S. coelicolor uses an active chromosome partitioning system for developmentally-regulated genome segregation, which is associated with spore formation. There are four known trans-acting segregation proteins (ParA, ParB, ParJ and Scy) and cis-acting centromere-like sites (parS). parA encodes a Walker-type ATPase that is required for efficient DNA segregation and proper placement of the ParB-parS nucleoprotein complexes. A paralogue of ParA is encoded by the S. coelicolor genome, SCO1772 (named ParH), that has 45% identical residues to ParA. In S. coelicolor aerial hyphae, a ∆parH mutant produces 5% of anucleate spores. In this study, ParH was identified as a novel interaction partner of S. coelicolor ParB. However, a Walker A motif K99E substitution in ParH and removal an N-terminal extension in ParH impaired interaction between ParH and ParB, as judged by bacterial two-hybrid analyses. ParH-EGFP localization resembles the evenly-spaced localization pattern of ParH-EGFP in aerial hyphae, which might suggest that ParH colocalizes with ParB. A parH-null mutant appears to be unable to properly organize the oriC regions within a subset of prespores, as judged by ParB-EGFP foci. In this study, through a random chromosomal library screening, a novel protein that interacts with ParA and ParH was also identified. HaaA (ParH and ParA Associated protein A) is required for proper chromosome segregation and is one of the 24 signature proteins of the Actinomycetes that are not found in other bacterial lineages. A bacterial two-hybrid analysis showed that HaaA interacts with itself and interaction between ParH and ParA was through the C-terminal unstructured region. Interaction between HaaA and ParA and ParA-like proteins was conserved in other Actinomycetes, such as S. venezuelae, C. glutamicum and M. smegmatis. There was no evidence for interaction with other tested segregation proteins. In addition, a haaA insertion-deletion mutant strain revealed that loss of HaaA affected chromosome segregation (6% anucleate spores) and HaaA-EGFP localizes within spores of the mature spore chains. Together these data revealed new information to further understand chromosome segregation in S. coelicolor. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;
| Identifer | oai:union.ndltd.org:DUQUESNE/oai:digital.library.duq.edu:etd/197203 |
| Date | 04 May 2017 |
| Creators | Hasipek, Metis |
| Contributors | Joseph McCormick, Valerie Oke, Jana Vogt, Michael Seaman |
| Source Sets | Duquesne University |
| Detected Language | English |
| Rights | One year embargo: no access to PDF file until release date by author request.; |
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