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1
Alcantara, O., S. V. Reddy, G. D. Roodman, and D. H. Boldt. "Transcriptional regulation of the tartrate-resistant acid phosphatase (TRAP) gene by iron." Biochemical Journal 298, no. 2 (March 1, 1994): 421–25. http://dx.doi.org/10.1042/bj2980421.
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Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting for increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 microM desferrioxamine, a potent iron chelator. The 5′ flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5′-flanking region of the TRAP gene contained iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5′ promoter region driving a luciferase reporter gene. Treatment of transfectants with 100 micrograms/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Constructs containing 1240 or 881 bp of the TRAP promoter gave only a 1.5- to 2-fold increase of luciferase activity with FeTF. In all cases, increase of luciferase activity was blocked by desferrioxamine. Cells transfected with another luciferase construct driven by a simian virus 40 promoter did not show any increase of luciferase activity with FeTF. These data indicate that expression of TRAP is regulated by iron and that this regulation is exerted at the level of gene transcription. The transfection experiments also suggest that the region of the TRAP 5′-flanking sequence between base pairs -1846 and -1240 contains an iron regulatory element.
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Casselli, Timothy, and Troy Bankhead. "Use of in vivo Expression Technology for the Identification of Putative Host Adaptation Factors of the Lyme Disease Spirochete." Journal of Molecular Microbiology and Biotechnology 25, no. 5 (2015): 349–61. http://dx.doi.org/10.1159/000439305.
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The causative agent of Lyme disease, <i>Borrelia burgdorferi,</i> is an obligate parasite that requires either a tick vector or a mammalian host for survival. Identification of the bacterial genes that are specifically expressed during infection of the mammalian host could provide targets for novel therapeutics and vaccines. In vivo expression technology (IVET) is a reporter-based promoter trap system that utilizes selectable markers to identify promoters of bacterial host-specific genes. Using previously characterized genes for in vivo and in vitro selection, this study utilized an IVET system that allows for selection of <i>B. burgdorferi</i> sequences that act as active promoters only during murine infection. This promoter trap system was able to successfully distinguish active promoter sequences both in vivo and in vitro from control sequences and a library of cloned <i>B. burgdorferi</i> genomic fragments. However, a bottleneck effect during the experimental mouse infection limited the utility for genome-wide promoter screening. Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of <i>B. burgdorferi,</i> and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with <i>B. burgdorferi.</i>
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Dunn, Anne K., Amy K. Klimowicz, and Jo Handelsman. "Use of a Promoter Trap To Identify Bacillus cereus Genes Regulated by Tomato Seed Exudate and a Rhizosphere Resident, Pseudomonas aureofaciens." Applied and Environmental Microbiology 69, no. 2 (February 2003): 1197–205. http://dx.doi.org/10.1128/aem.69.2.1197-1205.2003.
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ABSTRACT The goal of this study was to identify genes in Bacillus cereus, a bacterium commonly associated with plant seeds and roots, that are affected by compounds originating from a host plant, tomato, or another rhizosphere resident, Pseudomonas aureofaciens. We constructed a B. cereus chromosomal DNA library in a promoter-trap plasmid, pAD123, which contains a promoterless version of the green fluorescent protein (GFP) gene, gfpmut3a. The library was screened by using fluorescence-activated cell sorting for clones showing a change in GFP expression in response to either tomato seed exudate or culture supernatant of P. aureofaciens strain 30-84. We identified two clones carrying genes that were induced by the presence of tomato seed exudate and nine clones carrying genes that were repressed by P. aureofaciens culture supernatant. A clone chosen for further study contained an open reading frame, designated lipA, that encodes a deduced protein with a lipoprotein signal peptide sequence similar to lipoproteins in B. subtilis. Expression of gusA under control of the lipA promoter increased twofold when cells were exposed to tomato seed exudate and in a concentration-dependent manner when exposed to a mixture of amino acids. When the wild type and a 10-fold excess of a lipA mutant were applied together to tomato seeds, 2 days after planting, the wild type displayed medium-dependent culturability, whereas the lipA mutant was unaffected. This study demonstrates the power of a promoter trap to identify genes in a gram-positive bacterium that are regulated by the biotic environment and resulted in the discovery of lipA, a plant-regulated gene in B. cereus.
