Analysis of the virulence regulatory locus of Bordetella pertussis

McGillivray, Doreen Mary (1990) Analysis of the virulence regulatory locus of Bordetella pertussis. PhD thesis, University of Glasgow.

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The primary aim of this research was to characterize recombinant cosmid clone pRMB2, and subclone the vir locus. Clone pRMB2 was identified from a gene library of Bordetella pertussis TAB I DNA in Escherichia coli, by its ability to restore a Vir+ phenotype to Vir - Tn5 mutant B.pertussis BP347 (Brownlie et al., 1988). Restriction analysis with EcoRI revealed that the genomic insert of pRMB2 contained six fragments, designated El (l.lkb), E2 (2.5kb), E3 (2.7kb), E4 (4.7kb), E5 (5.1kb) and E6 (10.0kb). Hybridisation with pRMB2 and E3 probes showed that the Tn5 insertion in Vir- BP347 was located within an EcoRI fragment of 2.7kb. The inactivation of this region by Tn5 indicated that it was essential to the vir region. The E3 fragment of pRMB2 was subcloned in pLAFRl to give a construct designated pDM3. In-trans, pDM3 was unable to complement Vir BP347, which implied that the 2.7kb EcoRI fragment contained insufficient sequence to encode an active vir locus. In a small percentage of BP347 (pDM3) transconjugants, homologous recombination had occurred in the vir region, such that a fully functional vir locus was restored to BP347 with an attendant gain of expression of virulence-associated determinants. Clone pRMB2 was partially digested with Sau3A to generate random fragments of approximately 6kb. Ligation of these fragments into vector pRK310 yielded forty-eight recombinant clones, none of which contained sufficient sequence to complement Vir- BP347. Hybridisation analysis of a series of restriction digests of pRMB2 with E3 identified three putative vir-containing fragments, which each consisted of pLAFRl vector and the terminal portions of the genomic insert of pRMB2. One of these fragments was ligated to give a construct designated pDMl which, in-trans, restored a Vir+ phenotype to Vir- BP347. Restriction enzyme analysis of pDMl showed that the genomic insert comprised two noncontiguous fragments of 3.9kb and 8.0kb. The E3 fragment was contained within the latter, which demonstrated that this 8.0kb region encoded a functional vir locus. A restriction map was compiled for clone pRMB2, and comparison with a published map for the vir and fha region (Stibitz et al., 1988a) showed homology for a 15.2kb region (E3, E2 and E6) encoding vir and fhaB, but the remaining 10.9kb (E5, E4 and El) of pRMB2 showed a different restriction pattern. Hybridisation analysis confirmed that the genomic insert of pRMB2 comprised two non-contiguous regions of TAB I genomic DNA. Clone pRMB2 was unable to complement BP353 (Fha-) which contained a Tn5 insertion in fhaA, just downstream of fhaB. Hybridisation with pRMB2 and E3 probes was used to analyse regions homologous to vir in a number of strains of all four Bordetella species. These studies indicated that the vir locus was conserved in B.pertussis, B.parapertussis and B.bronchiseptica. However, the structure of the vir region in the latter two species differed from that of B.pertussis. Avirulent phase variant B.pertussis 11615 had a rearrangement in the vir region. B.avium chromosomal DNA also showed homology to the B.pertussis vir locus, but a markedly different hybridisation pattern was observed. This confirmed that B.avium was genetically divergent from the other Bordetella species. A slight variation in the hybridisation pattern was observed between virulent and avirulent B.avium strains. In-trans the vir locus encoded by pDMl was able to restore a Vir+ phenotype to avirulent phase variant strains of B.pertussis and B.bronchiseptica. This indicated that phase variation in both these species had occurred as a result of genotypic changes in vir. However, expression of virulence determinants in avirulent phase variant strains of B.bronchiseptica was restored to only a low level by pDMl, which implied that the mechanisms of regulation of expression of virulence determinants were different for the two species. The effect of multiple copies of vir in-trans in B.pertussis on the response to modulators was investigated. Multiple copies of vir, in clones pRMB2 and pDMl, had no effect on the response to modulation by low temperatures. Tohama-derived strains, including BP347 (pRMB2) and BP347 (pDMl) were resistant to modulation by nicotinic acid. The ability of vir to trans-activate expression in E.coli of B.pertussis virulence genes was investigated: no expression of fhaB, fim2, fim3 or cya was detected.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Dr. John Coote
Keywords: Microbiology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Supervisor, not known
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-72850
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 28 Jun 2021 15:48
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