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ARTICLE Comparative genome-scale analysis of niche-based stressresponsive genes in Lactobacillus helveticus strains

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Suja Senan, Jashbhai B. Prajapati, and Chaitanya G. Joshi

Abstract: Next generation sequencing technologies with advanced bioinformatic tools present a unique opportunity to compare genomes from diverse niches. The identification of niche-specific stress-responsive genes can help in characterizing robust strains for multiple applications. In this study, we attempted to compare the stress-responsive genes of a potential probiotic strain, Lactobacillus helveticus MTCC 5463, and a cheese starter strain, Lactobacillus helveticus DPC 4571, from a gut and dairy niche, respectively. Sequencing of MTCC 5463 was done using 454 GS FLX, and contigs were assembled using GS Assembler software. Genome analysis was done using BLAST hits and the prokaryotic annotation server RAST. The MTCC 5463 genome carried multiple orthologs of genes governing stress responses, whereas the DPC 4571 genome lacked in the number of major stressresponse proteins. The absence of the bile salt hydrolase gene in DPC 4571 and its presence in MTCC 5463 clearly indicated niche adaptation. Further, MTCC 5463 carried higher copy numbers of genes contributing towards heat, cold, osmotic, and oxidative stress resistance as compared with DPC 4571. Through comparative genomics, we could thus identify stress-responsive gene sets required to adapt to gut and dairy niches. Key words: probiotic, stress, genome, MTCC 5463. Résumé : Les nouvelles technologies de séquençage et les outils d’analyse bioinformatique offrent une opportunité unique de comparer les génomes retrouvés au sein de diverses niches. L’identification de gènes de réponse a` des stress spécifiques a` certaines niches pourrait aider a` caractériser des souches robustes pour diverses applications. Dans ce travail, les auteurs ont tenté de comparer les gènes de réponse aux stress chez une souche potentiellement probiotique, Lactobacillus helveticus MTCC 5463, et une souche d’amorçage en production fromagère, Lactobacillus helveticus DPC 4571, occupant respectivement des niches intestinale et laitière. Le séquençage de la souche MTCC 5463 a été réalisé sur un appareil 454 GS FLX et les contigs ont été assemblés au moyen du logiciel GS Assembler. L’analyse génomique a été faite par alignement BLAST et le serveur d’annotation procaryote RAST. Le génome de la souche MTCC 5463 présentait plusieurs orthologues de gènes impliqués dans la réponse aux stress, alors que plusieurs protéines importantes de réponse a` des stress étaient absentes de la souche DPC 4571. L’absence du gène codant pour l’hydrolase des sels biliaires au sein du génome de la souche DPC 4571 et sa présence au sein de la souche MTCC 5463 indiquent clairement une adaptation a` leurs niches respectives. De plus, la souche MTCC 5463 avait un plus grand nombre de copies des gènes impliqués dans la résistance aux stress thermiques (tant la chaleur que le froid), osmotique et oxydatif que la souche DPC 4571. Via la génomique comparée, il a ainsi été possible d’identifier des ensembles de gènes de réponse aux stress qui sont requis pour l’adaptation a` la niche laitière ou intestinale. [Traduit par la Rédaction] Mots-clés : probiotique, stress, génome, MTCC 5463.

Introduction The food biotransformation abilities of lactobacilli were known to man since ages. Recently, a great deal of commercial and academic interests has been generated because of their probiotic potential. Renewed interests in probiotic Lactobacillus strains have led to the characterization of robustness and functionality of the strains using whole genome analysis. Among lactobacilli, Lactobacillus helveticus strains, used largely in the cheese industry as a starter culture with high proteolytic activity, have been recently characterised at the genomic level (Klaenhammer et al. 2005; Callanan et al. 2008; Zhao et al. 2011; Prajapati et al. 2011; Tompkins et al. 2012). Although phylogenetically similar, owing to their small genomes and common metabolic pathways for sugar fermentation and lactic acid production, L. helveticus occupy diverse

niches such as dairy, oral, vaginal, and the gut milieu (Dellaglio and Felis 2005). Different niches offer diverse stressful conditions. In a dairy-based niche, lactobacilli strains are expected to resist adverse conditions encountered in industrial processes, for example, starter handling, storage, and preservation strategies. Whereas in a gut mileau, strains need robustness to survive the digestive tract, resist the intestinal flora, and colonize the digestive mucosa against perturbations and competition (van de Guchte et al. 2002). Lactobacilli have evolved defense mechanisms against stress in multiple niches (Marles-Wright and Lewis 2007). The adaptation responses may be similar or differ widely between species and strains. This could be due to gene loss or decay, horizontal gene transfer, gene up regulation, or mutation. Genomic analysis of Streptococcus thermophilus LMD-9 showed the presence of genes contributing to adaptation to the dairy environment in

