Publications
12 results found
Kotta-Loizou I, 2022, Molecular origins of transcriptional heterogeneity in diazotrophic Klebsiella oxytoca, mSystems, Vol: 7, Pages: 1-13, ISSN: 2379-5077
Phenotypic heterogeneity in clonal bacterial batch cultures has been shown for a range of bacterial systems; however, the molecular origins of such heterogeneity and its magnitude are not well understood. Under conditions of extreme low-nitrogen stress in the model diazotroph Klebsiella oxytoca, we found remarkably high heterogeneity of nifHDK gene expression, which codes for the structural genes of nitrogenase, one key enzyme of the global nitrogen cycle. This heterogeneity limited the bulk observed nitrogen-fixing capacity of the population. Using dual-probe, single-cell RNA fluorescent in situ hybridization, we correlated nifHDK expression with that of nifLA and glnK-amtB, which code for the main upstream regulatory components. Through stochastic transcription models and mutual information analysis, we revealed likely molecular origins for heterogeneity in nitrogenase expression. In the wild type and regulatory variants, we found that nifHDK transcription was inherently bursty, but we established that noise propagation through signaling was also significant. The regulatory gene glnK had the highest discernible effect on nifHDK variance, while noise from factors outside the regulatory pathway were negligible. Understanding the basis of inherent heterogeneity of nitrogenase expression and its origins can inform biotechnology strategies seeking to enhance biological nitrogen fixation. Finally, we speculate on potential benefits of diazotrophic heterogeneity in natural soil environments.
Waite C, Lindstrom-Battle A, Bennett M, et al., 2021, Resource allocation during the transition to diazotrophy in Klebsiella oxytoca, Frontiers in Microbiology, Vol: 12, Pages: 1-20, ISSN: 1664-302X
Free-living nitrogen-fixing bacteria can improve growth yields of some non-leguminous plants and, if enhanced through bioengineering approaches, have the potential to address major nutrient imbalances in global crop production by supplementing inorganic nitrogen fertilizers. However, nitrogen fixation is a highly resource-costly adaptation and is de-repressed only in environments in which sources of reduced nitrogen are scarce. Here we investigate nitrogen fixation (nif) gene expression and nitrogen starvation response signalling in the model diazotroph Klebsiella oxytoca (Ko) M5a1 during ammonium depletion and the transition to growth on atmospheric N2. Exploratory RNA-sequencing revealed that over 50% of genes were differentially expressed under diazotrophic conditions, among which the nif genes are among the most highly expressed and highly upregulated. Isotopically labelled QconCAT standards were designed for multiplexed, absolute quantification of Nif and nitrogen-stress proteins via multiple reaction monitoring mass spectrometry (MRM-MS). Time-resolved Nif protein concentrations were indicative of bifurcation in the accumulation rates of nitrogenase subunits (NifHDK) and accessory proteins. We estimate that the nitrogenase may account for more than 40% of cell protein during diazotrophic growth and occupy approximately half the active ribosome complement. The concentrations of free amino acids in nitrogen-starved cells were insufficient to support the observed rates of Nif protein expression. Total Nif protein accumulation was reduced 10-fold when the NifK protein was truncated and nitrogenase catalysis lost (nifK1-1203), implying that reinvestment of de novo fixed nitrogen is essential for further nif expression and a complete diazotrophy transition. Several amino acids accumulated in non-fixing ΔnifLA and nifK1-1203 mutants, while the rest remained highly stable despite prolonged N starvation. Monitoring post-translational uridylylation of the PII-type
Schumacher J, Waite C, Wang B, 2019, Synthetic transcription factors allowtuneable synthetic control of the complex bacterial nor regulon, EMBO: Creating is Understanding: Synthetic Biology Masters Complexity
Schumacher J, Waite C, 2018, In vivo absolute and relative Nif protein abundances of Klebsiella oxytoca, 13th European Nitrogen Fixation Conference
Gang S, Sarah M, Waite C, et al., 2018, Mutualism between Klebsiella SGM 81 and Dianthus caryophyllus in modulating root plasticity and rhizospheric bacterial density, Plant and Soil, Vol: 424, Pages: 273-288, ISSN: 0032-079X
AimsDianthus caryophyllus is a commercially important ornamental flower. Plant growth promoting rhizobacteria are increasingly applied as bio-fertilisers and bio-fortifiers. We studied the effect of a rhizospheric isolate Klebsiella SGM 81 strain to promote D. caryophyllus growth under sterile and non-sterile conditions, to colonise its root system endophytically and its impact on the cultivatable microbial community. We identified the auxin indole-3-acetic acid (IAA) production of Klebsiella SGM 81 as major bacterial trait most likely to enhance growth of D. caryophyllus.MethodsipdC dependent IAA production of SGM 81 was quantified using LC-MS/MS and localised proximal to D. caryophyllus roots and correlated to root growth promotion and characteristic morphological changes. SGM 81 cells were localised on and within the plant root using 3D rendering confocal microscopy of gfp expressing SGM 81. Using Salkowski reagent IAA production was quantified and localised proximal to roots in situ. The effect of different bacterial titres on rhizosphere bacterial population was CFU enumerated on nutrient agar. The genome sequence of Klebsiella SGM 81 (accession number PRJEB21197) was determined to validate PGP traits and phylogenic relationships.ResultsInoculation of D. caryophyllus roots with Klebsiella SGM 81 drastically promoted plant growth when grown in agar and soil, concomitant with a burst in root hair formation, suggesting an increase in root auxin activity. We sequenced the Klebsiella SGM 81 genome, identified the presence of a canonical ipdC gene in Klebsiella SGM 81, confirmed bacterial production and secretion of IAA in batch culture using LC-MS/MS and localised plant dependent IAA production by SGM 81 proximal to roots. We found Klebsiella SGM 81 to be a rhizoplane and endophytic coloniser of D. caryophyllus roots in a dose dependent manner. We found no adverse effects of SGM 81 on the overall rhizospheric microbial population unless supplied to soil in very high
