The John Innes Centre Publications Repository contains details of all publications resulting from our researchers.
The repository also includes Open Access publications, which can be identified by the icons found on search results.
|Green open access publications are marked by the PDF icon. Click on the publication title, or the PDF icon, and read a pre-print PDF version of the publication.||Gold open access publications have the gold open padlock icon. You can read the full version of these papers on the publishing journal’s website without a subscription.|
The creation of this publications repository was funded by BBSRC.
Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology.
Proceedings of the National Academy of Sciences of the United States of America (115) E8509-E8517
Publisher's version: 10.1073/pnas.1804992115
ID: 59833read more
Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have been made to simplify CBS by refactoring a large number of genes with rearranged polycistrons and synthetic regulatory circuits. Here, a posttranslational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system, where the expression stoichiometry is particularly important. Operon-based nif genes from Klebsiella oxytoca were regrouped into giant genes either by fusing genes together or by expressing polyproteins that are subsequently cleaved with Tobacco Etch Virus protease. After several rounds of selection based on protein expression levels and tolerance toward a remnant C-terminal ENLYFQ-tail, a system with only five giant genes showed optimal nitrogenase activity and supported diazotrophic growth of Escherichia coli This study provides an approach for efficient translation from an operon-based system into a polyprotein-based assembly that has the potential for portable and stoichiometric expression of the complex nitrogenase system in eukaryotic organisms.
Energy shifts induce membrane sequestration of DraG in Rhodospirillum rubrum independent of the ammonium transporters and diazotrophic conditions.
FEMS Microbiology Letters
Publisher's version: 10.1093/femsle/fny176
ID: 59474read more
Metabolic regulation of Rhodospirillum rubrum nitrogenase is mediated at the post- translational level by the enzymes DraT and DraG when subjected to changes in nitrogen or energy status. DraT is activated during switch-off whileDraG is inactivated by reversible membrane association. We confirm here that the ammonium transporter, AmtB1, rather than its paralog AmtB2, is required for ammonium induced switch off. Amongst several substitutions at the N100 position in DraG, only N100K, failed to locate to the membrane following ammonium shock, suggesting loss of interaction through charge repulsion. When switch off was induced by lowering energy levels, either by darkness during photosynthetic growth or oxygen depletion under respiratory conditions, reversible membrane sequestration of DraG was independent of AmtB proteins and occurred even under non-diazotrophic conditions. We propose that under these conditions, changes in redox status or possibly membrane potential induce interactions between DraG and another membrane protein in response to the energy status.
Frontiers in microbiology (9) 472
Publisher's version: 10.3389/fmicb.2018.00472
ID: 58412read more
The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a ?phaC1mutant strain of the diazotrophic bacteriumHerbaspirillum seropedicae. The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochromec-branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the ?phaC1mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.
Hierarchical interactions between Fnr orthologs allows fine-tuning of transcription in response to oxygen in Herbaspirillum seropedicae
Nucleic Acids Research (46 (8)) 39533966
Publisher's version: 10.1093/nar/gky142
ID: 58109read more
Bacteria adjust the composition of their electron transport chain (ETC) to efficiently adapt to oxygen gradients. This involves differential expression of various ETC components to optimize energy generation. In Herbaspirillum seropedicae, reprogramming of gene expression in response to oxygen availability is controlled at the transcriptional level by three Fnr orthologs. Here, we characterised Fnr regulons using a combination of RNA-Seq and ChIP-Seq analysis. We found that Fnr1 and Fnr3 directly regulate discrete groups of promoters (Groups I and II, respectively), and that a third group (Group III) is co-regulated by both transcription factors. Comparison of DNA binding motifs between the three promoter groups suggests Group III promoters are potentially co-activated by Fnr3-Fnr1 heterodimers. Specific interaction between Fnr1 and Fnr3, detected in two-hybrid assays, was dependent on conserved residues in their dimerization interfaces, indicative of heterodimer formation in vivo. The requirements for co-activation of the fnr1 promoter, belonging to Group III, suggest either sequential activation by Fnr3 and Fnr1 homodimers or the involvement of Fnr3-Fnr1 heterodimers. Analysis of Fnr proteins with swapped activation domains provides evidence that co-activation by Fnr1 and Fnr3 at Group III promoters optimises interactions with RNA polymerase to fine-tune transcription in response to prevailing oxygen concentrations.
Diazotrophic Growth Allows Azotobacter vinelandii To Overcome the Deleterious Effects of a glnE Deletion.
Applied and environmental microbiology (83) e00808-17
Publisher's version: 10.1128/AEM.00808-17
ID: 57689read more
Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance.IMPORTANCE In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.
Novel insights into ecological distribution and plant growth promotion by nitrogen-fixing endophytes - how specialised are they?
