison to wild type BH72. The candidate genes chosen for mutagenesis and functional analysis included previously uncharacterized genes like azo2916, encoding for a minor pilin PilX, azo2408 and azo1544, encoding proteins with GGDEF or GGDEF and EAL domains that are responsible for the turnover of a global secondary messenger cyclic-di-GMP and azo3888, encoding a serine threonine kinase responsible for assembly and activation of T6SS. The analysis revealed that these genes were relevant for rhizosphere competence and host colonization, thus suggesting that exudates prepare the organism for initiating an interaction with the host. To further explore the determinants required by Azoarcus sp. strain BH72 to colonize, adapt and proliferate in the host, transcriptomic analysis was performed using the Illumina Sequencing technology with RNA isolated from roots of Oryza sativa cv. Nipponbare infected with Azoarcus sp. strain BH72. Further studies based on the differentially regulated genes obtained from this analysis would certainly broaden our knowledge about the factors involved in plant-endophyte interactions. To summarize, the study provides valuable insight about many previously unknown factors responsible for successful Azoarcus-rice interaction and also opens new avenues to further expand our understanding of this field.doctoral thesis241 112 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Physiological and proteome analyses to investigate the nitrogen fixation mechanism particularly with respect tothe posttranslational regulation of nitrogenase in Azoarcus sp. BH72Nitrogenase activity control in the grass-endophyte Azoarcus sp. BH72 is subjected to a posttranslational regulation, which is accompanied by covalent modification of its nitrogenase Fe-protein that was characterized here. Nitrogen-fixing cells of the betaproteobacterium Azoarcus sp. BH72 inactivate their nitrogenase after stimulation by addition of an external nitrogen source or energy depletion through ADP-ribosylation of the arginine residue 102 inone subunit of the Fe-protein. The modification was detected in the modified peptide sequence applying high-performance liquid-chromatography electrospray ionization/tandem mass spectrometry and was verified by site-directed mutagenesis of the respective arginineresidue. In addition, analysis of the type of the modification led to the establishment of a MALDI-TOF MS (matrix assisted laser desorption/ionization-time of flight mass spectrometry) based method for the detection of a labile posttranslational modification.The Azoarcus sp. BH72 genome harbors a gene that encodes a protein exhibiting sequence similarity to known dinitrogenase reductase ADP-ribosyltransferases (DraT). It possesses two genes, coding for proteins with sequence similarity to dinitrogenase reductase activatingglycohydrolases (DraG). The expression of draT, which is co-transcribed with two genes encoding a hypothetical protein and a hemerythrin (DcrH1), was neither nitrogen nor oxygen regulated. Likewise, the expression of draG2 was independent on the availability of fixed nitrogen, while transcription of draG1 - probably located in an operon with nifQ - was nitrogen regulated. Physiological analysis of deletion mutants of the genes draT, draG1 and draG2 and of a draG1draG2 double mutant led to the characterization of the DraT/DraGsystem in Azoarcus sp. BH72. DraT was responsible for the inactivation of nitrogenase byADP-ribosylation after ammonium addition or anaerobiosis. However, a DraT-independent, ammonium-mediated nitrogenase "switch-off" was also observed. In addition, physiologicalexperiments of the draG-mutants as well as phylogenetic analysis of DraG amino acid sequences revealed that DraG1 mediates the reversibility of the nitrogenase inactivation by removal of the ADP-ribosylation, while DraG2 may have other functions as it only partially complemented a draG1 deletion mutation. Moreover, a putative role of DcrH1 in nitrogenase protection against oxygen damage under conditions of slightly elevated oxygen concentrations is proposed.Comparative proteomic studies led to the identification of proteins putatively important for the formation and function of intracytoplasmic membrane stacks (diazosomes) and for the interaction of Azoarcus sp. BH72 with a fungal endophyte of Kallar grass.doctoral thesis242 446 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Investigating the type VI secretion system in diazotrophic, grass endophyte Azoarcus sp. strain BH72(2014-09-23); ; ; The predicted T6SS gene clusters found within the genome of Azoarcus sp. BH72 were examined and found to share characteristics of T6SSs described in literature. The presence of Hcp in culture supernatants confirmed T6SS activity. Furthermore, deletion