Abstract
Lactic acid bacteria (LAB) have been widely used in starter cultures, notably in the dairy industry, for the fermentation of milk into yogurt and cheese. Domesticated lactococci, streptococci, and lactobacilli used in food manufacturing applications rely on a plethora of defense systems that allow them to survive bacteriophage (phage) predation. Unfortunately, phage exposure remains an issue throughout the dairy industry, and formulation and rotation strategies leverage natural phage defense mechanisms such as restriction-modification systems, abortive infection, and clustered regularly interspaced short palindromic repeats (CRISPR). This chapter highlights the most broadly used phage-resistance systems in LAB, with an emphasis on the novel CRISPR/Cas system, which provides acquired adaptive immunity against phages by targeting viral nucleic acid in a sequence-specific manner.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Alatossava T, Forsman P, Ritzenthaler P (1995) Genome homology and superinfection immunity between temperate and virulent Lactobacillus delbrueckii bacteriophages. Arch Virol 140:2261–2268
Anba J, Bidnenko E, Hillier A, Ehrlich D, Chopin MC (1995) Characterization of the lactococcal abiD1 gene coding for phage abortive infection. J Bacteriol 177:3818–3823
Andersson AF, Banfield JF (2008) Virus population dynamics and acquired virus resistance in natural microbial communities. Science 320:1047–1050
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712
Barrangou R, Fremaux C, Horvath P, Romero D, Boyaval P (2008) Cultures with improved phage resistance. Patent application WO 2008/108989, 12 Sept 2008
Blumenthal RM, Cheng X (2002) Restriction-modification systems. In: Yasbin RE and Streips UN (Eds.), Modern microbial genetics, 2nd ed. Wiley, New York, pp. 177–225
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151:2551–2561
Breitbart M, Rohwer F (2005) Here a virus, there a virus, everywhere the same virus? Trends Microbiol 13:278–284
Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964
Brüssow H (2001) Phages of dairy bacteria. Annu Rev Microbiol 55:283–303
Bruttin A, Desière F, Lucchini S, Foley S, Brüssow H (1997) Characterization of the lysogeny DNA module from the temperate Streptococcus thermophilus bacteriophage ΦSfi21. Virology 233:136–148
Burrus V, Bontemps C, Decaris B, Guédon G (2001) Characterization of a novel type II restriction-modification system, Sth368I, encoded by the integrative element ICESt1 of Streptococcus thermophilus CNRZ368. Appl Environ Microbiol 67:1522–1528
Chopin M-C, Chopin A, Bidnenko E (2005) Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8:473–479
Cluzel P-J, Chopin A, Ehrlich SD, Chopin MC (1991) Phage abortive infection mechanism from Lactococcus lactis subsp. lactis, expression of which is mediated by an Iso-ISS1 element. Appl Environ Microbiol 57:3547–3551
Coffey A, Ross P (2002) Bacteriophage-resistance systems in dairy starter strains: molecular Âanalysis to application. Antonie van Leeuwenhoek 82:303–321
Cui Y, Li Y, Gorgé O, Platonov ME, Yan Y, Guo Z, Pourcel C, Dentovskaya SV, Balakhonov SV, Wang X, Song Y, Anisimov AP, Vergnaud G, Yang R (2008) Insight into microevolution of Yersinia pestis by clustered regularly interspaced short palindromic repeats. PLoS One 3:e2652
Daly C, Fitzgerald GF, Davis R (1996) Biotechnology of lactic acid bacteria with special reference to bacteriophage resistance. Antonie van Leeuwenhoek 70:99–110
Deveau H, Labrie SJ, Chopin MC, Moineau S (2006) Biodiversity and classification of lactococcal phages. Appl Environ Microbiol 72:4338–4346
Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190:1390–1400
Djordjevic GM, Klaenhammer TR (1997) Bacteriophage-triggered defense systems: phage Âadaptation and design improvements. Appl Environ Microbiol 63:4370–4376
