A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species

Potato scab is a globally important disease caused by polyphyletic plant pathogenic Streptomyces species. Streptomyces acidiscabies, Streptomyces scabies and Streptomyces turgidiscabies possess a conserved biosynthetic pathway for the nitrated dipeptide phytotoxin thaxtomin. These pathogens also possess the nec1 gene which encodes a necrogenic protein that is an independent virulence factor. In this article we describe a large (325-660 kb) pathogenicity island (PAI) conserved among these three plant pathogenic Streptomyces species. A partial DNA sequence of this PAI revealed the thaxtomin biosynthetic pathway, nec1, a putative tomatinase gene, and many mobile genetic elements. In addition, the PAI from S. turgidiscabies contains a plant fasciation (fas) operon homologous to and colinear with the fas operon in the plant pathogen Rhodococcus fascians. The PAI was mobilized during mating from S. turgidiscabies to the non-pathogens Streptomyces coelicolor and Streptomyces diastatochromogenes on a 660 kb DNA element and integrated site-specifically into a putative integral membrane lipid kinase. Acquisition of the PAI conferred a pathogenic phenotype on S. diastatochromogenes but not on S. coelicolor. This PAI is the first to be described in a Gram-positive plant pathogenic bacterium and is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems.     

Characterization of Haemonchus contortus calreticulin suggests its role in feeding and immune evasion by the parasite

Haemonchus contortus, a gastrointestinal parasite of sheep and goat feeds on the blood of its host and causes bleeding at the biting site. In this report, we demonstrate that the Ca2+ binding protein, calreticulin (CalR), is present in excretory/secretory products of adult worms. The secreted CalR enhanced plasma coagulation time. Using recombinant fragments, this property has been mapped to C-terminal part of the molecule which has binding sites for Ca2+ as well as clotting factors. Complement protein C1q bound to immobilized CalR and C1q dependent lysis of sensitized sheep erythrocytes was inhibited by CalR, a function mapped to N-domain of the protein. Factor X and a 24 kDa polypeptide derived from prothrombin but not prothrombin bound to immobilized CalR. The binding site for 24 kDa polypeptide in the CalR molecule has been localized in the P-domain. Our results suggest at least two functions for secreted CalR: first, to prevent blood clotting by binding to Ca2+ and clotting factors thus enabling parasite to feed on the host blood and second to modulate the host immune response by binding to complement C1q thereby facilitating parasite's survival within the host.     

The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide

Sequence and predicted structural similarities between members of the Cys loop superfamily of ligand-gated ion channel receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops that intersect at subunit interfaces. We employed site-directed mutagenesis to investigate the role of amino acids from the loop C region of the murine 5-HT(3AS)R in interacting with two structurally different agonists, serotonin (5-HT) and m-chlorophenylbiguanide (mCPBG). Mutant receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies. Electrophysiological assays were employed to identify changes in response characteristics and relative efficacies of mCPBG and the partial agonist, 2-methyl 5-HT (2-Me5-HT). We have also constructed novel 5-HT and mCPBG docked models of the receptor binding site based on homology models of the AChBP. Both ligand-docked models correlate well with results from mutagenesis and electrophysiological assays. Four key amino acids were identified as being important to ligand binding and/or gating of the receptor. Among these, I228 and D229 are specific for effects mediated by 5-HT compared to mCPBG, indicating a differential interaction of these ligands with loop C. Residues F226 and Y234 are important for both 5-HT and mCPBG interactions. Mutations at F226, I228, and Y234 also altered the relative efficacies of agonists, suggesting a role in the gating mechanism.     

