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Chu, Chishih, Cheng-Hsun Chiu, Chi-Hong Chu, and Jonathan T. Ou. "Nucleotide and Amino Acid Sequences of oriT-traM-traJ-traY-traA-traL Regions and Mobilization of Virulence Plasmids of Salmonella enterica Serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium." Journal of Bacteriology 184, no. 11 (June 1, 2002): 2857–62. http://dx.doi.org/10.1128/jb.184.11.2857-2862.2002.
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ABSTRACT The virulence plasmid of Salmonella enterica serovar Gallinarum-Pullorum (pSPV) but not those of Salmonella enterica serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the oriT-traM-traJ-traY-traA-traL regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the oriT of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the oriT of the other two pSVs. The nucleotide sequences of the oriT-traM-traJ-traY-traA-traL region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in traJ, traY, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus βαα fragments in the TraY of pSPV and F plasmid, whereas there was only a single βαα fragment in that of pSEV and pSTV.
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Arutyunov, Denis, Barbara Arenson, Jan Manchak, and Laura S. Frost. "F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation." Journal of Bacteriology 192, no. 6 (January 15, 2010): 1730–34. http://dx.doi.org/10.1128/jb.00726-09.
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ABSTRACT F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.
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Miyazaki, Ryo, Yoshiyuki Ohtsubo, Yuji Nagata, and Masataka Tsuda. "Characterization of the traD Operon of Naphthalene-Catabolic Plasmid NAH7: a Host-Range Modifier in Conjugative Transfer." Journal of Bacteriology 190, no. 19 (August 1, 2008): 6281–89. http://dx.doi.org/10.1128/jb.00709-08.
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ABSTRACT Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to 105-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).
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He, Xuesong, William Chang, Deanne L. Pierce, Laura Ort Seib, Jennifer Wagner, and Clay Fuqua. "Quorum Sensing in Rhizobium sp. Strain NGR234 Regulates Conjugal Transfer (tra) Gene Expression and Influences Growth Rate." Journal of Bacteriology 185, no. 3 (February 1, 2003): 809–22. http://dx.doi.org/10.1128/jb.185.3.809-822.2003.
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ABSTRACT Rhizobium sp. strain NGR234 forms symbiotic, nitrogen-fixing nodules on a wide range of legumes via functions largely encoded by the plasmid pNGR234a. The pNGR234a sequence revealed a region encoding plasmid replication (rep) and conjugal transfer (tra) functions similar to those encoded by the rep and tra genes from the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens, including homologues of the Ti plasmid quorum-sensing regulators TraI, TraR, and TraM. In A. tumefaciens, TraI, a LuxI-type protein, catalyzes synthesis of the acylated homoserine lactone (acyl-HSL) N-3-oxo-octanoyl-l-homoserine lactone (3-oxo-C8-HSL). TraR binds 3-oxo-C8-HSL and activates expression of Ti plasmid tra and rep genes, increasing conjugation and copy number at high population densities. TraM prevents this activation under noninducing conditions. Although the pNGR234a TraR, TraI, and TraM appear to function similarly to their A. tumefaciens counterparts, the TraR and TraM orthologues are not cross-functional, and the quorum-sensing systems have differences. NGR234 TraI synthesizes an acyl-HSL likely to be 3-oxo-C8-HSL, but traI mutants and a pNGR234a-cured derivative produce low levels of a similar acyl-HSL and another, more hydrophobic signal molecule. TraR activates expression of several pNGR234a tra operons in response to 3-oxo-C8-HSL and is inhibited by TraM. However, one of the pNGR234a tra operons is not activated by TraR, and conjugal efficiency is not affected by TraR and 3-oxo-C8-HSL. The growth rate of NGR234 is significantly decreased by TraR and 3-oxo-C8-HSL through functions encoded elsewhere in the NGR234 genome.
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Geymonat, Ludovico V. "Il primo bagno di Gesù a Traù e Venezia." Hortus Artium Medievalium 20, no. 2 (May 2014): 854–60. http://dx.doi.org/10.1484/j.ham.5.102699.
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Harris, Robin L., and Philip M. Silverman. "Tra Proteins Characteristic of F-Like Type IV Secretion Systems Constitute an Interaction Group by Yeast Two-Hybrid Analysis." Journal of Bacteriology 186, no. 16 (August 15, 2004): 5480–85. http://dx.doi.org/10.1128/jb.186.16.5480-5485.2004.
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ABSTRACT Using yeast two-hybrid screens, we have defined an interaction group of six Tra proteins encoded by the F plasmid and required by F+ cells to elaborate F pili. The six proteins are TraH, TraF, TraW, TraU, TrbI, and TrbB. Except for TrbI, these proteins were all identified as hallmarks of F-like type IV secretion systems (TFSSs), with no homologues among TFSS genes of P-type or I-type systems (T. Lawley, W. Klimke, M. Gubbins, and L. Frost, FEMS Microbiol. Lett. 224:1-15, 2003). Also with the exception of TrbI, which is an inner membrane protein, the remaining proteins are or are predicted to be periplasmic. TrbI consists of one membrane-spanning segment near its N terminus and an 88-residue, hydrophilic domain that extends into the periplasm. Hence, the proteins of this group probably form a periplasmic cluster in Escherichia coli. The interaction network identifies TraH as the most highly connected node, with two-hybrid links to TrbI, TraU, and TraF. As measured by transcriptional activation of lacZ, the TrbI-TraH interaction in Saccharomyces cerevisiae requires the TraH amino acid segment from residues 193 to 225. The TraU and TraF interactions are localized to C-terminal segments of TraH (amino acids 315 to 458 for TraF and amino acids 341 to 458 for TraU). The TrbI-TraH interaction with full-length (less the signal peptide) TraH is weak but increases 40-fold with N-terminal TraH deletions; the first 50 amino acids appear to be critical for inhibiting TrbI binding in yeast. Previous studies by others have shown that, with the exception of trbB mutations, which do not affect the elaboration or function of F pili under laboratory conditions, a mutation in any of the other genes in this interaction group alters the number or length distribution of F pili. We propose a model whereby one function of the TraH interaction group is to control F-pilus extension and retraction.
