Membrane Topology of Outer Membrane Protein AlgE,Which Is Required for Alginate Production in Pseudomonas aeruginosa

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa secretes a viscous extracellular polysaccharide, called alginate, as a virulence factor during chronic infection of patients with cystic fibrosis. In the present study, it was demonstrated that the outer membrane protein AlgE is required for the production of alginate in P. aeruginosa. An isogenic marker-free algE deletion mutant was constructed. This strain was incapable of producing alginate but did secrete alginate degradation products, indicating that polymerization occurs but that the alginate chain is subsequently degraded during transit through the periplasm. Alginate production was restored by introducing the algE gene. The membrane topology of the outer membrane protein AlgE was assessed by site-specific insertions of FLAG epitopes into predicted extracellular loop regions.Pseudomonas aeruginosa is an ubiquitous opportunistic human pathogen responsible for chronic infections of the lungs of patients with cystic fibrosis (CF), in whom it is the leading cause of mortality and morbidity (9). The establishment of a chronic infection in the lungs of patients with CF coincides with the switch of P. aeruginosa to a stable mucoid variant, producing copious amounts of the exopolysaccharide alginate; this is typically a poor prognostic indicator for these patients (24, 31). Alginate is a linear unbranched exopolysaccharide consisting of 1,4-linked monomers of β-d-mannuronic acid and its C-5 epimer, α-l-guluronic acid, which is known to be produced by only two bacterial genera, Pseudomonas and Azotobacter (34). The switch to a mucoid phenotype coincides with the appearance of a 54-kDa protein in the outer membrane; this protein has been identified and has been designated AlgE (13, 31).The genes encoding the alginate biosynthesis machinery are located within a 12-gene operon (algD-alg8-alg44-algK-algE-algG-algX-algL-algI-algJ-algF-algA). AlgA and AlgD, along with AlgC (not encoded in the operon), are involved in precursor synthesis (34). Alg8 is the catalytic subunit of the alginate polymerase located at the inner membrane (35). AlgG is a C-5 mannuronan epimerase (19). AlgK contains four putative Sel1-like repeats, similar to the tetratricopeptide repeat motif often found in adaptor proteins involved in the assembly of multiprotein complexes (3, 10). AlgX shows little homology to any known protein, and its role is unclear (14). Knockout mutants of AlgK, AlgG, and AlgX have nonmucoid phenotypes, although they produce short alginate fragments, due to the activity of the alginate lyase (AlgL), which degrades the nascent alginate (1, 14, 19-21, 36). AlgF, AlgI, and AlgJ are involved in acetylation of alginate, but they are not ultimately required for its production (12). The membrane-anchored protein, Alg44, is required for polymerization and has a PilZ domain for the binding of c-di-GMP, a secondary messenger essential for alginate production (16, 25, 33). The periplasmic C terminus of Alg44 shares homology with the membrane fusion proteins involved in the bridging of the periplasm in multidrug efflux pumps (11, 43). The periplasmic alginate lyase, AlgL, appears to be required for the translocation of intact alginate across the periplasm (1, 26). AlgE is an outer membrane, anion-selective channel protein through which alginate is presumably secreted (30). A protein complex or scaffold through which the alginate chain can pass and be modified and which spans the periplasm bridging the polymerase located (Alg8) at the outer membrane pore (AlgE) has been proposed (21). Indeed, it has been demonstrated that both the inner and the outer membranes are required for the in vitro polymerization of alginate (35).The requirement of AlgE for the biosynthesis of alginate in P. aeruginosa was first observed by complementation of an alginate-negative mutant derived by chemical mutagenesis with a DNA fragment containing algE (8) Secondary structure predictions suggested that AlgE forms an 18-stranded β barrel with extended extracellular loops. Several of these loops show high densities of charged amino acids, suggesting a functional role in the translocation of the anionic alginate polymer (29, 30). Preliminary analysis of AlgE crystals has been reported (48).In this study, the role of AlgE in alginate biosynthesis was investigated and the membrane topology of AlgE was assessed by site-directed insertion mutagenesis.     

Toxin production by Fusarium species from sugar beets and natural occurrence of zearalenone in beets and beet fibers.