of predicted core components manipulated the on and off secretion status. In a secretome analysis, the secreted effector protein(s) could not be distinguished, but T6SS-2 components were more represented than T6SS-1 components. In plant experiments, rice inoculated with a T6SS deficient strain responded differently than when inoculated with wild type. Additionally, the T6SS deficient strain colonized rice seedlings in higher numbers than wild type. Finally, in a quest to identify T6SS transcriptional regulators, several potential regulators were screened. Azo0559 was determined to at least partially activate expression of T6SS-1.doctoral thesis373 139 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, From genomics to proteomics in Azoarcus sp. BH72 a N2-fixing endophytic bacteriumSince the first genome sequence was completed in 1995, the number of publicly available prokaryotic genomes increased exponentially up today, when more than 300 microbial projects had been completed. Within this 'genomic revolution', the complete genome analysis of environmentally and biotechnologically relevant microorganisms constitutes an emerging field of research. In this context the genome-sequencing project of Azoarcus sp. BH72 took place. The diazotrophic endophyte Azoarcus sp. BH72 posses a high biotechnological potential as it is capable of colonizing the interior of rice roots, which is one of the globally most important crops. Moreover, since the rice genome is also sequenced, the Azoarcus-rice system represents a great model for bacteria-grass interaction studies. In the present thesis three main topics, from genomics to functional genomic studies, were covered. The aim of the first part of this work was the characterization of an Azoarcus sp. BH72 bacterial artificial chromosome (BAC) library as well as the building of a physical map of strain BH72 chromosome. Both tools were used for an independent analysis of the genome structure in comparison to a shotgun library with small insert sizes, for contig assembly and gap closure of this shotgun library and for genome comparison analysis. The second main objective of this work was the annotation and analysis of a part of the genome sequence. In particular, genes which belong to the COG categories 'Ion Transporters and Metabolism', and 'Carbohydrates Transport and Metabolism', were studied. This analysis revealed several highlights in the genome sequence, particularly in the iron metabolism, which can be used as a starting point in future studies. Finally, in the last part of this work a functional genomic analysis of Azoarcus sp. BH72 grown under different conditions of N2-fixation was performed, using a differential-display proteomic approach. Proteomic patterns of strain BH72 N2-fixing cells, in pure- and co-culture with the endophytic fungus Acremonium alternatum, showed strongly significant differences. The identification of the major proteins showed that the nitrogen metabolism was very active in both conditions, as well as the carbon metabolism, which was adapted to the carbon sources available. Several membrane proteins were identified which most probably are involved in bacteria-fungus interaction, as well as in bacterial response to fungus metabolites. The results obtained contribute to a better understanding of the Azoarcus sp. BH72 physiology and ecology.doctoral thesis275 166 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Untersuchungen zu den Regulationsmechanismen der Typ IV-Pilingene aus Azoarcus sp. BH72 und der Involvierung von Quorum SensingAzoarcus sp. BH72 is a diazotrophic grass endophyte. The interaction with the rice plant is mediated by the type IV pilus, which is encoded by the pilAB operon. In the genome sequence pilT was identified, which encodes for a putative type IV pilus retraction protein. To gain more insights into the role of type IV pili during the colonization process, the pilT and pilA genes were inactivated by mutagenesis. Both mutants were abolished in twitching motility. Enhanced piliation of the pilT mutant indicates that indeed PilT has a role in pilus retraction. Studies on rice root colonization revealed that the retractile force mediated by PilT is not essential for the bacterial colonization of the plant surface, but that twitching motility is necessary for invasion and establishment inside the plant.Monitoring the pilAB expression with the reporter genes gusA and gfp at various time points revealed an induction of the pilAB gene expression in a cell density dependent manner. Experiments led to the assumption that Azoarcus doesn`t use an AHL based quorum sensing system, but probably uses a novel autoinducer (HSF, hydrophilic signal factor) for intercellular communication.doctoral thesis324 342