195 results found
Zhang X, Gao F, Ye F, et al., 2023, Structural Basis of s54 Displacement and Promoter Escape in Bacterial Transcription, Proceedings of the National Academy of Sciences of USA, ISSN: 0027-8424
Sharma S, Roy T, Kashyap Y, et al., 2023, Characterizing and demonstrating the role of <i>Klebsiella</i> SSN1 exopolysaccharide in osmotic stress tolerance using neutron radiography, SCIENTIFIC REPORTS, Vol: 13, ISSN: 2045-2322
Zhang X, Ye F, Gao F, et al., 2022, Mechanisms of DNA opening revealed in AAA+ transcription complex structures, Science Advances, Vol: 8, Pages: 1-12, ISSN: 2375-2548
Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ54), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo–electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ54 amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ54 inhibition while helping to open up DNA, using σ54 amino-terminal peptide as a pry bar.
Kotta-Loizou I, Giuliano MG, Jovanovic M, et al., 2022, The RNA repair proteins RtcAB regulate transcription activator RtcR via its CRISPR-associated Rossmann fold domain, iScience, Vol: 25, ISSN: 2589-0042
CRISPR-associated Rossmann fold (CARF) domain signaling underpins modulation of CRISPR-Cas nucleases; however, the RtcR CARF domain controls expression of two conserved RNA repair enzymes, cyclase RtcA and ligase RtcB. Here, we demonstrate that RtcAB are required for RtcR-dependent transcription activation and directly bind to RtcR CARF. RtcAB catalytic activity is not required for complex formation with CARF, but is essential yet not sufficient for RtcRAB-dependent transcription activation, implying the need for an additional RNA repair-dependent activating signal. This signal differs from oligoadenylates, a known ligand of CARF domains, and instead appears to originate from the translation apparatus: RtcB repairs a tmRNA that rescues stalled ribosomes and increases translation elongation speed. Taken together, our data provide evidence for an expanded range for CARF domain signaling, including the first evidence of its control via in trans protein-protein interactions, and a feed-forward mechanism to regulate RNA repair required for a functioning translation apparatus.
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.
Hofer M, Ren H, Hecker F, et al., 2022, Simulation, analysis and control of a self-propelling heat removal system using supercritical CO<inf>2</inf> under varying boundary conditions, Energy, Vol: 247, ISSN: 0360-5442
The supercritical carbon dioxide (sCO21) heat removal system, which is based on a closed Brayton cycle with sCO2 as a working fluid, is an innovative heat removal system for existing and future nuclear power plants. This paper provides the design, layout and control of the system based on assumptions developed in the project sCO2-4-NPP. A self-propelling operational readiness state enables a fast start-up and consumes only 12% of the design thermal power input. The system is analysed over a wide range of ambient and steam-side conditions in ATHLET, using performance maps for the turbomachinery, which were designed recently. The performance analysis suggests that it is a good option to operate the system at the design compressor inlet temperature of 55 °C at any boundary condition. With decreasing thermal power input, the rotational speed of the turbomachinery must be decreased to keep the system self-propelling. Moreover, the turbomachinery design with a higher surge margin is preferred. By controlling the compressor inlet temperature via the air mass flow rate and turbine inlet temperature via the turbomachinery speed, the heat removal system is successfully operated in interaction with a pressurized water reactor.
Hao M, Ye F, Jovanovic M, et al., 2022, Structures of Class I and Class II transcription complexes reveal the molecular basis of RamA-dependent transcription activation, Advanced Science, Vol: 9, Pages: 1-10, ISSN: 2198-3844
Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ70, together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ70 form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP-σ70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator-specific features in activator binding and uncovers distinct roles of the two C-terminal domains of RNAP α subunit.
Sharma S, Gang S, Schumacher J, et al., 2021, Genomic appraisal of <i>Klebsiella</i> PGPB isolated from soil to enhance the growth of barley, GENES & GENOMICS, Vol: 43, Pages: 869-883, ISSN: 1976-9571
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
Liu J, Tassinari M, Naskar S, et al., 2021, Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodelling superfamily, Cell, Vol: 184, Pages: 3660-3673.e18, ISSN: 0092-8674
Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.
