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Nishino, Kunihiko, and Akihito Yamaguchi. "Role of Histone-Like Protein H-NS in Multidrug Resistance of Escherichia coli." Journal of Bacteriology 186, no. 5 (March 1, 2004): 1423–29. http://dx.doi.org/10.1128/jb.186.5.1423-1429.2004.
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ABSTRACT The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulation of enteric bacteria. It is known that the expression of a variety of genes is repressed by H-NS, and mutations in hns result in various phenotypes, but the role of H-NS in the drug resistance of Escherichia coli has not been known. Here we present data showing that H-NS contributes to multidrug resistance by regulating the expression of multidrug exporter genes. Deletion of the hns gene from the ΔacrAB mutant increased levels of resistance against antibiotics, antiseptics, dyes, and detergents. Decreased accumulation of ethidium bromide and rhodamine 6G in the hns mutant compared to that in the parental strain was observed, suggesting the increased expression of some drug exporter(s) in this mutant. The increased drug resistance and decreased drug accumulation caused by the hns deletion were completely suppressed by deletion of the multifunctional outer membrane channel gene tolC. At least eight drug exporter systems require TolC for their functions. Among these, increased expression of acrEF, mdtEF, and emrKY was observed in the Δhns strain by quantitative real-time reverse transcription-PCR analysis. The Δhns-mediated multidrug resistance pattern is quite similar to that caused by overproduction of the AcrEF exporter. Deletion of the acrEF gene greatly suppressed the level of Δhns-mediated multidrug resistance. However, this strain still retained resistance to some compounds. The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of the MdtEF exporter. Double deletion of the mdtEF and acrEF genes completely suppressed Δhns-mediated multidrug resistance, indicating that Δhns-mediated multidrug resistance is due to derepression of the acrEF and mdtEF drug exporter genes.
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Jian, Huahua, Guanpeng Xu, Yingbao Gai, Jun Xu, and Xiang Xiao. "The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3." Applied and Environmental Microbiology 82, no. 8 (February 12, 2016): 2388–98. http://dx.doi.org/10.1128/aem.00297-16.
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ABSTRACTAlthough the histone-like nucleoid structuring protein (H-NS) is well known for its involvement in the adaptation of mesophilic bacteria, such asEscherichia coli, to cold environments and high-pressure stress, an understanding of the role of H-NS in the cold-adapted benthic microorganisms that live in the deep-sea ecosystem, which covers approximately 60% of the earth's surface, is still lacking. In this study, we characterized the function of H-NS inShewanella piezotoleransWP3, which was isolated from West Pacific sediment at a depth of 1,914 m. Anhnsgene deletion mutant (WP3Δhns) was constructed, and comparative whole-genome microarray analysis was performed. H-NS had a significant influence (fold change, >2) on the expression of a variety of WP3 genes (274 and 280 genes were upregulated and downregulated, respectively), particularly genes related to energy production and conversion. Notably, WP3Δhnsexhibited higher expression levels of lateral flagellar genes than WP3 and showed enhanced swarming motility and lateral flagellar production compared to those of WP3. The DNA gel mobility shift experiment showed that H-NS bound specifically to the promoter of lateral flagellar genes. Moreover, the high-affinity binding sequences of H-NS were identified by DNase I protection footprinting, and the results support the “binding and spreading” model for H-NS functioning. To our knowledge, this is the first attempt to characterize the function of the universal regulator H-NS in a deep-sea bacterium. Our data revealed that H-NS has a novel function as a repressor of the expression of genes related to the energy-consuming secondary flagellar system and to swarming motility.
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Kim, Eun A., and David F. Blair. "Function of the Histone-Like Protein H-NS in Motility of Escherichia coli: Multiple Regulatory Roles Rather than Direct Action at the Flagellar Motor." Journal of Bacteriology 197, no. 19 (July 20, 2015): 3110–20. http://dx.doi.org/10.1128/jb.00309-15.
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ABSTRACTA number of investigations ofEscherichia colihave suggested that the DNA-binding protein H-NS, in addition to its well-known functions in chromosome organization and gene regulation, interacts directly with the flagellar motor to modulate its function. Here, in a study initially aimed at characterizing the H-NS/motor interaction further, we identify problems and limitations in the previous work that substantially weaken the case for a direct H-NS/motor interaction. Nullhnsmutants are immotile, largely owing to the downregulation of the flagellar master regulators FlhD and FlhC. We, and others, previously reported that anhnsmutant remains poorly motile even when FlhDC are expressed constitutively. In the present work, we use better-engineered strains to show that the motility defect in a Δhns, FlhDC-constitutive strain is milder than that reported previously and does not point to a direct action of H-NS at the motor. H-NS regulates numerous genes and might influence motility via a number of regulatory molecules besides FlhDC. To examine the sources of the motility defect that persists in an FlhDC-constitutive Δhnsmutant, we measured transcript levels and overexpression effects of a number of genes in candidate regulatory pathways. The results indicate that H-NS influences motility via multiple regulatory linkages that include, minimally, the messenger molecule cyclic di-GMP, the biofilm regulatory protein CsgD, and the sigma factors σSand σF. The results are in accordance with the more standard view of H-NS as a regulator of gene expression rather than a direct modulator of flagellar motor performance.IMPORTANCEData from a number of previous studies have been taken to indicate that the nucleoid-organizing protein H-NS influences motility not only by its well-known DNA-based mechanisms but also by binding directly to the flagellar motor to alter function. In this study, H-NS is shown to influence motility through diverse regulatory pathways, but a direct interaction with the motor is not supported. Previous indications of a direct action at the motor appear to be related to the use of nonnull strains and, in some cases, a failure to effectively bypass the requirement for H-NS in the expression of the flagellar regulon. These findings call for a substantially revised interpretation of the literature concerning H-NS and flagellar motility and highlight the importance of H-NS in diverse regulatory processes involved in the motile-sessile transition.
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Paytubi, Sonia, Jesús García, and Antonio Juárez. "Bacterial Hha-like proteins facilitate incorporation of horizontally transferred DNA." Open Life Sciences 6, no. 6 (December 1, 2011): 879–86. http://dx.doi.org/10.2478/s11535-011-0071-3.
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AbstractHorizontal gene transfer (HGT), non-hereditary transfer of genetic material between organisms, accounts for a significant proportion of the genetic variability in bacteria. In Gram negative bacteria, the nucleoid-associated protein H-NS silences unwanted expression of recently acquired foreign DNA. This, in turn, facilitates integration of the incoming genes into the regulatory networks of the recipient cell. Bacteria belonging to the family Enterobacteriaceae express an additional protein, the Hha protein that, by binding to H-NS, potentiates silencing of HGT DNA. We provide here an overview of Hha-like proteins, including their structure and function, as well as their evolutionary relationship. We finally present available information suggesting that, by expressing Hha-like proteins, bacteria such as Escherichia coli facilitate HGT incorporation and hence, the impact of HGT in their genetic diversity.
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Castang, Sandra, and Simon L. Dove. "Basis for the Essentiality of H-NS Family Members in Pseudomonas aeruginosa." Journal of Bacteriology 194, no. 18 (July 20, 2012): 5101–9. http://dx.doi.org/10.1128/jb.00932-12.
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ABSTRACTMembers of the histone-like nucleoid-structuring (H-NS) family of proteins have been shown to play important roles in silencing gene expression and in nucleoid compaction. InPseudomonas aeruginosa, the two H-NS family members MvaT and MvaU are thought to bind the same AT-rich regions of the chromosome and function coordinately to control a common set of genes. Here we present evidence that the loss of both MvaT and MvaU cannot be tolerated because it results in the production of Pf4 phage that superinfect and kill cells or inhibit their growth. Using a ClpXP-based protein depletion system in combination with transposon mutagenesis, we identify mutants ofP. aeruginosathat can tolerate the depletion of MvaT in an ΔmvaUmutant background. Many of these mutants contain insertions in genes encoding components, assembly factors, or regulators of type IV pili or contain insertions in genes of the prophage Pf4. We demonstrate that cells that no longer produce type IV pili or that no longer produce the replicative form of the Pf4 genome can tolerate the loss of both MvaT and MvaU. Furthermore, we show that the loss of both MvaT and MvaU results in an increase in expression of Pf4 genes and that cells that cannot produce type IV pili are resistant to infection by Pf4 phage. Our findings suggest that type IV pili are the receptors for Pf4 phage and that the essential activities of MvaT and MvaU are to repress the expression of Pf4 genes.
