Secretion of nucleoside diphosphate kinase by mucoid Pseudomonas aeruginosa 8821: Involvement of a carboxy-terminal motif in secretion

Nucleoside diphosphate kinase (Ndk) is a ubiquitous enzyme which functions in balancing the nucleotide pool of the cell. We have recently reported tha t in addition to being intracellular in both mucoid and nonmucoid Pseudomon as aeruginosa, Ndk is also secreted into the extracellular environment by m ucoid P, aeruginosa cells. This secreted Ndk has biochemical activity simil ar to the intracellular Ndk and is 16 kDa in size. To demonstrate that Ndk is indeed secreted and to localize the secretion motif, we constructed an n nk knockout mutant, which lacks both intracellular and extracellular forms of Ndk, In this study, we report the construction of deletion derivatives m ade from the carboxy-terminal region of Ndk, These deletion derivatives wer e introduced into the ndk::Cm knockout mutant and were examined for the int racellular and extracellular presence of Ndk, It was observed that the carb oxy-terminal 8-amino-acid region is required for the secretion of Ndk into the extracellular region. This region has the sequence DXXX, where X is a p redominantly hydrophobic residue. Such sequences represent a conserved moti f in proteins secreted by the type I secretory pathway in gram-negative mic roorganisms, To investigate the significance of this motif in the secretion of Ndk, we constructed a fusion protein of Ndk and the blue fluorescent pr otein (BFP) as well as a fusion protein of mutated Ndk (whose DTEV motif ha s been changed to AAAA) and the BFP, The presence of extracellular Ndk was detected only in the ndk::Cm knockout mutant harboring the wild-type BFP-Nd k protein fusion, We could not detect the presence of extracellular Ndk in the ndk::Cm knockout mutant containing the mutated BBP-Ndk protein fusion, In addition, we have also used immunofluorescence microscopy to localize th e wild-type and mutated BFP-Ndk proteins in the cell. The significance of t hese observations is discussed.