Environmental microbiology reports (9) 179-181
Publisher's version: 10.1111/1758-2229.12529
ID: 57709read more
The genome sequence of Azoarcus olearis DQS-4T, reveals that it is highly similar to the model Azoarcus strain BH72, with almost 99% average nucleotide identity encoding a highly similar proteome, thus placing the two strains within the same species. The isolation and characterisation of a strain from an industrial location, highly similar to that of BH72, which was previously thought to exist only within the plant environment, is of considerable interest from the ecological point of view and raises the possibility that related plant endophytic bacteria are far more widespread in the environment than originally considered. Overall these findings raise a number of crucial questions concerning the habitat of Azoarcus strains, the relationship between nif gene organisation and plant growth promotion, the precise determinants required for plant colonisation and the importance of physiological traits to the ecology of Azoarcus species.
Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity
Proceedings of the National Academy of Sciences of the United States of America (114) E2460-E2465
Publisher's version: 10.1073/pnas.1620058114
ID: 55750read more
A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.
Biographical Memoirs of Fellows of the Royal Society (62) 483-504
Publisher's version: 10.1098/rsbm.2016.0006
ID: 55602read more
John Postgate was one of the foremost microbiologists of his generation. He is most famous for his lifelong research on sulfate-reducing bacteria and nitrogen fixation and for his seminal contributions to understanding the survival and death of bacteria. John Postgate is also known for his specialist and non-specialist books on science, most notably Microbes and man, first published in 1986 and now in several editions and translated into several languages. He played an important role in the development and dissemination of microbiology and in the leadership of science in the UK. John will also be remembered warmly as the supervisor and mentor of aspiring young microbiologists, several of whom have gone on to distinguished careers in microbiology. His other great love was jazz: he was an amateur cornet player of note, the leader of several jazz groups and a highly knowledgeable writer, reviewer and author of two books on the subject.
Major cereal crops benefit from biological nitrogen fixation when inoculated with the nitrogen-fixing bacterium Pseudomonas protegens Pf-5 X940
Environmental Microbiology (18) 3522-3534
Publisher's version: 10.1111/1462-2920.13376
ID: 55603read more
A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.
Deciphering the Principles of Bacterial Nitrogen Dietary Preferences: a Strategy for Nutrient Containment
mBio (7) e00792-16
Publisher's version: 10.1128/mBio.00792-16
ID: 53715read more
A fundamental question in microbial physiology concerns why organisms prefer certain nutrients to others. For example, among different nitrogen sources, ammonium is the preferred nitrogen source, supporting fast growth, whereas alternative nitrogen sources, such as certain amino acids, are considered to be poor nitrogen sources, supporting much slower exponential growth. However, the physiological/regulatory logic behind such nitrogen dietary choices remains elusive. In this study, by engineering Escherichia coli, we switched the dietary preferences toward amino acids, with growth rates equivalent to that of the wild-type strain grown on ammonia. However, when the engineered strain was cultured together with wild-type E. coli, this growth advantage was diminished as a consequence of ammonium leakage from the transport-and-catabolism (TC)-enhanced (TCE) cells, which are preferentially utilized by wild-type bacteria. Our results reveal that the nitrogen regulatory (Ntr) system fine tunes the expression of amino acid transport and catabolism components to match the flux through the ammonia assimilation pathway such that essential nutrients are retained, but, as a consequence, the fast growth rate on amino acids is sacrificed.
Microbiology & Molecular Biology Reviews (79) 419-35
Publisher's version: 10.1128/MMBR.00038-15
ID: 52636read more
The metabolite 2-oxoglutarate (also known as a-ketoglutarate, 2-ketoglutaric acid, or oxoglutaric acid) lies at the intersection between the carbon and nitrogen metabolic pathways. This compound is a key intermediate of one of the most fundamental biochemical pathways in carbon metabolism, the tricarboxylic acid (TCA) cycle. In addition, 2-oxoglutarate also acts as the major carbon skeleton for nitrogen-assimilatory reactions. Experimental data support the conclusion that intracellular levels of 2-oxoglutarate fluctuate according to nitrogen and carbon availability. This review summarizes how nature has capitalized on the ability of 2-oxoglutarate to reflect cellular nutritional status through evolution of a variety of 2-oxoglutarate-sensing regulatory proteins. The number of metabolic pathways known to be regulated by 2-oxoglutarate levels has increased significantly in recent years. The signaling properties of 2-oxoglutarate are highlighted by the fact that this metabolite regulates the synthesis of the well-established master signaling molecule, cyclic AMP (cAMP), in Escherichia coli.
Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation.
BMC Microbiology (15) 95
Publisher's version: 10.1186/s12866-015-0432-6
ID: 49933read more
Orthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation.
