APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2002, p. 3932–3942 Vol. 68, No. 8 0099-2240/02/$04.000 DOI: 10.1128/AEM.68.8.3932–3942.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. The Peptidyl-Prolyl Isomerase Motif Is Lacking in PmpA, the PrsA-Like Protein Involved in the Secretion Machinery of Lactococcus lactis Sophie Drouault, 1,2 Jamila Anba, 2 Sophie Bonneau, 2 † Alexander Bolotin, 2 S. Dusko Ehrlich, 2 and Pierre Renault 2 * Unite ´ d’Ecologie et de Physiologie du Syste `me Digestif 1 and Unite ´ de Ge ´ne ´tique Microbienne, 2 Institut National de la Recherche Agronomique, 78352 Jouy en Josas Cedex, France Received 16 November 2001/Accepted 16 May 2002 The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degra- dation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity. Lactococcus lactis is a gram-positive bacterium which is clas- sified as “generally regarded as safe” following its long history of use in the production of fermented milk products. The potential for using L. lactis for new applications, such as in secretion of heterologous proteins in fermented food products or in the digestive tract of humans, is currently under active study (3, 27, 49, 50, 52). In a previous work, we expressed the Staphylococcus hyicus lipase in L. lactis in order to use lacto- cocci as a lipase delivery vehicle (13). This goal could alleviate lipase deficiency in the digestive tract during digestion (ste- atorrhea) or improve flavor development in some cheese-mak- ing processes. In this study, we have identified several factors that affect the secretion of the S. hyicus lipase in L. lactis (13). In this heterologous host, about 50% of the lipase is not trans- located through the cytoplasmic membrane and remains bound to the inner side of the membrane as unprocessed preprolipase. Most of the translocated lipase is associated to the cell wall and partially degraded at its N-terminal end (13). Proteolysis is a natural process required not only for protein turnover but also for the degradation of misfolded proteins (15). In the case of the S. hyicus lipase secretion in L. lactis, it was suggested that most of the translocated lipase is not cor- rectly folded and is thus degraded (13). L. lactis has recently been sequenced, allowing the identifi- cation of at least eight genes implicated in protein secretion (6). Furthermore, the membrane protease HtrA, involved in the degradation of hybrid exported proteins, has also been characterized (41). In addition, L. lactis possesses a lipoprotein homologous to PrsA of Bacillus subtilis, encoded by nlp4 (40) or pmpA (6). In B. subtilis, the model organism for protein secretion in gram-positive bacteria, the overproduction of PrsA increases the yield of several heterologous proteins, such as -amylase (32) or antidigoxin single-chain antibody (55). This indicates that PrsA is a limiting factor in the secretion of heterologous proteins. Recently PrsA of B. subtilis has been shown to belong to a family of a class of proteins: the peptidyl cis/trans-isomerases (PPIases) (42, 46). PPIases are enzymes which catalyze the cis/trans isomerization of the peptidyl-pro- line bonds in oligopeptides and proteins. Such an isomeriza- tion could be important for the proper and efficient folding of proteins. The fact that several extracytoplasmic enzymes in- volved in the folding of external proteins, such as PrsA (31, 32), PrtM (carried on a plasmid) (24), SurA (4, 45), and PpiD (9), are members of this family suggests that PPIase activity is required for their activity. The PPIase activity was demon- * Corresponding author. Mailing address: Unite ´ de Ge ´ne ´tique Mi- crobienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas Cedex, France. Phone: 33-1 34 65 24 25 27. Fax: 33-1 34 65 24 25 21. E-mail: renault@jouy.inra.fr. † Present address: Unite ´ de Pathologie Ve ´ge ´tale, Institut National de la Recherche Agronomique, 49071 Beaucouze ´, Cedex, France. 3932