scholarly journals Identification of a TcpC-TcpQ Outer Membrane Complex Involved in the Biogenesis of the Toxin-Coregulated Pilus of Vibrio cholerae

2005 ◽  
Vol 187 (7) ◽  
pp. 2225-2232 ◽  
Author(s):  
Niranjan Bose ◽  
Ronald K. Taylor
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Sodium Dodecyl ◽  
Direct Interaction ◽  
High Molecular Weight Complex ◽  
Metal Affinity ◽  
Membrane Complex ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Toxin Coregulated Pilus

ABSTRACT The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis proteins revealed that TcpC was absent specifically in a tcpQ mutant. TcpQ is a predicted periplasmic protein required for TCP biogenesis. Fractionation studies revealed that the protein is not localized to the periplasm but is associated predominantly with the outer membrane fraction. An analysis of the amount of TcpQ present in the series of tcp mutants demonstrated the inverse of the TcpC result (absence of TcpQ in a tcpC deletion strain). Complementation of the tcpQ deletion restored TcpC levels and TCP formation, and similarly, complementation of tcpC restored TcpQ. Metal affinity pull-down experiments performed using His-tagged TcpC or TcpQ demonstrated a direct interaction between TcpC and TcpQ. In the presence of TcpQ, TcpC was found to form a high-molecular-weight complex that is stable in 2% sodium dodecyl sulfate and at temperatures below 65°C, a characteristic of secretin complexes. Fractionation studies in which TcpC was overexpressed in the absence of TcpQ showed that TcpQ is also required for proper localization of TcpC to the outer membrane.

scholarly journals Composition and Localization of Treponema denticola Outer Membrane Complexes

2011 ◽  
Vol 79 (12) ◽  
pp. 4868-4875 ◽  
Author(s):  
Valentina Godovikova ◽  
M. Paula Goetting-Minesky ◽  
J. Christopher Fenno
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Treponema Denticola ◽  
Content Type ◽  
Membrane Complex ◽  
Cell Extracts ◽  
Outer Membrane Lipoprotein ◽  
Extracellular Matrix Components ◽  
Active Protease ◽  
Membrane Lipoprotein

ABSTRACTTheTreponema denticolaouter membrane lipoprotein-protease complex (dentilisin) contributes to periodontal disease by degrading extracellular matrix components and disrupting intercellular host signaling pathways. We recently demonstrated thatprcB, located upstream of and cotranscribed withprcAandprtP, encodes a 22-kDa lipoprotein that interacts with PrtP and is required for its activity. Here we further characterize products of the protease locus and their roles in expression, formation, and localization of outer membrane complexes. PrcB migrates in native gels as part of a >400-kDa complex that includes PrtP and PrcA, as well as the major outer sheath protein Msp. PrcB is detectable as a minor constituent of the purified active protease complex, which was previously reported to consist of only PrtP and auxiliary polypeptides PrcA1 and PrcA2. Though it lacks the canonical ribosome binding site present upstream of bothprcAandprtP, PrcB is present at levels similar to those of PrtP in whole-cell extracts. Immunofluorescence microscopy demonstrated cell surface exposure of the mature forms of PrtP, PrcA1, PrcB, and Msp. The 16-kDa N-terminal acylated fragment of PrtP (predicted to be released during activation of PrtP) was present in cell extracts but was detected neither in the purified active protease complex nor on the cell surface. PrcA2, detectable on the surface of Msp-deficient cells but not that of wild-type cells, coimmunoprecipitated with Msp. Our results indicate that PrcB is a component of the outer membrane lipoprotein protease complex and that Msp and PrcA2 interaction may mediate formation of a very-high-molecular-weight outer membrane complex.

scholarly journals Structure-Function Analysis of BfpB, a Secretin-Like Protein Encoded by the Bundle-Forming-Pilus Operon of EnteropathogenicEscherichia coli

