The plasmid-maintenance functions of the P7 prophage

The region responsible for the maintenance of the prophage of bacteriophage P7 as a stable, unit-copy plasmid was isolated in a lambda att vector which lysogenizes Escherichia coli as a stable unit-copy plasmid under the control of the P7 replication origin. The P7 plasmid-maintenance region was shown to consist of adjacent replication and partition regions capable of functioning independently. The isolated replication region could support plasmid maintenance but the resulting plasmids were highly unstable unless the partition region was also included. Stable composite plasmids were isolated containing the putative P7 partition region and the origin of replication of the unrelated plasmid F, indicating that P7 encoded an active partition mechanism. The replication regions of P7 and P1 were shown to be highly homologous but the partition regions of the two plasmids appear to be unrelated in sequence. The incompatibility determinants associated with the two replication regions showed the same specificity, whereas the partition-region incompatibility determinants were different, showing no cross-specificity.     

Inhibition of gene expression of T7-related phages by prophage P1

Summary The gene expression of nine phages of the T7 group was compared after infection of Escherichia coli B(P1). With the exception of phage 13a which grew normally, all of them infected E. coli B(P1) abortively. Differences were found in the efficiency of host killing which ranged from 100% for phage 13a to 37% for phage A1122. Infection by T7 prevented colony formation by about 70% of the cells but they showed filamentous growth until about 2h after infection. It was shown by SDS-polyacrylamide gel electrophoresis and autoradiography of [³⁵S]methionine-labelled phage-coded proteins that all phages except for 13a showed measurable expression only of the early genes. No correlation was observed between killing capacity and the pattern of gene expression, and the ability to hydrolyse S-adenosyl-methionine (SAM, a cofactor for the P1 restriction endonuclease) by means of a phage-coded SAMase. Mixed infection of E. coli B(P1) with 13a and T7 yielded mixed progeny indistinguishable from that observed after mixed infection of the normal host E. coli B. Genetic crosses with amber mutants of 13a and T7 showed that the 13a marker opo ⁺ (overcomes P one), required for growth on B(P1), is located in the early region, to the left of gene 1 (RNA polymerase gene).     

Regulation of the ban gene containing operon of prophage P1

Summary A physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants. The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al. 1978). Evidence for additional member(s) of that operon is presented. Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive. The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac additional gene(s) — crr/ban in the clockwise direction of the circular map of P1.     

Replication of prophage P1 is cell-cycle specific.

P1 prophage replication during the Escherichia coli division cycle has been analyzed by using the membrane-elution technique to produce cells labelled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive prophage DNA. P1 prophage replicates during a restricted portion of the bacterial division cycle, like the minichromosome, but at a time during the division cycle different than the time at which the minichromosome replicates in the same cell. A high-copy mini-R6K plasmid present in the same cell replicates throughout the division cycle. Over a wide range of growth rates, the P1 prophage replicates approximately one-half generation after the minichromosome replicates. Thus, the mechanisms underlying P1 replication are similar to those for the F plasmid and the chromosome. Replication occurs when some property related to cell size or cell mass reaches a constant value per origin.     

Group Y incompatibility and copy control of P1 prophage

We have identified a restriction fragment (EcoRI-5) of bacteriophage P1 that, when cloned in a λ prophage, expresses incompatibility characteristic of the unit copy P1 plasmid prophage. Lysogens of λ-P1 chimeras in which the P1 fragment is EcoRI-5 fail to maintain P1 or P7 plasmids. In order to study the nature of this incompatibility, we isolated P1 mutants that overcome it. These mutants exhibit an elevated copy number. We provide evidence that the increased copy number results from a defect in a repressor of replication that can be furnished in trans by a chromosomally integrated P1, but not by EcoRI-5 itself. We, therefore, suggest that the incompatibility exerted by EcoRI-5 is not attributable to the represser of replication involved in the above copy control defect. Instead, it could be attributed to the presence of a DNA site required for proper plasmid partition at cell division. The elevated copy number of the P1 mutants would then enable them to compete favorably with the single copy of the cloned EcoRI fragment for a cellular component of the partition apparatus. Thus, incompatibility could be overcome.     

Replication of prophage P1 during the cell cycle of Escherichia coli.

Summary We have followed, by DNA-DNA hybridization, the variation in the number of copies of prophage P1 relative to two chromosomal markers when the doubling time of the host cells is modified by a change in carbon source. The ratio of P1/chromosome terminus undergoes a twofold decrease when the cell doubling time increases from 24 to 215 min, whereas the ratio of P1/chromosome origin increases 1.4 fold; both ratios tend towards unity at slow growth rates. This suggests that the replication of prophage P1 is not simultaneous with chromosome initiation or chromosome termination. The chromosome replication time is unaffected by the presence of P1, and remains constant over the range of doubling times studied, with a value of about 40 min. Following amino acid starvation, the P1/chromosome origin ratio increases from 0.7 to 0.9, suggesting that P1 retains the ability to replicate after chromosome initiation has stopped and in the absence of essential amino acids. The results are discussed with reference to similar studies done on F and R1.     

Physiological and pathological functions of P2X7 receptor in the spinal cord

ATP-mediated signaling has widespread actions in the nervous system from neurotransmission to regulation of proliferation. In addition, ATP is released during injury and associated to immune and inflammatory responses. Still, the potential of therapeutic intervention of purinergic signaling during pathological states is only now beginning to be explored because of the large number of purinergic receptors subtypes involved, the complex and often overlapping pharmacology and because ATP has effects on every major cell type present in the CNS. In this review, we will focus on a subclass of purinergic-ligand-gated ion channels, the P2X7 receptor, its pattern of expression and its function in the spinal cord where it is abundantly expressed. We will discuss the mechanisms for P2X7R actions and the potential that manipulating the P2X7R signaling pathway may have for therapeutic intervention in pathological events, specifically in the spinal cord.     

Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism

Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.     

New insights of P2X7 receptor signaling pathway in alveolar functions

Purinergic P2X7 receptor (P2X7R), an ATP-gated cation channel, is unique among all other family members because of its ability to respond to various stimuli and to modulate pro-inflammatory signaling. The activation of P2X7R in immune cells is absolutely required for mature interleukin -1beta (IL-1beta) and IL-18 production and release. Lung alveoli are lined by the structural alveolar epithelial type I (AEC I) and alveolar epithelial type II cells (AEC II). AEC I plays important roles in alveolar barrier protection and fluid homeostasis whereas AEC II synthesizes and secrete surfactant and prevents alveoli from collapse. Earlier studies indicated that purinergic P2X7 receptors were specifically expressed in AEC I. However, their implication in alveolar functions has not been explored. This paper reviews two important signaling pathways of P2X7 receptors in surfactant homeostatsis and Acute Lung Injury (ALI). Thus, P2X7R resides at the critical nexus of alveolar pathophysiology.     

Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages

Activation of the P2X7 receptor of macrophages plays an important role in inflammation. We recently reported that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-mediated cell death via Ca(2+) influx. However, it remained unclear whether P2X4 receptor is involved in P2X7 receptor-mediated inflammatory responses, such as cytokine production. Here, we present evidence that P2X4 receptor modulates P2X7 receptor-dependent inflammatory functions. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced high mobility group box 1 (HMGB1) release and IL-1β production via activation of P2X7 receptor. Knockdown of P2X4 receptor or removal of extracellular Ca(2+) suppressed ATP-induced release of both HMGB1 and IL-1β. On the other hand, knockdown of P2X4 receptor or removal of extracellular Ca(2+) enhanced P2X7-dependent LC3-II expression (an index of autophagy), suggesting that P2X4 receptor suppresses P2X7-mediated autophagy. Since LC3-II expression was inhibited by pretreatment with antioxidant and NADPH oxidase inhibitor, we examined P2X7-mediated production of reactive oxygen species (ROS). We found that activation of P2X7 receptor-mediated production of ROS was significantly facilitated in P2X4-knockdown cells, suggesting that co-expression of P2X4 receptor with P2X7 receptor may suppress anti-inflammatory function-related autophagy via suppression of ROS production. We conclude that co-expression of P2X4 receptor with P2X7 receptor enhances P2X7-mediated inflammation through both facilitation of release of cytokines and suppression of autophagy.     

Ant-mediated transactivation of early genes in Salmonella prophage P22 by superinfecting virulent P22 mutants

Summary The virulent mutants P22 vir B vy and P22 vy mutants, both insensitive to mnt-repressor, transactivate the early genes of a P22 prophage. The transactivation of early P22 prophage genes depends strictly on the expression of gene ant (antirepressor-protein) by the superinfecting P22 mutant and therefore occurs by derepression.     

Grazing protozoa and the evolution of the Escherichia coli O157:H7 Shiga toxin-encoding prophage

Humans play little role in the epidemiology of Escherichia coli O157:H7, a commensal bacterium of cattle. Why then does E. coli O157:H7 code for virulence determinants, like the Shiga toxins (Stxs), responsible for the morbidity and mortality of colonized humans? One possibility is that the virulence of these bacteria to humans is coincidental and these virulence factors evolved for and are maintained for other roles they play in the ecology of these bacteria. Here, we test the hypothesis that the carriage of the Stx-encoding prophage of E. coli O157:H7 increases the rate of survival of E. coli in the presence of grazing protozoa, Tetrahymena pyriformis. In the presence but not the absence of Tetrahymena, the carriage of the Stx-encoding prophage considerably augments the fitness of E. coli K-12 as well as clinical isolates of E. coli O157 by increasing the rate of survival of the bacteria in the food vacuoles of these ciliates. Grazing protozoa in the environment or natural host are likely to play a significant role in the ecology and maintenance of the Stx-encoding prophage of E. coli O157:H7 and may well contribute to the evolution of the virulence of these bacteria to colonize humans.     

Restoration of immunity in lysogens carrying prophage P2, derepressed at high temperature

Summary The possibility that a strain lysogenic for phage P2 could be brought into the so-called antiimmune state in which the synthesis of phage repressor is permanently turned off, was tested in the following way. Two lysogenic strains that could be derepressed at 42°C were prepared. In one, the prophage had, in addition to a temperature-sensitive repressor mutation (c5), amber mutations in the two early genes A and B. In the other, the prophage had an unknown defect that blocked expression of the A and B genes. Both strains could multiply at 42° C as well as or almost as well as a non-lysogen. After the strains had grown for several generations at 42° C, they were returned to 30° and the resynthesis of repressor was followed by measuring the restoration of immunity to super-infection. In both cases, the immunity returned slowly over a period of 2 to 3 h. In a strain made doubly lysogenic for two amA amB c5 prophages, immunity was restored at a more rapid rate, suggesting that the rate of restoration depended mainly on the number of copies of repressor gene present. Attempts to demonstrate channeling towards the lytic pathway in the derepressed lysogens was also negative. The temperature treatment tended instead to increase the frequency of lysogenization of superinfecting P2. Thus, the presence of a system, similar to the cro system described for phage lambda, to regulate repressor synthesis in phage P2 could not be demonstrated.