Curr Microbiol (2014) 68:551–557 DOI 10.1007/s00284-013-0512-5
Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli Iva Jebava • Victoria Chuat • Sylvie Lortal Florence Valence
•
Received: 10 October 2013 / Accepted: 29 October 2013 / Published online: 21 December 2013 Ó Springer Science+Business Media New York 2013
Abstract We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species ofl actobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-013-0512-5) contains supplementary material, which is available to authorized users. I. Jebava (&) Department of Dairy, Fat and Cosmetic Science, Institute of Chemical Technology, Prague, Czech Republic e-mail:
[email protected] V. Chuat S. Lortal F. Valence CIRM-BIA, UMR 1253—Science et Technologie du Lait et de l’OEuf, Institut National de la Recherche Agronomique, Rennes, France
Introduction Lactobacillus helveticus, a part of the L. acidophilus taxonomic subgroup, is hardly distinguishable by simple physiological and biochemical testing from other closely related thermophilic homofermentative lactobacilli such as L. acidophilus, L. amylovorus, L. crispatus, L. johnsonii, L. gasseri, and especially from the phylogenetically closest species L. gallinarum [10, 12, 17]. Accurate identification of this industrially relevant species is essential in basic and applied research [1]. Lactobacillus helveticus, known also as a probiotic and bioactive peptides producing culture, is mainly used as a starter or adjunct culture in cheese and some fermented drinks [15, 24, 34]. Contrary to L. helveticus which originates principally from dairy sources, L. gallinarum is mainly found in poultry and also in some dairy and non-dairy products like sourdough along with L. helveticus [11, 20, 21, 29, 32]. Nowadays, some simple molecular tools are available for identifying L. helveticus. The first reported DNA-based identification method was based on hybridization of a cloned 2 kb fragment from a 34 kb plasmid in L. helveticus CNRZ1094 [6]. Regarding PCR identification of L. helveticus, specific primers to amplify gene coding for the surface layer (S-layer) protein were also proposed [30]. Later, another PCR based on primers targeting genes coding for aminopeptidase C (pepC), aminopeptidase N (pepN), and trypsin-like serine protease (htrA) was described [9]. For simultaneous detection of L. helveticus and other thermophilic starter cultures in Grana Padano cheese, a multiplex PCR and a PCR assay targeting the genes coding for a cellenvelope associated proteinase (prtH) or the phenylalanyltRNA synthase alpha subunit (pheS) were developed [2, 5]. Remarkably, none of these methods used L. gallinarum as a negative control, except for the work of Ventura et al. [30].
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Table 1 Bacterial strains used in this study Species
Source
Strain
Equivalent
Biotope
Origin
L. helveticus
CCDM
BAII
nd
nd
Czech Republic
CCDM
112
nd
Highland isolate
Czech Republic
CCDM
121
nd
L. helveticus var. pragenesis, starter
Czech Republic
CCDM
125
nd
Starter
Czech Republic
CCDM
136
nd
Highland isolate
Czech Republic
CCDM
139
nd
Highland isolate
Czech Republic
CCDM
140
nd
Highland isolate
Czech Republic
CCDM
380
nd
nd
Czech Republic
CCDM
466
nd
Highland isolate
Czech Republic
CCDM
467
nd
Highland isolate
Czech Republic
CCDM CCDM
468 499
nd nd
Czech Republic Czech Republic France
L. acidophilus
CIRM-BIA
99
ITG LH77
Highland isolate Highland isolate Starter, Comte´ production
CIRM-BIA
100
CNRZ 303
Starter, Comte´ production
France
CIRM-BIA
101
CIP 103146T
CIRM-BIA
102
CNRZ 241
Starter, Emmental production Starter, Comte´ production
France France
France
CIRM-BIA
103
CNRZ 32
Starter, Comte´ production
CIRM-BIA
104
ISLC 5
Starter, Parmesan production
Italy
CIRM-BIA
105
CIP 615
nd
Japan
CIRM-BIA
106
CNRZ 414
Isolate from Koumis (cow’s milk)
Russia
