Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris

ABSTRACT An xps gene cluster composed of 11 open reading frames is required for the type II protein secretion in Xanthomonas campestris pv. campestris. Immediately upstream of thexpsD gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of X. campestris pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal xpsN gene, we demonstrated that it is required for the secretion of extracellular enzyme by X. campestrispv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)6-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two.

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Isolation and Characterization of a Porin-Like Outer Membrane Protein from Xanthomonas campestris pv. campestris

IUBMB Life ◽

10.1080/15216540213826 ◽

2002 ◽

Vol 54 (1) ◽

pp. 13-18 ◽

Cited By ~ 1

Author(s):

Lingyun Wang ◽

Ying Zheng ◽

Xujia Zhang

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Xanthomonas Campestris ◽

Isolation And Characterization ◽

Xanthomonas Campestris Pv Campestris

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Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

Microbiology ◽

10.1099/mic.0.27995-0 ◽

2005 ◽

Vol 151 (9) ◽

pp. 2975-2986 ◽

Cited By ~ 65

Author(s):

Bisweswar Nandi ◽

Ranjan K. Nandy ◽

Amit Sarkar ◽

Asoke C. Ghose

Keyword(s):

Membrane Protein ◽

Recombinant Protein ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Outer Membrane Protein ◽

Secondary Structure Prediction ◽

Sequence Data ◽

Insertion Mutagenesis ◽

Sds Page

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

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Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1987.tb13581.x ◽

1987 ◽

Vol 169 (1) ◽

pp. 65-71 ◽

Cited By ~ 18

Author(s):

Marja AGTERBERG ◽

Roland BENZ ◽

Jan TOMMASSEN

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Cell Surface ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Insertion Mutagenesis ◽

Exposed Region ◽

A Cell ◽

K 12

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Mutation of the gene encoding a major outer-membrane protein in Xanthomonas campestris pv. campestris causes pleiotropic effects, including loss of pathogenicity

Microbiology ◽

10.1099/mic.0.039420-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2842-2854 ◽

Cited By ~ 17

Author(s):

Yih-Yuan Chen ◽

Chieh-Hao Wu ◽

Juey-Wen Lin ◽

Shu-Fen Weng ◽

Yi-Hsiung Tseng

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Xanthomonas Campestris ◽

Extracellular Enzymes ◽

Surface Deformation ◽

Cell Signalling ◽

Cell Aggregation ◽

Major Outer Membrane Protein ◽

Gene Encoding

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by Clp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/dispersal, regulated by the effect of controlled synthesis of DSF on cell–cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo-β-1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mopB mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97 % identity with homologues from other members of Xanthomonas. Similarly to the rpfF mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo-β-1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria.

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