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Genomic organization of lactic acid bacteria phagesC Schouler

To cite this version:C Schouler. Genomic organization of lactic acid bacteria phages. Le Lait, INRA Editions, 1996, 76(1_2), pp.81-89. �hal-00929478�

Lait (1996) 76, 81-89© Elsevier/INRA

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Genomic organization of lactic acid bacteria phages

C Schouler

Laboratoire de génétique microbienne, Inra, Domaine de Vi/vert, 78352 Jouy-en-Josas, France

Summary - The infection of lactic acid bacteria starter cultures by bacteriophages is one of the mostcommon causes of slow or incomplete fermentation in the dairy industry. Given the magnitude of theproblem and its economic impact, considerable research has been undertaken on lactic acid bacteriaphages. In recent years, numerous projects for the determination of the complete nucleotide sequenceof lactobacilli and lactococci phage genomes were carried out. Functions have been assigned to theproducts of several open reading frames. The genome of the phages has a modular organization. It isdivided in several regions, each involved in one step of the phage Iife cycle: integration/excision, repli-cation, production of structural proteins, assembly, packaging of the DNA and release of the phage out-side the cell alter the host Iysis.

lactic acid bacteria / bacteriophage / genome organization

Résumé - Organisation génomique des bactériophages des bactéries lactiques. Le déroulementdes processus de fermentation par les bactéries lactiques peut être perturbé par le développement debactériophages inhibant partiellement ou totalement la croissance bactérienne. Vue l'importance despertes économiques résultantes, les bactériophages ont fait l'objet de nombreux projets de recherche.Ces dernières années, de nombreux projets de séquençage systématique du génome de plusieurs typesde phages de lactocoques et de lactobacilles ont été entrepris. Des fonctions ont pu être assignées àquelques produits de cadres ouverts de lecture. Le génome comprend des régions distinctes, chacuneétant impliquée dans des étapes du cycle de développement phagique : intégration/excision, réplica-tion, production des protéines structurales, assemblage, empaquetage de l'ADN dans la capside néoformée et lyse cellulaire.

bactérie lactique / bactériophage / organisation génomique

INTRODUCTION product quality (Lawrence, 1978). Given theimportance of the problem, considerableresearch has been conducted on lactic acidbacteria bacteriophages, especially onphages infecting lactococci and, in sornedegree, on those attacking lactobacilli. Initialstudies most commonly provided data on

The infection of lactic acid bacteria by bac-teriophages is of considerable economicimportance in the dairy industry. It resultsin decreased acid production and impaired

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morphological, serological and physiologicalcharacteristics. Combined with the com-parative DNA studies, these results led toa consistent classification of phages infect-ing lactococci in 12 different groups (Jarviset al, 1991). DNA homology analyses havealso been used to determine the relation-ships between Lactobacillus phages. Thoseof Lactobacillus delbrueckii subsp bulgaricusand lactis were classified in four groups(Sechaud et al, 1988), while ail Lactobaci/-lus helveticus phages examined so far wereassembled in a single homology group(Lahbib-Mansais et al, 1988).

ln recent years, increasing researchefforts have been devoted to the molecularbiology of lactic phages. The first require-ment for understanding phage biology is todetermine the organization of the genome.Studies on gene function and gene expres-sion wou Id subsequently provide knowl-edge on phage development. This willlead,in particular, to a better understanding ofthe regulation of the Iysogeny and of thesynthesis, assembly and release of virionparticles. The phages so far examined pos-sess double stranded linear DNA and haveeither 3' staggered cohesive ends or circu-larly permuted ends with terminal redun-dancy. The G+C content of their genomereflects that of the host. Thus, lactococcalphages have a G+C value ranging around36% (Schouler et al, 1994), while that forLactobacillus delbrueckii phages has beenreported to range near 50% (Mikkonen andAlatossava, 1994).

The origin of new lactococcal bacterio-phages in the dairy industry has been thesubject of speculation for many years (Hug-gins and Sandine, 1977). It has been sug-gested that Iytic phages may evolve fromone another by exchange of DNA modulesor that Iysogenic starter cultures may bethe source of new virulent phages (Jarvis,1989). Sequence analysis of related phagesprovided some knowledge on phage evo-lution.

