Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

Isolation of Tannin-Degrading Lactobacilli from Humans and Fermented Foods

Lactobacilli with tannase activity were isolated from human feces and fermented foods. A PCR-based taxonomic assay revealed that the isolates belong to Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Additional studies on a range of Lactobacillus species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species.     

Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.     

Extent of Genetic Lesions of the Arginine and Pyrimidine Biosynthetic Pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: Prevalence of CO2-Dependent Auxotrophs and Characterization of Deficient arg Genes in L. plantarum

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO₂. We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO₂ requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.     

Genome Sequence of Lactobacillus pentosus KCA1: Vaginal Isolate from a Healthy Premenopausal Woman

The vaginal microbiota, in particular Lactobacillus species, play an important role in female health through modulation of immunity, countering pathogens and maintaining a pH below 4.7. We report the isolation and genome sequence of Lactobacillus pentosus strain KCA1 (formally known as L. plantarum) from the vagina of a healthy Nigerian woman. The genome was sequenced using Illumina GA II technology. The resulting 16,920,226 paired-end reads were assembled with the Velvet tool. Contigs were annotated using the RAST server, and manually curated. A comparative analysis with the available genomes of L. pentosus IG1 and L. plantarum WCFS1 showed that over 15% of the predicted functional activities are found only in this strain. The strain has a chromosome sequence of 3,418,159 bp with a G+C content of 46.4%, and is devoid of plasmids. Novel gene clusters or variants of known genes relative to the reference genomes were found. In particular, the strain has loci encoding additional putative mannose phosphotransferase systems. Clusters of genes include those for utilization of hydantoin, isopropylmalate, malonate, rhamnosides, and genes for assimilation of polyglycans, suggesting the metabolic versatility of L. pentosus KCA1. Loci encoding putative phage defense systems were also found including clustered regularly interspaced short palindromic repeats (CRISPRs), abortive infection (Abi) systems and toxin-antitoxin systems (TA). A putative cluster of genes for biosynthesis of a cyclic bacteriocin precursor, here designated as pentocin KCA1 (penA) were identified. These findings add crucial information for understanding the genomic and geographic diversity of vaginal lactobacilli.     

In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.     

Diversity of lactic acid bacteria from Miang,a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041^T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.     

An in vitro study of Lactobacillus plantarum strains for the presence of plantaricin genes and their potential control of the table olive microbiota

Sixteen Lactobacillus plantarum strains, isolated from fermented table olives, were studied for the presence and expression of genes involved in the production of bacteriocins, pheromones and other peptides. The presence of 13 genes that belong to pln locus was monitored, while for the study of gene expression, producer strains were cultured in growth medium with variant salinity (0, 4, 6, and 8 % NaCl) and pH (3.5, 4.0, 4.5, and 6.4). The effect of producer strain on the growth of indicator microorganisms was evaluated using a well diffusion assay. In parallel, Real-Time PCR was employed to monitor the genetic expression of plnE/F and plnJ/K genes for strains that revealed the highest antimicrobial activity. The well diffusion assay showed that the growth of Lactobacillus pentosus was inhibited by six L. plantarum strains when cultured on control medium (0 % NaCl, pH 6.4). Moreover, when the same growth medium was supplemented with 4 and 6 % NaCl, the growth of L. pentosus was inhibited by three and two L. plantarum strains, respectively. Growth of L. pentosus was favoured when L. plantarum strains were cultured on a growth medium with lowered pH (3.5, 4.0, and 4.5). No inhibition of pathogens was observed, but in a few cases, inhibition of Aureobasidium pullulans was detected. The Real-Time PCR assay revealed that the expression of genes was dependent on producer strains and growth phase, whereas inhibition of indicator strains was enhanced in earlier stages of the growth curve in the presence of NaCl, although similar counts were obtained.     

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay

Background

Signature matching of nucleotide sequences in the V1 and V3 regions 16S rRNA genes using pyrosequencing technology is a powerful tool for typing vaginal Lactobacilli to the species level and has been used for investigating the vaginal microbial niche.

Methods

This study has characterized the normal cultivable vaginal Lactobacillus flora at varying estradiol levels in plasma; the study comprised 17 patients undergoing ovarian stimulation for In Vitro Fertilization (IVF) treatment. The vaginal status of each participant was initially assessed as normal according to Amsel and Nugent criteria.

Results

L. crispatus, L. gasseri and/or L. jensenii were present in 10 of the patients throughout the study period, and little variation among these three species was encountered in individual patients. The flora of three women was dominated by L. delbrüeckii, L. rhamnosus or L. vaginalis. One woman exhibited a dominance of L. iners. The flora of the remaining three women were initially dominated by L. rhamnosus or L. reuteri, but as their estrogen levels rose, their flora composition altered, to become dominated by one of the three species most common in a normal, healthy vagina.