CIRM-BIA
107
ITG LH1
Serum
France
DPC
4571
nd
Swiss cheese whey
Ireland
ICT
BROI
nd
Isolate from raw cow’s milk
Czech Republic
ICT
CH1
nd
Commercial starter
Czech Republic
ICT
KUM
nd
Isolate from Koumis (mare‘s milk)
Kazakhstan
CCDM
151
nd
nd
nd
CIRM-BIA CIRM-BIA
438 439
CNRZ 55 CNRZ 1880
Isolate from mouse excrement Human isolate
United Kingdom nd
CIRM-BIA
444
CNRZ 216
Isolate from mouse digestive tract
France
ICT
3006
nd
nd
nd
ICT
3007
nd
nd
nd
CIRM-BIA
773
CNRZ 449
Starter, Yogurt production
France
CIRM-BIA
776
nd
Starter, Yogurt production
France
CIRM-BIA
658T
CNRZ 208
Starter, Yogurt production
nd
ICT
30117
nd
nd
nd
CIRM-BIA
208
CNRZ 232
nd
nd
CIRM-BIA
210
CNRZ 226
nd
nd
CIRM-BIA
220T
CNRZ 207
Starter, Emmental production
nd
ICT
30139
nd
nd
nd
CCDM
1070T
nd
nd
nd
CIRM-BIA
523
CNRZ 1925
Isolate from chicken
USA
CIRM-BIA CIRM-BIA
664 668T
CNRZ 1924 CNRZ 1931
Conjunctivitis Isolate from chicken
France USA
CIRM-BIA
1322
nd
nd
nd
CIRM-BIA
1323
nd
nd
nd
L. amylolyticus
CIRM-BIA
669T
CNRZ 1928
Isolate from fermented corn
nd
L. johnsonii
CIRM-BIA
650
CNRZ 251
Starter, Gruyere production
France
CIRM-BIA
651
CNRZ218
Isolate from mouse digestive tract
France
L. delbruecki subsp. bulgaricus
L. delbruecki subsp. lactis
L. crispatus
L. gallinarum
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I. Jebava et al.: Peptidoglycan Hydrolases as Species-Specific Markers
553
Table 1 continued Species
L. rhamnosus
Source
Strain
Equivalent
CIRM-BIA
674T
CIRM-BIA
572
CIRM-BIA
604 T
Biotope
Origin
CNRZ 1937
Isolate from human blood
USA
nd
Raw cow0 s milk isolate
Brasil
nd
Raw cow0 s milk isolate
Brasil nd
CIRM-BIA
607
CNRZ 212
nd
ICT
30105
nd
nd
nd
CIRM-BIA
666T
CNRZ 209
Fermented sugar-beet
nd
CIRM-BIA
697
CNRZ 1627
Starter
Italy
CIRM-BIA
872
CNRZ 17
Starter, Gruyere production
France
L. plantarum
ICT
3011
nd
nd
nd
L. paracasei
ICT
30125
nd
nd
nd
L. reuteri
ICT
30111
nd
nd
nd
L. ultunensis
CCDM
838
nd
nd
nd
L. fermentum
CCDM Czech collection of dairy microorganisms, Prague, Czech Republic, CIRM-BIA Centre international de resources Microbiennes–Bacte´ries d0 Inte´reˆt Alimentaire, INRA Rennes, France, CNRZ Centre National de Recherche Zootechnique collection, INRA Jouy-en-Josas, France CIP Collection of Institut Pasteur, Paris, France ICT Institute of Chemical Technology, Prague, Czech Republic, ISLC Instituto Sperimentale Lattiero Caseario di Lodi, Italy, ITG Institut Technique du Gruye`re, La Roche-sur-Foron, France, nd not determined
To our knowledge, a simple PCR method to differentiate L. helveticus from its genomically closest species, L. gallinarum, has not been reported yet. Although the 16S rRNA gene sequencing is increasingly used to identify lactic acid bacteria, high sequence similarity between closely related species makes their discrimination at the genomic level difficult. Particularly, genotypic differentiation between L. helveticus and L. gallinarum is still not evident [7, 11, 14, 18, 23]. The first complete genome of L. helveticus DPC 4571 enabled to perform bioinformatic analysis of orthologous genes between related species [4]. Among the potentially genomic markers the peptidoglycan hydrolases, essential enzymes for cell growth and division, have shown important homology within L. helveticus strains [16, 28]. Peptidoglycan hydrolases are ubiquitous and contrary to cell wall structure [33]; they do not participate on different autolytic capacity among L. helveticus strains. Two conserved genes coding for peptidoglycan hydrolases in L. helveticus, Lhv_0190 and Lhv_0191, are known to be organized in an operon, but display different levels of sequence homology between themselves. The objective of this study was to develop a PCR method based on Lhv_0190 and Lhv_0191 genes to differentiate L. helveticus from other closely related homofermentative lactobacilli, especially from L. gallinarum.
