discovering lactic acid bacteria by genomics29 antonie van leeuwenhoek 82: 29-58, 2002. @ 2002...

29Antonie van Leeuwenhoek 82: 29-58, 2002.@ 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Discovering lactic acid bacteria by genomics

Todd Klaenhammer,*, Eric AltermannI, Fabrizio Arigoni3, Alexander Bolotin4, Fred Breidt5,

Jeffrey Broadbent6, Raul Cano7, Stephalle Chaillou24, Josef Deutscher8, Mike Gasson9,

Maarten van de Guchte4, Jean GuzzoIO, Axel Hartke25, Trevor HawkinsII, Pascal HolsI2,

Robert HutkinsI3, Michiel Kleerebezem2, Jan KokI4, Oscar KuipersI4, Mark LubbersI5, Em-

manuelle Maguin4, Larry McKayI6, David MillsI7, Arjen NautaI8, Ross OverbeekI9, Herman

Pel2o, David Pridmore3, Milton Saier2I, Douwe van Sinderen22, Alexei Sorokin4, James

Steele23, Daniel O'SullivanI6, Willem de Vos2, Bart Weimer6, Monique Zagorec24 & Roland

Siezen2,26,*I Department of Food Science, Southeast Dairy Foods Research Centel; North Carolina State University, Raleigh,

NC 27695-7624, USA; 2 Wageningen Center for Food Sciences, 6700AN Wageningen, The Netherlands; 3 Nest Ié

Research Centel; Vers-chez-Ies-Blanc, 1000 Lausanne 26, Switzerland; 4Génétique Microbienne, CRI INRA,

Domaine de Vilvert, 78352 Jouyen Iosas cedex, France; 5USDA Agricultural Research Service, Departmentof Food Science, North Carolina State University, Raleigh, NC 27695- 7624, USA; 6 Department of Nutritionand Food Sciences, Utah State University, Logan, UT 84322-8700, USA; 7 Environmental Biotechnology Insfi-

tute, Califomia Polytechnic State University, San Luis Obispo, CA 93407, USA; 8 Laboratoire de Génétique

des Microorganismes, INRA-CNRS URA1925, F-78850 Thiverval-Grignon, France; 9 Institute of Food Research,Norwich, UK; 10 Laboratoire de Microbiologie, UMR INRA-Université de Bourgogne, Equipe PG2MA ENSBANA

21000 Dijon, France; II Joint Genome Institute Production Genomic Facility, 2800 MitcheU Drive, Walnut Creek,

CA 94598, USA; 12 Unité de Génétique, Batiment Camoy, Place Croix du Sud, UCL, B-1348, Louvain-Ia Neuve,

Belgium; 13 University of Nebraska, Department of Food Science and Technology, Lincoln, NE 68583-09 19, USA;14 Department of Molecular Genetics, University of Groningen, 9751NN Haren, The Netherlands; 15 Fonterra

Research Centre [Formerly the New Zealand Dairy Research Institute], Private Bag 11 029, Palmerston North,New Zealand; 16 Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108, USA;

17 Department of Viticulture and Enology, University of Califomia, Davis, CA 95616-8749, USA; 18 Corporate

Research FCDF, PO Box 87, 7400AB Deventel; The Netherlands; 19 Interegated Genomics Inc, 2201 W Campbel!Park DI; Chicago, IL 600612, USA; 20 DSM Food Specialties, PO Box 1, 2600MA Delft, The Netherlands; 21

University Califomia, San Diego, La IoUa, CA 92093-0116, USA; 22 Department of Microbiology, University col-lege Cork, Cork, Republic of Ireland; 23 Department of Food Science, University of Wisconsin-Madison, Madison,WI53706-1565, USA; 24 Flore Lactique et Environnement Camé, INRA -CRI, Domaine de Vilvert, 78350 Iouy-en-

Iosas, France; 25 Laboratoire de Microbiologie de I' Environnement, EA 956 USC INRA, IRBA Université de Caen,

F- 14032 Caen Cedex, France; 26 Center for Molecular and Biomolecular Informatics, University of Nijmegen, PO

Box 9010, 6500GL Nijmegen, The Netherlands (* Authors for correspondence; E-mail; [email protected],

[email protected])

Key words: lactic acid bacteria, genomics, Gram-positive bacteria, food, health, Lactococcus, Lactobacillus,

Streptococcus, Pediococcus, Leuconostoc, Oenococcus, Propionibacterium, Bifidobacterium, Brevibacterium

Abstract

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing andanalysis. Summaries are presented on twent y different species, with each overview discussing the organismsfundamental and practical significance, environmental habitat, and its role in fermentation, bioprocessing, orprobiotics. Por those projects where genome sequence data were available by March 2002, summaries include

30

a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular viewof Gram-positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected tobe available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria win befundamentany changed as we rediscover the relationships and capabilities of these organisms through genomics.

Introduetion {eontributed by Todd Klaenhammer} 2.4 Mb in size and revealed a number of unexpectedfindings: biosynthetic pathways for all20 amino acids,albeit not all are functional, a complete set of late com-petence genes, five complete prophages, and partialcomponents for aerobic metabolism. Noting that someof these systems are not functional or complete, thegenomic analysis of Lactococcus suggests an evolu-tionary trend toward minimization of the chromosomeand elimination of unnecessary systems during adapt-ation to nutritionally complex environments, such asrnilk. At this writing, four other LAB genomes havebeen completed, (L. plantarum, L. johnsonii, L. acido-philus, S. thermophilus), and > 20 more are in progress(Table I) with expected completions for sequencingby the end of 2002. Additional genome sequencingis underway for microbes that are not considered asmembers of the LAB, but contribute important LAB-like properties either as probiotics (BifidobacteriumIongum, B. breve, and Brevibacterium linens) or fla-vor adjuncts (B. linens, Propionibacterium freuden-reichii). Among the total genome projects ongoing,there are several cases where genome sequences willbecome available for multiple strains of the same spe-cies, notably L. lactis (three strains), L. casei (twostrains), L. delbrueckii (three strains), S. thermophilus(three strains), Oenococcus oeni (two strains) and B.

Iongum (two strains).Of the 29 genomes listed in Table Ion LAB-

type microbes, II genomes are being sequencedby the Department of Energy-Joint Genome Insti-tute (JGI) in collaboration with the Lactic AcidBacteria Genome Consortium (LABGC), composedof 10 US scientists representing seven universit-ies in the U.S. As part of their microbial genomes

program (see http://www.jgi.doe.gov/JGI-microbia1/htm1/index.html) JGI will carry out genome sequen-cing of LAB species representing considerable di-versity in ecological habitat (milk, meat, plants, GItract) and roles (probiotic versus fermentation). Allof the microbial genomes will undergo the same pro-cessing; first to generate a 10x coverage of each gen-ome with a random shotgun small-insert library (3 kb ),and to supplement this with ""5 x coverage of a largeinsert (40 kb) cosrnid library .These data will be incor-porated into an assembly and the assembIed scaffolds

The lactic acid bacteria (LAB) represent a group ofbacteria that are functionally related by their abilityto produce lactic acid during homo- or heteroferment-ative metabolism. The acidificatioll and enzymaticprocesses accompanying the growth of LAB impartthe key flavor, texture, and preservative qualities toa variet y of fermented foods. lndustrial applicationsof the LAB rely oll six key belleficial and non-pathogenic species; Lactococcus (milk), Lactobacillus(milk, meat, vegetables, cereal), Leuconostoc (veget-ables, milk), Pediococcus (vegetables, meat), Oeno-coccus oeni (wille) and Streptococcus thermophilus(milk). Other members of the LAB, notably lacto-bacilli, occupy important niches in the gastrointest-illal tracts of humans and animals and are consideredto offer a number of probiotic bellefits to generalhealth and weIl being. These bellefits include a pos-itive illfluence oll the normal microflora, competitiveexclusioll of pathogens, and stimulation/modulatiollof mucosal immullity. More recently, LAB are be-ing used in the production of industrial chemicaland biological products including biopolymers (Leu-conostoc s pp.), bulk enzymes (Lactobacillus brevis),ethanol, and lactic acid (Lactobacillus casei, lactis,delbrueckii, brevis) (Gold et al. 1992; Hofvendahl &Hahn-Hagerdal 2000). The LAB are also strong can-didates for development as oral delivery vehicles fordigestive enzymes and vaccine antigens (Wells et al.1996; Pouwels et al. 1998; Steidler et al. 2000). Theirinnate acid tolerance, ability to survive gastric pas-sage, and safety record during humall consumption,are key features that call be exploited to effectivelydeliver biologics to targeted locations and tissues.

Realizing their practical significance in ferment-ation, bioprocessing, agriculture, food, and morerecently, medicine, the LAB have been the subjectof considerable research and commercial developmentover the past decade. Contributing to this explosiollhave been the recent efforts to determine the genomesequences of a representative collectioll of LAB spe-cies and strains. The first complete genome of the LABgroup was published oll Lactococcus lactis subsp. lac-tis ILl403 by Bolotill et al. (2001). The genome was

31

Table 1. Genome sequencing projects of lactic acid bacteria

Species Strain Contact persongenomesize (Mb)

lnstitution

Sequencing completed

Lactococcus lactis

Lactobacillus plantarum

Lactobacillus johnsonii

Lactobacillus acidophilus

IL1 403

WCFSl

NCCS33

ATCC700396

NCFM

NCC270S

2.3

3.3

2.0

2.0

A. Sarakin

M. Kleerebezem

D.Pridmare

T. Klaenbammer, R. Cana

INRA and Genascape, FR

WCFS, NL

Nestlé, CR

Narth Caralina State University and Cal.

Paly Technica! University, USA

Nestlé, CRBifidobacterium Iongum 2.3 F. Arigoni

MG1363 2.6 O.Kuipers, M.Gassan,D. van Sinderen

A. Sarakin, A. Balatin

University of Groningen, NL; IFR, UK;

UCC, IRL

INRA, FRMG1363 2.4

ATCC11842 2.3 E. Maguin, M. v d Guchte Genascape and INRA, FR

Sequencing ongoing

Lactococcus lactis

ssp. cremoris

Lactococcus lactis

ssp. cremoris

Lactobacillus delbrueckii

ssp. bulgaricus

Lactobacillus delbrueckii

ssp. bulgaricus

Lactobacillus sakei

Lactobacillus casei

Lactobacillus helveticus

Lactobacillus rhamnosus

DN-lOOlO7 2. T. Smokvina Danone Vitapole, FR

23K

BL23

CNRZ32

HNOOl

1.9

2.6

2.4

2.4

M. Zagorec, S. Chaillou

I. Deutscher, A. Hartke

I. Steele

M. Lubbers

Streptococcus thermophilus.

Streptococcus thermophilusOenococcus oeni

LMGl83ll

CNRZlO66

IOEB84.l3

1.9

1.8

1.8

P. Hols

A.Bolotin

J.Guzzo

INRA, FR.

INRA/CNRS, Caen University, FR

University of Wisconsin, USA

Fonterra Research Center (forrnerly NZDRI)

and ViaLactia BioSciences, NZ

UCL, Belginm

INRA, FR, Integrated Genomics Inc, USA

Universities Dijon and Bordeanx-INRA-

GENOME Express, FR

University College Cork (UCC), IRL

DSM Food Specialties, Friesland Coberco

Dairy Foods, NL

Bifidobacterium breve.

