The peptidyl-prolyl isomerase motif is lacking in PmpA, the PrsA-like protein involved in the secretion machinery of Lactococcus lactis

Abstract

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 degradation 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.

FIG. 1.

Comparative organization of different prsA-like genes. Arrows represent the open reading frames. Arrows with a common color or motif encode homologous products in the different genomes. Those discussed in the text are the following: plp (prsA-like protein), pepF (peptidase F), CAM (similar to a caffeoyl-CoA O-methyltransferase), alaRS (alanyl-tRNA synthetase), HIT (histidine triad proteins), speB (exotoxin SpeB), tpt (fibrial tuft organization from S. cristatus), plasmid-borne prtP (cell wall protease) and prtM (PtrP maturase), cbf1 and yhaM (similar to CMP-binding factor), spoVT (stage V sporulation protein T from B. subtilis), and mfd (transcription repair coupling factor mfd from B. subtilis). The double arrows indicate the conserved promoter region of plp2 and plp4 from C. acetobutylicum and C. difficile, respectively.

FIG. 2.

Multiple alignment of several PrsA-like proteins and PinA (A), more extensive alignment of PPIase domain of PLP with representative proteins belonging to the PPIase family (B), and phylogenetic dendrogram indicating the percentage of identity between the PLP of gram-positive bacteria (C). Protein sequences were aligned by using the PILEUP program (GCG). The alignments were edited manually and colored by Boxshade. Identical residues and conservative changes in more that half of the aligned sequences are on dark and light gray backgrounds, respectively. The boxed region contains the PPIase signature motif. Residues diverging from the PPIase signature are in white on a black background. The secondary structure of these proteins was predicted by the GORIV program (17). Alpha helix (gray arrows) and beta sheet (arrows with wave pattern) structures were indicated when predicted in more than 16 sequences at each position. The predicted structures fit with the known structure of the PPIase part of the human Pin1 protein is shown, for which the names of the domains are indicated under the arrows (α1 to α4 and β1 to β4) (6). The dendrogram was obtained by the successive use of the pileup, distances, and growtree programs of the GCG package. B_sub, B. subtilis; C_jej, C. jejuni; C_ace, C. acetobutylicum; C_dif, C. difficile; E_fae, E. faecalis; E_col, E. coli; L_lac, L. lactis; S_aur, S. aureus; S_mut, S. mutans; S_pne, S. pneumoniae; S_pyo, S. pyogenes.

FIG. 2.

Multiple alignment of several PrsA-like proteins and PinA (A), more extensive alignment of PPIase domain of PLP with representative proteins belonging to the PPIase family (B), and phylogenetic dendrogram indicating the percentage of identity between the PLP of gram-positive bacteria (C). Protein sequences were aligned by using the PILEUP program (GCG). The alignments were edited manually and colored by Boxshade. Identical residues and conservative changes in more that half of the aligned sequences are on dark and light gray backgrounds, respectively. The boxed region contains the PPIase signature motif. Residues diverging from the PPIase signature are in white on a black background. The secondary structure of these proteins was predicted by the GORIV program (17). Alpha helix (gray arrows) and beta sheet (arrows with wave pattern) structures were indicated when predicted in more than 16 sequences at each position. The predicted structures fit with the known structure of the PPIase part of the human Pin1 protein is shown, for which the names of the domains are indicated under the arrows (α1 to α4 and β1 to β4) (6). The dendrogram was obtained by the successive use of the pileup, distances, and growtree programs of the GCG package. B_sub, B. subtilis; C_jej, C. jejuni; C_ace, C. acetobutylicum; C_dif, C. difficile; E_fae, E. faecalis; E_col, E. coli; L_lac, L. lactis; S_aur, S. aureus; S_mut, S. mutans; S_pne, S. pneumoniae; S_pyo, S. pyogenes.

FIG. 3.

SDS-polyacrylamide gel electrophoresis and fluorography of total cell extracts radiolabeled with [³H]palmitic acid. The loaded samples correspond to 0.1 U (0.1 U: quantity of cells in 100 μl of L. lactis culture at an optical density at 600 nm of 1). (A) Overproduction of PmpA in L. lactis. Lanes 1 and 2, NZ9000 (control strain without pJIM2080) noninduced and induced by nisin, respectively; lanes 3 and 4, MG-P_nis::pmpA (NZ9000 with pJIM2080) noninduced and induced by nisin, respectively. (B) Visualization of pmpA mutation. Lane 1, MG1363; lane 2, MG-pmpA.

FIG. 4.

Western blot using anti-CtermLIP serum of total cell extract (C) (0.1 U) and supernatant (S) (0.5 U) of the control strain, MG-P_nis-Lip⁺, the strain overproducing PmpA with the lipase, MG-P_nis::pmpA-Lip⁺, and the pmpA mutant, MG-pmpA. The addition of the nisin inducer in the growing medium is indicated by -nis or +nis. See the legend to Fig. 3 for an explanation of U.

FIG. 5.

Growth of MG1363 (filled squares), MG-pmpA (open squares), MG-Lip⁺ (filled circles), and MG-pmpA-Lip⁺(open circles) at 30°C in CDM (A) and in CDM with NaCl 0.25 M (B).

FIG. 6.

Measure of the lux activity from the pmpA promoter in the strain MG-P_pmpA::lux grown in CDM with Casitone.