Genome Analysis of Lactobacillus plantarum LL441 and Genetic Characterisation of the Locus for the Lantibiotic Plantaricin C

Abstract

Bacteriocins are ribosomally synthesized peptides produced by bacteria with antimicrobial activity. The bacteriocins produced by lactic acid bacteria (LAB) may inhibit food-borne pathogens and spoilage organisms, and therefore have potential as natural preservatives. Lactobacillus plantarum LL441 produces a lantibiotic bacteriocin known as plantaricin C, a pore-forming antimicrobial peptide containing modified amino acids that inhibits cell wall synthesis by forming a complex with the peptidoglycan precursor lipid II. The present work describes the genome sequencing of L. plantarum LL441 and the characterisation of the plantaricin C locus. The draft genome sequence of L. plantarum LL441 consisted of 170 contigs and had a total 3,124,603 bp; the GC content was 44.52%. The plantaricin C locus was found in an 18 kbp-long contig, and consisted of six genes organized in an operon-like arrangement. This locus included the bacteriocin structural gene (plnC), followed by a gene encoding a LanM-like protein thought to be involved in the maturation of plantaricin C, and four downstream genes encoding ABC-type transporter components, probably belonging to its putative immunity and export machinery. plnC encodes a precursor of the bacteriocin, i.e., a 58-amino acid peptide containing a 31-amino acid double-glycine leader peptide and a 27-amino acid core peptide. In silico analysis and hybridisation experiments placed the plantaricin C locus to be located on pLL441-1, a large plasmid of L. plantarum LL441. Joining up the gaps between the contigs by conventional PCR, sequencing of the amplicons, and sequence assemblage, allowed the complete 55.3 kbp pLL441-1 molecule to be established. A portion of pLL441-1 larger than 34 kbp, which included the plantaricin C region, was identified in a plasmid-derived contig from the L. plantarum Nizo 3893 genome. Further, the plantaricin C coding locus (about 8.7 kbp) was shown to share 91% nucleotide identity with a portion of the plasmids pPECL-6 from Pediococcus claussenii ATCC BAA-344 and pL11995-4 from Lactobacillus paracollinoides TMW 1.1995. Knowledge of the sequence of the plantaricin C coding region will help in studying its molecular components and allow their involvement in bacteriocin synthesis to be investigated, facilitating the use of the bacteriocin or its genetic elements in new biotechnological applications.

Keywords: Lactobacillus plantarum; adjunct cultures; bacteriocins; genome analysis; genome sequencing; lactic acid bacteria; lantibiotics; plantaricin C.

FIGURE 1

Schematic representation of the plantaricin C locus in Lactobacillus plantarum LL441, in which the orientation and size of the different open reading frames (ORFs) and the proteins they encode are indicated. Faced arrows above the ORFs indicate the presence of inverted repeat sequences resembling ρ-independent terminators. The position of relevant restriction sites in the contig and the segments of DNA homologous to those found in plasmids pPECL-6 from Pediococcus claussenii ATCC BAA-344 and pL11995-4 from Lactobacillus paracollinoides TMW 1.1995 and a plasmid from L. plantarum Nizo 3893 are also indicated.

FIGURE 2

Sequence alignment of the precursor peptides of plantaricin C and those from selected Class Ia type II lantibiotics. Below the alignment, conserved amino acid positions (^∗) and the ELXXBXG motif are indicated.

FIGURE 3

Primary (down) and secondary (up) structures of plantaricin C. Lanthionine (Ala), methyllanthionine (Abu), and Dehydroalanine (Dha) residues are color-coded and indicated. (Modified from Turner et al., 1999).

FIGURE 4

PLACNET reconstruction of the L. plantarum LL441 genome. Contigs are represented by blue nodes, while gray nodes represent reference genomes. The sizes of contig nodes are proportional to the contig length, while those of reference nodes are fixed. The doted red oval embraces contigs containing genes of the core genome. Colored node outlines represent contigs containing plasmid-specific protein genes (yellow, plasmid replication proteins; green, relaxase proteins). The arrow points toward the node of the contig harboring the plantaricin C operon.

FIGURE 5

Gel electrophoresis of chromosomal and plasmid DNA from L. plantarum LL441 (A) and Southern blot results (B) of the gel in A after transferring of the DNA to a membrane and hybridizing with a digoxigenin-labeled probe (Roche) based on an internal 1-kbp fragment of the lanM gene (Figure 1) amplified by PCR. Order of the samples: 1, undigested chromosomal DNA from LL441; 2, 3, and 4, chromosomal DNA digested with PstI, EcoRI, and HindIII, respectively; 5, undigested plasmid DNA from LL441; 6, 7, and 8, plasmid DNA digested with PstI, EcoRI, and HindIII, respectively. A and B, pre-hybridized molecular weight markers: lambda DNA digested with PstI and lambda DNA digested with HindIII, respectively.

FIGURE 6

Genetic organization of plasmid pLL441-1, including position of relevant restriction enzymes and direction and length of genes and ORFs. Key of colors: in red, genes coding for proteins involved in plasmid replication, stability, and segregation; in yellow, ORFs of insertion sequences and integrase-related genes; in green, component genes of the plantaricin C locus; in pale blue, genes involved in conjugation; in pale brown, ORFs for other genes. ORFs overlapping to another by more than 20 amino acids or shorter than 50 amino acids in length were not taken into account.