traM Gene Research Articles

Differences in PGE2 production between primary human monocytes and differentiated macrophages: role of IL-1β and TRIF/IRF3.

Prostaglandin E2 (PGE2) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE2 is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE2 in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE2 production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE2 production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE2 production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE2 in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE2 production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.

LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium.

We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kappaB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kappaB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC.

Dual control of quorum sensing by two TraM-type antiactivators in Agrobacterium tumefaciens octopine strain A6.

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.

A rapid screen for functional mutants of TraM, an autoregulatory protein required for F conjugation.

TraM is an autoregulatory protein required for conjugative transfer of the F plasmid. A rapid screening procedure was developed to select for traM mutants constructed by random PCR mutagenesis. The mutated traM gene was cloned into pT7-5, without the traM promoters (collectively called P( traM)), such that these mutants were expressed from the downstream traJ promoter, resulting in constitutive, low-level, transcription of traM by polymerases that had circumnavigated the plasmid. P( traM) was cloned into pPR9tt as a translational fusion in which a DNA fragment containing P( traM), the ribosome binding site and first 24 codons of traM was fused to the 5' end of lacZ. To downregulate beta-galactosidase expression, a -1 frameshift mutation was introduced at the junction between traM and lacZ in the fusion. Selected TraM mutants were further characterized for their intracellular levels, electrophoretic mobility on nondenaturing gels, and activity in F conjugation. Point mutations throughout TraM were found to affect both autoregulation and conjugative function.

Transfer protein TraM stimulates TraI-catalyzed cleavage of the transfer origin of plasmid R1 in vivo

Factors contributing directly to the cleavage of the conjugative transfer origin of plasmid R1 in Escherichia coli were investigated. The essential transfer protein TraM was identified as a necessary positive effector of the catalytic activity of TraI relaxase at the R1 transfer origin in the absence of protein TraY. The stimulatory effect of TraM on the cleavage reaction in vivo correlated with the capacity of TraM to bind origin DNA. TraM was shown to be essential for heterologous mobilization of recombinant origin DNA. The requirement for TraM to promote mobilization was distinct from the protein’s positive effect on transfer gene regulation. Chimeric traM alleles, fusing heterologous amino and carboxyl coding sequences from the traM genes of the R1 and the IncFI plasmid P307, were used to localize the specificity determinant of TraM’s DNA binding activity. Use of the chimeric alleles also revealed that the requirement for TraM in mobilization is origin specific but transfer system independent. No evidence was found for a plasmid specific activity of TraM at a stage in the transfer process subsequent to the initial cleavage of origin DNA. In light of TraM’s regulatory functions in transfer gene expression, we propose that TraM could control conjugative DNA processing in response to intracellular levels of transfer proteins.

The finM promoter and the traM promoter are the principal promoters of the traM gene of the antibiotic resistance plasmid R100.

finP multicopy repression and traJ multicopy derepression indicate that the ratio of sense to antisense transcripts is important in the regulation of R100 conjugation. The extension of R100 traM transcripts into traJ shows that promoters in traM can affect this ratio, making the regulation of traM transcription important in the regulation of R100 conjugation. Since R100 traM, traY and tral proteins bind to the traM promoter region, we examined traM transcription in R100-1 traM, traY and tral mutants and compared it with traM transcription in both R100-1 and R100. We verified that the traM and finM promoters provide virtually all the transcripts originating in the R100-1 traM gene. When either is deleted, as in VAR22 or VAR30, the remaining promoter is highly active. We show here that traY positively regulates R100-1 traM transcription, as has been found for F. We found that tral did not regulate R100-1 traM transcription. The measured activity of the native R100 traM promoter was 12% of that in R100-1, whereas the native R100 finM promoter was 45% of that in R100-1. These data and data from the R100-1 traY and tral mutants show that the activities of the two promoters varied independently.

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network.

Site-directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a transfer-derepressed (finO-) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1-16 was 10(7)-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.

Identification of a pKM101 region which confers a slow growth rate and interferes with susceptibility to quinolone in Escherichia coli AB1157.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.

Regulation of the expression of the traM gene of the F sex factor of Escherichia coli.

