scholarly journals XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes

2002 ◽  
Vol 365 (1) ◽  
pp. 205-211 ◽  
Author(s):  
Nien-Tai HU ◽  
Wei-Ming LEU ◽  
Meng-Shiunn LEE ◽  
Avon CHEN ◽  
Shu-Chung CHEN ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Klebsiella Oxytoca ◽  
Subcellular Fractionation ◽  
Soluble Form ◽  
Wild Type ◽  
Outer Membranes ◽  
Extracellular Milieu ◽  
Large Complex ◽  
Xanthomonas Campestris Pv Campestris

GspG, -H, -I, -J and -K proteins are members of the pseudopilin family. They are the components required for the type II secretion pathway, which translocates proteins across the outer membrane of Gram-negative bacteria to the extracellular milieu. They were predicted to form a pilus-like structure, and this has been shown for PulG of Klebsiella oxytoca by using electron microscopy. In the present study, we performed biochemical analyses of the XpsG protein of Xanthomonas campestris pv. campestris and observed that it is a pillar-like structure spanning the cytoplasmic and outer membranes. Subcellular fractionation revealed a soluble form (SF) of XpsG, in addition to the membrane form. Chromatographic analysis of SF XpsG in the absence of a detergent indicated that it is part of a large complex (>440kDa). In vitro studies indicated that XpsG is prone to aggregate in the absence of a detergent. We isolated and characterized a non-functional mutant defective in forming the large complex. It did not interfere with the function of wild-type XpsG and was not detectable in the SF. Moreover, unlike wild-type XpsG, which was distributed in both the cytoplasmic and outer membranes, it appeared only in the cytoplasmic membrane. When wild-type XpsG was co-expressed with His6-tagged XpsH but not with untagged XpsH, SF XpsG bound to nickel and co-eluted with XpsH. This result suggests the presence of other pseudopilin components in the XpsG-containing large-sized molecules.

scholarly journals Role of Adhesin Release for Mucosal Colonization by a Bacterial Pathogen

2003 ◽  
Vol 197 (6) ◽  
pp. 735-742 ◽  
Author(s):  
Loïc Coutte ◽  
Sylvie Alonso ◽  
Nathalie Reveneau ◽  
Eve Willery ◽  
Brigitte Quatannens ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Wild Type ◽  
Early Step ◽  
Bacterial Surface ◽  
Extracellular Milieu ◽  
Wild Type Parent

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors called adhesins. To exert these functions, adhesins are typically surface-exposed, although, surprisingly, some are also released into the extracellular milieu, the relevance of which has previously not been studied. To address the role of adhesin release in pathogenesis, we used Bordetella pertussis as a model, since its major adhesin, filamentous hemagglutinin (FHA), partitions between the bacterial surface and the extracellular milieu. FHA release depends on its maturation by the specific B. pertussis protease SphB1. We constructed SphB1-deficient mutants and found that they were strongly affected in their ability to colonize the mouse respiratory tract, although they adhered even better to host cells in vitro than their wild-type parent strain. The defect in colonization could be overcome by prior nasal instillation of purified FHA or by coinfection with FHA-releasing B. pertussis strains, but not with SphB1-producing FHA-deficient strains, ruling out a nonspecific effect of SphB1. These results indicate that the release of FHA is important for colonization, as it may facilitate the dispersal of bacteria from microcolonies and the binding to new sites in the respiratory tract.

scholarly journals Mutagenesis of all Eight avr Genes in Xanthomonas campestris pv. campestris Had No Detected Effect on Pathogenicity, But One avr Gene Affected Race Specificity

2005 ◽  
Vol 18 (12) ◽  
pp. 1306-1317 ◽  
Author(s):  
Adriana Castañeda ◽  
Joseph D. Reddy ◽  
Basma El-Yacoubi ◽  
Dean W. Gabriel
Keyword(s):  
Xanthomonas Campestris ◽  
Subtractive Hybridization ◽  
Wild Type ◽  
Differential Host ◽  
Reading Frame ◽  
Race Specificity ◽  
Avr Gene ◽  
Xanthomonas Campestris Pv Campestris ◽  
Avr Genes ◽  
Insertion Mutations

Suppression subtractive hybridization (SSH) was used to identify genes present in the systemic crucifer black rot pathogen Xanthomonas campestris pv. campestris 528T but missing from the nonsystemic crucifer leaf spot pathogen, X. campestris pv. armoraciae 417. Among the DNA fragments unique to 528T was Xcc2109, one of eight putative avr genes identified in the published 528T genome (NC_003902). Individual and sequential deletion, insertion mutations, or both of all eight 528T avr gene loci were made, but no change in pathogenicity was observed with any combination of avr mutations, including a strain with all eight avr genes deleted. However, insertion or deletion mutants affecting the Xcc2109 locus lost avirulence (i.e., became virulent) on Florida Mustard, an X. campestris pv. campestris race-determining, differential host. The Xcc2109 open reading frame as annotated was cloned and found to be nonfunctional. A longer gene, encompassing Xcc2109 and here designated avrXccFM, was cloned and found to complement the Xcc2109 mutants and to confer avirulence to two additional wild-type X. campestris pv. campestris strains, thereby changing their races. Resistance in Florida Mustard to 528T strains carrying avrXccFM occurred without a typical hypersensitive response (HR) on leaves, although a vascular HR was observed in seedlings.

