Role of the Tat Transport System in Nitrous Oxide Reductase Translocation and Cytochrome cd ₁ Biosynthesis in Pseudomonas stutzeri

Abstract

By transforming N 2 O to N 2 , the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N 2 O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center Cu A rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N 2 O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N 2 O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd 1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D 1 macrocycle. The NirD protein as part of a complex required for heme D 1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

“…The highest level of NosZ was found with NosRa, including a small amount of pre-NosZ that was indicative of saturation of the Tat translocase (Fig. 3A) (18). The complemented systems with the truncated proteins NosRb and NosRc also overexpressed NosZ compared to the wild type but had less enzyme than the NosRa strain.…”

“…(46). Since NosZ biosynthesis depends on auxiliary gene products that affect its expression level and protein translocation efficiency (8,13,18,47), we cloned the entire nos cluster, including the tatE gene for protein transport, into pUCP22, which resulted in the nosR expression vector pPRE-3 (Fig. 1A).…”

“…Plasmid pPRE-3 was constructed by subcloning the complete nos gene cluster of pUCP22RE as an 8.8-kb EcoRI-XbaI fragment into pUCP22 that had been digested with SmaI (18). The orientation of the nos genes was inverted with respect to the lacZ promoter.…”

“…The plasmids pUC18 (Ap r ) (48) and pUCP22 (Ap r Gm r ) (46) were used as general cloning vectors. The plasmid pUCP22RZ was derived from pUCP22 and carries nosRZ within a 5.3-kb Eco47III-SmaI fragment (47); the plasmid pUCP22RE is another pUCP22 derivative which carries the entire nosRZDFYLtatE sequence within an 8.8-kb Eco47III-XbaI fragment (18). E. coli was grown in Luria-Bertani medium at 37°C.…”

“…Standard protocols were used for plasmid preparation and purification, agarose gel electrophoresis, dephosphorylation, and ligation of DNA (18). Restriction enzymes were used as recommended by the manufacturer (Fermentas).…”