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Schmidt-Eisenlohr, Heike, Natalie Domke, and Christian Baron. "TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures inEscherichia coli." Journal of Bacteriology 181, no. 18 (September 15, 1999): 5563–71. http://dx.doi.org/10.1128/jb.181.18.5563-5571.1999.
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ABSTRACT Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402–410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coliallowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery,traM, traB, traD, andtraE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essentialtra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.
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Karl, Wolfgang, Martina Bamberger, and Ellen L. Zechner. "Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed oriT Cleavage In Vivo." Journal of Bacteriology 183, no. 3 (February 1, 2001): 909–14. http://dx.doi.org/10.1128/jb.183.3.909-914.2001.
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ABSTRACT The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (oriT) in vivo was investigated. As expected, the cleavage reaction was not detected in Escherichia coli cells expressing tral and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at oriT in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1 oriT constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations,traY was dispensable for mobilization in the presence oftraM, but mobilization did not occur in the absence of bothtraM and traY. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA.
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Babić, Ivo. "Due colonnine con rilievi sul portale del duomo di Traù (Trogir)." IKON 2 (January 2009): 177–90. http://dx.doi.org/10.1484/j.ikon.3.41.
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Grahn, A. Marika, Jana Haase, Dennis H. Bamford, and Erich Lanka. "Components of the RP4 Conjugative Transfer Apparatus Form an Envelope Structure Bridging Inner and Outer Membranes of Donor Cells: Implications for Related Macromolecule Transport Systems." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1564–74. http://dx.doi.org/10.1128/jb.182.6.1564-1574.2000.
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ABSTRACT During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPα plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the twoE. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.
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Will, William R., and Laura S. Frost. "Hfq Is a Regulator of F-Plasmid TraJ and TraM Synthesis in Escherichia coli." Journal of Bacteriology 188, no. 1 (January 1, 2006): 124–31. http://dx.doi.org/10.1128/jb.188.1.124-131.2006.
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ABSTRACT The F plasmid of Escherichia coli allows horizontal DNA transfer between an F+ donor cell and an F− recipient. Expression of the transfer genes is tightly controlled by a number of factors, including the following plasmid-encoded regulatory proteins: TraJ, the primary activator of the 33-kb tra operon, and the autoregulators TraM and TraY. Here, we demonstrate that the host RNA binding protein, Hfq, represses TraJ and TraM synthesis by destabilizing their respective mRNAs. Mating assays and immunoblot analyses for TraM and TraJ showed that transfer efficiency and protein levels increased in host cells containing a disruption in hfq compared to wild-type cells in stationary phase. The stability of transcripts containing a putative Hfq binding site located in the intergenic untranslated region between traM and traJ was increased in hfq mutant donor cells, suggesting that Hfq destabilizes these transcripts. Electrophoretic mobility shift assays demonstrated that Hfq specifically binds this region but not the antisense RNA, FinP, encoded on the opposite strand. Together, these findings indicate that Hfq regulates traM and traJ transcript stability by a mechanism separate from FinOP-mediated repression.
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Lawley, Trevor D., Matthew W. Gilmour, James E. Gunton, Leah J. Standeven, and Diane E. Taylor. "Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27." Journal of Bacteriology 184, no. 8 (April 15, 2002): 2173–80. http://dx.doi.org/10.1128/jb.184.8.2173-2180.2002.
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ABSTRACT The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.
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Lin, Mei-Hui, and Shih-Tung Liu. "Stabilization of pSW100 from Pantoea stewartii by the F Conjugation System." Journal of Bacteriology 190, no. 10 (March 14, 2008): 3681–89. http://dx.doi.org/10.1128/jb.00846-07.
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ABSTRACT Plasmid pSW100 is 1 of the 13 plasmids from Pantoea stewartii subsp. stewartii SW2 which has a replicon that resembles that of ColE1. This work uses a pSW100 derivative, pSW140K, to study how the pSW100 replicon is stably maintained in its hosts. Our results indicate that although pSW140K is stable in Escherichia coli HB101, the plasmid is rapidly lost in another E. coli strain, DH5α, indicating that the genetic background of an E. coli strain affects the stability of pSW140K. Mutagenesis of E. coli HB101 with EZ::TN <DHFR-1> revealed that mutations in traC, traF, traG, traN, and traV, which encode the components of the sex pilus assembly, reduce plasmid stability. Furthermore, this work identified that a 38-bp region located immediately upstream of the RNAII promoter is critical to the maintenance of plasmid stability in E. coli HB101. TraC binds to the region, and in addition, deleting the region destabilizes the plasmid. Furthermore, inserting this 38-bp fragment into a plasmid that contains the minimal replicon from pSW200 stabilizes the plasmid in E. coli HB101. Fluorescence in situ hybridization and immunofluorescence staining also revealed that derivatives of pSW100, pSW128A, and TraC are colocalized in cells, suggesting that pSW100 may use the sex pilus assembly as a partition apparatus to ensure the even distribution of the plasmid during cell division, which may thus maintain the plasmid's stability.
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Tun-Garrido, Cristina, Patricia Bustos, Víctor González, and Susana Brom. "Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing." Journal of Bacteriology 185, no. 5 (March 1, 2003): 1681–92. http://dx.doi.org/10.1128/jb.185.5.1681-1692.2003.
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ABSTRACT Rhizobium etli CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the tra genes are highly similar to genes of Agrobacterium tumefaciens pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (traI, traR, and cinR) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although R. etli has been shown to produce different N-acyl-homoserine lactones, only one of them, a 3-oxo-C8-homoserine lactone encoded by the traI gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene, traM, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C8-homoserine lactone synthesized by TraI. The traI gene was autoregulated by these elements and positively regulated by CinR, while cinR expression required traI. Finally, we did not detect expression of traM, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a.
