Abstract
Bacteria possessing multiple copies of 16S rRNA (rrs) gene demonstrate high intragenomic heterogeneity. It hinders clear distinction at species level and even leads to overestimation of the bacterial diversity. Fifty completely sequenced genomes belonging to 19 species of Lactobacillus species were found to possess 4–9 copies of rrs each. Multiple sequence alignment of 268 rrs genes from all the 19 species could be classified into 20 groups. Lactobacillus sanfranciscensis TMW 1.1304 was the only species where all the 7 copies of rrs were exactly similar and thus formed a distinct group. In order to circumvent the problem of high heterogeneity arising due to multiple copies of rrs, 19 additional genes (732–3645 nucleotides in size) common to Lactobacillus genomes, were selected and digested with 10 Type II restriction endonucleases (RE), under in silico conditions. The following unique gene—RE combinations: recA (1098 nts)—HpyCH4 V, CviAII, BfuCI and RsaI were found to be useful in identifying 29 strains representing 17 species. Digestion patterns of genes—ruvB (1020 nts), dnaA (1368 nts), purA (1290 nts), dnaJ (1140 nts), and gyrB (1944 nts) in combination with REs—AluI, BfuCI, CviAI, Taq1, and Tru9I allowed clear identification of an additional 14 strains belonging to 8 species. Digestion pattern of genes recA,ruvB, dnaA, purA, dnaJ and gyrB can be used as biomarkers for identifying different species of Lactobacillus.
Electronic supplementary material
The online version of this article (doi:10.1007/s12088-016-0605-5) contains supplementary material, which is available to authorized users.
Keywords: Lactobacillus, Diagnosis, Biomarkers, Genome, In silico, Restriction endonuclease
Introduction
The diversity of microbial world has fascinated researchers since their discovery. Rapid progress in identifying bacteria has been made during the last 3 decades. Prof. Carl R. Woese developed an innovative strategy which brought around a revolutionary change in evolutionary biology and taxonomy. The most widely employed gene for bacterial identification has been 16S rDNA (rrs). Almost all laboratories around the world have mastered the technique of sequencing this gene. Ribosomal database project (RDP) (https://rdp.cme.msu.edu/), is a depository of 3,224,600 rrs sequences. Using the unique signatures and restriction endonuclease (RE) digestion patterns of rrs, it has been possible to re-classify bacteria, which were identified so far only up to genus level to species level [1–4]. In spite of such a roaring success of rrs, there have been cases where the gene sequences are quite similar and don’t prove effective in distinguishing closely related organisms. Another limitation generally encountered in the usage of rrs is in the case of organisms which harbour it in multiple copies: Clostridium, Yersinia, Vibrio, Staphylococcus, Streptococcus, etc. Here, high intragenomic heterogeneity leads to mis-identification and even overestimation of bacterial populations [5–10].
The Trouble with Lactobacillus Identification
Conventional methods of identifying Lactobacillus involve phenotypic and biochemical characterization. Nevertheless, with the advent of molecular biology, gene based methods were found to be more reliable, precise and consistent. Among the various genes used for identifying bacteria, rrs has been the most successful [11, 12]. It has completely revolutionized the way taxonomy has developed. A large number of genomic tools have proved helpful in establishing taxonomic and evolutionary relationships. All molecular methods developed so far have been proven to have some merits vis-a-vis others. The need to develop new genomic tools and find new biomarkers has been associated with the difficulties encountered while carrying out a particular assay or diagnosing a disease. The need for highly precise identification tool is especially connected with the economic importance of the bacteria. In case of food and waterborne contaminants and diseases caused by pathogens rapid diagnosis for prescribing a treatment is necessary. Use of Lactobacillus to control canine intestinal infections is an interesting proposal [13]. Lactobacillus is an economically important organism, especially as bioactive molecules, probiotic, preservative, fermentation and maturation of the sausages, milk products [14–18]. The dominance of Lactobacillus species such as L. iners,L. jensenii,L. gasseri, and L. crispatus in the vaginal region acts as a barrier for invasion of pathogenic bacteria and virus. Lactobacillus is an indicator of a healthy vaginal ecosystem, hence their rapid identification can prove helpful in diagnosing bacterial vaginosis [19–21].
