Identification Of A CDF Type Co～(2+)-Ni～(2+) Efflux Pump In Xanthomonas Campestris Pv. Campestris

Heavy metal ions have paradoxical biological effects. On one hand, many heavy metal ions are essential trace elements for almost all living organisms, but on the other hand, when its intracellular concentration exceeds a certain threshold, it forms unspecific complex compounds in the cell, which causes toxic effects to the organism. In consequence, the intracellular level of heavy metal ions must be precisely regulated. In bacteria, the balance between import and export of heavy metal ions, known as heavy metal ion homeostasis, is maintained mainly through the coordinated expression of export and import systems. When the intracellular metal ions exceed a certain concentration, the metal uptake regulator (usually as a repressor) binds to metal ions and represses the expression of import systems, also the metal efflux regulator (usually as an activator) binds to metal ions and activates the expression of export systems. In contrast, when the intracellular metal ions are deficient, the expression of import systems are induced by derepression of the metal uptake repressor, and the export systems are abolished by loss of the activation of the metal efflux activator, due to the release of metal ions from the regulator.Although the molecular mechanism of heavy metal ions homeostasis has been elucidated to some details in other bacteria, little is known about it in Xcc, the causal agent of the black rot disease of cruciferous plants. Genome annotation showed that ORF XC4064 encodes a putative heavy metal transporter. To determine whether XC4064 functions as a heavy metal efflux system, in this study, sensitivity of strain 158E03, a Tn5gusA5 insertion mutant of XC4046, to Zn²⁺,Co²⁺ Cd²⁺,Ni²⁺,Cu²⁺and Mn²⁺ was respectively tested. The results showed that the XC4064 mutant nearly failed to grow in the rich medium NYG supplemented respectively with Co²⁺ and Ni²⁺ to the final concentration of 200μM and 700μM, whereas the wild type strain 8004 and the complemented strain grew well in the same conditions. The growth rate of the XC4064 mutant was identical to that of the wild type strain 8004 in the rich medium supplemented respectively with Zn²⁺,Cd²⁺,Cu²⁺and Mn²⁺ to a serial of final concentrations. The GUS activity assays revealed that the transcription of XC4064 is induced by Co²⁺ and Ni²⁺. Bioinformatics analysis showed that the product of XC4064 is a membrane-bound protein with six transmembrane (TM) regions and a histidine-rich domain, which present a typical structure of a CDF type heavy metal efflux pump. These results collectively supported that the product of XC4064 functions as a CDF type Co²⁺-Ni²⁺ efflux pump.Because another CDF type heavy metal efflux pump czcD1 had been identified in Xcc 8004, we renamed XC4064 as czcD1. czcD1 had been shown to be regulated by the zinc uptake regulator Zur, however, we found that czcD1 is not regulated by Zur. Furthermore, the virulence of czcD1 mutant was identical to that of the wild type strain 8004, indicating that czcD1 is not involved in the pathogenesis of Acc.