Propionibacterium

freudenreichii

NCIMB8807

ATCC6207

2.4

2.6

D. van Sinderen

H. Pel, J. Sikkema

North Carolina State University, USA

Utah State University, USA

University of Wisconsin, Madison, USA

Joint Genome Institute/Lactic Acid Bacteria Genome Consortium (JGI/LABGC)

Lactobacillus gasseri ATCC33323 1.8 T. Klaenharnmer

Lactobacillus casei ATCC334 >2.2 J. Broadbent

Lactobacillus delbrueckii ATCCBAA-365 2.3 J. Steele

ssp. bulgaricusLactobacillus brevis ATCC367 2.0 M. Saier

Lactococcus lactis SKll 2.3 B. Weimer, L. McKay

ssp. cremoris

Leuconostoc mesenteroides ATCC 8293 F. Breidt. H.P. Fleming

Oenococcus oeni PSUl

AT CC BAA-331

ATCC25745

,3 D. Mills

University of Califomia, San Diego, USA

Utah State University and University of

Minnesota, USA

USDA, North Carolina State University,

USA

University of Califomia, Davis, USA

Pediococcus pentosaceus 2.0 J. Steele, J. Braadbent University of Wisconsin, Madison, Utah

State University, USA

University of Nebraska, USA

Utab State University, USA

University of Minnesota, USA

Streptococcus thennophilusBrevibacterium linens

Bifidobacterium Iongum

AT CC BAA-491

BL2/ AT CC 9174

DJO10A

1.8

3.0

2.1

R. Hutkins

B. Weimer

D. O'Sullivan

~2

istance, are encoded by conjugative plasmid DNA inthis species (Broadbent 2001).

With the availability of genomic maps of more than25 LAB, comparative genomic analysis will identifycritical similarities and differences and is expected toidentify 'islands of adaptability', defined as key ge-netic regions that may be instrumental in the evolutionof the various species to their specialized habitats, andtheir functions within those environments. Compar-isons of food-grade LAB with other related Gram-

positive pathogens (Enterococcus faecalis, Strepto-coccus agalactiae, Streptococcus equi, Streptococ-cus pneumoniae, Streptococcus mutans, Streptococcuspyogenes, Listeria monocytogenes) has already re-vealed many common features and will most certainlydefine the essential genetic differences between patho-gens, non-pathogens, and commensals. Over the nextyear, our scientific community will be fundamentallyempowered by the availability of numerous related,yet distinct genomes, to make these comparative ana-lyses and define the similarities and differences thatcharacterize the genomes of LAB.

This chapter presents a collection of summarieson the LAB organisms that are now in genomic se-quencing or analysis. Each summary describes theorganism and its roles in the environment or biopro-cessing, the status ofthe sequencing effort as ofMarch2002 and, for some, selected interesting features thathave been uncovered (Tables 2 and 3). Our view of theLAB will be fundamentally changed as we discoverthe relationships and capabilities of these organisms

through genomics.

ordered by P CR primer walking to fill gaps. The scaf -

folds will undergo an automated annotation which willbe performed by aak Ridge National Laboratories.

It is noteworthy that as these sequences are gener-ated, they will be placed in the public domain on theJGI website for common use. The timely and publicavailability of genome information for various LABspecies will catapult our collective efforts to carry onwith comparative and functional genomic analyses ofthe LAB group.

The explosion of available genome sequences forLAB will accelerate their exploitation in both tradi-tional and non-traditional arenas. While phylogenet-ically closely related by their small genomes ( ""2-4Mb), the LAB occupy a diverse set of ecologicalniches suggesting that considerable genetic adapta-tion has occurred during their evolution. Comparisonof the genome sequences of multiple LAB speciesand strains is expected to provide a critical view ofmicrobial adaptation and genetic events leading totheir adaptation to specialized environments. Compar-ative genomics among the microbes sequenced thusfar has already illustrated that essential housekeepinggene functions are widely conserved among microbesand horizontal gene transfer commonly occurs. Anexpected outcome of comparative genomics of LABwill be the definition of conserved and unique ge-netic functions in LAB that enable core functions,e.g., production of lactic acid, proteolytic and pepti-dase activities, survival at low pR, stress tolerance,production of antimicrobials, transport systems, cellsignaling, and attachmentlretention in dynamicallymobile environments.

It is anticipated that IS-elements, bacteriophages,and mobile genetic elements provide the major routesthrough which horizontal gene transfer occurs, and theroadmap to the most interesting and practically signi-ficant genetic regions that underscore the unique andbeneficial properties of the LAB. It is weIl documentedthat the LAB undergo conjugation, exist in phage-contaminated environments where gene transfer mayoccur by transduction, and harbor sets or remnantsof competence genes for transformation (Bolotin etal. 2001). Understanding gene transfer, particularlyin environments where LAB coexist, or compete,will provide one important view of their evolution,adaptation, and potential for unique applications. Con-jugation has played a key role in the evolution andadaptation of L. lactis to a milk environment notingthat many attributes for growth in milk, includinglactose and casein utilization and bacteriophage res-

SUMMARIES OF SEQUENCING PROJECTS

Lactococcus lactis subsp. cremoris SKll

( contributed by Larry L. McKay and Bart

Weimer)

Lactococci are mesophilic LAB that were first isol-ated from green pIants. However, today they are usedextensiveIy in food fermentations, which representabout 20% of the totaI economic vaIue of fermen-ted foods produced throughout the worId. This groupof bacteria, previously designated the Iactic strepto-cocci (Streptococcus lactis subsp. lactis or S. lac-tis subsp. cremoris) was pIaced in this new taxonin 1987 by Sch1eifer. Lactococci gained notabIe in-terest because many of their functions important forsuccessfuI fermentations are Iinked to p1asmid DNA

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35

proach. With this genome sequence, it will be possibleto confirm the metabolic and evolutionary differencesbetween subspecies of lactococci in order to identifythe important characteristics that define this genus.

Acknowledgments

The Joint Genome lnstitute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of K. Baldwin, L.-S. Chou,Y. Xie, T. Hawkins, S. Stilwagen, P. Richardson, andK. Kadner are gratefully acknowledged.

Lactococcus lactis ssp. cremoris MG1363( contributed by Douwe van Sinderen, MikeGasson, Jan Kok and Oscar Kuipers)

Lactococcus lactis ssp. cremoris MG1363 is a curedisolate of strain NCDO 712 (Gasson, 1983), and isthe most widely used model Lactococcus strain in ge-netic, physio1ogical and applied research allover theworld. L. lactis is a prominent bacterium in cheeseproduction and can also be used for various otherapplications such as a vehicle for oral vaccines ordelivery of health-promoting factors via the GI tract.Even before the genome sequencing projects star-ted already over 10% of its gene content had beenidentified and characterized by conventional methods.Recently the complete genome sequence of L. lac-tis ssp. lactis IL1403 was published (Bolotin et al.2001), providing a great opportunit y to compare thetwo genomes, with respect to their similarities anddifferences. Many important features, such as path-ways leading to the formation of flavour compounds,carbon and nitrogen metabolic routes, gene regula-tion mechanisms, prophage or sex-factor occurrence,and stress responses can be compared when the gen-ome sequence of MG1363 is also known. Functionalstudies using transcriptome and proteome analyses canalso be applied in order to speed up characterisation ofimportant novel targets.

(McKay 1985). P1asmids are commonly exchangedbetween strains via conjugation (McKay 1985; Dunny& McKay 1999) and with the chromosome by IS ele-ments (Rughes 2000). Presumably, these exchangesand rearrangements mediate rapid strain adaptationand evolution but also add to the instability of import-ant metabolic functions.

These bacteria are selected for use in fermenta-tions based on their metabolic stability, their resistanceto bacteriophage, and their ability to produce uniquecompounds -often from amino acid catabolism. Thestudy of their physiology in adverse conditions suchas low pR and high NaCl indicates that they adapt tothese environments quickly and change their metabol-ism as a re sult of carbohydrate starvation (Stuart &Weimer 1998). Recent genome studies and physicalmaps indicate that bacterial genomes are very dynamic(Rughes 2000). These rearrangements are mediated byIS elements and result in gene duplication, transloca-tion, inversion, deletion and horizontal transfer events.Por example, an inversion encompassing approxim-ately one-half of the chromosome of L. lactis ML3occurred by homologous recombination between twocopies of IS905 (Daveran-Mingot et al. 1998). Theresponse to these stresses, particularly to exposure tobacteriophage (Porde & Pitzgerald 1999), highlightsthe plasticity of the genome (Le Bourgeois et al. 1995 ;Delorme et al. 1994; Davidson et al. 1996). Estab-lishing the links between environmental conditions,genome organization, and cellular physiology in lacto-cocci will provide new and exciting information aboutthe molecular mechanisms of these important bacteria.Advances that define the fundamental knowledge ofthe genetics, molecular biology, physiology, and bio-chemistry of lactococci will provide new insights andapplications for these bacteria.

The importance of lactococci, specifically L. lactissubsp. cremoris, is demonstrated by its continual usein food fermentations (Garvie et al. 1981; Beimfohret al. 1997). L. lactis subsp. cremoris strains are pre-ferred over L. lactis subsp. lactis strains because oftheir superior contribution to product flavor via uniquemetabolic mechanisms (Sandine, 1988; Salama et al.1991). The DNA sequence divergence between thesubspecies is estimated to be between 20 and 30%(Godon et al. 1992). Of the many lactococcal strainsused, L. lactis subsp. cremoris SK11 is recognized forthe beneficia! flavor compounds it produces (Lawrenceet al. 1976). Although some progress in unlocking thisstrain's genetic secrets has been made, much more canbe accomplished by using a genomics/proteomics ap-

Genome sequencing status and results

To obtain the complete nucleotide sequence ofthe genome of Lactococcus lactis subsp. cremorisMGI363 a shotgun sequencing approach was used.The sequence of 15000 ends of inserts of pVC clones,ranging in size from 1 to 3 kb, have been determ-ined, y ielding 9 million bp of sequence. The obtained

36

Genome sequencing status and resultssequences were aligned with known sequences ofMG1363 present in the NCBI nucleotide database (:!:270 kb) aud sequences present within the consortium(:!: 150 kb) using the Staden and Sequencer softwarepackages. As the construction of a random librarywith inserts larger than 20 kb failed, the genome se-quence of L. lactis subsp. lactis IL1403 was used asa template to start completing the genome sequence.Af ter positioning of all the contigs on IL1403, forwardand reverse primers were designed using au in-housedeveloped software tool, in the ends of the contigs.The nucleotide sequences of the P CR fragments arecurrently being used to close the gaps. Annotation ofthe nucleotide sequence is being done with Glimmer,Blast, PFAM, Blocks.

Total DNA of L. lactis MG1363 was randomized byAlul in conditions of non-complete digestion or bysonication and extracted af ter separation in an 0.8%agarose gel to give an average fragment size of 1.5 kb.lt was then cloned into vector pSGMU2 cut by Smaland the inserts from approx. 1500 random clones weresequenced by forward and reverse primers. Sequencesof 513 of these clones, having a total non-redundantlength of 0.3 Mb, were used to construct a co-linearscaffold ofthe strain MG1363 over IL1403. The totalsize of the co-linear genome parts was estimated tobe around 1100 kb, that is approximately 45% of theentire genome (Bolotin et al. 2002). Oligonucleotide-directed sequencing over gaps amplified by LR P CRfragments of MG1363 and the entire sequence ofa few regions of interest resulted in the accumula-tion of 1038 kb non-redundant sequence distributedin 733 contigs. The contigs were compared for ho-mology, using BLASTx, with the complete proteinset of L. lactis IL1403. Sequences of 523 contigsencode proteins that are more than 80% identical toproteins from IL1403. The sequences of the 733 con-tigs are available from GenBank as dbGSS (acc. Nos.

BH770319-BH771051).

Acknowledgments

We thank Sinead Leahy, Gerald Fitzgerald, Udo Weg-mann, Claire Shearman, Girbe Buist, Richard Baer-ends, Aldert Zomer, Sacha van Hijurn, and Anne deJong for their contributions and support.

Lactococcus lactis ssp. cremoris MG1363

(contributed by Alexei Sorokin and Alexander

Bolotin)Acknowledgments

Two strains, IL1403 and MG1363, isolated from in-dustrial sources and cured from plasmids, are com-monly used to study genetics, physiology and mo-lecular biology of Lactococcus lactis. These strainsbelong to two L. lactis strain clusters, called lactisand cremoris, distinct by the spectra of dairy ferment-ation products. We sequenced the entire genome of thestrain IL1403 (Bolotin et al. 1999, 2001). To com-pare genomes of the two strains in the simplest waywe decided to perform a co-linear scaffolding of thechromosome of strain MG 1363. Co-linear scaffoldingconsists in random sequencing of a limited number ofclones and Long Range P CR mapping ofthe co-linearregions of two genomes, one of which was completelysequenced earlier. In the co-linear regions, establishedby P CR, gene content and order should be approxim-ately the same. The established co-linear scaffold ofthe MG1363 genome, which shares 85% identity withthat ofIL1403, allows easy access to 50% ofthe genesfrom this strain.

