Occurrence and roles of two integrative and conjugative elements in Klebsiella pneumoniae
thesisposted on 04.12.2017, 11:15 authored by Robeena Farzand
The genome of Klebsiella pneumoniae HS11286 carries two integrative and conjugative elements (ICEs), ICEKpnHS-1 and ICEKpnHS-2; the former belongs to the ICEKp1 family and the latter was unexplored. This study was designed to further characterise the two ICEs, and investigate their prevalence in local clinical K. pneumoniae isolates. Sequence analysis showed that ICEKpnHS-2 belonged to the subfamily of PAPI elements with XerC-like integrases and PCR screening revealed that ICEKpnHS-1 associated elements were more (30%) prevalent in local isolates compared to ICEKpnHS-2 (12.5%). Contributions of these elements to cell physiology, virulence and antibiotic resistance were investigated. Deletion of ICEKpnHS-1 reduced siderophore secretion and growth, particularly in iron restricted conditions. The ΔICEKpnHS-1 strain also showed reduced resistance to various antibiotics, especially those known to be affected by the ATP binding cassette (ABC) and major facilitator superfamily (MFS) exporters. Assays in Galleria mellonella provided preliminary evidence that deletion of ICEKpnHS-2 reduced the virulence of K. pneumoniae HS11286. Type IV secretion systems (T4SS) are major component modules of ICEs responsible for conjugative transfer of nucleoprotein complexes. Observed reduction of conjugative transfer of one of the ICEs following deletion of the other led us to study interactions between the two distinct T4SSs encoded on the ICEs in HS11286. A marker plasmid containing oriT of ICEKpnHS-1 (P-oriT1) was constructed and deletion of ICEKpnHS-2 was shown to produce a six–fold reduction in trans-conjugants. Deletion of the mobB (ICEKpnHS-1) orthologue in ICEKpnHS-2 (mob-2) and traI (relaxase of ICEKpnHS-2) both produced similar defects in transconjugants frequencies that could be complemented in-trans. The ATPase Mob-1 was bioinformatically shown to lack a Walker B and it was speculated that Mob-2 might compensate for this. Accordingly, point mutations in Walker motifs of mob-2 provided evidence supporting this complementary function in energy transduction contributing significantly to the crosstalk between the two non-homologous ICEs. Data on frequency of transfer of the native chromosomal ICEKpnHS-1 and its effects on the phenotypes of an E. coli were also shown. However, ICEKpnHS-2 self-conjugation was not detected.