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1
Frederick, Reid D., Musharaf Ahmad, Doris R. Majerczak, Angel S. Arroyo-Rodríguez, Shulamit Manulis, and David L. Coplin. "Genetic Organization of the Pantoea stewartii subsp. stewartii hrp Gene Cluster and Sequence Analysis of the hrpA, hrpC, hrpN, and wtsE Operons." Molecular Plant-Microbe Interactions® 14, no. 10 (October 2001): 1213–22. http://dx.doi.org/10.1094/mpmi.2001.14.10.1213.
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The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.
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Schmitt, Corinna, David Turner, Maria Boesl, Marion Abele, Matthias Frosch, and Oliver Kurzai. "A Functional Two-Partner Secretion System Contributes to Adhesion of Neisseria meningitidis to Epithelial Cells." Journal of Bacteriology 189, no. 22 (September 14, 2007): 7968–76. http://dx.doi.org/10.1128/jb.00851-07.
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ABSTRACT Neisseria meningitidis is a frequent commensal of the human nasopharynx causing severe invasive infections in rare cases. A functional two-partner secretion (TPS) system in N. meningitidis, composed of the secreted effector protein HrpA and its cognate transporter HrpB, is identified and characterized in this study. Although all meningococcal strains harbor at least one TPS system, the hrpA genes display significant C-terminal sequence variation. Meningococcal genes encoding the TPS effector proteins and their transporters are closely associated and transcribed into a single mRNA. HrpA proteins are translocated across the meningococcal outer membrane by their cognate transporters HrpB and mainly released into the environment. During this process, HrpA is proteolytically processed to a mature 180-kDa form. In contrast to other known TPS systems, immature HrpA proteins are stable in the absence of HrpB and accumulate within the bacterial cell. A small percentage of mature HrpA remains associated with the bacteria and contributes to the interaction of meningococci with epithelial cells.
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Blackwell, L. J., and J. A. Borowiec. "Human replication protein A binds single-stranded DNA in two distinct complexes." Molecular and Cellular Biology 14, no. 6 (June 1994): 3993–4001. http://dx.doi.org/10.1128/mcb.14.6.3993-4001.1994.
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Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.
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Blackwell, L. J., and J. A. Borowiec. "Human replication protein A binds single-stranded DNA in two distinct complexes." Molecular and Cellular Biology 14, no. 6 (June 1994): 3993–4001. http://dx.doi.org/10.1128/mcb.14.6.3993.
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Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.
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Neil, R. Brock, and Michael A. Apicella. "Role of HrpA in Biofilm Formation of Neisseria meningitidis and Regulation of the hrpBAS Transcripts." Infection and Immunity 77, no. 6 (March 16, 2009): 2285–93. http://dx.doi.org/10.1128/iai.01502-08.
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ABSTRACT Two-partner secretion systems of gram-negative organisms are utilized in adherence, invasion, and biofilm formation. The HrpAB proteins of Neisseria meningitidis are members of a two-partner secretion system, and HrpA is established as being important to adherence and intracellular escape. This study set out to determine the expression pattern of members of the hrpBAS putative operon and to find a functional role for the HrpA protein. The upregulation of these genes was found in situations of anaerobiosis and cell contact. These observations prompted the study of the function of HrpA in biofilms on human bronchial epithelial cells. HrpA mutants in encapsulated and unencapsulated NMB strains demonstrated biofilm growth equivalent to that of the wild-type strain at 6 h but a decreased ability to form biofilms at 48 h. Biofilms formed by hrpA mutants for 48 h on collagen-coated coverslips demonstrated significant reductions compared to those of wild-type strains. Taken together, these observations imply a role for HrpA in the biofilm structure. Further analysis demonstrated the presence of HrpA on the surface of the bacterium.
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Deng, Wen-Ling, Gail Preston, Alan Collmer, Chun-Jung Chang, and Hsiou-Chen Huang. "Characterization of the hrpC and hrpRSOperons of Pseudomonas syringae Pathovars Syringae, Tomato, and Glycinea and Analysis of the Ability of hrpF,hrpG, hrcC, hrpT, and hrpVMutants To Elicit the Hypersensitive Response and Disease in Plants." Journal of Bacteriology 180, no. 17 (1998): 4523–31. http://dx.doi.org/10.1128/jb.180.17.4523-4531.1998.
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The species Pseudomonas syringae encompasses plant pathogens with differing host specificities and corresponding pathovar designations. P. syringae requires the Hrp (type III protein secretion) system, encoded by a 25-kb cluster ofhrp and hrc genes, in order to elicit the hypersensitive response (HR) in nonhosts or to be pathogenic in hosts. DNA sequence analysis of the hrpC and hrpRSoperons of P. syringae pv. syringae 61 (brown spot of beans), P. syringae pv. glycinea U1 (bacterial blight of soybeans), and P. syringae pv. tomato DC3000 (bacterial speck of tomatos) revealed that the 13 genes comprising the right half of the hrp cluster (including those in the previously sequenced hrpZ operon) are conserved and identically arranged. The hrpC operon is comprised of hrpF,hrpG, hrcC, hrpT, and hrpV. hrcC encodes a putative outer membrane protein that is conserved in all type III secretion systems. The other four genes appear to be characteristic of group I Hrp systems, such as those possessed byP. syringae and Erwinia amylovora. The predicted products of these four genes in P. syringae pv. syringae 61 are HrpF (8 kDa), HrpG (15.4 kDa), HrpT (7.5 kDa), and HrpV (13.4 kDa). HrpT is a putative outer membrane lipoprotein. HrpF, HrpG, and HrpV are all hydrophilic proteins lacking N-terminal signal peptides. The HrpG, HrcC, HrpT, and HrpV proteins of P. syringae pathovars syringae and tomato (the two most divergent pathovars) had at least 76% amino acid identity with each other, whereas the HrpF proteins of these two pathovars had only 36% amino acid identity. The HrpF proteins of P. syringae pathovars syringae and glycinea also showed significant similarity to the HrpA pilin protein of P. syringae pathovar tomato. Functionally nonpolar mutations were introduced into each of the genes in thehrpC operon of P. syringae pv. syringae 61 by insertion of an nptII cartridge lacking a transcription terminator. The mutants were assayed for their ability to elicit the HR in nonhost tobacco leaves or to multiply and cause disease in host bean leaves. Mutations in hrpF, hrcC, andhrpT abolished or greatly reduced the ability of P. syringae pv. syringae 61 to elicit the HR in tobacco. ThehrpG mutant had only weakly reduced HR activity, and the activity of the hrpV mutant was indistinguishable from that of the wild type. Each of the mutations could be complemented, but surprisingly, the hrpV subclone caused a reduction in the HR elicitation ability of the ΔhrpV::nptIImutant. The hrpF and hrcC mutants caused no disease in beans, whereas the hrpG, hrpT, and hrpV mutants had reduced virulence. Similarly, thehrcC mutant grew little in beans, whereas the other mutants grew to intermediate levels in comparison with the wild type. These results indicate that HrpC and HrpF have essential functions in the Hrp system, that HrpG and HrpT contribute quantitatively but are not essential, and that HrpV is a candidate negative regulator of the Hrp system.
