Unusual genome complexity in Lactobacillus salivarius JCM1046

BMC Genomics Unusual genome complexity in Lactobacillus salivarius JCM1046 Emma J Raftis 0 1 Brian M Forde 0 1 Marcus J Claesson 0 1 Paul W O'Toole 0 1 0 Alimentary Pharmabiotic Centre, University College Cork , Cork , Ireland 1 School of Microbiology University College Cork , Cork , Ireland Background: Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046. Results: JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM. Conclusion: Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications. Lactobacillus salivarius; Megaplasmid; Multireplicon; Linear plasmid; Tn6224; Conjugative transposon - Background Lactobacillus salivarius [1] is a member of the indigenous microbiota of the oral cavity and the gastrointestinal tract (GIT) of both humans and animals [2,3], and has also been isolated from human breast milk [4]. The probiotic and immunomodulatory activity of L. salivarius strains has been recently reviewed [5] and are considered to be strain-specific traits [6]. Strains of L. salivarius are genetically diverse [7] and harbour distinctive multireplicon genomes. The first genome of this species to be published [8,9] was that of the well-characterised strain L. salivarius UCC118 [1,10-13] whose megaplasmid pMP118 (242 kb) encodes genes involved in GI tract survival, fitness and probiotic activity [9-11]. L. salivarius strains from a range of environmental sources harbour diverse circular megaplasmids [7,12]. At least 10 additional L. salivarius genomes have been sequenced since that of strain UCC118; three of these have been completed (strains CECT 5713 [14] NIAS840 [15] and SMXD51 [16]) with two being finished to a draft quality status [17,18]. Unlike circular plasmids, linear plasmids are rarely observed in lactobacilli [12] but often confer advantageous phenotypes to their hosts [19,20] and have been extensively studied in Streptomyces [21,22], Borrelia [23] and Bacillus [24]. Linear phage genomes are also harboured by strains of Escherichia coli [25], Yersinia enterocolitica [26], Klebsiella oxytoca [27] as well as the probiotic cheese strain Lactobacillus paracasei NFBC 338 [28]. Prior to the discovery of linear megaplasmids in L. salivarius [12], a 150 kb linear plasmid was identified in Lactobacillus gasseri CNRZ222 [29]; but no characterization of the plasmid was performed. We previously identified linear megaplasmids in two porcine L. salivarius isolates, JCM1046 and JCM1047, and one human intestinal isolate AH43348 [12]. The conjugative transposon (CTs) Tn916 (18.5 kb) [30] and other Tn916-like elements are highly promiscuous [31], both in the lab and in natural environments [32]. They have demonstrated intra- and interspecies transfer from Lactococcus lactis [33] and Lactobacillus paracasei [34] food strains; and between streptococcal species in dental biofilms [35]. There is a growing concern that commensal bacteria may act as natural reservoirs for antibiotic resistance determinants [36] and may be responsible for transfer of antibiotic resistance to pathogens and opportunistic pathogens [37]. In addition to the introduction of additional functional modules to the host cell, CTs have further potential to influence natural selection within a bacterial population [38]. There is therefore a growing need to characterize these mobile elements, particularly in species used in food or as probiotics. Here we present experimental evidence for a highly unusual genome architecture in L. salivarius JCM1046, a strain that harbours multiple extrachromosomal replicons of varying sizes and topologies and which has an enhanced ability to withstand the stresses associated with GIT survival [11]. The present study describes an unprecedented level of genome complexity in L. salivarius. Results and discussion Discovery of circular and linear extrachromosomal elements in L. salivarius JCM1046 Sequencing revealed that L. salivarius JCM1046 contains five replicons (Table 1): a 1.836 Mb chromosome, two Table 1 General genome features of L. salivarius JCM1046 % of genome size circular megaplasmids of 219 and 129 kb, a linear megaplasmid of 101 kb, and a 33 kb plasmid harbouring an integrated conjugative transposon (Figure 1). The complexity of this genome configuration presented extraordinary challenges for genome assembly, described below. Experimental validation of the genome structure is presented in Figure 2. L. salivarius strains JCM1047 and AH43348 were known to harbour linear megaplasmids that were presumed to be related to pLMP1046 [12] and were therefore included in these experiments. Our original study that identified pMP1046A (then designated pMP1046 [12]) in strain JCM1046 estimated its size as 230 kb, based on Pulsed Field Gel Electrophoresis (PFGE) [12]. However, the assembled sequence data revealed pMP1046A as closer to 220 kb in size. A combination of restriction digestion, PFGE and Southern hybridisation was used to validate the size (...truncated)