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Doree, Scott M., and Martha H. Mulks. "Identification of an Actinobacillus pleuropneumoniae Consensus Promoter Structure." Journal of Bacteriology 183, no. 6 (March 15, 2001): 1983–89. http://dx.doi.org/10.1128/jb.183.6.1983-1989.2001.
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ABSTRACT Actinobacillus pleuropneumoniaepromoter-containing clones were isolated from a genomic DNA library constructed in our lVET promoter trap vector pTF86. The promoter-containing clones were identified by their ability to drive expression of the promoterless luxAB genes of Vibrio harveyi. The degree of expression was quantifiable, and only high-expression or “hot” promoters were used for this study. Nine clones were sequenced, and their transcriptional start sites were determined by primer extension. The sequences upstream of the start site were aligned, and a consensus promoter structure for A. pleuropneumoniae was identified. The consensus promoter sequence for A. pleuropneumoniae was found to be TATAAT and TTG/AAA, centered approximately 10 and 35 bp upstream of the transcriptional start site, respectively. A comparison of the A. pleuropneumoniae consensus with other prokaryotic consensus promoters showed that the A. pleuropneumoniaeconsensus promoter is similar to that found in other eubacteria in terms of sequence, with an identical −10 element and a similar but truncated −35 element. However, the A. pleuropneumoniaeconsensus promoter is unique in the spacing between the −10 and −35 elements. The promoter spacing was analyzed by site-directed mutagenesis, which demonstrated that optimal spacing for an A. pleuropneumoniae promoter is shorter than the spacing identified for Escherichia coli and Bacillus subtilispromoters.
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KROJ, ANDREA, and HERBERT SCHMIDT. "Selection of In Vivo Expressed Genes of Escherichia coli O157:H7 Strain EDL933 in Ground Meat under Elevated Temperature Conditions." Journal of Food Protection 75, no. 10 (October 1, 2012): 1743–50. http://dx.doi.org/10.4315/0362-028x.jfp-11-453.
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Enterohemorrhagic Escherichia coli O157:H7 strains are important foodborne pathogens that are often transmitted to humans by the ingestion of raw or undercooked meat of bovine origin. To investigate adaptation of this pathogen during persistence and growth in ground meat, we established an in vivo expression technology model to identify genes that are expressed during growth in this food matrix under elevated temperatures (42°C). To improve on the antibiotic-based selection method, we constructed the promoter trap vector pAK-1, containing a promoterless kanamycin resistance gene. A genomic library of E. coli O157:H7 strain EDL933 was constructed in pAK-1 and used for promoter selection in ground meat. The 20 in vivo expressed genes identified were associated with transport processes, metabolism, macromolecule synthesis, and stress response. For most of the identified genes, only hypothetical functions could be assigned. The results of our study provide the first insights into the complex response of E. coli O157:H7 to a ground meat environment under elevated temperatures and establish a suitable vector for promoter studies or selection of in vivo induced promoters in foods such as ground meat.
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Dubnau, Eugenie, Patricia Fontán, Riccardo Manganelli, Sonia Soares-Appel, and Issar Smith. "Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages." Infection and Immunity 70, no. 6 (June 2002): 2787–95. http://dx.doi.org/10.1128/iai.70.6.2787-2795.2002.
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ABSTRACT We identified Mycobacterium tuberculosis genes preferentially expressed during infection of human macrophages using a promoter trap adapted for this pathogen. inhA encodes an enoyl-acyl carrier protein reductase that is required for mycolic acid biosynthesis (A. Quemard et al., Biochemistry 34:8235-8241, 1995) and is a major target for isoniazid (INH) in mycobacterial species (A. Banerjee et al., Science 263:227-230, 1994). Since overexpression of inhA confers INH resistance in Mycobacterium smegmatis (Banerjee et al., Science 263:227-230, 1994), we designed a promoter trap based on this gene. A library of clones, containing small fragments of M. tuberculosis DNA cloned upstream of inhA in a plasmid vector, was electroporated into M. tuberculosis, and the resulting culture was used to infect the human monocytic THP-1 cell line. Selection was made for clones surviving INH treatment during infection but retaining INH sensitivity on plates. The DNA upstream of inhA was sequenced in each clone to identify the promoter driving inhA expression. Thirteen genes identified by this method were analyzed by quantitative reverse transcription-PCR (R. Manganelli et al., Mol. Microbiol. 31:715-724, 1999), and eight of them were found to be differentially expressed from cultures grown in macrophages compared with broth-grown cultures. Several of these genes are presumed to be involved in fatty acid metabolism; one potentially codes for a unique DNA binding protein, one codes for a possible potassium channel protein, and the others code for proteins of unknown function. Genes which are induced during infection are likely to be significant for survival and growth of the pathogen; our results lend support to the view that fatty acid metabolism is essential for the virulence of M. tuberculosis.