Materials and Methods Bacterial Strains and Growth Conditions A total of 25 L. helveticus strains, characterized previously by Jebava et al. [16] from CIRM-BIA (Centre International
de Ressources Microbienne—Bacte´rie d0 Inte´reˆt Alimentaire, Rennes, France) and CCDM (Czech Collection of Dairy Microorganisms, Prague, Czech Republic), were used in this study. In addition, 35 strains of other Lactobacillus spp. were included as negative controls (Table 1). Cultures were grown in MRS broth (Difco Laboratories, MI, USA) at 42 °C for the following species: L. helveticus, L. delbrueckii, L. fermentum, L. acidophilus, and L. ultunensis; at 37 °C for L. johnsonii, L. amylolyticus, L. crispatus, and L. gallinarum, and at 30 °C for the other species. Bacterial growth was monitored at 650 nm using a spectrophotometer Beckman DU 7400 (Beckman Coulter, Nyon, Switzerland). Genomic DNA Extraction Genomic DNA was extracted from strains grown for 12 h at 42 °C in MRS broth by DNeasy Blood&Tissue Kit (QIAGEN, Hilden, Germany) as described previously [16]. In Silico Analysis of 16S rDNA Sequences All sequences were acquired from GenBank (NCBI, http:// www.ncbi.nlm.nih.gov/sites/gquery) and aligned using MEGA 5 software (http://www.megasoftware.net/) [22, 26] using the neighbor-joining method. Accession Nos of compared partial 16S rRNA gene sequences of type strains were: L. acidophilus DSM 20079T (equiv. NBRC 13951, GenBank ID: AB680529.1), L. amylovorus DSM 20531T (GenBank ID: AY944408.1), L. crispatus DSM 20584T (GenBank ID: AF257097.1), L. gallinarum DSM 10532T (equiv. JCM 2011T, GenBank ID: AB596947.1), L. gasseri DSM 20243T (equiv. CIP 102991T GenBank ID:
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Table 2 Specific primers designed for Lhv_0190 and Lhv_0191 using Primer3 software and applied in this work Sequence (50 –30 )
Position
PCR product length (bp)
Lhv_0190Fwd:
CAGTTGTGTTGACTTCCACAAT
35–57
542
Lhv_0190Re:
CAAATTGTGGCTGGTGATTCT
555–576
Lhv_0191Fwd:
GGGCTGATTACAGTGGCTAAT
43–64
Lhv_0191Re:
CTTGCCCTTTTCGGTGTAAA
769–789
Locus
Primer
Lhv_0190 Lhv_0191
HE573914.1), L. delbrueckii subsp. bulgaricus DSM 20081T (BCRC 10696T, GenBank ID: AY773948.1), L. helveticus DSM 20075T (GenBank ID: AY369116.1), L. johnsonii DSM 10533T (equiv. CIP 103602T, GenBank ID: AJ002515.1), L. salivarius DSM 20555T (equiv. JCM 1231, GenBank ID: AB370881.1), and L. ultunensis DSM 16047T (GenBank ID: AB370881.1). Primer Design Lhv_0190 and Lhv_0191 genes, coding for two peptidoglycan hydrolases of L. helveticus DPC 4571 (GenBank ID: NC_010080.1), were aligned with their ortholog genes in sequenced strains: L. helveticus DSM 20075 (GenBank ID: NZ_ACLM00000000.1), L. acidophilus NCFM (GenBank ID: NC_006814.3), L. delbrueckii subsp. bulgaricus ATCC 11842 (GenBank ID: NC_008054.1), L. crispatus 125-2-CHN (GenBank ID: NZ_ACPV00000000.1), L. crispatus ST1 (GenBank ID: NC_014106.1), and L. ultunensis DSM 16047 (GenBank ID: NZ_ACGU00000000.1). The comparison of two PGHs genes between L. helveticus and L. gallinarum cannot be performed due to the lack of L. gallinarum complete genome. The identical regions between ortholog genes of L. helveticus DPC 4571 and L. helveticus DSM 20075 as well as the different regions between L. helveticus DPC 4571 and other ortholog genes of other species (L. acidophilus, L. delbrueckii, L. crispatus, and L. ultunensis) were determined. Using Primer3Plus software [27], two pairs of specific primers (Table 