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Identification of CpxR as a Positive Regulator of icm and dot Virulence Genes of Legionella pneumophila. Ohad Gal-Mor, 2003.To date, 24 Legionella pneumophila genes (icm and dot genes) have been shown to be required for intercellular growth and host cell killing . A previous report indicated that the regulation of these genes is complicated and probably involves several regulatory proteins . In this study, a genetic screen performed in Escherichia coli identified the CpxR response regulator as an activator of the L . pneumophila icmR gene . Construction of an L . pneumophila cpxR insertion mutant showed that the expression of icmR is regulated by CpxR . In addition, a conserved CpxR binding site (GTAAA) was identified in the icmR regulatory region and L . pneumophila His-tagged CpxR protein was shown to bind to the icmR regulatory region using a mobility shift assay . Besides its dramatic effect on the icmR level of expression, the CpxR regulator was also found to affect the expression of the icmV-dotA and icmW-icmX operons, but to a lesser extent . The role of CpxA, the cognate sensor kinase of CpxR, was also examined and its effect on the icmR level of expression was found to be less pronounced than the effect of CpxR . The RpoE sigma factor, which was shown to coregulate genes together with CpxR, was examined as well, but it did not influence icm and dot gene expression . In addition, when the cpxR mutant strain, in which the expression of the icmR gene was dramatically reduced, and the cpxA and rpoE mutant strains were examined for their ability to grow inside Acanthamoeba castellanii and HL-60-derived human macrophages, no intracellular growth defect was observed . This study presents the first evidence for a direct regulator (CpxR) of an icm-dot virulence gene (icmR) . The CpxR regulator together with other regulatory factors probably concerts with the expression of icm and dot genes to result in successful infection . Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites. Frédéric H. Vaillancourt, 2003.Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp . strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB) . To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader . In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer . Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB  DHB
4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB . It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB . Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB
6-Cl DHB
3'-Cl DHB > DHB > 2'-Cl DHB
4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB) . Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB . The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III . As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2 . For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively . DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 ± 0.004 and 0.069 ± 0.002 s-1, respectively) . However, these isozymes had remarkably different apparent Km values for this compound (0.007 ± 0.001, 0.14 ± 0.01, and 3.9 ± 0.4 µM for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively) . The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R . globerulus P6 .
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