Conclusion

Signature matching of nucleotide sequences in the V1 and V3 regions of 16S rRNA genes is a discriminative tool for the study of vaginal Lactobacilli and can be used to track the Lactobacillus flora under a variety of physiological conditions.     

Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus plantarum and Lactobacillus pentosus

Lactobacillus plantarum and Lactobacillus pentosus grouped into one protein profile cluster at r ≥ 0.70, separate from Lactobacillus casei, Lactobacillus sake, and Lactobacillus curvatus. Similar sugar fermentation reactions were recorded for representative strains of L. plantarum and L. pentosus. Representative strains, including the type of each species, were selected from the different protein profile clusters and their genetic relatedness determined by using numerical analysis of random amplified polymorphic DNA (RAPD)-PCR. The type strains of L. plantarum (ATCC 14917^T) and L. pentosus (NCFB 363^T) displayed different RAPD profiles and grouped into two independent clusters, well separated from L. casei, L. curvatus, and L. sake. Numerical analysis of RAPD-PCR proved a reliable and accurate method to distinguish between strains of L. plantarum and L. pentosus.     

Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014.

Summary The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or DNA fragments in vectors derived from p353-2 or p8014-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be efficiently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.This paper is dedicated with great appreciation to Dr. Frits Berends on the occasion of his retirement as Head of the Biochemistry Department of the TNO Medical Biological Laboratory     

Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors

Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 10² to 10⁷ transformants per μg of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far.     

Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses

Non-starter lactic acid bacteria (NSLAB) were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced by different manufacturers. A total of 400 presumptive Lactobacillus isolates were obtained, and 123 isolates and 10 type strains were subjected to phenotypic, genetic, and cell wall protein characterization analyses. Phenotypically, the cheese isolates included 32% Lactobacillus plantarum isolates, 15% L. brevis isolates, 12% L. paracasei subsp. paracasei isolates, 9% L. curvatus isolates, 6% L. fermentum isolates, 6% L. casei subsp. casei isolates, 5% L. pentosus isolates, 3% L. casei subsp. pseudoplantarum isolates, and 1% L. rhamnosus isolates. Eleven percent of the isolates were not phenotypically identified. Although a randomly amplified polymorphic DNA (RAPD) analysis based on three primers and clustering by the unweighted pair group method with arithmetic average (UPGMA) was useful for partially differentiating the 10 type strains, it did not provide a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis cell wall protein profiles clustered by UPGMA were species specific and resolved the NSLAB. The only exceptions were isolates phenotypically identified as L. plantarum and L. pentosus or as L. casei subsp. casei and L. paracasei subsp. paracasei, which were grouped together. Based on protein profiles, Italian ewe cheeses frequently contained four different species and 3 to 16 strains. In general, the cheeses produced from raw ewe milk contained a larger number of more diverse strains than the cheeses produced from pasteurized milk. The same cheese produced in different factories contained different species, as well as strains that belonged to the same species but grouped in different RAPD clusters.     

Rapid identification of Lactobacillus plantarum group using the SNaPshot minisequencing assay

This study used SNaPshot minisequencing for species identification within the Lactobacillus plantarum group. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 35 strains of L. plantarum group species. The results showed that the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus fabifermentans. In conclusion, a rapid, accurate and cost-effective assay was successfully developed for species identification of the members of the L. plantarum group.     

Production of cholera toxin B subunit in Lactobacillus

The intracellular expression of the B subunit of cholera toxin (CTB) was first achieved in Lactobacillus paracasei LbTGS1.4 with an expression cassette including the P25 promoter of Streptococcus thermophilus combined with the translation initiation region from the strongly expressed L. pentosus d-lactate dehydrogenase gene (ldhD). Secretion of CTB was next attempted in L. paracasei LbTGS1.4 and L. plantarum NCIMB8826 with four different signal sequences from exported proteins of lactic acid bacteria (Lactococcus lactis Usp45 and PrtP, Enterococcus faecalis unknown protein and S. pyogenes M6 protein). Host-dependent secretion of CTB was clearly observed: whereas none of the secretion cassettes led to detectable CTB in the extracellular fraction of L. paracasei LbTGS1.4, secretion of CTB molecules was clearly achieved with three of the selected signal sequences in L. plantarum NCIMB8826.     

Rapid discrimination and classification of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> group based on a partial <Emphasis Type="Italic">dnaK</Emphasis> sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.     

Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences

The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.     

Decarboxylation of Substituted Cinnamic Acids by Lactic Acid Bacteria Isolated during Malt Whisky Fermentation

Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, and L. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation.