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Title: Efficiency of the V3 region of 16S rDNA and the rpoB gene for bacterial 1
community detection in Thai traditional fermented shrimp (Kung-Som) using PCR-2
DGGE techniques 3
Running title: Bacterial community detection in fermented shrimp (Kung-Som) using 4
PCR-DGGE 5
Authors: Chatthaphisuth Sanchart1, Soottawat Benjakul
2, Onnicha Rattanaporn
3, 6
Dietmar Haltrich4, Suppasil Maneerat
1* 7
Affiliations:1Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince 8
of Songkla University, Hat Yai, 90112, Thailand; 2Department of Food Technology, 9
Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, 10
Thailand; 3Department of Biochemistry, Faculty of Science, Prince of Songkla 11
University, Hat Yai, Songkhla 90112 Thailand; 4Department of Food Sciences and 12
Technology, Food Biotechnology Laboratory, BOKU University of Natural Resources 13
and Life Sciences, Vienna, Austria. 14
*Corresponding author E-mail: [email protected]; Tel.: +66-74-286379; Fax: +66-15
74-558866 16
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Abstract 25
Kung-Som is one of several Thai traditional fermented shrimp products, that is 26
especially in the southern part of Thailand. This is the first report to reveal the bacterial 27
communities in the finished product of Kung-Som. Ten Kung-Som samples were 28
evaluated using the polymerase chain reaction-denaturing gradient gel electrophoresis 29
(PCR-DGGE) methodology combined with appropriated primers to study the dynamics 30
of the bacterial population. Two primers sets (V3; 341f(GC)-518r and rpoB; 31
rpoB1698f(GC)-rpoB2014r primers) were considered as a possible tool for the 32
differentiation of bacteria and compared with respect to their efficiency of 16S rDNA 33
and rpoB gene amplification. PCR-DGGE analysis of both the V3-region and rpoB 34
amplicon was successfully applied to discriminate between lactic acid bacteria and 35
Gram positive strains in the bacterial communities of Kung-Som. In conclusion, the 36
application of these two primers sets using PCR-DGGE techniques is a useful tool for 37
analyzing the bacterial diversity in Kung-Som. Moreover, these preliminary results 38
provide useful information for further isolation of desired bacterial strains used as a 39
starter culture in order to improve the quality of Kung-Som. 40
Keywords: Kung-Som, rpoB gene, V3 region, PCR-DGGE, Bacterial community 41
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Introduction 49
Kung-Som is a traditional fermented shrimp product that is found widely 50
distributed in the south of Thailand. It is made from shrimp, sugar, salt and water and is 51
typically fermented with the natural, spontaneous microbial flora. The microbiology of 52
Kung-Som is diverse and complex. The principal microorganisms found in Kung-Som 53
are various lactic acid bacteria (LAB) (Tanasupawat et al., 1998; Hwanhlem et al., 54
2010). Species identification and population enumeration are critical in the study of 55
bacterial communities. Due to the limitations of conventional microbiological methods, 56
the identification of microorganisms that requires selective enrichment and subculturing 57
is problematic or impossible. Moreover, classical microbial techniques used have not 58
accurately analyzed the presence of the main bacterial species (Ben Omar and Ampe, 59
2000) and have not provided a completely accurate representation of these complex 60
communities. On the other hand, culture-independent molecular techniques have 61
provided better methods to give more information on the microbial diversity in complex 62
food samples (Cocolin et al., 2001; Ercolini, 2004). In addition, culture-independent 63
molecular techniques based on specific nucleotide sequences are widely used for 64
monitoring, detection, identification and classification of bacterial diversity. 65
In the recent decade, polymerase chain reaction-denaturing gradient gel 66
electrophoresis (PCR-DGGE) has been successfully applied to determine the 67
microbiology of fermented food such as fermented sausages (Fontana et al., 2005), 68
fermented grains (Chao et al., 2008), fermented meat (Hu et al., 2009), and fermented 69
dairy products (Liu et al., 2012), to name a few. This approach has provided new insight 70
