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Title: The molecular biology of chlamydiaphages
Author: Garner, Sarah Ann
ISNI:       0000 0001 3491 1724
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2003
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Chlamydiae are obligate intracellular bacteria that have a unique developmental cycle. They are responsible for causing a wide range of human and animal diseases. As there is no natural system for gene transfer in chlamydiae and due to the inaccessibility of the bacteria within the host cell, studies into the molecular biology of chlamydiae have been slow. Bacteriophages infecting chlamydiae may provide the basis for a gene transfer system. Therefore study into the molecular biology of these bacteriophages is extremely interesting. Chlamydiaphages belong to the Microviridae family of bacteriophages. These consist of lytic isometric phages with single-stranded circular DNA genomes. Members of the Microviridae family fall into two distinct sub-families. One sub-family that includes bacteriophages infecting enterobacteriaceae, and a second sub-family containing the chlamydiaphages, φMH2k and SpV4. Four chlamydiaphages have been discovered to date infecting various hosts; Chp1 (C. psitacci), Chp2 (C. abortus), φCPAR39 (C. pneumoniae) and φCPG1 (C. caviae). Whilst the genomes of Chp2, φCPAR39, φCPG1 remain highly conserved Chp1 shows high divergence throughout its genome. A novel bacteriophage was discovery during this study (Chp3). Chp3 infects a chlamydial species not previously known to carry bacteriophages (C. pecorum). The genome of Chp3 is 4,554 bp and encodes eight open reading frames that are organised in the same relative context as other chlamydiaphages. Chp3 is closely related to the Chp2-like bacteriophages. Interestingly while the VP1 of the Chp2-like bacteriophages remains highly conserved two regions show high divergence between Chp2/Chp3 and φCPG1/φCPAR39. It has been hypothesised that these regions are the chlamydiaphage receptor-recognition domain. The host range of Chp3 is identical to the host range of Chp2 (C. abortus, C. pecorum, C. felis and C. caviae). φCPAR39 is also able to infect C. abortus, C. caviae and C. pecorum, however, it is unable to infect C. felis and can additionally infect C. pneumoniae. This suggests that these two sub groups of chlamydiaphages use different surface receptor molecules. It has been predicted that the chlamydiaphage genome is arranged into eight major open reading frames (ORF1-8). Immuno-specific reagents were produced to the proteins encoded by ORF2-8 and a monoclonal antibody to VPl was identified. These reagents were then used to screen Chp2 infected C. abortus inclusions and semi-purified Chp2 particles. The expression of VP1, VP3, ORF5 protein ORF4 protein and ORF7 protein was demonstrated. It is likely that chlamydiaphages regulate their gene expression in a similar way to other bacteriophages in the Microviridae. By comparing the genome organisation of Chp2 to two members of this family of bacteriophages SpV4 and φX174 two promoter regions were identified in the Chp2 genome situated upstream of the ORF4 and ORF5 translational initiation codons. Using promoter selection vectors it was shown that these two promoter regions are functional in E.coli. The promoter situated upstream of the ORF5 translational initiation codon is a stronger E. coli promoter than the ORF4 promoter. This may be used as a mechanism for the regulation of gene expression and used to control when and how much of each protein is produced.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available