Djordjevic GM, O’Sullivan DJ, Walker SA, Conkling MA, Klaenhammer TR (1997) A triggered-suicide system designed as a defense against bacteriophages. J Bacteriol 179:6741–6748
Dupont K, Janzen T, Vogensen FK, Josephsen J, Stuer-Lauridsen B (2004) Identification of Lactococcus lactis genes required for bacteriophage adsorption. Appl Environ Microbiol 70:5825–5832
Durmaz E, Klaenhammer TR (1995) A starter culture rotation strategy incorporating paired restriction/modification and abortive infection bacteriophage defenses in a single Lactococcus lactis strain. Appl Environ Microbiol 61:1266–1273
Durmaz E, Klaenhammer TR (2007) Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis. Appl Environ Microbiol 189:1417–1425
Durmaz E, Madsen SM, Israelsen H, Klaenhammer TR (2002) Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor. J Bacteriol 184:6532–6544
Foley S, Lucchini S, Zwahlen MC, Brüssow H (1998) A short noncoding viral DNA element showing characteristics of a replication origin confers bacteriophage resistance to Streptococcus thermophilus. Virology 250:377–387
Forde A, Fitzgerald GF (1999) Bacteriophage defence systems in lactic acid bacteria. Antonie van Leeuwenhoek 76:89–113
Forde A, Daly C, Fitzgerald GF (1999) Identification of four phage resistance plasmids from Lactococcus lactis ssp. cremoris H2O. Appl Environ Microbiol 65:1540–1547
Fortier LC, Bouchard JD, Moineau S (2005) Expression and site-directed mutagenesis of the lactococcal abortive phage infection protein AbiK. J Bacteriol 187:3721–3730
Garvey P, Fitzgerald GF, Hill C (1995) Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40. Appl Environ Microbiol 61:4321–4328
Garvey P, Hill C, Fitzgerald GF (1996) The lactococcal plasmid pNP40 encodes a third bacteriophage resistance mechanism, one which affects phage DNA penetration. Appl Environ Microbiol 62:676–679
Godde JS, Bickerton A (2006) The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes. J Mol Evol 62:718–729
Guglielmotti DM, Deveau H, Binetti AG, Reinheimer JA, Moineau S, Quiberoni A (2009) Genome analysis of two virulent Streptococcus thermophilus phages isolated in Argentina. Int J Food Microbiol 136:101–109
Haft DH, Selengut J, Mongodin EF, Nelson KE (2005) A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 1:e60
Hale C, Kleppe K, Terns RM, Terns MP (2008) Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14:2572–2579
Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139:945–956
Heidelberg JF, Nelson WC, Schoenfeld T, Bhaya D (2009) Germ warfare in a microbial mat Âcommunity: CRISPRs provide insights into the co-evolution of host and viral genomes. PLoS One 4:e4169
Held NL, Whitaker RJ (2009) Viral biogeography revealed by signatures in Sulfolobus islandicus genomes. Environ Microbiol 11:457–466
Hill C, Pierce K, Klaenhammer TR (1989) The conjugative plasmid pTR2030 encodes two Âbacteriophage defense mechanisms in lactococci, restriction modification (R+/M+) and abortive infection (Hsp+). Appl Environ Microbiol 55:2416–2419
Hill C, Miller LA, Klaenhammer TR (1990) Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis. J Bacteriol 172:6419–6426
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of Bacteria and Archaea. Science 327:167–170
Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R (2008) Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol 190:1401–1412
Horvath P, Coûté-Monvoisin AC, Romero DA, Boyaval P, Fremaux C, Barrangou R (2009) Comparative analysis of CRISPR loci in lactic acid bacteria genomes. Int J Food Microbiol 131:62–70
Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433
Jansen R, van Embden JD, Gaastra W, Schouls LM (2002) Identification of genes that are Âassociated with DNA repeats in prokaryotes. Mol Microbiol 43:1565–1575