We thank Saulius Kulakauskas for giving us the L.lactis ssp cremoris MG1363 strain, Nathalie Galleronand Benois Quinquis for assisting in bank construc-tions and sequencing. Discussions with members ofLactococcus communities of Génetique Microbienneand UPLGA labs (CRJ INRA) and their interest to thiswork are very appreciated.

Lactobacillus plantarum WCFSl

( eontributed by Miehiel Kleerebezem and Roland

Sjezen)

The genus Lactobacillus encompasses a large num-ber of different species that display a relativelylarge degree of diversity. Among these, Lactobacillusplantarum is a flexible and versatile species that is en-countered in a large variet y of environmental niches,including some dairy, and many vegetable or plant fer-mentations. Some strains of Lactobacillus are foundas natural commensals of the gastrointestinal tract (GItract). the oral cavitv and the female uro-genital tract

37

inantly those encoded in the genomes of Listeria,Lactococcus, Bacillus and Streptococcus.

L. plantarum is a versatile and flexible organismand is able to grow on a wide variet y of sugar sources.This phenotypic trait is reflected by the high numberof predicted PEP-dependent sugar phosphotransferasesystems (PTS; 25 complete) and other sugar transport-ers encoded by WCFS I. Strikingly, over 60% of thesesugar transporters are Iocated within 250 kb in bothdirections from the origin of replication.

of animals and humans. The ability to adhere to spe-cific regions of the GI tract is a property which hasstimulated research aimed at the use of lactobacillias delivery-vehicles for therapeutic compounds suchas immunomodulators, antibodies, enzymes and vac-cines (Marteau & Rambaud 1993 Hols et al. 1997;Slos et al. 1998). Lactobacillus plantarum arrives inthe small intestine in an active state (Vesa et al. 2000)and is frequently encountered as a natural inhabit-ant of the human gastro-intestinal tract (Ahrne et al.1998). Some strains are being marketed as probiotic.Previous studies using gel electrophoresis have indic-ated that L. plantarum has one of the largest genomesknown among lactic acid bacteria (Chevalier et al.1994; Daniel 1995). The Wageningen Centre for FoodSciences (WCFS) in collaboration with Greenomicsin Wageningen, has determined the complete genomesequence of L. plantarum WCFSl, a single colonyisolate of strain NCIMB8826, a strain of human origin.

Acknowledgments

We thank Jos Boekhorst, Bjom Ursing, Richardvan Kranenburg, Douwe Molenaar and Willem deVos for their contribution to this work. Sequen-cing and assembly were performed by Greenomics

(www.greenomics.com).

Genome sequencing status and results Lactobacillus rhamnosus HNOOl

(contributed by James Dekker and Mark Lubbers)

Lactobacillus rhamnosus is one of the few species ofLactobacillus that have been used as probiotic organ-isms in functional foods. In addition, this species isone of the two most common non-starter lactic acidbacteria found in New Zealand cheddar cheese; theother is L. paracasei. We have identified a strain of L.rhamnosus, designated HNOOl, that has both flavour-enhancing and probiotic attributes. It can be used as anadjunct during cheese manufacture to reduce adventi-tious microflora, accelerate cheese ripening, and im-prove cheese flavour. We have also demonstrated thatHNO01 has the 'prerequisite' properties of a probiotic,inc1uding confirmation of taxonomic c1assification,acid and bile resistance, adherance to intestinal cells,transient colonisation, and lack of any toxicity or detri-mental effects (Prasad et al. 1998; Z hou et al. 2000a,b,2001; Gopal et al. 2001; Sheih et al. 2001). A num-ber of in vitro and in vivo tests, including and humanclinical trials, have demonstrated the ability of HNO01to influence specific cytokines, NK cell activity, T cellstimu1atory capacity and phagocytic ability, and influ-ence the balance of the intestinal microflora in humans(Gin et al. 2000, 2001a; Tannock et al. 2000). HNO01also has antimicrobial activity against important gutpathogens (Gin et al. 2001b). L. rhamnosus HNO01is genetical1y accessible and we have developed toolsand techniques for efficient gene disroption and over-expression. Therefore, HNOOl is an ideal candidate for

Sequencing was accomplished using a shotgun ap-proach, while À-clones and multiplex P CR were usedto fill the gaps. The circular chromosome of L.plantarum WCFS I consists of 3 308274 bp with anaverage G+C content of 44.5%, and is among thelargest of lactic acid bacteria. In addition, the strainharbours three plasmids of 36069 bp (G+C content:40.8%), 2365 bp (G+C content: 34.3%), and 1917 bp(G+C content: 39.5%), respectively. Automated pre-diction software (Glimmer, Genemark) was used toobtain a primary prediction of protein encoding gelles,which was manually improved to result in a current listcontaining about 3050 gelles.

The orientation of the majority of the gelles ishighly organized from the origin to the terminusof replication for both halyes of the chromosome.Five rRNA-encoding operons, with very few se-quence polymorphisms, could be identified scatteredaround the chromosome. Automated ORF allalysisand functional annotation was performed using Bio-Scout (LION) and Pedant-Pro (BioMax) softwarepackages, folIowed by extensive manual curation.Functions were predicted for over 65% of the geneproducts. Major categories are proteins involved inenergy metabolism (8% ), cell envelope (8% ), trans-port (13%) and regulation (9%). Over 2300 encodedproteins have expect scores better than I e-l 0 to data-base entries, and nearly 90% of these are most sirnilarto proteins of other Gram-positive bacteria, predom-

38

al. 1994; Schiffrin et al. 1995; Haller et al. 2000a,b),antipathogenic properties (Eernet et al. 1994; Eernet-Camard et al. 1997; Felley et al. 2001; Pérez et al.2001) and the ability to interact with the host (Granatoet al. 1999; Neeser et al. 2000). The determinationand analysis of the complete genome sequence of L.johnsonii Lal has provided valuable targets for theinvestigation of the above interactions. L. johnsoniiLal is amenable to genetic manipulation, an import-ant consideration in view of the wealth of informationrevealed in the genome sequence and the interest to in-vestigate the probiotic features of this bacterium at thegenetic level. Efficient transformation by plasmids, theability to use pG+host9 and its temperature sensitivereplication to produce genetic disroption of selectedgenes and the application of pG+host9:ISSl (Maguinet al. 1996) to produce random mutations are someof the tools available, making L. johnsonii Lal animportant host for advanced genetic analysis.

an in-depth study of factors that contribute to probioticand flavour-enhancing attributes of lactobacilli.


A draf t genome sequence of HNO01 was obtained byshotgun sequencing. Allalysis of the data indicates agenome size of about 2.4 Mb, and an average G+Ccontent of approximately 46.4%, which is within therange reported for other Lb. rhamnosus species. Inaddition, HNO01 harbours two plasmids of 8754 b p(G+C content: 41.9%) and approximately 40000 b p(G+C content: 43.7% ). Automated ORF predictionand annotation were not used. Rather, the whole gen-ome sequence was compared against publicly avail-able databases and the output manually examined forgelles of specific interest. In addition, metabo1ic path-ways of particular interest were also identified. Todate, over 900 candidate gelles have been examinedand almost 300 gelles with potential involvement inflavour, survival and probiotic activity (e.g. gut adhe-sion, immune and anti-microbial activity) have beenidentified. Of these, 39 HNO01 gelles have been func-tionally characterised using a number of approaches,including gene knockout by disruption, overexpres-sion, gene complementation and biochemica! assay ofpurified recombinant protein.


Random shotgun cloning and sequencing was sup-plemented with sequencing of BAC clones and long-range P CR products to finally produce a circular chro-mosome of approximately 2.022 Mbp. The computer-predicted Smal digest closely matches the physicaldigest pattem. Bioinformatic analysis has identifiedthe L. johnsonii Lal counterparts of the best-studiedgenes and pathways. The initia} analysis of these res-ults has not revea}ed any major surprises, except forthe complete lack of amino acid biosynthetic pathwayswhich appears to be compensated for by an increasednumber of amino acid transporters. Given the limitedliterature describing the natural habitat of this bac-terium, a more profound analysis of the genetic poten-tial of L. johnsonii Lal could provide important cluesas to the nutritional composition of its environment.The structure of the L. johnsonii Lal chromosome isunusual in that both the plot of GC skew and the dir-ection of the transcription of the majority of genes donot place the proposed terminus opposite the origin(approximately 1000 kb), but close to 1300 kb. Thischromosome structure may be explained by the struc-ture, orientation and position of two genes encodinglarge cell-wall anchored proteins in L. johnsonii Lal.Both genes predict proteins with more than 100 copiesof a conserved 10 amino acid repeat. This repeat isreflected in the gene sequences and may be the tar-get for recombination. Inversion of the approximately600 kb of sequence between the repeat would position

Acknowledgments

We acknowledge the valuable contributes of MichaelCollett, Christopher Pillidge, and Marie-Laure De-labre, and the financial support of our sponsors Vi-aLactia BioSciences NZ Ltd, the New Zealand Found-ation for Research Science and Technology, and theMarsden Fund of the Royal Society of New Zealand.

Lactobacillusjohnsonii Lal (NCC276l)

(contributed by David Pridmore)

Lactobacillus johnsonii strains have been mainly isol-ated from the feces ofhumans and animals (Johnson etal. 1980; Fujisawa et al. 1992), suggesting that thesebacteria constitute part of the natural intestinal flora.L. johnsonii Lal (formerly Lactobacillus acidophilusLal) is a Nestlé proprietary strain that has been extens-ively studied for its probiotic properties and is com-mercialised in the LCl fermented milk products. Lalshows immunomodulatory properties (Link-Amster et

39

the terminus opposite the origin. A second exampleof L. johnsonii Lal genome rearrangement has beenobserved by detining the direct repeats produced by ISelement transposition. One pair of elements showed aperfect exchange of the direct repeat sequences. Con-tirmed by P CR, this event has produced an inversionof approximately 800 kb of the L. johnsonii Lal gen-ome. Extension of this analysis to other L. johnsoniiisolates has allowed the identitication of some of theintermediates in the events leading to the L. johnsoniiLal genome contiguration.

variety of products and probiotic dairy foods, likeSweet AcidophilusTM milk. lts long history of safeuse in commercia1 products for and human consump-tion, and its close genetic relationship to the neotypestrain, ATCC4356 (Walker et al. 1996), were twomajor reasons that the L. acidophilus NCFM strain( deposited as ATCC700396) was selected for genome

sequencmg.L. acidophilus is an obligate homofermenter and

metabolizes hexoses primarily to lactic acid. Both theD- and L-isomers of lactate are produced. L. acido-philus NCFM metabolizes fructo-oligosaccharides(Kaplin & Hutkins 2000), which are prebiotic com-pounds that support the growth of beneficial gutbacterial. Some of the functional properties of thisorganism have been reviewed (Sanders & Klaenham-mer 2001) and include: survival through the GI tract(Conway et al. 1982), reduction offecal mutagenic en-zymes (Goldin & Gorbach 1980; Goldin et al. 1980),adherence to intestinal tissues (Conway et al. 1987;Greene & K1aenhammer 1994), stimulation of IgAin mice by a culture cocktail that includes L. acido-philus NCFM (Tejada-Simon & Pestka 1999), andproduction of the bacteriocin, lactacin B (Barefoot &K1aenhammer 1983). In contrast to the other mem-bers of the acidophilus complex, there is considerableinformation on L. acidophilus NCFM related to its

transformability by electroporation (Luchansky et al.1988; Walker et al. 1996) and the development of ge-netic tools, that will be used for the functional genomicana1ysis of this species (Kullen & K1aenhammer 1999;Russell & Klaenhammer 2001a,b).