Molecular tools used for distinguishing Lactobacillus species involve: Random amplified polymorphic DNA, repetitive element PCR, restriction fragment length polymorphism, pulse-field gel electrophoresis, denaturing/temperature gradient gel electrophoresis [22–26]. These techniques have helped to reclassify certain species such as L. brevis–L. hilgardii [22]. Multiplex polymerase chain reaction of the region between rrs and 23S rRNA was developed to identify seven probiotic Lactobacillus species [27, 28]. Amplification of rrs gene and its analysis with the help of amplified ribosomal DNA restriction analysis, by employing restriction enzymes (REs) such as: (1) HaeIII, DdeI, and HinfI, (2) ApaI, NotI and SmaI, (3) HaeIII, MspI, and HinfI was used for identifying 17, 24, and 42 lactobacilli, respectively [26, 29, 30]. In silico restriction digestion patterns (with 11 REs) were identified for distinguishing Lactobacillus only up to the species level. REs—SphI, NcoI and NheI could digest rrs, REs—DraI, EcoRI, HincII, SfuI, SspI, and VspI, and showed digestion patterns in the rrs—23S rRNA intergenic region, where as REs—AvrII and HindIII digested 23S rRNA gene [28]. Matrix-assisted laser desorption/ionization mass spectrometry, based on ribosomal subunit proteins as biomarkers was used to identify Lactobacillus plantarum and other species [12, 31]. In order to distinguish very closely related Lactobacillus species, rpoA gene—encoding for the alpha subunit of RNA polymerase, and pheS gene—encoding for the alpha subunit of phenylalanyl-tRNA synthase were sequenced. These two genes provided 10 and 5 % divergence for distinguishing Lactobacillus species. The usage of the two genes in combination proved more effective in distinguishing the species in comparison to rrs gene [32]. Lactobacillus plantarum was found to possess genes—mub, fbp and bsh, which encode for mucus-binding protein, fibronectin-binding protein and bile salt hydrolase, respectively. These genes were projected as probiotic identification markers [15]. Multiplex PCR using species-specific primers for amplifying recA gene of the L. plantarum group enabled identification of strains to be L. paraplantarum and L. pentosus [33]. Comparative genomic hybridization (CGH) has been recognized as a powerful technique for identifying Lactobacillus. Genes—recA, pheS, pyrG, tuf and rrs were sequenced to identify strains initially annotated as analyzed to belong to Lactobacillus taiwanensis. However, DNA–DNA hybridization assays revealed the five strains to be distinct and more close to Lactobacillus johnsonii and L. gasseri. CGH with a whole genome DNA microarray of L. johnsonii strain NCC533 allowed to place L. taiwanensis BL263 independent of L. johnsonii ATCC 33200T [34]. Evaluation of lactobacilli present in the cervix of the female genital tract was done initially using (GTG)(5)-PCR fingerprinting. The different clusters were then segregated based on pheS gene. This study revealed that although (GTG)(5)-PCR helps in identifying many isolates, however, a supplementary gene information is needed for reliable identification. An interesting finding of this study was the fact that Lactobacillus acidophilus is not a common inhabitant of female genital tract [35]. After an initial identification as Lactobacillus sp. DMDL 9010 done with rrs, the confirmatory test was performed using two fragments flanking the gene L-ldh1. The strain was identified as Lactobacillus pentosus or L. plantarum [36]. Molecular identification using minD gene, which encodes for an inhibitor cell division was carried out for Lactobacillus rhamnosus and L. acidophilus. The gene minD was observed to have high homology to that present in Escherichia coli, implying common evolution [37]. Horizontal gene transfer in Lactobacillus has been reported to be responsible for adapting to changes in lifestyles [38, 39]. Phylogenetic relationship was established through a multi-locus sequence typing using house keeping genes, which included: mutL, polA, ftsZ, pgm,metRS, and nrdD [39, 40].
The genus Lactobacillus is composed of 180 species [17]. Sequenced genomes of Lactobacillus allow comparative studies: L. acidophilus, L. johnsonii, L. plantarum, L. sakei, and L. salivarius [41]. Comparison of information deduced from sequenced genomes of Lactobacillus ruminis ATCC 27782 (2.06 Mb) isolated from bovine and L. ruminis strain, ATCC 25644 (2.138 Mb), isolated from human with L. salivarius core proteins showed that Lactobacillus species are categorized into 4 phylogenetically distinguishable groups [42]. Proteomics studies have proved helpful in identifying 6 proteins which can be used as biomarkers for selecting their probiotic potential since they are responsible for responding to bile salt and adaptation in L. plantarum [16]. Information on how Lactobacillus casei might have undergone diversity to adapt to the environment has been elucidated through completely sequencing its genome [39].
Lactobacillus have evolved from Bacillus and lost 600–1200 genes after the divergence. The loss of genes indicated that there has been a shift towards the nutrient rich atmosphere. The genomes of Lactobacillus species have sizes in the range of 1.8–3.3 Mb and a G+C content varying from 33 to 51 % [43]. Sequenced genomes of Lactobacillus species have revealed the evolution through gene degradation and horizontal gene transfer. Sequenced genomes also show that 55–60 % of proteins of Lactobacillus bulgaricus proteins show high homology with those present in L. acidophilus and L. johnsonii. L. bulgaricus has lost genes for mucin-binding proteins and bile salt hydrolase. L. plantarum has evolved to utilize diverse carbohydrates. L. salivarius has acquired genes for bile salt hydrolase and the pentose phosphate pathway. Comparative genomics done through Differential Blast Analysis (DBA), used for identifying specific genome regions, also elucidated information on regions which were present in some and absent in another set of organisms [18, 21, 43–50]. Lactobacillus helveticus DPC4571 show about 75 % homology with the genome of L. acidophilus NCFM [51]. This thus poses a major hurdle in identifying genes which may be common to all Lactobacillus species. Here, we have identified genes which are present in most of the Lactobacillus species. Of the large number of genes which can be potentially used for identifying novel biomarkers, a few were selected and subjected to in silico digestion with different type II restriction endonulcease (RE) enyzmes. RE digestion patterns unique to a given genome were identified.