Publisher's version: 10.1111/1462-2920.12887
ID: 52637read more
Molecular mechanisms of plant recognition and colonization by diazotrophic bacteria are barely understood. Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, to promote plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicae SmR1 recovered 1 and 3 days after inoculation. The results indicated that nitrogen metabolism was strongly activated in the rhizosphere and polyhydroxybutyrate storage was mobilized in order to assist the survival of H. seropedicae during the early stages of colonization. Epiphytic cells showed altered transcription levels of several genes associated with polysaccharide biosynthesis, peptidoglycan turnover and outer membrane protein biosynthesis, suggesting reorganization of cell wall envelope components. Specific methyl-accepting chemotaxis proteins and two-component systems were differentially expressed between populations over time, suggesting deployment of an extensive bacterial sensory system for adaptation to the plant environment. An insertion mutation inactivating a methyl-accepting chemosensor induced in planktonic bacteria, decreased chemotaxis towards the plant and attachment to roots. In summary, analysis of mutant strains combined with transcript profiling revealed several molecular adaptations that enable H. seropedicae to sense the plant environment, attach to the root surface and survive during the early stages of maize colonization.
The structural basis for enhancer-dependent assembly and activation of the AAA transcriptional activator NorR.
Molecular Microbiology (95) 1730
Publisher's version: 10.1111/mmi.12844
ID: 48829read more
s(54) -dependent transcription controls a wide range of stress-related genes in bacteria and is tightly regulated. In contrast to s(70) , the s(54) -RNA polymerase holoenzyme forms a stable closed complex at the promoter site that rarely isomerises into transcriptionally competent open complexes. The conversion into open complexes requires the ATPase activity of activator proteins that bind remotely upstream of the transcriptional start site. These activators belong to the large AAA protein family and the majority of them consist of an N-terminal regulatory domain, a central AAA domain and a C-terminal DNA binding domain. Here we use a functional variant of the NorR activator, a dedicated NO sensor, to provide the first structural and functional characterisation of a full length AAA activator in complex with its enhancer DNA. Our data suggest an inter-dependent and synergistic relationship of all three functional domains and provide an explanation for the dependence of NorR on enhancer DNA. Our results show that NorR readily assembles into higher order oligomers upon enhancer binding, independent of activating signals. Upon inducing signals, the N-terminal regulatory domain relocates to the periphery of the AAA ring. Together our data provide an assembly and activation mechanism for NorR.
Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli.
Proceedings of the National Academy of Sciences of the United States of America (111) E3718-3725
Publisher's version: 10.1073/pnas.1411185111
ID: 48406read more
All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium- or iron-dependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.
Proceedings of the National Academy of Sciences of the United States of America (PNAS) E2423-E2430
Publisher's version: 10.1073/pnas.1404097111
ID: 47912read more
To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA-PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria.
Current Opinion in Biotechnology (26) 19-24
Publisher's version: 10.1016/j.copbio.2013.08.006
ID: 45102read more
• The availability of nitrogen is a major limitation to crop production. • Tackling the nitrogen problem is vital for sustainable and secure food production. • Some bacteria fix atmospheric nitrogen to a form available to other organisms. • Crop plants could be engineered to associate with nitrogen-fixing bacteria. • Crop plants could be engineered to express nitrogenase, the nitrogen-fixing enzyme.
A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli.
PLoS Genetics (9) e1003865
Publisher's version: 10.1371/journal.pgen.1003865
ID: 45100read more
Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a s(70) (s(A))-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.
The Herbaspirillum seropedicae SmR1 Fnr orthologs controls the cytochrome composition of the electron transport chain.
Scientific Reports (3) 2544
Publisher's version: 10.1038/srep02544
ID: 43756read more
Using Synthetic Biology to Distinguish and Overcome Regulatory and Functional Barriers Related to Nitrogen Fixation
PLOS One (8) e68677
ID: 42235read more
PLoS ONE (7) e46651
Publisher's version: 10.1371/journal.pone.0046651
ID: 41012read more
The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of sigma54-Dependent Transcription.
Microbiology & Molecular Biology Reviews (76) 497-529
Publisher's version: 10.1128/MMBR.00006-12
ID: 40872read more
BMC Genomics (13) 162
Publisher's version: 10.1186/1471-2164-13-162
ID: 40555read more
Interaction of GlnK with the GAF domain of Herbaspirillum seropedicae NifA mediates NH(4)(+)-regulation.
Biochimie (94) 1041-1047
Publisher's version: 10.1016/j.biochi.2012.01.007
ID: 40088read more
Biochemical Society Transactions (39) 1293-1298
Publisher's version: 10.1042/BST0391293
ID: 39569read more