Fifty-five Fusarium isolates belonging to nine species were collected from fungus-invaded tissue of stored sugar beets and identified as F. acuminatum (11 isolates), F. avenaceum (1 isolate), F. culmorum (1 isolate), F. equiseti (23 isolates), F. graminearum (4 isolates), F. oxysporum (1 isolate), F. solani (4 isolates), F. sporotrichioides (7 isolates), and F. subglutinans (2 isolates). All isolates were cultured on autoclaved rice grains and assayed for toxicity by feeding weanling female rats the ground-rice cultures of the isolates in a 50% mixture with a regular diet for 5 days. Fifty-eight percent of the isolates were acutely toxic to rats, 26% caused hematuria, 18% caused hemorrhages, and 29% caused uterine enlargement. In most cases, toxicity could not be accounted for by the known toxins found. The following mycotoxins were found in extracts of the rice cultures: zearalenone (22 to 6,282 micrograms/g), chlamydosporol (HM-8) (68 to 4,708 micrograms/g), moniliformin (45 to 400 micrograms/g), deoxynivalenol (10 to 34 micrograms/g), 15-acetyldeoxynivalenol (5 to 10 micrograms/g), diacetoxyscirpenol (22 to 63 micrograms/g), monoacetoxyscirpenol (21 to 26 micrograms/g), scirpenetriol (24 micrograms/g), T-2 toxin (4 to 425 micrograms/g), HT-2 toxin (2 to 284 micrograms/g), neosolaniol (2 to 250 micrograms/g), and T-2 tetraol (4 to 12 micrograms/g). F. equiseti was the predominant species found on visibly molded beets in the field. Six of 25 moldy sugar beet root samples collected in the field contained zearalenone in concentrations ranging between 12 and 391 ng/g, whereas 10 samples from commercial stockpiles were negative for zearalenone.(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP-dependent protein kinases, their subunits, and cAMP-binding protein from four sources: a comparison of physical characteristics determined by polyacrylamide gel electrophoresis

The physical characteristics of cAMP-dependent protein kinases and their, regulatory subunits from calf uterus, human uterus, human mammary tumor, and rat pituitary and of cAMP-binding protein from calf uterus were determined by quantitative polyacrylamide gel electrophoresis in buffers containing the detergent, Triton X-100. In the four tissues, protein kinases of either type A¹, with molecular weight (M_r) = 200,000, or type B, of M_r = 80,000, or both, previously described were found. Trivial charge isomerism, or size isomerism, exists within each of the two classes, Protein Kinase A and B. The protein kinase recombined from the regulatory and catalytic subunits is not significantly different from the crude or isolated protein kinase. Protein Kinases A and B exist each in either one of the isozyme forms I and II but these are not reflected in polyacrylamide gel electrophoresis at pH 10.2. Protein Kinase B appears to be a product of the partial proteolysis of Protein Kinase A. The regulatory subunits of Protein Kinases A from the four tissues are distinct from those of Protein Kinases B. No physical distinction exists between regulatory subunits derived from isozyme forms I and II. cAMP-Binding Proteins A and B are physically indistinguishable, by polyacrylamide gel electrophoresis at pH 10.2, from the regulatory subunits of Protein Kinases A and B, respectively.     

Ultrastructure of cuticular exudations in parasitic juvenileHeterodera schachtii,as related to cuticle structure

Summary The development ofHeterodera schachtii inside roots of a cruciferous host plant grown under monoxenic conditions in an agar medium was observed with video-enhanced contrast light microscopy. One to 6 days after inoculation, roots were excised and processed for electron microscopic observations. Exudates were present on the cuticle surfaces of J 2 and early J 3 juveniles located at feeding sites. Fibrillar exudations were correlated with similar fibrillar patterns in the epicuticle, exocuticle, intermediate zone, and the striated endocuticle. Secretion vesicles assembled at many Golgi sites in the hypodermis, appeared to coalesce and form large electron translucent vesicles in the cytoplasm. We propose that secretion vesicles migrate toward the cuticle, contact the plasmalemma and transfer their contents by exocytosis or a similar mechanism to a secretion accumulation site. These contents are associated with cuticle structure and emerge as surface exudations.     

Herstellung künstlicher radioaktiver Teilchen

Zusammenfassung Es wurden künstliche radioaktive Teilchen in Form von im Durchmesser 5–20 m großen Plexiglasperlen hergestellt, in die eine organische Scandiumverbindung homogen eingelagert ist. Nach eintägiger Aktivierung der Perlen im Reaktor kann bei einem Teilchen von 10 m Durchmesser eine Scandiumaktivität bis zu 10^–10 Ci erreicht werden.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.