Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in nonmucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that eleven psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating pentasaccharide containing d-mannose, d-glucose, and l-rhamnose: false[→3false)−normalβ−normalD−Manp2↑1α−D−Manp−false(1→3false)−normalβ−normalD−Manp−false(1→3false)−normalα−normalL−Rhap−false(1→3false)−normalβ−normalD−Glcp−false(1→false]normaln− In addition we identified the sugar nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP-d-mannose, UDP-d-glucose, and dTDP-l-rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that wbpW restored Psl production in a pslB mutant and pslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of P. aeruginosa.

ARC1 is a novel U-box protein required in the Brassica pistil for the rejection of self-incompatible pollen; it functions downstream of the S receptor kinase (SRK). Here, we show that ARC1 has E3 ubiquitin ligase activity and contains several motifs that influence its subcellular localization. ARC1 can shuttle between the nucleus, cytosol, and proteasome/COP9 signalosome (CSN) when expressed in tobacco BY-2 suspension-cultured cells. However, ARC1 localization to the proteasome/ CSN occurs only in the presence of an active SRK. In the pistil, ubiquitinated protein levels increase specifically with incompatible pollinations, but they do not change in ARC1 antisense-suppressed pistils. In addition, inhibition of the proteasomal proteolytic activity disrupts the self-incompatibility response. We propose that ARC1 promotes the ubiquitination and proteasomal degradation of compatibility factors in the pistil, which in turn leads to pollen rejection.

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Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiaminrequiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470. Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

Summary Exopolysaccharide is a critical biofilm matrix component, yet little is known about how the synthesis of multiple exopolysaccharides is regulated. Pseudomonas aeruginosa can produce several biofilm matrix exopolysaccharides that include alginate, Psl and Pel. Here we demonstrated that AlgC, a key enzyme that provides sugar precursors for the synthesis of alginate and lipopolysaccharides (LPS) is also required for both Psl and Pel production. We showed that forced-synthesis of Psl in alginate-producing mucoid bacteria reduced alginate production but this was not due to transcription of the alginate biosynthesis-operon. Likewise, when either alginate or Psl were overproduced, levels of B-band LPS decreased. Induction of Pel resulted in a reduction of Psl levels. Because the effects of reduced exopolysaccharide synthesis when another is overproduced didn’t appear to be regulated at the transcriptional level, this suggests that the biosynthesis pathways of Psl, Pel, alginate, and LPS compete for common sugar precursors. As AlgC is the only enzyme that provides precursors for each of these exopolysaccharides, we propose that AlgC is a key checkpoint enzyme that coordinates the total amount of exopolysaccharide biosynthesis by controlling sugar precursor pool. Our data also provide a plausible strategy that P. aeruginosa utilizes to modulate its biofilm matrix exopolysaccharides.

FlaA1 from the human pathogen Helicobacter pylori is an enzyme involved in saccharide biosynthesis that has been shown to be essential for pathogenicity. Here we present five crystal structures of FlaA1 in the presence of substrate, inhibitors, and bound cofactor, with resolutions ranging from 2.8 to 1.9 Å . These structures reveal that the enzyme is a novel member of the short-chain dehydrogenase/reductase superfamily. Additional electron microscopy studies show the enzyme to possess a hexameric doughnut-shaped quaternary structure. NMR analyses of "real time" enzyme-substrate reactions indicate that FlaA1 is a UDP-GlcNAc-inverting 4,6-dehydratase, suggesting that the enzyme catalyzes the first step in the biosynthetic pathway of a pseudaminic acid derivative, which is implicated in protein glycosylation. Guided by evidence from site-directed mutagenesis and computational simulations, a three-step reaction mechanism is proposed that involves Lys-133 functioning as both a catalytic acid and base.Helicobacter pylori is a spiral shaped, motile, microaerophilic Gram-negative bacterium that resides in the gastric mucus layer or adheres to the epithelial lining of the stomach (1). It has been estimated that half to two-thirds of the world's population is chronically infected with H. pylori, and prevalence among adults in developing countries is typically 80 -90% (2). The bacterium can asymptomatically remain in the human stomach for decades; however, infections can lead to gastric inflammation and ulceration. In addition to its well established etiological role in several gastroduodenal diseases, H. pylori infection has also been implicated in the development of gastric cancer (3, 4). The significance of the discovery that H. pylori plays a causal role in gastritis and ulceration was recently recognized through the awarding of the 2005 Nobel Prize in Physiology or Medicine to Drs. Marshall and Warren.The pathogenicity of H. pylori can be attributed to numerous virulence factors that allow for host colonization, among these are urease, adhesins, flagella, and lipopolysaccharides (LPS) 6 (5-7). For example, the flagella enable H. pylori to penetrate the gastric mucus layer and reach the epithelial cells, and LPS O antigens employ mimicry to Lewis blood group antigens to facilitate subsequent cell adhesion and colonization (8). Because of drug resistance and an increase in treatment failure, the biochemical pathways responsible for the synthesis and delivery of these virulence factors have received much attention as they contain potential targets for drug development (9, 10).The flaA1 (HP0840) gene product has been shown to be involved in the synthesis of both flagella and LPS, and as such it plays a critical role in H. pylori pathogenesis and colonization. Specifically, disruption of the flaA1 gene results in bacteria devoid of flagella and with altered LPS that lacks most of the O antigen (9, 10). FlaA1 is a 37-kDa protein whose sequence suggests that it is a member of the short-chain dehydrogenase/ reductase (SDR) su...