7
Schneider, William P., Sun K. Ho, Jillian Christine, Monique Yao, Andrea Marra, and Alexander E. Hromockyj. "Virulence Gene Identification by Differential Fluorescence Induction Analysis of Staphylococcus aureus Gene Expression during Infection-Simulating Culture." Infection and Immunity 70, no. 3 (March 2002): 1326–33. http://dx.doi.org/10.1128/iai.70.3.1326-1333.2002.
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ABSTRACT We have employed a strategy utilizing differential fluorescence induction (DFI) in an effort to identify Staphylococcus aureus genes whose products can be targeted for antimicrobial drug development. DFI allows identification of promoters preferentially active under given growth conditions on the basis of their ability to drive expression of a promoterless green fluorescent protein gene (gfp). A plasmid-based promoter trap library was constructed of 200- to 1,000-bp fragments of S. aureus genomic DNA fused to gfp, and clones with active promoters were isolated under seven different in vitro growth conditions simulating infection. Six thousand two hundred sixty-seven clones with active promoters were screened to identify those that exhibited differential promoter activity. Bioinformatic analysis allowed the identification of 42 unique operons, containing a total of 61 genes, immediately downstream of the differentially active putative promoters. Replacement mutations were generated for most of these operons, and the abilities of the resulting mutants to cause infection were assessed in two different murine infection models. Approximately 40% of the mutants were attenuated in at least one infection model.
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Zaide, Galia, Haim Grosfeld, Sharon Ehrlich, Anat Zvi, Ofer Cohen, and Avigdor Shafferman. "Identification and Characterization of Novel and Potent Transcription Promoters ofFrancisella tularensis." Applied and Environmental Microbiology 77, no. 5 (December 30, 2010): 1608–18. http://dx.doi.org/10.1128/aem.01862-10.
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ABSTRACTTwo alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potentFrancisella tularensispromoters. Of the 26,000F. tularensisstrain LVSgfplibrary clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from thecatpromoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry uniqueF. tularensisDNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novelF. tularensispromoters reported here, along with the results ofin silicoand primer extension analyses, suggest thatF. tularensispossesses classicalEscherichia coliσ70-related promoter motifs. These motifs include the −10 (TATAAT) and −35 [TTGA(C/T)A] domains and an AT-rich region upstream from −35, reminiscent of but distinct from theE. coliupstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than theF. tularensis groELpromoter and is probably among the strongest promoters inF. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background ofF. tularensisintended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.
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Chen, S., M. Bagdasarian, M. G. Kaufman, and E. D. Walker. "Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System." Applied and Environmental Microbiology 73, no. 4 (December 22, 2006): 1089–100. http://dx.doi.org/10.1128/aem.01577-06.
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ABSTRACT We developed techniques for the genetic manipulation of Flavobacterium species and used it to characterize several promoters found in these bacteria. Our studies utilized Flavobacterium hibernum strain W22, an environmental strain we isolated from tree hole habitats of mosquito larvae. Plasmids from F. hibernum strain W22 were more efficiently (∼1,250-fold) transferred by electroporation into F. hibernum strain W22 than those isolated from Escherichia coli, thus indicating that an efficient restriction barrier exists between these species. The strong promoter, tac, functional in proteobacteria, did not function in Flavobacterium strains. Therefore, a promoter-trap plasmid, pSCH03, containing a promoterless gfpmut3 gene was constructed. A library of 9,000 clones containing chromosomal fragments of F. hibernum strain W22 in pSCH03 was screened for their ability to drive expression of the promoterless gfpmut3 gene. Twenty strong promoters were used for further study. The transcription start points were determined from seven promoter clones by the 5′ rapid amplification of cDNA ends technique. Promoter consensus sequences from Flavobacterium were identified as TAnnTTTG and TTG, where n is any nucleotide, centered approximately 7 and 33 bp upstream of the transcription start site, respectively. A putative novel ribosome binding site consensus sequence is proposed as TAAAA by aligning the 20-bp regions upstream of the translational start site in 25 genes. Our primary results demonstrate that at least some promoter and ribosome binding site motifs of Flavobacterium strains are unusual within the bacterial domain and suggest an early evolutionary divergence of this bacterial group. The techniques presented here allow for more detailed genetics-based studies and analyses of Flavobacterium species in the environment.
