The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation

Despite a very complex structure, the sugar composition of the rhamnogalacturonan II (RG-II) pectic fraction is extremely conserved. Among its constituting monosaccharides is the seldom-observed eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo), whose phosphorylated precursor is synthesized by Kdo-8-P synthase. As an attempt to alter specifically the RG-II structure in its sugar composition and assess the consequences on the function of RG-II in cell wall and its relationship with growth, Arabidopsis null mutants were sought in the genes encoding Kdo-8-P synthase. Here, the isolation and characterization of one null mutant for the isoform 1 (AtkdsA1-S) and two distinct null mutants for the isoform 2 of Arabidopsis Kdo-8-P synthase (AtkdsA2-V and AtkdsA2-S) are described. Evidence is provided that AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity, and that it accounts for 75% of the mRNAs to be translated into Kdo-8-P synthase. Furthermore, this predominant expression of AtKDSA2 over AtKDSA1 was confirmed by quantification of the cytosolic Kdo content in the mutants, in a variety of ecotypes. The inability to identify a double knockout mutant originated from pollen abortions, due to the inability of haploid pollen of the AtkdsA1- AtkdsA2- genotype to form an elongated pollen tube properly and perform fertilization

Xylella fastidiosa gene expression analysis by DNA microarrays

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants

The Airborne Metagenome in an Indoor Urban Environment

The indoor atmosphere is an ecological unit that impacts on public health. To investigate the composition of organisms in this space, we applied culture-independent approaches to microbes harvested from the air of two densely populated urban buildings, from which we analyzed 80 megabases genomic DNA sequence and 6000 16S rDNA clones. The air microbiota is primarily bacteria, including potential opportunistic pathogens commonly isolated from human-inhabited environments such as hospitals, but none of the data contain matches to virulent pathogens or bioterror agents. Comparison of air samples with each other and nearby environments suggested that the indoor air microbes are not random transients from surrounding outdoor environments, but rather originate from indoor niches. Sequence annotation by gene function revealed specific adaptive capabilities enriched in the air environment, including genes potentially involved in resistance to desiccation and oxidative damage. This baseline index of air microbiota will be valuable for improving designs of surveillance for natural or man-made release of virulent pathogens

Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths

Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide.

An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells. A. tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells. The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria. Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide. Anion-exchange chromatography followed by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar. Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide. Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant. This indicates that the acidic polysaccharide may play a role in the attachment of A. tumefaciens to host soma plant cells