User:Ben Pascoe

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 * Microbiology & Infection
 * Institute of Life Science
 * School of Medicine
 * Swansea University
 * Singleton Park
 * Swansea
 * SA2 8PP

I work in the Medical Microbiology and Infectious Disease research group led by Professor Mack at the University of Swansea.

Education

 * 2004-09: DPhil at the University of Sussex
 * 2000-03: BSc (Hons) at the University of Hertfordshire

Resuscitation Factor Proteins
Currently investigating the role of Resuscitation Protein Factors in recovering Staphylococcus aureus cells from 'dormant'/VBNC state and their possible role in increasing bacterial susceptibility to antibiotics.

Lab book

Functionl analysis of Zur, the zinc uptake regulator of Streptomyces coelicolor A3(2)
[Thesis Abstract]

Zinc is an essential metal that plays a structural or catalytic role in many cellular proteins. In response to zinc limitation, Streptomyces coelicolor produces a high affinity zinc uptake system (ZnuACB). Disruption of this system results in a ΔznuACB-zur mutant growing poorly in the absence of supplemental zinc, implying that the ZnuABC system is a key zinc uptake system for S. coelicolor. The Δzur mutant exhibited a sporulation defect, and appeared to inhibit sporulation of ΔznuA mutants, suggesting there might be cross-talk between the strains. Several important features are presented relating the structure of Zur to its function as a zinc-sensing regulatory protein and help differentiate Zur from the structurally similar iron uptake regulator, Fur. Zur was purified and the interaction between Zur and the promoter region of znuA investigated using electromobility shift assays (EMSA) and DNase I footprinting. S1 nuclease digestion assays were used to investigate the role of Zur in controlling expression of znuA.

In S. coelicolor, the major demand for zinc comes from the ribosome, with at least seven ribosomal (r-) proteins requiring structural zinc. Thus, a further adaptive response is the production of seven alternative zinc-free ribosomal proteins to replace the primary zinc-binding paralogues. The genome encodes paralogous versions of S14, S18, L28, L31, L32, L33, and L36, which are designated C- or C+ on the basis of the presence or absence of putative cysteine ligands. The zinc-responsive repressor Zur controls the expression of six C- r-protein genes. Surprisingly, two C- genes are instead controlled by the disulphide stress response sigma factor σR, and the activity of other σR-controlled promoters increase during zinc limitation. The incorporation of four C- r-proteins into the ribosome was confirmed using a proteomic approach. The RPC- cluster, containing five of the C- r-protein paralogues, was found to be common in the Actinobacteria and its evolution was used to speculate on a possible selective advantage for possessing paralogous forms of the r-proteins.

Global expression analysis identified 53 genes from 41 transcription units that were up-regulated more than 1.5x times in Δzur, including those of the ZnuABC system and the Zur-controlled C- r-proteins. A large non-ribosomal synthase cluster that encodes a putative secreted zinc siderophore (coelibactin) was also induced and differential expression confirmed by qRT-PCR. Upstream binding of Zur was shown by EMSAs suggesting it has a regulatory input in controlling coelibactin expression and possible involvement in zinc uptake. It may also be responsible for the crosstalk observed between zinc mutants. Members of the Zur regulon are proposed and the Zur binding operator sequence is defined.

Publications

 * 1) Paper1 pmid=17400736
 * 2) Paper2 pmid=19915027
 * 3) Thesis Functionl analysis of Zur, the zinc uptake regulator of Streptomyces coelicolor A3(2) DPhil thesis, University of Sussex, 2009