Milija Jovanovic

Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity

The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ54-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ54-RNA polymerase activation.

The role of the conserved phenylalanine in the σ54-interacting GAFTGA motif of bacterial enhancer binding proteins

σ54-dependent transcription requires activation by bacterial enhancer binding proteins (bEBPs). bEBPs are members of the AAA+ (ATPases associated with various cellular activities) protein family and typically form hexameric structures that are crucial for their ATPase activity. The precise mechanism by which the energy derived from ATP hydrolysis is coupled to biological output has several unknowns. Here we use Escherichia coli PspF, a model bEBP involved in the transcription of stress response genes (psp operon), to study determinants of its contact features with the closed promoter complex. We demonstrate that substitution of a highly conserved phenylalanine (F85) residue within the L1 loop GAFTGA motif affects (i) the ATP hydrolysis rate of PspF, demonstrating the link between L1 and the nucleotide binding pocket; (ii) the internal organization of the hexameric ring; and (iii) σ54 interactions. Importantly, we provide evidence for a close relationship between F85 and the −12 DNA fork junction structure, which may contribute to key interactions during the energy coupling step and the subsequent remodelling of the Eσ54 closed complex. The functionality of F85 is distinct from that of other GAFTGA residues, especially T86 where in contrast to F85 a clean uncoupling phenotype is observed.

Identification of the CysB-regulated gene, hslJ, related to the Escherichia coli novobiocin resistance phenotype

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance. Measurements of the HSLJ::lacZ gene fusion expression demonstrated that the hslJ gene is negatively regulated by CysB. In addition we observe the negative autogenous control of HslJ. When the control imposed by CysB is lifted in the cysB mutant, the elevation of Nov resistance can be achieved only in the presence of wild-type hslJ allele. A double cysB hslJ mutant restores the sensitivity to Nov. Overexpression of the wild-type HslJ protein either in a cysB(+) or a cysB(-) background increases the level of Nov resistance indicating that hslJ product is indeed involved in accomplishing this phenotype. The HSLJ::OmegaKan allele encodes the C-terminaly truncated mutant protein HslJ Q121Ter which is not functional in achieving the Nov resistance but when overexpressed induces the psp operon. Finally, we found that inactivation of hslJ does not affect the increased resistance to mecillinam in cysB mutants.

Activity Map of the Escherichia coli RNA Polymerase Bridge Helix

Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.

Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system.

Pseudomonas syringae pv. tomato DC3000, a plant pathogenic gram-negative bacterium, employs the type III secretion system (T3SS) to cause disease in tomato and Arabidopsis and to induce the hypersensitive response in nonhost plants. The expression of T3SS is regulated by the HrpL extracytoplasmic sigma factor. Expression of HrpL is controlled by transcriptional activators HrpR and HrpS and negative regulator HrpV. In this study, we analysed the organization of HrpRS and HrpV regulatory proteins and interplay between them. We identified one key residue I26 in HrpS required for repression by HrpV. Substitution of I26 in HrpS abolishes its interaction with HrpV and impairs interactions between HrpS and HrpR and the self-association of HrpS. We show that HrpS self-associates and can associate simultaneously with HrpR and HrpV. We now propose that HrpS has a central role in the assembly of the regulatory HrpRSV complex. Deletion analysis of HrpR and HrpS proteins showed that C-terminal parts of HrpR and HrpS confer determinants indispensable for their self-assembly.

Determination of the Self-Association Residues within a Homomeric and a Heteromeric AAA + Enhancer Binding Protein