Materials and Methods
Sequence Data and Comparative Genome Analysis
Genome sequences of the 50 strains of 19 species of the genus Lactobacillus were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/): L. acidophilus (2 strains), L. amylovorus (2 strains), L. brevis (2 strains), L. buchneri (2 strains), L. casei (5 strains), L. delbrueckii (4 strains), L. fermentum, L. gasseri, L. helveticus (4 strains), L. johnsonii (3 strains), L. kefiranofaciens, L. paracasei (2 strains), L. plantarum (6 strains), L. reuteri (5 strains), L. rhamnosus (6 strains), L. ruminisL. sakei, L. salivarius, L. sanfranciscensis (Table S1). Genomes characteristics of the Lactobacillus have been presented in Table S1. Genes common to most of the Lactobacillus genomes were selected with the help of GenBank data (Table S2). From the common gene pool, 19 genes were selected varying in size from 732 to 3645 nucleotides (nts) (Tables S1 and S2). Gene, rrs was used as reference, as it is generally employed for bacterial identification. Sequence analysis and their orientation were done using BioEdit [8].
Restriction Endonuclease Analysis of Common Genes
Based on our previous works 10 Type II REs were considered for generating digestion patterns: (1) 4 base cutters AluI, BfaI, BfuCI, CviAII, HpyCH4 V, RsaI, TaqI, Tru9I, and (2) 6 base cutters HaeI, and Hin1I [8]. RE digestion patterns of the 19 common gene sequences along with rrs was obtained using Cleaver (http://cleaver.sourceforge.net/) (Table S2). REs producing 5–15 fragments were considered for further analysis using BioEdit [8]. Unique gene-RE combinations were formed the basis for identifying Lactobacillus species.
Results
Analysis of rrs Gene
Multiple Sequence Alignment
Sequenced genomes of the 50 strains belonging to 19 species of Lactobacillus strains had 4–9 copies of rrs gene. The multiple sequence alignment of a total of 268 rrs copies from all the 50 genomes allowed us to segregate them into 20 groups (Table S3). Six of these 20 groups were represented by 5–61 rrs copies (in all 173) belonging to 2–3 Lactobacillus species each. These observations indicate high sequence similarity among rrs copies of different species. rrs copies of all the strains of L. delbrueckii (4 strains), L. reuteri (5 strains), L. buchneri (2 strains) and L. sanfranciscensis (1 strain) could be segregated into 5 independent groups. L. casei, presented a unique scenario, where rrs copies of (1) 3 strains were grouped along with L. paracasei (2 strains), L. rhamnosus (6 strains) and L. sakei, and (2) the rest 5 rrs copies in each of two strains were all distinct from each other and formed 10 groups. In summary, only L. sanfranciscensis (1 strain) could be segregated from all other genomes on the basis of its rrs gene sequences. Thus MSA proved that there is high level of intragenomic heterogeneity among different species of Lactobacillus.
In Silico RE Digestion
In silico RE digestion of all the 268 rrs copies belonging to 19 Lactobacillus species with 10 different REs revealed a few unique patterns (Table 1, Table S4). The only rrs-RE combinations, which could be used to distinguish different strains in an unambiguous manner were the following: (1) L. acidophilus La-14 with REs AluI, BfuCI, HpyCH4 V; (2) L. acidophilus NCFM and L. casei LC2 W with RE AluI; (3) L. amylovorus 30SC, L. brevis ATCC 367, and L. brevis KB290 with RE HpyCH4 V; (4) L. buchneri NRRL B-30929 with RE CviAII; (5) L. casei BL23, L. casei LOCK919, and L. rhamnosus ATCC 53103 with RE BfuCI; (6) L. fermentum IFO 3956 and L. kefiranofaciens ZW3 with REs BfuCI and Tru9I; (7) L. gasseri ATCC 33323 with RE Tru9I; (8) L. johnsonii DPC 6026, L. johnsonii N6.2 and L. paracasei ATCC 334 with RE CviAII; (9) L. plantarum subsp. plantarum P-8 with REs AluI and Tru9I; (10) L. reuteri TD1 with REs BfuCI, CviAII, and HpyCH4 V; (11) L. reuteri I5007 with RE BfaI; (12) L. rhamnosus ATCC 53103 with RE BfuCI; (13) L. ruminis ATCC 27782 with REs AluI, CviAI, HpyCH4,and Tru9I; (14) L. sakei subsp. sakei 23 K with REs AluI, BfuCI, CviAII, HpyCH4 V and Tru9I; (15) L. salivarius UCC118 with REs BfaI, BfuCI, CviAII, HpyCH4 V, RsaI and Tru9I; and (16) L. sanfranciscensis TMW 1.1304 with REs BfaI, BfuCI, CviAII, HpyCH4 V, TaqI, RsaI and Tru9I.
Table 1.