scholarly journals Uptake and metabolic fate of [HisA8,HisB4,GluB10,HisB27]insulin in rat liver in vivo

1998 ◽  
Vol 332 (2) ◽  
pp. 421-430 ◽  
Author(s):  
François AUTHIER ◽  
Gianni M. Di GUGLIELMO ◽  
Gillian M. DANIELSEN ◽  
John J. M. BERGERON
Keyword(s):  
Insulin Receptor ◽  
Subcellular Fractionation ◽  
Wild Type ◽  
Binding Assays ◽  
Insulin Degrading Enzyme ◽  
Direct Binding ◽  
Temporal Interaction ◽  
Kinetics Of Uptake

Receptor-mediated endocytosis and subsequent endosomal proteolysis of [125I]TyrA14-[HisA8,HisB4,GluB10,HisB27]insulin ([125I]TyrA14-H2 analogue), an insulin analogue exhibiting a high affinity for the insulin receptor, has been studied in liver parenchymal cells by quantitative subcellular fractionation and compared with that of wild-type [125I]TyrA14-insulin. Whereas the kinetics of uptake of the H2 analogue by liver was not different from that of insulin, the H2 analogue radioactivity after the 2 min peak declined significantly more slowly. A significant retention of the H2 analogue compared with insulin in both plasma membrane and endosomal fractions was observed and corresponded to decreased processing and dissociation of the H2 analogue. Cell-free endosomes preloaded in vivo with radiolabelled ligands and incubated in vitro processed insulin and extraluminally released insulin intermediates at a 2–3-fold higher rate than the H2 analogue. In vitro proteolysis of both non-radiolabelled and monoiodinated molecules by endosomal lysates showed a decreased response to the endosomal proteolytic machinery for the H2 analogue. However, in cross-linking and competition studies the H2 analogue exhibited an affinity for insulin-degrading enzyme identical with that of wild-type insulin. Brij-35-permeabilized endosomes revealed a 2-fold higher rate of dissociation of insulin from internalized receptors compared with the H2 analogue. After the administration of a saturating dose of both ligands, a rapid and reversible ligand-induced translocation of insulin receptor was observed, but without receptor loss. The H2 analogue induced a higher receptor concentration and tyrosine autophosphorylation of the receptor β subunit in endosomes. Moreover, a prolonged temporal interaction of the in vivo injected H2 analogue with receptor was observed by direct binding assays performed on freshly prepared subcellular fractions. These results indicate that endosomal proteolysis for the H2 analogue is slowed as a result of an increased residence time of the analogue on the insulin receptor and a low affinity of endosomal acidic insulinase for the dissociated H2 molecule.

scholarly journals Terapia fotodinâmica no controle de Xanthomonas campestris pv. campestris in vitro e no tratamento de sementes de canola naturalmente contaminadas

2020 ◽  
Vol 46 (4) ◽  
pp. 327-332
Author(s):  
Nayara Lima Baute Zancan ◽  
Nilvanira Donizete Tebaldi
Keyword(s):  
Brassica Napus ◽  
Xanthomonas Campestris ◽  
Brassica Napus L ◽  
Xanthomonas Campestris Pv Campestris

RESUMO A cultura da canola (Brassica napus L. var. oleifera) foi recentemente introduzida na região do Triângulo Mineiro e Alta Paranaíba, MG. A podridão negra causada pela bactéria Xanthomonas campestris pv. campestris (Xcc) é uma das principais doenças da cultura. A bactéria é disseminada pelas sementes e métodos alternativos de controle devem ser avaliados. Diante disso, o objetivo deste trabalho foi avaliar o uso da terapia fotodinâmica, com os corantes Azul de Metileno (AM) e Azul de Toluidina (AT), sob à irradiação, na inibição do crescimento de Xcc in vitro e no tratamento de sementes de canola naturalmente contaminadas com a bactéria. A suspensão bacteriana de Xcc foi tratada com os corantes AM, AT e associação deles (AM+AT) nas concentrações 25, 50 e 100 µmol L-1, irradiadas ou não, e cultivada em meio de cultura, em seguida foi avaliado o número de unidades formadoras de colônias. Sementes de três genótipos de canola foram tratadas com NaCl 0,45% (Testemunha), AM, AT e AM+AT, nas concentrações 100, 50 e 25 µmol L-1, respectivamente, irradiadas ou não. A porcentagem de germinação das sementes, índice de velocidade de emergência, porcentagem de emergência de plântulas e o controle da bactéria nas sementes foram avaliados. Os corantes AM, AT e AM+AT, nas concentrações 100, 50 e 25 µmol L-1 respectivamente, sob irradiação inibiram o crescimento de Xcc in vitro. A combinação dos corantes AM+AT a 25 µmol L-1 pode ser utilizada no tratamento das sementes de canola. O controle da bactéria Xcc em sementes de canola naturalmente contaminada não foi possível ser determinada com os diferentes corantes.

