# Genetic Heterogeneity ofAlicyclobacillus Strains Revealed by RFLP Analysis ofvdc Region andrpoB Gene

Authors: Agnieszka Dekowska; Jolanta Niezgoda; Barbara Sokołowska
Journal: BioMed Research International
 (2018)

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## Abstract

PCR-RFLP targeting of the 16S rDNA andrpoB genes, as well as thevdc region, was applied to identify and differentiate between the spoilage and non-spoilageAlicyclobacillus species. Eight reference strains and 75 strains isolated from spoiled juices, juice concentrates, drinks, its intermediates, and fresh apples were subject to study. Hin6I restriction patterns of the 16S rDNA gene enabled distinguishing between all the species analyzed, while therpoB gene andvdc gene cluster analysis also revealed that there were two major types among theA. acidoterrestris isolates, one similar to the reference strainA. acidoterrestris DSM 2498, and the other similar to the reference strainA. acidoterrestris ATCC 49025. Heterogeneity was also observed among theA. acidocaldarius isolates. RFLP analysis of the 16S rDNA andrpoB genes, as well asvdc region, can be used successfully in the identification and research of intraspecies heterogeneity of theAlicyclobacillus species.


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## Body

## 1. Introduction

The contamination of fruit juices byAlicyclobacillus has recently become one of the most important issues in the juice and beverage industry. These acidophilic, thermophilic, and spore-forming bacteria are very hard to eliminate from contaminated drinks.Alicyclobacillus are Gram positive, aerobic, soil borne bacteria that are able to grow within a range from pH 2.0 to 6.0 and at temperatures from 20 to 70°C [1–8]. The two main factors which prevent fruit products from spoilage with most other bacteria, which are thermal treatment and low pH values, are insufficient to eliminateAlicyclobacillus. The spores survive under typical pasteurization conditions and are able to germinate and grow in an acidic environment [9, 10]. Thermal treatment may even impel germination of the spores [11–13].Despite being nonpathogenic [14], someAlicyclobacillus species may cause the spoilage of juices and juice-containing products such as nectars and beverages.Alicyclobacillus species have been found all across the world, and their presence has been detected on fruit surfaces [15], in juices produced from several fruits: citrus, apple, banana, berry, and stone fruits [1, 8, 10, 16–24], in canned tomatoes [25], and in drinks, for example ice tea, and isotonic drinks [17, 26]. The spoilage mainly manifests itself as the formation of a medical, antiseptic off-odour, from compounds produced by the bacteria. The main compound associated with spoilage is guaiacol, produced from vanillin and vanillic acid [27–29], but halophenols, 2,6-dibromophenol and 2,6-dichlorophenol, have also been reported as spoilage agents. Among the 22 currently knownAlicyclobacillus species, five have been proven to produce an off-odour:A. acidoterrestris, A. acidiphilus, A. pomorum, A. cycloheptanicus,andA. herbarius. [1, 9, 14, 30–37].The classic method for isolating and characterizingAlicyclobacillus, devised by IFU (Internationale Fruchtsaft Union), which is commonly used in the juice and beverage industry, takes about 15 days. If present in the tested sample, guaiacol can be detected using the peroxidase method [27, 38], by sensory tests ([9, 10, 39]; Siegmund and Pöllinger-Zierler, 2006) or instrumental methods, for example, HPLC or gas chromatography [28, 35, 40, 41].The identification of theAlicyclobacillus species based on their ability to assimilate erythritol with acid production [19, 42] mainly allows differentiating between two species:A. acidoterrestris andA. acidocaldarius.Since the classic microbiologicalAlicyclobacillus detecting methods are time consuming, alternative approaches have been adopted, like flow cytometry ([30], Pieper et al. 2006), Fourier transform infrared spectroscopy [43–45], and genetic methods. Except for RAPD-PCR, (Yamazaki et al., 1997; [46–49]), most of the genetic methods used in the studies onAlicyclobacillus target the rDNA operon. These include 16S rDNA and ITS region sequencing, 16S rDNA RFLP, Real-Time PCR, and LAMP-PCR of the 16S rDNA fragment ([1, 50]; Durak et al., 2002; [6, 7, 51–54]).Guaiacol, the main spoilage agent, is produced by nonoxidative decarboxylation of vanillic acid. The ability to produce guaiacol is associated with presence ofvdc gene cluster, consisting of three genes,vdcB,vdcC, andvdcD. Chow et al. [55] describedvdc gene cluster inStreptomyces sp. Detection of thevdc genes ofA. acidoterrestris using RT-PCR was described by Niwa and Kawamoto [38]. Thevdc region sequence was published by Matsubara [56]. To date, there are no applications ofvdc region analysis for any microorganism.RpoB gene, encoding the β subunit of bacterial RNA polymerase, is one of the single-copy housekeeping genes and is widely used in studies on bacterial taxonomy. These studies include PCR-RFLP analyses ofrpoB gene fragments; however, so far, there are no reports on usingrpoB gene analysis in research on ofAlicyclobacillus.Our study focuses on the use ofrpoB gene,vdc region, and 16SrDNA gene as molecular markers for the identification and differentiation ofAlicyclobacillus.


## 2. Materials and Methods

### 2.1. Sample Acquisition and Bacterial Strains

Seventy-five strains analyzed in this study were isolated from concentrated apple juice (47), concentrated cherry juice (4), fresh apples (3), concentrated strawberry juice (2), concentrated black currant juice (2), tomato juice (2), orange juice (3), cloudy (1) and clear (1) apple juice, apple beverage (1), concentrated beetroot juice (1), concentrated raspberry juice (1), concentrated orange juice (1), orange beverage (1), banana nectar (1), cherry puree (1), and intermediates used in beverage production (3). All the strains were isolated according to the method described in IFU no. 12 September 2004/March 2007.Reference strains were obtained from the Leibniz InstituteDSMZ – German Collection of Microorganisms and Cell Cultures (Alicyclobacillus acidiphilusDSM 14558;Alicyclobacillus acidocaldarius DSM 446;Alicyclobacillus acidoterrestris DSM 2498;Alicyclobacillus herbariusDSM 13609;Alicyclobacillus hesperidum DSM 12489), and from the American Type Culture Collection (ATCC) (Alicyclobacillus acidoterrestrisATCC 49025;Geobacillus stearothermophilus ATCC 7953;Bacillus subtilisATCC 6655). Also,Alicyclobacillus acidocaldarius A1 from our own library collection was used as a reference strain, after biochemical and 16S rDNA sequencing confirmation.


### 2.2. Biochemical Tests

The strains were checked for erythritol utilization and guaiacol production. The ability of erythritol utilization was tested by plating the cultures on agar containing 1% erythritol and bromophenol blue as an indicator [42]. The ability to produce guaiacol was tested using the peroxidase method [27].