In silico restriction endonuclease digestion patterns (5′–3′) of rrs gene of Lactobacillus strains
| Lactobacillus spp. | GenBank ID | Copies of rrs (U/T)a | Unique restriction patternsb |
|---|---|---|---|
| AluI | |||
| L. plantarum WCFS1 | AL935263 | 2/5 | 222·51·615·105·102·207·269 |
| 3/5c | 273·615·105·102·207·269 | ||
| L. fermentum IFO 3956 | AP008937 | 5 | 273·186·429·105·102·473 |
| L. acidophilus NCFM | CP000033 | 4 | 69·146·20·33·186·429·207·221·44·217 |
| L. salivarius UCC118 | CP000233 | 7 | 843·207·207·248 |
| L. casei LC2 W | CP002616 | 1/5 | 1093·338·20·117 |
| 1/5 | 307·129·41·245·278·40·316·132·80 | ||
| 1/5 | 459·139·39·708·223 | ||
| 1/5 | 13·136·344·898·17·160 | ||
| 1/5c | 25·1222·321 | ||
| L. casei BD-II | CP002618 | 1/5 | 499·218·793·58 |
| 1/5 | 268·286·41·55·204·21·24·544·125 | ||
| 1/5 | 452·414·235·15·452 | ||
| 1/5 | 114·42·53·366·19·92·131·45·364·342 | ||
| 1/5 | 140·25·383·452·133·361·74 | ||
| L. reuteri SD2112 | CP002844 | 2/6 | 216·57·186·429·105·102·193·14·262 |
| 4/6c | 273·186·429·105·102·193·14·262 | ||
| L. ruminis ATCC 27782 | CP003032 | 6 | 54·37·775·207·207·247 |
| L. helveticus R0052 | CP003799 | 1/4 | 215·20·33·186·429·206·220·44·217 |
| 1/4 | 215·20·33·186·430·208·221·44·217 | ||
| 2/4 | 205·20·33·186·428·207·221·44·217 | ||
| L. acidophilus La-14 | CP005926 | 4 | 63·146·20·33·186·429·207·221·44·210 |
| L. plantarum subsp. plantarum P-8 | CP005942 | 5 | 63·201·615·105·102·207·262 |
| L. reuteri TD1 | CP006603 | 1/6 | 281·186·184·245·105·102·193·14·287 |
| 5/6 | 281·615·105·102·193·14·287 | ||
| L. sakei subsp. sakei 23 K | CR936503 | 3/7 | 85·801·160·47·207·271 |
| 4/7 | 665·160·47·207·271 | ||
| BfaI | |||
| L. salivarius UCC118 | CP000233 | 7 | 229·578·518·180 |
| L. casei BD-II | CP002618 | 1/5 | 570·268·581·149 |
| 4/5 | Not segregated | ||
| L. delbrueckii subsp. bulgaricus NDO2 | CP002341 | 2/9 | 104·159·578·323·195·201 |
| 7/9c | 104·159·578·185·333·202 | ||
| L. sanfranciscensis TMW 1.1304 | CP002461 | 7 | 242·33·578·323·195·199 |
| L. reuteri I5007 | CP006011 | 5/6 | 274·245·333·296·27·195·194 |
| 1/6c | 274·578·296·27·195·194 | ||
| BfuCI | |||
| L. reuteri JCM 1112 | AP007281 | 6 | 7·98·225·892·159·153 |
| L. rhamnosus ATCC 53103 | AP011548 | 5 | 15·315·699·12·340·177·16 |
| L. salivarius UCC118 | CP000233 | 2/7 | 162·123·119·1101 |
| 5/7 | 174·8·115·119·1101 | ||
| L. sanfranciscensis TMW 1.1304 | CP002461 | 2/7 | 7·98·1277·175·13 |
| 5/7 | 7·98·1277·176·13 | ||
| L. johnsonii DPC 6026 | CP002464 | 1/4 | 7·319·974·77·176·15 |
| 3/4c | 45·319·974·77·176·60 | ||
| L. kefiranofaciens ZW3 | CP002764 | 4 | 16·312·1051·174·21 |
| L. helveticus R0052 | CP003799 | 1/4 | 13·188·8·235·934·174·22 |
| 3/4 | 13·188·8·235·930·174·22 | ||
| L. casei LOCK919 | CP005486 | 5 | 16·67·116·132·699·12·340·167 |
| L. acidophilus La-14 | CP005926 | 4 | 7·188·8·116·119·932·174·15 |
| L. reuteri TD1 | CP006603 | 6 | 15·98·225·892·159·175·33 |
| L. sakei subsp. sakei 23 K | CR936503 | 3/7 | 8·439·46·886·176·16 |
| 4/7 | 226·46·886·176·16 | ||
| L. casei BL23 | FM177140 | 1/4 | 15·183·132·699·12·340·177·21 |
| 3/4c | 15·67·116·132·699·12·340·177·21 | ||
| CviAII | |||
| L. salivarius UCC118 | CP000233 | 7 | 15·153·493·269·106·148·125·34·50·112 |
| L. delbrueckii subsp. bulgaricus ATCC BAA-365 | CP000412 | 8/9 | 49·143·9·494·126·143·106·148·209·134 |
| 1/9c | 49·143·9·494·90·36·143·106·148·209·134 | ||
| L. paracasei ATCC 334 | CP000423 | 5 | 50·53·104·494·90·36·143·106·148·209·135 |
| L. sanfranciscensis TMW 1.1304 | CP002461 | 7 | 47·164·496·90·36·143·106·148·125·34·50·131 |
| L. johnsonii DPC 6026 | CP002464 | 1/4 | 47·162·493·90·36·143·106·148·209·134 |