2) were designed in the conserved regions of L. helveticus strains and, at same time, in the most variable regions of other species. The first set of primers (Lhv_0190Fwd and Lhv_0190Re) was used to amplify the sequence of the Lhv_0190 gene coding for the N-acetylglucosaminidase and the second set (Lhv_0191Fwd and Lhv_0191Re) to detect the Lhv_0191 gene coding for the N-acetylmuramyl-L-alanine amidase. PCR Amplification The composition of the PCR reaction mix was as follows: 20 ng genomic DNA, 2.5 lL 10 9 buffer containing MgCl2 (QIAGEN), 1 lL 10 mM dNTP Mix (QIAGEN), 1 lL of each primer (50 lM), and 2.5 U Taq DNA
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polymerase in a final volume of 25 lL. PCR was performed using a VeritiTM 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA). Similar conditions for single gene amplification and multiplex PCR were used. The PCR conditions were: denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 45 s. A final extension step was performed for 10 min at 72 °C before the storage of the samples at 4 °C. Negative (no DNA in sample) and positive (DNA from L. helveticus DPC 4571) controls were included in all PCR sets. All PCR products were analyzed by electrophoresis in a 1 % (w/v) agarose gel (Interchim, Montlucon, France) in a Mini-Sub cell GT system (BioRad, CA, USA). After electrophoresis, the gels were stained with ethidium bromide and visualized by UV light.
Results and Discussion In silico analysis of partial 16S rDNA gene was used to study genomic relationships among closely related obligatory homofermentative lactobacilli [25]. It showed that L. helveticus is mostly related to L. gallinarum (Fig. 1). Their genomic proximity explains the difficulty to perform accurate identification based only on 16S rRNA gene sequencing [8, 18, 31]. In order to perform the differentiation between these two closely related species, a PCR method based on the detection of genes coding for peptidoglycan hydrolases was designed. Since the complete genome of L. gallinarum is not available, the second closely related species, L. acidophilus (Fig. 1), was used to compare genes coding for peptidoglycan hydrolases. Lhv_0190 and Lhv_0191 sequences in L. helveticus DPC 4571 were compared to the orthologous genes, Lba_0176 and Lba_0177, in L. acidophilus NCFM. Lhv_0190 and Lhv_0191 and their orthologous genes in L. acidophilus NCFM showed 21.9 and 24.82 % variability in nucleotide sequences, respectively (for detail sequence analysis see Supplementary materials). This comparison revealed sufficient differences to construct species-specific primers to identify L. helveticus strains by PCR. Lhv_0190 and Lhv_0191 sequences in L. helveticus DPC 4571 were also compared to their orthologous genes, HMPREF0518_0918
I. Jebava et al.: Peptidoglycan Hydrolases as Species-Specific Markers
555
Fig. 1 Evolutionary relationship of selected homofermentative lactobacilli based on comparison of 16S rRNA. The analysis involved ten nucleotide sequences and, in total, 1,366 nucleotidic positions in the final dataset. All positions containing gaps and missing data were eliminated
Fig. 2 PCR products obtained by multiplex PCR for selected closely related lactobacilli. Lanes. 1-L. acidophilus CIRM-BIA 438, 2-L. delbrueckii subsp. bulgaricus CIRM-BIA 658T, 3-L. delbrueckii subsp. lactis CIRM-BIA 220T, 4-L. crispatus CIRM-BIA 1070T, 5-L. gallinarum CIRM-BIA 668T, 6-L. helveticus DPC 4571, 7-L.