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Stratmann, Thomas, S. Madhusudan, and Karin Schnetz. "Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO." Journal of Bacteriology 190, no. 3 (November 30, 2007): 926–35. http://dx.doi.org/10.1128/jb.01447-07.
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ABSTRACT The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ.
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Forns, Núria, Rosa C. Baños, Carlos Balsalobre, Antonio Juárez, and Cristina Madrid. "Temperature-Dependent Conjugative Transfer of R27: Role of Chromosome- and Plasmid-Encoded Hha and H-NS Proteins." Journal of Bacteriology 187, no. 12 (June 15, 2005): 3950–59. http://dx.doi.org/10.1128/jb.187.12.3950-3959.2005.
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ABSTRACT IncHI plasmids encode multiple-antibiotic resistance in Salmonella enterica serovar Typhi. These plasmids have been considered to play a relevant role in the persistence and reemergence of this microorganism. The IncHI1 plasmid R27, which can be considered the prototype of IncHI plasmids, is thermosensitive for transfer. Conjugation frequency is highest at low temperature (25 to 30°C), decreasing when temperature increases. R27 codifies an H-NS-like protein (open reading frame 164 [ORF164]) and an Hha-like protein (ORF182). The H-NS and Hha proteins participate in the thermoregulation of gene expression in Escherichia coli. Here we investigated the hypothetical role of such proteins in thermoregulation of R27 conjugation. At a nonpermissive temperature (33°C), transcription of several ORFs in both transfer region 1 (Tra1) and Tra2 from R27 is upregulated in cells depleted of Hha-like and H-NS-like proteins. Both chromosome- and plasmid-encoded Hha and H-NS proteins appear to potentially modulate R27 transfer. The function of R27-encoded Hha-like and H-NS proteins is not restricted to modulation of R27 transfer. Different mutant phenotypes associated with both chromosomal hha and hns mutations are compensated in cells harboring R27.
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Sonden, B., and B. E. Uhlin. "Coordinated and differential expression of histone-like proteins in Escherichia coli: regulation and function of the H-NS analog StpA." EMBO Journal 15, no. 18 (September 1996): 4970–80. http://dx.doi.org/10.1002/j.1460-2075.1996.tb00877.x.
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Wang, Yan, Yiquan Zhang, Zhe Yin, Jie Wang, Yongzhe Zhu, Haoran Peng, Dongsheng Zhou, Zhongtian Qi, and Wenhui Yang. "H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus." Canadian Journal of Microbiology 64, no. 1 (January 2018): 69–74. http://dx.doi.org/10.1139/cjm-2017-0315.
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Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ28-dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.
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Liu, Baomo, Lili Shui, Kai Zhou, Ying Jiang, Xiaoyu Li, Jing Guan, Qi Li, and Chao Zhuo. "Impact of Plasmid-Encoded H-NS–like Protein on blaNDM-1-Bearing IncX3 Plasmid in Escherichia coli." Journal of Infectious Diseases 221, Supplement_2 (March 16, 2020): S229—S236. http://dx.doi.org/10.1093/infdis/jiz567.
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Abstract Background This study was performed to assess the role of the histone-like nucleoid-structuring (H-NS)–like protein, carried by blaNDM-1-encoding IncX3-type plasmids, in the dissemination of IncX3 plasmids. Methods The blaNDM-1-encoding IncX3 plasmids were analyzed using southern blot, conjugation, and competition assays. Virulence was evaluated with a Galleria mellonella infection model. An hns-knockout IncX3 plasmid was also constructed to identify the functions of plasmid-borne H-NS–like protein in Escherichia coli. Results The assasys detected blaNDM-1-encoding IncX3-type plasmids with similar fingerprint patterns in all New Delhi metallo-β-lactamase (NDM) 1–producing carbapenem-resistant Enterobacteriaceae. The IncX3 plasmid conferred a fitness advantage to E. coli J53 but had no effect on host virulence. Moreover, the transconjugation frequency of the hns-null IncX3 plasmid pHN330-△hns was increased by 2.5-fold compared with the wild type. This was caused by up-regulation of conjugation-related plasmid-borne genes and the partition-related gene, in the J330-pHN330-△hns strain. In addition, decreased virulence was detected with this variant. Conclusions Our results highlight the important role of IncX3 plasmids in the dissemination of blaNDM-1 in south China. Plasmid-encoded H-NS–like protein can inhibit plasmid conjugation, partition, and the expression of related genes, in addition to promoting virulence in the host.
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Wagner, Karin, Jennifer Schilling, Stefan Fälker, M. Alexander Schmidt, and Gerhard Heusipp. "A Regulatory Network Controls Expression of the In Vivo-Expressed HreP Protease of Yersinia enterocolitica." Journal of Bacteriology 191, no. 5 (December 29, 2008): 1666–76. http://dx.doi.org/10.1128/jb.01517-08.
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ABSTRACT The human enteropathogen Yersinia enterocolitica survives and replicates in the lymphoid tissues of its host. Previous in vivo analyses of gene expression revealed that various chromosomal genes are expressed at this stage of infection, but not in vitro. One of these, termed hreP, encodes a protease that is necessary for full virulence of Y. enterocolitica. Using transposon mutagenesis, we identified three genes, pypA, pypB, and pypC, as positive regulators of hreP transcription. PypA is an inner membrane protein with no significant similarity to any known proteins; PypB is a ToxR-like transmembrane transcriptional regulator; and PypC is a cytoplasmic transcriptional regulator with an OmpR-like winged helix-turn-helix DNA binding motif. We show that all Pyp proteins are able to activate hreP independently of each other and that PypB and PypC interact directly with the hreP promoter region. Furthermore, pypB and pypC are autoregulated and regulate each other. Additional data indicate that transcription of hreP is repressed by the histone-like nucleoid-structuring protein H-NS in a temperature-dependent manner. Our data reveal a new regulatory network that might have implications for the controlled expression of further virulence-associated functions in Yersinia.
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Ohta, Tomoko, Chiharu Ueguchi, and Takeshi Mizuno. "rpoS Function Is Essential forbgl Silencing Caused by C-Terminally Truncated H-NS inEscherichia coli." Journal of Bacteriology 181, no. 20 (October 15, 1999): 6278–83. http://dx.doi.org/10.1128/jb.181.20.6278-6283.1999.
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ABSTRACT From evolutionary and physiological viewpoints, theEscherichia coli bgl operon is intriguing because its expression is silent (Bgl− phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in bgl silencing. However, we previously found that bgl expression is still silent in a certain subset of hns mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the cis-acting element of the bgl promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl+ phenotype in the background of hns60, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn10camtransposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be leuO,hns, and bglJ, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the rpoS gene and in front of the lrhA gene, respectively. The former encodes the stationary-phase-specific sigma factor, ςS, and the latter encodes a LysR-like DNA-binding protein. It was found that ςS is defective in both types of mutant cells. These results showed that the rpoS function is involved in the mechanism underlying bgl silencing, at least in thehns60 background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.
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Easton, Donna M., Luke P. Allsopp, Minh-Duy Phan, Danilo Gomes Moriel, Guan Kai Goh, Scott A. Beatson, Timothy J. Mahony, Rowland N. Cobbold, and Mark A. Schembri. "The Intimin-Like Protein FdeC Is Regulated by H-NS and Temperature in Enterohemorrhagic Escherichia coli." Applied and Environmental Microbiology 80, no. 23 (September 19, 2014): 7337–47. http://dx.doi.org/10.1128/aem.02114-14.
