Regulation of colony morphology and biofilm formation in Shewanella algae

Bacterial colony morphology can reflect different physiological stages such as virulence or biofilm formation. In this work we used transposon mutagenesis to identify genes that alter colony morphology and cause differential Congo Red (CR) and Brilliant Blue G (BBG) binding in Shewanella algae, a marine indigenous bacterium and occasional human pathogen. Microscopic analysis of colonies formed by the wild-type strain S. algae CECT 5071 and three transposon integration mutants representing the diversity of colony morphotypes showed production of biofilm extracellular polymeric substances (EPS) and distinctive morphological alterations. Electrophoretic and chemical analyses of extracted EPS showed differential patterns between strains, although the targets of CR and BBG binding remain to be identified. Galactose and galactosamine were the preponderant sugars in the colony biofilm EPS of S. algae. Surface-associated biofilm formation of transposon integration mutants was not directly correlated with a distinct colony morphotype. The hybrid sensor histidine kinase BarA abrogated surface-associated biofilm formation. Ectopic expression of the kinase and mutants in the phosphorelay cascade partially recovered biofilm formation. Altogether, this work provides the basic analysis to subsequently address the complex and intertwined networks regulating colony morphology and biofilm formation in this poorly understood species.     

Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules

Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.     

Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.     

Proteoglycans mediate malaria sporozoite targeting to the liver

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.     

The Arabidopsis repressor of light signaling SPA1 acts in the phloem to regulate seedling de-etiolation, leaf expansion and flowering time

Plants adjust their growth and development in response to the ambient light environment. These light responses involve systemic signals that coordinate differentiation of different tissues and organs. Here, we have investigated the function of the key repressor of photomorphogenesis SPA1 in different tissues of the plant by expressing GUS-SPA1 under the control of tissue-specific promoters in a spa mutant background. We show that SPA1 expression in the phloem vasculature is sufficient to rescue the spa1 mutant phenotype in dark-grown spa mutant seedlings. Expression of SPA1 in mesophyll, epidermis or root tissues of the seedling, by contrast, has no or only slight effects. In the leaf, SPA1 expression in both the phloem and the mesophyll is required for full complementation of the defect in leaf expansion. SPA1 in phloem and mesophyll tissues affected division and expansion of cells in the epidermal layer, indicating that SPA1 induces non-cell-autonomous responses also in the leaf. Photoperiodic flowering is exclusively controlled by SPA1 expression in the phloem, which is consistent with previous results showing that the direct substrate of the COP1/SPA complex, CONSTANS, also acts in the phloem. Taken together, our results highlight the importance of phloem vascular tissue in coordinating growth and development. Because the SPA1 protein itself is incapable of moving from cell to cell, we suggest that SPA1 regulates the activity of downstream component(s) of light signaling that subsequently act in a non-cell-autonomous manner. SPA1 action in the phloem may also result in mechanical stimuli that affect cell elongation and cell division in other tissues.     

Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells

Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, H?hn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More fundamentally, these observations shed new light on enigmatic issues such as the inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in heterologous agonist activation of the Ras/ERK pathway.     

Breast cancer risk and 6q22.33: combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2

Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361 BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341 (per-allele odds ratio (OR)?=?1.03, 95% CI 1.00-1.06, p?=?0.023). There was evidence for heterogeneity in the ORs among studies (I(2)?=?49.3%; p?=?<0.004). In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers (per-allele OR?=?0.89, 95%CI 0.80-1.00, p?=?0.048), indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.     

Fast Cytoplasmic pH Regulation in Acid-Stressed Leaves

Induction of photosynthesis in leaves was prolonged, and steadystate photosynthesis was inhibited by very high CO₂ concentrationswhich cause cytoplasmic acidification. Prolonged exposure tohigh CO₂ relieved initially observed inhibition of photosynthesisat least partially. The sensitivity of carbon assimilation tohigh CO₂ was different in different plant species. Acidificationby CO₂ (or subsequent alkalization) was detected by measuringrapid CO₂-release from the tissue and by monitoring fluorescenceof pH-indicating dyes which had been fed to the leaves throughthe petiole. The results indicate that two different mechanismsoperate in leaves to achieve and maintain pH homeostasis. Rapidand efficient pH-adjustment is provided by proton/cation exchangeacross the tonoplast. Slower and less efficient regulation occursby formation or consumption of base. In the presence of highCO₂ concentrations, protons are pumped from the cytosol intoalready acidic vacuoles. In turn, vacuolar cations replace exportedprotons in the cytosol permitting bicarbonate accumulation andincreasing the pH of the acidified cytosol. Similarly effectiveand fast proton/cation exchange relieves acid-stress in thechloroplast stroma and permits photosynthesis to proceed withhigh quantum efficiency or high light-saturated rates in thepresence of CO₂ concentrations which would, in the absence offast cytoplasmic pH regulation, inhibit photosynthesis. By inference,proton/cation exchange must also occur across the mitochondrialboundary. After cytoplasmic pH adjustment in the presence ofhigh CO₂, removal of CO₂ results in transient cytoplasmic alkalizationand, subsequently, in the return of cytoplasmic pH values tolevels observed prior to acid-stress. In addition to fast pHregulation by rapid proton/cation exchange across biomembranes,slow base production (e.g. NH₃-formation) also contributes torelieving acid stress. Base produced in the presence of highCO₂ is rapidly consumed after removal of CO₂. Implications of the findings in regard to forest damage by potentiallyacidic air pollutants such as SO₂ are briefly discussed. (Received November 8, 1993; Accepted February 3, 1994)     

Evolutionary origin of the medaka Y chromosome

Genetic sex determination in an XX-XY chromosome system can be realized through a locus on the Y chromosome that makes the undifferentiated gonad develop into a testis. Although this mechanism is widespread, only in two cases so far have the corresponding master male sex-determining genes been identified. One is Sry, which initiates testes determination in most mammals. The other is dmrt1bY (syn. dmy), from the fish medaka, Oryzias latipes. The mammalian Y is roughly estimated to be over 200 million years old. The medaka Y may be considerably younger. A comparative analysis of the genus Oryzias revealed that one sister species of the medaka has dmrt1bY on a homologous Y chromosome, whereas in another closely related species only a non-sex-linked pseudogene is present. In all other species, dmrt1bY was not detected. The divergence time for the different species was determined with mitochondrial DNA sequences. The timing was confirmed by independent calculations based on dmrt1 sequences. We show that the medaka sex-determining gene originated approximately 10 million years ago. This makes dmrt1bY and the corresponding Y chromosome the youngest male sex-determining system, at least in vertebrates, known so far.