Conjugative F-plasmid transfer is mediated by the transfer (tra) region which encodes nearly 40 genes, 25 of which are essential for this process in Escherichia coli. TraM is required for conjugation and is encoded on a separate operon between the origin of transfer and the traJ gene. The traJ gene product is the positive regulator of transcription of the 30 kb tra operon, the first gene of which is traY. Using primer-extension assays and immunoblots on the F plasmid itself and its derivatives, we demonstrate that F TraM regulates its own expression from two promoters and that it requires TraY as well as expression of the tra operon for maximal traM transcription. traY is the first gene in the tra operon under the control of the TraJ regulator, which is in turn negatively regulated by the antisense RNA, FinP, and the FinO protein. Thus, a control circuit has been established whereby traM is negatively regulated by the FinOP fertility inhibition system through its repression of TraJ expression, which adversely affects transcription of the traY gene.

Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene

The Agrobacterium Ti plasmid tra regulon was previously found to be positively regulated by the TraR protein in the presence of a diffusible N-acyl homoserine lactone designated Agrobacterium autoinducer (AAI). TraR and AAI are similar to LuxR from Vibrio fischeri and the Vibrio autoinducer (VAI), which regulate target bioluminescence (lux) genes in a cell density-dependent manner. We now show that tra genes are also regulated by a second protein, designated TraM, which acts to antagonize TraR-dependent activation. The traM gene is closely linked to traR, and the two genes are transcribed convergently. The predicted TraM proteins of two different Ti plasmids are 77% identical but are not significantly similar to other protein sequences in the database, and thus TraM may represent a novel regulatory protein. Null mutations in traM cause strongly increased conjugation, tra gene transcription, and AAI production. A functional copy of traM introduced into traM mutants decreased conjugation, tra gene transcription, and AAI synthesis. TraM inhibits transcription of traA, traI, and traM. Although traM was first identified by its octopine-inducible promoter, we now show that induction by octopine requires traR, strongly suggesting that TraR is the direct traM activator.

Regulation of R100 conjugation requires traM in cis to traJ.

Deletion mutants of R100-1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM-binding sites and the traM promoters. Complementation tests showed that traM was efficiently complemented only when the trans-acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences. The conclusion is that traM and traJ constitute a complex operon. A deletion mutant lacking all of the traJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer.

The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation

Transfer functions of the conjugative plasmid RP4 (IncP alpha) are distributed among distinct regions of the genome, designated Tra1 and Tra2. By deletion analyses, we determined the limits of the Tra1 region, essential for intraspecific Escherichia coli matings. The Tra1 core region encompasses approximately 5.8 kb, including the genes traF, -G, -H, -I, -J, and -K as well as the origin of transfer. The traM gene product, however, is not absolutely required for conjugation but significantly increases transfer efficiency. To determine the transfer phenotype of genes encoded by the Tra2 core region, we generated a series of defined Tra2 mutants. This revealed that at least trbB, -C, -E, -G, and -L are essential for RP4 conjugation. To classify these transfer functions as components of the DNA transfer and replication (Dtr) or of the mating pair formation (Mpf) system, we analyzed the corresponding derivatives with respect to mobilization of IncQ plasmids and donor-specific phage propagation. We found that all of the Tra2 genes listed above and the traG and traF genes of Tra1 are required for RSF1010 mobilization. Expression of traF from Tra1 in conjunction with the Tra2 core was sufficient for phage propagation. This implies that the TraG protein is not directly involved in pilus formation and potentially connects the relaxosome with proteins enabling the membrane passage of the DNA. The proposed roles of the RP4 transfer gene products are discussed in the context of virulence functions encoded by the evolutionarily related Ti T-DNA transfer system of agrobacteria.

Repression of the traM gene of plasmid R100 by its own product and integration host factor at one of the two promoters.

Plasmid R100 codes for the traM gene, which is required for DNA transfer and whose product has been shown to bind to the four sites, called sbmA to sbmD, upstream of traM. To determine whether the TraM protein regulates the expression of traM, we constructed the plasmids carrying various portions of the region upstream of the initiation codon ATG for traM, which was fused with lacZ in frame, and introduced them into the cells, which did or did not harbor another compatible plasmid carrying traM. We then assayed the beta-galactosidase (LacZ) activity to monitor the expression of the fusion genes and analyzed the traM-specific transcripts made in the cells. Two promoters for traM were identified and designated pM1 and pM2. Promoter pM2 lies upstream of pM1 and overlaps the sbmC-sbmD region. Promoter pM1 is constitutively expressed, while pM2 is much stronger but is repressed almost completely by the TraM protein and partially by integration host factor, whose binding site is near pM2. The traM gene is likely to be expressed from pM2 when the TraM protein is at low levels after dilution in the donor cell during cell growth or before its expression in the recipient cell which has just received R100 by conjugation. The expression from pM2 could maintain the amount of the TraM protein at a constant level needed to initiate DNA transfer at any time. Integration host factor, which can partially repress the traM gene, may play a role in forming an active complex with the TraM protein at the sbm region to facilitate DNA transfer.