Josephsen J, Neve H (1998) Bacteriophages and lactic acid bacteria. In: Salminen S, von Wright A (Eds.), Lactic acid bacteria: microbiology and functional aspects, 2nd ed. Marcel Dekker, New York, pp. 385–436
Josephsen J, Vogensen FK (1989) Identification of three different plasmid-encoded restriction/modification systems in Streptococcus lactis subsp. cremoris W56. FEMS Microbiol Lett 59:161–166
Kim JH, Kim SG, Chung DK, Bor YC, Batt CA (1992) Use of antisense RNA to confer bacteriophage resistance in dairy starter cultures. J Ind Microbiol 10:71–78
Klaenhammer TR, Sanosky RB (1985) Conjugal transfer from Streptococcus lactis ME2 of Âplasmids encoding phage resistance, nisin resistance and lactose-fermenting ability: evidence for a high-frequency conjugative plasmid responsible for abortive infection of virulent bacteriophage. J General Microbiol 131:1531–1541
Kunin V, He S, Warnecke F, Peterson SB, Martin HG, Haynes M, Ivanova N, Blackall LL, Breitbart M, Rohwer F, McMahon KD, Hugenholtz P (2008) A bacterial metapopulation adapts locally to phage predation despite global dispersal. Genome Res 18:293–297
Labrie SJ, Moineau S (2007) Abortive infection mechanisms and prophage sequences significantly influence the genetic makeup of emerging lytic lactococcal phages. Appl Environ Microbiol 189:1482–1487
Larbi D, Decaris B, Simonet JM (1992) Different bacteriophage resistance mechanisms in Streptococcus salivarius subsp. thermophilus. J Dairy Res 59:349–357
Lévesque C, Duplessis M, Labonté J, Labrie S, Fremaux C, Tremblay D, Moineau S (2005) Genomics organization and molecular analysis of virulent bacteriophage 2972 infecting an exopolysaccharide-producing Streptococcus thermophilus strain. Appl Environ Microbiol 71:4057–4068
Lillestøl RK, Redder P, Garrett RA, Brügger K (2006) A putative viral defence mechanism in archaeal cells. Archaea 2:59–72
Lillestøl RK, Shah SA, Brügger K, Redder P, Phan H, Christiansen J, Garrett RA (2009) CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol Microbiol 72:259–272
Lucchini S, Sidoti J, Brüssow H (2000) Broad-range bacteriophage resistance in Streptococcus thermophilus by insertional mutagenesis. Virology 275:267–277
Lucey M, Daly C, Fitzgerald GF (1992) Cell surface characteristics of Lactococcus lactis harbouring pCI528, a 46 kb plasmid encoding inhibition of bacteriophage adsorption. J Gen Microbiol 138:2137–2143
Madera C, GarcĂa P, Janzen T, RodrĂguez A, Suárez JE (2003) Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection. Int J Food Microbiol 86:213–222
Mahony J, McGrath S, Fitzgerald GF, van Sinderen D (2008) Identification and characterization of lactococcal-prophage-carried superinfection exclusion genes. Appl Environ Microbiol 74:6206–6215
Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV (2002) A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res 30:482–496
Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N, Karamychev V, Polouchine N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin K, Lee JH, DĂaz-Muñiz I, Dosti B, Smeianov V, Wechter W, Barabote R, Lorca G, Altermann E, Barrangou R, Ganesan B, Xie Y, Rawsthorne H, Tamir D, Parker C, Breidt F, Broadbent J, Hutkins R, O’Sullivan D, Steele J, Unlu G, Saier M, Klaenhammer T, Richardson P, Kozyavkin S, Weimer B, Mills D (2006a) Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci USA 103:15611–15616
Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV (2006b) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7
Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–1845
McGrath S, Seegers JF, Fitzgerald GF, van Sinderen D (1999) Molecular characterization of a phage-encoded resistance system in Lactococcus lactis. Appl Environ Microbiol 65:1891–1899