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Gat, O., I. Inbar, R. Aloni-Grinstein, E. Zahavy, C. Kronman, I. Mendelson, S. Cohen, B. Velan, and A. Shafferman. "Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines." Infection and Immunity 71, no. 2 (February 2003): 801–13. http://dx.doi.org/10.1128/iai.71.2.801-813.2003.
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ABSTRACT We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the α-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.
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Marra, Andrea, Jyoti Asundi, Magdalena Bartilson, Stacey Lawson, Flora Fang, Jillian Christine, Cedric Wiesner, Daniel Brigham, William P. Schneider, and Alexander E. Hromockyj. "Differential Fluorescence Induction Analysis of Streptococcus pneumoniae Identifies Genes Involved in Pathogenesis." Infection and Immunity 70, no. 3 (March 2002): 1422–33. http://dx.doi.org/10.1128/iai.70.3.1422-1433.2002.
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ABSTRACT Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.
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Brillard, Julien, Isabelle J�hanno, Claire Dargaignaratz, Isabelle Barbosa, Christian Ginies, Fr�d�ric Carlin, Sinda Fedhila, Christophe Nguyen-the, V�ronique Broussolle, and Vincent Sanchis. "Identification of Bacillus cereus Genes Specifically Expressed during Growth at Low Temperatures." Applied and Environmental Microbiology 76, no. 8 (February 26, 2010): 2562–73. http://dx.doi.org/10.1128/aem.02348-09.
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ABSTRACT The mechanisms involved in the ability of Bacillus cereus to multiply at low temperatures were investigated. It was assumed that many genes involved in cold acclimation would be upregulated at low temperatures. Recombinase-based in vivo expression technology (IVET) was adapted to the detection of the transient activation of B. cereus promoters during growth at 10�C. Four independent screenings of a promoter library from type strain ATCC 14579 were performed, and 17 clones were isolated. They corresponded to 17 promoter regions that displayed reproducibly elevated expression at 10�C relative to expression at 30�C. This analysis revealed several genes that may be important for B. cereus to grow successfully under the restrictive conditions of cold habitats. Among them, a locus corresponding to open reading frames BC5402 to BC5398, harboring a lipase-encoding gene and a putative transcriptional regulator, was identified three times. While a mutation in the putative regulator-encoding gene did not cause any particular phenotype, a mutant deficient in the lipase-encoding gene showed reduced growth abilities at low temperatures compared with the parental strain. The mutant did not change its fatty acid profiles in the same way as the wild type when grown at 12�C instead of 37�C. This study demonstrates the feasibility of a promoter trap strategy for identifying cold-induced genes. It outlines a first picture of the different processes involved in B. cereus cold acclimation.
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Saviola, Beatrice, Samuel C. Woolwine, and William R. Bishai. "Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology." Infection and Immunity 71, no. 3 (March 2003): 1379–88. http://dx.doi.org/10.1128/iai.71.3.1379-1388.2003.
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ABSTRACT A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.
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Li, W. W., Y. Hsiung, V. Wong, K. Galvin, Y. Zhou, Y. Shi, and A. S. Lee. "Suppression of grp78 core promoter element-mediated stress induction by the dbpA and dbpB (YB-1) cold shock domain proteins." Molecular and Cellular Biology 17, no. 1 (January 1997): 61–68. http://dx.doi.org/10.1128/mcb.17.1.61.
