Wheat starch contains two classes of associated proteins: proteins which are embedded within the granule and loosely associated surface proteins. The characterisation of the major proteins that are embedded in the granule are described. Gel electrophoresis on the basis of size resolved these proteins into five bands of molecular weights 60, 75, 85, 100 and 105 kDa. These polypeptides were demonstrated to be within the granule by their resistance to proteinase K digestion when granules were ungelatinised. The N-terminal sequences of these polypeptides are reported. The most prominent polypeptide is the 60 kDa granule-bound starch synthase. The N-terminal sequence obtained from the 75 kDa polypeptide shows homology to rice soluble starch synthase. The 85 kDa band was resolved into at least two types of polypeptides, one of which reacted with polyclonal antiserum to the maize branching enzyme IIb. The 100 and 105 kDa polypeptides were located only in the granule and are related, on the basis of N-terminal sequence similarity and cross-reactivity to monoclonal antibodies. SDS-PAGE and monoclonal antibody cross-reactivity experiments suggest that the 100 and 105 kDa polypeptides are absent from starch granules from all other species examined, including other cereals. It is speculated that all the major granule proteins are involved in starch biosynthesis.
Minimally processed fresh produce has been implicated as a major source of foodborne microbial pathogens globally. These pathogens must attach to the produce in order to be transmitted. Cut surfaces of produce that expose cell walls are particularly vulnerable. Little is known about the roles that different structural components (cellulose, pectin, and xyloglucan) of plant cell walls play in the attachment of foodborne bacterial pathogens. Using bacterial cellulose-derived plant cell wall models, we showed that the presence of pectin alone or xyloglucan alone affected the attachment of three Salmonella enterica strains (Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, and Salmonella enterica subsp. indica M4) and Listeria monocytogenes ATCC 7644. In addition, we showed that this effect was modulated in the presence of both polysaccharides. Assays using pairwise combinations of S. Typhimurium ATCC 14028 and L. monocytogenes ATCC 7644 showed that bacterial attachment to all plant cell wall models was dependent on the characteristics of the individual bacterial strains and was not directly proportional to the initial concentration of the bacterial inoculum. This work showed that bacterial attachment was not determined directly by the plant cell wall model or bacterial physicochemical properties. We suggest that attachment of the Salmonella strains may be influenced by the effects of these polysaccharides on physical and structural properties of the plant cell wall model. Our findings improve the understanding of how Salmonella enterica and Listeria monocytogenes attach to plant cell walls, which may facilitate the development of better ways to prevent the attachment of these pathogens to such surfaces.
The bran from polished rice grains can be used to produce rice bran oil (RBO). High oleic (HO) RBO has been generated previously through RNAi down-regulation of OsFAD2-1. HO-RBO has higher oxidative stability and could be directly used in the food industry without hydrogenation, and is hence free of trans fatty acids. However, relative to a classic oilseed, lipid metabolism in the rice grain is poorly studied and the genetic alteration in the novel HO genotype remains unexplored. Here, we have undertaken further analysis of role of OsFAD2-1 in the developing rice grain. The use of Illumina-based NGS transcriptomics analysis of developing rice grain reveals that knockdown of Os-FAD2-1 gene expression was accompanied by the down regulation of the expression of a number of key genes in the lipid biosynthesis pathway in the HO rice line. A slightly higher level of oil accumulation was also observed in the HO-RBO. Prominent among the down regulated genes were those that coded for FatA, LACS, SAD2, SAD5, caleosin and steroleosin. It may be possible to further increase the oleic acid content in rice oil by altering the expression of the lipid biosynthetic genes that are affected in the HO line.
Abstract Acinetobacter baumannii is a top-priority pathogen as classified by the World Health Organisation. It causes life-threatening infections in immunocompromised patients, resulting in prolonged hospitalisation and high mortality. Increasing cases of community-acquired A. baumannii infections with rapid progression and severe infections have been reported. This study used the previously described Galleria mellonella infection model to investigate the virulence mechanisms of the community strain C98 (Ab-C98) via transcriptomic analysis using direct RNA sequencing. This strain showed greater killing and more rapid colonisation in the larvae than a clinical reference strain (ATCC BAA1605). Differential gene expression analysis revealed the significant upregulation of three major iron clusters: the acinetobactin and baumannoferrin clusters for siderophore production and the Feo system for ferrous iron uptake. Targeted knockout of siderophore production genes ( basC , bfnD and isochorismatase family protein) significantly attenuated virulence in mutants with minimal impact on the bacterial growth in vivo . Overall, this study highlights the virulence of basC , isochorismatase family protein and bfnD in the pathogenicity of A. baumannii . As these targets are highly conserved in A. baumannii and the closely related A. pittii and A. lactucae , they could serve as potential therapeutic targets for developing new antivirulence agents to combat these pathogens.
The role of starch branching enzyme-I (SBE-I) in determining starch structure in the endosperm has been investigated. Null mutations of SBE-I at the A, B and D genomes of wheat were identified in Australian wheat varieties by immunoblotting. By combining individual null mutations at the B and D genomes through hybridisation, a double-null mutant wheat, which lacks the B and D isoforms of SBE-I, was developed. Wheat mutants lacking all the three isoforms of SBE-I were generated from a doubled haploid progeny of a cross between the BD double-null mutant line and a Chinese Spring (CS) deletion line lacking the A genome isoform. Comparison of starch from this mutant wheat to that from wild type revealed no substantial alteration in any of the structural or functional properties analysed. Further analysis of this triple-null mutant line revealed the presence of another residual peak of SBE-I activity, referred to as SBE-Ir, in wheat endosperm representing < 3% of the activity of SBE-I in wild type endosperm.
(1) Bulk mRNA from germinated wheat embryos was denatured with methylmercury and subjected to electrophoresis in agarose gel to obtain a fraction of mRNA that was modestly enriched with respect to its complement of translatable germin mRNA. This fraction of mRNA was used as a source of primary templates for preparing a cDNA library.(2) Escherichia coli JM101 was transfected with recombinant pUC8 plasmids containing cDNA inserts. Colonies of transformed bacteria (ca. 4 × 10 3 ) were differentially screened by hybridizing them with cDNA probes that were prepared from RNA populations containing different proportions of translatable germin mRNA.(3) A 160 base pair (bp) cDNA, which hybridized more strongly to the probe made from the RNA population containing the greater proportion of translatable germin mRNA in colony hybridizations, also hybridized more strongly to the RNA population containing the greater proportion of translatable germin mRNA when it was used as a probe for Northern analysis.(4) As judged by peptide mapping of a protein made by cell-free translation, the 160-bp cDNA selected virtually pure germin mRNA from the bulk mRNA of germinated wheat embryos when it was used in "hybrid release" experiments. The same 160-bp cDNA was used to select a "full length" germin cDNA from a library prepared by the Gubler–Hoffman method.
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