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ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) is a Shiga-toxigenic pathogen capable of inducing severe forms of enteritis (e.g., hemorrhagic colitis) and extraintestinal sequelae (e.g., hemolytic-uremic syndrome). The molecular basis of colonization of human and animal hosts by EHEC is not yet completely understood, and an improved understanding of EHEC mucosal adherence may lead to the development of interventions that could disrupt host colonization. FdeC, also referred to by its IHE3034 locus tag ECOK1_0290, is an intimin-like protein that was recently shown to contribute to kidney colonization in a mouse urinary tract infection model. The expression of FdeC is tightly regulatedin vitro, and FdeC shows promise as a vaccine candidate against extraintestinalE. colistrains. In this study, we characterized the prevalence, regulation, and function offdeCin EHEC. We showed that thefdeCgene is conserved in both O157 and non-O157 EHEC and encodes a protein that is expressed at the cell surface and promotes biofilm formation under continuous-flow conditions in a recombinantE. colistrain background. We also identified culture conditions under which FdeC is expressed and showed that minor alterations of these conditions, such as changes in temperature, can significantly alter the level of FdeC expression. Additionally, we demonstrated that the transcription of thefdeCgene is repressed by the global regulator H-NS. Taken together, our data suggest a role for FdeC in EHEC when it grows at temperatures above 37°C, a condition relevant to its specialized niche at the rectoanal junctions of cattle.
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Lippa, Andrew M., Michael J. Gebhardt, and Simon L. Dove. "1192. H-NS-like Proteins in Pseudomonas aeruginosa Coordinately Silence Intragenic and Antisense Transcription." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S618—S619. http://dx.doi.org/10.1093/ofid/ofaa439.1377.
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Abstract Background The H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome, which include horizontally acquired genes and numerous virulence factors. Although cells can tolerate the loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of the prophage Pf4 and subsequent superinfection of the cell. Although in other bacteria, H-NS promotes cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase, a role for MvaT and MvaU in repressing transcription from intragenic promoters has not been demonstrated. Methods Here we utilize a parental strain that cannot be infected by Pf4 phage to identify the combined MvaT and MvaU regulon. RNA-seq was utilized to identify genes differentially expressed in cells lacking MvaU or both MvaU and MvaT. ChIP-seq was utilized to identify genes directly regulated by MvaT and MvaU in wild-type cells. Further, ChIP-seq was performed in cells of the parental strain and cells lacking both MvaT and MvaU to map genome-wide σ 70-dependent promoters that were active in the presence or absence of both H-NS-like proteins. Results We demonstrate that the loss of both MvaT and MvaU, but not MvaU alone, leads to increased sense, antisense, and intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ 70 to those genomic regions vacated by these proteins. Loss of MvaT and MvaU causes global changes in gene expression Loss of MvaT and MvaU results in increased sense and antisense transcription Loss of both MvaT and MvaU results in genome-wide redistribution of RNA polymerase Conclusion Our findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and describe a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa. Disclosures All Authors: No reported disclosures
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Qu, Xueqi, Christiane Neuhoff, Mehmet Ulas Cinar, Maren Pröll, Ernst Tholen, Dawit Tesfaye, Michael Hölker, Karl Schellander, and Muhammad Jasim Uddin. "Epigenetic Modulation of TLR4 Expression by Sulforaphane Increases Anti-Inflammatory Capacity in Porcine Monocyte-Derived Dendritic Cells." Biology 10, no. 6 (May 31, 2021): 490. http://dx.doi.org/10.3390/biology10060490.
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Inflammation is regulated by epigenetic modifications, including DNA methylation and histone acetylation. Sulforaphane (SFN), a histone deacetylase (HDAC) inhibitor, is also a potent immunomodulatory agent, but its anti-inflammatory functions through epigenetic modifications remain unclear. Therefore, this study aimed to investigate the epigenetic effects of SFN in maintaining the immunomodulatory homeostasis of innate immunity during acute inflammation. For this purpose, SFN-induced epigenetic changes and expression levels of immune-related genes in response to lipopolysaccharide (LPS) stimulation of monocyte-derived dendritic cells (moDCs) were analyzed. These results demonstrated that SFN inhibited HDAC activity and caused histone H3 and H4 acetylation. SFN treatment also induced DNA demethylation in the promoter region of the MHC-SLA1 gene, resulting in the upregulation of Toll-like receptor 4 (TLR4), MHC-SLA1, and inflammatory cytokines’ expression at 6 h of LPS stimulation. Moreover, the protein levels of cytokines in the cell culture supernatants were significantly inhibited by SFN pre-treatment followed by LPS stimulation in a time-dependent manner, suggesting that inhibition of HDAC activity and DNA methylation by SFN may restrict the excessive inflammatory cytokine availability in the extracellular environment. We postulate that SFN may exert a protective and anti-inflammatory function by epigenetically influencing signaling pathways in experimental conditions employing porcine moDCs.
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Caballero-Flores, Gustavo G., Matthew A. Croxen, Verónica I. Martínez-Santos, B. Brett Finlay, and José L. Puente. "Identification and Regulation of a Novel Citrobacter rodentium Gut Colonization Fimbria (Gcf)." Journal of Bacteriology 197, no. 8 (February 9, 2015): 1478–91. http://dx.doi.org/10.1128/jb.02486-14.
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ABSTRACTThe Gram-negative enteric bacteriumCitrobacter rodentiumis a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagicEscherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. ForC. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novelC. rodentiumcolonization factor, calledgutcolonizationfimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. AgcfAmutant shows a severe colonization defect within the first 10 days of infection. Thegcfpromoter is not active inC. rodentiumunder severalin vitrogrowth conditions; however, it is readily expressed in aC. rodentiumΔhns1mutant lacking the closest ortholog of theEscherichia colihistone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region ofgcf, further supporting its direct role as a repressor of thegcfpromoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. Thegcfoperon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures.IMPORTANCEFimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection byC. rodentiumin mice, despite being poorly expressed underin vitrogrowth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection.
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Grainger, David C. "Structure and function of bacterial H-NS protein." Biochemical Society Transactions 44, no. 6 (December 2, 2016): 1561–69. http://dx.doi.org/10.1042/bst20160190.
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The histone-like nucleoid structuring (H-NS) protein is a major component of the folded chromosome in Escherichia coli and related bacteria. Functions attributed to H-NS include management of genome evolution, DNA condensation, and transcription. The wide-ranging influence of H-NS is remarkable given the simplicity of the protein, a small peptide, possessing rudimentary determinants for self-association, hetero-oligomerisation and DNA binding. In this review, I will discuss our understanding of H-NS with a focus on these structural elements. In particular, I will consider how these interaction surfaces allow H-NS to exert its different effects.
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Sun, Yaping, Elizabeth Weisiger, Tomomi Toubai, Charles Dinarello, James L. Ferrara, and Pavan Reddy. "A Critical Role for STAT-3 Dependent Induction of Indoleamine 2, 3 Dioxygenase in Histone Deacetylase Inhibitors Mediated the Regulation of GVHD." Blood 110, no. 11 (November 16, 2007): 351. http://dx.doi.org/10.1182/blood.v110.11.351.351.
