Genome Sequences of Lactobacillus sp. Strains wkB8 and wkB10, Members of the Firm-5 Clade, from Honey Bee Guts

Abstract

We sequenced two strains from the Lactobacillus Firm-5 clade, a dominant group of symbionts in the guts of honey bees and other social bees. The genome of strain wkB8, comprising a 1.93-Mb chromosome and a 6.4-kb plasmid, was fully closed, while strain wkB10 was assembled into 32 contigs. These genomes will provide insights into how gut symbionts evolve and interact with their host species.

GENOME ANNOUNCEMENT

Honey bees (Apis spp.) possess a highly specialized gut microbial community comprising about 8 bee-specific phylotypes (1). We recently sequenced the genomes of the major Gram-negative constituents, Snodgrassella alvi and Gilliamella apicola (2). Here, we present the genomes of two Lactobacillus strains of the Firm-5 clade, commonly identified as the most numerically abundant phylotype in honey bee guts (3, 4). This clade has also been reported in bumble bees (Bombus spp.) and stingless bees (Meliponini), which are close relatives of honey bees (5–7). Interestingly, host-associated Lactobacillus have not been found in other bee species, suggesting a uniquely coevolved symbiosis exists between Firm-5 and the social corbiculate bees (Apis, Bombus, and Meliponini) (8).

We isolated Firm-5 strains from the guts of the honey bee Apis mellifera as previously described (9), using Columbia agar with 5% sheep’s blood as a growth medium. DNA was extracted using phenol-chloroform and purified on DNeasy spin columns (Qiagen). Total genomic DNA was sequenced on the Illumina MiSeq platform from 2 × 250-bp paired-end libraries, returning 3,046,168 (strain wkB8) and 2,613,160 (strain wkB10) reads. Overlapping reads were combined using FLASH (10). All read types were then assembled with Velvet version 1.2.10 (11), producing a total of 18 and 32 contigs for wkB8 and wkB10, respectively. We successfully closed the wkB8 genome by in silico assembly inspection and combinatorial gap-spanning PCRs, although 45 ambiguous bases remain due to polymorphisms in the multicopy rRNA-encoding regions. Genomes were annotated with the RAST server (12).

The genome of wkB8 comprises a 1,926,135-bp chromosome and a 6,396-bp plasmid. It carries 1,772 predicted CDSs, 57 tRNAs, and 4 rRNA operons, and has a GC content of 36.7%. The wkB10 genome assembly was broken into 32 contigs with an N₅₀ of 165,078, but appeared to represent a single chromosome. Examination of read coverage depth uncovered several contigs representing genomic regions likely present in multiple copies: contig018 (19 copies), contig019 (13 copies), and contig028 (5 copies). Thus, we estimate the wkB10 genome to be between 2.08 and 2.30 Mb in size, with 35.4% GC content. We detected 1,957 CDSs, 55 tRNAs, and at least 4 rRNA operons in wkB10. Both strains possess pathways to ferment various carbohydrates (e.g., fructose, lactose, mannose, N-acetylglucosamine, sorbose, sucrose, trehalose, xylulose) to lactic acid, and both encode a large number of predicted extracellular proteins that may allow adhesion to and degradation of environmental substrates such as chitin (13).

Strains wkB8 and wkB10 are 98.9% identical at their 16S rRNA locus but have only 86.0% average nucleotide identity across orthologous genomic regions. The bee-associated Lactobacillus Firm-5 cluster clearly comprises a diverse, deeply branching group of symbionts. Based on recent efforts to classify this group (14, 15), wkB8 is likely most related to Lactobacillus helsingborgensis, while wkB10 is part of L. kullabergensis or L. kimbladii; however, more genomes are required to unravel the murky relationships within the Firm-5 clade.

Nucleotide sequence accession numbers.

The complete sequence of strain wkB8 has been deposited in GenBank under accession numbers CP009531 and CP009532, and the wkB10 whole-genome shotgun project has been deposited under accession number JRJB00000000.