Acknowledgments

Anne-Cecile Pillel, Marie-Camille Zwahlen, Bem-ard Berger, Mark Schell, David Vilanova, MariaKarmiranlzou, Frank Desiere and Josef Hermanns aresincerely lhanked for their conlribulion lo this work.

Lactobacillus acidophilus ATCC7003961 NCFM

{contributed by Todd R. Klaenhammer and Raul

Cano}

In 1900, Moro first iso1ated Lactobacillus acidophiZusfrom infant feces. Meaning 'acid loving', acidophiZuscan be found in the intestinal tract of humans and an-imals, as well as infants consuming high milk, lactose,or dextran diets. Metchnikoff's 1906 work on TheproZongation of Zife: optimistic studies, implicated alactic acid bacillus, found in Bulgarian yoghurts, asthe agent responsible for deterring intestinal putrefac-tion and aging. Later, Lactobacillus acidophiZus wasconsidered to be the most likely species to fulfill thebase criteria expected of a probiotic cultures; survivalthrough the GI tract, bile to1erance, acid tolerance,and antimicrobial production. Over the last centurya considerable amount of research has been carriedout on L. acidophiZus, and a group of closely-relatedspecies clustered in a group known as the 'acido-philus complex' (Klaenhammer & Russell, 1999).Of the six species in the group, L. acidophiZus con-tillues to be the bacterium most often implicated inproviding probiotic bellefits and remains to be the spe-cies most commonly found in foods or supplementsthat contain probiotic cultures. In a recent survey,Clark et al. (2001) found that when L. acidophiZuswas recovered from probiotics samples, the majorityof confirmed strains showed a genetic fingerprint re-sembling that of NCFM. The L. acidophiZus NCFMculture has been used commercially since 1972 in a


Genome sequencing was accomplished using a shot-gun random library approach and pUCI8. P CRand direct genome sequencing (Fidelity Systems,Inc.) were used to fill gaps and polisb selec-ted regions. Tbe genome bas been assembIedinto a single contig and consists of 1.99 Mbpwith an average GC content of 34.6%. Tbe gen-ome was automatically annotated by tbe predic-tion, database, and annotation software GAMOLA(Global annotation of multiplexed on-site blastedDNA sequences) to identify 1979 ORFs in a cod-ing density of 89.9%. Predicted ORFs were manuallyverified against the on-site Blast and Pfam databases.Some features encoded in tbe NCFM genome are sixcopies of an IS3-type element, two bacteriocins, threedistinct regions encoding competence gelles, two ldh

40

genes, two bile salt hydrolase genes, two operons forfructo-oligosaccharide (FOS) utilization, and no intactprophages. There is also a collection of genome se-quence information available in public databases forvarious strains of the L. acidophilus species, includingheat shock operons groELS and dnaK, a sigma factorropD, bacteriocins acidocins 8912 and B, bile salt hy-drolase, and f3- and phospho-f3-galactosidases. Com-parison of the NCFM genome sequence with theseavailable database sequences has revealed a numberof interesting similarities and marked differences.

that wi11 be essentia1 to functiona1 genomic analysis ofthis species (Kullen & Klaenhammer 1999; Russell &Klaenhammer 2001a,b).


Acknowledgments

This project was supported by Dairy Management,Inc, the Southeast Dairy Foods Research Center, theCalifornia Dairy Foods Research Center, the Environ-mental Biotechnology Institute and Rhodia, Inc. Theefforts of Eric Altermann, Mike RusselI, A. Harnrick,T. Cox, and D. Doherty are gratefully acknowledged.

Lactobacillus gasseri ATCC33323, neotype(contributed by Todd R. Klaenhammer, EricAltermann and Rodolphe Barrangou)

Recent developments in molecular taxonomy haverevealed six different Lactobacillus species that com-prise the 'acidophilus' group, which are widely con-sidered as model probiotic organisms (Klaenhammer& Russell 2000). Among these, Lactobacillus gas-seri appears to represent the major homofermentativeLactobacillus species that occupies the human GI tract(Mitsuoka, 1992; Kullen et al. 2000; Heilig et al.2002). L. gasseri demonstrates good survival in theGI tract (Conway et al. 1982; Pedrosa et al. 1995) andhas been associated with a variet y of probiotic activ-ities and roles including reduction of fecal mutagenicenzymes (Pedrosa et al. 1995), adherence to intestinaltissues (Conway et al. 1987; Greene & Klaenhammer1994), stimulation of macrophages (Kitazawa et al.1994; Kirjavainen et al. 1999; Kitazawa et al. 1994),and production of bacteriocins (Itoh et al. 1995).

Data are available on the transformability by elec-troporation (Russell & Klaenhammer 2001a,b), andexpression of heterologous gelles in L. gasseri (Choet al. 2000), as well as on the genome sequence of oneof its temperate bacteriophage (Altermann et al. 1999).L. gasseri is more amenable to DNA introduction andmanipulation than other members of the acidophiluscomplex, leading to the development of genetic tools

Genome sequencing of the L. gasseri neotype strain,ATCC33323, was carried out by the Department ofEnergy-Joint Genome Institute in collaboration withthe Lactic Acid Bacterial Genomics Consortium. Thestrategy for genome sequencing was based on a shot-gun random sequencing of a small genomic DNAinsert (average 2.5 kb) library cloned into pUCl8at approximately 8-fold coverage, complimented bysequencing a large insert (35-45 kb) Fosmid lib-rary at a lower coverage. Gap closing is ongoingusing a multiplex PCR approach. The genome as-sembly of L. gasseri currently consists of 67 contigs,representing a total of 1.84 Mbp, with an averageG+C content of 35.1 %. The sequence information isposted on the JGI Microbial Genomes web site at:(http:/ /www .jgi.doe.gov /JGI-microbial/html/ lactoba-cillus~as/lactob-gas-homepage.html) GAMOLA( GlobalAnnotation of On-site Basted DNA-sequences)software was developed by E. Altermann to automat-ically predict coding open reading frames (ORFs) andannotate the draft sequence (http://www.cals.ncsu.edu/food-science/trk/main.html). GAMOLA relies on theavailable software Glimmer2 (Delcher et al. 1999),NCBI tooIkit (National Center for Biotechnology In-formation Bldg 38A, NIH 8600 Rockville Pike Beth-esda, MD 20894), Primer3 (Rozen & Skaletsky, 1997)and HMMER2.2g (Eddy 1998) and combines the res-ults into functionally annotated DNA sequences inGenbank format.

For the L. gasseri sequence, the automated an-notation predicted 1803 ORFs larger than 100 bp.The average gene length is 930 bp with a codingpercentage of 89.1%. After the initial automated an-notation, the genome was predicted to encode: 31ABC transporters, 14 ion-ATPase transporters and twoproton-antiporters; 21 PTS-systems (some partial), 16stress responsive genes (sigma factor, chaperones, heatshock operons and chaperones), one intact prophage,14 mobile elements (transposases, integrases and IS-elements), 32 genes involved in cell-cycle or cell-shape, eight genes implied in aggregation/adhesion,seven genes related to late competence, 40 peptidases(proteolytic system plus general peptidases), 20 tRNAgenes, and at least two rRNA operons. Manual cur-ation of the automated annotation will be carried out

41

upon completion of genome sequencing and closureof the major gaps.

This project has revealed a number of gene systemsthat are likely to be important in the gastrointestinalsurvival and activity of this human probiotic species.The public availability of the L. gasseri genome winpromote comparative genomic allalysis among LAB.

Acknowledgment

The Joint Genome Institute of the US Department ofEnergy and the Lactic Acid Bacteria Genome Consor-tium is supporting this sequencing project. The effortsof R. Barrangou, E. Altermann, W. M. Russell, T.Hawkins, S. Stilwagen, P. Richardson, and K. Kadnerare gratefully acknowledged. Additional support wasprovided by Dairy Management, Inc, the SoutheastDairy Foods Research Center and Rhodia, Inc.

These cultures have an optimum growth temperat-ure of approximately 42 °C and contain Streptococcusthermophilus with L. delbrueckii subsp. bulgaricusand/or L. helveticus. These cultures are utilized inthe manufacture of Swiss-type and Italian-type cheesevarieties. There has been a 325% increase in the past20 years in the production of Mozzarella cheese inthe United States to more than 2244 million pounds,with an econoillic value of nearly $1 billion (Na-tional Cheese Institute 1998). Additionally, strains ofL. helveticus are commonly used as flavor adjunctcultures, where they are added to bacterial ripenedcheese varieties to reduce bitterness and accelerate thedevelopment of beneficial flavors.

To have utility in the manufacture of fermenteddairy products strains must be resistant to bacterio-phage, have stabIe fermentation properties, and con-sistently produce products with acceptable flavor andtexture attributes. A significant body of research isavailable concerning the proteolytic systern of theseorganisills, with at least 15 components of the pro-teolytic systern characterized at the nucleotide leveland numerous single and double mutants lacking spe-cific components studied in detail (Christensen et al.1999). Liillited inforillation is available concerning anuillber of other industrially relevant metabolic path-ways (i.e., carbohydrate fermentation) and enzymmes(i.e., esterases). Genoillic sequence analysis of L. hel-veticus wiIl aIlow researchers to fiIl the significantgaps present in our understanding of the physiologyof this organisill by providing a comprehensive viewof the enzymmes and metabolic pathways potentiaIly in-volved in industriaIly relevant phenotypes. This know-ledge wiIl aIlow researchers to develop more effectivestrategies to enhance the utility of these organisills inthe manufacture Swiss-type and Italian-type cheesevarieties as weIl as their utility as flavor adjunctcultures.

Lactobacillus helveticus CNRZ32

(contributed by: Jim Steele and JeffBroadbent)

Acknowledgment

This project was supported by Dairy Management,Inc. through the Wisconsin Center for Dairy Researchand the Western Dairy Center, College of Agricul-tural and Life Sciences and Chr. Hansen, Inc. FredBlattner and George F. Mayhew fiom the GenomeCenter of Wisconsin are thanked for their assistancewith genome assembly and annotation.

Within the genus Lactobacillus, L. helveticus is part ofthe obligately homofermentative ('Group I') cluster,which can produce D- and L-lactic acid from hex-ose sugars via the Embden-Meyerhof pathway andis incapable of fermenting pentoses (Axelsson 1998).L. helveticus grows on a relatively restricted numberof carbohydrates that includes lactose and ga1actoseand typically requires riboflavin, pantothenic acid andpyridoxal for growth (Hammes & Vogel1995).

Phylogenetically, L. helveticus is quite closelyrelated «10% sequence divergence) to L. amylo-vorus, L. acidophilus, L. delbrueckii, L. acetotoler-ans, L. gasseri, and L. amylophilus. Among thesespecies, L. helveticus is most closely related ( <2%sequence divergence) to L. amylovorus and L. acido-philus (Schleifer & Ludwig 1995). The genome sizeof L. helveticus has been determined to be 2.4 Mb by

pulse-field gel electrophoresis (unpublished data). Ap-proximately 40 chromosomal genes and four plasrnidshave been sequenced from L. helveticus. Methods forelectroporation and gene replacement have been de-veloped for L. helveticus CNRZ32 (Bhowmik & Steele1993; Bhowmik et a1. 1993); however, attempts toutilize these methods with other strains of L. helveticushave been unsuccessful.

L. helveticus is a component of 'thermophilic'starter cultures used in the manufacture of a numberof fermented dairy products (Hassan & Frank, 2001).

42

Acknowledgments

The Joint Genome lnstitute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of T. Hawkins, S. Stil-wagen, P. Richardson, and K. Kadner are gratefully

acknowledged.