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Abstract Histone deacetylase (HDAC) inhibitors reduce experimental acute graft-versus-host disease (GVHD) and recent work by us and others suggest that HDAC inhibitors regulate dendritic cell (DC) function. However, the critical cellular and molecular mechanisms underpinning these observations are not known. We investigated the mechanisms by utilizing two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and ITF 2357. Pretreatment of murine bone marrow (BM) and human peripheral blood mononuclear cell derived DCs with either HDAC inhibtors and stimulated with TLR ligands such as LPS caused a significant reduction in the secretion of TNF-α compared to the untreated controls (P< 0.01). Pre-treatment also significantly reduced the DC mediated in vitro and in vivo stimulation of allogeneic T cell proliferation (P<0.05). SAHA and ITF 2357 increased expression of indoleamine 2, 3-dioxygenase (IDO) at both mRNA and protein levels. Blockade of IDO induction with specific small interfering RNA (siRNA) in the wild type (WT) DCs and those derived from IDO deficient (IDO−/−) animals confirmed a functional role for IDO in the HDAC inhibitor mediated regulation TNFα secretion and allo-T cell proliferation. DNA-protein interaction analysis with ChIP assay demonstrated that both acetylated histone(H) 4 and STAT3 bound to murine IDO promoter. Using TESS DNA soft-wear analysis we found two potential STAT3 binding Gamma Activated Sites (GAS sites) in the IDO promoter and it was recently reported that acetylation of STAT3 is sufficient for its activation (Yuan, et al. Science 2005). We therefore sought to determine whether direct acetylation of STAT3 by the HDAC inhibitors is critical for the induction of IDO in DCs. SAHA or ITF2357 treatment induced acetylation, activation and dimerization of STAT-3 as determined by protein-protein interaction studies. Co-culture studies with JSI-124, an inhibitor of STAT3 signaling, demonstrated that STAT3 is critical for induction of IDO by the HDAC inhibitors. Functional relevance was confirmed by the lack of HDAC inhibitor induced suppression of DC function when co-treated with JSI-124. We next cloned 1500bp DNA fragment upstream of mouse IDO gene start codon and attached it to luciferase gene and peformed mutagenesis studies to evaluate for luciferase activity. Deletion of the GAS regions or treatment with JSI-124 impaired luciferase activity of the IDO promoter constructs demonstrating that STAT3 is necessary and sufficient for transcriptional induction of IDO by the HDAC inhibitors. To specifically address the in vivo relevance of IDO induction by HDAC inhibitors in only the host type DCs, we generated [B6 → B6] and [IDO−/−B6 → B6] BM chimeras and utilized them in a well characterized [BALB/c B6] mouse model of acute GVHD. [B6 → B6] and [IDO−/− B6 → B6] animals received 800 Gy on day −1 and were treated orally with of 50mg/kg of ITF 2357 or diluent on days −1 to +2. Mice were transplanted on day 0 with 3 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Treatment with ITF 2357 resulted in significantly better survival in the allogeneic [B6 → B6] animals (80% vs. 40%, P < 0.02) but did not confer any survival benefit to the [IDO−/− B6 → B6] animals when compared with diluent treated recipients [20% vs. 30%, P = NS]. Our data thus demonstrate a novel molecular pathway in modulation of GVHD through a STAT3 dependent induction of IDO in the host DCs.
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Madan, P., W. A. King, and D. H. Betts. "91 INHIBITION OF TELOMERASE REVERSE TRANSCRIPTASE (TERT) INDUCES EMBRYO ARREST IN BOVINE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 146. http://dx.doi.org/10.1071/rdv21n1ab91.
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Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. The objective of this study was to characterize the expression and function of the bovine telomerase reverse transcriptase (bTERT) subunit during bovine pre-implantation embryogenesis. Using RT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in pre-implantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and exhibited 94% sequence homology to the human hTERT cDNA sequence. In immature oocytes, the bTERT protein was localized within the germinal vesicle and after 18 to 20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctate staining, whereas others exhibited a more-uniform staining pattern within the cytoplasm. By the 8- to 16-cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, while in morulae and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 4′,6-diamidino-2-phenylindole (DAPI) staining. Treatment of zygotes with Telomerase Inhibitor III (telomere mimic that inhibits telomerase activity; Calbiochem, San Diego, CA) significantly induced embryos to permanently arrest in a senescence-like state at the 2- to 4-cell cleavage stage compared with nontreated controls. These arrested embryos showed abnormal nuclear phenotypes (elongated chromosomes, anaphase bridging) and phosphorylated histone γ-H2A.X foci suggesting a critical role(s) of TERT in chromosomal segregation and telomere integrity in early embryos. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the known telomere elongation event, whereas low levels of TERT at the 2- to 4-cell stage are associated with the previously documented state of permanent embryo arrest. Future studies would be directed toward understanding the relationship between TERT expression, telomere dysfunction, and permanent embryo arrest. Operating grants from the Canadian Institutes of Health Research (CIHR) to D.H.B. and W.A.K. supported this research.
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Hulton, Christopher S. J., Alexander Seirafi, Jay C. D. Hinton, Julie M. Sidebotham, Lesley Waddell, Graham D. Pavitt, Thomas Owen-Hughes, Annick Spassky, Henri Buc, and Christopher F. Higgins. "Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria." Cell 63, no. 3 (November 1990): 631–42. http://dx.doi.org/10.1016/0092-8674(90)90458-q.
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Duysak, Taner, Thanh Tuyen Tran, Aqeel Rana Afzal, and Che-Hun Jung. "Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein." International Journal of Molecular Sciences 22, no. 15 (July 23, 2021): 7871. http://dx.doi.org/10.3390/ijms22157871.
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The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.
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Ito, K., T. Oshima, T. Mizuno, and Y. Nakamura. "Regulation of lysyl-tRNA synthetase expression by histone-like protein H-NS of Escherichia coli." Journal of Bacteriology 176, no. 23 (1994): 7383–86. http://dx.doi.org/10.1128/jb.176.23.7383-7386.1994.
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Silva, Anisia J., Syed Zafar Sultan, Weili Liang, and Jorge A. Benitez. "Role of the Histone-Like Nucleoid Structuring Protein in the Regulation of rpoS and RpoS-Dependent Genes in Vibrio cholerae." Journal of Bacteriology 190, no. 22 (September 12, 2008): 7335–45. http://dx.doi.org/10.1128/jb.00360-08.
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ABSTRACT Production of the Zn-metalloprotease hemagglutinin (HA)/protease by Vibrio cholerae has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor σS (RpoS), encoded by rpoS. The histone-like nucleoid structuring protein (H-NS) has been shown to repress rpoS expression in Escherichia coli. In V. cholerae strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a Δhns mutant. The Δhns mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of hns resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in Δhns, ΔrpoS, and Δhns ΔrpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in ΔrpoS and ΔrpoS Δhns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The Δhns mutant exhibited a lower degree of motility and lower levels of expression of flaA, flaC, cheR-2, and motX mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, Δhns, ΔrpoS, and Δhns ΔrpoS strains revealed that deletion of rpoS had a more severe negative effect on their expression. Interestingly, deletion of hns in the rpoS background resulted in higher expression levels of flaA, flaC, and motX, suggesting that H-NS represses the expression of these genes in the absence of σS. Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.
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Müller, Claudia M., Ulrich Dobrindt, Gábor Nagy, Levente Emödy, Bernt Eric Uhlin, and Jörg Hacker. "Role of Histone-Like Proteins H-NS and StpA in Expression of Virulence Determinants of Uropathogenic Escherichia coli." Journal of Bacteriology 188, no. 15 (August 1, 2006): 5428–38. http://dx.doi.org/10.1128/jb.01956-05.
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ABSTRACT The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.
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Mertens, Daniel, Angela Philippen, Nupur Bhattacharya, Cordula Tschuch, Melanie Ruppel, Peter Lichter, Hartmut Doehner, and Stephan Stilgenbauer. "Epimutation of the Tumor Suppressor Mechanism in 13q14.3 Involves Monoallelic Expression, Non-Coding RNA Genes and Deregulation of NFkB Signalling." Blood 112, no. 11 (November 16, 2008): 783. http://dx.doi.org/10.1182/blood.v112.11.783.783.
