CHARACTERIZATION OF TYPE-II PROTEIN SECRETION (XCP) GENES IN THE PLANT-GROWTH STIMULATING PSEUDOMONAS-PUTIDA, STRAIN WCS358

In Pseudomonas aeruginosa, the products of the rep genes are required for the secretion of exoproteins across the outer membrane. Despite st ructural 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 rep ge nes of another fluorescent pseudomonad, the plant growth-promoting Pse udomonas putida strain WCS358, were cloned and characterized. Nucleoti de sequence analysis revealed the presence of at least five genes, i.e ., xcpP, Q, R, S, and T, with homology to rep genes of P. aeruginosa. Unlike the genetic organization in P. aeruginosa, where the rep cluste r consists of two divergently transcribed operons, the rep 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 rea ding frame, with no homolog in P, aeruginosa, was identified, which po ssibly encodes a lipoprotein. Mutational inactivation of rep genes in P. putida did not affect secretion, indicating that no proteins are se creted 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 protei n could be identified as flagellin. Mutations in the xcpQ and R genes of P. aeruginosa could not be complemented by introduction of the corr esponding xcp genes of P, putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third of P. aeruginosa XcpR a nd the C-terminal two-thirds of P, putida XcpR, did restore protein se cretion in a P. aeruginosa xcpR mutant.