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 ...
Aims: To determine whether horC confers beer‐spoilage ability and to evaluate the validity of horC as a trans‐species genetic marker for differentiating the beer‐spoilage ability of lactic acid bacteria (LAB). Methods and Results: Hop‐sensitive Lactobacillus brevis ABBC45cc was transformed with an expression plasmid, pHYchorBC, containing putative multidrug resistance gene horC and its putative regulator horB, and the transformant was designated as ABBC45cc/pHYchorBC. As a control, ABBC45cc was transformed with pHYchorB that contains horB, and the transformed strain was designated as ABBC45cc/pHYchorB. As a result of beer‐spoilage assay of these transformants, ABBC45cc/pHYchorBC exhibited beer‐spoilage ability, whereas ABBC45cc/pHYchorB did not. Furthermore ABBC45cc/pHYchorBC showed higher hop resistance than ABBC45cc/pHYchorB, accounting for the differences in beer‐spoilage ability observed between the two transformants. ABBC45cc/pHYchorBC also exhibited higher resistance to various structurally unrelated drugs, compared with ABBC45cc/pHYchorB. Conclusions: horC was shown to confer hop resistance and beer‐spoilage ability on ABBC45cc by presumably encoding a multidrug transporter. Significance and Impact of the Study: The finding that horC plays an important role in hop resistance and beer‐spoilage ability supports the validity of horC as a trans‐species genetic marker for differentiating the beer‐spoilage ability of LAB.
Abstract. We aim to test the feasibility of using near-infrared spectroscopy (NIRS) for indirect measurement of human saliva secretion in response to taste stimuli for potential application to organoleptic testing. We use an NIRS system to measure extracranial hemodynamics (Hb-signals around the temples) of healthy participants when taste stimuli are taken in their mouths. First, the Hb-signals and volume of expelled saliva (stimulated by distilled-water or sucrose-solution intake) are simultaneously measured and large Hb-signal changes in response to the taste stimuli (Hb-responses) are found. Statistical analysis show that both the Hb response and saliva volume are larger for the sucrose solution than for the distilled water with a significant correlation between them (r = 0.81). The effects of swallowing on the Hb-signals are investigated. Similar Hb responses, differing from the sucrose solution and distilled water, are obtained even though the participants swallow the mouth contents. Finally, functional magnetic resonance imaging is used to identify possible sources of the Hb signals corresponding to salivation. Statistical analysis indicates similar responses in the extracranial regions, mainly around the middle meningeal artery. In conclusion, the identified correlation between extracranial hemodynamics and the saliva volume suggests that NIRS is applicable to the measurement of hemodynamic signals accompanying stimulated saliva secretion. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).
J. Inst. Brew. 110(4), 276-283, 2004The horA homologues and adjacent DNA regions identified in beer-spoilage Lactobacillus lindneri DSM 20690 T and L. paracollinoides DSM15502 T were examined and compared with the corresponding DNA region of beer-spoilage L. brevis ABBC45, a strain in which the hop-resistance gene horA was originally identified. The PCR analysis suggests ORFB1-B5 regions surrounding horA are conserved in all of the strains. The nucleotide sequence comparison of the conserved DNA regions revealed extremely high levels of identities among the three beer-spoilage strains that are not typical for distinct species. The PCR methods using primers specific to the adjacent ORFs were found to be able to differentiate beer-spoilage Lactobacillus strains from non-spoilers, indicating these ORFs are also useful genetic markers for determining the beer-spoilage ability of lactobacilli. The presence or absence of the adjacent ORFs in 92 bacterial strains was completely identical with that of horA homologues, indicating the ORFB1-B5 regions are generally conserved in beer-spoilage Lactobacillus strains. Taken together, these results suggest the ORFB1-B5 regions have been acquired by beerspoilage lactobacilli through horizontal gene transfer and provide a theoretical basis for applying a trans-species genetic marker such as horA to deal with unencountered species of beerspoilage lactobacilli.
Aims:To conduct an inter-species comparative study on the nucleotide sequences of the conserved DNA regions surrounding ORF5, a genetic marker for differentiating beer-spoilage lactic acid bacteria. Methods and Results: The conserved DNA regions surrounding ORF5 were examined by PCR analysis, using three beer-spoilage strains, Lactobacillus brevis ABBC45 C , L. paracollinoides LA2 T and Pediococcus damnosus ABBC478. As a result, the DNA regions containing ORF1-7, originally found in ABBC45 C , appeared to be conserved among the three strains, while the downstream region was not found in L. paracollinoides LA2 T and P. damnosus ABBC478. The sequencing analysis of the conserved DNA regions of LA2 T and ABBC478 revealed ca 99% nucleotide sequence identities with that of ABBC45 C . Conclusions:The nucleotide sequences of the ca 8AE2 kb DNA regions containing ORF1-7 were virtually identical among the three strains belonging to different species. The internal organizations of the ORFs were found to be remarkably similar. Significance and Impact of the Study: The level of nucleotide sequence identities suggests the DNA regions surrounding ORF5 were horizontally acquired by these beer-spoilage strains belonging to the three different species of lactic acid bacteria.
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