JOURNAL OF BACrERIOLOGY, Oct. 1992, p. 6580-65890021-9193/92/206580-10$02.00/0Copyright X 1992, American Society for Microbiology

Vol. 174, No. 20

Branched-Chain Amino Acid Biosynthesis Genes inLactococcus lactis subsp. lactis

JEAN-JACQUES GODON,* MARIE-CHRISTINE CHOPIN, AND S. DUSKO EHRLICHLaboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert,

78352 Jouy-en-Josas Cedex, France

Received 21 April 1992/Accepted 12 August 1992

The genes for biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine in Lactococcuslactis subsp. lactis NCDO2118 were characterized by cloning, complementation in Escherichia coli and Bacilussubtilis, and nucleotide sequence analysis. Nine structural genes are clustered on a 12-kb DNA fragment in theorder leuABCD ilvDBNCA. Upstream of these genes, the nucleotide sequence suggests the existence ofregulation by transcriptional attenuation. Between the leuD and ilvD genes is an unexpected gene, encoding aprotein which belongs to the ATP-binding cassette protein superfamily.

The branched-chain amino acid (BCAA) pathway, bywhich leucine, isoleucine, and valine are synthesized, hasbeen widely studied in bacteria, fungi, and plants (8, 31, 59,60). Regulation of the expression ofBCAA genes is complexbecause of the common steps for synthesis of the threeamino acids, and the pathway is often presented as a modelfor organization and regulation studies. However, the se-quences of all of the genes from the same organism havenever been reported. Organization of the genes of the BCAApathway has been characterized for Escherichia coli (59),Salmonella typhimurium (59), Bacillus subtilis (37, 62, 63),Corynebacterium glutamicum (9), and Staphylococcus au-reus (44). On the E. coli chromosome, the genes are locatedin three clusters (3). The largest, at 85 min, is organized intoone large and two small transcription units, comprising theilvGMEDA, ilvY, and ilvC genes, respectively (36, 64).Another cluster, at 2 min, is composed of two transcriptionunits, comprising the ilvIH and leuACBD genes (17, 56), andthe last cluster, at 82 min, groups the ilvBN genes in a singletranscription unit (65). A similar organization is found inother enterobacteriaceae (12). In B. subtilis, ilvBNC andleuACBD genes are found in one chromosomal region (37)and ilvAD genes are found in another (46). Three noncontig-uous chromosomal fragments cloned from C. glutamicumcarry five BCAA genes, ilvCBN, ilvA, and ilvE (9). In S.aureus, eight structural genes are clustered in the orderilvABCD leuABCD, as found by genetic mapping (44). Herewe report the cloning, characterization, and sequences of L.lactis subsp. lactis BCAA genes. Similar analyses of thegenes of the tryptophan and histidine biosynthesis pathwaysare described in the accompanying reports (5, 10).

MATERIALS AND METHODSThe bacterial strains and plasmids used are described in

Table 1. Media and growth conditions and DNA cloning andmanipulation procedures are described in an accompanyingreport (5). The reported sequence was determined for bothstrands. Restrictionless B. subtilis strains mutated in the ilvAgene were constructed by transforming MT119-competentcells with pHV438 (41) to Cmr. Integration of this plasmidinto the chromosome by double crossing over replaces theilvA gene by the Cmr gene (42). A representative ilvA leuB6

* Corresponding author.

r- m- Cmr clone was designated IL2685. A restrictionless B.subtilis ilvD4 leuB6 double mutant was constructed bycongression, using GSY276 DNA to transform 1012 compe-tent cells to methionine independence. Transformants werefurther tested for isoleucine and leucine requirements andfor the absence of restriction by titration of phage rho. OneilvD4 leuB6 r- m+ clone was designated IL3151.

Nucleotide sequence accession number. The sequenceshown in Fig. 2 has been assigned GenBank accessionnumber M90761.

RESULTS

Cloning of BCAA biosynthetic genes. Total DNA from L.lactis subsp. lactis NCDO2118 was partially digested withendonuclease Sau3AI. Twenty micrograms of >10-kb DNAsegments was ligated to 10 ,ug of BamHI-cleaved plasmidvector pIL253 DNA at a final concentration of 500 ,ig/ml.The ligation mixture was used to transform competent cellsof the B. subtilis leuB6 ilvD4 mutant strain IL3151 toisoleucine independence. Four transformants were also Emrand contained plasmids of 18, 14.2, 13.5, and 9.5 kb,designated pIL384, pIL371, pIL374, and pIL373, respec-tively. In a similar experiment, XbaI-cleaved L. lactis subsp.lactis DNA was cloned into pIL253, using the B. subtilis ilvAmutant strain IL2685 as the recipient. Transformants wereselected on minimal medium supplemented with leucine andtryptophan but lacking isoleucine. Two Emr Ile' clones,containing an apparently identical 23.5-kb plasmid desig-nated pIL500, were obtained.

Complementation experiments. The cloned DNA segmentswere used for complementation of leu and ilv mutants in B.subtilis and, after subcloning in pBluescript, in E. coli. Theresults are summarized in Fig. 1. The E. coli nomenclature isused; leuB and leuC genes from E. coli correspond to leuCand leuB genes from B. subtilis, respectively, and the threeisoenzymes for acetolactate synthase and acetohydroxyacidsynthase activity, encoded by ilvBN, ilvIH, and ilvGM in E.coli, correspond to a single enzyme, encoded by ilvBN, in B.subtilis. Three genes (leuABC) were complemented in bothhosts, and three (leuD and ilvC in E. coli and ilvA in B.subtilis) were complemented in only one. One of the twogenes tested in one host only (ilvD) was complemented, andthe other (ilvB) was not. We have no explanation for thecomplementation pattern, which probably is due to the

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BRANCHED-CHAIN AMINO ACID GENES OF L. LACTIS 6581

TABLE 1. Strains and plasmids

Strain or plasmid Characteristics Source or reference

StrainsL. lactis subsp. lactis NCDO2118 Natural isolate National Collection of

Dairy OrganismsB. subtilisCU740 leuAS trpC2 (SPI) 63CU741 leuC7 trpC2 63CU315 leuDll7 trpC2 (SPO) 38IL2685 leuB6 trpC2 ilvA r- m- Cmr This workMT119 leuA80 trpC2 r- m- 57GSY184 ilvCl trpC2 4IL3151 ilvD4 leuB6 r- m+ This work1012 leuA8a metBS r- m+ 26GSY276 ilvD4 trpC2 4

