Mutants that overproduce TraTp, a plasmid-specified major outer membrane protein of Escherichia coli

Manning, Paul A.; Timmis, J. K.; Moll, A; Timmis, Kenneth N.

doi:10.1007/bf00332623

Cited by 15 publications

(9 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The strains used in this study are listed in Tables 1 and 2. E. coli K-12, C600 Rife(pKT146) (20), which produces the plasmid-specified outer membrane protein traTp, ECA (18), and LPS core, was used for immunization and for screening of hybridoma supernatant fluids. It was cultivated overnight at 37°C in tryptic soy broth containing tetracycline (10 Fg/ml).…”

Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Peters¹,

Jürs²,

Jann³

et al. 1985

Infect Immun

View full text Add to dashboard Cite

We established hybridoma cell lines producing monoclonal antibodies against enterobacterial common antigen (ECA) and a substructure of the outer core of different Escherichia coli lipopolysaccharides (LPSs). Anti-ECA antibodies 865 and 898 reacted with ECA in extracts of heated E. coli and with ECA-bound Rl and R4 core-containing LPS preparations, as well as with a purified sample of ECA from Salmonella montevideo. Antibody 865, but not antibody 898, cross-reacted with K5 capsular polysaccharide, suggesting that 4-linked ce-N-acetylglucosamine is part of an antigenic determinant shared by both K5 polysaccharide and ECA. Anti-LPS antibody 786 recognized an outer core structure common to E. coli K-12, B, R2, and R4 core type LPS, but not to Rl and R3 core type LPS. Its most probable target is the trisaccharide sequence Hexp(1->2)-a-D -Glcp(1-*3)a-D-Glcp->(Hepp) (where Hex is hexose, p is phosphate, Glc is glucose, and Hep is heptose), the first glucose being the immunodominant moiety. These monoclonal antibodies may be used not only for the detection of ECA, K5, and LPS core structures but also for analysis of the molecular forms resolved on polyacrylamide gels (banding patterns) of both ECA and LPS, independently of one another.on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

show abstract

Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Peters¹,

Jürs²,

Jann³

et al. 1985

Infect Immun

View full text Add to dashboard Cite

show abstract

“…the Gly->Ala change in the F and R6-5 proteins), it is likely that their effect is amplified because of the oligomeric nature of TraT. Under normal growth conditions an E. co//cell harbouring a conjugative F-like plasmid such as R6-5 contains about 20000 TraT monomers in its outer membrane (Manning et al, 1982). In the in vitro assay used in this work the addition of 8 (ig ml"^ of purified TraT protein caused a five-fold inhibition of transfer of the homologous plasmid.…”

Section: Discussionmentioning

confidence: 99%

“…Figure 4 shows a model for the interaction of a TraT protein oligomer with a pilus tip. TraT is represented as having five subunits as the pilin subunits are related by a fivefold rotation axis around the helix axis in the assembled pilus (Marvin and Folkhard, 1986). However, it should be stressed that the precise number of subunits present in the oligomer is not yet known.…”

Section: Discussionmentioning

confidence: 99%

Surface exclusion specificity of the TraT lipoprotein is determined by single alterations in a five‐amino‐acid region of the protein

Harrison

Taylor

Platt

et al. 1992

Molecular Microbiology

View full text Add to dashboard Cite

The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion. The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity. When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups. Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance. To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene. Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120). This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II). Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.

show abstract

“…Strains that express the traT gene show increased resistance to the bactericidal effects of serum and have a reduced ability to act as recipients when mated with a donor strain carrying a related plasmid, a phenomenon known as surface exclusion. Results with this protein and with several other outer membrane proteins have shown that their overexpression or alteration by mutation is frequently deleterious to bacterial growth (7,15).…”

mentioning

confidence: 99%

“…The TraT protein specified by the traT gene of F group conjugative plasmids such as F, R6-5, and R100 is a 26,000-dalton major outer membrane lipoprotein that is exposed on the cell surface of Escherichia coli (15). Strains that express the traT gene show increased resistance to the bactericidal effects of serum and have a reduced ability to act as recipients when mated with a donor strain carrying a related plasmid, a phenomenon known as surface exclusion.…”

mentioning

confidence: 99%

Highly repressible expression system for cloning genes that specify potentially toxic proteins

O’Connor

Timmis

1987

J Bacteriol

View full text Add to dashboard Cite

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, A PL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by mneasuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational.

show abstract

Mutants that overproduce TraTp, a plasmid-specified major outer membrane protein of Escherichia coli

Cited by 15 publications

References 16 publications

Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Surface exclusion specificity of the TraT lipoprotein is determined by single alterations in a five‐amino‐acid region of the protein

Highly repressible expression system for cloning genes that specify potentially toxic proteins

Contact Info

Product

Resources

About