Detection and classification of the integrative conjugative elements of Lactococcus lactis

Abstract

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

Keywords: Lactococcus lactis; Comparative genomics; Integrative conjugative element (ICE); Mobile genetic elements.

Fig. 1

Distribution of variations found in the conserved ICE region. A Using the integrase and coupling protein as boundaries we determined the size in base pairs and the amount of ORFs for each of the detected ICEs. B A distribution box plot for all of the conserved regions. C The amount of ORFs plotted against the length (base pairs) with two reference ICE sequences and an example listed

Fig. 2

Conserved region comparison of three ICEs (CV56_1 (Tn5276), KF147_2 (Tn6098) and KF67). Aligned conserved ICE regions of three ICEs of varied size. In dark blue the five conserved ICE functions used for initial detection. The variation in size can be attributed to gene insertion / deletion between the conserved functions. The red bar indicates the cutoff used for the region definition, which was set to encompass all conserved core ICE genes as identified in our comparative analysis (see materials and methods for details)

Fig. 3

Example of the composition for the selected core lactococcal ICE genes (L.ICE_CGs). A In dark blue the 5 core ICE functions used for the initial detection, in red the core L. lactis ICE genes and in light blue the variable genes. B Gene table for KF147_2 and the assigned identifiers. The identifiers are taken from literature nomenclature of MPF_FA type systems, in order to allow future comparisons and easier access. The identifiers describe as followed: tcpA = coupling protein, mobT = relaxase, tcpC and tcpE two MPF associated genes, virB4 = ATPase function, int = integrase. The listed locus tags (first column) refer to the standardized locus tag numbering in the NCBI database, which differ from those in the original genome assembly, to facilitate their correspondence we provide the original locus tags for the first and last gene: LLKF_RS11295 = LLKF_2255, and LLKF_11180 = LLKF_2229

Fig. 4

Example of the phylogenetic trees and clade definitions generated for each of the 17 core L. lactis ICE genes. In this example is the phylogenetic tree of the MobT function and the E-values of each clade specific HMM profile on each entry. A clear distribution and distinction of three clades can be discerned. Furthermore, the E-values are listed for the initial search for the ICE conserved features, illustrating that all lactococcal MobT proteins belong to the PF02486 protein family

Fig. 5

Heat map clustering of all identified L. lactis ICEs. Generated by compiling all the results of the clade definitions for each core L. lactis ICE gene. The colors correspond to different clades, and for exceptions (red for outliers, black for missing and grey for a truncation). By using the most obvious clustering (L.ICE_CG3 to CG6) the ICEs could be divided into three main families. Evidence of inter-family recombination is shown between family two and family three, by the interchange of at least the L.ICE_CG9 to CG10 functions. On the bottom of the heat map it is listed the total amount of clades in each L.ICE_CG function

Fig. 6

Phylogenetic tree and group definitions for VirB4s from both plasmids (P_) and ICEs. Each E-value is listed of each clade specific HMM profile on each entry. The plasmid derived VirB4 cluster definitely apart from the ICE derived VirB4s. Asterisk is the root of ICE derived VirB4 sequences

Fig. 7

Mapping of the detected VirB4s on representative genomes of the Firmicutes. Both the ICE (blue) and plasmid (green) entries of L. lactis are depicted in the bottom. One additional ICE derived VirB4 could be detected, and was found within the Lactobacillales order. Of all the plasmid derived VirB4 entries found outside L. lactis the majority is represented within the Lactobacillales and Clostridiales orders, with only one representative found in the Bacillales order