Lactobacillus delbrueckii subsp. bulgaricus AT CC

BAA-365

{contributed by James Steele)

Lactobacillus delbrueckii ssp. bulgaricusATCCl1842(contributed by Maarten van de Guchte andEmanuelle Maguin)

Lactobacillus delbrueckii ssp. bulgaricus is a thermo-philic lactic acid bacterium that is principally knownfor its use in yogurt production, where it assuresmilk fermentation in conjunction with Streptococcusthermophilus. As such, L. bulgaricus is one of theeconomically most important LAB.

For the type strain ATCCl1842 a reproducibletransformation procedure is available (Serror et al.2002) as wen as gene inactivation systems (unpub-lished) that win anow the exploitation of genomedata. A Proteome reference map has been established(unpublished) which win facilitate and benefit froma genome analysis. In this context, the determina-tion of the complete genome sequence of this strainwas undertaken in a joint effort of the Centre Na-tional de Séquen9age (CNS, Genoscope, Evry Fr) andthe Institut National de la Recherche Agronomique

(INRA).

Genome sequencing

Within the genus Lactobacillus, L. delbrueckii ispart of the obligately homofennentative ('Group I')cluster, which can produce D-lactic acid from hex-ose sugars via the Embden-Meyerhof pathway andis incapable of fennenting pentoses (Axelsson 1998).The L. delbrueckii species contains three subspe-cies, L. delbrueckii subsp. delbrueckii, L. delbrueckiisubsp. lactis, and L. delbrueckii subsp. bulgaricus. L.delbrueckii subsp. bulgaricus grows on a relativelyrestricted number of carbohydrates and typically re-quires pantothenic acid and niacin (Hammes & Vogel

1995).Phylogenetically, L. delbrueckii subsp. bulgaricus

is closely related ( < 10% sequence divergence) to L.amylovorus, L. acidophilus, L. helveticus, L. acet-otolerans, L. gasseri, and L. amylophilus (Schleifer& Ludwig 1995). The GC ratio of L. delbrueckiisubsp. bulgaricus (49-51 %) is somewhat higher thanthat found among other species (34-46% ) within thisphylogenetic tree (Hammes & Vogel1995). The gen-ome size of L. delbrueckii subsp. bulgaricus has beendetennined to be 2.3 Mb by pulse-field gel electro-phoresis (Leong-Morgentha1er 1990). Very few chro-mosoma1 genes ( < 15) have been sequenced from L.delbrueckii subsp. bulgaricus, however the completesequence of a sma11 cryptic plasmid and the par-tia1 sequence of a bacteriophage are known. Genetransfer systems for L. delbrueckii subsp. bulgaricusinclude two conjugation-based gene transfer systems(Rantsiou et a1. 1999; Thompson et a1. 1999) andelectroporation (Serror et a1. 2002).

L. delbrueckii subsp. bulgaricus is a component of'thennophillic' starter cultures used in the manufac-ture of a number of fennented dairy products (Hassan& Frank 2001). These cultures have an optimumgrowth temperature of approximately 42 °C and con-tain Streptococcus thermophilus with L. delbrueckiisubsp. bulgaricus and/or L. helveticus. These culturesare utilized in the yogurt, Swiss-type and lta1ian-typecheese varieties. There has been a 240% increase inthe past 20 years in the production of yogurt in theUnited States to 1371 million pounds in 1997, witha wholesa1e va1ue of over $1.1 billion (Milk IndustryFoundation 1998).

Although the genome size of L. bulgaricus was pre-viously estimated to be 2.3 Mbp, recent pulse fieldelectrophoresis analysis revealed that the genome sizeofthe type strain AT CC I 1842 was about 1.8 Mbp. Thegenome sequence has been determined using a shot-gun sequencing approach. Contigs were assembIedaf ter sequencing of two plasmid libraries with dif -

ferently sized inserts. Sequences from a mini-BAClibrary were subsequently used for scaffolding. Thescaffold structure was validated by long range P CR,and links between the initial scaffolds were establishedby multiplex long accurate P CR. The average ac con-tent of the L. bulgaricus genome is 50%, while regionsthat presumably result from horizontal transfer can bedetected with ac contents as low as 31 %.

43

Acknowledgments the genome content was carried out with the CDS pre-diction software SHOW (Nicolas et al. 2002) revealing1792 putative protein-encoding genes for 1772 kp se-quenced. Automated annotation folIowed by manualcuration will be done with a improved version ofArtemis software from the Sanger Center, UK.

We thank v. Barbe, S. Oztas, S. Mangenot, C. Robert,R. Eckenberg, R. Dervyn, A-M. Dudez, M. Zagorec,J. Weissenbach, and S.D. Ehrlich for their contributionand support. We thank Alexei Sorokin and AlexanderBolotin for helpful discussions.

Acknowledgments

This project was financed by the lnstitut National deIa Recherche Agronomique (INRA).

Lactobacillus sakei 23K

(contributed by Stéphane ChaiIlou and Monique

Zagorec)

Lactobacillus sakei is the predominant LAB found onfresh meat and is widely used as starter in fermentedmeat products in Western Europe. It is also occasion-ally found on silage, sourdough and smoked fish. Thephysiology of L. sakei is still poorly understood whencompared to dairy LAB. Recent studies seem to in-dicate that L. sakei has developed original metabolicand physiological traits to adapt to a meat environment(for a review see Champomier- Verges et al. 2002). Tobetter understand the ability and fitness of L. sakei togrow on meat we have chosen the genomic approach.The L. sakei genome sequencing project was launchedin year 2000, using strain 23K, isolated from sausage,as a model.

Lactobacillus casei AT CC 334

(contributed by Jeffrey Broadbent)


A physical and genetic mapping of the chromosomewas first established by pulse-field gel electrophoresisafter digestion with rare-cutting enzymes (Dudez etal. 2002). This preliminary map allowed to estim-ate the chromosome size to be about 1.85 Mb. Sevenrm operons including one doublet were identified andtheir flanking regions characterised. The sequencingof 90% of the genome was accomplished by a clas-sical shotgun strategy, where 8000 clones were firstsequenced on their forward side, followed by reads onthe reverse side for 2000 clones flanking contigs end,and 500 intemal reads. The gap closure phase wascarried out using ligation-mediated PCR and severalcustomised BLAST strategies against related genomessuch as Bacillus subtilis, Bacillus halodurans and Lis-teria monocytogenes. Remaining gaps are currentlyanalysed with a set of other strategies such as mul-tiplex PCR, sequencing on chromosoma1 DNA, andcontigs ordering based on the physical map. Currently,96% of the genome has been sequenced with an aver-age base redundancy of 4 x. A preliminary analysis of

Lactobacillus casei are rod-shaped LAB with a G+ C content of 45-47% (Kandler & Weiss, 1986).Within the genus Lactobacillus, L. casei is clusteredwith facultatively heterofermentative ('Group II') spe-cies which produce lactic acid from hexose sugarsvia the Embden-Meyerhofpathway and from pentosesby the 6-phosphogluconate/phosphoketolase pathway(Axelsson, 1998). Growth occurs at 15 °C but not45 °C, and requires riboflavin, folic acid, calciumpantothenate, and niacin (Kandler & Weiss 1986).Previously, four subspecies of L. casei were recog-nized: L. casei subsp. casei, L. casei subsp. pseudo-plantarum, L. casei subsp. rhamnosus, and L. caseisubsp. tolerans (Kandler & Weiss 1986). However,recent phylogenetic studies have led to proposals thatmembers of the L. casei group be divided into threespecies: L. rhamnosus, L. zeae and L. casei, with L.casei ATCC 334 as the neotype strain forthe latter spe-cies (Collins et al. 1989; Dellaglio et al. 1991; Dickset al. 1996; Mori et al. 1997; Chen et al. 2000).

Members of the L. casei cluster are remarkably ad-aptive, and may be isolated from raw and fermenteddairy products, fresh and fermented plant products,and the reproductive and intestinal tracts of humansand other animals (Kandler & Weiss 1986). Industri-ally, L. casei have application as human probiotics(health-promoting live cultures), as acid-producingstarter cultures for milk fermentation, and as specialtycultures for the intensification and acceleration of fla-vor development in certain bacterial-ripened cheesevarieties (Kosikowski 1982; Fox et al. 1998; Fondenet al. 2000).

L. casei ATCC 334 was originally isolated as anadventitious contarninant from Emmental cheese. Thisbacterium, which has an estimated genome size of

44

2.2 Mb (Ferrero et al. 1996; Tynkkynen et al. 1999),was selected for genome sequencing because: (a) itwas isolated from ripening cheese; (b) it is amen-able to transformation and genetic manipulation; and(c) it is the proposed type strain for L. casei. Se-quence analysis of the L. casei AT CC 334 genomeis expected to provide a comprehensive view of theenzymes and metabo1ic pathways that are potentialcontributors to cheese flavor development. In addi-tion, improved knowledge of global gene regulationand integrative metabolism in L. casei will help an-swer long-standing questions regarding mechanismsfor the health-promoting benefits of LAB, identifymeans by which LAB species grow in harsh environ-ments , highlight the most rational strategies for meta-bolic and genetic improvements to industrial strains,and improve molecular biology resources for geneticmanipulation of dairy lactobacilli.

features makes this organism a very interesting subjectfor further genetic and functional genomic studies.


Acknowledgment

The Joint Genome lnstitute of the US Departmentof Energy is supporting the sequencing effort forthis LAB genome. The efforts of B. McManus, T.Hawkins, S. Stilwagen, P. Richardson, and K. Kadnerare gratefully acknowledged.

The genome size of Lb. casei BL23 has previouslybeen estimated to be 2.3 Mb, but recent pulsed-field gel electrophoresis analysis (in collaboration withthe laboratory of M. Zagorec) revealed a genomesize between 2.5 and 2.7 Mb. In order to sequencethe genome of Lb. casei BL23 we used a shot-gun approach (collaboration between the Laboratoirede Génétique des Microorganismes, INRA-CNRS,Thiverval-Grignon and the Laboratoire Microbiologiede l'Environnement, Université de CAEN). Sequen-cing Lb. casei DNA fragments with a mean size of1.3 kb from both ends, present in about 8000 isolatedclones, allowed us to determine about 90% of the com-plete genome. We present I y at tempt to close the about560 remaining gaps. Lb. casei BL23 has been curedof plasmid pLZ 15 and therefore contains only a singlecircular chromosome.

Lb. casei seems to be a versatile organism capableof adapting to variabIe growth conditions. Most in-triguingly it seems to be able to utilize a large variet yof different carbon sources. This is reftected by therelatively large number of predicted phosphoenolpyr-

uvate:sugar phosphotransferase systems (PTS) (seeTable 3). In Lb. casei, the coordinate action of thevarious carbon utilization systems was found to becontrolled by a protein kinase/phosphatase (HprK/P)implicated in carbon catabolite repression and inducerexclusion (Dossonnet et al. 2000). Inactivation ofonly the phosphatase activity of this bifunctional en-zyme had a drastic effect on the growth behavior bypreventing the utilization of almost any carbohydrate(Monedero et al. 2001).

Lactobacillus casei BL23

(contributed by JosefDeutscher and Axel Hartke)

Lactobacillus casei is a heterofermentative organismwidely used in rnilk fermentation and is therefore ofgreat biotechnological importance. In addition, it be-longs to the few bacteria for which probiotic effectshave been unequivocally established (for a criticalreview on probiotics see Marchand & Vandenplas2000). 'Healthy' effects of this organism have beenobserved in numerous studies with men and anim-als. For example, Lb. casei, which survives transitthrough the gastrointestinal tract (Yuki et al. 1999),has been shown to positively affect the immune re-sponse (Matsuzaki & Chin 2000; Yasui et al. 1999),to enhance the resistance towards certain forms ofdiarrhea and to prevent infections by several patho-genic organisms (Wagner et al. 2000; Alvarez et al.2001). Genetic tools, which allow the transformationof many Lb. casei strains and the construction of pre-determined chromosomal mutants carrying deletionsor point mutations (Dossonnet et al. 2000; Viana et al.2000), are available, which together with its probiotic

Acknowledgments

We acknowledge the participation of Yanick Auf-fray, Alain Mazé, Gregory Boël, Ivan Mijakovic,Jean-Christophe Giard, Jean Marie Laplace, Abdel-lah Benachour, Alain Rincé, Vianney Pichereau andthe financial aide from the INRA, the Ministere dela Recherche and the Conseil Régionale de Basse-Normandie.

