Modification of cell-wall polymers of onion waste: III. Effect of extrusion-cooking on cell-wall material of outer fleshy tissues

The polymers of onion cell walls are known to be modified by heating, but there is little information on the effects of extrusion-cooking. This work investigates the effects of extrusion-cooking on the physico-chemical characteristics and microstructure of cell walls of onion waste in relation to cell-wall chemistry. Cell-wall material from white fleshy outer scale leaves of waste onions was extruded at a range of moisture contents, barrel temperatures and screw speeds through a co-rotating twin-screw extruder. Extrusion-cooking had little effect on the carbohydrate composition of cell-wall material. However, it resulted in an increase in the solubility of pectic polymers and hemicelluloses, and this was accompanied by an increase in swelling of the cell-wall material. The degree of solubility of the pectic polysaccharides was largely dependent on the barrel temperature, and involved depolymerisation.     

Butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes

Spinosyns, a novel class of insect active macrolides produced by Saccharopolyspora spinosa, are used for insect control in a number of commercial crops. Recently, a new class of spinosyns was discovered from S. pogona NRRL 30141. The butenyl-spinosyns, also called pogonins, are very similar to spinosyns, differing in the length of the side chain at C-21 and in the variety of novel minor factors. The butenyl-spinosyn biosynthetic genes (bus) were cloned on four cosmids covering a contiguous 110-kb region of the NRRL 30141 chromosome. Their function in butenyl-spinosyn biosynthesis was confirmed by a loss-of-function deletion, and subsequent complementation by cloned genes. The coding sequences of the butenyl-spinosyn biosynthetic genes and the spinosyn biosynthetic genes from S. spinosa were highly conserved. In particular, the PKS-coding genes from S. spinosa and S. pogona have 91–94% nucleic acid identity, with one notable exception. The butenyl-spinosyn gene sequence codes for one additional PKS module, which is responsible for the additional two carbons in the C-21 tail. The DNA sequence of spinosyn genes in this region suggested that the S. spinosa spnA gene could have been the result of an in-frame deletion of the S. pogona busA gene. Therefore, the butenyl-spinosyn genes represent the putative parental gene structure that was naturally engineered by deletion to create the spinosyn genes.     

Habitat specificity and home-range size as attributes of species vulnerability to extinction: a case study using sympatric rattlesnakes

Large home-range size and habitat specificity are two commonly cited ecological attributes that are believed to contribute to species vulnerability. The eastern diamondback rattlesnake Crotalus adamanteus is a declining species that occurs sympatrically with the more abundant canebrake rattlesnake Crotalus horridus in a portion of the south-eastern Coastal Plain, USA. In this study, we use the ecological similarities of the two species as experimental controls to test the role of home-range size and habitat specificity in the imperilment of the eastern diamondback rattlesnake. We used analysis of variance to investigate differences in home-range size between the two species, and home-range selection was modeled as habitat use versus availability with a case control sampling design using logistic regression. We failed to detect differences in home-range size between the two species; therefore, we could not identify home-range size as an attribute contributing to the imperilment of eastern diamondback rattlesnakes. Eastern diamondback rattlesnakes selected pine savannas to a degree that suggests that the species is a habitat specialist. Of the two factors examined, habitat specificity to the imperiled longleaf pine ecosystem may be a significant contributor to the decline of the eastern diamondback rattlesnake.     

Molecular basis for nucleotide-binding specificity: role of the exocyclic amino group "N2" in recognition by a guanylyl-ribonuclease