E. coliCU518 leuA371 54CU514 leuB401 54CU520 leuCl71 54CU526 leuD101 54AB1255 tonA42 lacYl tsx-S supE44 gal-6 A- hisGl rpsL8 malAlxyl-7 mtl-2 39

ilvA201 metBl argHl thi-1FD1062 ara-14 ilvI614 ilvH612 X- glyA18 reL4l spoTI ilvB619 bglR20 34

rbs-S::TnS ilvG468(ilvG+) thi-1JP58 galK2 X- rpsL704 xyl-S mtl-l ilvC7 argE3 thi-1 7TG1 supE thi A(lac-proAB) hsdDS (F'+ traD36proAB lacIqZAM15) 18a

PlasmidspIL253 EmT, 4.9 kb 52pHV438 Hybrid between pBR322, Cm' gene of pC194, and thyB and X 42

segments of B. subtilis DNApIL371 9.2-kb Sau3A fragment of L. lactis chromosome in pIL253 This workpIL373 4.5-kb Sau3A fragment of L. lactis chromosome in pIL253 This workpIL374 8.5-kb Sau3A fragment of L. lactis chromosome in pIL253 This workpIL384 13-kb Sau3A fragment of L. lactis chromosome in pIL253 This workpIL389 7.5-kb Sau3A-SmaI left fragment from pIL384 in pBluescript This workpIL500 18.5-kb XbaI fragment of L. lactis chromosome in pIL253 This workpIL505 6.5-kb SmaI-Sau3A right fragment from pIL384 in pBluescript This workpIL533 2.5-kb Sau3A-exonuclease III left fragment from pIL384 This workpBluescript Apr, M13 on, pBR322 on Stratagene

a This mutation is in fact leuB6 (68).

presence of active promoters upstream of the tested genesand the interaction of the gene products with the hostproteins. The L. lactis subsp. lactis genes for histidinebiosynthesis also gave inconsistent complementation pat-terns in B. subtilis and E. coli (10). Nevertheless, we wereable to tentatively identify seven genes required for BCAAbiosynthesis.

Nucleotide sequence of the genes. The nucleotide sequenceof a 12,720-bp region has been determined (Fig. 2). Acomputer analysis performed according to Gribskov et al.(22) revealed 10 open reading frames (ORFs) larger than 200bp. Each ORF is preceded by a putative ribosome bindingsite, complementary to the 3' end of the L. lactis subsp.lactis 16S rRNA (11) (Fig. 2). All ORFs begin with ATGexcept for the second, which starts with a TTG codon. Inaddition, four 72-bp direct repeats are present between thesecond and third ORFs.Assignment of the ORFs. The proteins deduced from the 10

ORFs were compared with those in the GENPRO andNBRF protein data bases. Significant homologies werefound for 10 ORFs, 9 of which correspond to the BCAAbiosynthetic genes (Table 2). In addition to genes detectedby complementation (see above), ilvB (homologous to E. coliilvB, vi, and idvG) and ilvN (homologous to E. coli ilvH)genes, for which complementation data were not obtained,

were identified. Most of the L. lactis proteins are similar insize to their homologs. However, three exceptions wereobserved. IlvA and IlvC lactococcal proteins lack 73 and 147carboxy-terminal amino acids, respectively, and IlvD had a36-amino-acid gap compared with the E. coli proteins. Oneof the ORFs, designated ORF2, encodes a protein which hasno homology with BCAA biosynthetic enzymes but carriestwo boxes conserved within a superfamily of closely relatedATP-binding cassette (ABC) proteins (Fig. 3) (23).

Organization of the sequenced fragment is shown in Fig. 1.All of the genes except the first, upstream of leuA (ORF1),are transcribed in the same direction. leu and ilv genes areclustered, and the two clusters are separated by 121 bp. Theleu genes are spaced by less than 19 bp except for leuB andleuC, which are separated by four direct repeats of 72 bp.The distance between ilv genes ranges from 10 to 42 bpexcept for ilvB and ilvN, which have a 9-bp overlap.

Transcription signals. Sequences which conform to theconsensus for lactococcus promoters (11) were found up-stream of the leu and ilv gene clusters (pl and p2, Fig. 1).The region between pl and leuA strongly resembles regula-tory regions of amino acid biosynthetic operons controlledby attenuation (32). The transcript initiated at pl can fold intwo ways. One leads to the formation of a rho-independenttranscription terminator (Fig. 4A), whereas the other does

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6582 GODON ET AL.

BIbuA lhuB leuC leuD lIvD IhB IWOCi/vA

+ + + - + nd _ ++ + + - + nd

+ + _ + nd - -

- + + - + nd- - - - + nd

+ + + + nd _ _ _+ - - - nd _ _ _-- - ind - +

'_-p1p_ p2FIG. 1. Structure of the DNA region carrying the L. lactis BCAA genes. (A) Segments used for complementation experiments in B. subtilis

and E. coli. These segments are carried on the indicated plasmids, which were constructed as described in Table 1 and the text. (B) Resultsof complementation experiments with mutants listed in Table 1, indicating growth (+) or no growth (-) on media lacking the correspondingamino acids. (C) Organization of the BCAA region as deduced from sequence results. pl and p2, putative transcription promoters; tl, putativetranscription terminator. The open box between pl and tl represents the leader peptide.

TABLE 2. Conservation of proteins involved in BCAA biosynthesis between L. lactis subsp. lactis and various organisms

% Identical amino acidsaOrganism compared Reference(s)

LeuA LeuB LeuC LeuD IlvD IlvB IlvN IlvC IlvA

Escherichia coli b 46 42 42C <20d 34 36 17, 36, 64, 6543e 37f 5540g <20" 36

Salmonella typhimurium 41 49 - 47, 49Bacillus subtilis 53 - 50 2, 27B. coagulans 53 - - 50Thermus aquaticus 42 - 29Saccharomyces cerevisiae 25 46 50' 52i 41 33 38 1, 6, 14, 30, 45, 53Phycomyces blakesleeanus 50' 49i - 28Mucor circinelloides 49 48

a Calculated by Kanehisa software (67) as the ratio of perfect matches to the shorter protein length.b _, Sequence not available.Comparison with IlvB.

d Comparison with IlvN.Comparison with IlvI.

f Comparison with IlvH.g Comparison with IlvG.h Comparison with IlvM.i NH2 end of Leul.C end of Leul.

