|
|
|
|
|
|
clpB, a Novel Member of the Listeria monocytogenes CtsR Regulon, Is Involved in Virulence but Not in General Stress Tolerance. Arnaud Chastanet, 2004. Clp-HSP100 ATPases are a widespread family of ubiquitous proteins that occur in both prokaryotes and eukaryotes and play important roles in the folding of newly synthesized proteins and refoldingof aggregated proteins . They have also been shown to participatein the virulence of several pathogens, including Listeria monocytogenes. Here, we describe a member of the Clp-HSP100 family of L . monocytogenes that harbors all the characteristics of the ClpB subclass, which is absent in the closely related gram-positive model organism, Bacillus subtilis. Transcriptional analysis of clpB revealed a heat shock-inducible  A-type
promoter . Potential binding sitesfor the CtsR regulator of stress
response were identified inthe promoter region . In vivo and in vitro
approaches were usedto show that expression of clpB is
repressed by CtsR, a findingindicating that clpB is a novel
member of the L . monocytogenesCtsR regulon . We showed that
ClpB is involved in the pathogenicityof L . monocytogenes
since the
 clpB
mutant is significantly affectedby virulence in a murine model of
infection; we also demonstratethat this effect is apparently not due
to a defect in generalstress resistance . Indeed, ClpB is not
involved in toleranceto heat, salt, detergent, puromycin, or cold
stress, even thoughits synthesis is inducible by heat shock .
However, ClpB wasshown to play a role in induced thermotolerance,
allowing increasedresistance of L . monocytogenes to lethal
temperatures . Thiswork gives the first example of a clpB gene
directly controlledby CtsR and describes the first role for a ClpB
protein in inducedthermotolerance and virulence in a gram-positive
organism.
Evolution of the C30 Carotenoid Synthase CrtM for Function in a C40 Pathway. Daisuke Umeno, 2002.The C30 carotene synthase CrtM from Staphylococcus aureus and the C40 carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C40 and C30 biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function . Each displayed negligible ability to synthesize the natural carotenoid product of the other . After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C40 carotenoids . In contrast, we failed to find a single variant of CrtB with detectable C30 activity . Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C40 pathway . These mutants showed significant variation in performance in their original C30 pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity . We discovered that Phe 26 alone determines the specificity of CrtM . The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