Gang S, Sharma S, Saraf M, et al., 2021, Bacterial indole-3-acetic acid influences soil nitrogen acquisition in barley and chickpea, Plants, Vol: 10, ISSN: 2223-7747
Farming of barley and chickpea is nitrogen (N) fertilizer dependent. Using strategies that increase the nitrogen use efficiency (NUE) and its components, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE) would reduce the N fertilizer application in the soil and its adverse environmental effects. We evaluated the effects of three different strains of diazotroph Klebsiella (K.p. SSN1, K.q. SGM81, and K.o. M5a1) to understand the role of biological nitrogen fixation (BNF) and bacterial indole-3-acetic acid (IAA) on NUE of the plants. A field study revealed that K.p. SSN1 results in profound increment of root surface area by eightfold and threefold compared to uninoculated (control) in barley and chickpea, respectively. We measured significant increase in the plant tissue nitrogen, chlorophyll content, protein content, nitrate reductase activity, and nitrate concentration in the inoculated plants (p ≤ 0.05). Treated barley and chickpea exhibited higher NUE and the components compared to the control plants (K.p. SSN1 ≥ K.q. SGM81> K.o. M5a1). Specifically, K.q. SGM81 treatment in barley increased NUpE by 72%, while in chickpea, K.p. SSN1 increased it by 187%. The substantial improvement in the NUpE and NUE by the auxin producers K.p. SSN1 and K.q. SGM81 compared with non-auxin producer K.o. M5a1 was accompanied by an augmented root architecture suggesting larger contribution of IAA over marginal contribution of BNF in nitrogen acquisition from the soil.
Wang Z, Zhao S, Li Y, et al., 2021, RssB-mediated σ<SUP>S</SUP> Activation is Regulated by a Two-Tier Mechanism via Phosphorylation and Adaptor Protein - IraD, JOURNAL OF MOLECULAR BIOLOGY, Vol: 433, ISSN: 0022-2836
Engl C, Jovanovic G, Brackston RD, et al., 2020, The route to transcription initiation determines the mode of transcriptional bursting in E. coli, Nature Communications, Vol: 11, ISSN: 2041-1723
Transcription is fundamentally noisy, leading to significant heterogeneity across bacterial populations. Noise is often attributed to burstiness, but the underlying mechanisms and their dependence on the mode of promotor regulation remain unclear. Here, we measure E. coli single cell mRNA levels for two stress responses that depend on bacterial sigma factors with different mode of transcription initiation (σ70 and σ54). By fitting a stochastic model to the observed mRNA distributions, we show that the transition from low to high expression of the σ70-controlled stress response is regulated via the burst size, while that of the σ54-controlled stress response is regulated via the burst frequency. Therefore, transcription initiation involving σ54 differs from other bacterial systems, and yields bursting kinetics characteristic of eukaryotic systems.
Gao F, Danson AE, Ye F, et al., 2020, Bacterial enhancer binding proteins - AAA+ proteins in transcription activation, Biomolecules, Vol: 10, ISSN: 2218-273X
Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.
Ye F, Kotta-Loizou I, Jovanovic M, et al., 2020, Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7., eLife, Vol: 9, ISSN: 2050-084X
Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.
Wang Z, Zhao S, Jiang S, et al., 2019, Resonance assignments of N-terminal receiver domain of sigma factor S regulator RssB from <i>Escherichia coli</i>, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 13, Pages: 333-337, ISSN: 1874-2718
Danson AE, Jovanovic M, Buck M, et al., 2019, Mechanisms of σ<SUP>54</SUP>-Dependent Transcription Initiation and Regulation, JOURNAL OF MOLECULAR BIOLOGY, Vol: 431, Pages: 3960-3974, ISSN: 0022-2836
Gang S, Sharma S, Saraf M, et al., 2019, Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method, BIO-PROTOCOL, Vol: 9
Danson A, Jovanovic M, Buck M, et al., 2019, Mechanisms of s54-dependent transcription initiation and regulation, Journal of Molecular Biology, ISSN: 0022-2836
Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation.
Glyde R, Ye F, Jovanovic M, et al., 2018, Structures of bacterial RNA polymerase complexes reveal mechanisms of DNA loading and transcription initiation, Molecular Cell, Vol: 70, Pages: 1111-1120.e3, ISSN: 1097-2765
Gene transcription is carried out by multi-subunit RNA polymerases (RNAP).Transcription initiation is a dynamic multi-step process that involves the opening of the double stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryo electron microscopy to a unique transcription system using 54 (N), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promotercomplex and an initial de novo transcribing complex at 3.4 and 3.7 Å respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilisation that involves coordinated, large scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by 54.
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
Glyde R, Ye F, Darbari V, et al., 2017, Structures of RNA polymerase closed and intermediate complexes revealmechanisms of DNA opening and transcription initiation, Molecular Cell, Vol: 67, Pages: 106-116, ISSN: 1097-2765
Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.
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.
Gosztolai A, Schumacher J, Behrends V, et al., 2017, GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation, Biophysical Journal, Vol: 112, Pages: 2219-2230, ISSN: 1542-0086
Ammonium assimilation in Escherichia coli is regulated by two paralogous proteins (GlnB and GlnK), which orchestrate interactions with regulators of gene expression, transport proteins, and metabolic pathways. Yet how they conjointly modulate the activity of glutamine synthetase, the key enzyme for nitrogen assimilation, is poorly understood. We combine experiments and theory to study the dynamic roles of GlnB and GlnK during nitrogen starvation and upshift. We measure time-resolved in vivo concentrations of metabolites, total and posttranslationally modified proteins, and develop a concise biochemical model of GlnB and GlnK that incorporates competition for active and allosteric sites, as well as functional sequestration of GlnK. The model predicts the responses of glutamine synthetase, GlnB, and GlnK under time-varying external ammonium level in the wild-type and two genetic knock-outs. Our results show that GlnK is tightly regulated under nitrogen-rich conditions, yet it is expressed during ammonium run-out and starvation. This suggests a role for GlnK as a buffer of nitrogen shock after starvation, and provides a further functional link between nitrogen and carbon metabolisms.