| L. buchneri NRRL B-30929 | CP002652 | 5 | 577·38·113·102·106·148·108·95·212·22·47 |
| L. reuteri SD2112 | CP002844 | 2/6 | 47·659·90·36·143·106·148·125·34·5·45·125 |
| L. ruminis ATCC 27782 | CP003032 | 3/6 | 37·142·11·9·485·269·106·148·125·34·50·111 |
| 3/6 | 39·142·11·267·227·269·106·148·125·34·50·109 | ||
| L. reuteri TD1 | CP006603 | 6 | 55·165·494·90·36·143·106·148·159·50·151 |
| L. johnsonii N6.2 | CP006811 | 4 | 53·162·493·90·36·143·106·148·209·140 |
| L. sakei subsp. sakei 23 K | CR936503 | 3/7 | 48·162·494·90·36·143·106·148·125·34·50·135 |
| 4/7 | 483·90·36·143·106·148·125·34·50·135 | ||
| HpyCH4 V | |||
| L. brevis KB290 | AP012167 | 5 | 58·35·565·25·201·195·262·234 |
| L. salivarius UCC118 | CP000233 | 1/7 | 40·35·562·25·396·262·208 |
| 6/7 | 18·35·561·25·396·262·208 | ||
| L. brevis ATCC 367 | CP000416 | 5 | 52·35·565·25·201·195·262·228 |
| L. sanfranciscensis TMW 1.1304 | CP002461 | 7 | 50·39·571·25·396·255·7·11·216 |
| L. amylovorus 30SC | CP002559 | 4 | 59·206·43·349·25·88·113·108·37·50·255·7·11·96·128 |
| L. ruminis ATCC 27782 | CP003032 | 6 | 40·35·562·25·201·195·255·7·11·196 |
| L. helveticus R0052 | CP003799 | 2/4 | 46·206·43·349·24·88·113·195·255·7·11·96·128 |
| 2/4c | 56·206·43·350·25·88·113·196·255·7·11·96·128 | ||
| L. acidophilus La-14 | CP005926 | 4 | 50·206·43·349·25·88·113·145·50·255·7·11·96·121 |
| L. reuteri TD1 | CP006603 | 6 | 58·39·178·43·349·25·88·308·262·247 |
| L. sakei subsp. sakei 23 K | CR936503 | 3/7 | 51·214·43·349·25·396·262·231 |
| 4/7 | 44·43·349·25·396·262·231 | ||
| RsaI | |||
| L. salivarius UCC118 | CP000233 | 1/7 | 896·253·102·146·131 |
| 6/7 | 873·355·146·131 | ||
| TaqI | |||
| L. sanfranciscensis TMW 1.1304 | CP002461 | 6/7 | 55·735·199·359·222 |
| 1/7c | 55·734·199·575 | ||
| L. helveticus R0052 | CP003799 | 4 | 51·722·199·359·230 |
| L. plantarum 16 | CP006033 | 1/5 | 65·725·159·41·582 |
| 4/5c | 64·724·199·583 | ||
| Tru9I | |||
| L. fermentum IFO 3956 | AP008937 | 5 | 223·245·22·25·130 |
| L. salivarius UCC118 | CP000233 | 7 | 225·349·26·338·134·44·150·239 |
| L. gasseri ATCC 33323 | CP000413 | 6 | 232·672·86·134·44·411 |
| L. sanfranciscensis TMW 1.1304 | CP002461 | 7 | 620·26·252·86·134·44·150·258 |
| L. buchneri NRRL B-30929 | CP002652 | 4/5 | 102·122·421·252·86·134·194·252 |
| 1/5c | 70·32·122·421·252·86·134·194·252 | ||
| L. kefiranofaciens ZW3 | CP002764 | 4 | 153·69·421·252·86·134·44·415 |
| L. reuteri SD2112 | CP002844 | 1/6 | 224·421·252·87·134·44·150·253 |
| 5/6c | 645·252·86·134·44·150·253 | ||
| L. ruminis ATCC 27782 | CP003032 | 6 | 248·349·26·252·86·134·44·150·238 |
| L. buchneri CD034 | CP003043 | 3/5 | 70·32·122·421·252·86·53·81·194·252 |
| 2/5c | 70·32·122·421·252·86·134·194·252 | ||
| L. helveticus R0052 | CP003799 | 1/4 | 640·252·85·177·416 |
| 3/4c | 630·251·86·134·44·416 | ||
| L. plantarum subsp. plantarum P-8 | CP005942 | 5 | 506·104·278·86·134·44·150·253 |
| L. plantarum 16 | CP006033 | 1/5 | 1·513·104·278·86·134·44·150·261 |
| 4/5c | 3·466·47·104·278·86·134·44·150·259 | ||
| L. sakei subsp. sakei 23 K | CR936503 | 3/7 | 617·278·86·134·194·262 |
| 4/7 | 396·278·86·134·44·150·262 | ||
Symbol (·) indicates RE site in the gene sequences
aUnique/total
bValues represent restriction fragments (nucleotides)
cThis pattern is not unique. It has been presented to indicate the RE digestion pattern of the rest of the rrs copies
In summary, only 22 strains belonging to 17 species can be identified without any discrepancy: L. acidophilus (2); L. amylovorus; L. brevis (2); L. buchneri; L. casei (2); L. fermentum; L. gasseri; L. johnsonii (2); L. kefiranofaciens; L. paracasei; L. plantarum; L. reuteri (2); L. rhamnosus; L. ruminis; L. sakei; L. salivarius; and L. sanfranciscensis.