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Fekete, Richard A., and Laura S. Frost. "Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity." Journal of Bacteriology 182, no. 14 (July 15, 2000): 4022–27. http://dx.doi.org/10.1128/jb.182.14.4022-4027.2000.
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ABSTRACT Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage atnic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage atnic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriTallowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).
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Gubbins, Michael J., Isabella Lau, William R. Will, Janet M. Manchak, Tracy L. Raivio, and Laura S. Frost. "The Positive Regulator, TraJ, of the Escherichia coli F Plasmid Is Unstable in a cpxA* Background." Journal of Bacteriology 184, no. 20 (October 15, 2002): 5781–88. http://dx.doi.org/10.1128/jb.184.20.5781-5788.2002.
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ABSTRACT The Cpx (conjugative plasmid expression) stress response of Escherichia coli is induced in response to extracytoplasmic signals generated in the cell envelope, such as misfolded proteins in the periplasm. Detection of stress is mediated by the membrane-bound histidine kinase, CpxA. Signaling of the response regulator CpxR by activated CpxA results in the expression of several factors required for responding to cell envelope stress. CpxA was originally thought to be required for the expression of the positive regulator of the F plasmid transfer (tra) operon, TraJ. It was later determined that constitutive gain-of-function mutations in cpxA led to activation of the Cpx envelope stress response and decreased TraJ expression. In order to determine the nature of the downregulation of TraJ, the level of expression of TraJ, TraM, and TraY, the F-encoded regulatory proteins of the F tra region, was determined both in a cpxA* background and in a wild-type background in which the Cpx stress response was induced by overexpression of the outer membrane lipoprotein, NlpE. Our results suggest that TraJ downregulation is controlled by a posttranscriptional mechanism that operates in the cytoplasm in response to upregulation of the Cpx stress response by both the cpxA* gain-of-function mutation and the overexpression of NlpE.
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Will, William R., and Laura S. Frost. "Characterization of the Opposing Roles of H-NS and TraJ in Transcriptional Regulation of the F-Plasmid tra Operon." Journal of Bacteriology 188, no. 2 (January 15, 2006): 507–14. http://dx.doi.org/10.1128/jb.188.2.507-514.2006.
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ABSTRACT The transfer (tra) operon of the conjugative F plasmid of Escherichia coli is a polycistronic 33-kb operon which encodes most of the proteins necessary for F-plasmid transfer. Here, we report that transcription from PY, the tra operon promoter, is repressed by the host nucleoid-associated protein, H-NS. Electrophoretic mobility shift assays indicate that H-NS binds preferentially to the tra promoter region, while Northern blot and transcriptional fusion analyses indicate that transcription of traY, the first gene in the tra operon, is derepressed in an hns mutant throughout growth. The plasmid-encoded regulatory protein TraJ is essential for transcription of the tra operon in wild-type Escherichia coli; however, TraJ is not necessary for plasmid transfer or traY operon transcription in an hns mutant. This indicates that H-NS represses transcription from PY directly and not indirectly via its effects on TraJ levels. These results suggest that TraJ functions to disrupt H-NS silencing at PY, allowing transcription of the tra operon.
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Kataoka, Masakazu, Takeshi Tanaka, Toshiyuki Kohno, and Yusuke Kajiyama. "The Carboxyl-Terminal Domain of TraR, a Streptomyces HutC Family Repressor, Functions in Oligomerization." Journal of Bacteriology 190, no. 21 (August 22, 2008): 7164–69. http://dx.doi.org/10.1128/jb.00843-08.
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ABSTRACT Efficient conjugative transfer of the Streptomyces plasmid pSN22 is accomplished by regulated expression of the tra operon genes, traA, traB, and spdB. The TraR protein is the central transcriptional repressor regulating the expression of the tra operon and itself and is classified as a member of the HutC subfamily in the helix-turn-helix (HTH) GntR protein family. Sequence information predicts that the N-terminal domain (NTD) of TraR, containing an HTH motif, functions in binding of DNA to the cis element; however, the function of the C-terminal region remains obscure, like that for many other GntR family proteins. Here we demonstrate the domain structure of the TraR protein and explain the role of the C-terminal domain (CTD). The TraR protein can be divided into two structural domains, the NTD of M1 to R95 and the CTD of Y96 to E246, revealed by limited proteolysis. Domain expression experiments revealed that both domains retained their function. An in vitro pull-down assay using recombinant TraR proteins revealed that TraR oligomerization depended on the CTD. A bacterial two-hybrid system interaction assay revealed that the minimum region necessary for this binding is R95 to P151. A mutant TraR protein in which Leu121 was replaced by His exhibited a loss of both oligomerization ability and repressor function. An in vitro cross-linking assay revealed preferential tetramer formation by TraR and the minimum CTD. These results indicate that the C-terminal R95-to-P151 region of TraR functions to form an oligomer, preferentially a tetramer, that is essential for the repressor function of TraR.
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Schröder, Gunnar, Sabine Krause, Ellen L. Zechner, Beth Traxler, Hye-Jeong Yeo, Rudi Lurz, Gabriel Waksman, and Erich Lanka. "TraG-Like Proteins of DNA Transfer Systems and of the Helicobacter pylori Type IV Secretion System: Inner Membrane Gate for Exported Substrates?" Journal of Bacteriology 184, no. 10 (May 15, 2002): 2767–79. http://dx.doi.org/10.1128/jb.184.10.2767-2779.2002.
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ABSTRACT TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.
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Paterson, E. Suzanne, Margret I. Moré, Gansen Pillay, Christina Cellini, Roger Woodgate, Graham C. Walker, V. N. Iyer, and Stephen C. Winans. "Genetic Analysis of the Mobilization and Leading Regions of the IncN plasmids pKM101 and pCU1." Journal of Bacteriology 181, no. 8 (April 15, 1999): 2572–83. http://dx.doi.org/10.1128/jb.181.8.2572-2583.1999.
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ABSTRACT The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within theirtra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tracomplementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination atoriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA,stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.