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Blackwell, L. J., J. A. Borowiec, and I. A. Mastrangelo. "Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase." Molecular and Cellular Biology 16, no. 9 (September 1996): 4798–807. http://dx.doi.org/10.1128/mcb.16.9.4798.
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Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.
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Sangeeta and Arnab Bhattacherjee. "Interdomain dynamics in human Replication Protein A regulates kinetics and thermodynamics of its binding to ssDNA." PLOS ONE 18, no. 1 (January 19, 2023): e0278396. http://dx.doi.org/10.1371/journal.pone.0278396.
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Human Replication Protein A (hRPA) is a multidomain protein that interacts with ssDNA intermediates to provide the latter much-needed stability during DNA metabolism and maintain genomic integrity. Although the ssDNA organization with hRPA was studied recently through experimental means, characterizing the underlying mechanism at the atomic level remains challenging because of the dynamic domain architecture of hRPA and poorly understood heterogeneity of ssDNA-protein interactions. Here, we used a computational framework, precisely tailored to capture protein-ssDNA interactions, and investigated the binding of hRPA with a 60 nt ssDNA. Two distinct binding mechanisms are realized based on the hRPA domain flexibility. For a rigid domain architecture of hRPA, ssDNA binds sequentially with hRPA domains, resulting in slow association kinetics. The binding pathway involves the formation of stable and distinct intermediate states. On contrary, for a flexible domain architecture of hRPA, ssDNA binds synergistically to the A and B domains followed by the rest of hRPA. The domain dynamics in hRPA alleviates the free energy cost of domain orientation necessary for specific binding with ssDNA, leading to fast association kinetics along a downhill binding free energy landscape. An ensemble of free energetically degenerate intermediate states is encountered that makes it arduous to characterize them structurally. An excellent match between our results with the available experimental observations provides new insights into the rich dynamics of hRPA binding to ssDNA and in general paves the way to investigate intricate details of ssDNA-protein interactions, crucial for cellular functioning.
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Haapalainen, Minna, Kristin van Gestel, Minna Pirhonen, and Suvi Taira. "Soluble Plant Cell Signals Induce the Expression of the Type III Secretion System of Pseudomonas syringae and Upregulate the Production of Pilus Protein HrpA." Molecular Plant-Microbe Interactions® 22, no. 3 (March 2009): 282–90. http://dx.doi.org/10.1094/mpmi-22-3-0282.
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Type III protein secretion is essential for the pathogenicity of Pseudomonas syringae on its host plants. Expression of HrpA, a major component of the type III secretion system (T3SS)-associated pilus, was studied both in plant leaves and in vitro using reporter genes. We found that induction of the hrpA promoter was stronger in plants than in vitro, and that the induction was enhanced by both host and nonhost plants of P. syringae pv. tomato. In vitro, the expression was enhanced by cell-free exudates from plant cell suspension cultures, added into the minimal medium. Further analysis of the plant-cell-derived, hrpA-inducing factors showed that they were small and water-soluble compounds, which could signal P. syringae the proximity of living plant cells. We also studied the production and secretion of native HrpA protein in vitro, and detected a plant-signal-dependent increase in HrpA secretion. In contrast to HrpA, the intracellular accumulation or secretion of the other T3SS-dependent proteins were not significantly increased, despite the presence of plant cell-derived, promoter-inducing factors. Thus, the accumulation of HrpA pilin seems to be subjected to a distinct post-transcriptional regulation.
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Grass, Lena M., Jan Wollenhaupt, Tatjana Barthel, Iwan Parfentev, Henning Urlaub, Bernhard Loll, Eberhard Klauck, Haike Antelmann, and Markus C. Wahl. "Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility." Proceedings of the National Academy of Sciences 118, no. 30 (July 21, 2021): e2100370118. http://dx.doi.org/10.1073/pnas.2100370118.
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Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5′ RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity. Structures of an expanded HrpA helicase cassette in the apo and RNA-bound states in combination with cross-linking/mass spectrometry revealed ratchet-like domain movements upon RNA engagement, much more pronounced than hitherto observed in related eukaryotic DEAH/RHA enzymes. Structure-based functional analyses delineated transient interdomain contact sites that support substrate loading and unwinding, suggesting that similar conformational changes support RNA translocation. Consistently, modeling studies showed that analogous dynamic intramolecular contacts are not possible in the related but helicase-inactive RNA-dependent nucleoside-triphosphatase, HrpB. Our results indicate that HrpA may be an interesting target to interfere with bacterial tolerance toward certain antibiotics and suggest possible interfering strategies.
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Daniely, Yaron, and James A. Borowiec. "Formation of a Complex between Nucleolin and Replication Protein a after Cell Stress Prevents Initiation of DNA Replication." Journal of Cell Biology 149, no. 4 (May 15, 2000): 799–810. http://dx.doi.org/10.1083/jcb.149.4.799.
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We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin–hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin–hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.
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Brown, Ian R., John W. Mansfield, Suvi Taira, Elina Roine, and Martin Romantschuk. "Immunocytochemical Localization of HrpA and HrpZ Supports a Role for the Hrp Pilus in the Transfer of Effector Proteins from Pseudomonas syringae pv. tomato Across the Host Plant Cell Wall." Molecular Plant-Microbe Interactions® 14, no. 3 (March 2001): 394–404. http://dx.doi.org/10.1094/mpmi.2001.14.3.394.
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The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 μm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.
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Huguet, Elisabeth, and Ulla Bonas. "hrpF of Xanthomonas campestris pv. vesicatoria Encodes an 87-kDa Protein with Homology to NolX of Rhizobium fredii." Molecular Plant-Microbe Interactions® 10, no. 4 (May 1997): 488–98. http://dx.doi.org/10.1094/mpmi.1997.10.4.488.