amylolyticus CIRM-BIA 669T, 8-L. johnsonii CIRM-BIA 674T, 9-L. rhamnosus CIRM-BIA 607T, 10-L. fermentum CIRM-BIA 666T, 11-L. ultunensis CCDM 838, 12 negative control, M molecular weight marker
and HMPREF0518_0919, in L. helveticus DSM 20075. The intra-species homology achieved 92.16 and 99.54 % identity for L. helveticus DPC 4571 and L. helveticus DSM 20075 genes. The search for orthologous genes in other closely related species of L. helveticus to perform in silico analyses was limited by the few data available in genomic databases. Nevertheless, orthologous genes in L. delbrueckii, L. crispatus, and L. ultunensis could be compared to L. helveticus DPC 4571 genes. For Lhv_0190, the lowest homology was found at the positions 30–60 and 550–580, and so the primers were designed at positions 35–57 and 555–576 (Lhv_0190Fwd and Lhv_0190Re, Table 2). A set of primers for Lhv_0191 was constructed within two regions showing minimal homology at the positions 43–64 and 769–789 (Lhv_0191Fwd and Lhv_0191Re, Table 2). Attention was paid to compatibility of these sets of primers regarding the size of the PCR products generated, preserving the possibility to carry out a multiplex PCR reaction.
When using a single PCR for each set of primers, only one PCR product of the predicted size, 542 bp for Lhv_0190 and 747 bp for Lhv_0191, was obtained (data not shown). Following a multiplex PCR, using the two sets of primers together, two PCR products were obtained: the first one of 542 bp corresponding to Lhv_0190 gene and the second one of 747 bp corresponding to Lhv_0191 gene. The PCR method was applied to 25 strains of L. helveticus previously identified by sequencing the whole 16S rRNA gene [16]. Specific amplifications at the expected size of 542 and 747 bp were observed for all tested strains of L. helveticus, with two pairs of primers used as either single or in multiplex PCR (Fig. 2). To confirm the specificity of the proposed PCR method, the primers were applied to 33 strains belonging to 13 closely related species of lactobacilli (Table 1). There was no amplification observed except for L. gallinarum (Fig. 2). For this species, one amplicon of 542 bp corresponding to Lhv_190 gene and one additional amplicon
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greater than 1,500 bp were observed. These two amplicons were consistently observed for three other tested strains of L. gallinarum. To our knowledge, this proposed multiplex PCR is the first using peptidoglycan hydrolase genes for species identification of Lactobacillus species, even though the taxonomic interest of peptidoglycan hydrolase zymograms was highlighted previously [19]. For other species, e.g., Vibrio anguillarum, a PCR method based on the detection of amiB encoding the Nacetylmuramyl-L-alanine amidase was successfully applied for species discrimination [13]. Bacterial identification based on conserved peptidoglycan hydrolase gene sequence could be certainly used more generally to distinguish closely related bacterial species. Considering the decreasing cost and increasing availability of sequencing data, determination of nucleotide sequences of peptidoglycan hydrolases and their use to define species-specific primers could become widespread complementing 16S rRNA sequencing. The targeted genes, i.e., peptidoglycan hydrolases, could be used analogically for identification of other homofermentative lactobacilli. The accurate identification of bacterial species has a great relevance in food analysis, microbial ecology, and also in clinical diagnosis. The proposed multiplex PCR offered sensitive and rapid identification of L. helveticus and could be applied to detect L. helveticus in dairy products, such as cheese and fermented milks. Lactobacillus gallinarum is not important from a technological point of view, but until now, its potential presence in dairy products has not been taken into account [3, 29]. However, L. gallinarum identification itself could become very important in future research because of its potential antimicrobial properties [3].