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Abstract Deletions in chromosomal band 13q14.3 occur in a variety of human neoplasms like chronic lymphocytic leukaemia (CLL), indicating a tumor suppressor mechanism (TSM) in this region. Intriguingly, several characteristics of the region of interest point to an epigenetic pathomechanism: candidate protein-coding genes and non-coding RNA genes including miR15a and miR16-1 lack point mutations in the majority of patients, yet these genes are significantly downregulated in almost all CLL patients the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active. We have recently shown that already in healthy tissue only one gene copy of 13q14.3 is active while one gene copy is randomly chosen for silencing. Thus, loss of the single active copy is sufficient for complete loss of gene function in tumor cells. In order to elucidate the epigenetic regulatory mechanism, we analysed DNA- and Histone-methylation of all CpG islands in the region in non-malignant B-cells and CLL cells. Using aPRIMES and ChIP-qPCR as screening tools, BioCOBRA as a quantitative high-throughput method and bisulfite sequencing for validation, we could identify two candidate regulatory elements with abnormal chromatin in CLL patients (n=80, median 57% DNA-methylation, range 0–100%) as compared to healthy probands (n=20, median 88% DNA-methylation, range 74–100%, p<0.003). Interestingly, this epimutation can be found in all cytogenetic subgroups of CLL patients and is independent of IgV(H) mutation status, making it a prime candidate for an underlying epigenetic defect in CLL. Pilot studies suggest that this epimutation regulates gene expression of the critical region via large non-coding RNA genes. In order to find out how loss of function of the 13q14 genes could result in the pathophenotype of CLL cells, we overexpressed and knocked-down RFP2, C13ORF1, KPNA3 and the largen non-coding RNA gene Dleu2 in two different cell lines and used custom oligonucleotide microarrays and timecourse experiments (n=68 array hybridizations) to identify genes that were subsequently deregulated and thus potential target genes. Less than 1% of genes represented on the arrays were significantly deregulated (median 211/25100 genes, range 44–370), showing the high specificity of the procedure. Using ingenuity pathway analyses, we found that modulation of the expression of 13q14.3 candidate genes deregulates most significantly NFkB target genes and components of the NFkB pathway itself. For a detailed validation analysis we focused on RFP2 and could show that it robustly and quickly induces NFkB activity in fibroblasts (HeLa), kidney cells (HEK-293) and CLL cell lines (Granta-591). However, analyses by oligonucleotide ELISA, Western Blot and EMSA-Band-Shift assays suggest that activation of NFkB occurs not via modulation of components of the canonical or non-canonical NFkB signalling pathways. Therefore, we propose a model for the TSM in 13q14.3 where in healthy B-cells, only one gene copy is active while the second is epigenetically silenced expression of candidate genes is deregulated in CLL cells by epimutation that is present in all cytogenetic subgroups and that this loss of function of 13q14 candidate genes results in deregulation of the NFkB signalling pathway which will change the activation level of CLL cells and their sensitivity to induction of apoptosis.
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Tippner, Detlev, Henning Afflerbach, Christiane Bradaczek, and Rolf Wagner. "Evidence for a regulatory function of the histone-like Escherichia coli protein H-NS in ribosomal RNA synthesis." Molecular Microbiology 11, no. 3 (February 1994): 589–604. http://dx.doi.org/10.1111/j.1365-2958.1994.tb00339.x.
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Franzon, J. H., and D. S. Santos. "A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens." Brazilian Journal of Medical and Biological Research 37, no. 12 (December 2004): 1763–69. http://dx.doi.org/10.1590/s0100-879x2004001200001.
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Shiga, Yasuyuki, Yasuhiko Sekine, Yasunobu Kano, and Eiichi Ohtsubo. "Involvement of H-NS in Transpositional Recombination Mediated by IS1." Journal of Bacteriology 183, no. 8 (April 15, 2001): 2476–84. http://dx.doi.org/10.1128/jb.183.8.2476-2484.2001.
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ABSTRACT IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.
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Ono, Shusuke, Martin D. Goldberg, Tjelvar Olsson, Diego Esposito, Jay C. D. Hinton, and John E. Ladbury. "H-NS is a part of a thermally controlled mechanism for bacterial gene regulation." Biochemical Journal 391, no. 2 (October 10, 2005): 203–13. http://dx.doi.org/10.1042/bj20050453.
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Temperature is a primary environmental stress to which micro-organisms must be able to adapt and respond rapidly. Whereas some bacteria are restricted to specific niches and have limited abilities to survive changes in their environment, others, such as members of the Enterobacteriaceae, can withstand wide fluctuations in temperature. In addition to regulating cellular physiology, pathogenic bacteria use temperature as a cue for activating virulence gene expression. This work confirms that the nucleoid-associated protein H-NS (histone-like nucleoid structuring protein) is an essential component in thermoregulation of Salmonella. On increasing the temperature from 25 to 37 °C, more than 200 genes from Salmonella enterica serovar Typhimurium showed H-NS-dependent up-regulation. The thermal activation of gene expression is extremely rapid and change in temperature affects the DNA-binding properties of H-NS. The reduction in gene repression brought about by the increase in temperature is concomitant with a conformational change in the protein, resulting in the decrease in size of high-order oligomers and the appearance of increasing concentrations of discrete dimers of H-NS. The present study addresses one of the key complex mechanisms by which H-NS regulates gene expression.
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Coker, Christopher, Olubunmi O. Bakare, and Harry L. T. Mobley. "H-NS Is a Repressor of the Proteus mirabilis Urease Transcriptional Activator GeneureR." Journal of Bacteriology 182, no. 9 (May 1, 2000): 2649–53. http://dx.doi.org/10.1128/jb.182.9.2649-2653.2000.
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ABSTRACT Expression of Proteus mirabilis urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A6TA2CA2TGGTA5GA6TGA5, is located 16 bp upstream of the ς70-likeureR promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured β-galactosidase activity of wild-typeEscherichia coli MC4100 (H-NS+) and its isogenic derivative ATM121 (hns::Tn10) (H-NS−) harboring a ureR-lacZ operon fusion plasmid (pLC9801). β-Galactosidase activity in the H-NS− host strain was constitutive and sevenfold greater (P < 0.0001) than that in the H-NS+ host. A recombinant plasmid containing cloned P. mirabilis hns was able to complement and restore repression of the ureR promoter in the H-NS−host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in β-galactosidase activity in the H-NS+ host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS−host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of P. mirabilis) was equivalent in both the H-NS−background and the H-NS+ background in the presence of urea but was eightfold greater (P = 0.0001) in the H-NS− background in the absence of urea. We conclude that H-NS represses ureR expression in the absence of urea induction.
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Eijkelkamp, Bart A., Uwe H. Stroeher, Karl A. Hassan, Liam D. H. Elbourne, Ian T. Paulsen, and Melissa H. Brown. "H-NS Plays a Role in Expression of Acinetobacter baumannii Virulence Features." Infection and Immunity 81, no. 7 (May 6, 2013): 2574–83. http://dx.doi.org/10.1128/iai.00065-13.
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ABSTRACTAcinetobacter baumanniihas become a major problem in the clinical setting with the prevalence of infections caused by multidrug-resistant strains on the increase. Nevertheless, only a limited number of molecular mechanisms involved in the success ofA. baumanniias a human pathogen have been described. In this study, we examined the virulence features of a hypermotile derivative ofA. baumanniistrain ATCC 17978, which was found to display enhanced adherence to human pneumocytes and elevated levels of lethality towardCaenorhabditis elegansnematodes. Analysis of cellular lipids revealed modifications to the fatty acid composition, providing a possible explanation for the observed changes in hydrophobicity and subsequent alteration in adherence and motility. Comparison of the genome sequences of the hypermotile variant and parental strain revealed that an insertion sequence had disrupted anhns-like gene in the variant. This gene encodes a homologue of the histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes seen in the autotransporter Ata, a type VI secretion system, and a type I pilus cluster. Interestingly, isolation and analysis of a second independent hypermotile ATCC 17978 variant revealed a mutation to a residue within the DNA binding region of H-NS. Taken together, these mutants indicate that the phenotypic and transcriptomic differences seen are due to loss of regulatory control effected by H-NS.
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Park, Kwon-Sam, Michiko Arita, Tetsuya Iida, and Takeshi Honda. "vpaH, a Gene Encoding a Novel Histone-Like Nucleoid Structure-Like Protein That Was Possibly Horizontally Acquired, Regulates the Biogenesis of Lateral Flagella in trh-Positive Vibrio parahaemolyticus TH3996." Infection and Immunity 73, no. 9 (September 2005): 5754–61. http://dx.doi.org/10.1128/iai.73.9.5754-5761.2005.