McGrath S, Fitzgerald GF, van Sinderen D (2001) Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis. Appl Environ Microbiol 67:608–616
McGrath S, Fitzgerald GF, van Sinderen D (2002a) Identification and characterization of phage-resistance genes in temperate lactococcal bacteriophages. Mol Microbiol 43:509–520
McGrath S, van Sinderen D, Fitzgerald GF (2002b) Bacteriophage-derived genetic tools for use in lactic acid bacteria. Int Dairy J 12:3–15
Moineau S (1999) Applications of phage resistance in lactic acid bacteria. Antonie van Leeuwenhoek 76:377–382
Moineau S, Walker SA, Holler BJ, Vedamuthu ER, Vandenbergh PA (1995) Expression of a Lactococcus lactis phage resistance mechanism by Streptococcus thermophilus. Appl Environ Microbiol 61:2461–2466
Mojica FJ, Ferrer C, Juez G, RodrĂguez-Valera F (1995) Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol Microbiol 17:85–93
Mojica FJ, DĂez-Villaseñor C, GarcĂa-MartĂnez J, Soria E (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60:174–182
Mojica FJ, DĂez-Villaseñor C, GarcĂa-MartĂnez J, Almendros C (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–740
Monteville MR, Ardestani B, Geller BL (1994) Lactococcal bacteriophages require a host cell wall carbohydrate and a plasma membrane protein for adsorption and ejection of DNA. Appl Environ Microbiol 60:3204–3211
Moscoso M, Suárez JE (2000) Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei. Virology 273:101–111
Nechaev S, Severinov K (2008) The elusive object of desire – interactions of bacteriophages and their hosts. Curr Opin Microbiol 11:186–193
Nyengaard N, Vogensen FK, Josephsen J (1995) Restriction-modification systems in Lactococcus lactis. Gene 157:13–18
O’Driscoll J, Glynn F, Cahalane O, O’Connell-Motherway M, Fitzgerald GF, Van Sinderen D (2004) Lactococcal plasmid pNP40 encodes a novel, temperature-sensitive restriction-Âmodification system. Appl Environ Microbiol 70:5546–5556
O’Sullivan DJ, Hill C, Klaenhammer TR (1993) Effect of increasing the copy number of bacteriophage origins of replication, in trans, on incoming-phage proliferation. Appl Environ Microbiol 59:2449–2456
O’Sullivan D, Coffey A, Fitzgerald GF, Hill C, Ross RP (1998) Design of a phage-insensitive lactococcal dairy starter via sequential transfer of naturally occurring conjugative plasmids. Appl Environ Microbiol 64:4618–4622
Pedersen MB, Jensen PR, Janzen T, Nilsson D (2002) Bacteriophage resistance of a ΔthyA mutant of Lactococcus lactis blocked in DNA replication. Appl Environ Microbiol 68:3010–3023
Pourcel C, Salvignol G, Vergnaud G (2005) CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151:653–663
Rousseau GM, Moineau S (2009) Evolution of Lactococcus lactis phages within a cheese factory. Appl Environ Microbiol 75:5336–5344
Sakamoto K, Agari Y, Agari K, Yokoyama S, Kuramitsu S, Shinkai A (2009) X-ray crystal structure of a CRISPR-associated RAMP superfamily protein, Cmr5, from Thermus thermophilus HB8. Proteins 75:528–532
Seegers JF, van Sinderen D, Fitzgerald GF (2000) Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain. Microbiology 146:435–443
Shah SA, Hansen NR, Garrett RA (2009) Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism. Biochem Soc Trans 37:23–28
Sijtsma L, Wouters JT, Hellingwerf KJ (1990) Isolation and characterization of lipoteichoic acid, a cell envelope component involved in preventing phage adsorption, from Lactococcus lactis subsp. cremoris SK110. J Bacteriol 172:7126–7130
Sing WD, Klaenhammer TR (1990) Plasmid-induced abortive infection in lactococci: a review. J Dairy Sci 73:2239–2251
Sorek R, Kunin V, Hugenholtz P (2008) CRISPR-a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 6:181–186