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The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. The main hrp (hypersensitive reaction and pathogenicity) gene cluster in X. campestris pv. vesicatoria spans a 23-kb chromosomal region, comprising six complementation groups designated hrpA to hrpF. Analysis of the hrpF locus revealed a single open reading frame encoding HrpF (86.4 kDa). HrpF is predominantly hydrophilic, and contains two hydrophobic domains in the C terminus. An interesting feature is the presence of two imperfect direct repeats in the N-terminal region. Deletion studies showed that one repeat is sufficient for function. Epitope tagging of HrpF allowed detection of the protein in X. campestris pv. vesicatoria. Subcellular localization studies suggest that HrpF is both in the soluble fraction and in the inner membrane. Interestingly, HrpF is 48% identical (67% similar) to the Rhizobium fredii NolX protein that is part of the host specificity locus. Since several Hrp proteins are believed to be components of the type III Hrp protein secretion apparatus, allowing export of proteins essential for the interaction with the plant, the possible role of hrpF and nolX in secretion is discussed.
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Bastas, Kubilay Kurtulus, and Ayşegül Gedük. "Elma ve Erwinia amylovora İnteraksiyonlarında Etkili Proteinler." Turkish Journal of Agriculture - Food Science and Technology 8, sp1 (December 14, 2020): 215–25. http://dx.doi.org/10.24925/turjaf.v8isp1.215-225.3923.
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Fire blight disease caused by Erwinia amylovora can infect almost 140 plants of the Rosaceae family and poses a great threat to pome fruits growing all over the world. It needs amylovoran and Type III secretion systems (T3SS) to cause disease in host plants. AmsB, AmsD, AmsE, AmsF, AmsG, AmsJ, AmsI and AmsK proteins are involved in the binding of different galactose, glucuronic acid and pyruvyl subunits to the lipid carrier to form an amylovoran unit. T3SS proteins secreted by E. amylovora are HrpA HrpN, HrpW, AvrRpt2EA, HopC1 and DspA/E. DspA/E, the sole effector of E. amylovora, is secreted by during the formation of pilus T3SS. The chaperone protein of E. amylovora is DsB/F, which is in the IA class. EopB (outer membrane protein) has been characterized as one of the secretory proteins of E. amylovora. In addition to the harpins, the pathogenicity protein DspE and OrfB proteins are secreted via the Hrp-secretory system of E. amylovora. E. amylovora forms a Hrp pilus, which is produced by the structural protein HrpA. Genes encoding antimicrobial proteins cloned and expressed in apples and pears for impart resistance to the pathogen, attacin E are cecropins and lysozymes. The expression of PR2, PR5 and PR8 proteins is increased with E. amylovora infection in apple. Again, the HIPM protein in apples interacts with the E. amylovora HrpN protein, and the HIPM protein is found in higher amounts in flowers than leaves and shoots. In addition, four apple proteins (DIPMs) that interact with E. amylovora effector protein DspA/E have an effective role in endurance. In order to understand the interaction between the plant and the pathogen, it will be possible to understand the proteins that recognize the pathogen in the host, as well as the signal system and plant defense mechanism resulting from the infection. In this study, the roles of proteins associated with pathogenesis as a result of infection of E. amylovora in apples were tried to be revealed.
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Iftode, C., and J. A. Borowiec. "Denaturation of the simian virus 40 origin of replication mediated by human replication protein A." Molecular and Cellular Biology 17, no. 7 (July 1997): 3876–83. http://dx.doi.org/10.1128/mcb.17.7.3876.
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The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.
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Ramos, Adela R., Joanne E. Morello, Sandeep Ravindran, Wen-Ling Deng, Hsiou-Chen Huang, and Alan Collmer. "Identification of Pseudomonas syringae pv. syringae 61 Type III Secretion System Hrp Proteins That Can Travel the Type III Pathway and Contribute to the Translocation of Effector Proteins into Plant Cells." Journal of Bacteriology 189, no. 15 (May 25, 2007): 5773–78. http://dx.doi.org/10.1128/jb.00435-07.
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ABSTRACT Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.
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Yang, Shihui, Nicole T. Perna, Donald A. Cooksey, Yasushi Okinaka, Steven E. Lindow, A. Mark Ibekwe, Noel T. Keen, and Ching-Hong Yang. "Genome-Wide Identification of Plant-Upregulated Genes of Erwinia chrysanthemi 3937 Using a GFP-Based IVET Leaf Array." Molecular Plant-Microbe Interactions® 17, no. 9 (September 2004): 999–1008. http://dx.doi.org/10.1094/mpmi.2004.17.9.999.
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A green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 E. chrysanthemi 3937 clones, 61 were confirmed as plant upregulated. On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown. Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors. Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes. Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants. Among these, mutants of a purM homolog from Escherichia coli (purM∷Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB∷Tn5, hrcJ∷Tn5, and hrpD∷Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host. Mutants of rhiT from E. chrysanthemi (rhiT∷ Tn5), and an eutR homolog from Salmonella typhimurium (eutR∷Tn5) showed decreased ability to cause systemic inva sion on African violet. However, compared with the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The pheno type of hrpB∷Tn5, hrcC∷Tn5, and hrpD∷Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937.
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Hu, Wenqi, Jing Yuan, Qiao-Ling Jin, Patrick Hart, and Sheng Yang He. "Immunogold Labeling of Hrp Pili of Pseudomonas syringae pv. tomato Assembled in Minimal Medium and In Planta." Molecular Plant-Microbe Interactions® 14, no. 2 (February 2001): 234–41. http://dx.doi.org/10.1094/mpmi.2001.14.2.234.
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Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseudomonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible tomato and Arabidopsis thaliana plants and to elicit the hypersensitive response in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, HrpW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in hrp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilus structural protein on the basis of copurification and mutational analyses. In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labeling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilus antibody revealed a characteristic lineup of gold particles around bacteria and/or at the bacterium-plant contact site. These results confirm that HrpA is the major structural protein of the Hrp pilus and provide evidence that Hrp pili are assembled in vitro and in planta.
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Yang, Shihui, Quan Peng, Qiu Zhang, Xuan Yi, Chang Jae Choi, Ralph M. Reedy, Amy O. Charkowski, and Ching-Hong Yang. "Dynamic Regulation of GacA in Type III Secretion, Pectinase Gene Expression, Pellicle Formation, and Pathogenicity of Dickeya dadantii (Erwinia chrysanthemi 3937)." Molecular Plant-Microbe Interactions® 21, no. 1 (January 2008): 133–42. http://dx.doi.org/10.1094/mpmi-21-1-0133.