Conclusion The detection of microorganisms is highly dependent on the specificity of the probes used for PCR. Most commonly used 16S rRNA, nucleotide sequences of small-subunit rRNA, may not always be high enough to distinguish closely related species even by subsequent sequencing. Therefore, the proposed multiplex PCR, highly specific for the identification of L. helveticus, should prove to be more powerful to discriminate between L. helveticus and L. gallinarum than the 16S rRNA sequencing used alone. A routine PCR technique with more accessible materials should increase the feasibility and accessibility of correct identification of these closely related species. Acknowledgments This work was supported by research grants from INRA and the Ministry of Education, Youth and Sport of the Czech Republic (Grant No. MSMT6046137305). We are grateful to Tom Beresford for providing the strain DPC 4571.
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References 1. Beresford TP, Fitzsimons NA, Brennan NL, Cogan TM (2001) Recent advances in cheese microbiology. Int Dairy J 11:259–274 2. Bottari B, Agrimonti C, Gatti M, Neviani E, Marmiroli N (2013) Development of a multiplex real-time PCR to detect thermophilic lactic acid bacteria in natural whey starters. Int J Food Microbiol 160:290–297 3. Bujnakova D, Kmet V (2012) Functional properties of Lactobacillus strains isolated from dairy products. Folia Microbiol 57:263–267 4. Callanan M, Kaleta P, O’Callaghan J, O0 Sullivan O, Jordan K, McAuliffe O, Sangrador-Vegas A, Slattery L, Fitzgerald GF, Beresford T, Ross RP (2008) Genome sequence of L. helveticus, an organism distinguished by selective gene loss and insertion sequence element expansion. J Bacteriol 190:727–735 5. Cremonesi P, Vanoni L, Morandi S, Silvetti T, Castiglioni B, Brasca M (2011) Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese. Int J Food Microbiol 146:207–211 6. De los Reyes-Gavilan CG, Limsowtin GKY, Tailliez P, Sechaud L, Accolas JP (1992) A L. helveticus—specific DNA probe detects restriction fragment length polymorphisms in this species. Appl Environ Microbiol 58:3429–3432 7. Du Plessis EM, Dicks LMT (1995) Evaluation of random amplified polymorphic DNA (RAPD)—PCR as a method to differentiate L. acidophilus, L. crispatus, L. amylovorus, L. gallinarum, L. gasseri, and L. johnsonii. Curr Microbiol 31:114–118 8. Felis GE, Dellaglio F (2007) Taxonomy of lactobacilli and bifidobacteria. Curr Issues Intest Microbiol 8:44–61 9. Fortina MG, Ricci G, Mora D, Parini C, Manachini PL (2001) Specific identification of L. helveticus by PCR with pepC, pepN and htrA targeted primers. FEMS Microbiol Lett 198:85–89 10. Fujisawa T, Benno Y, Yaeshima T, Mitsuoka T (1992) Taxonomic study of the L. acidophilus group, with recognition of L. gallinarum sp. nov. and L. johnsonii sp. nov. and synonymy of L. acidophilus Groupe A3 (Johnson et al. 1980) with the type strain of L. amylovorus (Nakamura et al. 1981). Int J Syst Bacteriol 42:487–491 11. Guan LL, Hagen KE, Tannock GW, Korver DR, Fasenko GM, Allison GE (2003) Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCRdenaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis. Appl Environ Microbiol 69:6750–6757 12. Holzapfel WH, Wood BJB (1998) The genera of lactic acid bacteria. Blackie Academic and Professional, London 13. Hong GE, Kim DG, Bae JY, Ahn SH, Bai SC, Kong IS (2007) Species-specific PCR detection of the fish pathogen, Vibrio anguillarum, using the amiB gene, which encodes N-acetylmuramoyl-L-alanine amidase. FEMS Microbiol Lett 269:201–206 14. Huang CH, Chang MT, Huang MC, Wang LT, Huang L, Lee FL (2012) Discrimination of the L. acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene. J Sci Food Agric 92:2703–2708 15. Jauhiainen T, Niittynen L, Oreisic M, Ja¨rvenpa¨a¨ S, Hiltunen TP, Ro¨nnback M, Vapaatalo H, Korpela R (2012) Effects of longterm intake of lactotripeptides on cardiovascular risk factors in hypertensive subjects. Eur J Clin Nutr 66:843–849 16. Jebava I, Plockova M, Lortal S, Valence F (2011) The nine peptidoglycan hydrolases genes in L. helveticus are ubiquitous and early transcribed. Int J Food Microbiol 148:1–7 17. Kandler O, Weiss N (1986) Regular nonsporing gram-positive rods. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds)
I. Jebava et al.: Peptidoglycan Hydrolases as Species-Specific Markers
18.