into the microbial diversity and allowed a more rapid, high-resolution description of 71
microbial communities than did the traditional approaches since it allows the separation 72
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of DNA molecules that differ by single bases (Ercolini, 2004). The use of appropriate 73
consensus primers is also a critical point in determining the resolution of DGGE 74
analysis in mixed microbial systems, especially in LAB differentiation (Chen et al., 75
2008). From the evidence of several published papers, the 16S rDNA seems to be by far 76
the most widely used as a molecular marker for the determination of the phylogenetic 77
relationships of bacteria. The hypervariable V3-region on the 16S rDNA is the most 78
frequently used to start the study of an unknown and complex bacterial community. In 79
addition, the V3-region is considered to have a high grade of resolution and to be highly 80
variable, and it is regarded as a good choice when it comes to length and inter-species 81
heterogeneity (Coppola et al., 2001; Florez and Mayo, 2006; Hovda et al., 2007; Chen 82
et al., 2008). Unfortunately, one problem related to the use of 16S rDNA in DGGE 83
analysis is the complexity created by the existence of multiple heterogeneous copies 84
within a genome (Dahllof et al., 2000; Crosby and Criddle, 2003; Rantsiou et al., 2004). 85
Consequently, a solution to the problem of 16S rDNA heterogeneity is provided 86
by the analysis of a gene that exists in only a single copy (Fogel et al., 1999). Certain 87
protein-coding genes, such as the gene encoding the beta-subunit of DNA-directed 88
RNA polymerase, rpoB, have been proposed to fulfill this criterion. rpoB is used as a 89
potential biomarker to overcome identification problems because it is considered a 90
housekeeping gene. Targeting the rpoB gene allowed the reliable discrimination of 91
species. The use of this gene as a marker was able to avoid the intraspecies 92
heterogeneity problem caused by the use of the 16S rDNA, which appears to exist in 93
one copy only in bacteria (Dahllof et al., 2000; Ko et al., 2002). In addition, in some 94
strains of bacteria, an internal region of rpoB is a more suitable sequence than 16S RNA 95
because of its higher nucleotide polymorphism (Khamis et al., 2005). However, the use 96
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of rpoB presents a taxonomic disavadantage: the database of the sequence is less well 97
documented than that of the 16S rDNA (Rantsiou et al., 2004; Renouf et al., 2006). 98
There are no data using the PCR-DGGE technique to characterize the dominant 99
bacteria in Kung-Som product. Consequently, the aim of this present study was to focus 100
on the use of the hypervariable V3-region on the 16S rDNA and rpoB gene by using 101
PCR-DGGE techniques as a tool to reveal the bacteria that commonly develop in the 102
Kung-Som product and to compare the efficiency of the 16S rDNA and rpoB gene 103
sequences for species discrimination in such a complex food sample. The obtained 104
results provide preliminary information for study to apply in the microbial starter 105
cultures in Kung-Som fermentation. 106
Materials and Methods 107
Lactic acid determination in Kung-Som 108
Kung-Som samples were purchased from different local markets in Songkhla 109
Province, Thailand. The pH value was measured by a pH meter (420A ORION, USA). 110
Total acidity as lactic acid was determined according to the AOAC (AOAC, 1995). 111
Three independent measurements were made for each sample. Data presented are the 112
means and standard deviations calculated. 113
DNA extraction from Kung-Som 114
DNA was extracted from the juice sample of Kung-Som by the method Cocolin 115
et al. (2004), with slight modification. One millilitre of juice sample of each sample was 116
centrifuged at 14,000×g for 10 min at 4 °C to pellet the cells. The pellet was washed 117
twice with 1 ml of sterile 0.85% (w/v) NaCl. The pellet was resuspended in 50 µl of 20 118
mgml-1
lysozyme (Fluka, USA). After 30 min incubation at 37 °C, 30 µl of 25 mgml-1
119
proteinase K (AMRESCO®
, USA) and 150 µl proteinase K buffer were added. The 120
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tubes were incubated at 65 °C for 90 min before the addition of 400 µl breaking buffer 121