Stanley E, Walsh L, van der Zwet A, Fitzgerald GF, van Sinderen D (2000) Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance. FEMS Microbiol Lett 182:271–277
Sturino JM, Klaenhammer TR (2002) Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages. Appl Environ Microbiol 68:588–596
Sturino JM, Klaenhammer TR (2004a) Bacteriophage defense systems and strategies for lactic acid bacteria. In: Laskin AI, Bennett JW, Gadd GM (Eds.), Advances in applied microbiology, Vol. 56. Academic, San Diego, pp. 331–378
Sturino JM, Klaenhammer TR (2004b) Antisense RNA targeting of primase interferes with bacteriophage replication in Streptococcus thermophilus. Appl Environ Microbiol 70:1735–1743
Sturino JM, Klaenhammer TR (2006) Engineered bacteriophage-defence systems in bioprocessingÂ. Nat Rev Microbiol 4:395–404
Sturino JM, Klaenhammer TR (2007) Inhibition of bacteriophage replication in Streptococcus thermophilus by subunit poisoning of primase. Microbiology 153:3295–3302
Suárez V, Zago M, Giraffa G, Reinheimer J, Quiberoni A (2009) Evidence for the presence of restriction/modification systems in Lactobacillus delbrueckii. J Dairy Res 76:433–440
Sun X, Göhler A, Heller KJ, Neve H (2006) The ltp gene of temperate Streptococcus thermophilus phage TP-J34 confers superinfection exclusion to Streptococcus thermophilus and Lactococcus lactis. Virology 350:146–157
Tangney M, Fitzgerald GF (2002) AbiA, a lactococcal abortive infection mechanism functioning in Streptococcus thermophilus. Appl Environ Microbiol 68:6388–6391
Trotter M, Ross RP, Fitzgerald GF, Coffey A (2002) Lactococcus lactis DPC5598, a plasmid-free derivative of a commercial starter, provides a valuable alternative host for culture improvement studies. J Appl Microbiol 93:134–143
Tyson GW, Banfield JF (2008) Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ Microbiol 10:200–207
van der Ploeg JR (2009) Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology 155:1966–1976
Vergnaud G, Li Y, Gorgé O, Cui Y, Song Y, Zhou D, Grissa I, Dentovskaya SV, Platonov ME, Rakin A, Balakhonov SV, Neubauer H, Pourcel C, Anisimov AP, Yang R (2007) Analysis of the three Yersinia pestis CRISPR loci provides new tools for phylogenetic studies and possibly for the investigation of ancient DNA. Adv Exp Med Biol 603:327–338
Walker SA, Klaenhammer TR (2000) An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage. Appl Environ Microbiol 66:310–319
Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA (2009) Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure 17:904–912
Yang JM, Deurraza PJ, Matvienko N, O’Sullivan DJ (2006) Involvement of the LlaKR2I methylase in expression of the AbiR bacteriophage defense system in Lactococcus lactis subsp. lactis biovar diacetylactis KR2. J Bacteriol 188:1920–1928
Acknowledgments
We would like to acknowledge our colleagues and collaborators Patrick Boyaval, Christophe Fremaux, Dennis Romero, Anne-Claire Coûté-Monvoisin, Hélène Deveau, Josiane Garneau, Jessica Labonté, Manuela Villion, and Egon Bech Hansen for their support and many scientific contributions. We would also like to specifically thank Sylvain Moineau, Virgis Siksnys, and Jill Banfield for their insights and expertise on CRISPR.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this chapter
Cite this chapter
Barrangou, R., Horvath, P. (2011). Lactic Acid Bacteria Defenses Against Phages. In: Tsakalidou, E., Papadimitriou, K. (eds) Stress Responses of Lactic Acid Bacteria. Food Microbiology and Food Safety. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-92771-8_19
Download citation
DOI: https://doi.org/10.1007/978-0-387-92771-8_19
Published:
Publisher Name: Springer, Boston, MA
Print ISBN: 978-0-387-92770-1
Online ISBN: 978-0-387-92771-8
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)