Received 1 February 2014. Accepted 30 April 2014. Corresponding Editor: J.B. Bell. S. Senan and J.B. Prajapati. Department of Dairy Microbiology, Sheth MC College of Dairy Science, Anand Agricultural University, Anand 388110, India. C.G. Joshi. Department of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand 388110, India. Corresponding author: Suja Senan (e-mail: [email protected]). Genome 57: 1–8 (2014) dx.doi.org/10.1139/gen-2014-0020

Published at www.nrcresearchpress.com/gen on 6 May 2014.

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addition to alien genes such as exopolysaccharide and peptide transporters that reflect the multiple transient habitats of LMD-9 beyond the restricted dairy environment (Goh et al. 2011). Functional and comparative genomic analyses has revealed key gene systems that direct functions and correlate them to important phenotypic behaviors such as stress tolerance and adaptation (Azcarate-Peril et al. 2005; Siezen and van Hylckama Vlieg 2011). Awareness of the key gene sets that could promote a gut or dairy lifestyle could be a useful tool for strain selection (Cai et al. 2009). Lactobacillus helveticus MTCC 5463 strain is a potential probiotic strain and the first in India to be fully sequenced. MTCC 5463 displays antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, and Escherichia coli (Khedkar et al. 1990), immunomodulating effects in a chicken model (Patidar and Prajapati 1999) and hypocholesteromic effect in human volunteers (Ashar and Prajapati 1998). Lactobacillus helveticus DPC 4571, a Swiss cheese isolate, is used as a starter and adjunct culture for rapid autolysis, reduced bitterness, and increased flavor notes in cheese (Hannon et al. 2003). The most intriguing feature of L. helveticus is a remarkable similarity in gene content with many intestinal lactobacilli (Cremonesi et al. 2012). Our objective was to compare the stress-response gene sets in closely related strains of L. helveticus DPC4571 and L. helveticus MTCC 5463 that explains their niche adaptability to a dairy or human gut environment.

Materials and methods Strains Lactobacillus helveticus MTCC 5463 strain, earlier known as Lactobacillus acidophilus V3 (based on biochemical characteristics), was originally isolated from the vaginal tract of a healthy adult female at the Department of Dairy Microbiology, Sheth MC College of Dairy Science, Anand Agricultural University, Anand, Gujarat, India. Pyrosequencing A DNA sample was subjected to pyrosequencing (454 Life Sciences technology) based on a high throughput sequencer (GS FLX, Roche) and according to manufacturer’s instructions. In brief, the DNA was nebulized to generate fragments. The sequencing library was prepared by applying adapters to both ends of the fragments as described by the manufacturer. Emulsion PCR was carried out to amplify cloned fragments on sequencing beads, followed by recovery and loading onto a picotitre plate along with enzyme beads. Pyrosequencing was carried out for 200 cycles with the flow of A, T, G, and C nucleotides sequentially, and images were captured. Further, these captured images were processed by image processing software to get sequencing reads. The resultant sequencing reads were generated by GS Run Browser and assembled using GS De Novo Assembler V.2.6. The unmapped reads were separated using a Perl script developed in-house and were assembled using GS De Novo Assembler. Sequence analysis 16S rRNA sequence comparison was performed by local BLAST of the assembled contigs with the 16S rDNA database downloaded from the National Center for Biotechnology Information (NCBI). Further confirmation of homology was performed using the Ribosomal Database Project classifier. The genome annotation of MTCC 5463 was performed with the Rapid Annotation using Subsystem Technology V.4.0 (http://rast.nmpdr.org/rast.cgi) service. The complete sequence of the L. helveticus MTCC 5463 genome can be accessed under GenBank accession numbers AEYL01000001– AEYL01000593. To identify putative protein-encoding sequences, the sequences were also analyzed using the SEED platform (http:// www.theseed.org/), which comprises all available genomic data from genome sequencing centers. Genome sequences of MTCC 5463 and DPC 4571 were uploaded to the SEED Viewer server (http://rast. nmpdr.org/seedviewer.cgi) independently. Functional roles of RAST