Characterization of the beta-carbon processing reactions of the mammalian cytosolic fatty acid synthase: role of the central core

The properties of the beta-ketoacyl reductase, dehydrase, and enoyl reductase components of the animal fatty acid synthase responsible for the reduction of the beta-ketoacyl moiety formed at each round of chain elongation have been studied by engineering and characterizing mutants defective in each of these three catalytic domains. These "beta-carbon processing" mutants leak the stalled four-carbon intermediates by direct transfer to CoA. However, enoyl reductase mutants leak beta-ketobutyryl, beta-hydroxybutyryl, and crotonyl moieties, a finding explained, at least in part, by the observation that the equilibrium and rate constant for the dehydrase reaction favor the formation of beta-hydroxy rather than enoyl moieties. In this regard, the type I animal fatty acid synthase resembles its type II counterpart in Escherichia coli in that both systems rely on the enoyl reductase to pull the beta-carbon processing reactions to completion. Kinetic and nucleotide binding measurements on fatty acid synthases mutated in either of the two nucleotide binding domains revealed that the NADPH binding sites are nonidentical, the enoyl reductase exhibiting higher affinity. Surprisingly, NADPH binding is also completely compromised by certain deletions and mutations in the central core region distant from the nucleotide binding sites. Comparable central core sequences are present in the structurally related modular polyketide synthases, except in those modules that lack all three beta-carbon processing enzymes. These findings suggest that the central core region of fatty acid and polyketide synthases plays an important role in facilitating the beta-carbon processing reactions.     

Peptides identified through phage display direct immunogenic antigen to dendritic cells

Dendritic cells (DC) play a critical role in adaptive immunity by presenting Ag, thereby priming naive T cells. Specific DC-binding peptides were identified using a phage display peptide library. DC-peptides were fused to hepatitis C virus nonstructural protein 3 (NS3) while preserving DC targeting selectivity and Ag immunogenicity. The NS3-DC-peptide fusion protein was efficiently presented to CD4+ and CD8+ T cells derived from hepatitis C virus-positive blood cells, inducing their activation and proliferation. This immunogenic fusion protein was significantly more potent than NS3 control fusion protein or NS3 alone. In chimeric NOD-SCID mice transplanted with human cells, DC-targeted NS3 primed naive CD4+ and CD8+ T cells for potent NS3-specific proliferation and cytokine secretion. The capacity of peptides to specifically target immunogenic Ags to DC may establish a novel strategy for vaccine development.     

The Caenorhabditis elegans pvl-5 gene protects hypodermal cells from ced-3-dependent, ced-4-independent cell death

Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. Here we describe pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway.     

Combining ability in the F1 and F2 generations of diallel cross in hexaploid wheat (Triticum aestivum L. em. Thell)

The F(1) and F(2) progenies of a ten-parent diallel cross (excluding reciprocals) of hexaploid wheat (Triticum aestivum L. em. Thell) were analyzed for combining ability for quantitative and quality traits. The results indicated significant differences among the parents for general combining ability (gca) and crosses for specific combining ability (sca) for all the characters studied. The gca and sca components of variance were significant for all the traits. However, the gca component of variance was predominant indicating the predominance of additive gene effects for the traits studied. Among the parents Durgapura 65, HD 2285, Lok-1, Raj 1972 and HD 2329 were the best general combiners for grain yield and average to high combiners for tillers per plant, grain yield per spike, grains per spike and 1000-grain weight. The best specific crosses for grain yield were Sonalika x WH 157, HD 2428 x Durgapura 65, Durgapura 65 x Sonalika, HD 2428 x Lok-1 and CPAN 3004 x Raj 1972. The parent Raj 1972, Lok-1 and HD 2285 were the best general combiners for grain yield and protein content, however, Raj 3077 was the best general combiner for protein content. The most suitable specific crosses for protein content were HD 2329 x HD 2285, HD 2428 x Raj 1972 and CPAN 3004 x WH 157. Most of the specific crosses for grain yield as well as protein content involved high x average, average x average and average x poor general combiners. To ensure further increase in grain yield along with high protein, combinations of desirable yield components is advocated. Inclusion of F(1) hybrids showing high sca and having parents with good gca, into multiple crosses and/or bi-parental mating, or diallel selective mating could prove a worthwhile approach for further improvement of grain yield in bread wheat.