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Zhu, Jun, John W. Beaber, Margret I. Moré, Clay Fuqua, Anatol Eberhard, and Stephen C. Winans. "Analogs of the Autoinducer 3-Oxooctanoyl-Homoserine Lactone Strongly Inhibit Activity of the TraR Protein ofAgrobacterium tumefaciens." Journal of Bacteriology 180, no. 20 (October 15, 1998): 5398–405. http://dx.doi.org/10.1128/jb.180.20.5398-5405.1998.
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ABSTRACT The TraR and TraI proteins of Agrobacterium tumefaciensmediate cell-density-dependent expression of the Ti plasmidtra regulon. TraI synthesizes the autoinducer pheromoneN-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C8-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type A. tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C8-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with tra gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C8-HSL significantly stimulates tra gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.
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Mihajlovic, Sanja, Silvia Lang, Marta V. Sut, Heimo Strohmaier, Christian J. Gruber, Günther Koraimann, Elena Cabezón, Gabriel Moncalián, Fernando de la Cruz, and Ellen L. Zechner. "Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface." Journal of Bacteriology 191, no. 22 (September 18, 2009): 6877–87. http://dx.doi.org/10.1128/jb.00918-09.
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ABSTRACT Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔLk = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.
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Yonezawa, L. A., T. S. Barbosa, M. J. Watanabe, C. L. Marinho, J. L. Knaut, and A. Kohayagawa. "Efeito da suplementação com vitamina E sobre os metabolismos oxidativo e cardíaco em equinos submetidos a exercício de alta intensidade." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 67, no. 1 (February 2015): 71–79. http://dx.doi.org/10.1590/1678-7019.
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A suplementação antioxidante visa prevenir os danos oxidativos induzidos pelo exercício físico em diversos tecidos, como o miocárdio. Nesse contexto, este estudo objetivou avaliar os marcadores cardíacos e a lipoperoxidação em equinos no teste de exercício de rápida aceleração e curta duração (TRA), em esteira de alta velocidade, antes e após a suplementação com vitamina E. Para tanto, foram utilizados 10 equinos sem treinamento, que realizaram o primeiro TRA (TRA1) com carga de trabalho fundamentada no consumo máximo de oxigênio individual (VO2max) e que induziu a concentração de lactato maior que 4mmol/L, sendo considerado predominantemente anaeróbico. Em seguida, os equinos receberam vitamina E (dl-alfa-tocoferol) na dose de 1.000UI/dia, por via oral, durante 52 dias, e, posteriormente, realizaram um segundo TRA (TRA2) com o mesmo protocolo de TRA1. As amostras de sangue foram colhidas nos momentos antes do exercício, imediatamente após o término do teste e em 1h, 3h, 6h, 12h e 24h subsequentes. Determinou-se o malondialdeído (MDA) plasmático como índice de lipoperoxidação, e as concentrações séricas de troponina I cardíaca (cTnI), isoenzima MB da creatinoquinase (CK-MB) e mioglobina, como marcadores cardíacos. Como efeito do exercício, observou-se aumento discreto de MDA, de cTnI e de CK-MB, sendo significativo apenas para CK-MB. A suplementação foi capaz de amenizar a produção das espécies reativas de oxigênio, evidenciada pela menor concentração de MDA em TRA2, em 24h, além de causar um efeito protetor no miocárdio, devido ao menor valor de cTnI em 6h no TRA2 em relação ao TRA1. Não houve grandes alterações na concentração de mioglobina. Concluiu-se que o exercício de alta intensidade promoveu estresse no miocárdio nos equinos avaliados, bem como houve efeito benéfico da vitamina E na proteção miocárdica e sobre a lipoperoxidação.
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Audette, Gerald. "Structural, Functional and Dynamic Studies of F Plasmid T4SS Proteins." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1285. http://dx.doi.org/10.1107/s2053273314087142.
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The transfer of genetic material within a bacterial population through the process of conjugation distributes novel genetic elements for survival in unique environments. Bacterial conjugation is important to public health as the spread antibiotic resistance genes among bacteria results in multi-drug resistance. Indeed, approximately 70% of bacteria that cause hospital-acquired infections are resistant to at least one antibiotic. Conjugative systems, such as the F plasmid of Escherichia coli, consist of proteins that share similarities to type IV secretion systems (T4SS). T4SS proteins of the F plasmid form a membrane spanning protein complex and surface exposed pilus. The periplasmic T4SS proteins TraF, TraW and TrbC play important roles during the F pilus assembly and DNA transfer. Functional analysis of a series of TraF mutants has shown that modification to TraF abolishes pilus synthesis and in turn F plasmid conjugation. In addition, dynamic analysis of TraF using time-resolved hydrogen-deuterium exchange mass spectrometry has revealed a well structured C-terminal thioredoxin-like domain and a more dynamic N-terminal domain that is predicted to interact with companion T4SS protein TraH. In addition, interaction analysis of the putative pore forming proteins TraW and TrbC indicate that the C-terminal domain of TrbC is not required for interaction with TraW, unlike previous models of F T4SS assembly. Rather the C-terminal domain of TraW preferentially interacts with the N-terminal domain of TrbC. These studies are providing a clearer picture of the structures and interactions that occur within the F T4SS assembly during the conjugative process.
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Su, Shengchang, Sharik R. Khan, and Stephen K. Farrand. "Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal." Journal of Bacteriology 190, no. 13 (January 18, 2008): 4398–407. http://dx.doi.org/10.1128/jb.01684-07.
