Draft genome comparison of representatives of the three dominant genotype groups of dairy Bacillus licheniformis strains

Abstract

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.

FIG 1

Whole-genome comparison of B. licheniformis strains ATCC 14580, G-1, F2-1, and F1-1 using Mauve, version 2.3.1. The pairwise alignment of draft genomes is shown. The similarly colored blocks are presumably homologous (and internally free of rearrangements) among genomes. White areas within blocks indicate sequences which were not aligned to other genomes and represent nonhomologous regions.

FIG 2

Evolutionary relationship between B. licheniformis strains using the MLST database. An unrooted phylogenetic tree of B. licheniformis strains was based on concatenated sequences of six housekeeping genes in the absence (top) and presence (bottom) of B. subtilis 168 using the neighbor-joining method. It can be inferred that the lineage of strain G-1 separated from the lineage of other strains earlier, but, as expected, it is still very close to other B. licheniformis strains when a B. subtilis 168 outgroup is included.

FIG 3

Minimum spanning tree showing the position of the nodes representing the 27 sequence types of B. licheniformis and location of strains G-1, F1-1, and F2-1. This tree was constructed using PHYLOViZ with the sequences of six housekeeping genes (adk, ccpA, recF, rpoB, spo0A, and sucC) from all of the 27 sequence types of B. licheniformis present in the MLST database (http://www.pubmlst.org) and those from strains G-1, F1-1, and F2-1. The tree shows that strains G-1, F1-1, and F2-1 are different sequence types individually and that groups F and G fall in different clusters.

FIG 4

Group G isolates, but not group F1 or F2 isolates, possess antimicrobial activity against M. luteus. Using a disk diffusion assay, the zone of inhibition of M. luteus ATCC 10240 generated from 50 μl of supernatant of B. licheniformis is expressed as the total diameter of the clearing zone minus the diameter of the filter paper disk (8 mm).

FIG 5

Variable resistance to erythromycin of different groups of B. licheniformis isolates. Using a disk diffusion assay, the erythromycin sensitivity of different B. licheniformis isolates is expressed as the total diameter of the clearing zone minus the diameter of the filter paper disk (8 mm).

FIG 6

RAPD profiles of B. licheniformis strains G-1, F2-1, and F1-1. The RAPD bands A to F were successfully cloned and sequenced.