### 2.3. DNA Isolation

SelectedAlicyclobacillus strains were cultured in BAT medium (pH 4.0±0.2) at 45°C for 2–3 days.Bacillus subtilis was cultured in TSB medium (pH 7.1±0.2) at 30°C for 2 days, andGeobacillus stearothermophilus was cultured in TSB medium at 45°C for 2 days.Bacterial chromosomal DNA was purified using a Genomic Mini Kit (A&A Biotechnology), following the manufacturer’s instructions.


### 2.4. Primer Designing and Amplification

All PCR reactions were performed using a Pequstar 2x Gradient thermocycler (Pequlab).


#### 2.4.1. 16S rDNA Amplification

A fragment of the 16S rDNA gene was amplified using universal primers, similar to that used by Wang et al., 2010 [57]. The primer sequences were 8F (5′– AGAGTTTGATCCTGGCTCAG),E. coli positions 8-27 and 1512R, shortened by 1 nucleotide at 3′ end (5′ – ACGGCTACCTTGTTACGACT),E. coli positions 1512–1493. The size of the amplification product was 1495 bp.PCR reactions were performed in a total volume of 50μl, containing 5 ng of the template DNA, 50 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 40 cycles of denaturation at 94°C for 30 sec, annealing at 51°C for 35 sec, elongation 72°C for 1 min 40 sec, and the final elongation at 72°C for 2 min.


#### 2.4.2.vdc Region Amplification

Thevdc gene cluster was amplified using primers vdc fr (5′ – CTGTTGGCTCAATGGCGGCTGAGCGAT), vdc rev (5′ – TTATCAGCGGTTTATCCGCGGTGGAACAGTC), vdc1 fr (5′ – AACGACGCAGGTGTGGAAAC), vdc1 rev (5′ – AGCGTGGGCAAGTTGTCATGTG), vdc K (5′ – TTGGCAACGGAGAAGTGGGAG) and vdc S (5′ – AATCACGCGCTGATGATGGG). The 1586 fragment ofvdc region, containing fragments ofvdcB andvdcC genes, was used as a template for PCR-RFLP and was amplified using the primers Bur 5 (5′ GCCGACGTGATGCTCAARGAGCGCA) and Bur 6 (5′ GTSGCRTCGAGAATCATCTTGTG). The primers were designed based on a comparison of the raw genome sequences derived fromAlicyclobacillus acidoterrestris ATCC 49025 ([58]; GenBank number AURB01000113.1), andAlicyclobacillus herbarius DSM 13609 (GenBank number AUMH01000032.1), the sequence published by Matsubara (GenBank number BD187778.1), and sequences obtained from severalAlicyclobacillus acidoterrestris strains analyzed in this study. Sequence alignments were performed using the Serial Cloner program. The positions and directions of thevdc primers are described in Table 1 and shown in Figure 1.Table 1
The positions and directions of vdc primers.
Primer name Primer position (bp) Primer direction vdc fr 1-27 forward  vdc rev 2493-2523 reverse  vdc1 fr 314-333 forward  vdc1 rev 2287-2308 reverse  vdc K 2202-2222 forward  vdc S 1044-1063 forward  Bur5 560-584 forward  Bur6 2124-2146 reverseFigure 1
Vdc region, positions of genetic elements and primers.The 2523 bp vdc fr – vdc rev amplification product contained the wholevdc region. The size of the Bur5-Bur6 amplification product was 1586 bp. PCR reactions were performed in a total volume of 50 μl, containing 2.5 ng of the template DNA, 5 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 59°C for 50 sec, elongation 72°C for 1 min 40 sec, and the final elongation at 72°C for 5 min.


#### 2.4.3.rpoB Gene Amplification

A fragment ofrpoB gene was amplified using Gru3–Gru6 primers. The primers were designed based on a comparison of therpoB gene sequences ofAlicyclobacillus acidocaldarius,Bacillus subtilis, andGeobacillus stearothermophilus, as well as sequences derived fromA. acidoterrestris 49025,A. hesperidum URH17-3-68, andA. herbarius DSM 13609 raw genomic sequences. Gru5 and Gru6 primers are nondegenerate versions of the Gru3 and Gru4 primers, respectively. The primer sequences were Gru3 (CGYGACGTDCACTAYTCBCACTA), Gru4 (5′ – GCCCANACYTCCATCTCRCCRAA) Gru5 (5′ – CGCGACGTACACTATTCGCACTA), and Gru6 (5′ – GCCCAAACCTCCATCTCACCAAA). The size of the amplification product was 1735 bp. PCR reactions were performed in a total volume of 50 μl, containing 30 ng of the template DNA, 40 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 57°C (for the Bur5 and Bur6 primer pairs) or at 59°C (for the Bur3 and Bur4 primers) for 35 sec, elongation 72°C for 1 min 45 sec, and the final elongation at 72°C for 5 min.


### 2.5. PCR-RFLP

5-15μl of the PCR products were digested with BsuRI, Hin6I, and HphI (Thermo Scientific) in 20 μl volumes. The samples were incubated at 37°C for 1-2 hours, and the enzymes were then inactivated with ten-minute incubation at 80°C. The digests were analyzed on 2.5–3% agarose gel.


### 2.6. DNA Sequencing

DNA samples were sequenced using 8F and shortened 1512R primers for the 16S rDNA gene; vdc fr, vdc rev, vdc1 fr, vdc1 rev, Bur5, Bur6, and two additional primers to fill the gaps, vdcS (5′ – AATCACGCGCTGATGATGGG) and vdcK (5′ – TTGGCAACGGAGAAGTGGGAG) for thevdc region; and Gru3 – Gru6 for therpoBgene.The contig assemblies, sequence alignments, and phylogenetic analysis were performed using Serial Cloner software (SerialBasic) and Clustal Omega. The sequences were compared to the GenBank sequences database using BLAST tools.


## 2.1. Sample Acquisition and Bacterial Strains

Seventy-five strains analyzed in this study were isolated from concentrated apple juice (47), concentrated cherry juice (4), fresh apples (3), concentrated strawberry juice (2), concentrated black currant juice (2), tomato juice (2), orange juice (3), cloudy (1) and clear (1) apple juice, apple beverage (1), concentrated beetroot juice (1), concentrated raspberry juice (1), concentrated orange juice (1), orange beverage (1), banana nectar (1), cherry puree (1), and intermediates used in beverage production (3). All the strains were isolated according to the method described in IFU no. 12 September 2004/March 2007.Reference strains were obtained from the Leibniz InstituteDSMZ – German Collection of Microorganisms and Cell Cultures (Alicyclobacillus acidiphilusDSM 14558;Alicyclobacillus acidocaldarius DSM 446;Alicyclobacillus acidoterrestris DSM 2498;Alicyclobacillus herbariusDSM 13609;Alicyclobacillus hesperidum DSM 12489), and from the American Type Culture Collection (ATCC) (Alicyclobacillus acidoterrestrisATCC 49025;Geobacillus stearothermophilus ATCC 7953;Bacillus subtilisATCC 6655). Also,Alicyclobacillus acidocaldarius A1 from our own library collection was used as a reference strain, after biochemical and 16S rDNA sequencing confirmation.