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Dickeya dadantii (Erwinia chrysanthemi 3937) secretes exoenzymes, including pectin-degrading enzymes, leading to the loss of structural integrity of plant cell walls. A type III secretion system (T3SS) is essential for full virulence of this bacterium within plant hosts. The GacS/GacA two-component signal transduction system participates in important biological roles in several gram-negative bacteria. In this study, a gacA deletion mutant (Ech137) of D. dadantii was constructed to investigate the effect of this mutation on pathogenesis and other phenotypes. Compared with wild-type D. dadantii, Ech137 had a delayed biofilm-pellicle formation. The production of pectate lyase (Pel), protease, and cellulase was diminished in Ech137 compared with the wild-type cells. Reduced transcription of two endo-Pel genes, pelD and pelL, was found in Ech137 using a green fluorescence protein-based fluorescence-activated cell sorter promoter activity assay. In addition, the transcription of T3SS genes dspE (an effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) was reduced in Ech137. A lower amount of rsmB regulatory RNA was found in gacA mutant Ech137 compared with the wild-type bacterium by quantitative reverse-transcription polymerase chain reaction. Compared with wild-type D. dadantii, a lower amount of hrpL mRNA was observed in Ech137 at 12 h grown in medium. Although the role of RsmA, rsmB, and RsmC in D. dadantii is not clear, from the regulatory pathway revealed in E. carotovora, the lower expression of dspE, hrpA, and hrpN in Ech137 may be due to a posttranscriptional regulation of hrpL through the Gac-Rsm regulatory pathway. Consequently, the reduced exoenzyme production and Pel gene expression in the mutant may be partially due to the regulatory role of rsmB-RsmA on exoenzyme expression. Similar to in vitro results, a lower expression of T3SS and pectinase genes of Ech137 also was observed in bacterial cells inoculated into Saintpaulia ionantha leaves, perhaps accounting for the observed reduction in local maceration. Interestingly, compared with the wild-type D. dadantii, although a lower concentration of Ech137 was observed at day 3 and 4 postinoculation, there is no significant difference in bacterial concentration between the wild-type bacterium and Ech137 in the early stage of infection. Finally, the nearly abolished systemic invasion ability of Ech137 suggests that GacA of D. dadantii is essential for the pathogenicity and systemic movement of the bacterium in S. ionantha.
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Preston, Gail, Wen-Ling Deng, Hsiou-Chen Huang, and Alan Collmer. "Negative Regulation of hrp Genes inPseudomonas syringae by HrpV." Journal of Bacteriology 180, no. 17 (September 1, 1998): 4532–37. http://dx.doi.org/10.1128/jb.180.17.4532-4537.1998.
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ABSTRACT Mutations in the five hrp and hrc genes in the hrpC operon of the phytopathogen Pseudomonas syringae pv. syringae 61 have different effects on bacterial interactions with host and nonhost plants. The hrcC gene within the hrpC operon encodes an outer membrane component of the Hrp secretion system that is conserved in all type III protein secretion systems and is required for most pathogenic phenotypes and for secretion of the HrpZ harpin to the bacterial milieu. The other four genes (in order), hrpF, hrpG, (hrcC), hrpT, and hrpV, appear to be unique to the group I hrp clusters found in certain phytopathogens (e.g., P. syringae and Erwinia amylovora) and are less well understood. We initiated an examination of their role in Hrp regulation and secretion by determining the effects of functionally nonpolar nptIIcartridge insertions in each gene on the production and secretion of HrpZ, as determined by immunoblot analysis of cell fractions. P. syringae pv. syringae 61 hrpF, hrpG, andhrpT mutants were unable to secrete HrpZ, whereas thehrpV mutant overproduced and secreted the protein. This suggested that HrpV is a negative regulator of HrpZ production. Further immunoblot assays showed that the hrpV mutant produced higher levels of proteins encoded by all three of the majorhrp operons tested—HrcJ (hrpZ operon), HrcC (hrpC operon), and HrcQB (hrpUoperon)—and that constitutive expression of hrpV intrans abolished the production of each of these proteins. To determine the hierarchy of HrpV regulation in the P. syringae pv. syringae 61 positive regulatory cascade, which is composed of HrpRS (proteins homologous with ς54-dependent promoter-enhancer-binding proteins) and HrpL (alternate sigma factor), we tested the ability of constitutively expressed hrpV to repress the activation of HrcJ production that normally accompanies constitutive expression of hrpL or hrpRS. No repression was observed, indicating that HrpV acts upstream of HrpRS in the cascade. The effect of HrpV levels on transcription of thehrpZ operon was determined by monitoring the levels of β-glucuronidase produced by ahrpA′::uidA transcriptional fusion plasmid in different P. syringae pv. syringae 61 strains. The hrpV mutant produced higher levels of β-glucuronidase than the wild type, a hrcU (type III secretion) mutant produced the same level as the wild type, and the strain constitutively expressing hrpV in trans produced low levels equivalent to that of a hrpS mutant. These results suggest that HrpF, HrpG, and HrpT are all components of the type III protein secretion system whereas HrpV is a negative regulator of transcription of the Hrp regulon.
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Weber, Ernst, and Ralf Koebnik. "Domain Structure of HrpE, the Hrp Pilus Subunit of Xanthomonas campestris pv. vesicatoria." Journal of Bacteriology 187, no. 17 (September 1, 2005): 6175–86. http://dx.doi.org/10.1128/jb.187.17.6175-6186.2005.
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ABSTRACT The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3Δ2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.
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Ramilo, Cecilia, Liya Gu, Shuangli Guo, Xiping Zhang, Steve M. Patrick, John J. Turchi, and Guo-Min Li. "Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A." Molecular and Cellular Biology 22, no. 7 (April 1, 2002): 2037–46. http://dx.doi.org/10.1128/mcb.22.7.2037-2046.2002.
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ABSTRACT DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.
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Morello, Joanne E., and Alan Collmer. "Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein." Journal of Bacteriology 191, no. 9 (March 6, 2009): 3120–31. http://dx.doi.org/10.1128/jb.01623-08.
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ABSTRACT Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.
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Tabira, Yasuhiro, Naoya Ohara, and Takeshi Yamada. "Identification and characterization of the ribosome-associated protein, HrpA, of Bacillus Calmette-Guérin." Microbial Pathogenesis 29, no. 4 (October 2000): 213–22. http://dx.doi.org/10.1006/mpat.2000.0384.
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Jovanovic, Milija, Christopher Waite, Ellen James, Nicholas Synn, Timothy Simpson, Ioly Kotta-Loizou, and Martin Buck. "Functional Characterization of Key Residues in Regulatory Proteins HrpG and HrpV of Pseudomonas syringae pv. tomato DC3000." Molecular Plant-Microbe Interactions® 30, no. 8 (August 2017): 656–65. http://dx.doi.org/10.1094/mpmi-03-17-0073-r.