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The highly conserved grp78 core promoter element plays an important role in the induction of grp78 under diverse stress signals. Previous studies have established a functional region in the 3' half of the core (stress-inducible change region [SICR]) which exhibits stress-inducible changes in stressed nuclei. The human transcription factor YY1 is shown to bind the SICR and transactivate the core element under stress conditions. Here we report that expression library screening with the core element has identified two new core binding proteins, YB-1 and dbpA. Both proteins belong to the Y-box family of proteins characterized by an evolutionarily conserved DNA binding motif, the cold shock domain (CSD). In contrast to YY1, which binds only double-stranded SICR, the Y-box/CSD proteins much prefer the lower strand of the SICR. The Y-box proteins can repress the inducibility of the grp78 core element mediated by treatment of cells with A23187, thapsigargin, and tunicamycin. In gel shift assays, YY1 binding to the core element is inhibited by either YB-1 or dbpA. A yeast interaction trap screen using LexA-YY1 as a bait and a HeLa cell cDNA-acid patch fusion library identified YB-1 as a YY1-interacting protein. In cotransfection experiments, the Y-box proteins antagonize the YY1-mediated enhancement of transcription directed by the grp78 core in stressed cells. Thus, the CSD proteins may be part of the stress signal transduction mechanism in the mammalian system.
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Marco, Maria L., Jennifer Legac, and Steven E. Lindow. "Conditional Survival as a Selection Strategy To Identify Plant-Inducible Genes of Pseudomonas syringae." Applied and Environmental Microbiology 69, no. 10 (October 2003): 5793–801. http://dx.doi.org/10.1128/aem.69.10.5793-5801.2003.
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ABSTRACT A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of Pseudomonas syringae that are induced during growth on bean leaves. This strategy is based on the complementation of metXW, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of P. syringae on bean leaves exposed to conditions of low humidity. We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type P. syringae metXW genes. Only with an active promoter fused to metXW on pTrap did this plasmid restore methionine prototrophy to the P. syringae metXW mutant B7MX89 and survival of this strain on bean leaves. To test this method, a partial library of P. syringae genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves. This resulted in the enrichment of five clones, each with a unique RsaI restriction pattern of their DNA insert. Sequence analysis of these clones revealed those P. syringae genes for which putative plant-inducible activity could be assigned. Promoter activity experiments with a gfp reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium. Based on green fluorescent protein fluorescence levels, it appears that many P. syringae genes have relatively low expression levels and that the metXW HIRS strategy is a sensitive method to detect weakly expressed P. syringae genes that are active on plants. Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for P. syringae expressing metXW than exposed sites. Thus, the metXW HIRS strategy should lead to the identification of P. syringae genes that are expressed primarily in these areas on the leaf.
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Mason, Kevin M., Robert S. Munson, and Lauren O. Bakaletz. "Nontypeable Haemophilus influenzae Gene Expression Induced In Vivo in a Chinchilla Model of Otitis Media." Infection and Immunity 71, no. 6 (June 2003): 3454–62. http://dx.doi.org/10.1128/iai.71.6.3454-3462.2003.
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ABSTRACT The gram-negative bacterium nontypeable Haemophilus influenzae (NTHI) is the predominant pathogen in chronic otitis media with effusion and, with Streptococcus pneumoniae and Moraxella catarrhalis, is a causative agent of acute otitis media. To identify potential virulence determinants, bacterial gene expression was monitored by differential fluorescence induction during early disease progression in one specific anatomical niche of a chinchilla model of NTHI-induced otitis media. Genomic DNA fragments from NTHI strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene. NTHI strain 86-028NP served as the host for the promoter trap library. Pools of 2,000 transformants were inoculated into the left and right middle ear cavities of chinchillas. Middle ear effusions were recovered by epitympanic tap at 24 and 48 h, and clones containing promoter elements that were induced in vivo and producing green fluorescent protein were isolated by two-color fluorescence-activated cell sorting. Insert DNA was sequenced and compared to the complete genome sequence of H. influenzae strain Rd. In a screen of 16,000 clones, we have isolated 44 clones that contain unique gene fragments encoding biosynthetic enzymes, metabolic and regulatory proteins, and hypothetical proteins of unknown function. An additional eight clones contain gene fragments unique to our NTHI isolate. Using quantitative reverse transcription-PCR, we have confirmed that 26 clones demonstrated increased gene expression in vivo relative to expression in vitro. These data provide insight into the response of NTHI bacteria as they sense and respond to the middle ear microenvironment during early events of otitis media.