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ABSTRACT Conjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of TraR and its signal N-(3-oxo-octanoyl)-l-homoserine lactone. This system is, in turn, controlled by the conjugative opines produced by crown gall tumors induced on plants by the bacteria. Using nonpolar traI mutants, we examined the kinetics of induction of conjugative transfer in response to exogenous acyl-homoserine lactone. In the absence of the antiactivator TraM, onset of induction of transfer requires about 30 min, 15 to 20 min of which is needed for expression and construction of the conjugative apparatus. TraM delays the onset of conjugation by 30 min. While the rate of development of conjugative competence was not significantly affected by levels of TraR, maximum efficiencies of transfer were correlated with amounts of the activator in the donors. Donors harboring Ti plasmids lacking TraM were fully induced by the quormone at concentrations as low as 100 pM. TraM raised the concentration of signal required for maximum activity to 1 nM. Donors grown in batch culture retained conjugative competence following signal removal, even when in stationary phase. However, donors kept in balanced growth rapidly lost transfer ability following signal removal. Loss of transfer was mirrored by a decrease in levels of active TraR. Decreases in TraR activity and conjugative competence could be accounted for by dilution associated with cell division, suggesting that while induction of Ti plasmid conjugation is an active process, the cells lack a mechanism for disassembling the conjugative apparatus when signals become limiting.
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Muscholl-Silberhorn, Albrecht B. "Pheromone-Regulated Expression of Sex Pheromone Plasmid pAD1-Encoded Aggregation Substance Depends on at Least Six Upstream Genes and a cis-Acting, Orientation-Dependent Factor." Journal of Bacteriology 182, no. 13 (July 1, 2000): 3816–25. http://dx.doi.org/10.1128/jb.182.13.3816-3825.2000.
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ABSTRACT Conjugative transfer of Enterococcus faecalis-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an α-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated α-amylase activity, a total of at least six genes, traB, traC, traA,traE1, orfY, and orf1, are required: TraB efficiently represses asa1 (by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence oftraC); only traC can relievetraB-mediated repression in a pheromone-dependent manner. In addition to traB, traA is required but not sufficient for negative control. Mutational inactivation oftraE1, orfY, or orf1, respectively, results in a total loss of α-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment coveringtraA, P0, and traE1 without disrupting any gene or control element switches off amyL orasa1 expression, indicating the involvement of acis-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in trans, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed.
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M�ndez, J., L. Fern�ndez, A. Men�ndez, P. Reimundo, D. P�rez-Pascual, R. Navais, and J. A. Guijarro. "A Chromosomally Located traHIJKCLMN Operon Encoding a Putative Type IV Secretion System Is Involved in the Virulence of Yersinia ruckeri." Applied and Environmental Microbiology 75, no. 4 (December 16, 2008): 937–45. http://dx.doi.org/10.1128/aem.01377-08.
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ABSTRACT Nucleotide sequence analysis of the region surrounding the pIVET8 insertion site in Yersinia ruckeri 150RiviXII, previously selected by in vivo expression technology (IVET), revealed the presence of eight genes (traHIJKCLMN [hereafter referred to collectively as the tra operon or tra cluster]), which are similar both in sequence and organization to the tra operon cluster found in the virulence-related plasmid pADAP from Serratia entomophila. Interestingly, the tra cluster of Y. ruckeri is chromosomally encoded, and no similar tra cluster has been identified yet in the genomic analysis of human pathogenic yersiniae. A traI insertional mutant was obtained by homologous recombination. Coinfection experiments with the mutant and the parental strain, as well as 50% lethal dose determinations, indicate that this operon is involved in the virulence of this bacterium. All of these results suggest the implication of the tra cluster in a virulence-related type IV secretion/transfer system. Reverse transcriptase PCR studies showed that this cluster is transcribed as an operon from a putative promoter located upstream of traH and that the mutation of traI had a polar effect. A traI::lacZY transcriptional fusion displayed higher expression levels at 18�C, the temperature of occurrence of the disease, and under nutrient-limiting conditions. PCR detection analysis indicated that the tra cluster is present in 15 Y. ruckeri strains from different origins and with different plasmid profiles. The results obtained in the present study support the conclusion, already suggested by different authors, that Y. ruckeri is a very homogeneous species that is quite different from the other members of the genus Yersinia.
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Cheah, Keat-Chye, Animesh Ray, and Ron Skurray. "Expression of F plasmid trat: Independence of tray → z promoter and traJ control." Plasmid 16, no. 2 (September 1986): 101–7. http://dx.doi.org/10.1016/0147-619x(86)90068-5.
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Bates, Steven, Annette M. Cashmore, and Brian M. Wilkins. "IncP Plasmids Are Unusually Effective in Mediating Conjugation of Escherichia coli and Saccharomyces cerevisiae: Involvement of the Tra2 Mating System." Journal of Bacteriology 180, no. 24 (December 15, 1998): 6538–43. http://dx.doi.org/10.1128/jb.180.24.6538-6543.1998.
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ABSTRACT Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10−5 transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage oforiT in cis and the provision intrans of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.
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Bragagnolo, Nicholas, and Gerald F. Audette. "Solution characterization of the dynamic conjugative entry exclusion protein TraG." Structural Dynamics 9, no. 6 (November 2022): 064702. http://dx.doi.org/10.1063/4.0000171.
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The R100 plasmid and the secretion system it encodes are representative of F-like conjugative type IV secretion systems for the transmission of mobile DNA elements in gram-negative bacteria, serving as a major contributor to the spread of antibiotic resistance in bacterial pathogens. The TraG protein of F-like systems consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain, denoted TraG*. TraG* is essential in preventing redundant DNA transfer through a process termed entry exclusion. In the donor cell, it interacts with TraN to facilitate mating pair stabilization; however, if a mating pore forms between bacteria with identical plasmids, TraG* interacts with its cognate TraS in the inner membrane of the recipient bacterium to prevent redundant donor–donor conjugation. Structural studies of TraG* from the R100 plasmid have revealed the presence of a dynamic region between the N- and C-terminal domains of TraG. Thermofluor, circular dichroism, collision-induced unfolding–mass spectrometry, and size exclusion chromatography linked to multiangle light scattering and small angle x-ray scattering experiments indicated an N-terminal truncation mutant displayed higher stability and less disordered content relative to full-length TraG*. The 45 N-terminal residues of TraG* are hypothesized to serve as part of a flexible linker between the two independently functioning domains.