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ABSTRACT A histone-like nucleoid structure (H-NS) is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Here, we cloned and characterized the gene for the H-NS-like protein VpaH in Vibrio parahaemolyticus. vpaH encodes a protein of 134 amino acids that shows approximately 55%, 54%, and 41% identities with VicH in Vibrio cholerae, H-NS in V. parahaemolyticus, and H-NS in Escherichia coli, respectively. The vpaH gene was found in only trh-positive V. parahaemolyticus strains and not in Kanagawa-positive or in trh-negative environmental strains. Moreover, the G+C content of the vpaH gene was 38.6%, which is lower than the average G+C content of the whole genome of this bacterium (45.4%). These data suggest that vpaH was transmitted to trh-possessing V. parahaemolyticus strains by lateral transfer. The vpaH gene was located about 2.6 kb downstream of the trh gene, in the convergent direction of the trh transcription. An in-frame deletion mutant of vpaH lacked motility on semisolid motility assay plates. Western blot analysis and electron microscopy observations revealed that the mutant was deficient in lateral flagella biogenesis, whereas there was no defect in the expression of polar flagella. Additionally, the vpaH mutant showed a decreased adherence to HeLa cells and a decrease in biofilm formation compared with the wild-type strain. Introduction of the vpaH gene in the vpaH-negative strain increased the expression of lateral flagella compared with the wild-type strain. In conclusion, our findings suggest that VpaH affects lateral flagellum biogenesis in trh-positive V. parahaemolyticus strain TH3996.
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Yun, Choong-Soo, Chiho Suzuki, Kunihiko Naito, Toshiharu Takeda, Yurika Takahashi, Fumiya Sai, Tsuguno Terabayashi, et al. "Pmr, a Histone-Like Protein H1 (H-NS) Family Protein Encoded by the IncP-7 Plasmid pCAR1, Is a Key Global Regulator That Alters Host Function." Journal of Bacteriology 192, no. 18 (July 16, 2010): 4720–31. http://dx.doi.org/10.1128/jb.00591-10.
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ABSTRACT Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.
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Gowrishankar, J., and A. J. Pittard. "Superimposition of TyrR Protein-Mediated Regulation on Osmoresponsive Transcription of Escherichia coli proUIn Vivo." Journal of Bacteriology 180, no. 24 (December 15, 1998): 6743–48. http://dx.doi.org/10.1128/jb.180.24.6743-6748.1998.
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ABSTRACT Osmotic regulation of proU expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the ς70-controlled promoter of proU inEscherichia coli, we were able to demonstrate a superposed TyrR-mediated activation by l-phenylalanine (Phe), as well as repression by l-tyrosine, of proU expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of proU at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in E. coli.
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Cabello, Olga A., Elena Eliseeva, WeiGong He, Hagop Youssoufian, Sharon E. Plon, B. R. Brinkley, and John W. Belmont. "Cell Cycle-dependent Expression and Nucleolar Localization of hCAP-H." Molecular Biology of the Cell 12, no. 11 (November 2001): 3527–37. http://dx.doi.org/10.1091/mbc.12.11.3527.
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Condensin is a conserved 13S heteropentamer composed of two nonidentical structural maintenance of chromosome (SMC) family proteins, in Xenopus XCAP-C and XCAP-E, and three regulatory subunits, XCAP-D2, XCAP-G, and XCAP-H. Both biochemical and genetic analyses have demonstrated an essential role for the 13S condensin complex in mitotic chromosome condensation. Further, a potential requirement for condensin in completion of chromatid arm separation in early anaphase is demonstrated by the mutational phenotypes of the Drosophila homologues ofXCAP-H, barren and XCAP-C,DmSMC4. In this study we have investigated the expression and subcellular distribution of hCAP-H, the human homolog of XCAP-H, in order to better understand its cellular functions. Transcription of hCAP-H was restricted to proliferating cells with highest expression during the G2 phase of the cell cycle. In contrast, cellular hCAP-H protein levels were constant throughout the cell cycle. hCAP-H was found to be associated with mitotic chromosomes exhibiting a nonuniform but symmetric distribution along sister chromatids. The symmetry of hCAP-H association with sister chromatids suggests that there are sequence-dependent domains of condensin aggregation. During interphase hCAP-H, -C, and -E, have distinct punctate nucleolar localization, suggesting that condensin may associate with and modulate the conformation and function of rDNA. hCAP-H association with condensed chromatin was not observed in the early phase of chromosome condensation when histone H3 phosphorylation has already taken place. This finding is consistent with the hypothesis that histone H3 phosphorylation precedes condensin-mediated condensation.
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Japaridze, Aleksandre, Sylvain Renevey, Patrick Sobetzko, Liubov Stoliar, William Nasser, Giovanni Dietler, and Georgi Muskhelishvili. "Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein." Journal of Biological Chemistry 292, no. 18 (March 18, 2017): 7607–18. http://dx.doi.org/10.1074/jbc.m117.780239.
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Structural differentiation of bacterial chromatin depends on cooperative binding of abundant nucleoid-associated proteins at numerous genomic DNA sites and stabilization of distinct long-range nucleoprotein structures. Histone-like nucleoid-structuring protein (H-NS) is an abundant DNA-bridging, nucleoid-associated protein that binds to an AT-rich conserved DNA sequence motif and regulates both the shape and the genetic expression of the bacterial chromosome. Although there is ample evidence that the mode of H-NS binding depends on environmental conditions, the role of the spatial organization of H-NS-binding sequences in the assembly of long-range nucleoprotein structures remains unknown. In this study, by using high-resolution atomic force microscopy combined with biochemical assays, we explored the formation of H-NS nucleoprotein complexes on circular DNA molecules having different arrangements of identical sequences containing high-affinity H-NS-binding sites. We provide the first experimental evidence that variable sequence arrangements result in various three-dimensional nucleoprotein structures that differ in their shape and the capacity to constrain supercoils and compact the DNA. We believe that the DNA sequence-directed versatile assembly of periodic higher-order structures reveals a general organizational principle that can be exploited for knowledge-based design of long-range nucleoprotein complexes and purposeful manipulation of the bacterial chromatin architecture.
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Feng, Jia-Xun, Zhi-Zhong Song, Cheng-Jie Duan, Shuai Zhao, Ying-Qiao Wu, Chao Wang, J. Maxwell Dow, and Ji-Liang Tang. "The xrvA gene of Xanthomonas oryzae pv. oryzae, encoding an H-NS-like protein, regulates virulence in rice." Microbiology 155, no. 9 (September 1, 2009): 3033–44. http://dx.doi.org/10.1099/mic.0.028910-0.
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Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice, one of the most serious rice diseases. The xrvA gene from Xoo strain 13751 encodes a protein containing a histone-like nucleoid-structuring protein (H-NS) domain. The expression of xrvA in strain 13751 was enhanced in XOM2 minimal medium. Mutation of the xrvA gene of strain 13751 led to a significant reduction in virulence in the host plant rice, a delayed hypersensitive response in the nonhost castor-oil plant, a decrease in extracellular polysaccharide and diffusible signal factor production, and an increase in intracellular glycogen accumulation. Northern hybridization analyses revealed that the virulence-associated genes hrpG, hrpX, rpfC, rpfF, rpfG and gumB were downregulated in the xrvA mutant compared to the wild-type and complemented strains. Interestingly, increase of copy number of xrvA in the wild-type strain 13751 resulted in a strain showing similar phenotypes as the xrvA mutant and a reduction of the expression of gumB, hrpX, rpfC, rpfF and rpfG. These findings indicate that the xrvA gene, which is highly conserved in the sequenced strains of Xanthomonas, encodes an important regulatory factor for the virulence of Xoo.
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Salomon, Dor, John A. Klimko, and Kim Orth. "H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1." Microbiology 160, no. 9 (September 1, 2014): 1867–73. http://dx.doi.org/10.1099/mic.0.080028-0.
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The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity.
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Ricci, Dante P., Michael D. Melfi, Keren Lasker, David L. Dill, Harley H. McAdams, and Lucy Shapiro. "Cell cycle progression inCaulobacterrequires a nucleoid-associated protein with high AT sequence recognition." Proceedings of the National Academy of Sciences 113, no. 40 (September 19, 2016): E5952—E5961. http://dx.doi.org/10.1073/pnas.1612579113.