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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.
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Iftode, Cristina, and James A. Borowiec. "5‘ → 3‘ Molecular Polarity of Human Replication Protein A (hRPA) Binding to Pseudo-Origin DNA Substrates†." Biochemistry 39, no. 39 (October 2000): 11970–81. http://dx.doi.org/10.1021/bi0005761.
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Ohara, N., N. Ohara, M. Naito, C. Miyazaki, S. Matsumoto, Y. Tabira, and T. Yamada. "HrpA, a new ribosome-associated protein which appears in heat-stressed Mycobacterium bovis bacillus Calmette-Guérin." Journal of bacteriology 179, no. 20 (1997): 6495–98. http://dx.doi.org/10.1128/jb.179.20.6495-6498.1997.
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Pizzolante, Graziano, Roberta Romano, Carla Palumbo, and Pietro Alifano. "Molecular cloning, over-expression, purification, and functional characterization of the meningococcal HrpA protein C-terminal domain." Journal of Biotechnology 185 (September 2014): S98—S99. http://dx.doi.org/10.1016/j.jbiotec.2014.07.335.
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Plener, Laure, Pablo Manfredi, Marc Valls, and Stéphane Genin. "PrhG, a Transcriptional Regulator Responding to Growth Conditions, Is Involved in the Control of the Type III Secretion System Regulon in Ralstonia solanacearum." Journal of Bacteriology 192, no. 4 (December 11, 2009): 1011–19. http://dx.doi.org/10.1128/jb.01189-09.
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ABSTRACT The ability of Ralstonia solanacearum to cause disease in plants depends on its type III secretion system (T3SS). The expression of the T3SS and its effector substrates is coordinately controlled by a regulatory cascade, at the bottom of which is HrpB. Transcription of the hrpB gene is activated by a plant-responsive regulator named HrpG, which is a master regulator of a wide array of pathogenicity functions in R. solanacearum. We have identified in the genome of strain GMI1000 a close paralog of hrpG (83% overall similarity at the protein level) that we have named prhG. Despite this high similarity, the expression pattern of prhG is remarkably different from that of hrpG: prhG expression is activated after growth of bacteria in minimal medium but not in the presence of host cells, while hrpG expression is specifically induced in response to plant cell signals. We provide genetic evidence that prhG is a transcriptional regulator that, like hrpG, controls the expression of hrpB and the hrpB-regulated genes under minimal medium conditions. However, the regulatory functions of prhG and hrpG are distinct: prhG has no influence on hrpB expression when the bacteria are in the presence of plant cells, and transcriptomic profiling analysis of a prhG mutant revealed that the PrhG and HrpG regulons have only one pathogenicity target in common, hrpB. Functional complementation experiments indicated that PrhG and HrpG are individually sufficient to activate hrpB expression in minimal medium. Rather surprisingly, a prhG disruption mutant had little impact on pathogenicity, which may indicate that prhG has a minor role in the activation of T3SS genes when R. solanacearum grows parasitically inside the plant. The cross talk between pathogenicity regulatory proteins and environmental signals described here denotes that an intricate network is at the basis of the bacterial disease program.
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Hwang, In, Patrick Mailliet, Virginie Hossard, Jean-Francois Riou, Anthony Bugaut, and Lauréline Roger. "Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA)." Molecules 24, no. 3 (February 6, 2019): 577. http://dx.doi.org/10.3390/molecules24030577.
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Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods.
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Weber, Ernst, Tuula Ojanen-Reuhs, Elisabeth Huguet, Gerd Hause, Martin Romantschuk, Timo K. Korhonen, Ulla Bonas, and Ralf Koebnik. "The Type III-Dependent Hrp Pilus Is Required for Productive Interaction of Xanthomonas campestris pv. vesicatoria with Pepper Host Plants." Journal of Bacteriology 187, no. 7 (April 1, 2005): 2458–68. http://dx.doi.org/10.1128/jb.187.7.2458-2468.2005.
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ABSTRACT The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation.
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Jovanovic, Milija, Edward Lawton, Jörg Schumacher, and Martin Buck. "Interplay amongPseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system." FEMS Microbiology Letters 356, no. 2 (June 19, 2014): 201–11. http://dx.doi.org/10.1111/1574-6968.12476.
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Yang, Ching-Hong, Marina Gavilanes-Ruiz, Yasushi Okinaka, Regine Vedel, Isabelle Berthuy, Martine Boccara, Jason Wei-Ta Chen, Nicole T. Perna, and Noel T. Keen. "hrp Genes of Erwinia chrysanthemi 3937 Are Important Virulence Factors." Molecular Plant-Microbe Interactions® 15, no. 5 (May 2002): 472–80. http://dx.doi.org/10.1094/mpmi.2002.15.5.472.
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We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN Δpel (Δpel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.
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Kim, Jihyun F., and Steven V. Beer. "HrpW of Erwinia amylovora, a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class." Journal of Bacteriology 180, no. 19 (October 1, 1998): 5203–10. http://dx.doi.org/10.1128/jb.180.19.5203-5210.1998.
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ABSTRACT Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream ofhrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwiniaspecies, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.
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Petnicki-Ocwieja, Tanja, Karin van Dijk, and James R. Alfano. "The hrpK Operon of Pseudomonas syringae pv. tomato DC3000 Encodes Two Proteins Secreted by the Type III (Hrp) Protein Secretion System: HopB1 and HrpK, a Putative Type III Translocator." Journal of Bacteriology 187, no. 2 (January 15, 2005): 649–63. http://dx.doi.org/10.1128/jb.187.2.649-663.2005.
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ABSTRACT Pseudomonas syringae is a gram-negative bacterial plant pathogen that is dependent on a type III protein secretion system (TTSS) and the effector proteins it translocates into plant cells for pathogenicity. The P. syringae TTSS is encoded by hrp-hrc genes that reside in a central region of a pathogenicity island (Pai). Flanking one side of this Pai is the exchangeable effector locus (EEL). We characterized the transcriptional expression of the open reading frames (ORFs) within the EEL of P. syringae pv. tomato DC3000. One of these ORFs, PSPTO1406 (hopB1) is expressed in the same transcriptional unit as hrpK. Both HopB1 and HrpK were secreted in culture and translocated into plant cells via the TTSS. However, the translocation of HrpK required its C-terminal half. HrpK shares low similarity with a putative translocator, HrpF, from Xanthomonas campestris pv. vesicatoria. DC3000 mutants lacking HrpK were significantly reduced in disease symptoms and multiplication in planta, whereas DC3000 hopB1 mutants produced phenotypes similar to the wild type. Additionally, hrpK mutants were reduced in their ability to elicit the hypersensitive response (HR), a programmed cell death associated with plant defense. The reduced HR phenotype exhibited by hrpK mutants was complemented by hrpK expressed in bacteria but not by HrpK transgenically expressed in tobacco, suggesting that HrpK does not function inside plant cells. Further experiments identified a C-terminal transmembrane domain within HrpK that is required for HrpK translocation. Taken together, HopB1 is a type III effector and HrpK plays an important role in the TTSS and is a putative type III translocator.
