Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening?closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA

Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice

BACKGROUND: Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models. METHODS: The antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays. RESULTS: C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas. CONCLUSIONS: The combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies

Transcriptional activity of transposable elements in maize

<p>Abstract</p> <p>Background</p> <p>Mobile genetic elements represent a high proportion of the Eukaryote genomes. In maize, 85% of genome is composed by transposable elements of several families. First step in transposable element life cycle is the synthesis of an RNA, but few is known about the regulation of transcription for most of the maize transposable element families. Maize is the plant from which more ESTs have been sequenced (more than two million) and the third species in total only after human and mice. This allowed us to analyze the transcriptional activity of the maize transposable elements based on EST databases.</p> <p>Results</p> <p>We have investigated the transcriptional activity of 56 families of transposable elements in different maize organs based on the systematic search of more than two million expressed sequence tags. At least 1.5% maize ESTs show sequence similarity with transposable elements. According to these data, the patterns of expression of each transposable element family is variable, even within the same class of elements. In general, transcriptional activity of the <it>gypsy</it>-like retrotransposons is higher compared to other classes. Transcriptional activity of several transposable elements is specially high in shoot apical meristem and sperm cells. Sequence comparisons between genomic and transcribed sequences suggest that only a few copies are transcriptionally active.</p> <p>Conclusions</p> <p>The use of powerful high-throughput sequencing methodologies allowed us to elucidate the extent and character of repetitive element transcription in maize cells. The finding that some families of transposable elements have a considerable transcriptional activity in some tissues suggests that, either transposition is more frequent than previously expected, or cells can control transposition at a post-transcriptional level.</p

Derepression of the Plant Chromovirus LORE1 Induces Germline Transposition in Regenerated Plants

Transposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposable elements in plants. Recent studies have identified chromoviruses to be a widely distributed lineage of Gypsy elements. These elements contain chromodomains in their integrases, which suggests a preference for insertion into heterochromatin. In turn, this preference might have contributed to the patterning of heterochromatin observed in host genomes. Despite their potential importance for our understanding of plant genome dynamics and evolution, the regulatory mechanisms governing the behavior of chromoviruses and their activities remain largely uncharacterized. Here, we report a detailed analysis of the spatio-temporal activity of a plant chromovirus in the endogenous host. We examined LORE1a, a member of the endogenous chromovirus LORE1 family from the model legume Lotus japonicus. We found that this chromovirus is stochastically de-repressed in plant populations regenerated from de-differentiated cells and that LORE1a transposes in the male germline. Bisulfite sequencing of the 5′ LTR and its surrounding region suggests that tissue culture induces a loss of epigenetic silencing of LORE1a. Since LTR promoter activity is pollen specific, as shown by the analysis of transgenic plants containing an LTR::GUS fusion, we conclude that male germline-specific LORE1a transposition in pollen grains is controlled transcriptionally by its own cis-elements. New insertion sites of LORE1a copies were frequently found in genic regions and show no strong insertional preferences. These distinctive novel features of LORE1 indicate that this chromovirus has considerable potential for generating genetic and epigenetic diversity in the host plant population. Our results also define conditions for the use of LORE1a as a genetic tool

The Tnt1 Retrotransposon Escapes Silencing in Tobacco, Its Natural Host

Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host

The in vivo division and death rates of Salmonella typhimurium in the spleens of naturally resistant and susceptible mice measured by the superinfecting phage technique of Meynell.

Salmonella typhimurium appears to divide faster in the spleen of naturally susceptible BALB/c than in resistant (B10 x A/J)F1 mice. S. typhimurium M526 is an LT2 derivative lysogenic for a non-excluding P22 mutant which allows superinfection with a second, non-replicating, P22 phage so that the proportion of superinfected organisms halves at each division. The true in vivo division and death rates can be calculated from successive determinations of the proportion of superinfected organisms and the viable count. It was found that the division time was 2.86 h in BALB/c and 5.02 h in (B10 x A/J)F1; the death rate was low and actually greater in the susceptible BALB/c strain. These results suggest that the gene controlling in vivo salmonella net growth rate, which is very important in natural resistance to salmonella infection, acts very early by regulating the division rate, perhaps inside macrophages. The actual mechanism remains unknown

Genetics of natural resistance to salmonellae in mice.