I..actobacillus brevis AT CC 367

(contributed by Milton Saier}Pediococcus pentosaceus AT CC 25745

(contributed by Jeffery Broadbent and James

Steele)Lactobacillus brevis is a heterofermentative bacteriumthat can be isolated from many different environments.ft is frequently used as a starter culture in silage fer-mentation, sourdough production and LAB-type beerfermentation. In beverages obtained by alcoholic fer-mentation, lactobacilli may contribute to the qualityof the product but may also cause spoilage. CertainL. brevis strains are resistant to hop bittering sub-stances such as isohumulone and are able to growin beer. Their growth changes the turbidity, flavorand aroma of the beer (Richards & Macrae 1964). L.brevis strains involved in wine fermentation may pro-duce biogenic amines by decarboxylation of precursoramino acids through the action of substrate-specificenzymes (Moreno-Arribas & Lonvaud-Funel 2001).The ingestion of foods containing high levels of sucharnines, particularly histamine and tyrarnine, can leadto several toxicological disturbances (ten Brink et al.1990; Mariné-Font et al. 1995).

We have studied the involvement of HPr(ser-P)in the regulation of non-PTS permeases in L. brevis(Ye et al. 1994, 1995). When provided with anexogenous energy source soch as arginine, galactosegrown cells of L. brevis transport galactose and thenon-metabolizable galactose analogue, thiomethyl-f3-galactoside (T MG), by a sugar:H+ symport mechan-ism (Romano et al. 1987; Djordjevic et al. 2001). L.brevis shows very low transformation efficiency byelectroporation rendering this bacteria difficult to ma-nipulate. The cryptic plasmid of 14 kb from L. brevishas recently been sequenced and will be used in thedevelopment of novel vectors and essential genetictools.

Phylogenetically Pediococcus and Lactobacillus forma super-cluster that can be divided in to two sub-clusters, a!l species of Pediococcus fall within theLactobacillus casei-Pediococcus sub-cluster. Mor-phologically, pediococci and lactobacilli (rods) aredistinct. The formation of tetrads via cell division intwo perpendicular directions in a single plane is a dis-tinctive characteristic of pediococci. Pediococcus canbe described as 'the only acidophilic, homoferment-ative, LAB that divide altematively in two perpendic-ular directions to form tetrads' (Simpson & Taguchi1995). Lactic acid is produced from hexose sugarsvia the Embden-Meyerhof pathway and from pentosesby the 6-phosphogluconate/phosphoketolase pathway

(Axelsson, 1998).Strains of P. pentosaceus have been reported to

contain between three and five resident plasmids (Gra-ham & McKay 1985). Plasmid-linked traits includethe ability to ferment raffinose, melibiose, and sucrose,as well as, the production of bacteriocins (Daeschel& Klaenhammer 1985; Gonzalez & Kunka 1986).Plasmids can be conjugally transferred between Pe-diococcus and Enterococcus, Streptococcus, or Lacto-coccus (Gonzalez & Kunka 1983). Electroporationhasbeen utilized to introduce plasmids into pediococci,including P. pentosaceus (Kim et al. 1992; CaldweIl

1996).P. pentosaceus can be isolated from a variet y of

plant materials and bacterial-ripened cheeses. This or-ganism is used as an acid-producing starter culturein sausage fermentations, cucumber and green beanfermentations, soya milk fermentations, and silage(Simpson & Taguchi 1995). P. pentosaceus are alsoa typica! component of the adventitious or non-startermicroflora of most cheese varieties during ripening(Beresford et al. 2001). In addition, it has been sug-gested that this organism may have value as an acid-producing starter culture in the dairy fermentations(CaldweIl et al. 1996, 1998).

Genetic studies of P. pentosaceus have generated alimited quantity of information on plasmid and chro-mosoma! encoded gelles. With only one plasmid andeight unique chromosomal regions sequenced, the vastmajority of gelles encoding industriaIly important at-tributes have yet to be described. Genomic sequenc~analysis of P. pentosaceus genome win help fin keyknowledge gaps by providing a comprehensive view~

Acknowledgment

The Joint Genome lnstitute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of G. Lorca, T. Hawkins, S.Stilwagen, P. Richardson, and K. Kadner are gratefully

acknowledged.

46

of the enzymes and metabolic pathways related to:(I) acid and flavor production in fermented meat andvegetable foods; (2) mechanisms by which by P.pentosaceus and other non-starter LAB grow and dir-ect flavor development in ripening cheese; and (3)mechanisms by which P. pentosaceus and related LABspoil wine and other alcoholic beverages.

polysaccharides that act as natural thickening agentshas also attracted significant attention. Finally, S.thermophilus has an important role as a probiotic, al-leviating symptoms of lactose intolerance and othergastrointestinal disorders.

The genome of S. thermophilus is 1.8 Mb, makingit among the smallest genomes of all LAB. Althougha moderate thermophile, it is phylogenetically relatedto the more mesophilic lactococci and has a compar-able low G+C ratio (40% ). Genes coding for metabolicpathways involved in sugar catabolism (Poolman etal. 1989; Vaughan et al. 2001), protein and peptideutilization (Femandez-Espla et al. 2000; Garault etal. 2002), polysaccharide production (Almirón-Roiget al. 2000), the stress response system (Perrin et al.1999), and phage resistance mechanisms (Solow &Somkuti 2000; Burrus 2001) have been sequenced andcharacterized. Although most strains do not harborplasmids, other mobile elements have been reported(Guedon et. al. 1995), and techniques for gene trans-fer and mutagenesis have been developed (Coderre &Somkuti 1999; Baccigalupi et al. 2000).

Acknowledgments

The Joint Genome lnstitute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of T. Hawkins, S. Stil-wagen, P. Richardson, and K. Kadner are gratefully

acknowledged.

Streptococcus thermophilus LMD-9

(coDtributed by Robert HutkiDS)

Research during the past two decades has revealedthat Streptococcus thermophilus has properties thatmake it one of the most commercially important ofall LAB. S. thermophilus is used, along with Lacto-bacillus s pp., as a starter culture for the manufactureof several important fermented dairy foods, includingyogurt and Mozzarella cheese. lt's use has increasedsignificantly during the past two decades as a re sultof the tremendous increase in consumption of theseproducts. According to USDA statistics, in 1998, morethan 2.24 billion pounds Mozzarella cheese and 1.37billion pounds of yogurt were produced, respectively,with a combined economic value of nearly $5 billion.This increase has led to new demands on the per-formance and production requirements of such startercultures. lndustrial strains, for example, should be in-sensitive to bacteriophage, have stabIe fermentationcharacteristics, and produce products having consist-ent flavor and texture properties. Although research onthe physiology of S. thermophilus has revealed import-ant information on some of these properties, includingsugar and protein metabolism, polysaccharide pro-duction, and flavor generation, only recently has thegenetic basis for many of these traits been determined.

Currently, several traits in S. thermophilus havebeen targeted for strain improvement programs (Del-cour et al. 2000). Since bacteriophage are responsiblefor considerable economic losses during cheese man-ufacture, efforts are underway to engineer restrictionand other phage-resistance systems into commercialstrains. Enhancing stability and expression of exo-

Acknowledgments

The Joint Genome lnstitute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of L. Durso, J. Goh, T.Hawkins, S. Stilwagen, P. Richardson, and K. Kadnerare gratefully acknowledged.

Streptococcus thermophilus LMGl83ll and

CNRZlO66

( contributed by Alexander Bolotin, Ross Overbeek

and Pascal Hols)

Streptococcus thermophilus is one of the most eco-nomically important LAB used in the manufactureof yogurt and some Swiss- or Italian-type hardcooked cheeses. This Gram-positive, non-sporulating,catalase-negative, facultative-anaerobe coccus pro-duces remarkable quantities of polysaccharides con-tributing to the rheological properties of fermentedproducts. The comprehensive knowledge of S. thermo-philus biology is important for the dairy industry butis rather limited (Delcour et al. 2000). The availabilityof the whole genome sequence of this species wouldstrongly improve the knowledge of its metabolism andpave the way for engineering of new starter culturesand better control of existing fermentation processes.

47

The Life Science Institute at the Catholic University ofLouvain (Belgium), the Laboratory of Genetic Micro-biology, Jouyen Josas, INRA (France) and IntegratedGenomics Inc, Chicago (USA) joined their efforts indetermining the genome sequences oftwo strains of S.

thermophilus.

de Génétique, UCL who participated in the sequencingprojects of the two S. thennophilus genomes.

Leuconostoc mesenteroides subsp. mesenteroidesLA81

(contributed by Fred Breidt)

Genome sequencing status and resultsLeuconostoc species are epiphytic bacteria that arewide spread in the natural environment and play animportant role in several industrial and food ferment-ations. Leuconostoc mesenteroides is a facultative an-aerobe requiring complex growth factors and aminoacids (Reiter & Oram 1982; Garvie 1986). Moststrains in liquid culture appear as cocci, occuringsingly or in pairs and short chains; however, mor-phology can vary with growth conditions; cells grownin glucose or on solid media may have an elongatedor rod-shaped morphology. Cells are Gram-positive,asporogenous and non-motile. A variet y of LAB, in-cluding Leuconostoc species are commonly found oncrop plants (Mundt et al. 1967; Mundt 1970). L.mesenteroides is perhaps the most predominant LABspecies found on fruits and vegetables and is respons-ible for initiating the sauerkraut and other vegetablefermentations (Pederson & Albury 1969). L. mes-enteroides starter cultures also used in some dairyand bread dough fermentations (Server-Busson et al.1999). Under microaerophilic conditions, a hetero-lactic fermentation is carried out. Glucose and otherhexose sugars are converted to equimolar amount ofD-Iactate, ethanol and CO2 via a combination of thehexose monophosphate and pentose phosphate path-ways (Demoss et al. 1951; Garvie 1986; Gottschalk1986). Other metabolic pathways include conversionof citrate to diacetyl and acetoin (Cogan et al 1981)and production of dextrans and levan from sucrose(AIsop 1983; Broker 1977). Viscous polysaccharidesproduced by L. mesenteroides are widely recognizedas causing product losses and processingproblems inthe production of sucrose from sugar cane and sugarbeets (TalIgren et al. 1999). The first observation ofthe production of polysaccharide 'slime' from sugar,dates to the earliest days of the science of microbio-logy; Pasteur (1861) attributed this activity to smallcocci, presumably Leuconostoc species. Commercialproduction dextrans and levans by L. mesenteroides,for use in the biochemical and pharmaceutical in-dustry, has been carried out for more than 50 years(AIsop 1983; Sutherland 1996). Dextrans are usedin the manufacture of blood plasma extenders, hep-

The complete sequences were determined by the ran-dom shotgun sequencing strategy folIowed by mul-tiplex P CR described earlier (Bolotin et al. 2001).Sequences of the two strains were determined byconstruction of two independent sequence datasetscontaining 20000 random and 1500 primer directedreads for LMGl8311 strain and 28000 random and2000 primer directed reads for CNRZl066 strain. TheassembIed genomes were analysed using the ERGObioinformatics suite. Both genomes contain '"'"' 1.8 Mb

encoding six rRNA, a compete set of tRNAs, sev-eral types of IS, and about 1800 open reading frames.They are organized as a single circular chromosomewith 39% GC content, and show 95% nucleotideidentity. Although the S. thermophilus species is char-acterized as GRAS (Generally Recognized As Safe),several features of its metabolism are similar to thatof pathogenic Streptococci. Analysis of the genomehas revealed several key aspects of the pathwaysof carbohydrates, amino acids, nucleotides, poly-sacharides and lipids metabolism. More than 100genes involved in the transport of a variet y of sub-strates such as peptides, sugars, metal ions, andcofactors have been identified. The presence of sev-eral phage and plasmid related genes indicates theimportance of horizontal gene transfer for evolutionof S.thermophilus. More information can be foundat web sites: http://www.biol.ucl.ac.be/gene/genome/,

http://spooky.jouy.inra.fr (restricted access), http://www.integratedgenomics.com (restricted access)

Acknowledgments

We thank Benoit Quinquis, Pierre Renault, AlexeiSorokin, and S. Dusko Ehrlich from Génetique Mi-crobienne, INRA; A1la Lapidus, Eugene Goltsman,Mikhail Mazur, Gordon D. Pusch, and Michael Fon-stein from Interegated Genomics Inc.; Bénédicte Pur-ne1le, Deborah Prozzi, Katrina Ngui, David Masuy,Frédéric Hancy, Anne Bemard, Sophie Burteau, MarcBoutry, André Goffeau, and Jean Delcour from Unité

48

arin substitutes for anticoagulant therapy, cosmetics,and other products (A;sp[ 1983; Kim & Day 1994;Leathers et al 1995; Sutherland 1996). Another useof dextrans is the manufacture of Sephadex gels orbeads, which are widely used for industrial and labor-atory protein separations (Sutherland 1996). We haveselected L. mesenteroides subsp. mesenteroides LA8lfor sequencing. The strain is widely available from avariety of culture collections listed as: ATCC 8293,DSM 20343, NCIB 8023, CCM 1803, NCDO 523,and NRRL B-1l18. The strain is generally accepted asa neotype strain for this species; it produces dextran,and was originally isolated from fermenting olives.