and incubated futher at 65 °C for 15 min. Then, 400 µl of phenol:chloroform:isoamyl 122
alcohol (25:24:1, pH 6.7) was added for extracting DNA, RNA and protein. The tubes 123
were centrifuged at 12,000×g at 4 °C for 10 min, the aqueous phase was collected and 124
the nucleic acid was precipitated with 1 ml of ice-cold absolute isopropanol. The DNA 125
was obtained by centrifugation at 14,000×g at 4 °C for 10 min, washed briefly with 70% 126
(v/v) ice-cold ethanol and centrifuged again. The DNA was dried at room temperature, 127
resuspended in 20 µl of RNase-DNase-free sterile water, and treated with 5 µl of 10 128
mgml-1
DNase-free Rnase (Vivantis, USA). After 5 min incubation at 37 °C, genomic 129
DNA was stored at -20 °C. 130
The V3 region of 16S rDNA and rpoB gene amplification 131
Primers 341f (5´-CCTACGGGAGGCAGCAG-3´) and 518r 132
(5´ATTACCGCGGCTGCTGG-3´) were used to amplify a region of approximately 200 133
bp of the V3 region of 16S rDNA (Muyzer et al., 1993). Primers rpoB1698f (5’-134
AACATCGGTTTGATCAAC-3’) and rpoB2014r (5’-CGTTGCATGTTGGTACCCAT 135
-3’) were used to amplify a region of approximately 350 bp of the rpoB gene (Dahllof et 136
al., 2000). Amplification reactions were carried out in volumes of 50 µl. In addition, a 137
GC clamp was added to the forward primer to improve the sensitivity in the detection of 138
mutations by DGGE (Sheffield et al., 1989). PCR products were examined by 2% (w/v) 139
agarose gel electrophoresis. These were used to check the quality and size of PCR 140
products before being subjected to DGGE analysis. 141
DGGE analysis 142
DGGE analysis was performed using the Dcode universal mutation detection 143
system apparatus (Cleaver Scientific, UK) according to Fontana et al. (2005) with slight 144
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modification. Thirty millilitres of PCR product was mixed with loading dye and applied 145
to 8% (w/v) polyacrylamide gels. A 28% to 55% denaturing gradient (100% of 146
denaturant corresponded to 7 mol l-1
urea and 40% formamide) were used for both the 147
341f(GC)-518r and rpoB1698f(GC)-rpoB2014r primer sets. Electrophoresis was run in 148
1X TAE buffer at constant temperature (60 ºC) for 10 min at 20 V and subsequently for 149
16 h at 85 V. After electrophoresis, the gel was stained for 30 min with 1X (final 150
concentration) SYBR Gold (Invitrogen, USA) in 1X TAE buffer, rinsed in water, and 151
then visualized and photographed under UV illumination with the Gel Documentation 152
(UVI-TECH, England). 153
After running the DGGE analysis, relevant bands were punched from the gel 154
with sterile pipette tips. Each piece was transferred into 20 µl of RNase-DNase-free 155
sterile water and incubated overnight at 4 ºC to allow the diffusion of the DNA. Then, 156
the eluted DNA was used as a template and re-amplification took place with primers 157
without the GC clamp. The PCR products were purified by using the HiYieldGel/PCR 158
DNA Fragments Extraction Kit (RBC, Taiwan), and sequenced by a DNA sequencer 159
(Ward Medic Ltd., Malaysia). 160
Construction of phylogenetic tree 161
Searches in GenBank with the BLAST program on the NCBI website were 162
performed to determine the closest known relatives of the determined 16S V3 region 163
and rpoB gene sequences. Multiple sequence alignments were created by using the 164
BioEdit version 3.3.19.0. The phylogenetic tree was obtained to compare similarities 165
among the sequences by the neighbor-joining method (Saitou and Nei, 1987) using the 166
MEGA software version 5. Kimura’s method was followed and 1,000 repetitions were 167
made for bootstrap (Tamura et al., 2011). 168
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Results and Discussion 169
Kung-Som is a one of the traditional fermented food products from southern 170
Thailand. The production process traditionally relies on a spontaneous fermentation 171
initiated by natural and fortuitous microorganisms, mainly various LAB and coagulase-172
negative cocci (CNC). Kung-Som is made from the main raw materials shrimp, sugar, 173
salt and water. These raw materials and personal hygiene can also be the possible 174
sources of pathogenic microorganisms or spoilage bacteria such as Escherichia coli, 175
Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus and Salmonella sp. In 176