Jovanovic M, Waite C, James E, et al., 2017, Functional Characterization of Key Residues in Regulatory Proteins HrpG and HrpV of Pseudomonas syringae pv. tomato DC3000, Molecular Plant-Microbe Interactions, Vol: 30, Pages: 656-665, ISSN: 0894-0282
The plant pathogen Pseudomonas syringae pv. tomato DC3000 uses a type III secretion system (T3SS) to transfer effector proteins into the host. The expression of T3SS proteins is controlled by the HrpL σ factor. Transcription of hrpL is σ54-dependent and bacterial enhancer-binding proteins HrpR and HrpS coactivate the hrpL promoter. The HrpV protein imposes negative control upon HrpR and HrpS through direct interaction with HrpS. HrpG interacts with HrpV and relieves such negative control. The sequence alignments across Hrp group I-type plant pathogens revealed conserved HrpV and HrpG amino acids. To establish structure–function relationships in HrpV and HrpG, either truncated or alanine substitution mutants were constructed. Key functional residues in HrpV and HrpG are found within their C-terminal regions. In HrpG, L101 and L105 are indispensable for the ability of HrpG to directly interact with HrpV and suppress HrpV-dependent negative regulation of HrpR and HrpS. In HrpV, L108 and G110 are major determinants for interactions with HrpS and HrpG. We propose that mutually exclusive binding of HrpS and HrpG to the same binding site of HrpV governs a transition from negative control to activation of the HrpRS complex leading to HrpL expression and pathogenicity of P. syringae.
Waite C, Schumacher J, Jovanovic M, et al., 2017, Evading plant immunity: feedback control of the T3SS in Pseudomonas syringae., Microbial Cell, Vol: 4, Pages: 137-139, ISSN: 2311-2638
Microbes are responsible for over 10% of the global yield losses in staple crops such as wheat, rice, and maize. Understanding the decision-making strategies that enable bacterial plant pathogens to evade the host immune system and cause disease is essential for managing their ever growing threat to food security. Many utilise the needle-like type III secretion system (T3SS) to suppress plant immunity, by injecting effector proteins that inhibit eukaryotic signalling pathways into the host cell cytoplasm. Plants can in turn evolve resistance to specific pathogens via recognition and blocking of the T3SS effectors, so leading to an ongoing co-evolutionary 'arms race' between pathogen and host pairs. The extracytoplasmic function sigma factor HrpL co-ordinates the expression of the T3SS regulon in the leaf-dwelling Pseudomonas syringae and similar pathogens. Recently, we showed that association of HrpL with a target promoter directly adjacent to the hrpL gene imposes negative autogenous control on its own expression level due to overlapping regulatory elements. Our results suggest that by down-regulating T3SS function, this fine-tuning mechanism enables P. syringae to minimise effector-mediated elicitation of plant immunity.
Waite CJ, Schumacher J, Jovanovic M, et al., 2017, Negative autogenous control of the master type III secretion system regulator HrpL in Pseudomonas syringae, mBio, Vol: 8, ISSN: 2150-7511
The type III secretion system (T3SS) is a principal virulence determi-nant of the model bacterial plant pathogenPseudomonas syringae. T3SS effectorproteins inhibit plant defense signaling pathways in susceptible hosts and elicitevolved immunity in resistant plants. The extracytoplasmic function sigma factorHrpL coordinates the expression of most T3SS genes. Transcription ofhrpLis depen-dent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpSforhrpLpromoter activation.hrpLis oriented adjacently to and divergently from theHrpL-dependent genehrpJ, sharing an intergenic upstream regulatory region. Weshow that association of the RNA polymerase (RNAP)-HrpL complex with thehrpJpromoter element imposes negative autogenous control onhrpLtranscription inP. syringaepv.tomatoDC3000. ThehrpLpromoter was upregulated in a ΔhrpLmu-tant and was repressed by plasmid-bornehrpL. In a minimalEscherichia coliback-ground, the activity of HrpL was sufficient to achieve repression of reconstitutedhrpLtranscription. This repression was relieved if both the HrpL DNA-binding func-tion and thehrp-box sequence of thehrpJpromoter were compromised, implyingdependence upon thehrpJpromoter. DNA-bound RNAP-HrpL entirely occluded theHrpRS and partially occluded the integration host factor (IHF) recognition elementsof thehrpLpromoterin vitro, implicating inhibition of DNA binding by these factorsas a cause of negative autogenous control. A modest increase in the HrpL concen-tration caused hypersecretion of the HrpA1 pilus protein but intracellular accumula-tion of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses.
Buck M, Engl C, Joly N, et al., 2015, In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription., Methods Mol Biol, Vol: 1276, Pages: 53-79
Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ(54)), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ(54)-dependent gene expression.
Schumacher J, 2014, Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics, Frontiers in Plant Science, Vol: 5
Engl C, Waite CJ, McKenna JF, et al., 2014, Chp8, a Diguanylate Cyclase from <i>Pseudomonas syringae</i> pv. Tomato DC3000, Suppresses the Pathogen-Associated Molecular Pattern Flagellin, Increases Extracellular Polysaccharides, and Promotes Plant Immune Evasion, MBIO, Vol: 5, ISSN: 2150-7511
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Lawton E, Jovanovic M, Joly N, et al., 2014, Determination of the Self-Association Residues within a Homomeric and a Heteromeric AAA plus Enhancer Binding Protein, JOURNAL OF MOLECULAR BIOLOGY, Vol: 426, Pages: 1692-1710, ISSN: 0022-2836
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