Acknowledgments

The Joint Genome lnstitute of the US Departmentof Energy is supporting the sequencing effort forthis LAB genome. The efforts of v. Plengvidhya, T.Hawkins, S. Stilwagen, P. Richardson, and K. Kadnerare gratefully acknowledged.

(Tonon et al. 2001) and rDNA genes (Martinez-Murcia& Co1lins 1990). Several genetic elements from 0.oeni have been characterized including bacteriophages(Gindreau et al. 1997; Gindreau & Lonvaud-Funel1999), and plasmids (Fremaux et al. 1993). In ad-dition, the conjugative transposon Tn916 has beenmobilized into the 0. oeni genome (Zuniga et al.

1996b).Many researchers have examined the diversity of

0. oeni strains within and around wineries. Stud-ies have employed various molecular typing methods

(protein profiling, plasmid profiling, RAPD, PFGE,rDNA RFLP, etc.) to discem regional differences instrains. An outcome of this ana1ysis is the generalview that 0. oeni is a genetica1ly homogenous species(Zapparoli et a1. 2000).

The strain sequenced in this project, 0. oeni PSU-1 (ATCC BAA-331), was origina1ly isolated at PennState University from red wine undergoing a spontan-eous malolactic fermentation (Beelman et al. 1977).PSU-1 was shown to reliably induce the malolacticfermentation in wines and is currently employed com-mercia11y (Beelman et al. 1980). While the 0. oeniis considered homogenous species, the strain PSU-1was previously shown to be representative of the largerof two divergent groupings (Tenreiro et a1. 1994). Anextensive physical map of PSU-1 generated by Ze-Zeet al. 1998, 2000) has tremendously aided sequencecompilation and genome scaffolding operations.

Oenococcus oeni PSU-l (AT CC BAA-331)

(contributed by David Mills)

Acknowledgments

This project was supported by the US Departmentof Energy-Joint Genome Institute, the AmericanVineyard Foundation, and the California CompetitiveGrants Program for Research in Viticulture and En-ology. The efforts of C. M. Joseph, K. Ranstiou, T.Hawkins, S. Stilwagen, P. Richardson, and K. Kadnerare gratefully acknowledged.

Oenococcus oeni (formerly called Leuconostoc oenos)is a lactic acid bacterium that occurs naturally infruit mashes and related habitats (Van Vuuren & Dicks1993).0. oeni is employed commercially to carry outthe malolactic conversion, an important secondary fer-mentation in the production of wine (Kunkee 1991).0. oeni is a facultative anaerobe and one of the mostacid- and alcohol-tolerant LAB. 0. oeni shares relat-ively little DNA homology with the other genera inthe Leuconostoc branch of the LAB (Dellaglio et al.

1995).Perhaps the most studied aspect of 0. oeni is its

ability to carry out the malolactic conversion. This in-volves uptake of malate, decarboxylation to L-lacticacid and CO2, and subsequent export of end products.The malolactic conversion generates energy for thecell in the form of a proton motive force (Salema etal. 1996). Recently the genes encoding the malate de-carboxylase (mleA) and malate permease (mleP) havebeen cloned and characterized (Labarre et al. 1996a,b ).Other chromosomal genes from 0. oeni that havebeen characterized include: histidine decarboxylase(Coton et al. 1998), a-acetolactic acid decarboxylase(Garmyn et al. 1996), stress-related genes (Jobin et al.1997, 1999), genes involved in arginine metabolism

Oenococcus oeni IOEB 8413

(contributed by Jean Guzzo)

Oenococcus oeni is a LAB most often responsiblefor an important step in the winemaking process, thema1olactic fermentation (MLF). The main transform-ation is decarboxylation of ma1ic acid to lactic acid.MLF leads to a natural decrease of acidity, togetherwith an enhancement of stability and quality of wine.Norma1ly, it occurs spontaneously after a1coholic fer-

49

Brevibacterium linens RL2 Rio

(contributed by Hart Weimer)

mentation, but in some wines the environment is sohostile to bacterial growth that MLF is delayed or eventotally prevented. To solve this problem, winemakersuse malolactic starters prepared by the industrial cul-ture of selected strains. The laboratory of rnicrobi-ology, UMR INRA, from Dijon University and thelaboratory of biotechnology and applied microbiology,UA INRA, from Bordeaux University, in collabor-ation with GENOME Express, have determined thecomplete nucleotide sequence of the chromosome ofO.oeni IOEB8413, a strain isolated from Bordeauxwine. The knowledge of the 0. oeni genome repres-ents a considerable value for developing new tools forthe control of malolactic fermentation in wine.


We first sequenced two random small insert andlarge insert libraries to favour the scaffold formationbetween sequence contigs during the assembly stage.Subsequently primers were designed for gap clos-ure sequencing by gene walking and multiplex P CRapproaches. The sequenced contigs were assembIedusing the Phred and Phrap software packages. Thecurrent assembly represents 1 753879 nucleotides anda G+C content of 37.9%. At this stage, early an-notation has been undertaken in order to improve thefinishing step. The annotation was performed using theGenoAnnofm software (Geno*tm platform), an integ-rated computer environment specialised in large-scalesequence annotation. A first pass in an automatic modewith well-dedicated annotation strategies revealed atotal of 1784 putative coding sequences, 43 tRNAand 588 predicted rho-independant terminators. Func-tional annotation was then based on similarity searcheswith the Blast programs family. Preliminary Blast ana-lysis of the current assembly revealed two ribosomalRNA operons, the presence of three different IS ele-ments in several copies, and protein-coding genesbelonging to prophages. Some functional categoriesof proteins are being studied in detail: stress pro-teins, transcriptional regulators, central carbon meta-bolism enzymes and transporters, proteins involvedin nitrogen assimilation and the membrane anabolic

pathway.

The Brevibacterium genus is a heterogeneous mixtureof coryneform organisms that have particular applica-tion to industrial production of vitamins, amino acidsfor fine chemical production, and are commonly usedin cheese production (Amador et al. 1999; Rattray& Fox 1999). This genus contains nine species fromdiverse habitats, such as soil, poultry, fish, humanskin, and food. While Brevibacterium linens is pheno-typically similar to Arthrobacter globiformis, cellularpigmentation, cell wall composition, DNAIDNA hy-bridization and 5s RNA analysis show that Brevibac-terium is distinctly different (Park et al. 1987). PFGEanalysis indicates that diversity within the species isrelated to polymorphisms in the 16S rRNA genes withgenome si zes that range from 3.2 and 3.9 Mbp (Lima& Correia 2000).

B. linens is a non-motile, non-spore forming, non-acid fast, Gram-positive coryneform that tolerates highsalt concentrations (8-20% ) and is capable of grow-ing in a broad pH range (5.5-9.5), with an optimumof pH 7.0. They also survive carbohydrate starva-tion and drying for extended periods (Boyaval et al.1985). B. linens is unusual as they produce baseas they grow, raising the pH to ""9.5 within 24-36h. Recent interest in B. linens has focused aroundtheir ability to produce a self -processing extracellularprotease (Rattray et al. 1997; Weimer et al. 2000),their ability to produce high levels of volatile sulfurcompounds (Ferchichi et al. 1985; Dias & Weimer1998a,b ), bacteriocin production (Valdes-Stauber &Scherer 1996), cell-membrane-associated carotenoidpigment production (Arrachet al. 2001), and aromaticamino acid metabolism (Leuschner & Hammes 1998;Ummadi & Weimer 2001). These organisms are alsonoted for their metabolize heterocyclic and polycyclicring structures-a trait that is not associated with otherbacteria but is common in fungi. Of particular noteis the degradation of insecticides (including DTT andDDE). These organisms also metabolize amino acids,particularly aromatic amino acids, to produce plantgrowth hormones.

Acknowledgments Acknowlegments

We thank Aline Lonvaud-Funel (University of Bor-deaux) and Yves Vandenbrouck (GENOME Express)for their contribution to this work.

The Joint Genome Institute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of T. Hawkins, S. Stil-

49

mentation, but in some wines the environment is sohostile to bacteria1 growth that MLF is delayed or eventotally prevented. To solve this problem, winemakersuse malolactic starters prepared by the industrial cul-ture of selected strains. The laboratory of rnicrobi-ology, UMR INRA, from Dijon University and thelaboratory of biotechnology and applied microbiology,UA INRA, from Bordeaux University, in collabor-ation with GENOME Express, have determined thecomplete nucleotide sequence of the chromosome ofO.oeni IOEB8413, a strain isolated from Bordeauxwine. The knowledge of the 0. oeni genome repres-ents a considerable value for developing new tools forthe control of malolactic fermentation in wine.

Brevibacterium linens RL2 Rio

(contributed by Rart Weimer)


We first sequenced two random small insert andlarge insert libraries to favour the scaffold formationbetween sequence contigs during the assembly stage.Subsequently primers were designed for gap clos-ure sequencing by gene walking and multiplex P CRapproaches. The sequenced contigs were assembIedusing the Phred and Phrap software packages. Thecurrent assembly represents I 753879 nucleotides anda G+C content of 37.9%. At this stage, early an-notation has been undertaken in order to improve thefinishing step. The annotation was performed using theGenoAnnottm software (Geno*tm platform), an integ-rated computer environment specialised in large-scalesequence annotation. A first pass in an automatic modewith well-dedicated annotation strategies revealed atotal of 1784 putative coding sequences, 43 tRNAand 588 predicted rho-independant terminators. Func-tional annotation was then based on sirnilarity searcheswith the Blast programs family. Preliminary Blast ana-lysis of the current assembly revealed two ribosomalRNA operons, the presence of three different IS ele-ments in several copies, and protein-coding genesbelonging to prophages. Some functional categoriesof proteins are being studied in detail: stress pro-teins, transcriptional regulators, central carbon meta-bolism enzymes and transporters, proteins involvedin nitrogen assirnilation and the membrane anabolic

pathway.

The Brevibacterium genus is a heterogeneous mixtureof coryneforlli organisllis that have particular applica-tion to industrial production of vitamins, amino acidsfor fine chellical production, and are commonly usedin cheese production (Allador et al. 1999; Rattray& Fox 1999). This genus contains nine species frolldiverse habitats, such as soil, poultry, fish, humanskin, and food. While Brevibacterium linens is pheno-typically sillilar to Arthrobacter globiformis, cellularpigmentation, cell wall composition, DNA/DNA hy-bridization and 5s RNA analysis show that Brevibac-terium is distinctly different (Park et al. 1987). PFGEanalysis indicates that diversity within the species isrelated to polylliorphisllis in the 16S rRNA genes withgenome sizes that range froll 3.2 and 3.9 Mbp (Lima& Correia 2000).