all Kung-Som samples of this study, it was found that the pH and lactic acid 177
concentration ranged from 3.58 to 4.04 and 1.78% to 3.12%, respectively (Fig.1). The 178
pH of all samples was below 4.5 because LAB utilizes the carbohydrate substrates 179
available to produce organic acids, and especially lactic acid, as part of their 180
metabolites. These acids not only contribute to the taste, aroma and texture of the 181
product but also lower the pH of the product which is one of the important key factors 182
to ensure quality and safety (Visessanguan et al., 2006; Kopermsub and Yunchalard, 183
2010). Generally, a pH lower than 4.4 can inhibit growth of E. coli (Alvarado et al., 184
2006) and Salmonella sp. (Sorrells and Speck, 1970); a pH lower than 3.7 can inhibit S. 185
aureus (Alvarado et al., 2006); a pH lower than 4.0 inhibits B. cereus (Yang et al., 186
2008) and a pH of 4.5-5.0 has been demonstrated to inhibit V. parahaemolyticus 187
(Adams and Moss, 2008). Consequently, the pathogenic or spoilage bacteria were 188
inhibited by organic acids that affected the bacterial growth and extracted DNA 189
concentration in our samples. This finding is probably related to the PCR-DGGE 190
detection limit (104 cfuml
-1). In accordance with these results, no DNA bands 191
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corresponding to pathogenic and spoilage bacteria were detected in Kung-Som samples 192
of our study. 193
Renouf et al. (2006) reported that the first step for finding suitable primers is to 194
assume that the primers must be present in all the species and delimit variable 195
sequences to separate each species. The last step is to find the most suitable and 196
accurate gradient and the best DGGE conditions (temperature, time). Two sets of 197
primers (V3-region of 16S rDNA and rpoB gene) were considered suitable in this study 198
since it is a housekeeping gene. Consequently, the bacterial diversity of the Kung-Som 199
product was revealed by DGGE analysis. Figure 2 and 3 show the DGGE profiles 200
obtained by the DNA directly extracted from Kung-Som in different regions in 201
Songkhla Province, Thailand. Both the V3 region of 16S rDNA and the rpoB gene were 202
amplified from these DNA templates. The V3 region of 16S rDNA and rpoB gene 203
profiles displayed different patterns. The DGGE profile of the rpoB gene amplification 204
showed that the numbers of the bands were lower than those of the V3 region of 16S 205
rDNA amplification. For the DGGE profile and the phylogenetic relationship of the V3 206
region of 16S rDNA amplification (Fig. 2A and 2B), bands corresponding to 207
Tetragenococcus halophilus (band b), Lactobacillus farciminis (band d) and 208
L. plantarum (band l and k) were prominent in all Kung-Som samples, which is in 209
agreement with data published by Hwanhlem et al. (2010). Although band k was found 210
in all samples, it was faint. L. acetotolerans (band g) and L. rapi (band j, o, p) were 211
present in all samples except samples 3 and 6. Salinivibrio sharmensis (band a) and 212
Macrococcus sp. (band e) appeared only in sample 3 and L. crustorum (band h and i) 213
was only found in sample 6. Staphylococcus piscifermentans (band c), Weissella 214
thailandensis (band m) and W. cibaria (band n) were exhibited in some samples but with 215
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very weak intensity. Salinivibrio sharmensis (band a) was found only in sample 3. 216
Several bands originating from a single species were observed on the DGGE gels, and 217
these were from L. crustorum, L. rapi and L. plantarum. The reason for this is the 218
sequence heterogeneity as described by Crosby and Criddle (2003). 219
For the DGGE profile and the phylogenetic relationship of rpoB gene 220
amplification (Fig. 3A and 3B), bands 4 and 5, corresponding to W. thailandensis and 221
S. piscifermentans, were predominating in every sample. Furthermore, L. fermentum 222
(band 2) and L. reuteri (band 3) were present in all samples except sample 3 and 6. 223
Band 1 was only detected in sample 3. 224
Species-specific DGGE bands from two sets of primers for the main members of 225
LAB and CNC were exhibited. LAB (Lactobacillus, Tetragenococcus and Weissella), 226
including CNC species (Macrococcus and Staphylococcus), are the most commonly 227