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Meng, Jiang-Ping, Yi-Bing Yin, Xue-Mei Zhang, Yuan-Shuai Huang, Kai Lan, Fang Cui, and Song-Xiao Xu. "Identification of Streptococcus pneumoniae genes specifically induced in mouse lung tissues." Canadian Journal of Microbiology 54, no. 1 (January 2008): 58–65. http://dx.doi.org/10.1139/w07-117.
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To identify Streptococcus pneumoniae genes expressed specifically during infections, a selection system based on the in vivo expression technology (IVET) was established. galU, which is critical for capsular polysaccharide biosynthesis, and lacZY encoding β-galactosidase were employed as dual reporter genes to screen in-vivo-induced (ivi) genes of S. pneumoniae. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose, thus failing to synthesize capsular polysaccharide, and therefore loses its ability to survive in the host. A promoter-trap library was constructed in S. pneumoniae, which was used to infect BALB/c mice in an intranostril model. Those strains recovered from lung tissue of mice and exhibiting a white colony phenotype on tryptic soy agar containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) were collected and identificated. A total of 15 unique sequences were obtained through in vivo screening. The ivi genes of S. pneumoniae are involved in many processes, such as colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, and cell wall synthesis. There are some hypothetical proteins whose functions are not clear. This novel IVET is a useful tool for identifying ivi genes in S. pneumoniae.
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Meier, Matthew J., E. Suzanne Paterson, and Iain B. Lambert. "Use of Substrate-Induced Gene Expression in Metagenomic Analysis of an Aromatic Hydrocarbon-Contaminated Soil." Applied and Environmental Microbiology 82, no. 3 (November 20, 2015): 897–909. http://dx.doi.org/10.1128/aem.03306-15.
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ABSTRACTMetagenomics allows the study of genes related to xenobiotic degradation in a culture-independent manner, but many of these studies are limited by the lack of genomic context for metagenomic sequences. This study combined a phenotypic screen known as substrate-induced gene expression (SIGEX) with whole-metagenome shotgun sequencing. SIGEX is a high-throughput promoter-trap method that relies on transcriptional activation of a green fluorescent protein (GFP) reporter gene in response to an inducing compound and subsequent fluorescence-activated cell sorting to isolate individual inducible clones from a metagenomic DNA library. We describe a SIGEX procedure with improved library construction from fragmented metagenomic DNA and improved flow cytometry sorting procedures. We used SIGEX to interrogate an aromatic hydrocarbon (AH)-contaminated soil metagenome. The recovered clones contained sequences with various degrees of similarity to genes (or partial genes) involved in aromatic metabolism, for example,nahG(salicylate oxygenase) family genes and their respective upstreamnahRregulators. To obtain a broader context for the recovered fragments, clones were mapped to contigs derived fromde novoassembly of shotgun-sequenced metagenomic DNA which, in most cases, contained complete operons involved in aromatic metabolism, providing greater insight into the origin of the metagenomic fragments. A comparable set of contigs was generated using a significantly less computationally intensive procedure in which assembly of shotgun-sequenced metagenomic DNA was directed by the SIGEX-recovered sequences. This methodology may have broad applicability in identifying biologically relevant subsets of metagenomes (including both novel and known sequences) that can be targeted computationally byin silicoassembly and prediction tools.
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Stamminger, Thomas, Matthias Gstaiger, Konstanze Weinzierl, Kerstin Lorz, Michael Winkler, and Walter Schaffner. "Open Reading Frame UL26 of Human Cytomegalovirus Encodes a Novel Tegument Protein That Contains a Strong Transcriptional Activation Domain." Journal of Virology 76, no. 10 (May 15, 2002): 4836–47. http://dx.doi.org/10.1128/jvi.76.10.4836-4847.2002.
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ABSTRACT A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.
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Vidal, F., P. Lopez, L. A. Lopez-Fernandez, F. Ranc, J. C. Scimeca, F. Cuzin, and M. Rassoulzadegan. "Gene trap analysis of germ cell signaling to Sertoli cells: NGF-TrkA mediated induction of Fra1 and Fos by post-meiotic germ cells." Journal of Cell Science 114, no. 2 (January 15, 2001): 435–43. http://dx.doi.org/10.1242/jcs.114.2.435.