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annotated genes were assigned and grouped in subsystem feature categories. Genome information has been previously reported by the authors (Prajapati et al. 2011). The choice of DPC 4571 as the stain to be compared with MTCC 5463 is guided by its availability in a public database, whole genome information, and proven starter culture attributes (L. helveticus DPC 4571 (NCBI:CP000517)). Identification of niche-based stress-response genes A gene list contributing to stress responses was prepared followed by its identification by manual comparison of the two genomes. This led to the identification of key gene sets that differentiated niche adaptability of the dairy and probiotic strains. Phylogenetic analysis The phylogenetic tree of the 16S rRNA sequences was constructed via the CBRG (Computational Biochemistry Research Group) server (http://cbrg.inf.ethz.ch/) by using the Newick format.

Results and discussion The decoding of microbial genomes offers the opportunity to understand the mechanisms of action, metabolic capabilities, and defining traits relevant to adaptation and performance of the organism. The subsystem analysis using the SEED platform revealed the common structure between L. helveticus MTCC 5463 and L. helveticus DPC 4571 for subsystems like iron acquisition and metabolism, motility, chemotaxis, secondary metabolism, stress response, nitrogen metabolism, and dormancy and sporulation as shown in Figs. 1 and 2. Major subsystems and metabolic pathways are conserved between the strains; however, the numbers of genes are higher in MTCC 5463 for certain functional categories compared with DPC 4571. The presence of biotin synthesis genes and difference in cofactors, vitamins, prosthetic groups, and pigments suggest the differential ability of the strain in production of bioactive compounds in contrast to the DPC 4571 strain. The unrooted phylogenetic tree of MTCC 5463, and other sequenced L. helveticus strains, is shown in Fig. 3 where the PAM distances indicated at the branches of the tree show similarity among L. helveticus strains. The presence of a putative gene for alkaline shock protein in MTCC 5463 is a clue of the vaginal origin of MTCC 5463, as this gene has also been identified in the vaginal isolate Lactobacillus iners AB-1. Hence, comparative genomics throw light on the phylogeny and developmental biology of the strain. We can further identify genes that can act as biomarkers for selection of strains with improved stress resistance and enhanced effectiveness. The absence of identified stress-adaptive genes in a strain can be supplemented with genetic manipulation measures to enhance the strain’s fitness. A comprehensive list of stressresistance genes can act as criteria for prescreening of a strain’s ability to survive the gastric transit in case of probiotic cultures or storage/preservation stability for a starter strain. The locus tags of the genes found to be distinctly present or absent in MTCC 5463 in comparison with DPC 4571 are shown in Table 1. Acid stress resistance Lactobacilli strains categorized as probiotic bacteria have to survive shifting pH encountered in the gut (Cotter and Hill 2003). When a probiotic strain passes through the stomach it gets exposed to enzymes and an acidic pH. To transcend the colon, the strains must possess a number of strategies. These include F0F1ATPase, glutamate decarboxylase system, general stress chaperones to repair proteins and DNA, cell envelope composition, and alkalization of the external environment (De Angelis and Gobbetti 2004). The acid tolerance ability of MTCC 5463 was estimated by comparing the growth of viable cell counts in MRS agar plates after 24 h that showed a tolerance up to pH 3 and with no growth at pH 2 (Ashar and Prajapati 1998), which is in accordance to standards for acid tolerance of probiotic culture set at pH 3 (Liong and Shah 2005). The acid tolerance of MTCC 5463 is further Published by NRC Research Press

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Fig. 1. Comparison of subsystem features between Lactobacillus helveticus MTCC 5463 and L. helveticus DPC 4571.

Fig. 2. Comparison of subsystem feature counts of stress response between Lactobacillus helveticus MTCC 5463 and L. helveticus DPC 4571.

revealed in its capacity to increase the titratable acidity from 0.17 to 1.68; furthermore, 2.22% lactic acid in a skimmed milk medium product when fermented at 37 °C for 0 h, 12 h, and 24 h showed a significant (P < 0.05) increase in titratable acidity (R. Goswami, personal communications). F0F1-ATPase is a multiple-subunit enzyme that functions as a membranous channel for proton translocation (Sebald et al. 1982), while the atpBEFHAGDC operon is up regulated as a result of acid stress in lactobacilli (Kullen and Klaenhammer 1999). The probiotic strain MTCC 5463 and starter strain DPC 4571 displayed six subunits of the atp operon including atpB, atpE, atpF, atpH, atpA, atpD, and atpC. The atpB and atpG

