Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion

Craig-Mylius, Kathleen A.

doi:10.1016/s0378-1097(99)00447-4

Cited by 16 publications

(43 citation statements)

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“…The excess VirB complexes are used for the transport of the pilin protein from the membrane to the site of pilus assembly in a virD4-independent manner. Two studies suggest that the VirB proteins without VirD4 can form a functional transport apparatus (Craig-Mylius and Weiss, 1999;Lai et al, 2000). The formation of the T-pilus does not require VirD4 but is absolutely dependent on the VirB proteins, and a VirD4 homologue is not necessary for the export of pertussis toxin in B. pertussis.…”

Section: Discussionmentioning

confidence: 99%

Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4

Kumar

Das

2002

Molecular Microbiology

103

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Summary Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants. VirD4 presumably functions as a coupling factor that facilitates communication between a substrate and the transport pore. To serve as a coupling protein, VirD4 may be required to localize near the transport apparatus. In a previous study, we observed that several constituents of the transport apparatus localize to the cell membranes. In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence microscopy, cells probed with anti‐VirD4 antibodies had foci of fluorescence primarily at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location of VirD4 was not dependent on T‐DNA processing, the formation of the transport apparatus and the presence of other Vir proteins. VirD4 is an integral membrane protein with one periplasmic domain. The large cytoplasmic region contains a nucleotide‐binding domain. To investigate the role of these domains in DNA transfer, we introduced mutations in virD4 and studied the effect of a mutation on substrate transfer. A deletion of most of the periplasmic domain as well as the alterations of glycine 151 to serine and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that both domains are essential for substrate transfer. Subcellular localization of the mutant proteins indicated that both the periplasmic and the nucleotide‐binding domains are required for polar localization of VirD4. The periplasmic domain mutant VirD4Δ36–61 was distributed throughout the cell membrane, whereas the nucleotide binding site mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.

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Section: Discussionmentioning

confidence: 99%

Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4

Kumar

Das

2002

Molecular Microbiology

103

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“…Modulation or suppression of the Bvg-regulated transcription of the ptx-ptl operon (26) was achieved by growth on BG agar plates containing 40 mM MgSO 4 . Strains were streaked onto modulating plates and incubated at 37°C for 72 h, and then they were restreaked onto modulating plates and incubated at 37°C for 24 h. Suspensions of modulated cells were made in 7 ml of Stainer-Scholte broth at an optical density at 600 nm (OD 600 ) of 0.1 and overlaid onto BG plates containing appropriate antibiotics as previously described (10). The plates were incubated at 37°C, and 1-ml aliquots were harvested by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…The amount of secreted toxin was determined by using filtered-sterilized culture supernatants. The amount of periplasmic pertussis toxin was determined as previously described (10,42). Briefly, periplasmic toxin was released from the cell suspensions by shock treatment with lysozyme and EDTA.…”

Section: Methodsmentioning

confidence: 99%

“…Chinese hamster ovary (CHO) cell assays were performed as previously described (10). Briefly, serial twofold dilutions of samples were made in Ham's F-12 medium containing 1% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

“…The A subunit of the toxin is the S1 polypeptide, while the pentameric B subunit is comprised of S2, S3, S4, and S5, assembled in a ratio of 1:1:2:1 (36). Pertussis toxin is secreted past the outer membrane of Bordetella pertussis by a type IV secretion system comprised of the products of the nine ptl (for "pertussis toxin liberation") genes (8,10,42). The ptl genes are located immediately downstream of the pertussis toxin genes (42) and are transcribed from the same promoter (2, 18, 31).…”

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confidence: 99%

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Temporal Expression of Pertussis Toxin and Ptl Secretion Proteins byBordetella pertussis

Rambow-Larsen

Weiss

2004

J Bacteriol

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Pertussis toxin is an AB 5 toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.Pertussis toxin is an AB 5 toxin comprised of the products of five genes, S1 through S5 (25, 27). The A subunit of the toxin is the S1 polypeptide, while the pentameric B subunit is comprised of S2, S3, S4, and S5, assembled in a ratio of 1:1:2:1 (36). Pertussis toxin is secreted past the outer membrane of Bordetella pertussis by a type IV secretion system comprised of the products of the nine ptl (for "pertussis toxin liberation") genes (8,10,42). The ptl genes are located immediately downstream of the pertussis toxin genes (42) and are transcribed from the same promoter (2, 18, 31).It is not known how many secretion complexes are present in one bacterium during active secretion, nor is the stoichiometry of the proteins in the complex known. The structures of other type IV systems, such as the Agrobacterium tumefaciens virB system (11,38,40), which secretes a tumerogenic T-DNA and effector proteins into host plant cells, and the P-plasmid tra conjugation genes (23), have been more thoroughly studied than the Ptl system. The extensive similarity suggests that the Ptl proteins also form a large complex spanning both the inner and outer membranes. Nevertheless, the Ptl secretion system and the DNA transport systems have substantially different substrates and functions. The most intriguing difference is that the same basic machinery transports protein-coated DNA between the cytoplasm of two cells for the conjugation systems or a periplasmic protein complex across the outer membrane in the case of the Ptl system. Functionally, the DNA t...

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Bordetella protein toxins

Mašín

Osička

Bumba

et al. 2015

The Comprehensive Sourcebook of Bacterial Protein Toxins

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Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion

Cited by 16 publications

References 11 publications

Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4

Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4

Temporal Expression of Pertussis Toxin and Ptl Secretion Proteins byBordetella pertussis

Bordetella protein toxins

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