2001 ◽  
Vol 183 (16) ◽  
pp. 4848-4859 ◽  
Author(s):  
Sarah A. Schmidt ◽  
David Bieber ◽  
Sandra W. Ramer ◽  
Jaiweon Hwang ◽  
Cheng-Yen Wu ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Function Analysis ◽  
Sodium Dodecyl ◽  
Functional Studies ◽  
Membrane Complex ◽  
Type Iv ◽  
Outer Membrane Lipoprotein ◽  
Multimeric Complex ◽  
Pilus Biogenesis ◽  
Gated Channel

ABSTRACT Production of type IV bundle-forming pili by enteropathogenicEscherichia coli (EPEC) requires BfpB, an outer-membrane lipoprotein and member of the secretin protein superfamily. BfpB was found to compose a ring-shaped, high-molecular-weight outer-membrane complex that is stable in 4% sodium dodecyl sulfate at temperatures of ≤65°C. Chemical cross-linking and immunoprecipitation experiments disclosed that the BfpB multimeric complex interacts with BfpG, and mutational studies showed that BfpG is required for the formation and/or stability of the multimer but not for the outer-membrane localization of BfpB. Formation of the BfpB multimer also does not require BfpA, the repeating subunit of the pilus filament. Functional studies of the BfpB-BfpG complex revealed that its presence confers vancomycin sensitivity, indicating that it may form an incompletely gated channel through the outer membrane. BfpB expression is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may support both pilus biogenesis and protein secretion.

scholarly journals Pseudomonas aeruginosa fur Overlaps with a Gene Encoding a Novel Outer Membrane Lipoprotein, OmlA

1999 ◽  
Vol 181 (4) ◽  
pp. 1099-1109 ◽  
Author(s):  
Urs A. Ochsner ◽  
Adriana I. Vasil ◽  
Zaiga Johnson ◽  
Michael L. Vasil
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Iron Concentration ◽  
Sodium Dodecyl ◽  
Attachment Site ◽  
Signal Peptidase ◽  
Cell Fractionation ◽  
Promoter Regions ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein

ABSTRACT A novel outer membrane lipoprotein in Pseudomonas aeruginosa is encoded by the omlA gene, which was identified immediately upstream of the fur (ferric uptake regulator) gene. The omlA and fur genes were divergently transcribed and had overlapping promoter regions. The proximal fur P2 promoter and the omlA promoter shared a 5-bp DNA motif for their −10 promoter elements. The distalfur P1 promoter was located within the omlAcoding sequence, and the omlA and fur T1 mRNAs overlapped by 154 nucleotides. Optimal expression of bothfur and omlA required roughly 200 bp of DNA upstream of the promoter regions, suggesting the presence ofcis-acting transcriptional activation elements located within the omlA and fur genes, respectively. The levels of Fur and OmlA proteins had no influence onomlA or fur expression, excluding anytrans-acting cross-regulation between fur andomlA. Expression of omlA was constitutive regardless of growth phase, oxygen tension, iron concentration, pH, and temperature. OmlA contained a signal sequence typical of bacterial lipoproteins, with a cysteine as a putative cleavage and lipid attachment site. Inhibition of signal peptidase II by globomycin resulted in failure to process OmlA, thus giving strong evidence that OmlA is a lipoprotein. Cell fractionation followed by Western blot analysis indicated that all OmlA protein is localized in the outer membrane. Mature OmlA was an acidic (pI = 4.5) protein of 17.3 kDa and had close to 40% amino acid sequence identity to SmpA (small protein A) of Escherichia coli, Vibrio cholerae, and Haemophilus influenzae, a protein of unknown function. All P. aeruginosa strains tested as well as Pseudomonas fluorescens were found to produce OmlA. A mutant strain with impaired production of OmlA but no change in the expression of the overlapping fur gene was constructed. TheomlA mutant was hypersusceptible to anionic detergents such as sodium dodecyl sulfate and deoxycholate, and it showed increased susceptibility to various antibiotics, including nalidixic acid, rifampin, novobiocin, and chloramphenicol. A structural role of OmlA in maintaining the cell envelope integrity is proposed.

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