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, Mcdonald C, Jovanovic G, et al., 2016, Structure and function of PspA and Vipp1 N-terminal peptides:Insights into the membrane stress sensing and mitigation, BBA Biomembranes, Vol: 1859, Pages: 28-39, ISSN: 0005-2736
The phage shock protein (Psp) response maintains integrity ofthe innermembrane (IM) in responsetoextracytoplasmic stress conditionsand is widely distributed amongstenterobacteria. Its central componentPspA, a member of the IM30 peripheral membrane protein family, acts asa major effector of the systemthrough its direct association with the IM. Under non-stress conditions PspA also negatively regulates its own expression via direct interaction with the AAA+ ATPase PspF. PspA hasa counterpart in cyanobacteria calledVipp1, which is implicated in protection of the thylakoid membranes. PspA’s and Vipp1’s conservedN-terminal regions contain a putative amphipathic helix a (AHa) required for membranebinding.Anadjacent amphipathic helix b (AHb) in PspAis required for imposing negative control uponPspF.Here, purified peptides derived from the putative AH regions of PspA and Vipp1were used to directly probe their effectorand regulatory functions.We observed direct membrane-binding of AHaderived peptides and an accompanying change in secondary structure from unstructuredto alpha-helical establishing them as bonafidemembrane-sensing AH’s. The peptide-binding specificitiesand theireffects on membrane stability depend onmembrane anionic lipid content and stored curvature elastic stress,in agreement withfull length PspA and Vipp1 proteinfunctionalities. AHbof PspA inhibited the ATPase activity of PspF demonstratingits direct regulatory role. These findings provide new insight into the membrane binding and function of PspA and Vipp1and establish that synthetic peptides can be used to probe the structure-function of the IM30 protein family.
Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.
Bonato P, Alves LR, Osaki JH, et al., 2016, The NtrY/NtrX two-component system is involved in controlling nitrate assimilation in Herbaspirillum seropedicae strain SmR1., FEBS Journal, Vol: 283, Pages: 3919-3930, ISSN: 1742-4658
Herbaspirillum seropedicae is a diazotrophic β-Proteobacterium found endophytically associated with gramineae (Poaceae or graminaceous plants) such as rice, sorghum and sugar cane. In this work we show that nitrate-dependent growth in this organism is regulated by the master nitrogen regulatory two-component system NtrB/NtrC, and by NtrY/NtrX which functions to specifically regulate nitrate metabolism. NtrY is a histidine kinase sensor protein predicted to be associated with the membrane and NtrX is the response regulator partner. The ntrYntrX genes are widely distributed in Proteobacteria. In α-Proteobacteria they are frequently located downstream from ntrBC, whereas in β-Proteobacteria these genes are located downstream from genes encoding a RNA methyltransferase and a proline-rich protein with unknown function. The α-Proteobacteria NtrX protein has an AAA+ domain, absent in those from β-Proteobacteria. An ntrY mutant of H. seropedicae showed wild type fixing nitrogen phenotype, but the nitrate dependent growth was abolished. Gene fusion assays indicated that NtrY is involved in the expression of genes coding for the assimilatory nitrate reductase as well as the nitrate-responsive two-component system NarX/NarL (narK and narX promoters, respectively). The purified NtrX protein was capable of binding the narK and narX promoters, and the binding site at the narX promoter for the NtrX protein was determined by DNA footprinting. In silico analyses revealed similar sequences in other promoter regions of H. seropedicae that are related to nitrate assimilation, supporting the role of the NtrY/NtrX system in regulating nitrate metabolism in H. seropedicae. This article is protected by copyright. All rights reserved.
Bradley RW, Buck M, Wang B, 2016, Recognizing and engineering digital-like logic gates and switches in gene regulatory networks, Current Opinion in Microbiology, Vol: 33, Pages: 74-82, ISSN: 1879-0364
A central aim of synthetic biology is to build organisms that can perform useful activities in response to specified conditions. The digital computing paradigm which has proved so successful in electrical engineering is being mapped to synthetic biological systems to allow them to make such decisions. However, stochastic molecular processes have graded input-output functions, thus, bioengineers must select those with desirable characteristics and refine their transfer functions to build logic gates with digital-like switching behaviour. Recent efforts in genome mining and the development of programmable RNA-based switches, especially CRISPRi, have greatly increased the number of parts available to synthetic biologists. Improvements to the digital characteristics of these parts are required to enable robust predictable design of deeply layered logic circuits.
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.