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Analysis of complex signalisation networks involving distinct cell types is required to understand most developmental processes. Differentiation of male germ cells in adult mammals involves such a cross-talk between Sertoli cells, the somatic component which supports and controls germinal differentiation, and germ cells at their successive maturation stages. We developed a gene trapping strategy to identify genes, which, in Sertoli cells, are either up- or down-regulated by signals emitted by the germinal component. A library of approximately 2,000 clones was constituted from colonies independently selected from the Sertoli line 15P-1 by growth in drug-containing medium after random integration of a promoter-less (beta)geo transgene (neo(r)-lacZ fusion), which will be expressed as a fusion transcript from a ‘trapped’ cellular promoter, different in each clone. A first screen conducted on 700 events identified six clones in which beta-galactosidase activity was increased and one in which it was repressed upon addition of germ cells. The targeted loci were identified by cloning and sequencing the genomic region 5′ of the insert. One of them was identified as the gene encoding Fra1, a component of the AP1 transcription regulatory complex. Accumulation of Fra1 mRNA was induced, both in 15P-1 and in freshly explanted Sertoli cells, by addition of either round spermatids or nerve growth factor (NGF). The effect of NGF was mediated by the TrkA receptor and the ERK1-ERK2 kinase kinase pathway. Fos and Fra1 transcription were induced within the first hour after addition of the neurotrophin, but, unlike what is observed after serum induction in the same cells, a second wave of transcription of Fra1, but not of Fos, started 16 hours later and peaked at higher levels at about 20 hours. These results suggest that AP1 activation may be an important relay in the Sertoli-germ cell cross-talk, and validate the gene trapping approach as a tool for the identification of target genes in cell culture systems.
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Fujita, Natsuko, Kenji Oritani, Michiko Ichii, Norimitsu Saitoh, Kengo Yamawaki, Kazuma Tomizuka, and Yuzuru Kanakura. "Osteoblast Stimulating Factor-5 Regulates B Lymphopoiesis Via Inhibiting Pre B Cell Proliferation." Blood 124, no. 21 (December 6, 2014): 2926. http://dx.doi.org/10.1182/blood.v124.21.2926.2926.
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Abstract B lymphopoiesis is a complex and multistep process originated from hematopoietic stem cells (HSC). Recent studies showed that the microenvironment surrounding progenitor cells affects the development of B cells. Soluble factors that bone marrow (BM) stromal cells produce are crucial to the process. For example, SDF1α regulates the survival and homing of HSC and the lineage progenitor cells. IL7 is produced by osteoblasts and promotes the differentiation and proliferation of pre B cells. Although various molecules have been reported for the important roles, there are many proteins, which are secreted from stromal cells and function as a direct regulator of progenitors, remained unknown. In this study, we aimed to find novel secreted or membrane proteins, which modulate B cell differentiation via the microenvironment. We used MS-5 stromal cells to identify the novel secreted regulators because the cell line is known to have the potential to support HSC and B lymphopoiesis. The secreted proteins have signal sequence to pass into endoplasmic reticulum, followed by cell surface expression. Thus, we applied the modified signal sequence trap method (Tashiro et al, Science, 1993). Briefly, the cDNA library from MS-5 was inserted to the HPC4-TF/pEFBOS vector to produce fusion proteins, composed of proteins from cDNA, a HPC4-epitope, and a tissue factor transmembrane. With the screening of each plasmid based on the capacity to express HPC4-epitope on cell surface, we successfully identified 21 secreted or transmembrane proteins, which MS-5 cells produce. Among these proteins, pleiotrophin, proliferin-2 and osteoblast stimulating factor-5 (OSF-5) were selected because of the limited mRNA expression within stromal cell lines. For the functional analysis without the effects on the process of fetus development, we generated transgenic chimera mice (Tg), which produce the indicated protein under the control of kappa chain promoter. As a result, OSF-5, but not pleiotrophin or proliferin-2, Tg