scholarly journals The Prosequence of Pro-ςK Promotes Membrane Association and Inhibits RNA Polymerase Core Binding

1998 ◽  
Vol 180 (9) ◽  
pp. 2434-2441 ◽  
Author(s):  
Bin Zhang ◽  
Antje Hofmeister ◽  
Lee Kroos
Keyword(s):  
Rna Polymerase ◽  
Mother Cell ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Membrane Association ◽  
Cell Fractionation ◽  
Wild Type ◽  
Large Complex ◽  
Triton X ◽  
Mutant Cells

ABSTRACT Pro-ςK is the inactive precursor of ςK, a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis. Upon subcellular fractionation, the majority of the pro-ςK was present in the membrane fraction. The rest of the pro-ςKwas in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the ςK was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-ςE was analyzed. Pro-ςK was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-ςK did not require spoIVFgene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-ςKprocessing in the mother cell. Furthermore, pro-ςKassociated with the membrane when overproduced in vegetative cells. Overproduction of pro-ςK in sporulating cells resulted in more pro-ςK in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-ςK was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-ςK. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-ςK and ςK from other cell constituents. After salt removal, ςK, but not pro-ςK, reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-ςK to the membrane, where it may interact with the processing machinery.

scholarly journals Enzymic oxidation of unconjugated bilirubin by rat liver

1986 ◽  
Vol 236 (3) ◽  
pp. 625-633 ◽  
Author(s):  
R Cardenas-Vazquez ◽  
O Yokosuka ◽  
B H Billing
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Mitochondrial Protein ◽  
Subcellular Fractionation ◽  
Sprague Dawley ◽  
Bilirubin Oxidase ◽  
Outer Membranes ◽  
The Media ◽  
The Mean

The presence of the enzyme bilirubin oxidase, which degrades bilirubin in vitro, was demonstrated in the liver. Subcellular-fractionation experiments indicate that bilirubin oxidase is located in both the inner and outer membranes of the mitochondria. The mean rate of the reaction is 1.57 +/- 0.38 (S.D.) nmol of bilirubin degraded/min per mg of mitochondrial protein (munits/mg of protein). With respect to the overall breakdown of bilirubin, the enzyme has a Km' of 136 microM-bilirubin and a Vmax.' of 9.13 munits/mg of protein. Its activity is influenced by the ionic strength of the media and is inhibited by KCN, thiol reagents, NADH and albumin. The enzyme is aerobic, and between 1 and 1.5 mol of O2 are consumed per mol of bilirubin degraded. The products of the reaction include propentdyopents. The hepatic bilirubin oxidase activity of the jaundiced Gunn-rat liver is not significantly different from that of the Sprague-Dawley rat, and it is not induced by beta-naphthoflavone.

scholarly journals The Zur of Xanthomonas campestris Is Involved in Hypersensitive Response and Positively Regulates the Expression of the hrp Cluster Via hrpX But Not hrpG

2009 ◽  
Vol 22 (3) ◽  
pp. 321-329 ◽  
Author(s):  
Dong-Liang Huang ◽  
Dong-Jie Tang ◽  
Qing Liao ◽  
Xiao-Qian Li ◽  
Yong-Qiang He ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Hypersensitive Response ◽  
Reverse Transcription ◽  
Xanthomonas Campestris ◽  
Zinc Homeostasis ◽  
Wild Type ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Xanthomonas Campestris Pv Campestris

In bacteria, Zur is a key regulator for zinc homeostasis. Our previous work has shown that, in the phytopathogen Xanthomonas campestris pv. campestris, in addition to regulating zinc homeostasis, Zur is essential for full virulence. Here, we demonstrate that the X. campestris pv. campestris Zur is involved in hypersensitive response (HR) and positively regulates the transcription of hrpA to hrpF operons and hrpX but not hrpG. Constitutively expressing hrpX but not hrpG in the zur mutant could bypass the requirement of Zur for the expression of hrpA to hrpF operons and the induction of wild-type HR, indicating that Zur controls the expression of hrp cluster via hrpX. Promoter-gusA reporter and semiquantitative reverse-transcription polymerase chain reaction analyses revealed that HrpG controls the expression of hrpX and HrpX regulates the expression of all the six hrp operons (hrpA to hrpF) in X. campestris pv. campestris.

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