No unique RE digestion patterns with any of the REs employed were observed in the following strains: L. amylovorus GRL1118, L. casei str. Zhang, L. delbrueckii subsp. bulgaricus 2038, L. helveticus CNRZ32, L. helveticus DPC 4571, L. helveticus H10, L. paracasei subsp. paracasei 8700.2, L. plantarum JDM1, L. plantarum subsp. plantarum ST-III, L. plantarum ZJ316, L. reuteri DSM 20016, L. rhamnosus ATCC 8530, L. rhamnosus GG ATCC 53103, L. rhamnosus Lc 705, L. rhamnosus LOCK900, and L. rhamnosus LOCK908.
It may be concluded that with MSA and RE digestions only 22 strains can be identified. For the rest of the 28 strains we may need to resort to other genes.
Analysis of Common Genes
In Silico RE Digestion Analysis of Common Genes
A list of genes chosen for study from the common gene pool from sequenced genomes of Lactobacillus strains has been presented in Table S2. Of the 19 selected genes (732–3645 nts in length) only 9 genes (cysS, dnaA, dnaJ, dnaK, gyrB, polA, pyrB, pyrG, and recA) were present in all the 47–50 genomes, where as the rest of the 10 genes were present in 44–46 genomes. All the 19 genes were present in single copies. In silico RE digestion of all the 19 genes present in Lactobacillus species with 10 different REs revealed a few unique digestion patterns.
Analysis of recA Gene
Among the genes which were considered as suitable for identifying Lactobacillus strains, RE digestion patterns of recA (1098 nts) have been presented in Tables 2 and 3. In silico RE digestion of recA, revealed a few unique patterns in 29 strains representing 17 species. Unique RE digestion patterns could not be identified for L. amylovorus (2 strains) and L. johnsonii (3 strains). RE—HpyCH4 V, proved to be the most effective by generating unique digestion patterns in 19 strains belonging to 14 species. On the other hand, REs—CviAII, BfuCI and RsaI were found to be useful in identifying 12–15 strains representing 12–14 species each (Tables 2, 3). Hence, through the use of complementary REs, most of the strains can be identified in an unambiguous manner.
Table 2.
Unique in silico restriction endonuclease digestion patterns (5′-3′) of recA gene of Lactobacillus strains
| Lactobacillus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| CviAII | BfuCI | HpyCH4 V | RsaI | |
| L. acidophilus La-14 | 299·255·226·148·78·42·38·6 | 565·297·120·87·23 | – | – |
| L. acidophilus NCFM | 299·255·226·148·78·42·38·11·6 | 565·308·120·87·23 | – | – |
| L. brevis ATCC 367 | –a | – | 639·228·120·79·68 | 555·215·132·116·116 |
| L. brevis KB290 | – | 633·315·207·9 | 639·258·120·79·68 | 555·215·146·132·116 |
| L. buchneri CD034 | – | – | 389·256·162·145·132·56 | – |
| L. buchneri NRRL B-30929 | – | – | 645·162·145·132·56 | – |
| L. casei BL23 | – | 302·297·250·90·87·36 | – | – |
| L. casei str. Zhang | 299·255·228·200·42·38 | – | 568·249·111·83·33·18 | 564·260·195·43 |
| L. delbrueckii subsp. bulgaricus ATCC 11842 | 309·216·183·168·78·42 | 186·162·108·107·106·90·72·72·54·39 | 618·231·129·18 | 790·81·74·51 |
| L. delbrueckii subsp. bulgaricus NDO2 | – | 220·186·162·108·107·90·72·72·54·39 | – | – |
| L. fermentum IFO 3956 | 708·159·120·75 | 265·223·165·150·98·90·65·6 | – | 758·163·114·27 |
| L. gasseri ATCC 33323 | – | 392·297·210·185·11 | 686·241·107·30·24·7 | 449·341·156·127·22 |
| L. helveticus CNRZ32 | – | – | – | – |
| L. helveticus DPC 4571 | – | – | – | – |
| L. helveticus H10 | 299·232·232·231·80·24 | – | – | – |
| L. helveticus R0052 | – | – | – | 331·276·248·227·16 |
| L. kefiranofaciens ZW3 | 379·255·244·232 | 380·348·202·132·24·24 | 612·237·86·41·34·33·31·21·15 | 250·239·185·129·119·91·81·16 |
| L. paracasei ATCC 334 | – | – | 564·206·111·83·43·37·18 | – |
| L. plantarum subsp. plantarum P-8 | 420·411·155·82·75 | 366·306·174·115·93·63·26 | 539·469·126·9 | – |
| L. plantarum ZJ316 | – | – | – | – |
| L. reuteri I5007 | – | – | – | – |
| L. reuteri SD2112 | 379·255·229·147·79 | – | 426·232·116·111·75·75·48·6 | – |
| L. reuteri TD1 | – | 546·250·245·48 | 537·232·116·81·75·48 | – |
| L. rhamnosus ATCC 53103 | 433·359·144·93·24 | 250·237·166·111·88·72·31·28·27·27·16 | 296·242·228·202·52·33 | 296·244·190·156·91·76 |
| L. rhamnosus GG ATCC 53103 | 354·232·180·115·68·51·38·11·4 | – | 299·270·165·136·84·83·12·4 | – |
| L. rhamnosus LOCK900 | – | – | – | – |
| L. ruminis ATCC 27782 | 371·229·183·159·147 | 392·392·294·11 | 452·223·188·142·42·33·5·4 | – |
| L. sakei subsp. sakei 23 K | 554·285·252·45 | 633·201·118·109·75 | 808·259·61·8 | 467·341·124·112·81·11 |
| L. salivarius UCC118 | 299·262·255·182·148 | 819·190·107·30 | 562·111·93·89·81·69·64·56·12·9 | 467·294·185·109·91 |
| L. sanfranciscensis TMW 1.1304 | 489·226·216·99·47·36 | – | 452·231·136·126·126·42 | 607·221·198·87 |
Symbol (·) indicates RE site in the gene sequences
a No unique pattern was observed
Table 3.
