High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn 1546 Transposon

Abstract

Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA^2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA^2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA^2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA^2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain.

Keywords: Tn1546 transposon; genome analysis; heat resistance; spoVA operon; spores.

Figure 1

(A) Overview of the Tn1546 transposons found in B. subtilis B4146, B. amyloliquefaciens B425 and DSM7, and B. licheniformis B4090, B4092, and B4090. The predicted gene functions are indicated for the transposon of B. subtilis B4146: a transposase gene (tnpA), a resolvase gene (res), an operon of N-acetylmuramoyl-L-alanine amidase, gerKA and ger(X)C (Operon 1), an operon of a gene with unknown function and a manganese catalase (Operon2), an operon of two genes with unknown functions, spoVAC, spoVAD, spoVAEb and two genes with unknown functions (Operon 3, spoVA^2mob), a fragmented yetF gene (Gene 4), and a cardiolipin synthase gene (Gene 5). The transposons in B. amyloliquefaciens are smaller, probably due to site specific recombination events, whereby operon 1 and operon 2 were lost. In B. licheniformis B4092 and B4094 the resolvase gene was not present, which is most likely a result of gene loss. (B) Maximum likelihood phylogenetic tree based on core genome of single genes of 15 strains of the B. subtilis group. The presence and copy number of the Tn1546 transposon was indicated behind the corresponding strains. B. subtilis B4146, B. licheniformis B4090, B4092, and B4094 carry a single transposon. The B. amyloliquefaciens strain DSM7 carries three copies of the transposon, while for strain B425 the copy number could not be determined.

Figure 2

(A) Overview of the spoVA^2mob operon, as initially found in B. subtilis strain B4146. The spoVA^2mob operon has a predicted sigma G binding site upstream of the first gene. The operon consists of seven genes: a gene of unknown function with a predicted DUF1657 domain, a gene of unknown function with a predicted YhcN/YlaJ domain, spoVAC, spoVAD, spoVAEb, a gene of unknown function with a predicted DUF1657 domain, and a gene of unknown function with a predicted DUF421 and a DUF1657 domain. (B) Spore counts after heating at 80°C for 10 min and at 100°C for 60 min for strains of B. subtilis 168, B. subtilis 168 amyE::spoVA^2mob [B4067, data from Berendsen et al. (2016a)], B. subtilis 168 amyE::spoVA^2mob (B4090), and B. subtilis 168 amyE::spoVA^2mob (DSM7). The downward arrow indicates that the spores were inactivated below detection limit.