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Niyukuri, David, Denis Sinzinkayo, Emma V. Troth, Colins O. Oduma, Mediatrice Barengayabo, Mireille Ndereyimana, Aurel Holzschuh, et al. "Performance of highly sensitive and conventional rapid diagnostic tests for clinical and subclinical Plasmodium falciparum infections, and hrp2/3 deletion status in Burundi." PLOS Global Public Health 2, no. 7 (July 28, 2022): e0000828. http://dx.doi.org/10.1371/journal.pgph.0000828.
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Rapid diagnostic tests (RDTs) are a key tool for the diagnosis of malaria infections among clinical and subclinical individuals. Low-density infections, and deletions of the P. falciparum hrp2/3 genes (encoding the HRP2 and HRP3 proteins detected by many RDTs) present challenges for RDT-based diagnosis. The novel Rapigen Biocredit three-band Plasmodium falciparum HRP2/LDH RDT was evaluated among 444 clinical and 468 subclinical individuals in a high transmission setting in Burundi. Results were compared to the AccessBio CareStart HRP2 RDT, and qPCR with a sensitivity of <0.3 parasites/μL blood. Sensitivity compared to qPCR among clinical patients for the Biocredit RDT was 79.9% (250/313, either of HRP2/LDH positive), compared to 73.2% (229/313) for CareStart (P = 0.048). Specificity of the Biocredit was 82.4% compared to 96.2% for CareStart. Among subclinical infections, sensitivity was 72.3% (162/224) compared to 58.5% (131/224) for CareStart (P = 0.003), and reached 88.3% (53/60) in children <15 years. Specificity was 84.4% for the Biocredit and 93.4% for the CareStart RDT. No (0/362) hrp2 and 2/366 hrp3 deletions were observed. In conclusion, the novel RDT showed improved sensitivity for the diagnosis of P. falciparum.
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Fu, Zheng Qing, Ming Guo, and James R. Alfano. "Pseudomonas syringae HrpJ Is a Type III Secreted Protein That Is Required for Plant Pathogenesis, Injection of Effectors, and Secretion of the HrpZ1 Harpin." Journal of Bacteriology 188, no. 17 (September 1, 2006): 6060–69. http://dx.doi.org/10.1128/jb.00718-06.
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ABSTRACT The bacterial plant pathogen Pseudomonas syringae requires a type III protein secretion system (TTSS) to cause disease. The P. syringae TTSS is encoded by the hrp-hrc gene cluster. One of the genes within this cluster, hrpJ, encodes a protein with weak similarity to YopN, a type III secreted protein from the animal pathogenic Yersinia species. Here, we show that HrpJ is secreted in culture and translocated into plant cells by the P. syringae pv. tomato DC3000 TTSS. A DC3000 hrpJ mutant, UNL140, was greatly reduced in its ability to cause disease symptoms and multiply in Arabidopsis thaliana. UNL140 exhibited a reduced ability to elicit a hypersensitive response (HR) in nonhost tobacco plants. UNL140 was unable to elicit an AvrRpt2- or AvrB1-dependent HR in A. thaliana but maintained its ability to secrete AvrB1 in culture via the TTSS. Additionally, UNL140 was defective in its ability to translocate the effectors AvrPto1, HopB1, and AvrPtoB. Type III secretion assays showed that UNL140 secreted HrpA1 and AvrPto1 but was unable to secrete HrpZ1, a protein that is normally secreted in culture in relatively large amounts, into culture supernatants. Taken together, our data indicate that HrpJ is a type III secreted protein that is important for pathogenicity and the translocation of effectors into plant cells. Based on the failure of UNL140 to secrete HrpZ1, HrpJ may play a role in controlling type III secretion, and in its absence, specific accessory proteins, like HrpZ1, may not be extracellularly localized, resulting in disabled translocation of effectors into plant cells.
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Ham, Jong Hyun, Yaya Cui, James R. Alfano, Pablo Rodríguez-Palenzuela, Clemencia M. Rojas, Arun K. Chatterjee, and Alan Collmer. "Analysis of Erwinia chrysanthemi EC16 pelE∷uidA, pelL∷uidA, and hrpN∷uidA Mutants Reveals Strain-Specific Atypical Regulation of the Hrp Type III Secretion System." Molecular Plant-Microbe Interactions® 17, no. 2 (February 2004): 184–94. http://dx.doi.org/10.1094/mpmi.2004.17.2.184.
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The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL∷ΩCmr and hrpS∷ΩCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative σ54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE∷uidA, pelL∷uidA, and hrpN∷uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.
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Meyer, Damien, Sébastien Cunnac, Mareva Guéneron, Céline Declercq, Frédérique Van Gijsegem, Emmanuelle Lauber, Christian Boucher, and Matthieu Arlat. "PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family." Journal of Bacteriology 188, no. 13 (July 1, 2006): 4903–17. http://dx.doi.org/10.1128/jb.00180-06.
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ABSTRACT Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum.
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Wei, Zhongmin, Jihyun F. Kim, and Steven V. Beer. "Regulation of hrp Genes and Type III Protein Secretion in Erwinia amylovora by HrpX/HrpY, a Novel Two-Component System, and HrpS." Molecular Plant-Microbe Interactions® 13, no. 11 (November 2000): 1251–62. http://dx.doi.org/10.1094/mpmi.2000.13.11.1251.