Unique in silico restriction endonuclease digestion patterns (5′-3′) of recA gene of Lactobacillus strains
| Lactobacillus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| AluI | TaqI | Tru9I | BfaI | |
| L. acidophilus La-14 | –a | – | – | – |
| L. acidophilus NCFM | – | – | – | – |
| L. brevis ATCC 367 | – | 720·313·70·31 | 366·323·192·181·48·24 | – |
| L. brevis KB290 | – | 720·313·100·31 | 366·337·192·181·48·24·16 | – |
| L. buchneri CD034 | – | 501·453·159·22·5 | 535·292·117·87·48·37·24 | – |
| L. buchneri NRRL B-30929 | 453·432·170·63·22 | 501·258·195·159·22·5 | 535·379·117·48·37·24 | – |
| L. casei BL23 | – | – | – | – |
| L. casei str. Zhang | – | 580·242·181·59 | 504·381·110·67 | – |
| L. delbrueckii subsp. bulgaricus ATCC 11842 | 290·241·241·224 | 315·166·136·98·81·72·54·36·15·14·9 | – | – |
| L. delbrueckii subsp. bulgaricus NDO2 | – | – | – | – |
| L. fermentum IFO 3956 | – | 360·215·205·117·70·51·18·14·12 | – | – |
| L. gasseri ATCC 33323 | 425·138·136·132·130·98·36 | – | 300·244·168·87·82·68·67·40·24·15 | – |
| L. helveticus CNRZ32 | – | – | 508·334·87·69·67·33 | – |
| L. helveticus DPC 4571 | 270·261·144·144·97·95·87 | – | – | – |
| L. helveticus H10 | – | – | – | – |
| L. helveticus R0052 | – | 454·435·159·50 | – | – |
| L. kefiranofaciens ZW3 | 366·330·162·136·90·18·8 | 784·228·50·48 | 353·259·184·114·111·41·33·15 | – |
| L. paracasei ATCC 334 | – | – | – | – |
| L. plantarum subsp. plantarum P-8 | 353·256·216·108·72·47·39·28·24 | – | 308·270·258·153·53·43·33·21·4 | – |
| L. plantarum ZJ316 | – | – | – | 725·267·102·25·24 |
| L. reuteri I5007 | 368·268·231·193·29 | – | – | – |
| L. reuteri SD2112 | – | 567·247·228·47 | – | – |
| L. reuteri TD1 | – | – | – | – |
| L. rhamnosus ATCC 53103 | 466·166·129·119·100·73 | 370·266·235·103·40·27·12 | 383·240·216·95·70·49 | 541·422·73·17 |
| L. rhamnosus GG ATCC 53103 | – | – | 535·289·165·40·24 | – |
| L. rhamnosus LOCK900 | 478·339·133·54·49 | 824·124·56·49 | – | – |
| L. ruminis ATCC 27782 | 491·249·174·94·81 | 437·227·220·109·96 | – | – |
| L. sakei subsp. sakei 23 K | – | 372·298·172·150·108·36 | 352·329·219·165·30·24·14·3 | – |
| L. salivarius UCC118 | 420·344·207·63·55·45·12 | – | 535·225·160·117·49·39·21 | – |
| L. sanfranciscensis TMW 1.1304 | 903·74·46·43·42·5 | 266·206·193·157·123·118·50 | 344·184·174·156·138·61·43·10·3 | – |
Additional unique patterns were also recorded for L. fermentum IFO 3956 - Hin1I - 552·189·124·108·89
Symbol (·) indicates RE site in the gene sequences
aNo unique pattern was observed
Analysis of Other Common Genes
There were 21 strains, which could not be distinguished on the basis of RE digestion pattern of recA gene. Analysis of digestion patterns of genes—ruvB (1020 nts), dnaA (1368 nts), purA (1290 nts), dnaJ (1140 nts), and gyrB (1944 nts) in combination with REs—AluI, BfuCI, CviAI, Taq1,and Tru9I proved instrumental in providing information for clear identification of 16 strains belonging to 8 species.