## 2.2. Biochemical Tests

The strains were checked for erythritol utilization and guaiacol production. The ability of erythritol utilization was tested by plating the cultures on agar containing 1% erythritol and bromophenol blue as an indicator [42]. The ability to produce guaiacol was tested using the peroxidase method [27].


## 2.3. DNA Isolation

SelectedAlicyclobacillus strains were cultured in BAT medium (pH 4.0±0.2) at 45°C for 2–3 days.Bacillus subtilis was cultured in TSB medium (pH 7.1±0.2) at 30°C for 2 days, andGeobacillus stearothermophilus was cultured in TSB medium at 45°C for 2 days.Bacterial chromosomal DNA was purified using a Genomic Mini Kit (A&A Biotechnology), following the manufacturer’s instructions.


## 2.4. Primer Designing and Amplification

All PCR reactions were performed using a Pequstar 2x Gradient thermocycler (Pequlab).


### 2.4.1. 16S rDNA Amplification

A fragment of the 16S rDNA gene was amplified using universal primers, similar to that used by Wang et al., 2010 [57]. The primer sequences were 8F (5′– AGAGTTTGATCCTGGCTCAG),E. coli positions 8-27 and 1512R, shortened by 1 nucleotide at 3′ end (5′ – ACGGCTACCTTGTTACGACT),E. coli positions 1512–1493. The size of the amplification product was 1495 bp.PCR reactions were performed in a total volume of 50μl, containing 5 ng of the template DNA, 50 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 40 cycles of denaturation at 94°C for 30 sec, annealing at 51°C for 35 sec, elongation 72°C for 1 min 40 sec, and the final elongation at 72°C for 2 min.


### 2.4.2.vdc Region Amplification

Thevdc gene cluster was amplified using primers vdc fr (5′ – CTGTTGGCTCAATGGCGGCTGAGCGAT), vdc rev (5′ – TTATCAGCGGTTTATCCGCGGTGGAACAGTC), vdc1 fr (5′ – AACGACGCAGGTGTGGAAAC), vdc1 rev (5′ – AGCGTGGGCAAGTTGTCATGTG), vdc K (5′ – TTGGCAACGGAGAAGTGGGAG) and vdc S (5′ – AATCACGCGCTGATGATGGG). The 1586 fragment ofvdc region, containing fragments ofvdcB andvdcC genes, was used as a template for PCR-RFLP and was amplified using the primers Bur 5 (5′ GCCGACGTGATGCTCAARGAGCGCA) and Bur 6 (5′ GTSGCRTCGAGAATCATCTTGTG). The primers were designed based on a comparison of the raw genome sequences derived fromAlicyclobacillus acidoterrestris ATCC 49025 ([58]; GenBank number AURB01000113.1), andAlicyclobacillus herbarius DSM 13609 (GenBank number AUMH01000032.1), the sequence published by Matsubara (GenBank number BD187778.1), and sequences obtained from severalAlicyclobacillus acidoterrestris strains analyzed in this study. Sequence alignments were performed using the Serial Cloner program. The positions and directions of thevdc primers are described in Table 1 and shown in Figure 1.Table 1
The positions and directions of vdc primers.
Primer name Primer position (bp) Primer direction vdc fr 1-27 forward  vdc rev 2493-2523 reverse  vdc1 fr 314-333 forward  vdc1 rev 2287-2308 reverse  vdc K 2202-2222 forward  vdc S 1044-1063 forward  Bur5 560-584 forward  Bur6 2124-2146 reverseFigure 1
Vdc region, positions of genetic elements and primers.The 2523 bp vdc fr – vdc rev amplification product contained the wholevdc region. The size of the Bur5-Bur6 amplification product was 1586 bp. PCR reactions were performed in a total volume of 50 μl, containing 2.5 ng of the template DNA, 5 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 59°C for 50 sec, elongation 72°C for 1 min 40 sec, and the final elongation at 72°C for 5 min.


### 2.4.3.rpoB Gene Amplification

A fragment ofrpoB gene was amplified using Gru3–Gru6 primers. The primers were designed based on a comparison of therpoB gene sequences ofAlicyclobacillus acidocaldarius,Bacillus subtilis, andGeobacillus stearothermophilus, as well as sequences derived fromA. acidoterrestris 49025,A. hesperidum URH17-3-68, andA. herbarius DSM 13609 raw genomic sequences. Gru5 and Gru6 primers are nondegenerate versions of the Gru3 and Gru4 primers, respectively. The primer sequences were Gru3 (CGYGACGTDCACTAYTCBCACTA), Gru4 (5′ – GCCCANACYTCCATCTCRCCRAA) Gru5 (5′ – CGCGACGTACACTATTCGCACTA), and Gru6 (5′ – GCCCAAACCTCCATCTCACCAAA). The size of the amplification product was 1735 bp. PCR reactions were performed in a total volume of 50 μl, containing 30 ng of the template DNA, 40 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 57°C (for the Bur5 and Bur6 primer pairs) or at 59°C (for the Bur3 and Bur4 primers) for 35 sec, elongation 72°C for 1 min 45 sec, and the final elongation at 72°C for 5 min.


## 2.4.1. 16S rDNA Amplification

A fragment of the 16S rDNA gene was amplified using universal primers, similar to that used by Wang et al., 2010 [57]. The primer sequences were 8F (5′– AGAGTTTGATCCTGGCTCAG),E. coli positions 8-27 and 1512R, shortened by 1 nucleotide at 3′ end (5′ – ACGGCTACCTTGTTACGACT),E. coli positions 1512–1493. The size of the amplification product was 1495 bp.PCR reactions were performed in a total volume of 50μl, containing 5 ng of the template DNA, 50 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 40 cycles of denaturation at 94°C for 30 sec, annealing at 51°C for 35 sec, elongation 72°C for 1 min 40 sec, and the final elongation at 72°C for 2 min.


