Characterisation and Regulation of cutF (nlpE) Homologue of Salmonella typhi
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Date
2003-01
Authors
Leong, Lau Kwok
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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.
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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.
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Keywords
cutF (nlpE) Homologue