AB. subtils

pIL500pIL384ptL371 I

pIL374pIL373

E. coli

pIL389pIL533pIL505

C, ,t1

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TAAAACTCGATAATCTTGAGTCATAATTTCTCCTTAATCTTATTAGTACATTAGAATCCATTATAATTTAATCATTTTATGTCTACCTAAAGCAACAAAATTGCTTGTATATTTTCTAAC 120L V R Y D Q T M

4- orflAAGCTTAATTATGTGGATTTAATTGAATATTAAAGGGAGAAGTTGTAATCTATTTGTTGTTAAATTCTTGTTAATACAAATAMAATTTATTAAATATTATTATTTTATTGACAATTTAAAA 240

-35

TATTAAGAGTATTATAATGTAAATTAACAAAAAAAAGAGGAACTTGAAATGACATACACACAATTTTCATTGTTGTTGATCAAGGTGGACCTACATTAGCTTTTTTGGCTAAAATATGTG 360M T Y T Q F S L L L I K V D LH *

-10 leader p.ptlde-+___ _ ~ ~~~~~~~~- ~RES

GGTCCTGTTTGGCGAT MAGGAC ACGTCCTCGAGGGCGTCTTTTTTGTTTCTTAATAAAAAATAGAGGTAATATTATGCGAAAAATTGAATTCTTTGACACAAGTT 4803 4 M R K I E F F D T S

l1uA-*TGAGAGATGGCGAACAGACACCGGGCGTTAGTTTCTCCATTTCAGAAAAAGTAACGATTGCTAAACAACTGGAAAAATGGAGGATTTCTGTCATAGAGGCTGGTTTTTCTGCGGCAAGTC 600L R D G E Q T P G V S F S I S E K V T I A K Q L E K W R I S V I E A G F S A A S

CAGATAGTTTTGAAGCAGTAAAGCAAATTGCTGATTCTTTGAATGATACGGCTGTCACTGCATTAGCTCGCTGTGTTATTTCAGATATCGATAAAGCGGTTGAAGCGGTAAAGGGGGCTA 720P D S F E A V K Q I A D S L N D T A V T A L A R C V I S D I D K A V E A V K G A

AATATCCGCAAATTCATGTTTTCATTGCAACTTCACCTATTCACATGAAATATAAACTTAAAATCAGTCCCGAAGAAGTTTTGAAAAATATTGATAAGTGTGTGAGATACGCACGTGAAC 840K Y P Q I H V F I A T S P I H M K Y K L K I S P E E V L K N I D K C V R Y A R E

GGGTCGAGGTTGTTGAGTTTTCTCCAGAGGATGCAACAAGAACGGAGTTGAATTTTCTTTTAGAGGCTGTTCAAACGGCTGTCGATGCTGGAGCAACTTATATTAATATTCCTGACACTG 960R V E V V E F S P E D A T R T E L N F L L E A V Q T A V D A G A T Y I N I P D T

TCGGTTATACGACACCAGAAGAATATGGAAAAATTTTTAAATTTTTGATTGATAATACTAAGTCTGACCGAGAAATTATTTTTAGTCCACATTGTCATGATGATTTAGGAATGGCTGTAG 1080V G Y T T P E E Y G K I F K F L I D- N T K S D R E I I F S P H C H D D L G M A V

CTAATTCATTAGCTGCAATTAAAGCTGGGGCTGGGAGAGTTGAAGGAACTGTCAATGGTATTGGAGAGCGAGCTGGGAATGCTGCTCTTGAAGAAATTGCTGTGGCACTACATATTCGTA 1200A N S L A A I K A G A G R V E G T V N G I G E R A G N A A L E E I A V A L H I R

AAGATTTTTATCAGGCACAAAGTCCTTTAAAACTTTCAGAAACTGCTGCAACGGCAGAACTAATTTCACAATTTTCAGGAATTGCTATTCCAAAAAATAAAGCAATTGTTGGTGCTAATG 1320K D F Y Q A Q S P L K L S E T A A T A E L I S Q F S G I A I P K N K A I V G A N

CTTTTGCACACGAATCAGGAATTCATCAAGATGGTGTCCTTAAAAATGCTGAAACTTATGAAATTATTACACCAGAACTTGTCGGAATAAAGCATAATTCGTTGCCTTTAGGTAAACTTT 1440A F A H E S G I H Q D G V L K N A E T Y E I I T P E L V G I K H N S L P L G K L

CTGGTCGTCATGCTTTTAGTGAAAAATTGACGGAACTTAATATTGCTTATGACGATGAAAGTCTTGCAATTTTATTTGAAAAATTTAAAAAATTAGCTGACAAGAAAAAAGAAATTACTG 1560S G R H A F S E K L T E L N I A Y D D E S L A I L F E K F K K L A D K K K E I T

ACGCAGATATTCATGCCTTGTTTACAGGAGAAACGGTAAAAAATCTAGCTGGATTTATACTTGATAATGTTCAAATTGATGGGCACAAGGCATTGGTGCAACTAAAAAATCAAGAAGAGG 1680D A D I H A L F T G E T V K N L A G F I L D N V QI D G H K A L V Q L K N Q E E

AAATTTATGTTAGCCAAGGAGAGGGGTCAGGTTCAGTGGATGCAATTTTTAAAGCTATTGATAAAGTCTTTAATCATCAACTAAAATTAATTTCCTATTCAGTTGATGCTGTAACTGATG 1800E I Y V S Q G E G S G S V D A I F K A I D K V F N H Q L K L I S Y S V D A V T D

GAATTGATGCACAAGCAACGACTTTGGTTTCTGTTGAAAATCTATCTACAGGCACTATATTTAATGCTAAAGGTGTTGATTATGATGTATTGAAAGGAAGCGCCATTGCTTACATGAACG 1920G I D A Q A T T L V S V E N L S T G T I F N A K G V D Y D V L K G S A I A Y M N

CTAATGTTTTAGTTCAAAAAGAAAATTTACAAGGAAAGGTTGAACAAATTTCAGCTCATGATGGAATTTAAGGTGAAAAATATTGTCTAAAAAAATTGTGACACTTGCGGGAGATGGAAT 2040A N V L V Q K E N L Q G K V E Q I S A H D G I * L S K K I V T L A G D G I

TGGGCCAGAAATTATGTCAGCTGGTTTAAGTGTTTTAAAAGCTGTCAGTAAAAAAATTGATTTTGAGTATGAATTAGAAGCTAAAGATTTTGGAGGAATTGCAATTGATAAGCATGGTCA 2160G P E I M S A G L S V L K A V S KR I D F E Y E L E A K D F G G I A I D K H G H

TCCTTTACCAGAAGAAACTTTGCAAGCAGTTAAAAATGCTGACGCAATCTTGCTCGCTGCAATTGGTCATCCTAAATACAACAATGCAAAAGTTAGACCAGAACAAGGGCTACTTGCTTT 2280P L P E E T L Q A V K N A D A I L L A A I G H P K Y N N A K V R P E Q G L L A L

ACGAAAAGAATTAGGACTGTATGCTAATGTTCGTCCATTAAAAATTTATCCGGCTCTAAAAAAACTTTCTCCCATACGAAATGTTGAAAATGTTGATTTCCTAGTGATTCGCGAACTTAC 2400R K E L G L Y A N V R P L K I Y P A L K K L S P I R N V E N V D F L V I R E L T