The σ(54)-dependent transcription in bacteria requires specific activator proteins, bacterial enhancer binding protein (bEBP), members of the AAA+ (ATPases Associated with various cellular Activities) protein family. The bEBPs usually form oligomers in order to hydrolyze ATP and make open promoter complexes. The bEBP formed by HrpR and HrpS activates transcription from the σ(54)-dependent hrpL promoter responsible for triggering the Type Three Secretion System in Pseudomonas syringae pathovars. Unlike other bEBPs that usually act as homohexamers, HrpR and HrpS operate as a highly co-dependent heterohexameric complex. To understand the organization of the HrpRS complex and the HrpR and HrpS strict co-dependence, we have analyzed the interface between subunits using the random and directed mutagenesis and available crystal structures of several closely related bEBPs. We identified key residues required for the self-association of HrpR (D32, E202 and K235) with HrpS (D32, E200 and K233), showed that the HrpR D32 and HrpS K233 residues form interacting pairs directly involved in an HrpR-HrpS association and that the change in side-chain length at position 233 in HrpS affects self-association and interaction with the HrpR and demonstrated that the HrpS D32, E200 and K233 are not involved in negative regulation imposed by HrpV. We established that the equivalent residues K30, E200 and E234 in a homo-oligomeric bEBP, PspF, are required for the subunit communication and formation of an oligomeric lock that cooperates with the ATP γ-phosphate sensing PspF residue R227, providing insights into their roles in the heteromeric HrpRS co-complex.

Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae

ABSTRACT The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae. T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL. In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation 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. IMPORTANCE The United Nations Food and Agriculture Organization has warned that agriculture will need to satisfy a 50% to 70% increase in global food demand if the human population reaches 9 billion by 2050 as predicted. However, diseases caused by microbial pathogens represent a major threat to food security, accounting for over 10% of estimated yield losses in staple wheat, rice, and maize crops. Understanding the decision-making strategies employed by pathogens to coordinate virulence and to evade plant defenses is vital for informing crop resistance traits and management strategies. Many plant-pathogenic bacteria utilize the needle-like T3SS to inject virulence factors into host plant cells to suppress defense signaling. Pseudomonas syringae is an economically and environmentally devastating plant pathogen. We propose that the master regulator of its entire T3SS gene set, HrpL, downregulates its own expression to minimize elicitation of plant defenses. Revealing such conserved regulatory strategies will inform future antivirulence strategies targeting plant pathogens. The United Nations Food and Agriculture Organization has warned that agriculture will need to satisfy a 50% to 70% increase in global food demand if the human population reaches 9 billion by 2050 as predicted. However, diseases caused by microbial pathogens represent a major threat to food security, accounting for over 10% of estimated yield losses in staple wheat, rice, and maize crops. Understanding the decision-making strategies employed by pathogens to coordinate virulence and to evade plant defenses is vital for informing crop resistance traits and management strategies. Many plant-pathogenic bacteria utilize the needle-like T3SS to inject virulence factors into host plant cells to suppress defense signaling. Pseudomonas syringae is an economically and environmentally devastating plant pathogen. We propose that the master regulator of its entire T3SS gene set, HrpL, downregulates its own expression to minimize elicitation of plant defenses. Revealing such conserved regulatory strategies will inform future antivirulence strategies targeting plant pathogens.

The Design and Construction of a Set of Modular Synthetic BioLogic Devices for Programming Cells

Modularity is an essential property for rationally engineered standard parts and devices. This principle is now being extended to biological based parts and devices for programming cells. However, the design principles and building blocks which are currently in Synthetic Biology are somewhat limited. In addition, it is important to explore the underlying mechanisms of existing, natural biological systems in order to utilise them in designing novel genetic circuit modules. In this paper, we will describe a set of modular synthetic biological parts and devices that are based in rational design. Particularly, a modular tight-controlled and hypersensitive genetic circuit with digital logic AND function is rationally designed and engineered. They use a sigma factor 54(σ54) dependent hetero-regulation module in the hrp (hypersensitive response and pathogenicity) gene regulatory system for Type III secretion in Pseudomonas syringae. Their inputs and outputs are both promoters and thus do not rely on specific inducible promoters and could drive various cellular responses. It shows that the hrp system has significant potential for building a range of biological parts and devices with good performance and flexibility.

Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation.

Summary Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). 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 cryoelectron microscopy to a unique transcription system using σ54 (σN), 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 promoter complex 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 stabilization 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 σ54.

Functional Characterization of Key Residues in Regulatory Proteins HrpG and HrpV of Pseudomonas syringae pv. tomato DC3000

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.