isolated bacteria from fermented foods, especially meat and fish (Hu et al., 2008; 228
Kopermsub and Yunchalard, 2010; Hwanhlem et al., 2011). In addition, the genus 229
Salinivibrio was found to be the dominant species in sample 3 of the V3 region 230
amplification. These species are generally isolated from fermented fish samples 231
(Chamroensaksri et al., 2009). The differences in the results obtained, such as the 232
DGGE pattern from the rpoB gene amplification, indicated the occurrence of very low 233
bacterial diversity when compared with the V3-region amplification. These results 234
depended on the specificity of the primers (Endo and Okada, 2005; Renouf et al., 2006) 235
and the PCR conditions (Hongoh et al., 2003). 236
Rantsiou et al. (2004) and Renouf et al. (2006) reported that the use of rpoB 237
gene amplification combined with PCR-DGGE can only reveal the predominant species 238
in a sample. Moreover, Chen et al. (2008) suggested that the use of appropriate 239
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consensus primers is a critical point in influencing the resolution of DGGE analysis in 240
mixed microbial systems, especially in LAB differentiation. Endo and Okada (2005) 241
indicated that LAB could not be detected by universal bacterial PCR primer but were 242
detected when groups of LAB-specific primer were used. This is because the DGGE 243
profile can demonstrate only the diversity of bacteria present at more than 1% of the 244
target bacteria. Therefore, the detection of numerous different species present at low 245
concentrations appeared to be difficult using PCR-rpoB/DGGE. Thus, the rpoB gene 246
pattern exhibited the different species which did not appear in the V3-region pattern. 247
In both the rpoB gene and V3-region patterns, samples 3 and 6 showed different 248
DGGE patterns from those of compared to other samples. This is because these samples 249
were originated from different recipes or processes of preparation which could vary the 250
initial food matrix, fermentation process, personal hygiene, local tradition or local 251
geographic preferences. All of these are crucial factors in determining the growth of 252
specific microbial communities (Cocolin et al., 2004; Ercolini, 2004; Chen et al., 2008). 253
This outcome was related to the higher pH and lower lactic acid content of samples 3 254
and 6 (Fig.1). In accordance with this lower lactic acid concentration, a difference in the 255
LAB groups of these samples was detected (Fig. 2 and 3). 256
A number of faint bands could not be identified because of their low content 257
which might be related to the heterogeneous distribution of microorganism in the food 258
matrix (Florez and Mayo, 2006). In a detection limit analysis, an individual species 259
(Pediococcus pentosaceus DMST 18752) was identified by PCR-DGGE when its 260
number was higher than 104 cfuml
-1 (data not shown). The detection limit of PCR-261
DGGE depends on the species or perhaps even the strain considered. Furthermore, the 262
number and the concentration of the other members of the microbial community, along 263
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with the nature of the food matrix, all represent variables influencing the detection limit 264
of DGGE. These factors affect both the efficiency of DNA extraction and the PCR 265
amplification due to possible competition among templates (Ercolini, 2004; 266
Temmerman et al., 2004; De Vero et al., 2006). 267
Conclusion 268
The suitability of the primers used was based on the discriminatory efficiency of 269
the hypervariable V3-region and the rpoB gene that allowed species differentiation from 270
the dominant groups of bacteria in Kung-Som. Although the applications of PCR-DGGE 271
techniques combined with appropriate consensus primers to study complex microbial 272
communities originating from food samples have been shown to be an efficient tool for 273
detection of complex bacteria populations, we believe that our findings represent a 274
preliminary analysis. The data can not be considered sufficient to achieve a confident 275
identification at species level, but should suggest that the relevant genes together with 276
other target sequences such as other region of 16S rDNA, gyrA, gyrB, recA or rpoC, 277
