Role of plasmids in Lactobacillus brevis BSO 464 hop tolerance and beer spoilage

Abstract

Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.

FIG 1

Growth of the parent strain (L. brevis BSO 464^OG [LbOG]) and plasmid variants on HGA plates. The hop gradient ranged from 0 to 135 BU. Higher hop tolerance is reflected by a greater growth distance across the gradient of hops. Control plates without hops showed growth across the entire 6-cm length of the plate. n = 5 for all strains; medians and ranges of values are shown.

FIG 2

Growth of L. brevis BSO 464^OG (parent strain), L. brevis BSO 464²⁴⁵, L. brevis BSO 464⁴⁵⁸, L. brevis BSO 464⁴⁵, and L. brevis BSO 464⁵ in degassed beer. Despite the loss of pLb464-1, -3, -6, -7, and -8 from the parent strain in L. brevis BSO 464²⁴⁵, there is maintenance of a short lag phase, albeit with a decrease in the robustness (height) of the growth curve and the length of the stationary phase (i.e., cell death occurs more rapidly). With the loss of pLb464-2, there is a dramatic increase in the lag phase (for L. brevis BSO 464⁴⁵⁸, L. brevis BSO 464⁴⁵, and L. brevis BSO 464⁵). The presence of pLb464-8 results in a shortened lag phase and more robust exponential growth for L. brevis BSO 464⁴⁵⁸ compared to L. brevis BSO 464⁴⁵ (L. brevis BSO 464⁴⁵ exhibits only a 0.5-log-unit increase in growth). L. brevis BSO 464⁵ is not capable of establishing growth in degassed beer but does remain bacteriostatic in beer.