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Two novel regulatory components, hrpX and hrpY, of the hrp system of Erwinia amylovora were identified. The hrpXY operon is expressed in rich media, but its transcription is increased threefold by low pH, nutrient, and temperature levels—conditions that mimic the plant apoplast. hrpXY is autoregulated and directs the expression of hrpL; hrpL, in turn, activates transcription of other loci in the hrp gene cluster (Z.-M. Wei and S. V. Beer, J. Bacteriol. 177:6201-6210, 1995). The deduced amino -acid sequences of hrpX and hrpY are similar to bacterial two-component regulators including VsrA/VsrD of Pseudomonas (Ralstonia) solanacearum, DegS/DegU of Bacillus subtilis, and UhpB/UhpA and NarX/NarP, NarL of Escherichia coli. The N-terminal signal-input domain of HrpX contains PAS domain repeats. hrpS, located downstream of hrpXY, encodes a protein with homology to WtsA (HrpS) of Erwinia (Pantoea) stewartii, HrpR and HrpS of Pseudomonas syringae, and other σ54-dependent, enhancer-binding proteins. Transcription of hrpS also is induced under conditions that mimic the plant apoplast. However, hrpS is not autoregulated, and its expression is not affected by hrpXY. When hrpS or hrpL were provided on multicopy plasmids, both hrpX and hrpY mutants recovered the ability to elicit the hypersensitive reaction in tobacco. This confirms that hrpS and hrpL are not epistatic to hrpXY. A model of the regulatory cascades leading to the induction of the E. amylovora type III system is proposed.
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Ancona, Veronica, Jae Hoon Lee, Tiyakhon Chatnaparat, Jinrok Oh, Jong-In Hong, and Youfu Zhao. "The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora." Journal of Bacteriology 197, no. 8 (February 9, 2015): 1433–43. http://dx.doi.org/10.1128/jb.02551-14.
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ABSTRACTThe hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor inErwinia amylovora. Previous studies have demonstrated that the T3SS inE. amylovorais transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence ofE. amylovorawas investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp0) as well as adksAmutant ofE. amylovorawas completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence ofE. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp0anddksAmutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp0anddksAmutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size ofE. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activatedhrpAandhrpLpromoter activities inhrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles inE. amylovoravirulence and indicated thatE. amylovorautilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence.IMPORTANCEThe type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of anspoTnull mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.
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Hutcheson, Steven W., Jamie Bretz, Thomas Sussan, Songmu Jin, and Kyong Pak. "Enhancer-Binding Proteins HrpR and HrpS Interact To Regulate hrp-Encoded Type III Protein Secretion inPseudomonas syringae Strains." Journal of Bacteriology 183, no. 19 (October 1, 2001): 5589–98. http://dx.doi.org/10.1128/jb.183.19.5589-5598.2001.
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ABSTRACT In Pseudomonas syringae strains, thehrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity.hrpR and hrpS were shown to be expressed as an operon: a promoter was identified 5′ to hrpR, and reverse transcriptase PCR detected the presence of anhrpRS transcript. The hrpR promoter and coding sequence were conserved among P. syringaestrains. The coding sequences for hrpR andhrpS were cloned into compatible expression vectors, and their activities were monitored in Escherichia colitransformants carrying an hrpL′-lacZfusion. HrpS could function as a weak activator of thehrpL promoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain. This finding is consistent with a requirement for both HrpR and HrpS in the activation of the hrpL promoter. By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex. Physical interaction of HrpR and HrpS was confirmed by column-binding experiments. The results show that HrpR and HrpS physically interact to regulate the ς54-dependenthrpL promoter in P. syringae strains.
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Li, Yu-Rong, Yi-Zhou Che, Hua-Song Zou, Yi-Ping Cui, Wei Guo, Li-Fang Zou, Eulandria M. Biddle, Ching-Hong Yang, and Gong-You Chen. "Hpa2 Required by HrpF To Translocate Xanthomonas oryzae Transcriptional Activator-Like Effectors into Rice for Pathogenicity." Applied and Environmental Microbiology 77, no. 11 (April 8, 2011): 3809–18. http://dx.doi.org/10.1128/aem.02849-10.
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ABSTRACTXanthomonas oryzaepv. oryzicola, the causative agent of bacterial leaf streak, injects a plethora of effectors through the type III secretion system (T3SS) into rice cells to cause disease. The T3SS, encoded by thehrpgenes, is essential for the pathogen to elicit the hypersensitive response (HR) in nonhost tobacco and for pathogenicity in host rice. Whether or not a putative lytic transglycosylase, Hpa2, interacts with a translocon protein, HrpF, to facilitate bacterial pathogenicity remains unknown. Here we demonstrated that both thehpa2andhrpFgenes are required for the pathogenicity ofX. oryzaepv. oryzicola strain RS105 in rice but not for HR induction in tobacco. The expression ofhpa2was positively regulated by HrpG and HrpD6 but not by HrpX.In vivosecretion and subcellular localization analyses confirmed that Hpa2 secretion is dependent on HpaB (a T3SS exit protein) and that Hpa2 binds to the host cell membrane. Protein-protein assays demonstrated that Hpa2 interacts with HrpF.In plantatranslocation of AvrXa10 indicated that the mutation inhpa2andhrpFinhibits the injection of the HpaB-dependent transcriptional activator-like (TAL) effector into rice. These findings suggest that Hpa2 and HrpF form a complex to translocate T3S effectors into plant cells for pathogenesis in host rice.
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Büttner, Daniela, Dirk Nennstiel, Birgit Klüsener, and Ulla Bonas. "Functional Analysis of HrpF, a Putative Type III Translocon Protein from Xanthomonas campestris pv. vesicatoria." Journal of Bacteriology 184, no. 9 (May 1, 2002): 2389–98. http://dx.doi.org/10.1128/jb.184.9.2389-2398.2002.
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ABSTRACT Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell. The TTSS of the plant pathogen Xanthomonas campestris pv. vesicatoria, encoded by the hrp (hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant. One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation. In this study, complementation analyses of an hrpF null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region. Deletion of the N terminus abolished type III secretion of HrpF. Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal. Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation. These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane.
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Noël, Laurent, Frank Thieme, Dirk Nennstiel, and Ulla Bonas. "Two Novel Type III-Secreted Proteins of Xanthomonas campestris pv. vesicatoria Are Encoded within the hrp Pathogenicity Island." Journal of Bacteriology 184, no. 5 (March 1, 2002): 1340–48. http://dx.doi.org/10.1128/jb.184.5.1340-1348.2002.
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ABSTRACT The Hrp type III protein secretion system (TTSS) is essential for pathogenicity of gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. cDNA-amplified fragment length polymorphism and reverse transcription-PCR analyses identified new genes, regulated by key hrp regulator HrpG, in the regions flanking the hrp gene cluster. Sequence analysis revealed genes encoding HpaG, a predicted leucine-rich repeat-containing protein, the lysozyme-like HpaH protein, and XopA and XopD, which are similar in sequence to Hpa1 from Xanthomonas oryzae pv. oryzae and PsvA from Pseudomonas syringae, respectively. XopA and XopD (Xanthomonas outer proteins) are secreted by the Xanthomonas Hrp TTSS and thus represent putative effector proteins. Mutations in xopA, but not in xopD, resulted in reduced bacterial growth in planta and delayed plant reactions in susceptible and resistant host plants. Since the xopD promoter contains a putative hrp box, which is characteristic of hrpL-regulated genes in P. syringae and Erwinia spp., the gene was probably acquired by horizontal gene transfer. Interestingly, the regions flanking the hrp gene cluster also contain insertion sequences and genes for a putative transposase and a tRNAArg. These features suggest that the hrp gene cluster of X. campestris pv. vesicatoria is part of a pathogenicity island.