C Schouler

PHAGES OF LACrOBACILLI

Characterization of phages

Only a few Lactobacillus phages have beencharacterized in detail. For most of them,the only data available at the molecular levelconsist of the physical map. The nucleotidesequence of some genes or genomic ele-ments has been determined: an insertionsequence in phage <t>FSV(Shimizu-Kadotaet al, 1985), the attachment site (attP) (Rayaet al, 1992) and the integrase gene of phageoadh (Fremaux et al, 1993), the cohesiveends of phage PL-1 (Nakashima et al, 1994)and the Iysis genes of phage oadh (Hen-rich et al, 1995). The characterization ofgenetic elements required for the integra-tion of phage oadh into the Lactobacillusgasseri chromosome led to the construc-tion of an integration vector (Raya et al,1992).

The best studied are the Lactobacillusdelbrueckii subsp temperate phage mv4and the virulent phage LL-H. Both areclosely related and belong to the samehomology group (Mata et al, 1986). The36 kb genome of phage mv4 has beenmapped physically and its DNA is circularlypermuted (Lahbib-Mansais et al, 1992). Sev-eral genes have been characterized, suchas those involved in cell Iysis (Dupont,unpublished results, GenBank Z26590), thegenes encoding structural proteins (Vasalaet al, 1993) and the genetic elementsrequired for site-specific integration (attP,integrase, excisionase) (Dupont et al, 1995).The segments sequenced cover about 23%of the mv4 phage genome.

It has been shown that integration of thephage mv4 DNA in the host chromosomeoccurs at the tRNASer gene but an intactgene is preserved (Dupont et al, 1995). Anon-replicative vector based on phage inte-gration elements has been constructed andwas shown to integrate into the tRNASer

Bacteriophage of lactic acid bacteria

gene of Lactobacillus plantarum (Dupont etal,1995).

Phage LL-H has a circularly permutedand terminally redundant double strandedDNA genome (Trautwetter et al, 1986). Itslength is 34.6 kb with terminal repeats of2.8 ± 0.2 kb (Forsman and Alatossava,1991). The genes encoding the structuralproteins (Mikkonen and Alatossava, 1994),the terminase (Mikkonen and Alatossava,1995) and the Iysin (Alatossava, personalcommunication) have been characterized.This sequenced segment represents about67% of the genome. A group 1 intron hasbeen discovered in the gene encoding thelarge subunit of the terminase (Mikkonenand Alatossava, 1995).

Comparison studies

Extensive studies on the homology betweenphages LL-H and mv4 have been con-ducted. Firstly, DNA hybridization studiesbetween these two phages and sorne othershave shown that they are closely relatedand form one group of DNA homology (Mataet al, 1986). Comparison at the nucleotidelevel of the c1uster encoding structural pro-teins for both phages revealed that they arevery similar (Vasala et al, 1993). More inter-estingly, it has been found that the virulentphage LL-H genome has a partially deletedintegrase gene homologous to the 3' endof the mv4 integrase gene. In the LL-Hgenome also sorne traces of site-specificintegration elements remain. These datasuggest that the virulent phage LL-H andthe temperate phage mv4 could haveevolved from a corn mon temperate an ces-tor (Mikkonen and Dupont, personal com-munication). It was previously shown thatthe virulent phage <I>FSVhas been derivedfrom the prophage <I>FSW,in Lactobacilluscasei strain S-1, by acquisition of a newISL 1 insertion sequence (Shimizu-Kadotaet al, 1985).

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Comparison studies of two highly homo-logo us phages of Lactobacillus delbrueckiisubsp lactis, LL-H and LL-K, revealed theexistence of an 1.5 kb insertion in the genomeof the phage LL-K, called KIS-element (Fors-man and Alatossava, 1994). This element,composed of two putative open readingframes flanked by perfect 19 bp directrepeats, is absent in the phage LL-H genomewhich retained only one copy of the repeats.

These results indicate that related lacto-bacilli phages, like lambdoid phages(Campbell, 1994) are able to evolve byexchange. insertion or deletion of modules.