AGGGGGAATCTATTTCGGTCAGCATGAATTGGCAGATGATAAAGCACGAGATGTCAATGATTATTCTGCTGATGAAATAAGGAGAATTCTTCATTTTGCTTTCAAAAGTGCTCAAAGTCG 2520G G I Y F G Q H E L A D D K A R D V N D Y S A D E I R R I L H F A F K S A Q S R

GCCCAGAAAATTACTGACTTCGGTTGATAAACAAAATGTTCTTGCAACTTCTAAATTATGGCGAAAAATGGCTGATGAAATTGCTGACGAATATCCTGATGTACGATTAGAGCACCAATT 2640P R K L L T S V D K Q N V L A T S K L W R K M A D E I A D E Y P D V R L E H Q L

GGTCGATTCTTGTGCGATGTTACTGATTACTAATCCGCAACAATTTGATGTGATAGTCACTGAAAATCTATTTGGTGATATTCTCTCTGATGAAGCAAGTAGTTTGGCCGGTAGCTTAGG 2760V D S C A M L L I T N P Q Q F D V I V T E N L F G D I L S D E A S S L A G S L G

AGTGATGCCTTCGAGTTCGCATGGATTTAACGGTTTAGCACTCTATGAGCCAATTCATGGTTCGGCACCAGATATTGCAGGAAAAGGAATTGCGAACCCTGTTTCGATGATTCTATCAAT 2880V M P S S S H G F N G L A L Y E P I H G S A P D I A G K G I A N P V S M I L S I

TGCCATGATGCTAAGAGAATCTTTTGGGCAAGAAGATGGGGCTGCGATGATTGAAAAAGCCGTAACCCAAACTTTTACTGACGGAATTTTGACTAAAGATTTAGGTGGGACTGCAACAAC 3000A M M L R E S F G Q E D G A A M I E K A V T Q T F T D G I L T K D L G G T A T T

TAAAGAAATGACAGAAGCAATCCTGAAAAATZgGTAAAAATGCGATTGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACCGTCAGCATAG@m9cAATTC3GTcaaTAAATGCGAT 3120K E M T E A I L K N C Q *

TGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACCGTCAGCAT G5ACIAATTC3GTCATAATTGCGATTGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACCGTCAGCATAG= 3240

__------------>--------------- - - - - - - - - - - ->

gkW,jATTC3:GTcaGTAATTGCGATTGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACTATCAGCGTAAk3AnCAATT mscGAAATATTA GL^AATACAAAATTA=UG 3360

aaGAATTTGTCAGAATAAATTTTTAAAAAAGGAAATAAAAAAATGTCAGGTAAAACAATATTTGATAAACTTTGGGATCAGCATGTGATTGCTGGAAATGAGGGAGAACCTCAACTGCTT 3480M S G K T I F D K L W D Q H V I A G N E G E P Q L Ll-uC-+

TATATTGACCTTCATGTTATTCATGAGGTTACGAGTCCGCAAGCATTTCAGGGCTTACGTGAAGCAGGACGTCGTGTTCGGAGAAAAGATTTGACATACGGAACTCTTGACCACAATGTT 3600Y I D L H V I H E V T S P Q A F Q G L R E A G R R V R R K D L T Y G T L D H N V

CCAACACAAAATATTTTTAATATTCAAGATTTGATTTCTAAAAAACAAATTGATACTTTTACTAAAAATGTCAAAGAATTTGATGTTCCAGCGGAGACTCATGGTGGAAAAGGACAAGGA 3720P T Q N I F N I Q D L I S K K Q I D T F T K N V K E F D V P A E T H G G K G Q G

ATTGTTCACATGGTAGCACCTGAATCTGGCAGAACTCAACCGGGAAAAACAATTGTTTGTGGCGATAGTCATACCGCAACAAATGGAGCATTTGGTGCAATTGCTTTTGGAATTGGTACA 3840I V H M V A P E S G R T Q P G K T I V C G D S H T A T N G A F G A I A F G I G T

AGTGAAGTTGAACATGTTCTTGCAACTCAAACCATTTGGCAAGTTAAACCCAAGCGTATGAAAATTGAATTTCAAGGTCATCCACAAAAAGGAATTTATAGCAAAGACTTTATCCTCGCA 3960S E V E H V L A T Q T I W Q V K P K R M K I E F Q G H P Q K G I Y S K D F I L A

TTAATTGCTAAATATGGTGTGGATGCAGGTGTAGGTTATGCGGTTGAATATAGTGGGGATGCTATCAGTGATTTAAGCATGGAAGAACGGATGACAATCTGTAACATGTCAATTGAATTT 4080L I A K Y G V D A G V G Y A V E Y S G D A I S D L S M E E R M T I C N M S I E F

GGGGCAAAAATTGGCCTGATGAATCCTGATGAAAAAACTTATGACTATGTCAAAGGGCGTGAACATGCACCTAAAAACTTTGATGAAGCTGTCAGTAAATGGGAAAAACTTGTCAGTGAT 4200G A K I G L M N P D E K T Y D Y V K G R E H A P K N F D E A V S K W E K L V S D

TCTGATGCACAATACGATAAGATTTTAAGTCTTGATGTCAGCCAGTTGAAACCAATGGTGACATGGGGAACAAATCCCGGAATGGGCCTAGAATTTGGCGAAAAGTTTCCGGAAATTAAC 4320S D A Q Y D K I L S L D V S Q L K P M V T W G T N P G M G L E F G E K F P E I N

AATGATTTGAATTATGAACGTGCTTATCAGTACATGGATTTAAAGCCAGGCCAAACCGCTTCTGACATAGATTTAGGCTATATTTTCATTGGTTCTTGTACGAATGCTAGACTTGGTGAT 4440N D L N Y E R A Y Q Y M D L K P G Q T A S D I D L G Y I F I G S C T N A R L G DFIG. 2. Nucleotide and deduced amino acid sequences of the L. lactis NCD02118 BCAA region. Numbers at the right refer to nucleotides.

Gene names are indicated at the beginning of each amino acid sequence. Translational stop codons (*) and putative ribosome binding sites(RBS) are indicated. The -10 and -35 boxes of the putative promoters are underlined. Inverted repeats which might form the preemptor(numbered 1 and 2) and terniinator (numbered 5 and 6) and those which might form the antiterminator (numbered 3 and 4) are indicated (seeFig. 4). Long dashed arrows indicate the 72-bp direct repeats; bold characters show the 6-bp inverted repeats contained within them.

