Characterisation and Regulation of cutF (nlpE) Homologue of Salmonella typhi

dc.contributor.author	Leong, Lau Kwok
dc.date.accessioned	2018-08-02T07:24:19Z
dc.date.available	2018-08-02T07:24:19Z
dc.date.issued	2003-01
dc.description.abstract	Studies on the cutF (nlpE) gene from E. coli or other bacteria were very limited. To date, its :>unction still remained unclear. One study indicated that it is probably involved in tolerance to copper, while another hypothesis postulated that it plays an important role in suppression of extracytoplasmic toxicity. The objective of this research was to clone the cutF (nlpE) homologue from S. typhi for characterisation and gene regulation studies. A genomic library of S. typhi was constructed in A.200 1 and screening of the library with a specific DNA probe resulted in the s·uccessful cloning of the cutF (nlpE) homologue in a 6.3 kb Sall-Hindiii fragment. Analysis of the sequence data showed that the order of the S. typhi cutF (nlpE) homologue and its neighbouring ORFs (!deC, mesJ, yaeQ) is the same as in E. coli K-12. However, these four ORFs in S. typhi are flanked by two IS200 insertion sequences, an arrangement resembling a genomic island, which is not found in E. coli. The S. typhi cutF (nlpE) is very similar to its homologue in E. coli with respect to high values of identity and similanty, size, ribosome-binding-site (RBS) sequence, and distance separating the RBS from the putative initiation codon. Although the similarity value between the two homologues is 82%, S. typhi CutF(NlpE) does not contain the motifM-X-X-M-X-X-X-X-M-X-X-XX- M, a metal-binding site that exists in E. coli CutF (NlpE). This raised doubt concerning its function in copper homeostasis. Subsequently, an S. typhi cutF (nlpE) insertion mutant was constructed to study its gene regulation. It involved firstly, the construction of a new vector suitable for fusion of cutF (nlpE) with the E. coli lacZ gene (which encodes p-galactosidase and serves as reporter), and secondly, the development of a genetic system for chromosomal mutagenesis and allele replacement in S. typhi. xviii Characterisation of the 11cutF(nlpE)::lacZ-aph mutant thus constructed showed that, as in E. coli cutF (nlpE) mutant, cutF (nlpE) mutation in S. typhi did not alter the permeability properties of the cells. There was also no difference in the sensitivity of the mutant to copper sulphate compared to wild-type S. typhi. It thus reinforced the suspicion that cutF (nlpE) may not function in copper homeostasis. Subsequent induction tests with various factors showed that high concentrations of heavy metal ions (such as copper, cobalt, and silver), high osmolarity, and iron deficiency which suppressed normal growth were able to cause an increased transcription of cutF (nlpE). These observations indicated that cutF (nlpE) S. typhi probably constitutes part of a stress regulon involved in sensing of extracytoplasmic stresses.	en_US
dc.identifier.uri	http://hdl.handle.net/123456789/6141
dc.subject	cutF (nlpE) Homologue	en_US
dc.title	Characterisation and Regulation of cutF (nlpE) Homologue of Salmonella typhi	en_US
dc.type	Thesis	en_US