Secretome

The L. plantarum pan-genome specifies a total of 317 predicted secreted proteins, of which 91 are absent in the reference strain WCFS1 (Table 10). More than half of the secretome-associated proteins are tentatively plasmid-encoded, whereas 75 of the chromosomally encoded genes appear to be part of the L. plantarum core genome. The secretome proteins have been sorted and categorized into functional categories (Table 11).

More than 50 proteins fall in the cell-wall metabolism category, and the majority of these are highly conserved as they belong to the core functions of L. plantarum. Forty proteins belong to the cell surface complexes (csc) category.Gene clusters encoding csc have been identified in various Gram-positive bacteria. These consist of characteristic cscA, cscB, cscC and cscD family proteins, which are hypothesized to form cell-surface bound complexes of unknown function. L. plantarum WCFS1 was shown to contain 9 csc gene clusters (42). Among the 40 csc proteins, 16 are present in all strains, while the remaining clusters are differently distributed across the strains (Table 10).            

In addition to the two main variable secretome regions described above, the comparative genome analysis allowed to detect new proteins in L. plantarum. L. plantarum is not previously known to encode an extracellular serine proteinase of the subtilisin-like family (also called subtilase), as found in several other LAB (43). This cell-envelope subtilase allows the strain to use proteins and oligopeptides as growth substrates. In the present study, 7 L. plantarum strains (NIZO1837, NIZO2259, NIZO2260, NIZO2806, NIZO3400, NIZO3893 and P8) were found to have a plasmid-encoded, extracellular cell-envelope bound subtilase (OG_3299). These strains are of different origins (Table 10).

There are 23 proteins classified as adhesion proteins, which could be involved in binding or adherence to mucus, collagen, mannose, etc. Most of the adhesion proteins of strain WCFS1 are also present in the other L. plantarum strains, but there are some exceptions. The mannose-specific adhesion protein (OG_2571) appears to be absent in most strains and is present in 12 strains of different origins. Two large mucus-binding proteins (OG_2486, OG_2483) are absent in 14 strains of various origins, but intact in the other strains.

Molenaar et al. (44) suggested that L. plantarum WCFS1 contained a unique genetic region involved in the non-ribosomal peptide biosynthesis (NRPS), which remained undetected in any other L. plantarum strain by CGH. This study principally confirms these results, although the 19.1 strain was found to contain exactly the same cluster as WCFS1 (100% nucleotide sequence identity). The two strains are isolated from different environments but they belong to the same clade, which supports the finding of the same NRPS. This result suggests that the cluster was obtained from the same unknown donor through horizontal gene transfer.

Lantibiotics are a class of ribosomally synthesized peptide antibiotics that are produced by a large number of Gram-positive bacteria (45). So far, only the L. plantarum LL441 strain has been shown to produce a lantibiotic called plantaricin C (46). In this study, L. plantarum strain NIZO3893 of human origin was found to contain a lantibiotic biosynthesis gene cluster (putatively on a 48 kb conjugative plasmid). The encoded proteins of this cluster are highly alike (88-95% identity) to those encoded in lantibiotic gene clusters of Lactobacillus parafarraginis, Pediococcus classenii and Tetragenococcus halophilus.

Thiopeptides are a class of polythiazolyl antibiotics (47). The clinical interest in this protein family was recently renewed since many members show potent activity against various drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP) and vancomycin-resistant enterococci (VRE) (48,49). We found that L. plantarum strain NIZO3892 of human origin contains a gene cluster for the biosynthesis of such thiopeptide, which is flanked by mobile-element elements, suggesting that it has been acquired by horizontal gene transfer. Parts of this cluster have also been found in Streptococcus pneumoniae, Streptococcus equinus and Dyadobacter crusticola, but the gene sequences are highly different from those belonging to NIZO3892 strain.

42.      Siezen R, Boekhorst J, Muscariello L, Molenaar D, Renckens B, Kleerebezem M. Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria. BMC Genomics. 2006;7:126.

43.      Siezen RJ. Multi-domain, cell-envelope proteinases of lactic acid bacteria. Antonie Van Leeuwenhoek. 1999 Jul;76(1-4):139–55.

44.      Molenaar D, Bringel F, Schuren FH, de Vos WM, Siezen RJ, Kleerebezem M. Exploring Lactobacillus plantarum genome diversity by using microarrays. Journal of Bacteriology. 2005 Aug 31;187(17):6119–27.

45.      van Kraaij C, de Vos WM, Siezen RJ, Kuipers OP. Lantibiotics: biosynthesis, mode of action and applications. Nat Prod Rep. 1999 Oct;16(5):575–87.

46.      Delgado S, Mayo B. Development of Lactobacillus plantarum LL441 and its plasmid-cured derivatives in cheese. J Ind Microbiol Biotechnol. 2003 Apr;30(4):216–9.

47.      Bagley MC, Dale JW, Merritt EA, Xiong X. Thiopeptide antibiotics. Chem Rev. 2005 Feb;105(2):685–714.

48.      Haste NM, Thienphrapa W, Tran DN, Loesgen S, Sun P, Nam S-J, et al. Activity of the thiopeptide antibiotic nosiheptide against contemporary strains of methicillin-resistant Staphylococcus aureus. J Antibiot. 2012 Dec;65(12):593–8.

49.       Liao R, Duan L, Lei C, Pan H, Ding Y, Zhang Q, et al. Thiopeptide biosynthesis featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. Chem Biol. 2009 Feb 27;16(2):141–7.