## 2.4.2.vdc Region Amplification

Thevdc gene cluster was amplified using primers vdc fr (5′ – CTGTTGGCTCAATGGCGGCTGAGCGAT), vdc rev (5′ – TTATCAGCGGTTTATCCGCGGTGGAACAGTC), vdc1 fr (5′ – AACGACGCAGGTGTGGAAAC), vdc1 rev (5′ – AGCGTGGGCAAGTTGTCATGTG), vdc K (5′ – TTGGCAACGGAGAAGTGGGAG) and vdc S (5′ – AATCACGCGCTGATGATGGG). The 1586 fragment ofvdc region, containing fragments ofvdcB andvdcC genes, was used as a template for PCR-RFLP and was amplified using the primers Bur 5 (5′ GCCGACGTGATGCTCAARGAGCGCA) and Bur 6 (5′ GTSGCRTCGAGAATCATCTTGTG). The primers were designed based on a comparison of the raw genome sequences derived fromAlicyclobacillus acidoterrestris ATCC 49025 ([58]; GenBank number AURB01000113.1), andAlicyclobacillus herbarius DSM 13609 (GenBank number AUMH01000032.1), the sequence published by Matsubara (GenBank number BD187778.1), and sequences obtained from severalAlicyclobacillus acidoterrestris strains analyzed in this study. Sequence alignments were performed using the Serial Cloner program. The positions and directions of thevdc primers are described in Table 1 and shown in Figure 1.Table 1
The positions and directions of vdc primers.
Primer name Primer position (bp) Primer direction vdc fr 1-27 forward  vdc rev 2493-2523 reverse  vdc1 fr 314-333 forward  vdc1 rev 2287-2308 reverse  vdc K 2202-2222 forward  vdc S 1044-1063 forward  Bur5 560-584 forward  Bur6 2124-2146 reverseFigure 1
Vdc region, positions of genetic elements and primers.The 2523 bp vdc fr – vdc rev amplification product contained the wholevdc region. The size of the Bur5-Bur6 amplification product was 1586 bp. PCR reactions were performed in a total volume of 50 μl, containing 2.5 ng of the template DNA, 5 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 59°C for 50 sec, elongation 72°C for 1 min 40 sec, and the final elongation at 72°C for 5 min.


## 2.4.3.rpoB Gene Amplification

A fragment ofrpoB gene was amplified using Gru3–Gru6 primers. The primers were designed based on a comparison of therpoB gene sequences ofAlicyclobacillus acidocaldarius,Bacillus subtilis, andGeobacillus stearothermophilus, as well as sequences derived fromA. acidoterrestris 49025,A. hesperidum URH17-3-68, andA. herbarius DSM 13609 raw genomic sequences. Gru5 and Gru6 primers are nondegenerate versions of the Gru3 and Gru4 primers, respectively. The primer sequences were Gru3 (CGYGACGTDCACTAYTCBCACTA), Gru4 (5′ – GCCCANACYTCCATCTCRCCRAA) Gru5 (5′ – CGCGACGTACACTATTCGCACTA), and Gru6 (5′ – GCCCAAACCTCCATCTCACCAAA). The size of the amplification product was 1735 bp. PCR reactions were performed in a total volume of 50 μl, containing 30 ng of the template DNA, 40 pM of each of the primers, and 25 μl of the DreamTaq™ Green PCR Master Mix (Thermo Scientific). PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 57°C (for the Bur5 and Bur6 primer pairs) or at 59°C (for the Bur3 and Bur4 primers) for 35 sec, elongation 72°C for 1 min 45 sec, and the final elongation at 72°C for 5 min.


## 2.5. PCR-RFLP

5-15μl of the PCR products were digested with BsuRI, Hin6I, and HphI (Thermo Scientific) in 20 μl volumes. The samples were incubated at 37°C for 1-2 hours, and the enzymes were then inactivated with ten-minute incubation at 80°C. The digests were analyzed on 2.5–3% agarose gel.


## 2.6. DNA Sequencing

DNA samples were sequenced using 8F and shortened 1512R primers for the 16S rDNA gene; vdc fr, vdc rev, vdc1 fr, vdc1 rev, Bur5, Bur6, and two additional primers to fill the gaps, vdcS (5′ – AATCACGCGCTGATGATGGG) and vdcK (5′ – TTGGCAACGGAGAAGTGGGAG) for thevdc region; and Gru3 – Gru6 for therpoBgene.The contig assemblies, sequence alignments, and phylogenetic analysis were performed using Serial Cloner software (SerialBasic) and Clustal Omega. The sequences were compared to the GenBank sequences database using BLAST tools.


## 3. Results

### 3.1. RFLP Analysis of 16S rDNA Fragment

The 1495 bp fragment of the 16S rDNA gene, amplified using 8F and shortened 1512R primers, was digested by BsuRI, Hin6I, and HphI.While BsuRI and HphI digestions did not allow to distinguish between all the species analyzed, the patterns obtained by Hin6I digestion were species specific (Figure2).Figure 2
Restriction patterns of a 1495 bp fragment of the 16S rDNA gene, digested with Hin6I. Lanes 6, 7, 9, 14, and 18,A. acidoterrestris; 2, 3, 4, 8, 10, 11, 12, 13, 15, 16, and 17,A. acidocaldarius; 5,Brevibacillus agri; 19A. acidoterrestris DSM 2498; 20,A. acidoterrestris ATCC49025; 21,A. acidiphilus; 22,A.hesperidum; 23,A.herbarius; 24,A. acidocaldarius; 25,B. subtilis; 26,G. stearothermophilus; lane 1, molecular marker.All analyzed isolates with their features and RFLP profiles are described in Table2.Table 2
List of isolates and their RFLP profiles. AAT,A. acidoterrestris; AAC, A. acidocaldarius; BA, Brevibacillus agri; BG, bacillus ginsengihumi.
Isolate Source Guaiacol production Erythritol utilization RFLP analysis 16S/Hin61 vdc/BsuRI vdc/Hin61 vdc/HphI rpoB/BsuRI rpoB/Hin61 rpoB/HphI 1 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 2 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 3 intermediate for beverage production yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 4 intermediate for beverage production no no AAC - - - AAC(I) AAC(I) AAC(I) 5 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 6 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 7 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 8 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 9 orange beverage yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 10 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 11 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 12 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 13 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 14 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 15 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 16 concentrated apple juice yes non typical AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 17 banana nectar yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 18 orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 19 concentrated orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 20 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 21 orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 22 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 23 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 24 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 25 fresh apples yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 26 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 27 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 28 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 29 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 30 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 31 concentrated black currant juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 32 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 33 concentrated apple juice yes non typical AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 34 fresh apples yes yes AAT AAT(I) AAT(I) AAT(IA) AAT(I) AAT(I) AAT(I) 35 fresh apples  yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 36 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 37 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 38 intermediate for beverage production no no AAC - - - AAC(I) AAC(I) AAC(IA) 39 concentrated apple juice yes - BG - - - BG BG BG 40 concentrated apple juice yes - BG - - - BG BG BG 41 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 42 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(IIA) 43 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 44 spoiled apple beverage yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 45 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 46 spoiled apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 47 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 48 concentrated strawberry juice no no AAC - - - AAC(II) AAC(II) AAC(II) 49 concentrated cherry juice no no AAC - - - AAC(IA) AAC(I) AAC(IA) 50 tomato juice from fresh tomatoes no no AAC - - - AAC(II) AAC(II) AAC(II) 51 concentrated beetroot juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 52 concentrated cherry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 53 concentrated cherry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 54 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(IIA) AAT(II) AAT(II) 55 concentrated cherry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 56 concentrated raspberry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 57 concentrated apple juice yes non typical AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 58 orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 59 concentrated apple juice no non typical AAC - - - AAC(IA) AAC(I) AAC(I) 60 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 61 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 62 tomato juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 63 concentrated apple juice yes - BA - - - BA BA BA 64 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II) 65 cherry puree yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 66 concentrated black currant juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 67 concentrated strawberry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 68 cloudy apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 69 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 70 concentrated apple juice no no AAC - - - AAC(IA) AAC(I) AAC(I) 71 concentrated apple juice no no AAC - - - AAC(IA) AAC(I) AAC(I) 72 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II) 73 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 74 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II) 75 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II)