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Wistner, Sarah C., Ian A. MacDonald, Karly A. Stanley, and Nathaniel A. Hathaway. "Characterization of Hepatoma-Derived Growth Factor-Related Protein 2 Interactions with Heterochromatin." Cells 12, no. 2 (January 14, 2023): 325. http://dx.doi.org/10.3390/cells12020325.
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The expression of genetic information is tightly controlled by chromatin regulatory proteins, including those in the heterochromatin gene repression family. Many of these regulatory proteins work together on the chromatin substrate to precisely regulate gene expression during mammalian development, giving rise to many different tissues in higher organisms from a fixed genomic template. Here we identify and characterize the interactions of two related heterochromatin regulatory proteins, heterochromatin protein 1 alpha (HP1α) and M-phase phosphoprotein 8 (MPP8), with hepatoma-derived growth factor-related protein 2 (HRP2). We find in biochemical experiments that HRP2 copurifies and co-sediments with heterochromatin-associated proteins, including HP1α and MPP8. Using the Chromatin in vivo Assay in multiple cell types, we demonstrate that HP1α-mediated gene repression dynamics are altered by the presence of HRP2. Furthermore, the knockout of HRP2 in MDA-MB-231 cells results in significant changes to chromatin structure and stability, which alter gene expression patterns. Here, we detail a mechanism by which HRP2 contributes to epigenetic transcriptional regulation through engagement with heterochromatin-associated proteins to stabilize the chromatin landscape and influence gene expression.
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Susin, Michelle F., Humberto R. Perez, Regina L. Baldini, and Suely L. Gomes. "Functional and Structural Analysis of HrcA Repressor Protein from Caulobacter crescentus." Journal of Bacteriology 186, no. 20 (October 15, 2004): 6759–67. http://dx.doi.org/10.1128/jb.186.20.6759-6767.2004.
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ABSTRACT A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a σ32-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity.
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van Dijk, Karin, Derrick E. Fouts, Amos H. Rehm, Angela R. Hill, Alan Collmer, and James R. Alfano. "The Avr (Effector) Proteins HrmA (HopPsyA) and AvrPto Are Secreted in Culture from Pseudomonas syringaePathovars via the Hrp (Type III) Protein Secretion System in a Temperature- and pH-Sensitive Manner." Journal of Bacteriology 181, no. 16 (August 15, 1999): 4790–97. http://dx.doi.org/10.1128/jb.181.16.4790-4797.1999.
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ABSTRACT We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways fromPseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22°C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel theP. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name ofhrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.
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Roncarati, Davide, Eva Pinatel, Elisabetta Fiore, Clelia Peano, Stefany Loibman, and Vincenzo Scarlato. "Helicobacter pylori Stress-Response: Definition of the HrcA Regulon." Microorganisms 7, no. 10 (October 11, 2019): 436. http://dx.doi.org/10.3390/microorganisms7100436.
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Bacteria respond to different environmental stresses by reprogramming the transcription of specific genes whose proper expression is critical for their survival. In this regard, the heat-shock response, a widespread protective mechanism, triggers a sudden increase in the cellular concentration of different proteins, including molecular chaperones and proteases, to preserve protein folding and maintain cellular homeostasis. In the medically important gastric pathogen Helicobacter pylori the regulation of the principal heat-shock genes is under the transcriptional control of two repressor proteins named HspR and HrcA. To define the HrcA regulon, we carried out whole transcriptome analysis through RNA-sequencing, comparing the transcriptome of the H. pylori G27 wild type strain to that of the isogenic hrcA-knockout strain. Overall, differential gene expression analysis outlined 49 genes to be deregulated upon hrcA gene inactivation. Interestingly, besides controlling the transcription of genes coding for molecular chaperones and stress-related mediators, HrcA is involved in regulating the expression of proteins whose function is linked to several cellular processes crucial for bacterial survival and virulence. These include cell motility, membrane transporters, Lipopolysaccharide modifiers and adhesins. The role of HrcA as a central regulator of H. pylori transcriptome, as well as its interconnections with the HspR regulon are here analyzed and discussed. As the HrcA protein acts as a pleiotropic regulator, influencing the expression of several stress-unrelated genes, it may be considered a promising target for the design of new antimicrobial strategies.
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Tamura, Naoyuki, Yukio Murata, and Takafumi Mukaihara. "Isolation of Ralstonia solanacearum hrpB constitutive mutants and secretion analysis of hrpB-regulated gene products that share homology with known type III effectors and enzymes." Microbiology 151, no. 9 (September 1, 2005): 2873–84. http://dx.doi.org/10.1099/mic.0.28161-0.
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The Hrp type III secretion system (TTSS) is essential for the pathogenicity of the Gram-negative plant pathogen Ralstonia solanacearum. To examine the secretion of type III effector proteins via the Hrp TTSS, a screen was done of mutants constitutively expressing the hrpB gene, which encodes an AraC-type transcriptional activator for the hrp regulon. A mutant was isolated that in an hrp-inducing medium expresses several hrpB-regulated genes 4·9–83-fold higher than the wild-type. R. solanacearum Hrp-secreted outer proteins PopA and PopC were secreted at high levels into the culture supernatants of the hrpB constitutive (hrpB c) mutant. Using hrpB c mutants, the extracellular secretion of several hrpB-regulated (hpx) gene products that share homology with known type III effectors and enzymes was examined. Hpx23, Hpx24 and Hpx25, which are similar in sequence to Pseudomonas syringae pv. tomato effector proteins HopPtoA1, HolPtoR and HopPtoD1, are also secreted via the Hrp TTSS in R. solanacearum. The secretion of two hpx gene products that share homology with known enzymes, glyoxalase I (Hpx19) and Nudix hydrolase (Hpx26), was also examined. Hpx19 is accumulated inside the cell, but interestingly, Hpx26 is secreted outside the cell as an Hrp-secreted outer protein, suggesting that Hpx19 functions intracellularly but Hpx26 is a novel effector protein of R. solanacearum.