PHAGES OF LACTOCOCCI

Much more data are available on phagesactive against lactococci th an on thoseactive against lactobacilli. Lactococcalphages have been classified in 12 groupsbased on morphology, serological studiesand DNA homology (Jarvis et al, 1991). Themajority of phages encountered in dairyplants belong to three groups. In France,48% of the phages analyzed were virulentsmall isometric-headed (P008 type phage),29% were virulent prolate-headed (c6a typephage) and 21% were either virulent or tem-perate small isometric-headed (P335 typephage) (Prevots et al, 1990). This distributionis grossly the same in New Zealand (Jarvis,1977) and in Ireland (Casey et al, 1993). Incontrast, in Germany, 60% of the phagesisolated were prolate-headed (c6a typephage) and 40% were small isometric-headed (P008 and P335 type phages)(Lembke et al, 1980). The best character-ized phages belong to one of these threeprevaling groups of homology.

P335 phage species

These phages are isometric-headed andhave a genome size ranging around 36 kb.

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This group of homolgy is the only one, inthe lactococci phage classification, whichcontains both virulent and temperatephages. The phage genome possessescohesive ends or circularly permuted endswith terminal redundancy (Jarvis et al, 1991).Extensive studies have been carried out onthe characterization of the temperate phagesessentially with the aim to develop genetictools, such as chromosomal integration sys-tems and to have a better understanding ofthe mechanisms governing the conversionbetween the Iytic cycle and the Iysogeniza-tion step. The complete genome sequenceof phage rtt (van Sinderen et al, 1995) andTuc2009 (Oaly and Fitzgerald, personalcommunication) was determined. In con-trast, few data are available on virulentphages.

The genetic elements required for phageintegration into the host chromosome (theattachment site which is the site of recom-bination between the phage and the hostchromosome, and the integrase which cata-Iyzes the recombination event) have beenidentified for several phages. The attPsequence, 5'-nCnCATG-3', and the inte-grase from phages Tuc2009 (van de Guchteet al, 1994a), <l>LC3(Lillehaug and Birke-land, 1993) and r1 t (van Sinderen et al,1995) are almost identical. In contrast, theTP901-1 genome possesses a different attPsequence, 5'-TCAAT-3', (Christiansen et al,1994), suggesting an integration mecha-nism different from the one used byTuc20009, <l>LC3and rtt. The genetic ele-ments required for phage integration havebeen used to develop integrative vectors(Lillehaug and Birkeland, 1993; van deGuchte et al, 1994a).

Maintenance of Iysogeny depends onthe synthesis of a repressor which blockstranscription of the ly1ic genes. A gene cod-ing for a protein homologous to lambdaphage repressor CI has been identified inTuc2009 (van de Guchte et al, 1994b). Ahighly homologous gene, rra, identified in

C Schouler

phage rtt (van Sinderen et al, 1995), wasshown to encode a repressor of the Iyticgenes (Nauta et al, 1995). The repressorprotein Rro binds to three operatorsequences of 21 bp which repress tran-scription of the Iytic genes (Nauta et al,1995). These data have been used todevelop an inducible gene expression vec-tor (Nauta et al, 1995).

Some genes involved in the Iytic cyclehave also been identified by homology withsequences from the databases: proteinsinvolved in ONA replication, a dUTPase (vanSinderen et al, 1995). Genes coding forstructural proteins were identified by homo-logy with the N terminal sequence of phageproteins isolated on SOS-PAGE elec-trophoresis (Arendt et al, 1994; van Sinderenet al, 1995). The functions of the genesinvolved in cell Iysis have been confirmedby expression in E coli (Arendt et al, 1994;Birkeland, 1994).

The presence of terminally redundantends in the Tuc2009 genome (Arendt et al,1994) suggests a headful packaging mech-anism. <l>LC3and r1t have instead identi-cal 3' staggered cohesive ends (Lillehauget al, 1991; van Sinderen et al, 1995). Bothtypes of known phage packaging mecha-nisms seem therefore to be present in thisfamily of lactococcal phages. A transduc-ing vector based on the cohesive end regionof phage <l>LC3 has been constructed(Birkeland and Holo, 1993).

