Mutations in TNFRSF13B, better known as transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), contribute to common variable immunodeficiency and autoimmunity in humans. How TACI regulates these two opposing conditions is unclear, however. TACI binds the cytokines BAFF and APRIL, and previous studies using gene KO mice indicated that loss of TACI affected only T-cell-independent antibody responses. Here we demonstrate that Taci −/− mice have expanded populations of T follicular helper (T fh ) and germinal center (GC) B cells in their spleens when immunized with T-cell-dependent antigen. The increased numbers of T fh and GC B cells in Taci −/− mice are largely a result of up-regulation of inducible costimulator (ICOS) ligand on TACI-deficient B cells, given that ablation of one copy of the Icosl allele restores normal levels of T fh and GC B cells in Taci −/− mice. Interestingly, despite the presence of increased T fh and antigenspecific B cells, immunized Taci −/− mice demonstrate defective antigen-specific antibody responses resulting from significantly reduced numbers of antibody-secreting cells (ASCs). This effect is attributed to the failure to down-regulate the proapoptotic molecule BIM in Taci −/− plasma cells. Ablation of BIM could rescue ASC formation in Taci −/− mice, suggesting that TACI is more important for the survival of plasma cells than for the differentiation of these cells. Thus, our data reveal dual roles for TACI in B-cell terminal differentiation. On one hand, TACI modulates ICOS ligand expression and thereby limits the size of T fh and GC B-cell compartments and prevents autoimmunity. On the other hand, it regulates the survival of ASCs and plays an important role in humoral immunity.costimulation | T-cell-dependent humoral immunity | TNF receptor T ransmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI/TNFRSF13B), B-cell activating factor (BAFF) receptor (BAFF-R/TNFRSF13C), and B-cell maturation antigen (BCMA/TNFRSF17) are closely related members of the TNF receptor superfamily and bind B-cell survival cytokines BAFF and APRIL (1). Although BAFF-R and BCMA have been shown to mediate the survival of follicular B cells (2) and plasma cells (3), respectively, a similar role for TACI in mediating the survival of B lymphocytes at any particular stage of B-cell differentiation has not been demonstrated definitively.However, mutations in TACI are thought to contribute to ∼10% of common variable immunodeficiency (CVID) in humans (4, 5). This syndrome is characterized by antibody deficiency in late childhood and early adulthood. Paradoxically, some patients with TACI-mutated CVID also develop autoimmune diseases (6). How TACI deficiency leads to antibody deficiencies on one hand and autoimmunity on the other hand is not well understood.Mice lacking TACI have been generated (7,8), and initial characterizations of these mice revealed modest phenotypes. Taci −/− mice had increased numbers of B cells but exhibited decreased antibody respons...

The Streptomyces phage BT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the BT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra-and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the BT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.Site-specific recombinases have been widely used in genetic engineering: for example, in vitro cloning, plant and mammalian cell genome modification, and gene therapy (3,11,12,15,16). Nearly all site-specific recombinases can be classified as tyrosine recombinases, also known as the integrase family, or serine recombinases, also known as the resolvase/invertase family, based on comparisons of amino acid sequences and different mechanisms of catalysis; these two types use tyrosine or serine, respectively, to attack the DNA sugar-phosphate backbone (14,18,20). The process of strand exchange catalyzed by tyrosine recombinases involves a Holliday junction intermediate and cleavage and rejoining of the strands one by one. In contrast, serine-mediated recombination involves a process of double-strand breakage, followed by rotation and religation (9). The well-known tyrosine recombinase integrase recognizes two different attachment substrate sites, the 25-bp attB gene (in the bacterial chromosome) and the 240-bp attP gene (in the phage genome), which are inverted into attL and attR with the aid of the integration host factor (a chaperone). Furthermore, excision between attL and attR requires recombination directionality factor (RDF), and recombinations mediated by tyrosine recombinases require supercoiling of the substrate DNAs (10). In contrast, the extensively studied large serine recombinase from Streptomyces phage C31 is thought to catalyze unidirectional recombination between DNA substrates attB and attP, independent of DNA supercoiling and host cofactors (18). In addition, the attachment sites of the C31 integrase are smaller than those of most tyrosine recombinases, with minimal sizes of 34 bp for attB and 39 bp for attP (11,18).Streptomyces phage BT1, a homoimmune relative of C31, is inserted into the chromosomes of a wide range of Streptomyces spp. for lysogeny and encodes a large serine recombinase (5, 8). Similar to those of the C31 site-specific recombination system, the attB and attP sites of BT1 ...

Streptomyces not only exhibits complex morphological differentiation but also produces a plethora of secondary metabolites, particularly antibiotics. To improve our general understanding of the complex network of undecylprodigiosin (Red) biosynthesis regulation, we used an in vivo transposition system to identify novel regulators that influence Red production in Streptomyces coelicolor M145. Using this screening system, we obtained 25 Red-deficient mutants. Twenty-four of these mutants had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite, actinorhodin (Act). One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized gene rrdA (regulator of redD, sco1104), which encodes a putative TetR family transcription factor. Compared with wild-type strain M145, the rrdA null mutant exhibited increased Red production and decreased Act production. A high level of rrdA expression resulted in a severe reduction in Red production and Act overproduction. Reverse transcription-PCR analysis showed that RrdA negatively regulated Red production by controlling redD mRNA abundance, while no change was observed at the transcript level of the Act-specific activator gene, actII-orf4. The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.In addition to its complex morphological differentiation, the gram-positive genus Streptomyces is notable for its ability to produce a wide range of secondary metabolites during its life cycle. These metabolites include the majority of known pharmaceutically important secondary metabolites that exhibit antibacterial, anticancer, and immunosuppressive activities (6, 7). The genes responsible for the biosynthesis of secondary metabolites are often physically clustered in the genome and coordinately regulated by pathway-specific transcriptional activators (1,4,10,12,29,33). These specific regulators are controlled by various higher-level pleiotropic regulators, and their expression is typically affected by a variety of environmental and physiological cues, including the nature and levels of carbon and nitrogen sources and the availability of phosphate and small signaling molecules, such as ppGpp and ␥-butyrolactone (5).Streptomyces coelicolor A3 (2) has been used for many years as a model organism in morphological and physiological differentiation studies, particularly in studies of the regulation of antibiotic biosynthesis (7). S. coelicolor produces four antibiotics: actinorhodin (Act), undecylprodigiosin (Red), methylenomycin, and calcium-dependent antibiotic. It has been shown that certain regulators are involved in the pleiotropic control of antibiotic production in S. coelicolor, including AbsA1/A2, AfsR/K, AtrA, and PhoR/P (18, 24, 31, 32). Recently, mutational analysis and adventitious overexpression of key regulators in S. coelicolor revealed cross-regulation at the transcriptional level a...

Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.