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TTAGAAGAAGCTGCAAAAATTATTGGAGACAGACATATTGCTGATGGACTGACAGGAATTGTCGTCCCTGGAAGCAGACCTGTGAAAGAAGCGGCTGAAGCACAAGGGCTTGATAAAATTL E E A A K I I G D R H I A D G L T G I V V P G S R P V K E A A E A Q G L D K I

TTTAAAGAAGCTGGTTTTGAATGGCGGGAACCGGGTTGCTCAGCCTGTCTTGGAATGAATCCTGACCAAATTCCAGAATACGTTCATTGTGCTTCAACCTCTAATCGAAATTTTGAAGGTF K E A G F E N R E P G C S A C L G M N P D Q I P E Y V H C A S T S N R N F E G

,RBSCGTCAAGGACATAATGCAAGAACGCACCTGTGCTCTCCAGCTATGGCTGCTGCCGCCGCAATCGCTGGTAAATTTGTAGATGTTAGAATGCTCGTAACAGATTAGTCTGTAGAAAGAAAAR Q G H N A R T H V C S P A M A A A A A I A G K F V D V R M L V T D *

AAAGATGGAAAAATTCACGATTTACAAAGGGACAAGTGTTCCAGTCATGAACGATAATATTGACACAGACCAAATTATTCCTAAACAATTTTTGAAAGCAATCGATAAAAAGGGCTTTGGM E K F T I Y K G T S V P V M N D N I D T D Q I I P K Q F L K A I D K K G F Gl-uD-+

GAAAAATTTATTTTATGAATGGCGTTATCTTAAAGATTACGATGAGAATCCTGATTTTATTTTGAATGCTCCAAAATACAAAAAAGCTTCTCTGTTAATTTCAGGAGATAATTTTGGTTCK N L F Y E W R Y L K D Y D E N P D F I L N A P K Y K K A S L L I S G D N F G S

GGGTTCTTCAAGAGAACATGCGGCATGGGCCTTATCAGATTACGGCTTTCGGGCAATTATTGCTGGCTCTTACTCAGATATTTTTTATAATAATGCTTTAAAAAATGGCTTGTTACCAATG S S R E H A A W A L S D Y G F R A I I A G S Y S D I F Y N N A L K N G L L P I

TAAACAACCAAGAGAAGTTCTAAATCAACTGACAAAACTGTCAAGTCAAGAAGAAATTACAATTGATTTACCCCATCAGCTAATCATCACAAGCCTTGGTGACTTTCATTTTGAGATTGAK Q P R E V L N Q L T K L S S Q E E I T I D L P H Q L I I T S L G D F H F E I D

CCCCATTTGGAAAGACAAATTAATTAATGGCTTAGATGATATTGGAATAACTTTGCAATATGAAGAAGCAATCTCAGCTTACGAACAAAAAAATCAATAAGAGCGAGCCTAAAATGACAAP I N K D K L I N G L D D I G I T L Q Y E E A I S A Y E Q KNQ * M T

o.r22TTATTAATTTAAAGAATGTAAATCTTACTCGAAATAAAAAAGAAATTCTTAAAGATATTACTTGGAAAGTAAATCCCGGCGAAAATTGGGTTATTCTGGGCCTCAACGGCTCTGGAAAATI I N L K N V N L T R N K K E I L K D I T W K V N P G E N W V I L G L N G S G K

CAAGTCTTTTGAAATTGATTTTAGCAGAAGAATGGAAAACTTCTGGTGAAATCACTGTTTTAAATACTCAATTTAGAAATGGAGAAATTCCTAAGTTGAGAAAAAGAATCAGCGTAGTTGS S L L K L I L A E E W K T S G E I T V L N T Q F R N G E I P K L R K R I S V V

GCTCATTTATTGCTGAAAGATTTCAACCAAATATTAAGGCTGAAAACCTTGTTTATACTGGGAAATTTAATTCGAGCATGCTCTATAAACCCTACACAGATCAGGAACTTGATGAGGCCCG S F I A E R F Q P N I K A E N L V Y T G K F N S S M L Y K P Y T D Q E L D E A

GTCAGCTTTTAAGACAAATGGGCGCAAAATCACTTATTGGCCGAAATTATGCCAGCCTTTCTCAAGGGGAAAAGCAAGTTCTTCTTATTGCTAGGAGCTTAATTTTAAAGCCTGAGCTTTR Q L L R Q M G A K S L I G R N Y A S L S Q G E K Q V L L I A R S L I L K P E L

TAATTTTGGACGAAGCAACGAACGGTTTAGATTTATTTGCTAAAGAAAAATTATTAAAGCAACTGCAGCAGATTAATCAATTAAAAACCGCACCAACACTAATTTATATTTCTCATCATCL I L D E A T N G L D L F A K E K L L K Q L Q Q I N Q L K T A P T L I Y I S H H

CCGATGAAATCACTGATATTTTTACTCACCTTTTACTTTTAAGAGAAGGAAAAGTGATTCAATCAGGGAAAAAAGAAAACTTATTAAATGAAAAGATACTTACTGATTTTTATCAAGAAAP D E I T D I F T H L L L R E G K V I Q S G R K E N L L N E K I L T D F Y Q E

AAGTAGAAGTTCACCGTTTTGAGCAGAAATATTTTGTAATTCCTGCTAACTGAGAAAGGAAAGCAAAAGTATTTTATATACTATATAGAATATTCTGACAGATTATTGTATTTTCATTTTK V E V H R F E Q K Y F V I P A N*------

RBS -35TTTAGTGATAAAATAGCTCTATGTAAATTTACGGGGAGGTCAAAAAGATAACATATGGAATTCAAATATAACGGAAAAGTTGAATCAGTGGAACTCAATAAATATTCTAAGACATTGACT

M E F K Y N G K V E S V E L N K Y S K T L T-10 Ilv<-+

CCAAGATCAACACAACCAGCGACTCAAGCGATGTACTACGGCATTGGTTTTAAAGATGAGGATTTCAAAAAAGCTCAGGTCGGAATCGTCAGCATGGATTGGGACGGAAATCCATGTAATP R S T Q P A T Q A M Y Y G I G F K D E D F K A Q V G I V S M D W D G N P C N

ATGCACTTGGGAACACTTGGGAGTAAAATCAAAAGTTCTGTCAACCAAACTGACGGATTGATTGGACTTCAATTTCATACTATTGGAGTTTCTGATGGAATTGCTAACGGAAAGCTTGGCM H L G T L G S S V N Q T D G L I G L Q F H T I G V S D G I A N G K L G

ATGAGATATTTCACTGTTCAGTGAAGTTATTGCTGACAGCATCGATTTCAACGCTGGCGCAGAATATTATGATGCCATCGTTGCCATTCCCGGTTGTGATAAAAATATGCCCGGGTCAM R Y S L V S R E V I A D S I E T N A G A E Y Y D A I V A I P G C L K N M P G S

ATTATCGGAATGGCTCGCTTAAATCGTCCGTCAATTATGGTCTATGGTGGAACGATTGAACATGGCGAATATAAAGGTGAAAAATTAAATATTGTTTCGGCCTTTGAAGCTCTGGGGCAAI I GK A R L N R P S I M V Y G G T I E H G E Y K G E K L N I V S A F E A L GT