### 3.2. RFLP Analysis of thevdc Region Fragment

The fragment ofvdc region was amplified using Bur5 and Bur6 primers. The product was a single band of 1586 bp and was observed only for guaiacol producing strains:A. acidoterrestris,A. acidophilus, andA. herbarius. The PCR product was digested by BsuRI (Figure 3), Hin6I (Figure 4), and HphI (Figure 5).Figure 3
Restriction patterns of a 1586 bp fragment of thevdc region, digested with BsuRI. Lanes 1–3, 5, 7–11, 13, and 14,A. acidoterrestris type I; 4, 6, 12, and 15,A. acidoterrestris type II; 16A. acidoterrestris DSM 2498; 17,A. acidoterrestris ATCC49025; 18,A. acidiphilus; 19,A.herbarius; 20, molecular marker.Figure 4
Restriction patterns of a 1586 bp fragment of thevdc region, digested with Hin6I. Lanes 1–3, 5, 7–11, 13, and 14,A. acidoterrestris type I; 4, 6, 12, and 15,A. acidoterrestris type II; 16A. acidoterrestris DSM 2498; 17,A. acidoterrestris ATCC49025; 18,A. acidiphilusDSM 14558; 19,A.herbariusDSM 13609; lane 20, molecular marker.Figure 5
Restriction patterns of a 1586 bp fragment of thevdc region, digested with HphI. Lanes 2–4, 6, 8–12, 14, and 15,A. acidoterrestris type I; 5, 7, 13, and 16,A. acidoterrestris type II; 17A. acidoterrestris DSM 2498; 18,A. acidoterrestris ATCC49025; 19,A. acidiphilus; 20,A.herbarius; lane 1, molecular marker.BsuRI and Hin6I RFLP patterns enabled distinguishing between all the species analyzed, and divided theA. acidoterrestris group into two clusters. Type I pattern was identical to the pattern given byA. acidoterrestris DSM 2498, and type II pattern was identical toA. acidoterrestrisATCC 49025. The HphI patterns confirm this rule with the exception of one strain, which represented the type I pattern in the BsuRI and Hin6I analysis.


### 3.3. RFLP Analysis of therpoB Gene Fragment

The 1735 bp fragment of therpoB gene was amplified using Gru3 and Gru4, or Gru5 and Gru6 primers. Gru3 and Gru4 primers were degenerated versions of Gru5 and Gru6 (respectively) and were used for amplification of isolates other thanA. acidoterrestris.A acidoterrestris isolates were amplified either with Gru3–Gru4, or Gru5–Gru6 primers, and the Gru5–Gru6 primer pair was chosen due to the better efficiency of the reaction. Considering the individual isolates, the RFLP patterns were identical for both pairs of primers.The patterns obtained by all of the nucleases used (Figures6–8) enabled distinguishing between the species analyzed. With exception of two strains, the BsuRI patterns divided theA. acidoterrestris group into two clusters, analogical to the clusters revealed by thevdc region analysis. The pattern (IIA) is shown by two exceptional strains, most resembling the type II pattern ofA. acidoterrestris, and those strains were classified as type II in other analyses. The reference strain ATCC 49025 shows this type of pattern. TheA. acidocaldarius group was represented by three types of patterns, two of them resembling each other.A acidocaldariusDSM 446 belongs to the cluster II ofA. acidocaldarius group, whileA. acidocaldarius A1 was assigned to cluster I.Figure 6
Restriction patterns of a 1735 bp fragment of therpoB gene, digested with BsuRI. Lanes 1–4, 6, and 8–10,A. acidoterrestris type I; 5, 7, 21, 22, and 24-26,A. acidoterrestris type II; 23,A. acidoterrestris type IIA; 27,A. acidocaldarius type II, 28,A. acidocaldariustype IA; 11,A. acidoterrestris DSM 2498; 12,A. acidoterrestris ATCC49025; 13,A. acidiphilus; 14,A.hesperidum; 15,A.herbarius; 16,A. acidocaldarius A1; 17,B. subtilis; 18,G. stearothermophilus; lane 19, molecular marker.Figure 7
Restriction patterns of a 1735 bp fragment ofrpoB gene, digested with Hin6I. Lanes 3 and 5,A. acidoterrestris type I; 1, 2, 4, and 6,A. acidoterrestris type II; 17,A. acidocaldarius type I; 18,A. acidocaldarius type II; 8,A. acidoterrestris DSM 2498; 9,A. acidoterrestris ATCC49025; 10,A. acidiphilus; 11,A.hesperidum; 12,A.herbarius; 13,A. acidocaldarius A1; 14,B. subtilis; 15,G. stearothermophilus; lanes 7 and 16, molecular marker.Figure 8
Restriction patterns of a 1735 bp fragment ofrpoB gene, digested with HphI. Lanes 1-4, 6, 8-11, 13, 25, and 27,A. acidoterrestris I; 5, 7, 12, 26, and 28,A. acidoterrestris II; 24,A. acidoterrestris IIA; 29,A. acidocaldarius IA; 30, A. acidocaldarius type II; 15,A. acidoterrestris DSM 2498; 16,A. acidoterrestris ATCC49025; 17,A. acidiphilus; 18,A.hesperidum; 19,A.herbarius; 20,A. acidocaldariusA1; 21,B. subtilis; 22,G. stearothermophilus; lanes 14 and 23, molecular marker.For Hin6I there were two types of patterns for theA. acidoterrestris andA. acidocaldarius.A. acidoterrestrisDSM 2498 belongs to the cluster I, whileA. acidoterrestris ATCC 49025 belongs to the cluster II. A. acidocaldarius DSM 446 represents cluster II of theA. acidocaldarius group, whileA. acidocaldarius A1 represents cluster I.HphI endonuclease produces two types of patterns forA. acidoterrestris, analogically as before, and three types of patterns forA. acidocaldarius. One sample ofA. acidoterrestriscluster II has a slightly different pattern and has been classified as type II in other analyses. Two of the three patterns ofA. acidocaldarius differ with only one band.A. acidocaldarius DSM 446 represents cluster II of theA. acidocaldarius group, whileA. acidocaldarius A1 represents cluster I.