B. linens is a non-llotile, non-spore forming, non-acid fast, Grall-positive coryneform that tolerates highsalt concentrations (8-20% ) and is capable of grow-ing in a broad pH range (5.5-9.5), with an optimumof pH 7.0. They also survive carbohydrate starva-tion and drying for extended periods (Boyaval et al.1985). B. linens is unusual as they produce baseas they grow, raising the pH to ""'9.5 within 24-36h. Recent interest in B. linens has focused aroundtheir ability to produce a self-processing extracellularprotease (Rattray et al. 1997; Weillier et al. 2000),their ability to produce high levels of volatile sulfurcompounds (Ferchichi et al. 1985; Dias & Weillier1998a,b ), bacteriocin production (Valdes-Stauber &Scherer 1996), cell-llellibrane-associated carotenoidpigment production (Arrachet al. 2001), and aromaticallino acid metabolislli (Leuschner & Hammes 1998;Ummadi & Weillier 2001). These organisllis are alsonoted for their metabolize heterocyclic and polycyclicring structures-a trait that is not associated with otherbacteria but is common in fungi. Of particular noteis the degradation of insecticides (including DTT andDDE). These organisllis also metabolize allino acids,particularly aromatic allino acids, to produce plantgrowth hormones.

Acknowledgments Acknowlegments

We thank Aline Lonvaud-Funel (University of Bor-deaux) and Yves Vandenhrouck (GENOME Express)for their contrihution to this work.

The Joint Genome Institute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of T. Hawkins, S. Stil-

50

wagen, Po Richardson, and Ko Kadner are gratefully

acknowledgedoAcknowledgments

Jan Sikkema (FCDF), Jan Hunik (DSM Food Spe-ciaties) and Albert van Ooyen (DSM Food Special-ties) are greatfully acknowledged for their contri-bution and support to this work. The Propionibac-terium freudenreichii genome was sequenced by Qia-gen (www.qiagen.com) The genome was annotated byBiomax Informatics (www.biomax.de).

Propionibacterium freudenreichii AT CC 6207

{contributed by Herman Pel)

Propionibacteria are high G+C Gram-positive bac-teria belonging to the class of Actinobacteria thatprefer anaerobic growth conditions and have a peculiarphysiology. They produce propionate as their majorfermentation product. Propionate fermentation yieldsmore energy and, consequently, biomass than anyother anaerobic microbial fermentation. Furthermore,propionibacteria utilize polyphosphate and pyrophos-phate instead of ATP for several energy-dependentreactions and their metabolism is tuned to synthesizehigh levels of porphyrins, in particular B12.

DSM Food Specialties uses a classical dairy mi-croorganism Propionibacterium freudenreichii to pro-duce vitamin Bl2 for feed applications. Vitamin Bl2is a highly complex but essential vitamin that canonly be produced economically through the ferment-ation of microorganisms. Friesland Coberco DairyFoods (FCDF) is a world player in cheese produc-tion. Propionibacteria have long been employed in theproduction process of Swiss-type cheeses for whichthey are indispensable for the typical eye-formationand production of characteristic taste components.

Bifidobacterium breve NCIMB 8807

(contributed by: Douwe van Sinderen)

Species of the genus Bifidobacterium are Gram-positive bacteria, strictly anaerobic, fermentative rods,often Y-shaped or clubbed at the end and containDNA with a relatively high G+C content. They rep-resent a major element in the microflora of the humangastrointestinal tract. They are considered to have asignificant role in maintaining the good health of thehuman host, while there is mounting evidence pointingto the activity of these bacteria in mediating other pos-itive health attributes such as the alleviation of lactosetolerance, stimulation/potentiation of the immune sys-tem, and production of vitamins and antimicrobials.Bifidobacteria have been shown to be the predominantspecies in the gastrointestinal tract of infants, and rep-resent the third most numerous species encountered inthe colon of adult humans, considerably outnumberingother groups such as Lactobacillus species. The role ofthese bacteria in human health has stimulated signific-ant interest in the health care and food industries, andhas highlighted the position of these bacteria in thedevelopment of functional and pharma foods, whichwould contain these bacteria as active ingredients.

Despite growing consumer interest, key aspects re-garding Bifidobacterium species, such as metabolicactivities (particularly relating to catabolism of pre-biotics) and physiology are still poorly understood.The deterrnination of the complete genome of the B.breve strain NCIM 8807 (National Collection of In-dustrial and Marine bacteria, Aberdeen, Scotland), anisolate from nursling stools, was undertaken as a firststep towards the molecular analysis of a probiotic Bi-fidobacterium species. This plasmid-free strain wasselected because it is reasonably easy to transform(:J:: 104 transformants per .ug ofplasmid DNA), showsgood adherence properties to epithelial cells, and ex-hibits reproducible growth properties and moderatetolerance to oxidative. osmotic and acid stresses.


The lack of fundamental physiological knowledgeof propionibacteria hampers their efficient industrialexploitation. DSM Food Specialties and FrieslandCoberco Dairy Foods have therefore in a combinedeffort sequenced and annotated the genome of theP. freudenreichii type strain AT CC 6207. A total of2641522 base pairs were sequenced using a shot-gun approach resulting in 12 contigs. The genomesequence revealed a ac content of 67% and was foundto contain at least 2552 open reading frames. The an-notated genome, including the resulting blueprint ofthe metabolic capabilities of the cell, forms a solidbasis for the use of powerlul tools such as transcrip-tomics and metabolomics to analyze the metabolicresponse of P. freudenreichii to genetic and environ-mental changes.

51

Genome sequencing status and results they were classified in the genus Lactobacillus, due totheir rod-like shapes and obligate fermentative char-acteristics. However, the accumulation of studies de-tailing DNA hybridizations, G+C content and uniquemetabolic capabilities resulted in the resurrection ofthe Bijidobacterium genus. They are characterized bya unique hexose metabolism that occurs via a phos-phoketolase pathway often termed the 'bifid shunt'.Fructose-6-phosphate phosphoketolase (F6PPK) is akey enzyme of the 'bifid shunt' and its presence is themost common diagnostic test for this genus, as it is notpresent in other Gram-positive intestinal bacteria.

The genus is comprised of 31 characterized spe-cies, 11 of which have been detected in human feces(Tannock 1999). B. longum is often the dominant spe-cies detected in humans and is the only species toregularly harbor plasmids. It is a leading member ofthe probiotic bacteria due to numerous studies thathave provided a growing body of evidence for its rolein a myriad of potential health bellefits. These in-clude diarrhea prevention in antibiotic treated patiellts(Black et al. 1991), cholesterol reduction (Dambekodi& Gilliland 1998), alleviation of lactose intolerancesymptoms (Jiang et al. 1996), immune stimulation(Takahashi et al. 1998), and cancer prevention (Reddy& Rivenson 1993). This myriad of potential healthbellefits attributed to the B. longum species clearly il-lustrates that this species possesses many very interest-ing characteristics. It is anticipated that identificationand functional analysis ofthe genetic determinants in-volved in these activities will strengthen the evidencefor the involvement of B. longum in these signific-ant health bellefits. Selection of suitable strains forprobiotic purposes is very difficult as inherent charac-teristics of strains of B. longum that are necessary forits survival and competition in the human large intest-ine are currently very poorly understood (0' Sullivan2001). The use ofthe sequenced genome in microarrayallalysis should reveal the pertinent traits that are im-portant for these bacteria to attain dominance in these

complex ecosystems.

Random sequences were obtained using a small-insert(2-4 kb) plasmid bank and a large insert (20-35kb) cosmid library generating a total of roughly 10.5million of raw sequence data (approximately 4.4-fold redundancy). These sequences have each beenchecked for quality and contamillating cloning vectorsequences, and subsequently been used for an overallassembly by means of a combination of the Stadensoftware package, and PHRED/PHRAP. This resultedin an initial assembly into 376 contigs, which werethen examined individually for intemal joints to re-duce the total number of contigs to 250. Gap closureby primer walking is now ongoing and has reduced thenumber of contigs to 140, representing a total DNAsequence of 2.43 Mb. Preliminary allalysis of the ob-tained sequence has revealed quite a high number of ISelements, and a large number of gelles predicted to beinvolved in the degradation of poly- and oligosacchar-ides, e.g., arabinogalactan endo-l,4-f3-galactosidaseand a-mannosidase. Four rRNA-encoding operonshave sofar been identified, while the calculated G+Ccontent of the genome is roughly 53%.

Acknowledgments

Funding of this work was obtained through the HigherEducation Authority Programme for Research in ThirdLevel Institutions, Cycle 1 (HEA PRTLI1), andBioResearch Ireland. Sinead Leahy, Sinead Ryan,Jose Antonio Moreno Mufioz, Des Higgins and Ger-ald Fitzgerald are gratefully acknowledged for theircontributions to this work.

Bifidobacterium Iongum DJOlOA

{contributed by Daniel O'Sullivan}

Bifidobacteria are anaerobic, Gram-positive, irregu-lar or branched rod-shaped bacteria that are com-monly found in the intestines of humans and mostanimals and insects. They were first isolated and de-scribed over 100 years ago from human feces and werequickly associated with a healthy GI tract due to theirnumerical dominance in breast-fed infants comparedto bottle-fed infants (Tissier 1899, 1906). While theywere first grouped in the genus Bacillus, the genus Bi-fidobacterium was proposed in the 1920s (Orla-Jensen1924). However, there was not a taxonomic consensusfor this new genus and for much of the 20th century,

Acknowledgment;

The Joint Genome lnstitute of the US Department ofEnergy is supporting the sequencing effort for thisLAB genome. The efforts of m-H. Lee, J. Halgersen,T. Hawkins, S. Stilwagen, P. Richardson, and K.Kadner are gratefully acknowledged.

52

Bifidobacterium Iongum NCC270S

(contributed by Fabrizio Arigoni)

an unusual number (>8.5%) assigned to the carbo-hydrate transport-metabolism category. This suggeststhat B. Iongum is wen adapted to take advantage ofthe wide diversity and fluctuations in the nutrient com-position in the colon. In addition, we observed thatgene duplication and horizontal gene transfer haveplayed an important part in this physiological ad-aptation. Using SignalP (Nielsen et al. 1997), weidentified approximately 200 proteins with probableSec-type signal peptides. Of these 59 were predictedas surface-associated lipoproteins (PROSITE acces-sion PSOO013) and 26 as solute-binding proteins ofABC transporter systems.

Bifidobacteria are obligate anaerobes in the Actin-omycetales branch of the high G+C Gram-positivebacteria. There are at least 32 species of bifidobac-teria, largely isolated from the GITs of many mammalsas well as chickens and honeybees (Biavati & Mat-tarelli 2001). They are among the first colonizers ofthe sterile GITs of newboms and predominate un-til weaning when they are surpassed by other groups(Harmsen et al. 2000; Favier et al. 2002). Althoughthe bifidobacteria represent <6% of the adult fecalflora, their presence has been associated with be-neficial health effects (Biavati & Mattarelli 2001).For example, some studies showed that infant for-mula containing bifidobacteria reduces incidence ofdiarrhea (Saavedra et al. 1994). These types of stud-ies have led to widespread use of bifidobacteria ascomponents of health-promoting foods (probiotics).Although foods containing bifidobacteria are widelyconsumed probiotics, there is only fragmentary in-formation about the physiology, ecology, and geneticsof any one species. To rapidly increase knowledgeand understanding of bifidobacterial biology and theircomplex interactions with their human hosts and GITmicroflora, we determined and extensively analyzedthe genome sequence of a B. Iongum strain isolatedfrom infant feces.

Acknowledgments

MoAo Schell, Mo Karmirantzou, Bo Snel, Bo Berger, Do

Vilanova, Go Pessi, Po Bork, To Pohl, Go Bothe, MoC

Zwahlen, Mo Delley are sincerely thanked for their

contrihution to this worko

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