'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.     

5'-methylthioadenosine nucleosidase is implicated in playing a key role in a modified futalosine pathway for menaquinone biosynthesis in Campylobacter jejuni

Menaquinone (vitamin K(2)) serves as an electron carrier in the electron transport chain required for respiration in many pathogenic bacteria. Most bacteria utilize a common menaquinone biosynthetic pathway as exemplified by Escherichia coli. Recently, a novel biosynthetic pathway, the futalosine pathway, was discovered in Streptomyces. Bioinformatic analysis strongly suggests that this pathway is also operative in the human pathogens Campylobacter jejuni and Helicobacter pylori. Here, we provide compelling evidence that a modified futalosine pathway is operative in C. jejuni and that it utilizes 6-amino-6-deoxyfutalosine instead of futalosine. A key step in the Streptomyces pathway involves a nucleosidase called futalosine hydrolase. The closest homolog in C. jejuni has been annotated as a 5'-methylthioadenosine nucleosidase (MTAN). We have shown that this C. jejuni enzyme has MTAN activity but negligible futalosine hydrolase activity. However, the C. jejuni MTAN is able to hydrolyze 6-amino-6-deoxyfutalosine at a rate comparable with that of its known substrates. This suggests that the adenine-containing version of futalosine is the true biosynthetic intermediate in this organism. To demonstrate this in vivo, we constructed a C. jejuni mutant strain deleted for mqnA2, which is predicted to encode for the enzyme required to synthesize 6-amino-6-deoxyfutalosine. Growth of this mutant was readily rescued by the addition of 6-amino-6-deoxyfutalosine, but not futalosine. This provides the first direct evidence that a modified futalosine pathway is operative in C. jejuni. It also highlights the tremendous versatility of the C. jejuni MTAN, which plays key roles in S-adenosylmethionine recycling, the biosynthesis of autoinducer molecules, and the biosynthesis of menaquinone.     

Singlet oxygen-mediated and EXECUTER-dependent signalling and acclimation of Arabidopsis thaliana exposed to light stress

Plants respond to environmental changes by acclimation that activates defence mechanisms and enhances the plant''s resistance against a subsequent more severe stress. Chloroplasts play an important role as a sensor of environmental stress factors that interfere with the photosynthetic electron transport and enhance the production of reactive oxygen species (ROS). One of these ROS, singlet oxygen (¹O₂), activates a signalling pathway within chloroplasts that depends on the two plastid-localized proteins EXECUTER 1 and 2. Moderate light stress induces acclimation protecting photosynthetic membranes against a subsequent more severe high light stress and at the same time activates ¹O₂-mediated and EXECUTER-dependent signalling. Pre-treatment of Arabidopsis seedlings with moderate light stress confers cross-protection against a virulent Pseudomonas syringae strain. While non-pre-acclimated seedlings are highly susceptible to the pathogen regardless of whether ¹O₂- and EXECUTER-dependent signalling is active or not, pre-stressed acclimated seedlings without this signalling pathway lose part of their pathogen resistance. These results implicate ¹O₂- and EXECUTER-dependent signalling in inducing acclimation but suggest also a contribution by other yet unknown signalling pathways during this response of plants to light stress.     

The role of EDS1 (enhanced disease susceptibility) during singlet oxygen-mediated stress responses of Arabidopsis

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2) that is restricted to the plastid compartment. Distinct sets of genes are activated that are different from those induced by hydrogen peroxide/superoxide. One of the genes that is rapidly upregulated is EDS1 (enhanced disease susceptibility). The EDS1 protein has been shown to be required for the resistance to biotrophic pathogens and the accumulation of salicylic acid (SA) that enhances the defenses of a plant by inducing the synthesis of pathogen-related (PR) proteins. Because of the similarity of its N-terminal portion to the catalytic site of lipases, EDS1 has also been implicated with the release of polyunsaturated fatty acids and the subsequent formation of various oxylipins. The release of singlet oxygen in the flu mutant triggers a drastic increase in the concentration of free SA and activates the expression of PR1 and PR5 genes. These changes depend on the activity of EDS1 and are suppressed in flu/eds1 double mutants. Soon after the beginning of singlet oxygen production, the synthesis of oxylipins such as jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) also start and plants stop growing and induce a cell-death response. The inactivation of EDS1 does not affect oxylipin synthesis, growth inhibition and the initiation of cell death, but it does allow plants to recover much faster from singlet oxygen-mediated growth inhibition and it also suppresses the spread of necrotic lesions in leaves. Hence, singlet oxygen activates a complex stress-response program with EDS1 playing a key role in initiating and modulating several steps of it. This program includes not only responses to oxidative stress, but also responses known to be activated during plant-pathogen interactions and wounding.     