### 3.4. DNA Sequencing

DNA sequencing was performed for selected strains. Sequencing of the 16S rDNA gene was performed to confirm the proper identification of the isolates and to identify the non-Alicyclobacillus isolates. The 16S rDNA sequence of strains 31 and 34, classified as type I, showed the greatest similarity toA. acidoterrestris DSM 2498, also classified as type I. The sequences of strains 33, 51, 52, 53, 55, and 56, classified as type II, showed the greatest similarity toA. acidoterrestris ATCC 49025, also classified as type II. The sequence of strain 41, classified as type II, showed the greatest similarity toA. acidoterrestrisDSM 3922.Figure9 shows the phylogenetic tree constructed from the 16S rDNA sequences of analyzed strains ofA. acidoterrestris. The sequence analysis indicates thatA. acidoterrestris DSM 2498 and two other type I strains are closely related and it shows greater variation within class II strains. The isolates selected for sequencing represent both groups and different sources which they were isolated from: concentrated apple juice (isolates 33 and 41), fresh apples (isolate 34), concentrated blackcurrant juice (isolate 31), concentrated raspberry juice (isolate 56), concentrated beetroot juice (isolate 51), and concentrated cherry juice (isolates 52, 53, and 55). The sequence analysis shows no apparent correlation between diversity of the 16S rDNA sequence and the sources of the isolates.Figure 9
Phylogenetic tree of partial 16S rRNA sequence of 11A. acidoterrestris isolates. The tree was constructed by neighbor joining method using Clustal Omega software.Five wholevdc gene clusters were sequenced. Two of them were isolated from the reference strains,A. acidoterrestris DSM 2498, andA. acidoterrestris ATCC 49025. 2416 bp fragments of thevdc regions were aligned. Thevdc region sequence of strains 15 and 34, classified as type I, showed 99.9% identity with thevdc sequence ofA. acidoterrestrisDSM 2498; the sequence of strain 41, classified as type II, showed 99.3% identity with thevdc sequenceA. acidoterrestris ATCC 49025.Vdc sequences ofA. acidoterrestrisDSM 2498 andA. acidoterrestrisDSM 49025 showed 94.5% identity. For the last pair, protein sequences of the gene products showed 98.5% positives and 96.5% identities for VdcB, 99.4% and 98.9% for VdcC, and 100% and 97.4% for VdcD.RpoB gene sequencing was performed to confirm that both primer pairs, Gru3–Gru4 and Gru5–Gru6, enabled to amplify the correct DNA fragment and that degeneration of the primers did not affect the sequence specificity, although the degenerated primers produced significantly lower amount of PCR product. All of the sequences obtained showed greatest similarity to the appropriaterpoB genes.The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences; KX453673-KX453677 for fullvdc region sequences; KX453678 and KX453679 for partialvdc sequences ofA. herbarius andA. acidiphilus, respectively; and KX453680- KX453682 for partialrpoB sequences.


## 3.1. RFLP Analysis of 16S rDNA Fragment

The 1495 bp fragment of the 16S rDNA gene, amplified using 8F and shortened 1512R primers, was digested by BsuRI, Hin6I, and HphI.While BsuRI and HphI digestions did not allow to distinguish between all the species analyzed, the patterns obtained by Hin6I digestion were species specific (Figure2).Figure 2
Restriction patterns of a 1495 bp fragment of the 16S rDNA gene, digested with Hin6I. Lanes 6, 7, 9, 14, and 18,A. acidoterrestris; 2, 3, 4, 8, 10, 11, 12, 13, 15, 16, and 17,A. acidocaldarius; 5,Brevibacillus agri; 19A. acidoterrestris DSM 2498; 20,A. acidoterrestris ATCC49025; 21,A. acidiphilus; 22,A.hesperidum; 23,A.herbarius; 24,A. acidocaldarius; 25,B. subtilis; 26,G. stearothermophilus; lane 1, molecular marker.All analyzed isolates with their features and RFLP profiles are described in Table2.Table 2
List of isolates and their RFLP profiles. AAT,A. acidoterrestris; AAC, A. acidocaldarius; BA, Brevibacillus agri; BG, bacillus ginsengihumi.
Isolate Source Guaiacol production Erythritol utilization RFLP analysis 16S/Hin61 vdc/BsuRI vdc/Hin61 vdc/HphI rpoB/BsuRI rpoB/Hin61 rpoB/HphI 1 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 2 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 3 intermediate for beverage production yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 4 intermediate for beverage production no no AAC - - - AAC(I) AAC(I) AAC(I) 5 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 6 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 7 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 8 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 9 orange beverage yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 10 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 11 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 12 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 13 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 14 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 15 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 16 concentrated apple juice yes non typical AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 17 banana nectar yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 18 orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 19 concentrated orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 20 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 21 orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 22 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 23 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 24 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 25 fresh apples yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 26 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 27 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 28 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 29 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 30 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 31 concentrated black currant juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 32 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 33 concentrated apple juice yes non typical AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 34 fresh apples yes yes AAT AAT(I) AAT(I) AAT(IA) AAT(I) AAT(I) AAT(I) 35 fresh apples  yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 36 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 37 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 38 intermediate for beverage production no no AAC - - - AAC(I) AAC(I) AAC(IA) 39 concentrated apple juice yes - BG - - - BG BG BG 40 concentrated apple juice yes - BG - - - BG BG BG 41 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 42 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(IIA) 43 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 44 spoiled apple beverage yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 45 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 46 spoiled apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 47 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 48 concentrated strawberry juice no no AAC - - - AAC(II) AAC(II) AAC(II) 49 concentrated cherry juice no no AAC - - - AAC(IA) AAC(I) AAC(IA) 50 tomato juice from fresh tomatoes no no AAC - - - AAC(II) AAC(II) AAC(II) 51 concentrated beetroot juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 52 concentrated cherry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 53 concentrated cherry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 54 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(IIA) AAT(II) AAT(II) 55 concentrated cherry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 56 concentrated raspberry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 57 concentrated apple juice yes non typical AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 58 orange juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 59 concentrated apple juice no non typical AAC - - - AAC(IA) AAC(I) AAC(I) 60 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 61 concentrated apple juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 62 tomato juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 63 concentrated apple juice yes - BA - - - BA BA BA 64 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II) 65 cherry puree yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 66 concentrated black currant juice yes yes AAT AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) AAT(I) 67 concentrated strawberry juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 68 cloudy apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 69 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 70 concentrated apple juice no no AAC - - - AAC(IA) AAC(I) AAC(I) 71 concentrated apple juice no no AAC - - - AAC(IA) AAC(I) AAC(I) 72 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II) 73 concentrated apple juice yes yes AAT AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) AAT(II) 74 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II) 75 concentrated apple juice no no AAC - - - AAC(II) AAC(II) AAC(II)