TraM of plasmid R1 regulates its own expression

Regulation of the traM gene, which encodes a factor essential for conjugation of resistance plasmid R1, was studied in vivo using translational gene fusion. traM"lacZ fusion constructs were transferred to the chromosome via the recombinant phage lambda RZ5. The level of beta-galactosidase expressed by the lysogens indicates that the traM promoters are very active. Expression of traM was diminished five- to sixfold when the single-copy plasmids R1 or R1-19 were present in trans. When recombinant plasmids carrying traM were present at higher copy numbers, traM expression was reduced as much as 45-fold. The negative effect of R1 plasmids on traM expression in trans, which we interpret as autoregulation, was observed regardless of whether the plasmids were conjugatively repressed or derepressed. Site-specific mutagenesis of the region encoding the N-terminus of the TraM protein eliminated the autoregulative effect indicating that the N-terminal amino acids of the protein are important to its DNA-binding function. The autoregulatory behaviour of TraM is allele specific. R1- or P307-encoded TraM molecules were found to recognize only the cognate DNA.

Specific DNA binding of the TraM protein to the oriT region of plasmid R100

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.

The TraM protein of plasmid R1 is a DNA‐binding protein

The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA-binding studies. Both gel retardation- and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene. Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist. The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat. DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences. At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene. In one region this extension leads to binding of TraM protein at its own promoters.

Origin of transfer of IncF plasmids and nucleotide sequences of the type II oriT, traM, and traY alleles from ColB4-K98 and the type IV traY allele from R100-1.

The complete nucleotide sequences of the ColB4-K98 (ColB4) plasmid transfer genes oriT, traM, and traY as well as the traY gene of R100-1 are presented and compared with the corresponding regions from the conjugative plasmids F, R1, and R100. The sequence encoding the oriT nick sites and surrounding inverted repeats identified in F was conserved in ColB4. The adenine-thymine-rich sequence following these nick sites was conserved in R1 and ColB4 but differed in F and R100, indicating that this region may serve as the recognition site for the traY protein. A series of direct repeats unique to the ColB4 plasmid was found in the region of dyad symmetry following this AT-rich region. This area also encodes 21-base-pair direct repeats which are homologous to those in F and R100. The traM gene product may bind in this region. Overlapping and following these repeats is the promoter(s) for the traM protein. The traM protein from ColB4 is similar to the equivalent products from F, R1, and R100. The traY protein from ColB4 is highly homologous to the R1 traY gene product, while the predicted R100-1 traY product differs at several positions. These differences presumably define the different alleles of traM and traY previously identified for IncF plasmids by genetic criteria. The translational start codons of the ColB4 and R100-1 traY genes are GUG and UUG, respectively, two examples of rare initiator codon usage.

The traM gene of the resistance plasmid R1: comparison with the corresponding sequence of the Escherichia coli F factor

The 7.7-kb EcoRI fragment of the resistance plasmid R1 contains the gene for the TraM protein. The sequence was identified by the presence of an open reading frame (ORF) which is preceded upstream by two promoter sequences. Both these promoters were found to be active, although the more distant one predominates, as was judged by the relative abundance of mRNA of the expected length. The TraM protein could be synthesized in an in vitro DNA-dependent protein synthesis system if the DNA of the corresponding region was supplied as template. Comparison of the traM genes of R1 and the F factor showed a high degree of similarity, although a number of mutations, especially near the 5' terminus, introduce specific amino acid (aa) changes. Two-thirds of the 3' sequences differ mainly in silent mutations; hence the aa sequence of the corresponding carboxy-terminal portion of the protein is highly conserved. The 5' and 3' untranslated regions of the mRNAs show little homology. One of the promoter regions, the ribosome-binding sequences, and the transcription termination sites are located at comparable positions but differ in details.