Does a light-harvesting protochlorophyllide a/b-binding protein complex exist?

Recent in vitro studies have led to speculation that a novel light-harvesting protochlorophyllide a/b-binding protein complex (LHPP) might exist in dark-grown angiosperms. Structurally, it has been suggested that LHPP consists of a 5:1 ratio of dark-stable ternary complexes of the light-dependent NADPH: protochlorophyllide oxidoreductases A and B containing nonphotoactive protochlorophyllide b and photoactive protochlorophyllide a, respectively. Functionally, LHPP has been hypothesized to play major roles in establishing the photosynthetic apparatus, in protecting against photo-oxidative damage during greening, and in determining etioplast inner membrane architecture. However, the LHPP model is not compatible with other studies of the pigments and the pigment-protein complexes of dark-grown angiosperms. Protochlorophyllide b, which is postulated to be the major light-harvesting pigment of LHPP, has, for example, never been detected in etiolated seedlings. This raises the question: does LHPP exist?     

Generation of single-chain bispecific green fluorescent protein fusion antibodies for imaging of antibody-induced T cell synapses

There is growing interest in the development of novel single-chain bispecific antibodies for retargeting of immune effector T cells to tumor cells. Until today, functional fusion constructs consisting of a single-chain bispecific antibody and a fluorescent protein were not reported. Such molecules could be useful for an in vivo visualization of this retargeting process. Recently, we established two novel single-chain bispecific antibodies. One is capable of retargeting T cells to CD33, and the other is capable of retargeting T cells to the prostate stem cell antigen (PSCA). CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). The PSCA is a potential target on prostate cancer cells. Flanking the reading frame encoding the green fluorescent protein (GFP) with a recently described novel helical linker element allowed us to establish novel single-chain bispecific fusion antibodies. These fluorescent fusion antibodies were useful to efficiently retarget T cells to the respective tumor cells and visualize the formation of immune synapses between effector and target cells.     

Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.     

Characterization of the Snowy Cotyledon 1 Mutant of Arabidopsis Thaliana: The Impact of Chloroplast Elongation Factor G on Chloroplast Development and Plant Vitality

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco).One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.     

A Global Metabolic Shift Is Linked to Salmonella Multicellular Development

Bacteria can elaborate complex patterns of development that are dictated by temporally ordered patterns of gene expression, typically under the control of a master regulatory pathway. For some processes, such as biofilm development, regulators that initiate the process have been identified but subsequent phenotypic changes such as stress tolerance do not seem to be under the control of these same regulators. A hallmark feature of biofilms is growth within a self-produced extracellular matrix. In this study we used metabolomics to compare Salmonella cells in rdar colony biofilms to isogenic csgD deletion mutants that do not produce an extracellular matrix. The two populations show distinct metabolite profiles. Even though CsgD controls only extracellular matrix production, metabolite signatures associated with cellular adaptations associated with stress tolerances were present in the wild type but not the mutant cells. To further explore these differences we examine the temporal gene expression of genes implicated in biofilm development and stress adaptations. In wild type cells, genes involved in a metabolic shift to gluconeogenesis and various stress-resistance pathways exhibited an ordered expression profile timed with multicellular development even though they are not CsgD regulated. In csgD mutant cells, the ordered expression was lost. We conclude that the induction of these pathways results from production of, and growth within, a self produced matrix rather than elaboration of a defined genetic program. These results predict that common physiological properties of biofilms are induced independently of regulatory pathways that initiate biofilm formation.