Finally, a site-specific ONA endonucle-ase, homologous to those found in group 1introns was identified in phage r1t (van Sin-deren et al, 1995).

The only study on virulent P335 phagesconcerns the presumed origin of replicationof the phage <1>50(Hill et al, 1990). It is con-stituted of long direct repeats. This locus isable to affect <1>50ONA replication in trans,and when cloned in a plasmid, the resultedrecombinant has an increased copy num-ber during infection by the phage.

Bacteriophage of lactic acid bacteria

However, studies on both types (tem-perate and virulent) of phages are of par-ticular interest due to the fact they may c1ar-ifY their relationships.

POOBphage species

Phages related to P008 are virulent smallisometric-headed with a genome size rang-ing around 30 kb and cohesive ends (Jarviset al, 1991). Only few genes from this typehave been clonee and sequenced. A con-served region has been found in thegenomes of several of these phages (Kimand Batt, 1991 a). An open reading framehas been identified within the region, andanalysis of the deduced protein sequenceencoded by the gene indicated that it hasa high Iysin content and that it shares homol-ogy with eucaryotic translation initiation fac-tor. It was suggested that this conservedprotein may have a role in either regulatingphage replication or specifying expression ofits own gene (Kim and Batt, 1991a). Sev-eral genes encoding structural proteins fromphage F4-1 have been characterized(Chung et al, 1991; Kim and Batt, 1991 b).The sequence of the region surrounding thecohesive ends of phage sk1 has been deter-mined (Chandry et al, 1994a). It presentsstructural features that may be involved inrecognition by the terminase. The <1>US3Iysin gene has been identified and its prod-uct, contrary to the other Iysins characterizedto date, seems to be an amidase instead ofa muramidase (Platteeuw and de Vos,1992).

Transcriptional studies on phage sk1established that transcription is temporallyregulated into three distinct phases con-cerning the early, middle and late stage ofinfection (Beresford et al, 1993; Chandry etal, 1994b). Sequence analysis of the blL66middle expressed region revealed an operonformed of four open reading frames andtranscribed 10 min after infection (Bidnenko

85

et al, 1995). Characterization of the blL66middle promoter has been reported. lt isconstituted of an extended -10 promotersequence consensus and no consensus-35 region was observed (Bidnenko et al,1995). This obervation suggests the possi-bility that transcription could be initiated fromthis promoter by an activator. Identificationof such kind a factor could be useful for thedesign of a controlled expression vector andfor the understanding of the temporal regu-lation of transcription.

c6a phage species

Phages related to c6a are virulent prolate-headed with a genome size ranging around22 kb and cohesive ends (Jarvis et al, 1991).Transcriptional studies have been done onphage c2 (Beresford et al, 1993). Two fam-ilies of time dependent transcripts have beendetected at early and late stage of infection.Until recently, the complete nucleotidesequence was available only for phageblL67 (Schouler et al, 1994). Thirty-sevenopen reading frames, organized in two c1us-ters, were identified. Functions wereassigned to the putative products of six ofthem. These were a DNA polymerase, aprotein involved in recombination, a Iysin,a terminase subunit, a structural protein anda holin. By Northern analysis, transcriptscorresponding to the region including thegene encoding Iysin were detected 20 minafter infection (data not shown) and the c1us-ter was referred to as the late region. Lysisof the host cell (IL 1403, Chopin et al, 1984)occurs within 70 min after infection. In com-parison with the amino acid sequence ofknown proteins, the second late gene hasbeen identified as the gene encoding theIysin. It shares very high homology, up to95%, with the Iysin gene of <1>VML3(Shearman et al, 1989), P001 (Hertwig,1990) and c2 (Ward et al, 1993). It seemsthat the start codon for ail these genes is

-----------------~ ----

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not an AUG but an AUA (encodingisoleucine) in phages c2 and <l>VML3, andan AUC (encoding isoleucine) in P001 andb1L67. AUA has already been found as astart codon in E coli (Belin et al, 1979), andrecently, AUC has also been described as astart codon (Chalut and Egly, 1995). lt hasbeen suggested that the use of a rare startcodon could be a way to decrease transla-tion of Iysin during phage multiplication(Schouler et al, 1994). Low levels of the<l>VML3 Iysin were praduced even underthe inducible T7 promoter (Shearman et al,1994).