subunits were predicted to have undergone frameshift mutations in the MTCC 5463 genome, whereas both subunits were intact in the DPC 4571 genome. A mutated atpG subunit, however, does not reduce the function of the gene because the substitution at position Met-23 by Arg or Lys has little effect on membrane ATPase activity or H+ pumping (Shin et al. 1992). A mutation, leading to a reduced ATPase activity in the strain, may be beneficial from an industrial point of view as it may lead to reduced post-acidification during storage (Liu et al. 2009). Lactobacillus helveticus membranes contain an inwardly directed K+ pump with a putative role in cytoplasmic pH regulation (Solari et al. 1997). Both Published by NRC Research Press

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Fig. 3. Unrooted phylogenetic tree of the 16S rRNA genes of closely related Lactobacillus helveticus strains with PAM distances indicated at the branches of the tree.

the dairy and probiotic strains carry a single ORF (open reading frame) for H+/K+ ATPase exchange. The conservation of this gene in both genomes could be due to its role in preservative-resistance mechanisms, especially, against weak acids. Another mechanism of maintaining pH homeostasis in bacteria are the Na(+)/H(+) antiporters. We identified two ORFs of Na(+)/H(+) antiporters in the probiotic strain, whereas the dairy strain harbored a single homolog. Na(+)/H(+) antiporter was recently identified to be a bacterial robustness indicator because of its role in salt tolerance (van Bokhorst-van de Veen et al. 2012). This justifies the maintenance of Na(+)/H(+) antiporter genes in DPC 4571, a cheese starter where salting is an essential step in cheese making. Apart from pH homeostasis, the expression of Na(+)/H(+) exchangers play a significant role in the antidiarrheal mechanism of action in probiotics (Singh et al. 2012). Copper (Cu(+)) ATPases are membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+) contributing towards maintaining pH homeostasis. Data mining for the core elements of copper homesostasis operon, we could identify the presence of a copper transporting ATPase in the MTCC 5463 and DPC 4571 genomes. The gene for a copper chaperone was present only in the MTCC 5463 genome. There is no identified function for copper or selenium in any member of the Lactobacillales, hence the presence of a copper chaperone gene suggests its role in defense mechanism. Both genomes revealed

the presence of cadmium-translocating P-type ATPases, ABC-type cobalt transport system, zinc ABC transporter ATP-binding subunit, and nickel transport system permease protein. Although lactobacilli do not require iron for growth, MTCC 5463 contained genes for high-affinity chelators (siderophores), which were found to be absent in the DPC 4571 genome. Traditionally, cheese was being made in copper kettles, which must have led the starter strains to maintain genes involved in copper homeostasis. Although copper kettles are no longer being used in cheese manufacturing, the presence of genes involved in copper homeostasis in both genomes suggest that the genes may have been conserved due to its role in acid tolerance (Penaud et al. 2006). The presence of heavy metal resistance genes in MTCC 5463 improves the functionality of the strain as a heavy metal detoxifying agent by binding and effluxing heavy metals from food and water (Halttunen et al. 2007). Strains MTCC 5463 and DPC 4571 exhibited homologs of ornithine decarboxylase known to generate secondary metabolic energy and provide resistance against acid stress. The glutamate-dependent acid resistance system components identified in the probiotic MTCC 5463 include L-2,4-diaminobutyrate decarboxylase and related PLP-dependent proteins with a frameshifted glutamate/gamma-aminobutyrate antiporter. The glutamate decarboxylase gene was totally absent in the dairy genome. Thus, the presence of gene clusters encoding amino acid Published by NRC Research Press

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Table 1. List of unique genes conferring stress resistance in Lactobacillus helveticus MTCC 5463 and reference strain L. helveticus DPC 4571.