## 3.2. RFLP Analysis of thevdc Region Fragment

The fragment ofvdc region was amplified using Bur5 and Bur6 primers. The product was a single band of 1586 bp and was observed only for guaiacol producing strains:A. acidoterrestris,A. acidophilus, andA. herbarius. The PCR product was digested by BsuRI (Figure 3), Hin6I (Figure 4), and HphI (Figure 5).Figure 3
Restriction patterns of a 1586 bp fragment of thevdc region, digested with BsuRI. Lanes 1–3, 5, 7–11, 13, and 14,A. acidoterrestris type I; 4, 6, 12, and 15,A. acidoterrestris type II; 16A. acidoterrestris DSM 2498; 17,A. acidoterrestris ATCC49025; 18,A. acidiphilus; 19,A.herbarius; 20, molecular marker.Figure 4
Restriction patterns of a 1586 bp fragment of thevdc region, digested with Hin6I. Lanes 1–3, 5, 7–11, 13, and 14,A. acidoterrestris type I; 4, 6, 12, and 15,A. acidoterrestris type II; 16A. acidoterrestris DSM 2498; 17,A. acidoterrestris ATCC49025; 18,A. acidiphilusDSM 14558; 19,A.herbariusDSM 13609; lane 20, molecular marker.Figure 5
Restriction patterns of a 1586 bp fragment of thevdc region, digested with HphI. Lanes 2–4, 6, 8–12, 14, and 15,A. acidoterrestris type I; 5, 7, 13, and 16,A. acidoterrestris type II; 17A. acidoterrestris DSM 2498; 18,A. acidoterrestris ATCC49025; 19,A. acidiphilus; 20,A.herbarius; lane 1, molecular marker.BsuRI and Hin6I RFLP patterns enabled distinguishing between all the species analyzed, and divided theA. acidoterrestris group into two clusters. Type I pattern was identical to the pattern given byA. acidoterrestris DSM 2498, and type II pattern was identical toA. acidoterrestrisATCC 49025. The HphI patterns confirm this rule with the exception of one strain, which represented the type I pattern in the BsuRI and Hin6I analysis.


## 3.3. RFLP Analysis of therpoB Gene Fragment

The 1735 bp fragment of therpoB gene was amplified using Gru3 and Gru4, or Gru5 and Gru6 primers. Gru3 and Gru4 primers were degenerated versions of Gru5 and Gru6 (respectively) and were used for amplification of isolates other thanA. acidoterrestris.A acidoterrestris isolates were amplified either with Gru3–Gru4, or Gru5–Gru6 primers, and the Gru5–Gru6 primer pair was chosen due to the better efficiency of the reaction. Considering the individual isolates, the RFLP patterns were identical for both pairs of primers.The patterns obtained by all of the nucleases used (Figures6–8) enabled distinguishing between the species analyzed. With exception of two strains, the BsuRI patterns divided theA. acidoterrestris group into two clusters, analogical to the clusters revealed by thevdc region analysis. The pattern (IIA) is shown by two exceptional strains, most resembling the type II pattern ofA. acidoterrestris, and those strains were classified as type II in other analyses. The reference strain ATCC 49025 shows this type of pattern. TheA. acidocaldarius group was represented by three types of patterns, two of them resembling each other.A acidocaldariusDSM 446 belongs to the cluster II ofA. acidocaldarius group, whileA. acidocaldarius A1 was assigned to cluster I.Figure 6
Restriction patterns of a 1735 bp fragment of therpoB gene, digested with BsuRI. Lanes 1–4, 6, and 8–10,A. acidoterrestris type I; 5, 7, 21, 22, and 24-26,A. acidoterrestris type II; 23,A. acidoterrestris type IIA; 27,A. acidocaldarius type II, 28,A. acidocaldariustype IA; 11,A. acidoterrestris DSM 2498; 12,A. acidoterrestris ATCC49025; 13,A. acidiphilus; 14,A.hesperidum; 15,A.herbarius; 16,A. acidocaldarius A1; 17,B. subtilis; 18,G. stearothermophilus; lane 19, molecular marker.Figure 7
Restriction patterns of a 1735 bp fragment ofrpoB gene, digested with Hin6I. Lanes 3 and 5,A. acidoterrestris type I; 1, 2, 4, and 6,A. acidoterrestris type II; 17,A. acidocaldarius type I; 18,A. acidocaldarius type II; 8,A. acidoterrestris DSM 2498; 9,A. acidoterrestris ATCC49025; 10,A. acidiphilus; 11,A.hesperidum; 12,A.herbarius; 13,A. acidocaldarius A1; 14,B. subtilis; 15,G. stearothermophilus; lanes 7 and 16, molecular marker.Figure 8
Restriction patterns of a 1735 bp fragment ofrpoB gene, digested with HphI. Lanes 1-4, 6, 8-11, 13, 25, and 27,A. acidoterrestris I; 5, 7, 12, 26, and 28,A. acidoterrestris II; 24,A. acidoterrestris IIA; 29,A. acidocaldarius IA; 30, A. acidocaldarius type II; 15,A. acidoterrestris DSM 2498; 16,A. acidoterrestris ATCC49025; 17,A. acidiphilus; 18,A.hesperidum; 19,A.herbarius; 20,A. acidocaldariusA1; 21,B. subtilis; 22,G. stearothermophilus; lanes 14 and 23, molecular marker.For Hin6I there were two types of patterns for theA. acidoterrestris andA. acidocaldarius.A. acidoterrestrisDSM 2498 belongs to the cluster I, whileA. acidoterrestris ATCC 49025 belongs to the cluster II. A. acidocaldarius DSM 446 represents cluster II of theA. acidocaldarius group, whileA. acidocaldarius A1 represents cluster I.HphI endonuclease produces two types of patterns forA. acidoterrestris, analogically as before, and three types of patterns forA. acidocaldarius. One sample ofA. acidoterrestriscluster II has a slightly different pattern and has been classified as type II in other analyses. Two of the three patterns ofA. acidocaldarius differ with only one band.A. acidocaldarius DSM 446 represents cluster II of theA. acidocaldarius group, whileA. acidocaldarius A1 represents cluster I.