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Csitkovits, Vanessa C., Damir Ðermić, and Ellen L. Zechner. "Concomitant Reconstitution of TraI-catalyzed DNA Transesterase and DNA Helicase Activityin Vitro." Journal of Biological Chemistry 279, no. 44 (August 17, 2004): 45477–84. http://dx.doi.org/10.1074/jbc.m407970200.
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TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5′-3′ DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiledoriTDNA with site and strand specificityin vitrobut fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI withoriTDNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1oriTsequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing ≤30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY andEscherichia coliintegration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding fromnic.
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Luo, Zhao-Qing, and Stephen K. Farrand. "The Agrobacterium tumefaciens rndHomolog Is Required for TraR-Mediated Quorum-Dependent Activation of Ti Plasmid tra Gene Expression." Journal of Bacteriology 183, no. 13 (July 1, 2001): 3919–30. http://dx.doi.org/10.1128/jb.183.13.3919-3930.2001.
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ABSTRACT Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-l-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation oftra genes on pTiC58ΔaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd ofEscherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant,traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect intra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.
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Klimke, William A., and Laura S. Frost. "Genetic Analysis of the Role of the Transfer Gene,traN, of the F and R100-1 Plasmids in Mating Pair Stabilization during Conjugation." Journal of Bacteriology 180, no. 16 (August 15, 1998): 4036–43. http://dx.doi.org/10.1128/jb.180.16.4036-4043.1998.
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ABSTRACT Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the traN gene on an F plasmid derivative, pOX38, to produce pOX38N1::CAT. pOX38N1::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1 traN were capable of complementing pOX38N1::CAT transfer equally well when wild-type recipients were used. FtraN, but not R100-1 traN, supported a much lower level of transfer when there was an ompA mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1traN gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of FtraN suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA.
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Beranek, Andreas, Markus Zettl, Klaus Lorenzoni, Alexandra Schauer, Michael Manhart, and Günther Koraimann. "Thirty-Eight C-Terminal Amino Acids of the Coupling Protein TraD of the F-Like Conjugative Resistance Plasmid R1 Are Required and Sufficient To Confer Binding to the Substrate Selector Protein TraM." Journal of Bacteriology 186, no. 20 (October 15, 2004): 6999–7006. http://dx.doi.org/10.1128/jb.186.20.6999-7006.2004.
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ABSTRACT Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (ΔN155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 × 107 liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.
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Lu, Jun, and Laura S. Frost. "Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation." Journal of Bacteriology 187, no. 14 (July 2005): 4767–73. http://dx.doi.org/10.1128/jb.187.14.4767-4773.2005.
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ABSTRACT Conjugation is a major mechanism for disseminating genetic information in bacterial populations, but the signal that triggers it is poorly understood in gram-negative bacteria. F-plasmid-mediated conjugation requires TraM, a homotetramer, which binds cooperatively to three binding sites within the origin of transfer. Using in vitro assays, TraM has previously been shown to interact with the coupling protein TraD. Here we present evidence that F conjugation also requires TraM-TraD interactions in vivo. A three-plasmid system was used to select mutations in TraM that are defective for F conjugation but competent for tetramerization and cooperative DNA binding to the traM promoter region. One mutation, K99E, was particularly defective in conjugation and was further characterized by affinity chromatography and coimmunoprecipitation assays that suggested it was defective in interacting with TraD. A C-terminal deletion (S79*, where the asterisk represents a stop codon) and a missense mutation (F121S), which affects tetramerization, also reduced the affinity of TraM for TraD. We propose that the C-terminal region of TraM interacts with TraD, whereas its N-terminal domain is involved in DNA binding. This arrangement of functional domains could in part allow TraM to receive the mating signal generated by donor-recipient contact and transfer it to the relaxosome, thereby triggering DNA transfer.
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Chen, Guozhou, Chao Wang, Clay Fuqua, Lian-Hui Zhang, and Lingling Chen. "Crystal Structure and Mechanism of TraM2, a Second Quorum-Sensing Antiactivator of Agrobacterium tumefaciens Strain A6." Journal of Bacteriology 188, no. 23 (September 22, 2006): 8244–51. http://dx.doi.org/10.1128/jb.00954-06.
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ABSTRACT Quorum sensing is a community behavior that bacteria utilize to coordinate a variety of population density-dependent biological functions. In Agrobacterium tumefaciens, quorum sensing regulates the replication and conjugative transfer of the tumor-inducing (Ti) plasmid from pathogenic strains to nonpathogenic derivatives. Most of the quorum-sensing regulatory proteins are encoded within the Ti plasmid. Among these, TraR is a LuxR-type transcription factor playing a key role as the quorum-sensing signal receptor, and TraM is an antiactivator that antagonizes TraR through the formation of a stable oligomeric complex. Recently, a second TraM homologue called TraM2, not encoded on the Ti plasmid of A. tumefaciens A6, was identified, in addition to a copy on the Ti plasmid. In this report, we have characterized TraM2 and its interaction with TraR and solved its crystal structure to 2.1 Å. Like TraM, TraM2 folds into a helical bundle and exists as homodimer. TraM2 forms a stable complex (Kd = 8.6 nM) with TraR in a 1:1 binding ratio, a weaker affinity than that of TraM for TraR. Structural analysis and biochemical studies suggest that protein stability may account for the difference between TraM2 and TraM in their binding affinities to TraR and provide a structural basis for L54 in promoting structural stability of TraM.
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Samuels, A. Lacey, Erich Lanka, and Julian E. Davies. "Conjugative Junctions in RP4-Mediated Mating ofEscherichia coli." Journal of Bacteriology 182, no. 10 (May 15, 2000): 2709–15. http://dx.doi.org/10.1128/jb.182.10.2709-2715.2000.
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ABSTRACT The physical association of bacteria during conjugation mediated by the IncPα plasmid RP4 was investigated. Escherichia colimating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.
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Ham, Lindsay M., David Cram, and Ron Skurray. "Transcriptional analysis of the F plasmid surface exclusion region: Mapping of traS, traT, and traD transcripts." Plasmid 21, no. 1 (January 1989): 1–8. http://dx.doi.org/10.1016/0147-619x(89)90081-4.
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Li, Pei-Li, and Stephen K. Farrand. "The Replicator of the Nopaline-Type Ti Plasmid pTiC58 Is a Member of the repABC Family and Is Influenced by the TraR-Dependent Quorum-Sensing Regulatory System." Journal of Bacteriology 182, no. 1 (January 1, 2000): 179–88. http://dx.doi.org/10.1128/jb.182.1.179-188.2000.
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ABSTRACT The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034–1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, andrepC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids fromRhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region betweentraI and repA contained two copies of thetra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from thetra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal repplasmid increased five- to sevenfold in strains expressingtraR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.
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Wang, Chao, Hai-Bao Zhang, Guozhou Chen, Lingling Chen, and Lian-Hui Zhang. "Dual Control of Quorum Sensing by Two TraM-Type Antiactivators in Agrobacterium tumefaciens Octopine Strain A6." Journal of Bacteriology 188, no. 7 (April 1, 2006): 2435–45. http://dx.doi.org/10.1128/jb.188.7.2435-2445.2006.
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ABSTRACT Agrobacterium tumefaciens wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the traM gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated traM was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second traM homologue. The homologue, designated traM2, has 64% and 65% identities with traM at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the tra gene promoters. Deletion of traM2 in strain A6 harboring the mutated traM confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.
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Alonso, Guillermina, Kelly Baptista, Trinh Ngo, and Diane E. Taylor. "Transcriptional organization of the temperature-sensitive transfer system from the IncHI1 plasmid R27." Microbiology 151, no. 11 (November 1, 2005): 3563–73. http://dx.doi.org/10.1099/mic.0.28256-0.
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One of the characteristic features of IncHI1 plasmids is a thermosensitive process of conjugation, which is optimal between 22 °C and 30 °C but inhibited at 37 °C. R27, the prototypical IncHI1 plasmid, contains transfer genes clustered in two regions of the plasmid, Tra1 and Tra2. In the present study, transcriptional analyses of the tra genes were undertaken at both 30 °C and 37 °C. Screening of 38 tra genes showed that tra genes are transcriptionally linked in six operons, three in each Tra region. RT-PCR analysis showed that gene expression was reduced at 37 °C relative to that observed at 30 °C. The transcription start sites of the six transcripts were identified, promoters and upstream regions were cloned, and transcription was tested at both temperatures. In cells grown at 37 °C, in the presence of R27, the promoters were inhibited, except for promoters of the H operon and AN operon. Conditions that influenced DNA topology, such as osmolarity, anaerobiosis, quorum sensing and acidity, showed no significant influence on transfer frequency. These results should facilitate future understanding of the basis of temperature-sensitive transfer in this large conjugative plasmid.
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Kurenbach, Brigitta, Jolanta Kopeć, Marion Mägdefrau, Kristin Andreas, Walter Keller, Christine Bohn, Mouhammad Y. Abajy, and Elisabeth Grohmann. "The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501." Microbiology 152, no. 3 (March 1, 2006): 637–45. http://dx.doi.org/10.1099/mic.0.28468-0.
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The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR. The tra genes are expressed throughout the life cycle of Ent. faecalis, and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the tra operon, binds to the tra promoter P tra , which partially overlaps with the origin of transfer (oriT). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. β-Galactosidase assays with P tra–lacZ fusions proved P tra promoter activity, which was strongly repressed when TraA was supplied in trans. Thus, it is concluded that the pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.
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Harris, Robin L., Veronica Hombs, and Philip M. Silverman. "Evidence that F-plasmid proteins TraV, TraK and TraB assemble into an envelope-spanning structure in Escherichia coli." Molecular Microbiology 42, no. 3 (July 7, 2008): 757–66. http://dx.doi.org/10.1046/j.1365-2958.2001.02667.x.
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Finlay, B. B., L. S. Frost, and W. Paranchych. "Nucleotide sequences of the R1-19 plasmid transfer genes traM, finP, traJ, and traY and the traYZ promoter." Journal of Bacteriology 166, no. 2 (1986): 368–74. http://dx.doi.org/10.1128/jb.166.2.368-374.1986.
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Jasrotia, P., J. Yadav, B. Singh, S. D. Patil, S. Kumar, and G. P. Singh. "Efficiency of sticky traps for monitoring aphids in wheat under North-Western Plains and Peninsular zones of India." Journal of Environmental Biology 43, no. 6 (November 15, 2022): 794–800. http://dx.doi.org/10.22438/jeb/43/6/mrn-1952.
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Aim: To evaluate the efficiency of most effective trap among two types of sticky traps of, i.e., card or tray of two colours (yellow and blue) and their placement height within wheat crop for catching alate of R. maidis, the most abundant aphid species in selected locations. Methodology: Sticky card and tray traps of two colours; yellow and blue were placed at different heights within wheat crop and the effects of trap parameters (type, colour and height) were evaluated to determine the trapping efficiency of R. maidis alate. The traps were installed at two heights above ground level; 100 cm and 150 cm at Karnal and Ludhiana and at 60 cm and 120 cm above ground level at Niphad location. Alate aphid counts were recorded weekly to make comparisons. Results: The highest number of R. maidis alate were caught on yellow coloured sticky card traps placed within the crop canopy at 100 cm height above ground and the lowest on blue coloured sticky tray traps at 150 cm height above ground at Ludhiana and Karnal. At Niphad, the highest population was caught on yellow coloured sticky card traps placed at 60 cm height above ground and the lowest number on blue coloured sticky tray traps at 120 cm height above ground. Correlation analysis revealed that all three trap parameters (trap type, colour and placement height) were correlated with mean alate trap catches. Backward stepwise regression modelling indicated that trap type and placement height had a maximum influence on the R. maidis alate trapping. Interpretation: This study indicated that the yellow sticky card traps were most effective in catching R. maidis alate as compared to other tested traps. Key words: Aphids, Insect sampling, Trapping efficiency, Trap parameters, Triticum aestivum
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Salgado-Pabón, Wilmara, Ying Du, Kathleen T. Hackett, Katelynn M. Lyons, Cindy Grove Arvidson, and Joseph P. Dillard. "Increased Expression of the Type IV Secretion System in Piliated Neisseria gonorrhoeae Variants." Journal of Bacteriology 192, no. 7 (February 5, 2010): 1912–20. http://dx.doi.org/10.1128/jb.01357-09.