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Locus tag Subsystem

Gene*

Gene name

MTCC 5463

DPC 4571

Acid stress resistance

atpB(−) napA(+) copZ(+) nikB(+) ddc(+) arcB(+) arc(+) dnaK(+) GrpE(+) hrcA(+) pspC(+) uvrA(−)

F0F1-ATP synthase Na(+)/H(+) antiporter Copper chaperone Nickel transport system permease protein L-2,4-diaminobutyrate decarboxylase Ornithine carbamoyltransferase Carbamate kinase Chaperone protein GrpE protein (hsp-70 cofactor hsp20) Heat-inducible transcription repressor Polymorphic protein Excinuclease abc subunit a

— AAULH_03086, AAULH_01088 AAULH_13076 AAULH_01205 AAULH_14036 AAULH_07591 AAULH_07596 AAULH_07741 AAULH_07746 AAULH_07756 AAULH_03153 —

LHV_0806 — — — — — — — — — — LHV_1132, LHV_0730

Bile stress resistance

Cbsh(+)

Conjugated bile salt hydrolase

AAULH_13111, AAULH_05404, AAULH_05409

—

Starvation response

cstA(+) PsiE(+) yugI(+)

Starvation protein a Phosphate starvation-inducible protein General stress-like protein

AAULH_14511 AAULH_08703 AAULH_12636, AAULH_12641

— — —

Heat shock resistance

HtpX(+) Hsp33(+) Hsp10(+)

Cytoplasmic membrane metalloprotease Heat shock protein 33 10 kda chaperonin heat shock protein

AAULH_00653 AAULH_01627 AAULH_02323

— — —

Cold stress resistance

CheY(+)

Cold stress response involved in chemotaxis

AAULH_00584, AAULH_08643, AAULH_09183, AAULH_10577, AAULH_04400

—

Osmotic stress resistance

Dps(+) infA(+) PepX(+)

Dna-binding ferritin-like protein Protein chain initiation factor X-prolyl dipeptidyl aminopeptidase

AAULH_07011 AAULH_07776 AAULH_08346

— — —

*(+) indicates presence and (−) indicates absence of that gene in L. helveticus MTCC 5463 as compared to reference strain L. helveticus DPC 4571.

decarboxylation pathways are strain specific rather than species specific, indicating that the clusters have been spread through horizontal gene transfer (Rossi et al. 2011). The glutamate decarboxylase gene is known to be present in very high levels in lactic acid bacteria originally isolated from fermented foods, hence it was not surprising that MTCC 5463, which is extensively used in the manufacture of synbiotic foods, carried the gene (De Biase and Pennacchietti 2012). Foods fermented with strains that harbor the glutamate decarboxylase gene (GAD) are known to exhibit increased angiotensin-converting enzyme inhibition activity, 1,1-diphenyl2-picylhydrazyl radical-scavenging ability, and oxygen radical absorbance capacity compared with strains absent for GAD (Chiu et al. 2013). Whole genome analysis can help identify such functional genes in the genome that can be exploited for enhanced health benefit attributes of the strain. The arginine deiminase (ADI) pathway and the resulting basic compounds have an established role in maintenance of pH homeostasis. The pathway consists of three cytoplasmic enzymes: (i) arginine deiminase (arcA), converting arginine into citrulline and ammonia; (ii) catabolic ornithine carbamoyltransferase (arcB), converting citrulline into carbamoyl phosphate and ornithine; and (iii) carbamate kinase (arcC), converting carbamoyl phosphate into ammonia, carbon dioxide, and ATP. Thus, the ADI pathway results in ATP for additional energy, whereas ammonia production offers an advantage under acid stress conditions. The probiotic MTCC 5463 carried frameshifted genes of arcB and arcC while harboring functional carbamate kinase homologs. DPC 4571 carried none of the genes involved in the ADI pathway, this may be because only obligate heterofermenters possessed all three genes for ADI pathway as in Lactobacillus sanfranciscensis (Liu et al. 2009). The probotic strain has been adapted to a milk-based medium for regular passages and in milk-based synbiotic formulations since the last 25 years. This presents a selective pressure for maintaining carbamate kinase in dairy environments where the natural

