SUMMARY The PII family of signal transduction proteins are among the most widely distributed signal proteins in the bacterial world. First identified in 1969 as a component of the glutamine synthetase regulatory apparatus, PII proteins have since been recognized as playing a pivotal role in control of prokaryotic nitrogen metabolism. More recently, members of the family have been found in higher plants, where they also potentially play a role in nitrogen control. The PII proteins can function in the regulation of both gene transcription, by modulating the activity of regulatory proteins, and the catalytic activity of enzymes involved in nitrogen metabolism. There is also emerging evidence that they may regulate the activity of proteins required for transport of nitrogen compounds into the cell. In this review we discuss the history of the PII proteins, their structures and biochemistry, and their distribution and functions in prokaryotes. We survey data emerging from bacterial genome sequences and consider other likely or potential targets for control by PII proteins.

Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3′-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3′ untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation.

X-ray crystallographic analysis of the Escherichia coli P II protein paralogues GlnB and GlnK has shown that they share a superimposable structural core but can differ in conformation of the T-loop, a region of the protein (residues 37-54) that has been shown to be important for interaction with other proteins. In Klebsiella pneumoniae GlnK has been shown to have a clearly defined function in regulating NifL-mediated inhibition of NifA activity in response to the nitrogen status, and GlnB, when expressed from the chromosome, does not substitute for GlnK. Because the T-loops of K. pneumoniae and E. coli GlnB and GlnK differ at just three residues, 43, 52, and 54, we have used a previously constructed heterologous system, in which K. pneumoniae nifLA is expressed in E. coli, to investigate the importance of GlnK residues 43, 52, and 54 for regulation of the NifLA interaction. By site-directed mutagenesis of glnB we have shown that residue 54 is the single most important amino acid in the T-loop in the context of the regulation of NifA activity. Furthermore, a combination of just two changes, in residues 54 and 43, allows GlnB to function as GlnK and completely relieve NifL inhibition of NifA activity.

In Streptococcus pneumoniae, stkP and phpP, encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called "soluble" protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone "competence stimulating peptide." We show that PhpP activity is essential in a stkP Signal transduction via the transfer of phosphoryl groups and transient protein phosphorylation involves the concerted activities of kinases and phosphatases and controls various cellular functions (for a review, see reference 18). In eubacteria, the histidine kinases of two-component systems (TCSs) and the phosphatases with which they interact are involved in cellular adaptation to environmental conditions (for a review, see reference 44). Serine-threonine kinases and PP2C phosphatases also contribute to regulatory and developmental processes, as described in Bacillus subtilis (1,2,13,23,29,33,43). In mycobacteria, a membrane-associated PP2C phosphatase controls the activity of several serine-threonine kinases (4, 6, 52), and the transcriptional activator EmbR has been identified as a phosphorylation target for PknH kinase (3, 4, 42). In Streptococcus agalactiae, the signaling pair Stk1-Stp1 plays a role in virulence, by modifying cytotoxin production and purine metabolism (34,35,36). In Streptococcus pyogenes, a histonelike protein is phosphorylated by the serine-threonine kinase, generating a substrate for the kinase-related phosphatase (21). The human pathogen Streptococcus pneumoniae has only one gene encoding a PP2C-type phosphatase, PhpP, located upstream from the stkP gene, which encodes the only membrane-associated serine-threonine kinase, StkP (17). The phpP and stkP genes overlap by 4 bp and form an operon (31). In a mouse model of infection, null mutations affecting StkP greatly attenuate tissue and bloodstream invasion (17). In cultures, these mutations are highly pleiotropic, presenting a notably important impact on competence development for genetic transformation (17, 40). In vitro studies have shown that autophosphorylated recombinant StkP (StkP-P) is a substrate for recombinant PhpP, suggesting that StkP and PhpP may function in a coordinated manner (31). However, the regulators controlling the cellular level of StkP-P and the network involving StkP in growing cultures remain ill defined. Recent studies of strain TIGR4 revealed that the transcriptional regulator RitR (45) is phosphorylated by StkP and dephosphorylated by PhpP (50). In order to get better insight into StkP signaling, we investigated the specific role of PhpP phosphatase on bacterial growth and on the development of competence for genetic transformation in cultures, by using genetic analysis. Specific null mutation in ...

Two photorespiratory mutants from Lotus japonicus, namely Ljgln2-1 and Ljgln2-2, deficient in plastidic glutamine synthetase (GLN2), were analysed at the molecular level. Both mutants showed normal levels of Gln2 mRNA, indicating that they were affected post-transcriptionally. Complete sequencing of full-length Gln2 cDNAs revealed the presence of a single point mutation on each mutant, leading to G85R and L278H amino acid replacements, respectively. Different types of experimental approaches, including heterologous expression and complementation tests in Escherichia coli, showed that both GLN2 mutant proteins completely lacked of biosynthetic and transferase enzyme activities. Moreover, it was also shown that while GLN2-1 mutant protein was assembled into a less stable inactive octamer, GLN2-2 mutant protein was unable to acquire a proper quaternary structure and was rapidly degraded. Therefore, the mutations analysed are the first of their type affecting the stability and/or the quaternary structure of the GLN2 enzyme. The kinetic parameters of purified recombinant GLN2 were determined. The enzyme showed positive cooperativity towards ammonium and Mg(2+). Thiol compounds stimulated by twofold the biosynthetic activity but not the transferase activity of recombinant GLN2 and were able to alter the kinetics towards glutamate of the enzyme. Moreover, the biosynthetic activity of recombinant GLN2 was stimulated by more than tenfold by the presence of free Mg(2+).

Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable PI, protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable PI,. In free-living conditions, PI, is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. PI, is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that PI, controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the PI, protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.

BackgroundThe E2f transcription factor family has a pivotal role in controlling the cell fate in general, and in particular cancer development, by regulating the expression of several genes required for S phase entry and progression through the cell cycle. It has become clear that the transcriptional activation of at least one member of the family, E2F1, can also induce apoptosis. An appropriate balance of positive and negative regulators appears to be necessary to modulate E2F1 transcriptional activity, and thus cell fate.Methodology/Principal FindingsIn this report, we show that Api5, already known as a regulator of E2F1 induced-apoptosis, is required for the E2F1 transcriptional activation of G1/S transition genes, and consequently, for cell cycle progression and cell proliferation. Api5 appears to be a cell cycle regulated protein. Removal of Api5 reduces cyclin E, cyclin A, cyclin D1 and Cdk2 levels, causing G1 cell cycle arrest and cell cycle delay. Luciferase assays established that Api5 directly regulates the expression of several G1/S genes under E2F1 control. Using protein/protein and protein/DNA immunoprecipitation studies, we demonstrate that Api5, even if not physically interacting with E2F1, contributes positively to E2F1 transcriptional activity by increasing E2F1 binding to its target promoters, through an indirect mechanism.Conclusion/SignificanceThe results described here support the pivotal role of cell cycle related proteins, that like E2F1, may act as tumor suppressors or as proto-oncogenes during cancer development, depending on the behavior of their positive and negative regulators. According to our findings, Api5 contributes to E2F1 transcriptional activation of cell cycle-associated genes by facilitating E2F1 recruitment onto its target promoters and thus E2F1 target gene transcription.

To evaluate the role of uridylyl-transferase, the Sinorhizobium meliloti glnD gene was isolated by heterologous complementation in Azotobacter vinelandii. The glnD gene is cotranscribed with a gene homologous to Salmonella mviN. glnD1::⍀ or mviN1::⍀ mutants could not be isolated by a powerful sucrose counterselection procedure unless a complementing cosmid was provided, indicating that glnD and mviN are members of an indispensable operon in S. meliloti.Sinorhizobium meliloti forms a symbiosis with Medicago sativa (alfalfa) in which nitrogen is fixed by the bacteria and released to the plant in exchange for photosynthates. After infection of plants, bacterial cells differentiate into bacteroids contained within plant membrane-enclosed organelles, symbiosomes, located within root nodules. Establishment of a successful symbiosis involves a shift in bacterial metabolism from assimilation of ammonia to export of nitrogenous compounds to the host plant. Therefore, ascertaining the features regulating this particular metabolic switch may provide valuable insight into symbiotic nitrogen fixation. As in many other bacteria, S. meliloti assimilates ammonia through the glutamine synthetase (GS)/glutamate synthase cycle. Unusually, members of the Rhizobiaceae carry three genes encoding isoforms of GS at separate loci (8). The major enzyme, GSI, is similar to GS of the enteric bacteria and is susceptible to posttranslational adenylylation, which reduces the rate of ammonia assimilation in vivo (1). As in enteric bacteria, a specialized protein called P II regulates the level of GSI adenylylation (2). In Escherichia coli, a model organism for studies on nitrogen regulation, the interactions of P II with its targets depend on the uridylylation state of P II , which responds to intracellular concentrations of the key metabolites glutamine and ␣-ketoglutarate (for current reviews, see references 10 and 12). Depletion of glutamine is sensed by the GlnD protein (9), which carries uridylyl-transferase/uridylyl-removing (UTase) activities, resulting in uridylylation of P II (10).The role of P II in S. meliloti was previously examined by construction of two alleles of the corresponding glnB gene: ⌬glnB10, a nonpolar null mutation, and glnBP5, a second allele that encodes a protein altered at the site of uridylylation. With respect to symbiosis, P II was required to efficiently transfer fixed nitrogen to the plant but not for nitrogenase expression (2). Predictions were that glnD mutants would exhibit phenotypes similar to glnBP5 mutants; however, this is complicated by the occurrence of multiple P II -like proteins in S. meliloti (D. Kahn and P. Rudnick, unpublished data) as in many other organisms (reviewed in reference 12). Therefore, the focus of this work was to directly address the role of the nitrogensensing UTase in the regulation of P II and its homologues in S. meliloti. We report here the cloning, sequencing, and mutagenesis of the S. meliloti glnD gene as well as evidence that this region of the chromosome is essential.Clo...