AAAATCACTGGAAATATTTCTGATGAAGATTATCATGGCGTTATTTGCAATGCCATTCCAGGACAAGGTGCTTGCGGAGGAATGTACACTGCCAATACCCTGGCTGCTGCTATTGAAACTK I T G N I S D E D Y E G V I C N A I P G Q G A C G G M Y T A N T L A A A I E T

TTGGGAATGAGTTTACCTTATTCCTCTTCCAATCCAGCAGTTCAAGAAAAACAAGAAGAGTGTGATGACATTGGTTTAGCCATCAAAAATTTATTAGAAAAAGATATTAAACCAAGTL G M S L P Y S S S N P A V S Q E K Q E E C D D I G L A I K N L E K D I K P S

GATATCATGACCAAAGAAGCTTTTGAAAATGCCATAACAATTGTCATGGTCCTTGGAGGCTCAACCAATGCTGTGCTTCATATCATTGCAATGGCAAATGCCATTGGTGTAGAAATTACGD I M T K E A F E N A I T N A V L H I I A M A N A I G V E I T

CAAGATGATTTCCAACGTATTTCAGATATTATCCCTGTTCTTGGCGATTTCAAACCGAGCGGAAAATATATGATGGAAGATCTGCACAAAATTGGTGGCCTTCCTGCTGTTTTGAAATACQ D D F KR I S D I P V L G D F K P S G K Y M M E D L H K I G G L P A V L K Y

CTACTTAAAGAAGGAAAACTTCACGGTGATTGTTTGACCGTCACAGGTAAAACTTTGGCTGAAAATGTTGAAACAGCATTAGATTTGGACTTTGACAGTCAAGATATTATGCGACCACTAL L V E G K L H G D C L T V T G K T L A E N V E T A L D L D F D S Q D I M R P L

AAAAATCCAATTAAAGCTACTGGACATTTACAAATTTTGTACGGTAATCTTGCCCAAGGGGGTTCTGTTGCAAAAATTTCTGGTAAAGAAGGCGAATTTTTCAAAGGAACAGCTCGTGTTK N P I K A T G H L Q I L Y G N L A Q G G S V AG I S G K E G E F F K G T A R V

TTTGACGGAGAACAACACTTTATCGATGGCATTGAGTCTGGCCGATTGCATGCCGGTGATGTTGCGGTCATTAGAAATATTGGCCCAGTCGGAGGTCCGGGAATGCCAGAGATGTTAAAAF D G E Q H F I D G I E S G R L H A G D V A V I R N I G P V G G P G M P E M L K

CCAACCTCAGCATTAATTGGAGCAGGACTTGGAAAATCTTGTGCCCTAATTACTGACGGAAGATTTTCTGGTGGCACACACGGCTTTGTTGTGGGTCATATCGTCCCTGAAGCAGTTGAAP T S A L I G A G L G K S C A L I T D G R F S G G T H G F V V G H I V P E A V E

GGTGGGTTGATTGGTTTAGTTGAAGATGATGATATTATCGAAATTGATGCGGTGAATAATAGTATTAGTTTAAAAGTTTCTAATGAAGAAATTGCTAAACGACGTGCCAATTATCAAAAAG G L I G L V E D D D I I E I D A V N N S I D L IVK DA K R R A N Y Q K

CCAACCCCTAAAGCAACGCGTGGTGTTCTTGCAAAATTTGCCAAACTTACGCGCCCCGCTAGTGAAGGTTGCGTTACAGATTTATAGAAAGGTTTGAATGAAAAAAATAAAGTTAGAAAAP T P K A T R G V L A K F A K L T R P A S E G C V T D L *M K K I K L E K

ACCTACTTCCGGTTCCCAACTTGTTCTCCAAACCTTAAAAGAACTTGGAGTAGAAATTATTTTTGGTTATCCTGGTGGGGCCATGCTCCCCTTGTATGATGCGATTCATAATTTTGAAGGP T S G S Q L V L Q T L K E L G V E I I F G Y P G G A M L P L Y D A I H N F E G

AATTCAACATATTTTAGCCCGTCATGAGCAAGGAGCAACGCATGAAGCCGAAGGTTACGCTAAATCGTCTGGTAAAGTTGGTGTCGTCGTTGTTACGTCAGGACCAGGAGCGACTAATGCI Q H I L A R H E Q G A T H E A E G Y A KS S G K V G V V V V T S G P G A T N A

AGTAACCGGAATTGCTGACGCTTATCTTGATTCAGTCCCATTGTTAGTTTTCACAGGTCAAGTTGGCCGTCAGTCAATTGGTAAAGATGCTTTTCAAGAAGCAGATACTGTTGGAATTACV T G I A D A Y L D S V P L L V F T G QV G R Q S I G K D A F Q E A D T V G I T

AGCCCCAATTACAAAATATAATTATCAAATTAGGGAAACCGCAGATATTCCAAGAATTGTTACAGAAGCCTATTATTTGGCAAGGACAGGACGTCCTGGCAGTAGAAATTGATTTACCA P I T K Y N Y Q I R E T A D I P R I V T E A Y Y L A R T G R P G P V E I D L P

AAAAGATGTTTCCACCCTTGAAGTCACTGAAATTAATGACCCAAGCTTGAATCTTCCTCATTATCACGAAAGTGAAAAAGCGACTGATGAACAATTGCAAGAATTACTGACAGAACTTTCK D V S T L E V T E I N D P S L N L P H Y H E S E K A T D E Q L Q E L L T E L S

TGTCAGTAAAAAACCAGTCATTATTGCTGGCGGAGGAATTAATTATTCTGGCTCAGTTGATATTTTCAGAGCATTTGTCGAAAAATATCAAATTCCAGTTGTTTCTACATTGCTTGGCTTV S K K P V I I A G G G I N Y S G S V D I F R A F V E K Y Q I P V V S T L L G L

AGGAACATTACCAATCAGCCACGAATTGCAACTAGGAATGGCAGGAATGCACGGTTCATACGCTGCAAATATGGCTTTAGTTGAAGCTGACTATATTATTAATTTGGGATCACGTTTTGAG T L P I S H E L Q L G M A G M H G S Y A A N M A L V E A D Y I I N L G S R F D

CGATAGAGTTGTATCCAATCCTGCAAAATTTGCTAAAAATGCTGTCGTTGCTCATATTGATATTGACGCTGCTGAACTTGGCAAAATTGTAAAAACCGATATTCCAATCCTTTCTGATTTD R V V S N P A K F A K N A V V A H I D I D A A E L G K I V K T D I P I L S D L

FIG 2Continued.

