Explore the vast range of titles available.

The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli . Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.

Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase. We show that DNA replication within this but not subsequent cycles is required for a differentiation switch comprising rapid and simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the β-globin locus. Specifically, S-phase progression is required for the formation of DNase I hypersensitive sites and for DNA demethylation at this locus. Mechanistically, we show that S-phase progression during this key committal step is dependent on downregulation of the cyclin-dependent kinase p57(KIP2) and in turn causes the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel role for a cyclin-dependent kinase inhibitor in differentiation, distinct to their known function in cell cycle exit. Furthermore, we show that a novel, mutual inhibition between PU.1 expression and S-phase progression provides a \"synchromesh\" mechanism that \"locks\" the erythroid differentiation program to the cell cycle clock, ensuring precise coordination of critical differentiation events.

Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, 'EryA', negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune-deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response.

Summary Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast‐two‐hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP‐22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface.

We investigated the origin and diversification of the high-affinity nitrate transporter NRT2 in fungi and other eukaryotes using Bayesian and maximum parsimony methods. To assess the higher-level relationships and origins of NRT2 in eukaryotes, we analyzed 200 amino acid sequences from the Nitrate/Nitrite Porter (NNP) Family (to which NRT2 belongs), including 55 fungal, 41 viridiplantae (green plants), 11 heterokonts (stramenopiles), and 87 bacterial sequences. To assess evolution of NRT2 within fungi and other eukaryotes, we analyzed 116 amino acid sequences of NRT2 from 58 fungi, 40 viridiplantae (green plants), 1 rhodophyte, and 5 heterokonts, rooted with 12 bacterial sequences. Our results support a single origin of eukaryotic NRT2 from 1 of several clades of mostly proteobacterial NNP transporters. The phylogeny of bacterial NNP transporters does not directly correspond with bacterial taxonomy, apparently due to ancient duplications and/or horizontal gene transfer events. The distribution of NRT2 in the eukaryotes is patchy, but the NRT2 phylogeny nonetheless supports the monophyly of major groups such as viridiplantae, flowering plants, monocots, and eudicots, as well as fungi, ascomycetes, basidiomycetes, and agaric mushrooms. At least 1 secondary origin of eukaryotic NRT2 via horizontal transfer to the fungi is suggested, possibly from a heterokont donor. Our analyses also suggest that there has been a horizontal transfer of nrt2 from a basidiomycete fungus to an ascomycete fungus and reveal a duplication of nrt2 in the ectomycorrhizal mushroom genus, Hebeloma. [PUBLICATION ABSTRACT]

Acute gastroenteritis caused by Salmonella enterica serovar typhimurium is a significant public health problem. This pathogen has very sophisticated molecular machinery encoded by the two pathogenicity islands, namely Salmonella Pathogenicity Island 1 and 2 (SPI-1 and SPI-2). Remarkably, both SPI-1 and SPI-2 are very tightly regulated in terms of timing of expression and spatial localization of the encoded effectors during the infection process within the host cell. This regulation is governed at several levels, including transcription and translation, and by post-translational modifications. In the context of a finely tuned regulatory system, we will highlight how these effector proteins co-opt host signaling pathways that control the ability of the organism to infect and survive within the host, as well as elicit host pro-inflammatory responses.

S almonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from S almonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast‐two‐hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP‐22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface.

Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase. We show that DNA replication within this but not subsequent cycles is required for a differentiation switch comprising rapid and simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the β-globin locus. Specifically, S-phase progression is required for the formation of DNase I hypersensitive sites and for DNA demethylation at this locus. Mechanistically, we show that S-phase progression during this key committal step is dependent on downregulation of the cyclin-dependent kinase p57KIP2 and in turn causes the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel role for a cyclin-dependent kinase inhibitor in differentiation, distinct to their known function in cell cycle exit. Furthermore, we show that a novel, mutual inhibition between PU.1 expression and S-phase progression provides a \"synchromesh\" mechanism that \"locks\" the erythroid differentiation program to the cell cycle clock, ensuring precise coordination of critical differentiation events.

The gene, encoding Recombinase A (RecA) is one of three (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb within . Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.

Bacterial pathogens possess virulence factors that distinguish them from their non-pathogenic counterparts, and enable them to induce pathogenesis. Typically, these unique genes are encoded on specialized regions of the bacterial chromosome termed pathogenicity islands. Acquired through horizontal transmission, pathogenicity islands are large sections of the chromosome that differ in nucleotide content and in the presence of genetic elements compared to the core genome, and contain genes that promote pathogenesis. Pathogenicity islands were first discovered in bacteria belonging to the Enterobacteriaceae family, but are now known to exist in various animal and plant pathogens. As pathogenicity islands are unique to pathogenic bacteria, it is likely their presence permitted the emergence of bacterial pathogens and continues to shape their evolution. This chapter highlights the genetic organization and content of pathogenicity islands, their regulation, and their impact on the evolution of pathogenic bacteria.