Genome sequencing and analysis of the first complete genome ofLactobacillus kunkeeistrain MP2, anApis melliferagut isolate

Abstract

Background. The honey bee (Apis mellifera) is the most important pollinator in agriculture worldwide. However, the number of honey bees has fallen significantly since 2006, becoming a huge ecological problem nowadays. The principal cause is CCD, or Colony Collapse Disorder, characterized by the seemingly spontaneous abandonment of hives by their workers. One of the characteristics of CCD in honey bees is the alteration of the bacterial communities in their gastrointestinal tract, mainly due to the decrease of Firmicutes populations, such as the Lactobacilli. At this time, the causes of these alterations remain unknown. We recently isolated a strain of Lactobacillus kunkeei (L. kunkeei strain MP2) from the gut of Chilean honey bees. L. kunkeei, is one of the most commonly isolated bacterium from the honey bee gut and is highly versatile in different ecological niches. In this study, we aimed to elucidate in detail, the L. kunkeei genetic background and perform a comparative genome analysis with other Lactobacillus species.Methods. L. kunkeei MP2 was originally isolated from the guts of Chilean A. mellifera individuals. Genome sequencing was done using Pacific Biosciences single-molecule real-time sequencing technology. De novo assembly was performed using Celera assembler. The genome was annotated using Prokka, and functional information was added using the EggNOG 3.1 database. In addition, genomic islands were predicted using IslandViewer, and pro-phage sequences using PHAST. Comparisons between L. kunkeei MP2 with other L. kunkeei, and Lactobacillus strains were done using Roary.Results. The complete genome of L. kunkeei MP2 comprises one circular chromosome of 1,614,522 nt. with a GC content of 36,9%. Pangenome analysis with 16 L. kunkeei strains, identified 113 unique genes, most of them related to phage insertions. A large and unique region of L. kunkeei MP2 genome contains several genes that encode for phage structural protein and replication components. Comparative analysis of MP2 with other Lactobacillus species, identified several unique genes of L. kunkeei MP2 related with metabolism, biofilm generation, survival under stress conditions, and mobile genetic elements (MGEs).Discussion. The presence of multiple mobile genetic elements, including phage sequences, suggest a high degree of genetic variability in L. kunkeei. Its versatility and ability to survive in different ecological niches (bee guts, flowers, fruits among others) could be given by its genetic capacity to change and adapt to different environments. L. kunkeei could be a new source of Lactobacillus with beneficial properties. Indeed, L. kunkeei MP2 could play an important role in honey bee nutrition through the synthesis of components as isoprenoids.

Metabolism of Fructophilic Lactic Acid Bacteria Isolated from the Apis mellifera L. Bee Gut: Phenolic Acids as External Electron Acceptors

“…Apiaries located in Mottola and Valenzano showed the highest diversity at both the species and strain levels, although a relative high diversity at the strain level was found among all the five geographical locations, as shown by RAPD-PCR and phenotypic microarray analyses. Consistent with this finding, a polymorphism at strain level has been previously described in L. kunkeei and F. fructosus (53)(54)(55). Representative FLAB strains of each cluster group obtained by combined random amplification of polymorphic DNA patterns were phenotypically characterized using the OmniLog phenotype microarray (PM) Technology (Biolog).…”

Alterations in honey bee gut microorganisms caused by Nosema spp. and pest control methods

“…Six of the 10 isolated Lactobacillus species identified in the study had been previously reported as honey bee endophytes including L. plantarum , L. buchneri , L. gasseri , L. fermentum , L. pentosaceus and L. sanfranciscensis . These Lactobacillus species are linked with bee health due to their antimicrobial properties, high lactic acid yield and biofilm formation, among others …”

“…As expected, oxytetracycline was one of the most inhibitory compounds in vitro , confirming the results of Vásquez et al ., who reported high sensitivity to antibiotics in Lactobacillus isolated from bees. In tolerant strains, this would be related to antibiotic resistance genes, as seen in L. sanfranciscensis , which is closely related genetically to L. kunkeei in which inhibition of growth was not significant. Thymol is well known for its antimicrobial properties .…”

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“…While initial genome sequencing strategies embraced the traditional Sanger sequencing methods 25 , more advanced sequencing technologies that are collectively referred to as the Next Generation Sequencing (NGS) technologies have been eventually adopted 26,[33][34][35] . The genome sequencing of probiotic species until or before 2010 was accomplished by the traditional Sanger sequencing method and shotgun sequencing technique.…”

“…lactis AD011 25,27 . Post 2010, NGS technologies have been adopted for genome sequencing with the prominent ones being 454 pyrosequencing technology, Illumina/ Solexa paired end sequencing technology, Ion Torrent sequencing technology, and Pacific BioSciences sequencing technology 26,[33][34][35] .…”