should be used for unequivocal identification of individual species. Moreover, these 278
preliminary results provide useful information for improving product quality. An 279
improved understanding of the changing microflora in Kung-Som fermentations could 280
be used to develop a starter culture in the future. 281
Acknowledgements 282
This study was financially supported by the Thailand Research Fund through the 283
Royal Golden Jubilee Ph.D Program (Grant No. PHD/0060/2556), the Graduate School 284
and the Thailand Research Fund Senior Scholar Program Prince of Songkla University. 285
References 286
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415
Figure captions 416
Fig. 1 The pH value and titratable acidity of Kung-Som samples. 417
Fig. 2 DGGE profiles (A) and the phylogenetic tree based on V3-region on 16S rDNA 418
(B) of the bacteria community obtained from DNA directly extracted from Kung-Som 419
samples. The scale bar represents the number of inferred substitutions per site. 420
Fig. 3 DGGE profiles (A) and the phylogenetic tree based on rpoB gene (B) of the 421
bacteria community obtained from DNA directly extracted from Kung-Som samples. 422
The scale bar represents the number of inferred substitutions per site. 423
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Fig. 1
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a
e
h i
k
c
d
f g
j
l
n
o p
b
m
A
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
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Fig. 2
CS-d
Lactobacillus farciminis KCTC 3681T (AEOT01000034)
Lactobacillus crustorum LMG 23699T (AM285450)
CS-k, CS-l
Lactobacillus plantarum HK01 (JN671595)
CS-h, CS-i
Lactobacillus pentosus ATCC 8041T (D79211)
CS-j, CS-o, CS-p
Lactobacillus rapi YIT 11204T (AB366389)
Lactobacillus buchneri JCM 1115 (NR 041293)
Lactobacillus parabuchneri ATCC 49374 (AB205056)
CS-g
Lactobacillus acetotolerans JCM 3825T (AB303841)
Lactobacillus homohiochii NBRC 13120 (AB680370)
CS-b
Tetragenococcus halophilus NBRC 12172 (AP012046)
Tetragenococcus halophilus IAM 1676T (D88668)
Tetragenococcus koreensis JS T (AY690334)
Staphylococcus xylosus ATCC 29971T (D83374)
CS-c
Staphylococcus carnosus ATCC 51365T (AB009934)
Staphylococcus piscifermentans ATCC 51136T (AB009943)
CS-e
Macrococcus brunensis LMG 21712 (AY119686)
Macrococcus bovicus ATCC 51825 (Y15714)
Macrococcus hajekii CCM 4809 (AY119685)
Weissella confusa JCM 1093T (AB023241)
Weissella kimchii DSM 14295 T (AF312874)
CS-m
Weissella sp. NBRC 107204 (AB682503)
Weissella thailandensis FS61-1T (AB023838)
Weissella thailandensis 5-5 (HQ384297)
Weissella thailandensis fsh4-2 (HE575179)
CS-n
Weissella cibaria BJ31-3C (AY341572)
Weissella cibaria LMG 17699T (AJ295989)
Weissella paramesenteroides NRIC 1542 (AB023238)
Weissella paramesenteroides ATCC 33313T (ACKU01000017)
CS-a
Salinivibrio costicola ATCC 35508T (X74699)
Salinivibrio costicola SI3 (HQ641751)
Salinivibrio proteolyticus AF-2004 (DQ092443)
Salinivibrio sharmensis BAGTT (AM279734)
Salinivibrio budaii MML 1937 (JN848571)
Escherichia coli20-10-1-9 (JF919881)
100
98
71
95
84
93
59
89
89
88
68
87
86
69
61
58
63
50
55
78
0.1
B
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1 2
4
3
5
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
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Fig. 3
Lactobacillus fermentum CECT 5716 (CP002033)
Lactobacillus fermentum IFO 3956 (AP008937)
Lactobacillus fermentum CNRZ 246 (AF515648)
CS2-rpo
CS3-rpo
Lactobacillus reuteri BR11 (GU191835)
Lactobacillus reuteri SD2112 (CP002844)
Lactobacillus reuteri JCM 1112 (AP007281)
CS1-rpo
Lactobacillus pentosus IG1 (FR874854)
Lactobacillus pentosus MP-10 (FR871825)
Lactobacillus plantarum JDM1 (CP001617)
Lactobacillus plantarum DSM 20174 (AF515652)
CS4-rpo
Weissella thailandensis fsh4-2 (HE575158)
Weisella paramesenteroides (Y16471)
Weissella paramesenteroides DSM 20288 (AF531265)
Staphylococcus simulans DSM 20322 (JN190408)
Staphylococcus piscifermentans DSM 7373 (DQ120745)
Staphylococcus piscifermentans CCM 4789 (HM146318)
CS5-rpo
Staphylococcus condimenti DSM 11675 (DQ120734)
Staphylococcus carnosus subsp. utilisDSM 11677 (DQ120731)
Staphylococcus carnosus subsp. carnosus DSM 20501 (JN190401)
Escherichia coli CIP 54.8 (EU010107)
98
87
92
83
94
90
97
98
93
100
84
93
87
100
60
57 70
98
95
0.1
B
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