showed impaired B lymphocyte development. In OSF-5 chimera mice, the number of B lineage cells was decreased. B220+ cells in the spleen as well as pre B cells and immature B cells in the BM were significantly decreased (B220low CD43low IgM- pre B cells: 2.2 ± 0.2 x 105 cells in OSF-5 Tg vs. 6.7 ± 1.9 x 105 cells in control, B220+ CD43- IgM+ immature B cells: 2.3 ± 0.6 x 105 cells in OSF-5 Tg vs. 5.3 ± 0.8 x 105 cells in control). OSF-5 is widely expressed in mice BM, spleen, thymus, liver, kidney and lung, and the effects on hematopoiesis have never been examined. OSF-5 includes two splicing variants. Variant 1 is a secreted protein and known as aortic carboxypeptidase like protein. Variant 2 is a non-secreted, intracellular protein, known as adipocyte enhancer binding protein. In mice BM, OSF-5 variant 1 is secreted only from stromal cells, while OSF-5 variant 2 is expressed in hematopoietic cells. First, to exclude the cell-intrinsic effects of OSF-5, we ectopically expressed variant 2 in lineage- Sca-1+ c-Kit+ Flt3- HSC and co-cultured them with MS-5. As a result, the generated number of CD19+ B cells was not changed. In contrast, when we knocked down the secreted type of OSF-5 in OP9 stromal cell line to mimic the BM environment, the modified OP9 cells could support the proliferation of pre B cell line, 2E8 more efficiently, compared to control (7.3 ± 1.1 x 105 cells in KD vs. 4.4 ± 0.7 x 105 cells in control). In addition, the knock-down (KD) of variant 1 protein increased the recovered number of CD19+ cells in co-cultures of BM mononuclear cells (6.6 ± 2.8 x 105 cells in KD vs. 4.7 ± 2.8 x 105 cells in control). Finally, we found that colony-forming unit of pre B cells was decreased in the existence of OSF-5 variant 1 (17 ± 8 colonies in variant 1 vs. 198 ± 22 colonies in control). This result indicated that OSF-5 produced by BM stromal cells had a direct effect to inhibit the proliferation of pre B cells. In conclusion, we identified OSF-5 as a BM stromal cell-derived secreted protein, which has an ability to inhibit B lymphopoiesis via regulating the pre B cell proliferation. Our findings could help us to understand molecular regulatory mechanisms of normal B lymphopoiesis as well as causes of B lymphocyte dysregulation, such as change during aging. Disclosures No relevant conflicts of interest to declare.
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Wang, Congying, Shen Chen, Aiqing Feng, Jing Su, Wenjuan Wang, Jinqi Feng, Bing Chen, et al. "Xa7, a Small Orphan Gene Harboring Promoter Trap for AvrXa7, Leads to the Durable Resistance to Xanthomonas oryzae Pv. oryzae." Rice 14, no. 1 (May 30, 2021). http://dx.doi.org/10.1186/s12284-021-00490-z.
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Abstract Background The rice (Oryza sativa) gene Xa7 has been hypothesized to be a typical executor resistance gene against Xanthomonas oryzae pv. oryzae (Xoo), and has conferred durable resistance in the field for decades. Its identity and the molecular mechanisms underlying this resistance remain elusive. Results Here, we filled in gaps of genome in Xa7 mapping locus via BAC library construction, revealing the presence of a 100-kb non-collinear sequence in the line IRBB7 compared with Nipponbare reference genomes. Complementary transformation with sequentially overlapping subclones of the BACs demonstrated that Xa7 is an orphan gene, encoding a small novel protein distinct from any other resistance proteins reported. A 27-bp effector binding element (EBE) in the Xa7 promoter is essential for AvrXa7-inducing expression model. XA7 is anchored in the endoplasmic reticulum membrane and triggers programmed cell death in rice and tobacco (Nicotiana benthamiana). The Xa7 gene is absent in most cultivars, landraces, and wild rice accessions, but highly homologs of XA7 were identified in Leersia perrieri, the nearest outgroup of the genus Oryza. Conclusions Xa7 acts as a trap to perceive AvrXa7 via EBEAvrXa7 in its promoter, leading to the initiation of resistant reaction. Since EBEAvrXa7 is ubiquitous in promoter of rice susceptible gene SWEET14, the elevated expression of which is conducive to the proliferation of Xoo, that lends a great benefit for the Xoo strains retaining AvrXa7. As a result, varieties harboring Xa7 would show more durable resistance in the field. Xa7 alleles analysis suggests that the discovery of new resistance genes could be extended beyond wild rice, to include wild grasses such as Leersia species.