The last late gene located near the cossite, orf37, has ail the structural featuresdefining a holin (Schouler et al, 1994). Holinsare membrane proteins which form poresto allow access of the Iysin to the peptide-glycan substrate (Young, 1992). Orf37, sub-cloned in E coli, is able to complement abacteriophage À defective for its holin gene(ÀSam) (data not shown). This experimentgives the first indication that the gene reallyencodes a holin. The location of the blL67holin gene, 12.6 kb downstream of the Iysingene is very unusual. It ressembles theorganization of T7 (Dunn and Studier, 1983),while other holin genes map immediatelyupstream of the Iysin gene. In any case, thislocation seems to be conserved for phagesrelated to c6a, since by homology research,it has also been found in phage P6 (Ermel etal, 1994) P001 and c2 (Perrin et al, unpub-lished results, GenBank L37090 andL37091).

The recent publication of the completenucleotide sequence of phage c2 (Polzin,personal communication) will allow a moredetailed comparison of c6a phage speciesgenome organization and evolution.

CONCLUSION

Accumulated data arising from the charac-terization of the lactobacilli and lactococci

C Schouler

phage genomes provided information ontheir global organization. Functions havebeen assigned to the products of severalgenes. Even though many functions remainunknown, these studies pravide some infor-mation on the phage life cycle, such as tem-poral expression of the genes, genetic con-trol between Iytic and Iysogen cycle andrelease of the virion capsid outside the host.Bacteriophages represent a very dynamicpopulation which is constantly evolving toaccomodate changes. Some studies havegiven information on how the phages couldevolve by acquisition of modules throughrecombination to overcome certain phageresistance mechanisms. Phage <1>50acquired the methylase gene LIai from theR/M region of pTR2030 to circumvent thedefense mechanisms of lactococci(Alatossava and Klaenhammer, 1991; Hillet al, 1991). Interestingly, van Sinderen et al(1995) reported homologies between <1>50and rlt at the borders of insertion of themethylase gene in the phage <1>50genome.Phage ul36 acquired a region from the chro-mosome of the infected host to overcomean abortive infection mechanism encodedon conjugative plasmid pTN20. This recom-bination led to the formation of a new Iyticphage, u137, resistant to the abortive infec-tion mechanism, but, also with different mor-phology and origin of replication fram thatof the original phage (Moineau et al, 1994).These two examples of DNA cassetteexchange between the infecting and theresident DNA are of particular interest. Elu-cidation of the recombination event shouldpravide insight into the process of phageevolution and would lead to the elaborationof more performing starter cultures.

A short sequence could act as a recom-bination hot spot as it was found for rtt (vanSinderen et al, 1995). Two direct repeatsof 25 and 31 bp have been found in blL67(Schouler et al, 1994). These repeats flanka c1uster of genes, suggesting an exchange-able module.

Bacteriophage of lactic acid bacteria

Evidence is now available on the emer-gence of Iytic phages from a Iysogenicancestor phage (Mikkonen and Dupont, per-sonal communication).

The presence of a group 1 intron wasalso reported in the genome of unrelatedphages (Mikkonen and Alatossava, 1995;van Sinderen et al, 1995). Such mobileintrons are phylogenetically diverse, beingfound in both procaryotic and eucaryoticgenomes (Derbyshire et al, 1995).

Phage encoded traits on cloned frag-ments may interfere with the normal Iyticcycle by overproduction or titration of essen-tial regulatory signais necessary for phagepropagation (Hill et al, 1990). Another pos-sible approach is the use of antisensemRNA to inhibit expression of essentialphage genes (Kim and Batt, 1991 c; Chunget al, 1992). We could then expect that char-acterization of the phage genome will allowthe design of new strategies to preventphage infection.

ACKNOWLEDGMENTS

1 am indebted to L Dupont, P Ritzenthaler,M Mikkonen, T Alatossava, A Nauta, D van Sin-deren, J Kok and G Venema for results commu-nicated before publication. 1also thank C Anag-nostopoulos and MC Chopin for critical reading ofthe manuscript.

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