19.
20.
21.
22.
23.
24.
25.
26.
Bergey0 s manual of systematic bacteriology, vol 2., Williams and Wilkins CoBaltimore, MD, pp 1208–1234 Kullen MJ, Sanozky-Dawes RB, Crowell DC, Klaenhammer TR (2000) Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the L. acidophilus complex. J Appl Microbiol 89:511–516 Lortal S, Valence F, Bizet C, Maubois JL (1997) Electrophoretic pattern of peptidoglycan hydrolases, a new tool for bacterial species identification: application to 10 Lactobacillus species. Res Microbiol 148:461–474 Meroth CB, Hammes WP, Hertel C (2004) Characterisation of the microbiota of rice sourdoughs and description of L. spicheri sp. nov. Syst Appl Microbiol 27:151–159 Moroni AV, Arendt EK, Bello FD (2011) Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs. Food Microbiol 28:497–502 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Singh S, Goswami P, Singh R, Heller KJ (2009) Application of molecular identification tools for Lactobacillus, with a focus on discrimination between closely related species: a review. LWT Food Sci Technol 42:448–457 Slattery L, O’Callaghan J, Fitzgerald GF, Beresford T, Ross RP (2010) Invited review: L. helveticus—a thermophilic dairy starter related to gut bacteria. J Dairy Sci 93:4435–4454 Stackebrandt E, Frederiksen W, Garrity GM, Grimont PAD, Kampfer P, Maiden MCJ, Nesme X, Rossello-Mora R, Swings J, Truper HG, Vauterin L, Ward AC, Whitman BW (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52:1043–1047 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using
557
27.
28.
29.
30.
31.
32.
33.
34.
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74 Valence F, Lortal S (1995) Zymogram and preliminary characterization of L. helveticus autolysins. Appl Environ Microbiol 61:3391–3399 Van Hoorde K, Verstraete T, Vandamme P, Huys G (2008) Diversity of lactic acid bacteria in two Flemish artisan raw milk Gouda-type cheeses. Food Microbiol 25:929–935 Ventura M, Callegari ML, Morelli L (2000) S-layer gene as a molecular marker for identification of L. helveticus. FEMS Microbiol Lett 189:275–279 Ventura M, Canchaya C, Meylan V, Klaenhammer TR, Zink R (2003) Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 69:6908–6922 Viiard E, Mihhalevski A, Ru¨hka T, Paalme T, Sarand I (2013) Evaluation of the microbial community in industrial rye sourdough upon continuous back-slopping propagation revealed L. helveticus as the dominant species. J Appl Microbiol 114: 404–412 Vinogradov E, Valence F, Maes E, Jebava I, Chuat V, Lortal S, Grard T, Guerardel Y, Sadovskaya I (2013) Structural studies of the cell wall polysaccharides from three strains of L. helveticus with different autolytic properties: DPC4571, BROI, and LH1. Carbohydr Res 379:7–12 Wine E, Gareau MG, Johnson-Henry K, Sherman PM (2009) Strain-specific probiotic L. helveticus inhibition of Campylobacter jejuni invasion of human intestinal epithelial cells. FEMS Microbiol Lett 300:146–152
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