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Faithful cell cycle progression in the dimorphic bacteriumCaulobacter crescentusrequires spatiotemporal regulation of gene expression and cell pole differentiation. We discovered an essential DNA-associated protein, GapR, that is required forCaulobactergrowth and asymmetric division. GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Despite constitutive synthesis, GapR accumulates preferentially in the swarmer compartment of the predivisional cell. Homologs of GapR, which are ubiquitous among the α-proteobacteria and are encoded on multiple bacteriophage genomes, also accumulate in the predivisional cell swarmer compartment when expressed inCaulobacter. TheEscherichia colinucleoid-associated protein H-NS, like GapR, selectively associates with AT-rich DNA, yet it does not localize preferentially to the swarmer compartment when expressed exogenously inCaulobacter, suggesting that recognition of AT-rich DNA is not sufficient for the asymmetric accumulation of GapR. Further, GapR does not silence the expression of H-NS target genes when expressed inE. coli, suggesting that GapR and H-NS have distinct functions. We propose thatCaulobacterhas co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression.
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Brunet, Yannick R., Ahmad Khodr, Laureen Logger, Laurent Aussel, Tâm Mignot, Sylvie Rimsky, and Eric Cascales. "H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing." Infection and Immunity 83, no. 7 (April 27, 2015): 2738–50. http://dx.doi.org/10.1128/iai.00198-15.
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The secretion of bacterial toxin proteins is achieved by dedicated machineries called secretion systems. The type VI secretion system (T6SS) is a widespread versatile machine used for the delivery of protein toxins to both prokaryotic and eukaryotic cells. InSalmonella entericaserovar Typhimurium, the expression of the T6SS genes is activated during macrophage or mouse infection. Here, we show that the T6SS gene cluster is silenced by the histone-like nucleoid structuring H-NS protein using a combination of reporter fusions, electrophoretic mobility shift assays, DNase footprinting, and fluorescence microscopy. We further demonstrate that derepression of theS. Typhimurium T6SS genes induces T6SS-dependent intoxication of competing bacteria. Our results suggest that relieving T6SS H-NS silencing may be used as a sense-and-kill mechanism that will helpS. Typhimurium to homogenize and synchronize the microbial population to gain efficiency during infection.
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Soutourina, O., A. Kolb, E. Krin, C. Laurent-Winter, S. Rimsky, A. Danchin, and P. Bertin. "Multiple Control of Flagellum Biosynthesis in Escherichia coli: Role of H-NS Protein and the Cyclic AMP-Catabolite Activator Protein Complex in Transcription of the flhDC Master Operon." Journal of Bacteriology 181, no. 24 (December 15, 1999): 7500–7508. http://dx.doi.org/10.1128/jb.181.24.7500-7508.1999.
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ABSTRACT Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of fliA and fliC, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the flhDC master operon restored, at least in part, motility in crp andhns mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates flhDC expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the flhDC promoter was decreased in thecrp strain and increased in the hns mutant. In contrast, the activity of a transcriptional fusion encompassing the entire flhDC regulatory region extending to the ATG translational start codon was strongly reduced in both hnsand crp mutants. These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in acrp mutant by activation-deficient CAP mutated proteins and characterization of cfs, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activatesflhDC transcription by binding to its promoter and interacting with RNA polymerase.
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Hekmat, Azadeh, Mojtaba Sadeghi Manesh, Zahra Hajebrahimi, and Shadie Hatamie. "Microgravity-Induced Alterations in the H3.3B (H3F3B) Gene Expression and the Histone H3 Structure." Advanced Science, Engineering and Medicine 12, no. 8 (August 1, 2020): 1084–94. http://dx.doi.org/10.1166/asem.2020.2672.
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It has been believed that microgravity directly can modify the structure, function, and morphology of biosystems and numerous researches have been performed to recognize these alterations. Since histone H3 is an essential protein in the field of epigenetics, this research aimed to evaluate the effects of simulated microgravity on the human H3.3B (H3F3B) gene expression and histone H3 structure. The two-dimensional clinostat was applied for simulating microgravity. Analysis of the gene expression by real-time quantitative PCR revealed that simulated microgravity diminished the expression level of H3.3B considerably (P < 0.001). The UV-Visible absorption and extrinsic fluorescence emission results displayed that after 72 h of simulated microgravity the tertiary structure of histone H3 changed and the surface hydrophobicity of the protein incremented remarkably. Nevertheless, circular dichroism (CD) data showed that simulated microgravity did not perturb the secondary structure of histone H3. Collectively, microgravity can strictly affect the gene expression level of H3.3. Furthermore, histone H3 72 h after subjecting to simulated microgravity can exhibit a molten globule structure. The significance of this research lied in the fact that simulating microgravity can be an effective physical force in gene expression regulation and the protein folding process. This finding could help astrobiologists to realize major health risks for astronaut crews and space travelers and reduce these harmful effects. Furthermore, our observations can open fascinating research lines in astrobiology, biophysics, and exobiology.
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Erdő-Bonyár, Szabina, Judit Rapp, Tünde Minier, Gábor Ráth, József Najbauer, László Czirják, Péter Németh, Timea Berki, and Diána Simon. "Toll-Like Receptor Mediated Activation of Natural Autoantibody Producing B Cell Subpopulations in an Autoimmune Disease Model." International Journal of Molecular Sciences 20, no. 24 (December 6, 2019): 6152. http://dx.doi.org/10.3390/ijms20246152.
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Altered expression and function of the Toll-like receptor (TLR) homologue CD180 molecule in B cells have been associated with autoimmune disorders. In this study, we report decreased expression of CD180 at protein and mRNA levels in peripheral blood B cells of diffuse cutaneous systemic sclerosis (dcSSc) patients. To analyze the effect of CD180 stimulation, together with CpG (TLR9 ligand) treatment, on the phenotype defined by CD19/CD27/IgD/CD24/CD38 staining, and function (CD69 and CD180 expression, cytokine and antibody secretion) of B cell subpopulations, we used tonsillar B cells. After stimulation, we found reduced expression of CD180 protein and mRNA in total B cells, and CD180 protein in B cell subpopulations. The frequency of CD180+ cells was the highest in the CD19+CD27+IgD+ non-switched (NS) B cell subset, and they showed the strongest activation after anti-CD180 stimulation. Furthermore, B cell activation via CD180 induced IL-6 and natural autoantibody secretion. Treatment with the combination of anti-CD180 antibody and CpG resulted in increased IL-6 and IL-10 secretion and natural autoantibody production of B cells. Our results support the role of CD180 in the induction of natural autoantibody production, possibly by NS B cells, and suggest an imbalance between the pathologic and natural autoantibody production in SSc patients.
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Martínez-Santos, Verónica I., Abraham Medrano-López, Zeus Saldaña, Jorge A. Girón, and José L. Puente. "Transcriptional Regulation of theecpOperon by EcpR, IHF, and H-NS in Attaching and Effacing Escherichia coli." Journal of Bacteriology 194, no. 18 (July 13, 2012): 5020–33. http://dx.doi.org/10.1128/jb.00915-12.
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ABSTRACTEnteropathogenic (EPEC) and enterohemorrhagic (EHEC)Escherichia coliare clinically important diarrheagenic pathogens that adhere to the intestinal epithelial surface. TheE. colicommon pili (ECP), or meningitis-associated and temperature-regulated (MAT) fimbriae, are ubiquitous among both commensal and pathogenicE. colistrains and play a role as colonization factors by promoting the interaction between bacteria and host epithelial cells and favoring interbacterial interactions in biofilm communities. The first gene of theecpoperon encodes EcpR (also known as MatA), a proposed regulatory protein containing a LuxR-like C-terminal helix-turn-helix (HTH) DNA-binding motif. In this work, we analyzed the transcriptional regulation of theecpgenes and the role of EcpR as a transcriptional regulator. EHEC and EPECecpRmutants produce less ECP, while plasmids expressing EcpR increase considerably the expression of EcpA and production of ECP. Theecpgenes are transcribed as an operon from a promoter located 121 bp upstream of the start codon ofecpR. EcpR positively regulates this promoter by binding to two TTCCT boxes distantly located upstream of theecppromoter, thus enhancing expression of downstreamecpgenes, leading to ECP production. EcpR mutants in the putative HTH DNA-binding domain are no longer able to activateecpexpression or bind to the TTCCT boxes. EcpR-mediated activation is aided by integration host factor (IHF), which is essential for counteracting the repression exerted by histone-like nucleoid-structuring protein (H-NS) on theecppromoter. This work demonstrates evidence about the interplay between a novel member of a diverse family of regulatory proteins and global regulators in the regulation of a fimbrial operon.