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ABSTRACT Neisseria gonorrhoeae produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either traD expression or growth rate. A mutant with a deletion in traD was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that traD is transcriptionally and translationally coupled to traI, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing traI and traD. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of traD and traI.
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Wathugala, N. Dulmini, Kasuni M. Hemananda, Cynthia B. Yip, and Michael F. Hynes. "Defining the requirements for the conjugative transfer of Rhizobium leguminosarum plasmid pRleVF39b." Microbiology 166, no. 3 (March 1, 2020): 318–31. http://dx.doi.org/10.1099/mic.0.000885.
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Rhizobium leguminosarum strain VF39 contains a plasmid, pRleVF39b, which encodes a distinctive type of conjugation system (rhizobial type IVa) that is relatively widespread among rhizobial genomes. The cluster of genes encoding the transfer functions lacks orthologs to genes such as traCD, traF and traB, but contains 15 conserved genes of unknown function. We determined the importance of these genes in conjugation by constructing marked and unmarked mutations in each gene, and established that six genes, now designated trcA-F, played a significant role in plasmid transfer. Like the relaxase gene, traA, and the genes encoding the MPF system (trb genes), five of these genes, located in two divergently transcribed operons, are regulated by the Xre family repressor TrbR. The other gene, trcF encodes a protein with similarity to histidinol phosphatases, and its role in conjugation is unclear, but mutations in trcF are severely impaired for conjugation. TrcF does not play a role in regulation of other conjugation genes.
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White, Catharine E., and Stephen C. Winans. "Cell–cell communication in the plant pathogen Agrobacterium tumefaciens." Philosophical Transactions of the Royal Society B: Biological Sciences 362, no. 1483 (March 13, 2007): 1135–48. http://dx.doi.org/10.1098/rstb.2007.2040.
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The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease strategy benefits the bacteria as the infected plant tissue produces novel nutrients, called opines, that the colonizing bacteria can use as nutrients. Almost all of the genes that are required for virulence, and all of the opine uptake and utilization genes, are carried on large tumour-inducing (Ti) plasmids. The observation more than 25 years ago that specific opines are required for Ti plasmid conjugal transfer led to the discovery of a cell–cell signalling system on these plasmids that is similar to the LuxR–LuxI system first described in Vibrio fischeri . All Ti plasmids that have been described to date carry a functional LuxI-type N -acylhomoserine lactone synthase (TraI), and a LuxR-type signal receptor and transcriptional regulator called TraR. The traR genes are expressed only in the presence of specific opines called conjugal opines. The TraR–TraI system provides an important model for LuxR–LuxI-type systems, especially those found in the agriculturally important Rhizobiaceae family. In this review, we discuss current advances in the biochemistry and structural biology of the TraR–TraI system.
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Zhao, Bao-Sheng, Hai-Ru Huo, Yue-Ying Ma, Hong-Bin Liu, Lan-Fang Li, Feng Sui, Cang-Hai Li, Shu-Ying Guo, and Ting-Liang Jiang. "Effects of 3-Phenyl-Propenal on the Expression of Toll-Like Receptors and Downstream Signaling Components on Raw264.7 Murine Macrophages." American Journal of Chinese Medicine 36, no. 01 (January 2008): 159–69. http://dx.doi.org/10.1142/s0192415x08005679.
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3-phenyl-propenal is one of the principle compounds isolated from Guizhi (Ramulus Cinnamomi), the principal drug in Guizhi-Tang (GZT), a famous traditional Chinese medical formula. The aim of the present study was to investigate the effects of 3-phenyl-propenal on the expression of toll-like receptor 3 (TLR3), TLR4 and the downstream signaling components on Raw264.7 murine microphages. Raw264.7 cells were cultured in RPMI-1640 medium containing LPS (lipopolysaccharide) or poly (I:C) in the presence or absence of 3-phenyl-propenal. After 24-hour incubation, the medium was collected and the amount of TNF-α and IFN-β was measured by ELISA. mRNA expression of TLR3, TLR4, myeloid differentiation factor (MyD88), TRAF-6 (tumor necrosis factor receptor-associated), TRAM (toll-like receptor-associated molecule) and TRIF (TIR domain-containing adaptor inducing IFN-β) were analyzed by real-time PCR with SYBR green dye. Protein expression of TLR3 and TLR4 was analyzed by Western blotting and that of MyD88 and TRAF-6 was analyzed by immunofluorescence assay. The results indicate that LPS increased the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF, but had no influence on TLR3, while poly (I:C) up-regulated the expression of TLR3, MyD88, TRAM and TRIF. 3-phenyl-propenal significantly decreased the expression of LPS-induced TLR4, MyD88, TRAF-6, while possessing no effect on LPS-induced TRAM and TRIF expression in Raw264.7 cells. When cells were stimulated by poly (I:C), 3-phenyl-propenal significantly decreased TLR3 and MyD88 expression. In conclusion, 3-phenyl-propenal blocked the over-expression of TLR3, TLR4, their downstream signaling components MyD88 and TRAF-6, which indicate that it had an antagonistic effect on TLR3 and TLR4.
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Kurenbach, Brigitta, Dagmar Grothe, María Eugenia Farías, Ulrich Szewzyk, and Elisabeth Grohmann. "The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase." Journal of Bacteriology 184, no. 6 (March 15, 2002): 1801–5. http://dx.doi.org/10.1128/jb.184.6.1801-1805.2002.
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ABSTRACT The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriTpIP501 . The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg2+ or Mn2+ and is highest at temperatures of between 42 and 45°C.