free amino acid concentrations are very low. Carbamae kinase plays a role in cellular amino acid biosynthetic process. Both genomes, MTCC 5463 and DPC 4571, lack the genes of the urease gene cluster ureABIEFGH. Utilization of such urease-deficient mutants in dairy processes is known to improve regularity of acidification and lower production of ammonia in whey (Monnet et al. 2004). Protection and repair of macromolecules The MTCC 5463 genome carried genes for the triad, DnaK, GrpE, and DnaJ, whereas DPC 4571 carried homologs for only DnaJ. Acid adaptation is supported by the induction of the chaperones GroES, GroEL, HrcA, ClpE, ClpP, and ClpL, trigger factor, GrpE, DnaK, DnaJ, and the repression of ClpC. Both genomes showed the presence of GroES, GroEL, and a large number of ClpATPase family of stress proteins. MTCC 5463 expressed heat-inducible transcription repressor hrcA and a stress-responsive transcriptional regulator pspC. The dairy strain genome lacked the CtsR, HrcA, PspC, or CcpA regulators. A large number of conserved sequences of DNA repair proteins have been identified in both genomes, including the DNA mismatch repair protein, DNA damage inducible protein, and excinuclease ABC subunit A, subunit B, and subunit C. The uvrA gene of MTCC 5463, coding for subunit A of the excinuclease ABC complex and involved in the nucleotide excision repair mechanism, was frameshifted and hence rendered nonfunctional. The other genes contributing to repair and protection of macromolecules included DNA repair protein RecO, RadA, transcription-repair coupling factor, and recombination protein RecR. The comparative genomic data reveal an abundance of molecular chaperones in both genomes conferring the ability to recognize and bind with stress-induced denatured/unfolded polypeptides, leading to partial unfolding of the misfolded polypeptide substrates. ATP hydrolysis can induce further unfolding Published by NRC Research Press

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and release from the chaperone, leading to spontaneous refolding into native proteins (Priya et al. 2013). Bile stress resistance Bile tolerance has often been used as a selection criterion for probiotic bacterial strains. When challenged with bile, bacteria are known to modify cell envelope properties to counter the deleterious effects, including protein misfolding and denaturation, DNA damage, the formation of secondary structure in RNA, and intracellular acidification (Lebeer et al. 2008). Bile did not inhibit the growth of MTCC 5463 when subjected to 2% of bile, suggestive of stress-adaptation mechanisms (Ashar and Prajapati 1998). Common bile stress-response factors include chaperones and bile salt hydrolases (bsh) or conjugated bile acid hydrolases (cbsh) genes belonging to the choloylglycine hydrolase family. The MTCC 5463 genome exhibited multiple coding sequences for choloylglycine hydrolase. Multiple copies of bsh were also annotated in probiotic Lactobacillus acidophilus NCFM (bshA and bshB), Lactobacillus johnsonii NCC533 (three genes), and Lactobacillus gasseri ATCC33323 (two genes) that promote the excretion of lipids such as cholesterol. This could be the mechanism behind the hypocholesterolemic ability of MTCC 5463 as proved by clinical trials (Ashar and Prajapati 1998). Evidently, the cheese starter DPC 4571 adapted to the dairy niche and displayed a total lack of bsh genes. A frameshift at nucleotide position 417 introduced a stop codon in the bile salt hydrolase gene of DPC 4571 rendering the gene inactive (O’Sullivan et al. 2009). This loss is mainly due to the transition of DPC 4571 to a nutritionally rich environment, which allowed a metabolic simplification (Callanan et al. 2008). The absence of bsh in the DPC 4571 genome and its presence in the probiotic genome is due to the selective pressure of bile in the gut environment (Cremonesi et al. 2012). Proteomic analysis of Lactobacillus plantarum identified cyclopropane-fatty-acyl-phospholipid synthase (cfa) as a potential marker for bile tolerance (Hamon et al. 2011). Both genomes under study exhibited single homologs of cfa, establishing their ability to tolerate bile. The enzyme cyclopropane fatty acid synthase is again implicated in maintenance of cell envelope integrity and in synthesis of lactobacillic acid that plays an indirect role in immunomodulatory activities (Jones et al. 2011). The expression of cfa could have contributed towards the immunomodulatory effects of the probiotic strain (Patidar and Prajapati 1999). Starvation response and stress tolerance Starvation is one of the most common stresses faced by lactobacilli, leading to nutrient exhaustion and subsequent starvation. In a nutritious niche, such as dairy, a cheese starter needs to adapt under the physiological and nutritional conditions in the cheesecurd ecosystem while competing with other lactic acid bacteria (Derzelle et al. 2000). Glucose and lactose starvation has been shown to induce resistance to many stresses (heat, low pH, bile, and oxidative and osmotic stress) (Chervaux et al. 2000). The MTCC 5463 genome harbors genes for carbon starvation protein A, phosphate starvation-inducible stress-related protein, phosphate starvation-inducible protein PsiE, and phosphate starvationinducible protein PhoH gene. The DPC 4571 genome carried a functional homolog of PhoH along with a phosphate starvationinducible stress-related protein but lacked the carbon starvation protein (cstA). Unlike DPC 4571, MTCC 5463 harbors cstA, which could be for peptide utilization and to influence host microbe interactions (Rasmussen et al. 2013). The higher number of homologs of starvation-induced genes in the gut strain could be to counter the competition with numerically dominant gut microflora for the adhesion receptors and available nutrients. Induction of guanosine tetraphosphate pool upon amino acid starvation also plays a role in acid stress resistance (Wehmeier et al. 2001). Both genomes carry guanosine tetraphosphate synthetase genes, and universal stress protein (UspA) induced by response to heat,