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VOL. 174, 1992 BRANCHED-CHAIN AMINO ACID GENES OF L. LACTIS 6585

GA,AAGCGGCTTTAAGCAGACTTTTGCAATTAAATAAGGTCAGGACTGACTTTAATGATTGGATTAAAACTGTCATTGAAAATAAAGAGAAAGCACCATTTACTTATGAGCCCCAAAACCA 9120K A A L S R L L Q L N K V R T D F N D W I K T V I E N K E K A P F T Y E P Q N H

TGATATCCGTCCACAGGAAACAATTAAATTAATTGGAGAATACACTCAAGGAGATGCAATCATTGTAACTGACGTTGGGCAACATCAAATGTGGGTGGCGCAATATTATCCTTATAAAAA 9240D I R P Q E T I K L I G E Y T Q G D A I I V T D V G Q H Q M W V A Q Y Y P Y K N

TGCAAGGCAACTTATTACTTCTG,GGGGAATGGGAACGATGGGCTTTGGCATTCCTGCAGCAATCGGTGCAAAGCTGGCACAGCCAAATAAAAATGTCATTGTTTTTGTTGGCGATGGTGG 9360A R Q L I T S G G M G T M G F G I P A A I G A K L A Q P N K N V I V F V G D G G

CTTTCAAATGACTAATCAAGAATTAGCATTACTTAATGGCTACGGTATTGCAATCAAAGTTGTGCTGATTAATAATCATTCATTGGGAATGGTACGTCAATGGCAAGAATCATTCTATGA 9480F Q M T N Q E L A L L N G Y G I A I K V V L I N N H S L G M V R Q W Q E S F Y E

AGAGCGACGTTCACAATCGGTTTTTGATGTTGAACCCAATTTTCAATTGTTAGCCGAAGCTTATGGCATCAAACATGTTAAGTTAGATAATCCAAAAACTTTGGCTGATGATTTAAAMAT 9600E R R S Q S V F D V E P N F Q L L A E A Y G I K H V K L D N P K T L A D D L K I

TATTACAGAAGATGAGCCAATGCTTATTGAAGTTCTAATTTCAAAATCTGAGCATGTTTTACCAATGATACCAGCTGGATTACACAATGACGAAATGATTGGACTTCATTTTACTGATAA 9720I T E D E P M L I E V L I S K S E H V L P M I P A G L H N D E M I G L H F T D K

RisGAATGAGGAGATAGATAATGCGTAGAATGATTATCGCAAAACTTCATAACGTGACAGGAATTATGAATCGATTTACCGCCGTTCTCAATCGAAGGCAAGTGAACATTCTCTCAATTACCG 9840N E E I D N A *

M R R M I I A K L H N V T G I M N R F T A V L N R R Q V N I L S I T

CTGGAGTTACAGAAAGTCAAGACTTAACTCATACCACTTTTGTTATTGAAGTTGATCATCTTGATGAAGTAGAACAAATCATTAAMCAATTAAATCGCTTAATAGATGTAATTGAAGTAG 9960A G V T E S Q D L T H T T F V I E V D H L D E V E Q I I K Q L N R L I D V I E V

CTGATATTACTGATTTTCCTCATGTAGAACGTGAAGTCGTCTTGATTAAAGTATCAGCTCCACCGACCATTAGGGCAGAAATTTTTACAATGATTGAACCTTTTAGAGTAAATGTAGTTG 10080A D I T D F P H V E R E V V L I K V S A P P T I R A E I F T M I E P F R V N V V

ATGTCAATCTGGAAAATGTCACCATTCAATTAACGGGTGATTCAGCAAAAATCGAAGCACTTATTGAGGTTGTTAGTCCTTATGGCATTCTAAATATGGCTCGGACAGGTAGTGCAGGTT 10200D V N L E N V T I Q L T G D S AK I E A L I E V V S P Y G I L N M A R T G S A G

TTGAGCGTGGCTAAATTTAAATAAGTTAACAAATAAATAGAAAAsATAGAGGAAACAAAAATGGCAGTTACAATGTATTATGAAGATGATGTAGAAGTATCAGCACTTGCTGGAAAGCAAA 10320F E R G * M A V T M Y Y E D D V E V S A L A G KQ

TTGCAGTAATCGGTTATGGTTCACAAGGACATGCTCACGCACAGAATTTGCGTGATTCTGGTCACAACGTTATCATTGGTGTGCGCCACGGAAAATCTTTTGATAAAGCAAAAGAAGATG 10440I A V I G Y G S Q G H A H A Q N L R D S G H N V I I G V R H G K S F D K A K E D

GCTTTGAAACATTTGAAGTAGGAGAAGCAGTAGCTAAAGCTGATGTTATTATGGTTTTGGCACCAGATGAACTTCAACAATCCATTTATGAAGAGGACATCAAACCAAACTTGAAAGCAG 10560G F E T F E V G E A V A K A D V I M V L A P D E L Q Q S I Y E E D I K P N L K A

GTTCAGCACTTGGTTTTGCTCACGGATTTAATATCCATTTTGGCTATATTAAAGTACCAGAAGACGTTGACGTCTTTATGGTTGCGCCTAAGGCTCCAGGTCACCTTGTCCGTCGGACTT 10680G S A L G F A H G F N I H F G Y I K V P E D V D V F M V A P K A P G H L V R R T

ATACTGAAGGTTTTGGTACACCAGCTTTGTTTGTTTCACACCAAAATGCAAGTGGTCATGCGCGTGAAATCGCAATGGATTGGGCCAAAGGAATTGGTTGTGCTCGAGTGGGAATTATTG 10800Y T E G F G T P A L F V S H Q N A S G H A R E I A M D W A K G I G C A R V G I I

AAACAACTTTTAAAGAAGAAACAGAAGAAGATTTGTTTGGAGAACAAGCTGTTCTATGTGGAGGTTTGACAGCACTTGTTGAAGCCGGTTTTGAAACACTGACAGAAGCTGGATACGCTG 10920E TT FK Z E T E E D L F G E Q A V L C G G L T A L V E A G F E T L T E A G Y A

GCGAATTGGCTTACTTTGAAGTTTTGCACGAAATGAAATTGATTGTTGACCTCATGTATGAAGGTGGTTTTACTAAAATGCGTCAATCCATCTCAAATACTGCTGAGTTTGGCGATTATG 11040G E L A Y F E V L H E M K L I V D L M Y E G G F T K M R Q S I S N T A E F G D Y

TGACTGGTCCACGGATTATTACTGACGAAGTTAAAAAGAATATGAAGCTTGTTTTGGCTGATATTCAATCTGAATTTGCTCAAGATTTCGTTGATGACTTCAAAGCGGGGCGTCCAA 11160V T G P R I I T D E V K K N M K L V L A D I Q S G K F A Q D F V D D F K A G R P

