scholarly journals Genetic and Sequence Analysis of the pTiC58 trb Locus, Encoding a Mating-Pair Formation System Related to Members of the Type IV Secretion Family

1998 ◽  
Vol 180 (23) ◽  
pp. 6164-6172 ◽  
Author(s):  
Pei-Li Li ◽  
Dawn M. Everhart ◽  
Stephen K. Farrand
Keyword(s):  
Sequence Analysis ◽  
Pair Formation ◽  
Type Iv Secretion ◽  
Ti Plasmid ◽  
Open Reading Frames ◽  
Conjugal Transfer ◽  
Mating Pair ◽  
Secretion Systems ◽  
Type Iv ◽  
And Function

ABSTRACT Conjugal transfer of pTiC58 requires two regions, trawhich contains the oriT and several genes involved in DNA processing and a region of undefined size and function that is located at the 2-o’clock position of the plasmid. Using transposon mutagenesis with Tn3HoHo1 and a binary transfer system, we delimited this second region, called trb, to an 11-kb interval between the loci for vegetative replication and nopaline catabolism. DNA sequence analysis of this region identified 13 significant open reading frames (ORFs) spanning 11,003 bp. The first, encodingtraI, already has been described and is responsible for the synthesis of Agrobacterium autoinducer (AAI) (I. Hwang, P.-L. Li, L. Zhang, K. R. Piper, D. M. Cook, M. E. Tate, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 91:4639–4643, 1994). Translation products of the next 11 ORFs showed similarities to those of trbB, -C, -D,-E, -J, -K, -L,-F, -G, -H, and -I of the trb region of the octopine-type Ti plasmid pTi15955 and of the tra2 core region of RP4. In RP4, these genes encode mating-pair formation functions and are essential for the conjugal transfer of the IncP plasmid. Each of the trb gene homologues is oriented counterclockwise on the Ti plasmid. Expression of these genes, as measured by using the lacZ fusions formed by Tn3HoHo1, required the traI promoter and the transcriptional activator TraR along with its coinducer, AAI. While related to that of RP4, the trb system of pTiC58 did not allow propagation of the trb-specific bacteriophages PRD1, PRR1, and Pf3. The products of several trb genes of the Ti plasmid are similar to those of other loci that encode DNA transfer or protein secretion systems, all of which are members of the type IV secretion family.

scholarly journals Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

2009 ◽  
Vol 191 (9) ◽  
pp. 2985-2992 ◽  
Author(s):  
Gisèle Bourg ◽  
Romain Sube ◽  
David O'Callaghan ◽  
Gilles Patey
Keyword(s):  
Critical Role ◽  
Type Iv Secretion ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iv ◽  
Brucella Suis ◽  
Periplasmic Domain ◽  
Tm Domain ◽  
And Function

ABSTRACT The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.

scholarly journals Fluorescence assays for F-pili and their application

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3541-3548 ◽  
Author(s):  
Katrin Daehnel ◽  
Robin Harris ◽  
Lucinda Maddera ◽  
Philip Silverman
Keyword(s):  
Primary Structure ◽  
Type Iv Secretion ◽  
Filament Formation ◽  
Secretion Systems ◽  
Type Iv ◽  
Bacteriophage Infection ◽  
And Function ◽  
Domain I ◽  
Donor Activity

Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell–cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F+ cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0·1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions.

scholarly journals A Homologue of an Operon Required for DNA Transfer in Agrobacterium Is Required in Brucella abortusfor Virulence and Intracellular Multiplication

2000 ◽  
Vol 182 (17) ◽  
pp. 4849-4855 ◽  
Author(s):  
Rodrigo Sieira ◽  
Diego J. Comerci ◽  
Daniel O. Sánchez ◽  
Rodolfo A. Ugalde
Keyword(s):  
Critical Role ◽  
Genome Project ◽  
Type Iv Secretion ◽  
Open Reading Frames ◽  
Secretion Systems ◽  
Cell Infection ◽  
Type Iv ◽  
Intergenic Regions ◽  
Correct Function ◽  
Intracellular Multiplication

ABSTRACT As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virBoperon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment.

scholarly journals BIOGENESIS, ARCHITECTURE, AND FUNCTION OF BACTERIAL TYPE IV SECRETION SYSTEMS

2005 ◽  
Vol 59 (1) ◽  
pp. 451-485 ◽  
Author(s):  
Peter J. Christie ◽  
Krishnamohan Atmakuri ◽  
Vidhya Krishnamoorthy ◽  
Simon Jakubowski ◽  
Eric Cascales
Keyword(s):  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iv ◽  
Type Iv Secretion Systems ◽  
And Function ◽  
Bacterial Type

scholarly journals A New Type IV Secretion System Promotes Conjugal Transfer in Agrobacterium tumefaciens

2002 ◽  
Vol 184 (17) ◽  
pp. 4838-4845 ◽  
Author(s):  
Lishan Chen ◽  
Yuching Chen ◽  
Derek W. Wood ◽  
Eugene W. Nester
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Bartonella Henselae ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Ti Plasmid ◽  
Dna Transfer ◽  
Conjugal Transfer ◽  
Type Iv Secretion System ◽  
Plant Host ◽  
Type Iv

ABSTRACT Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.

Sign in / Sign up

Export Citation Format

Share Document