The genetics of natural resistance to salmonellae were studied in F1 hybrid and backcross mice. Overall resistance to Salmonella typhimurium C5 is complex, but one parameter, the early net growth rate of the organism in vivo, is controlled by a single autosomal gene or cluster of genes. 'Slow' net growth rate is necessary but insufficient, for resistance to S. typhimurium C5. Resistance requires the participation of other mechanisms, detectable by the end of the first week, which presumably involve an immune response. F1 hybrids bred from parents of low, intermediate and high natural resistance showed either high or low resistance. Most of the F1 hybrids were of a similar high resistance, and were bred from pairs in which at least one parent showed slow net growth rate. Hybrids of low resistance were only obtained when neither parent showed slow net growth rate. No hybrid was less resistant than the parents, many were more resistant. Backcross analysis on two hybrids challenged with S. typhimurium C5 supports the hypothesis of complex genetic control of overall resistance but with single gene control of the early net growth rate of the organism. Similar experiments were performed using a much more virulent organism, S. enteritidis 5694. All mouse strains were very susceptible (LD 50 less than ten organisms) to this strain given either i.v. or s.c. This organism produced an overwhelming infection which did not allow the cell-mediated immune response time to develop. This, however, did not interfere with the mechanism controlling early net growth rate, and genetic analysis using this organism gave similar results to those obtained with S. typhimurium C5. These results suggest that the mechanism regulating early net growth rate does not operate via the cell-mediated immune response, which develops later in the course of the infection

The natural resistance of radiation chimeras to S. typhimurium C5.

Differences in the in vivo net growth rate of Salmonella typhimurium C5 during the first week of infection in different mouse strains are controlled by a single autosomal gene. In lethally irradiated mice repopulated with semi-allogeneic bone marrow, the early net growth rate shows the phenotype of the donor of the bone marrow cell and not the phenotype of the irradiated recipient. Thus, genetically controlled differences in in vivo bacterial net growth rate are a consequence of mechanisms operating in cells which have originated from bone marrow precursors. Natural resistance to S. typhimurium C5 requires, in addition to slow net growth rate, other mechanisms which come into operation at the end of the first week of the infection. These later acting processes are more complex and can not be transferred to susceptible mice using bone marrow cells alone

Natural resistance to salmonella typhimurium in different inbred mouse strains.

The mechanisms of natural resistance to intravenous challenge with Salmonella typhimurium C5 are complex. LD50 determinations showed inbred mouse strains of low, intermediate and high natural resistance, with BALB/c and B10 strains the most susceptible, A/J the most resistant. Delayed (footpad) hypersensitivity was not by itself a measure of natural resistance. Resistant mouse strains sensitized either s.c. or i.v. with an attenuated salmonella strain showed positive 48 h footpad reactions when tested 8 days later with a salmonella extract, but three very susceptible strains also showed positive reactions. Determinations of the in vivo net growth rate of salmonellae in the liver and spleen during the first phase of the infection (up to day 4) arrange the different mouse strains into two categories of fast and slow net growth rate. All fast net growth rate strains are susceptible, but not all slow net growth rate strains are resistant. Besides slow net growth rate, resistance requires the participation of other factors appearing in the second phase of the infection (towards the end of the first week) probably involving the cellular immune response, which halts further bacterial growth. Not all slow net growth rate strains are equally capable of suppressing bacterial growth in this second phase. The host mechanism determining slow net growth rate is inherited as a dominant trait, and appears to be operating before the main cellular immune response. The influence of this mechanism on net growth rate is reflected in the time to death following a given dose of salmonellae. The present results suggest that overall resistance to salmonellae is polygenic, but that the mechanism responsible for the differences in early net growth rate is less complex