Characterization of type II protein secretion (xcp) genes in the plant growth-stimulating Pseudomonas putida, strain WCS358

In Pseudomonas aeruginosa, the products of the xcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, the xcp genes of another fluorescent pseudomonad, the plant growth-promoting Pseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e., xcpP, Q, R, S, and T, with homology to xcp genes of P. aeruginosa. Unlike the genetic organization in P. aeruginosa, where the xcp cluster consists of two divergently transcribed operons, the xcp genes in P. putida are all oriented in the same direction, and probably comprise a single operon. Upstream of xcpP in P. putida, an additional open reading frame, with no homolog in P. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation of xcp genes in P. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in the xcpQ and R genes of P. aeruginosa could not be complemented by introduction of the corresponding xcp genes of P. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third of P. aeruginosa XcpR and the C-terminal two-thirds of P. putida XcpR, did restore protein secretion in a P. aeruginosa xcpR mutant.