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substrate starvation, exposure to antimicrobial agents, and oxidative stress. A general stress-like protein gene was found only in the probiotic genome, whereas both genomes expressed the stressresponsive transcriptional regulator PspC and stress-induced DNA-binding protein. Heat shock response A previous study by the authors focused on enhancing the heat tolerance of MTCC 5463 by subjecting it to repeated heat shocks to improve its viability during spray drying. The spray-dried milkcereal blend slurry had 2.1 × 109 cfu/g of live MTCC 5463, which reduced by two log cycles after four months of refrigerated storage (Prajapati et al. 1987). The heat shock related negative regulators CtsR and HrcA, and chaperones GrpE, DnaK, DnaJ, GroEL, and GroES play a major role in bacterial responses to sudden increases of environmental temperature by assisting protein folding. The probiotic genome carries hrcA, whereas the dairy strain lacks the repressor. For heat responses, the MTCC 5463 genome had DnaK, GrpE, and DnaJ chaperones, whereas DPC 4571 carried homologs for only DnaJ. Apart from the molecular protein chaperones mentioned above, small heat shock proteins function as mediators to correct protein folding in the context of a multi-chaperone network. Small heat shock proteins were found to be differently distributed in the MTCC 5463 and DPC 4571 genomes as shown in Table 1. This observation is similar to the view presented by Broadbent et al. (1997) that DnaJ and GrpE may not be specifically induced in response to heat shock in species of dairy lactobacilli. HtrA proteases, like serine proteases, that ensure efficient removal of misfolded or damaged proteins have been identified in both the strains. Heat shock proteins, such as GroES and GroEL, also affect immunomodulation, and their presence in the probiotic strain genome, while deleted in the dairy strain, proves the functional characteristics of MTCC 5463. During the manufacturing of cheeses, the lactic acid bacteria are exposed to elevated temperatures during cooking, which explains why DPC 4571 maintains heat shock proteins in its genome. Expression of heat stress resistance conferring genes can be beneficial to probiotic strains to withstand preservation techniques like spray drying and freeze drying. Cold stress resistance Freeze dried MTCC 5463 formulation with favourable reducing and bulking agents in a standardized ratio displayed viable counts of 8.1 and 9.32 log CFU/g at 25 ± 2 °C storage temperature (up to 2 months) and refrigerated temperature storage (up to 1 year), respectively (U.K. Panchal, personal communications, 2013). Bacteria are able to adapt to cold temperatures through a set of cold shock proteins. A well orchestrated response can prove beneficial to a dairy strain for increased recovery of cells after freeze drying. Bacteria respond to heat shock and osmotic shock within minutes compared with cold shock that takes a few hours (Derzelle et al. 2000). Comparative proteomic analysis of adaptation mechanisms of cold-adapted prokaryotes revealed relative abundance of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases (Watanabe et al. 2012). Data mining the genomes under study revealed the presence of cold shock proteins and ATPdependent RNA helicase, DEAD/DEAH box in both the strains. Other cold-induced proteins have also been identified in the genomes that function at various levels of cellular physiology such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, Ilvc, Gap, triosephosphate isomerase). Thus, both strains under study carry a diverse set of cold adaptation genes to adapt to frozen storage, low temperature fermentation during cheese ripening, and below optimal storage conditions. Information from data mining could contribute to optimize the fermentation process and conservation conditions for these strains. Published by NRC Research Press

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Senan et al.