Gene ruvB in combination with different REs was effective in segregating different phylogenetically close strains (Table 4): (1) RE AluI helped to segregate L. amylovorus 30SC, L. amylovorus GRL1118, L. johnsonii DPC 6026, L. johnsonii N6, and L. johnsonii NCC 533, (2) RE BfuCI could distinguish L. paracasei subsp. paracasei 8700.2 from L. plantarum 16, and (3) RE TaqI provided unique digestion pattern for L. rhamnosus Lc 705. In addition, the following unique gene—RE combinations can also be used in the following cases: (1) dnaA- AluI for L. delbrueckii subsp. bulgaricus 2038; (2) purA- Tru9I for L. plantarum subsp. plantarum ST-III, and purA-AluI for L. plantarum JDM1; (3) dnaJ- AluI for L. casei LOCK919; dnaJ- BfuCI for L. delbrueckii subsp. bulgaricus ATCC BAA-365; dnaJ- CviAII for L. plantarum WCFS1; and (4) gyrB- TaqI for L. reuteri DSM 20016; gyrB- AluI for L. reuteri JCM 1112.
Table 4.
Unique in silico restriction endonuclease digestion patterns (5′-3′) of common genes of Lactobacillus strains (other than recA)
| Lactobacillus spp. | Gene | RE | RE digestion patterns |
|---|---|---|---|
| L. amylovorus 30SC | ruvB | AluI | 423·252·220·92·30 |
| L. amylovorus GRL1118 | 408·252·220·92·30·15 | ||
| L. johnsonii DPC 6026 | 561·169·141·108·41 | ||
| L. johnsonii N6 | 366·195·169·141·108·41 | ||
| L. johnsonii NCC 533 | 561·169·108·80·61·41 | ||
| L. paracasei subsp. paracasei 8700.2 | BfuCI | 351·185·128·93·88·63·56·24·18·11 | |
| L. plantarum 16 | 546·262·169·34 | ||
| L. rhamnosus Lc 705 | Taq1 | 344·271·184·171·50·27 | |
| L. delbrueckii subsp. bulgaricus 2038 | dnaA | AluI | 29·386·228·320·96·150·156 |
| L. plantarum subsp. plantarum ST-III | purA | Tru9I | 348·216·173·134·106·86·73·69·52·33 |
| L. plantarum JDM1 | AluI | 574·297·197·196·26 | |
| L. casei LOCK919 | dnaJ | AluI | 84·268·66·330·109·287·20 |
| L. delbrueckii subsp. bulgaricus ATCC BAA-365 | BfuCI | 491·66·147·52·110·141·130 | |
| L. plantarum WCFS1 | CviAII | 112·408·78·545 | |
| L. reuteri DSM 20016 | gyrB | TaqI | 776·461·294·234·190·70 |
| L. reuteri JCM 1112 | AluI | 1041·391·280·238 |
Symbol (·) indicates RE site in the gene sequences
With the available set of gene-RE combinations in this study, 4 strains could not be distinguished. Here, we may have to resort to additional gene-RE combinations.
Discussion
Bacterial identification with the help of rrs gene is practiced around the world. However, in cases where bacterial genomes contain multiple copies of rrs, such as in Clostridium, Staphylococcus, Streptococcus, Vibrio, and Yersinia species, a high level of heterogeneity is seen. It is difficult to identify these bacterial species on the basis of their rrs gene alone [5–10]. The same problem has been faced in the case of Lactobacillus as well. A large number of functional genes have been employed for identifying Lactobacillus sp., however, no consensus has been reached as yet. Hence, we identified genes which were common to almost all the Lactobacillus species. Of the 19 common genes which were processed for RE digestion patterns, pheS, polA, pyrG, recA, and rpoA have been reported in literature as markers for Lactobacillus [32–34, 39, 40]. Of these five genes, we could use only recA with effectiveness; where as the other 4 genes didn’t prove helpful, because they generated a large number of fragments on digestion with REs. In our case, the following additional genes could be used to distinguish very closely related species: ruvB (1020 nts), dnaJ (1140 nts), purA (1290 nts), dnaA (1368 nts), and gyrB (1944 nts) in combination with REs—AluI, BfuCI, CviAI, Taq1,and Tru9I. Our comparison of recA from Lactobacillus and Staphylococcus gave very distinct RE digestion patterns, allowing easy and reliable distinction [8]. It may be proposed here that the biomarkers identified in this study can be used for developing rapid and reliable protocol for diagnostic purposes.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgments
We are thankful to the Director of CSIR-Institute of Genomics and Integrative Biology (IGIB), and CSIR projects GENESIS (BSC0121) and INDEPTH (BSC0111) for providing the necessary funds, facilities and moral support. Authors are also thankful to Academy of Scientific & Innovative Research (AcSIR), New Delhi.
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