Proteins interact with nucleotides to perform a multitude of functions within cells. These interactions are highly specific; however, the molecular basis for this specificity is not well understood. To identify factors critical for protein-guanine nucleotide recognition the binding of two closely related ligands, guanosine 3'-monophosphate (3'GMP) and inosine 3'-monophosphate (3'IMP), to Ribonuclease Sa (RNase Sa), a small, guanylyl-endoribonuclease from Streptomyces aureofaciens, was compared using isothermal titration calorimetry, NMR, X-ray crystallography and molecular dynamics simulations. This comparison has allowed for the determination of the contribution of the exocyclic amino group "N2" of the guanine base to nucleotide binding specificity. Calorimetric measurements indicate that RNase Sa has a higher affinity for 3'GMP (K=(1.5+/-0.2)x10(5)) over 3'IMP (K=(3.1+/-0.2)x10(4)) emphasizing the importance of N2 as a key determinant of RNase Sa guanine binding specificity. This result was unexpected as the published structural data for RNase Sa in complex with 3'GMP showed only a potential long-range interaction (>3.3A) between N2 and the side-chain of Glu41 of RNase Sa. The observed difference in affinity is largely due to a reduction in the favorable enthalpy change by 10 kJ/mol for 3'IMP binding as compared to 3'GMP that is accompanied by a significant difference in the heat capacity changes observed for binding the two ligands. To aid interpretation of the calorimetric data, the first crystal structure of a small, guanylyl ribonuclease bound to 3'IMP was determined to 2.0 A resolution. This structure has revealed small yet unexpected changes in the ligand conformation and differences in the conformations of the side-chains contacting the sugar and phosphate moieties as compared to the 3'GMP complex. The structural data suggest the less favorable enthalpy change is due to an overall lengthening of the contacts between RNase Sa and 3'IMP as compared to 3'GMP. The long-range interaction between N2 and Glu41 is critical for positioning of the nucleotide in the binding cleft for optimal contact formation. Thus, combined, the data demonstrate how a long-range interaction can have a significant impact on nucleotide binding affinity and energetics.     

Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages

The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.     

Production of feruloyl esterases and xylanases by Talaromyces stipitatus and Humicola grisea var. thermoidea on industrial food processing by-products

Feruloyl esterase (FAE) and xylanase activities were detected in culture supernatants from Humicola grisea var. thermoidea and Talaromyces stipitatus grown on brewers' spent grain (BSG) and wheat bran (WB), two agro-industrial by-products. Maximum activities were detected from cultures of H. grisea grown at 150 rpm, with 16.9 U/ml and 9.1 U/ml of xylanase activity on BSG and WB, respectively. Maximum FAE activity was 0.47 U/ml and 0.33 U/ml on BSG and WB, respectively. Analysis of residual cell wall material after microbial growth shows the preferential solubilisation of arabinoxylan and cellulose, two main polysaccharides present in BSG and WB. The production of low-cost cell-wall-deconstructing enzymes on agro-industrial by-products could lead to the production of low-cost enzymes for use in the valorisation of food processing wastes.     

Salmonella must be viable in order to attach to the surface of prepared vegetable tissues

Aims: The aims of the current study were to explore the site of bacterial attachment to vegetable tissues and to investigate the hypothesis that Salmonella must be living in order to attach to this site(s). Methods and Results: Scanning electron micrographs of intact potato cells showed that Salm. serotype Typhimurium attached to cell-wall junctions; suggesting a high-level of site selectivity. Inactivation of Salm. Typhimurium using heat, ethanol, formalin or Kanamycin resulted in cells that could be no longer attached to these sites. Attachment of a Gfp⁺ strain of Salm. Typhimurium to cell-wall material (CWM) was examined via flow cytometric analysis. Only live Salm. Typhimurium attached to the CWM. Conclusions: Salmonella serotype Typhimurium must be metabolically active to ensure attachment to vegetable tissues. Attachment preferentially occurs at the plant cell-wall junction and the cell-wall components found here, including pectate, may provide a receptor site for bacterial attachment. Significance and Impact of the Study: Further studies into individual plant cell-wall components may yield the specific bacterial receptor site in vegetable tissues. This information could in turn lead to the development of more targeted and effective decontamination protocols that block this site of attachment.     

Protein kinase D isozymes activation and localization during mitosis

The protein kinase D (PKD) family consists of three serine/threonine protein kinases involved in the regulation of fundamental biological processes in response to their activation and intracellular redistribution. Although a substantial amount of information is available describing the mechanisms regulating the activation and intracellular distribution of the PKD isozymes during interphase, nothing is known of their activation status, localization and role during mitosis. The results presented in this study indicate that during mitosis, PKD3 and PKD are phosphorylated at Ser⁷³¹ and Ser⁷⁴⁴ within their activation loop by a mechanism that requires protein kinase C. Mitosis-associated PKD3 Ser⁷³¹ and PKD Ser⁷⁴⁴ phosphorylation is related to the catalytic activation of these kinases as evidenced by in vivo phosphorylation of histone deacetylase 5, a substrate of PKD and PKD3. Activation loop-phosphorylated PKD3 and PKD, as well as PKD2, associate with centrosomes, spindles and midbody suggesting that these activated kinases establish dynamic interactions with the mitotic apparatus. Thus, this study reveals a connection between the PKD isozymes and cell division, suggesting a novel role for this family of serine/threonine kinases.