Molecular evolution of Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi, and characterization of temperate bacteriophages

Lee, InKyoung (2005) Molecular evolution of Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi, and characterization of temperate bacteriophages. PhD thesis, University of Glasgow.

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Comparative nucleotide sequence analysis of nineteen genes of different functional classes was carried out in representative isolates of Mannheimia haemolytica (10 to 32 isolates), Mannheimia glucosida (1 to 6 isolates), and Pasteurella trehalosi (1 to 4 isolates). The genes include one DNA repair and recombination gene (recA), nine metabolic enzyme genes aroA, asd, galE, gap, gnd, g6pd, mdh, mtlD, and pmm), one secreted protein gene (gcp), four periplasm-associated lipoprotein genes (plpA, plpB, plpC, and plpD), and four outer membrane protein genes (ompA, tbpB, tbpA, and wza). This study was inspired by previous work which had revealed that recombination involving M. glucosida and P. trehalosi, together with host switching of isolates from cattle to sheep, have played important roles in the evolution of the M. haemolytica leukotoxin operon (Davies et al., 2001; 2002). Comparative nucleotide sequence analysis of the nineteen genes indicated that only single nucleotide substitutions had occurred in the recA, asd, gnd, g6pd, mtlD, and gcp genes of M haemolytica. A phylogenetic tree based on their concatenated sequences supported the evolutionary relationships of isolates of M. haemolytica based on previous MLEE studies. In contrast, the aroA, gap, mdh, pmm, plpA, plpB, plpC, plpD, ompA, tbpB, tbpA, and wza genes showed evidence of varying degrees of mosaic structure which suggests that horizontal DNA transfer and intragenic recombination have played important roles in the evolution of these genes. Recombinant segments of M haemolytica genes have been derived from M glucosida and other unidentified sources indicating that DNA from other bacterial species has contributed to the evolution of M haemolytica. Extensive recombinant exchanges have occurred in the outer membrane protein genes ompA, tbpB, tbpA, and wza. The different tree topologies and different patterns of nucleotide and amino acid diversity of these four genes indicate that they have different evolutionary histories. However, the evolutionary histories of these four genes support the previously proposed view that M. haemolytica diversity has been influenced by the transmission of isolates from cattle to sheep and vice versa, which is probably linked to the domestication of these species. The temperate bacteriophages of representative isolates of M. haemolytica, M. glucosida, and P. trehalosi were also investigated in this study since phages are known to mediate horizontal DNA transfer. Induction of bacteriophages with mitomycin C and examination by electron microscopy revealed that temperate phages were more prevalent in M. haemolytica (24 of 32 isolates) than M. glucosida (one of six isolates) and P. trehalosi (one of four isolates). Genetic variation of the phages was assessed by restriction endonuclease analysis and host range was determined by plague assay. Phage DNA was successfully isolated from 15 M. haemolytica, one M glucosida, and one P. trehalosi isolates and restriction endonuclease analysis identified nine different RE types (A to I) in M. haemolytica, one RE type (J) in M. glucosida, and one RE type (K) in P. trehalosi. Plaque assay revealed that 13 M. haemolytica, one M. glucosida, and one P. trehalosi lysates could produce lysis zones on one to six M. haemolytica, two M. glucosida, and one P. trehalosi indicator isolates, respectively. Using restriction endonuclease analysis and genomic analysis of the M. haemolytica genome sequence, phages of RE type A were shown to be very similar to P2 phages of E. coli. The annotated genome of this phage type was derived from the bacterial unannotated genomic sequence and compared with the genome of P2 phages.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology, microbiology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Davies, Dr. Robert
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-71452
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 14:38
Last Modified: 04 Aug 2021 09:13
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