Isolation of a Hop-Sensitive Variant of Lactobacillus lindneri and Identification of Genetic Markers for Beer Spoilage Ability of Lactic Acid Bacteria

Abstract

We have isolated a hop-sensitive variant of the beer spoilage bacterium Lactobacillus lindneri DSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designated horB L and horC L that encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs of horB and horC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those of horA and ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use of horB/horC and horA is able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison of horB and horC homologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution of horB and horC homologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.Beer has been recognized as a beverage with high microbiological stability. Among the beer spoilers, several species of lactic acid bacteria (LAB) are reported to be responsible for approximately 70% of spoilage incidents caused by microorganisms (2, 3). For this reason, species-specific identification methods based on PCR have been widely evaluated for potential applications to microbiological quality control (6,19,41,42,44). Although species-specific PCR tests are rapid and reasonably accurate, there are two problems for applying this approach to the quality control of breweries.One problem is that the species-specific method is unable to distinguish intraspecies differences between beer spoilage strains and nonspoilage strains (9,26,28,35). Hop compounds added to confer bitter flavor are reported to exert an antibacterial effect by acting as proton ionophores and dissipate transmembrane pH gradient, which prevents gram-positive bacteria, including most LAB, from growing in beer (24,25,27,28,40). Hop resistance ability has been known as the distinguishing character of beer spoilage strains of LAB and nonspoilage strains typically exhibit hop resistance ability considerably weaker than that of beer spoilage strains belonging to the same species (1,9,28,34,35). The presence of nonspoilage strains within a beer spoilage species inevitably leads to false-positive results as long ...

Isolation and Identification of Potential Beer-Spoilage Pediococcus inopinatus and Beer-Spoilage Lactobacillus backi Strains Carrying the horA and horC Gene Clusters

“…The strains were subjected to the horA-or horCspecific PCR as described previously 15,17 . The primers for PCR are listed in Table I.…”

“…Although this approach can rapidly determine whether detected isolates belong to the wellknown beer-spoilage species, the species-specific methods are not only unable to determine the intra-species differences in beer-spoilage ability but are also incapable of detecting previously uncharacterized beer-spoilage species. Based on our previous results, that all of the investigated beer-spoilage strains belonging to four beer-spoilage species have either or both of the horA and horC homolog, we have recently suggested that the combined use of horA and horC is useful for evaluating the beer-spoilage ability of LAB in a species-independent manner 15,17 . Therefore P. inopinatus LA20 and the three L. backi strains isolated in this study were examined for the presence or absence of the horA and horC genes.…”

“…In the course of our studies, we identified two hop resistance genes, horA and horC, in L. brevis ABBC45 8,12,14,15,17,18,21 . The horA-and horC-carrying regions found in L. lindneri, L. paracollinoides and P. damnosus are approximately 99% identical with each corresponding region of L. brevis ABBC45 despite their distinct species status, indicating the emergence of beerspoilage LAB by horizontal transfer of the hop resistance genes 17,18,[22][23][24] .…”

Sake and Beer Spoilage Lactic Acid Bacteria - A Review

“…As for species-independent genetic markers, horA and horC have been demonstrated to confer hop resistance on LAB 46,90 , and it has been suggested that these two hop-resistance genes transfer horizontally between LAB, transforming nonspoilage strains into spoilage ones 109 . The horA and horC genes are highly specific genes to beer spoilage LAB at the strain level, and the presence or absence of these genetic markers was shown to highly correlate with the beer spoilage ability of LAB strains 36,92,107 . In fact, it was proposed that the horizontal acquisitions of hop resistance genes have been implicated in the emergence and spread of beer spoilage LAB through the history of beer brewing 102 .…”

Effects of Lysozyme on the Microbiological Stability and Organoleptic Properties of Unpasteurized Beer

“…Among them the most important spoilage organisms are, according to brewing literature, L. brevis, L. lindneri, L. paracollinoides and P. damnosus 2,30,32,34 . In addition, L. backi and P. inopinatus have been reported as potential beer spoilers 7,22,33 .…”