## 3.4. DNA Sequencing

DNA sequencing was performed for selected strains. Sequencing of the 16S rDNA gene was performed to confirm the proper identification of the isolates and to identify the non-Alicyclobacillus isolates. The 16S rDNA sequence of strains 31 and 34, classified as type I, showed the greatest similarity toA. acidoterrestris DSM 2498, also classified as type I. The sequences of strains 33, 51, 52, 53, 55, and 56, classified as type II, showed the greatest similarity toA. acidoterrestris ATCC 49025, also classified as type II. The sequence of strain 41, classified as type II, showed the greatest similarity toA. acidoterrestrisDSM 3922.Figure9 shows the phylogenetic tree constructed from the 16S rDNA sequences of analyzed strains ofA. acidoterrestris. The sequence analysis indicates thatA. acidoterrestris DSM 2498 and two other type I strains are closely related and it shows greater variation within class II strains. The isolates selected for sequencing represent both groups and different sources which they were isolated from: concentrated apple juice (isolates 33 and 41), fresh apples (isolate 34), concentrated blackcurrant juice (isolate 31), concentrated raspberry juice (isolate 56), concentrated beetroot juice (isolate 51), and concentrated cherry juice (isolates 52, 53, and 55). The sequence analysis shows no apparent correlation between diversity of the 16S rDNA sequence and the sources of the isolates.Figure 9
Phylogenetic tree of partial 16S rRNA sequence of 11A. acidoterrestris isolates. The tree was constructed by neighbor joining method using Clustal Omega software.Five wholevdc gene clusters were sequenced. Two of them were isolated from the reference strains,A. acidoterrestris DSM 2498, andA. acidoterrestris ATCC 49025. 2416 bp fragments of thevdc regions were aligned. Thevdc region sequence of strains 15 and 34, classified as type I, showed 99.9% identity with thevdc sequence ofA. acidoterrestrisDSM 2498; the sequence of strain 41, classified as type II, showed 99.3% identity with thevdc sequenceA. acidoterrestris ATCC 49025.Vdc sequences ofA. acidoterrestrisDSM 2498 andA. acidoterrestrisDSM 49025 showed 94.5% identity. For the last pair, protein sequences of the gene products showed 98.5% positives and 96.5% identities for VdcB, 99.4% and 98.9% for VdcC, and 100% and 97.4% for VdcD.RpoB gene sequencing was performed to confirm that both primer pairs, Gru3–Gru4 and Gru5–Gru6, enabled to amplify the correct DNA fragment and that degeneration of the primers did not affect the sequence specificity, although the degenerated primers produced significantly lower amount of PCR product. All of the sequences obtained showed greatest similarity to the appropriaterpoB genes.The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences; KX453673-KX453677 for fullvdc region sequences; KX453678 and KX453679 for partialvdc sequences ofA. herbarius andA. acidiphilus, respectively; and KX453680- KX453682 for partialrpoB sequences.


## 4. Discussion

Among more than one thousand samples of concentrated apple juice tested in our laboratory between 2004 and 2012, 67% was contaminated withAlicyclobacillus sp., and 31% of the isolates were identified asA. acidoterrestris[59]. The statistics show thatAlicyclobacillus spoilage is still a major concern in the fruit processing industry.In this study, 75 guaiacol producing and non-guaiacol producing strains were isolated from various fruit juices, concentrated fruit juices, and fresh apples. Additionally, 8 reference strains were used. The isolates and reference strains were examined using classic methods, and PCR-RFLP focusing on 16S rDNA andrpoB gene fragments as well as thevdcregion fragment. For selected strains, DNA sequencing of the 16S rDNA gene was performed. Sixty of the isolates analyzed were identified asA. acidoterrestris, 12 asA. acidocaldarius, one asBrevibacillus agri, and two asBacillus ginsengihumi. Four of the isolates, which gave ambiguous results during classic identification (nontypical colour on an erythritol medium or lack of growth at 65°C connected with lack of guaiacol production), were identified by genetic methods asA. acidoterrestris orA. acidocaldarius.A. acidoterrestris isolates were grouped in two major clusters. 27 of the isolates belonged to the cluster I, and 33 to the cluster II. Also,A. acidocaldarius isolates were grouped into two clusters, but showed more intracluster diversity.These results support the observations made by Osopale at al. [60] and Durak et al. [23] who also reported two genetic clusters among analyzedA. acidoterrestris samples, by RAPD analysis,and by 16S rDNA sequencing, respectively.Most of the strains analyzed in this study were isolated from concentrated apple juice, as this is the main subject of theAlicyclobacillus focused screening made or outsourced by polish fruit industry; however, it is one of the main subjects of similar screenings performed by fruit industry worldwide.A. acidoterrestris representing both clusters have been found in concentrated apple juice. For other sources, threeA. acidoterrestris cluster II isolates have been found in orange juices, two cluster I in concentrated black currant juice, and three cluster II in concentrated cherry juice; however, these are only single observations and further research is needed to establish ifA. acidoterrestris strains representing both clusters are found in other juices.Genetic methods are broadly used in the detection, characterization, and differentiation of microorganisms. The application of PCR-RFLP in the characterization ofAlicyclobacillus and other thermoacidophilic bacteria isolated from the apple juice processing environment has been described by Chen et al. [52], although only the 16S rDNA gene was the subject of this study and the Hin6I enzyme was not used.RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis ofAlicyclobacillus so far, although the value of this gene in taxonomy has been confirmed by many studies on other microorganisms. To date, there are no reports on the use of thevdc gene cluster in taxonomic studies of microorganisms, although its sequence may be proven valuable for research both on the vectors and diversity and evolution of the element itself. Hin6I restriction patterns for 16S rDNA were sufficient to differentiate between all the species analyzed, but provided no closer data on intraspecies diversity. The diversity was revealed both by therpoB gene andvdc region RFLP analyses.Vdc gene cluster ofStreptomyces sp., described by Chow et al. [55], consists of three genes:vdcB (0.6kb),vdcC (1.4 kb), andvdcD (0.2 kb), transcribed as single, policistronic mRNA molecule. All three genes were essential to produce guaiacol from vanillic acid. Niwa and Kawamoto [38] described analogical gene cluster inA. acidoterrestris, consisting of ORF1 (597 bp), ORF2 (1425 bp), and ORF3 (321 bp). Three respective open reading frames were found in all five analyzedvdc sequences.Alicyclobacillus acidoterrestris RFLP patterns of both therpoB gene andvdc region showed consistently that there were two major types among the isolates, one similar to the reference strainA. acidoterrestris DSM 2498, and the other similar to the reference strainA. acidoterrestris ATCC 49025. Obtaining such consistent data concerning two genetic elements of distant function reveals that there is deeper intraspecies genetic diversity in theA. acidoterrestris species.This division may be considered when examining other features ofAlicyclobacillus such as susceptibility to temperature or high hydrostatic pressure, in order to establish if there are any differences between the groups.Both therpoB gene andvdc region seem to be single-copy genetic elements and showed no signs of intragenomic heterogeneity, which often makes the RFLP comparison of rDNA genes harder to perform.The sequence analysis of the 16S rDNA fragment of selected isolates shows no apparent correlation between diversity of the 16S rDNA sequence and the sources which the strains were isolated from.In conclusion, the application of PCR-RFLP has been proven to be a fast and reliable method forAlicyclobacillus identification and differentiation. The method is also technically less difficult than most of other molecular techniques. Two major groups ofA. acidoterrestrishave been identified. The primers designed for this study could be useful in further research onAlicyclobacillus.



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