Oxygen stress resistance Aerobic conditions are present during the process of manufacture and storage of functional foods, and aerotolerance is a desired trait in probitoic bacteria intended for use in industry. The strains carried homologs for thioredoxin and thioredoxin reductase that contribute towards oxygen detoxification. Both genomes lacked antioxidative enzymes, such as catalase, superoxide dismutase, peroxidases, and oxidases, resulting in inefficient cellular H2O2 detoxification under aerobic conditions. Oxidative stressrelated genes support the successful survival of intestinal bacteria under osmotic stress conditions caused by carbohydrates in the diet and cellular response of phagocytes. The probiotic and dairy starter strains employ osmotic stress resistance conferring genes for protection against high concentrations of sugar and salt in synbiotic formulations and cheeses. The MTCC 5463 strain exhibits abundant growth in aerobic condition, although traditionally being a facultatively anaerobic strain. We can suggest that the thioredoxin-thioredoxin reductase system is the major thiol/ disulfide redox system that supports L. helveticus growth under aerobic conditions. This system can be further exploited to enhance the oxidative stress response in MTCC 5463 for robustness and recovery after freeze drying, the primary preservation format. Osmotic stress resistance The ability of MTCC 5463 to survive the mammalian gastrointestinal tract anaerobically at high osmolarity could be due to the presence of DNA-binding ferritin-like protein (Dps) identified in the genome. Surprisingly, this protein was absent in the dairy strain DPC 4571. Other genes present in both genomes that might contribute towards osmotic stress resistance include periplasmicbinding protein of the oligopeptide ABC transporter (suggestive of an uptake system for glycine-betaine and proline), protein chain initiation factor, aspartate kinase, single-stranded DNA-binding protein, and glutathione reductase. Both genomes do not carry genes coding for glycine betaine and (or) proline transporters that are known to contribute towards tolerance to high osmolarity in lactic acid bacteria. The presence of PepQ (a cytoplasmic prolidase which specifically liberate proline from di-peptides), PepX (Xprolyl dipeptidyl aminopeptidase), and PepI (iminopeptidase) in MTCC 5463 can contribute towards osmotic stress resistance. The dairy starter DPC 4571 exhibited PepQ and PepI but lacked the PepX gene. In conclusion, traditional methods of analysing functionality of a strain is replaced by cost effective next generation sequencing revealing the maximum potential risk and benefit. There is a great interest in analyzing the genomes of L. helveticus strains because of their widespread use in cheese technology. By comparing genomes we can identify unique gene sets for stress adaptation that could help in identifying fitness factors that enhances the probiotic features of a strain. In this study, we decided to concentrate on only two highly related dairy strains. It was observed that both strains shared an abundance of common stress adaptation mechanisms. The striking niche-based gene set comprised of multiple homologs of the bile salt hydrolase (bsh) gene in the probiotic genome, whereas DPC 4571 adapted to the nonbile-containing dairy niche that displayed a total lack of bsh genes. The incorporation of bile tolerance genes will be an essential mediation to use dairy strains as functional agents. The molecular chaperones are found more in the probiotic genome. This is not surprising because the strain faces much more physiological stress in the human gut than the dairy strain propagated in an aseptic nutrient-rich atmosphere. The probiotic genome with a few subunits of F0F1-ATPase operon missing is supported by a complete heavy metal homeostasis system, glutamate decaroxylase system. The dairy genome in turn carries a complete F0F1-ATPase operon but a loss in heavy metal homeostasis and glutamate decaroxylase system. A categorical comparison of other available genomes, such as L. helveticus H10, L. helveticus R0052, and L. helveticus CNRZ 32

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strains with completed genome sequences, could give more decisive information on the niche-specific gene sets. It needs to be reiterated that the conclusions drawn on the gene sets along with the presence of missing genes and frameshifts depended entirely on the extent to which the genome has been completely assembled and annotated. The precision of the comparative genomic analysis is based on the 593 contig sequences of MTCC 5463. There could be a number of hidden genes in contig gaps. The two genome sequences in this study were annotated using two different approaches, suggesting that some differences might actually be due to technical biases rather than true biological differences. Frameshifts are frequently found in the assembly using reads obtained by GS-FLX due to single-nucleotide indel errors, especially in oligomeric nucleotide region such as GGGGGG or CCCCCC. Thus, for conclusive evidence on the frameshift of the nucleotide sequence of the genes, experimental confirmation is needed. The frameshifts identified in genes could be further confirmed by Sanger sequencing methods. Attempts to prove the expression of the identified genes under the specific stress conditions could provide experimental evidence to support the suggested outcomes. We have attempted to relate molecular function of genes to their physiological importance. We conclude that the presence and number of genetic determinants of stress resistance are species and strain specific, which can be suggestive of the suitability of a strain for a particular application. Comparative genomics could be a tool for applying a quality by design approach to the food industry.

Acknowledgements The authors are grateful to Manisha Sajnani for her assistance in performing the genome bioinformatic analysis. Additionally, thank you to Arpita Patel for her contributions towards phylogenetic analysis and literature search.

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