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An, Beum-Soo, Luz E. Tavera-Mendoza, Vassil Dimitrov, Xiaofeng Wang, Mario R. Calderon, Hui-Jun Wang, and John H. White. "Stimulation of Sirt1-Regulated FoxO Protein Function by the Ligand-Bound Vitamin D Receptor." Molecular and Cellular Biology 30, no. 20 (August 23, 2010): 4890–900. http://dx.doi.org/10.1128/mcb.00180-10.
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ABSTRACT Hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), signals through the nuclear vitamin D receptor (VDR). 1,25D regulates cell proliferation and differentiation and has been identified as a cancer chemopreventive agent. FoxO proteins are transcription factors that control cell proliferation and survival. They function as tumor suppressors and are associated with longevity in several organisms. Accumulating data have revealed that 1,25D and FoxO proteins regulate similarly common target genes. We show here that the ligand-bound VDR regulates the posttranslational modification and function of FoxO proteins. 1,25D treatment enhances binding of FoxO3a and FoxO4 within 4 h to promoters of FoxO target genes and blocks mitogen-induced FoxO protein nuclear export. The VDR associates directly with FoxO proteins and regulators, the sirtuin 1 (Sirt1) class III histone deacetylase (HDAC), and protein phosphatase 1. In addition, phosphatase activity and trichostatin A-resistant HDAC activity coimmunoprecipitate with the VDR. 1,25D treatment rapidly (in <4 h) induces FoxO deacetylation and dephosphorylation, consistent with activation. In contrast, ablation of VDR expression enhances FoxO3a phosphorylation, as does knockdown of Sirt1, consistent with the coupling of FoxO acetylation and phosphorylation. 1,25D regulation of common VDR/FoxO target genes is attenuated by blockade of phosphatase activity or by small interfering RNA (siRNA)-mediated knockdown of Sirt1 or FoxO protein expression. Finally, 1,25D-dependent cell cycle arrest is blocked in FoxO3a-deficient cells, indicating that FoxO proteins are key downstream mediators of the antiproliferative actions of 1,25D. These studies link 1,25D signaling through the VDR directly to Sirt1 and FoxO function and provide a molecular basis for the cancer chemopreventive actions of 1,25D.
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Chatterjee, Arpita, Pradeep K. Dutta, and Rukhsana Chowdhury. "Effect of Fatty Acids and Cholesterol Present in Bile on Expression of Virulence Factors and Motility of Vibrio cholerae." Infection and Immunity 75, no. 4 (January 29, 2007): 1946–53. http://dx.doi.org/10.1128/iai.01435-06.
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ABSTRACT Bile induces pleiotropic responses that affect production of virulence factors, motility, and other phenotypes in the enteric pathogen Vibrio cholerae. Since bile is a heterogeneous mixture, crude bile was fractionated, and the components that mediate virulence gene repression and enhancement of motility were identified by nuclear magnetic resonance, gas chromatography (GC), and GC-mass spectrometry analyses. The unsaturated fatty acids detected in bile, arachidonic, linoleic, and oleic acids, drastically repressed expression of the ctxAB and tcpA genes, which encode cholera toxin and the major subunit of the toxin-coregulated pilus, respectively. The unsaturated fatty acid-dependent repression was due to silencing of ctxAB and tcpA expression by the histone-like nucleoid-structuring protein H-NS, even in the presence of the transcriptional activator ToxT. Unsaturated fatty acids also enhanced motility of V. cholerae due to increased expression of flrA, the first gene of a regulatory cascade that controls motility. H-NS had no role in the fatty acid-mediated enhancement of motility. It is likely that the ToxR/ToxT system that negatively regulates motility is rendered nonfunctional in the presence of unsaturated fatty acids, leading to an increase in motility. Motility and flrA expression were also increased in the presence of cholesterol, another component of bile, in an H-NS- and ToxR/ToxT-independent manner.
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Choi, Garam, Kyung Ku Jang, Jong Gyu Lim, Zee-Won Lee, Hanhyeok Im, and Sang Ho Choi. "The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus." Journal of Biological Chemistry 295, no. 16 (March 13, 2020): 5350–61. http://dx.doi.org/10.1074/jbc.ra120.012724.
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For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.
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Naz, R. K., K. Ahmad, and P. Kaplan. "Expression and function of ras proto-oncogene proteins in human sperm cells." Journal of Cell Science 102, no. 3 (July 1, 1992): 487–94. http://dx.doi.org/10.1242/jcs.102.3.487.
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The presence and role of c-ras proteins were investigated in mature human sperm cells. The v-H-ras monoclonal antibody (mAb) against the c-ras protein, p21, reacted specifically with the acrosomal region of methanol-fixed as well as unfixed-live capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The v-H-ras mAb predominantly recognized c-ras protein of 21 kDa on the Western blot of lithium diiodosalicylate (LIS)-solubilized human sperm preparation. The incubation of sperm cells with v-H-ras mAb affected the sperm cell function in the human sperm penetration assay. The antibody significantly reduced the acrosome reaction and release of acrosin activity from the sperm cells. There was no effect of the mAb on percentage motility, although the mAb significantly affected various motility characteristics such as linearity, amplitude of lateral head displacement (ALH) and beat frequency, the motility parameters involved in the hyperactivation phenomenon of sperm cells leading to capacitation and acrosome reaction. These results suggest that the c-ras or c-ras-like proteins are present in mature sperm cell and may have a role in capacitation and/or acrosome reaction of human sperm cell.
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Atlung, T., S. Sund, K. Olesen, and L. Brøndsted. "The histone-like protein H-NS acts as a transcriptional repressor for expression of the anaerobic and growth phase activator AppY of Escherichia coli." Journal of bacteriology 178, no. 12 (1996): 3418–25. http://dx.doi.org/10.1128/jb.178.12.3418-3425.1996.
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Purtov, Yuri A., Olga A. Glazunova, Sergey S. Antipov, Viktoria O. Pokusaeva, Eugeny E. Fesenko, Elena V. Preobrazhenskaya, Konstantin S. Shavkunov, Maria N. Tutukina, Viktor I. Lukyanov, and Olga N. Ozoline. "Promoter islandsas a platform for interaction with nucleoid proteins and transcription factors." Journal of Bioinformatics and Computational Biology 12, no. 02 (April 2014): 1441006. http://dx.doi.org/10.1142/s0219720014410066.
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Seventy-eight promoter islands with an extraordinarily high density of potential promoters have been recently found in the genome of Escherichia coli. It has been shown that RNA polymerase binds internal promoters of these islands and produces short oligonucleotides, while the synthesis of normal mRNAs is suppressed. This quenching may be biologically relevant, as most islands are associated with foreign genes, which expression may deplete cellular resources. However, a molecular mechanism of silencing with the participation of these promoter-rich regions remains obscure. It has been demonstrated that all islands interact with histone-like protein H-NS — a specific sentinel of foreign genes. In this study, we demonstrated the inhibitory effect of H-NS using Δhns mutant of Escherichia coli and showed that deletion of dps, encoding another protein of bacterial nucleoid, tended to decrease rather than increase the amount of island-specific transcripts. This observation precluded consideration of promoter islands as sites for targeted heterochromatization only and a computer search for the binding sites of 53 transcription factors (TFs) revealed six proteins, which may specifically regulate their transcriptional output.