AATTAATAGCCTATCGCGAAGCTGCAAAATCTTGAAATTGAAAAAAsTTGGGGAGAGCACGTCAAGCAATGCCATTCACACAATCTGGTGATGACGATGCCTTTAAAATCTATCAGTA 11280K L I A Y R E A A K N L E I E K I G A E H V K Q C H S H N L V M T M P L K S I S

ATTTCTCTTATTGATTGAACAAAAACATAAAAGCATTTTATGGAGGAATGACATAAATGATAAGTGCCAAAGAGGTTGAAGATGCCTATGATTTGTTAAAAGCAGTTGTCACTAAAACAC 11400N F S Y M I S A K E V E D A Y D L L K A V V T K T

IlvA-+CTTTACAATTAGACCCTTACCTTTCCAATAAATATCAAGCAAATATTTACTTAAAAGAAGTTGTCACTAAAACACCTTTACAATTAGACCCTTACCTTTCCAATAAATATCAAGCAAATA 11520P L Q L D P Y L S N K Y Q A N I Y L K E V V T K T P L Q L D P Y L S N K Y Q A N

TTTACTTAAAAGAAGAAAACTTACAGAAAGTTCGTTCTTTTAAATTACGAGGAGCTTATTATTCTATCAGTAAATTATCTGATGAGCAACGCTCTAAAGGAGTGGTTTGTGCCTCAGCAG 11640I Y L K E E N L Q K V R S F K L R G A Y Y S I S K L S D E Q R S K G V V C A S A

GAAATCATGCACAAGCGGG.-TTGCTTTTGCTGCAAATCAATTAAATATTTCTGCGACAATTTTTATGCCCGTTACCACACCTAACCAAAAAATTTCACAAGTTAAATTTTTTGGCGAAAGTC 11760G N H A Q G V A F A A N Q L N I S A T I F M P V T T P N Q K I S Q V K F F G E S

ACGTAACAATTCGTTTAATTGGTGATACTTTTGATGAATCAGCCAGAGCAGCALAAAGCTTTTTCTCAAGATAATGACAAACCATTTATAGACCCTTTTGATGATGAAAATGTAATTGCTG 11880H V T I R L I G D T F D E S A R A A K A F S Q D N D K P F I D P F D D E N V I A

GTCAAGGGACAGTGGCTTTAGAAATTTTTGCGCAAGCTAAAAAACAAGGAATAAGTTTAGATAAGATTTTTGTACAGATTGGTGGAGGTGGTTTAATTGCAGGAATTACTGCCTACAGTA 12000G Q G T V A L E I F A Q A K K Q G I S L D K I F V Q I G G G G L I A G I T A Y S

AGGAGCGCTATCCCCAAACTGAAATTATCGGAGTTGAAGCAAAAGGGGCA1ACAAGTATGAAAGCTGCCTACTCTGCTGGTCAGCCCGTCACCTTGGAACACATTGATAAATTTGCTGACG12120K E R Y P Q T E I I G V E A K G A T S M K A A Y S A G Q P V T L E H I D K F A D

GAATTGCGGTTGCGACTGTCGGTCAGAAAACTTACCAACTTATTAATGACAAAGTGAAACAATTGCTTGCGGTTGATGAAGGTTTAATTTCTCAAACCATACTCGAATTGTATTCAAAAT 12240G I A V A T V G Q K T Y Q L I N D K V K Q L L A V D E G L I S Q T I L E L Y S K

TAGGAATTGTCGCCGAGCCAGCAGGTGCAACATCTGTTGCCGCACTTGAACTTATTAAAGATGAAATCAAGGGTAAAAATATCGTCTGTATCATCAGCGGCGGAAATAATGATATTAGTC 12360L G I V A E P A G A T S V A A L E L I K D E I K G K N I V C I I S G G N N D I S

GAATGCAAGAAATTGAAGAAAGAGCTTTGGTTTATGAAGGTCTAAAACATTATTTTGTCATTAACTTTCCTCAAAGACCAGGATCCTTACGAACTTTTGTCAGTGATATTTTAGGGCCAA 12480R M Q E I E E R A L V Y E G L K H Y F V I N F P Q R P G S L R T F V S D I L G P

ATGATGATATCACCCGATTTGAGTACATCAAAAGGGCTGATAAAGGTAAAGGACCTTGTCTTGTTGGGATTTTACTTTCAGATGCTAGTGATTATGATTCATTGATTAATCGGATTGAAA 12600N D D I T R F E Y I K R A D K G K G P C L V G I L L S D A S D Y D S L I N R I E

GATTTGATAATCGTTATGTTAACTTACGTGGAAATGATAGTTTATACGAACTTTTGGTCTAACTAACCAATTGGTTTGAGCCATTTTCTAGTTTCAATTCTCTTTAAATCACTAGAAATT 12720R F D N R Y V N L R G N D S L Y E L L V *

FIG. 2-Continued.

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PROTEIN RESIDUE

ORF2Nodl1MalleGlnQ3ProV4HlyB5CyaB6

ORF2NodlMalKGlnQProVHlyBCyaB

ORF2NodlMalKGlnQProV

3627181643

484487

1151039594

124561564

193181169172200

CONSERVED SEQUENCE

o-w b- L vj.nLz w--\-n%m j-ruvm-vvzwjAr wra yc

VVNDLSFTIAAQZCFGLLQPNGAGQ8BITRMILGMTSPSVQKITV----LGAQEPGQVRLARAKIGIVSQFDNLDL--EFTVVSKDINLDIHEGZFVVFVQPSGCCKBTLLRNIAGLETITSGDLFIGEKRMNDTPPAE------R-GVGNVFQSYALYPHLSVVLHNIDLNIAQCEVVVIIGP8G8GKSTLLRCINKLEEITSGDLIVDGLKVND-PKVDERLIRQE-A-GMVtQQFYLFPHLTAGVKDASLAIEEGZIFVIMGLBG8GKBTMVRLLNRLIEPTRGQVLIDGVDIAKISDAELREVRRK-KIAMVWQSFALMPHMTVILDNINLSIKQGZVIGIVGRSGS8kBTLTKLIQRFYIPENGQVLIDGHDLALADPNWLR--RQ---VGVVLQDNVLLNRSIIALRNVSLRIAPGEVVGVVCRS fGlPSSLTRLXRMFVADRGRVLIDGHDIGTVDSASLR--RO--- 'vvLORSTY.RSUR

* *** * *

NB1

ENLVYTGKFNSSMLYKPYTDQELDEARQLLRQM--GAKSLIGRNYASLBQGEKQVLLIARSLILKPELLILDIATNGLDLFARENLLV--YGRYFRMSTR--EIETVIPSLLEFA--RLESKANTRVADLSOGMKRRLTLAGALINDPQLLILDZPTTGWPHAAENM-S--FG-LKPAGAKKEVINQRVNQVAEVL--QLAHLLDRKPKAISCCQRQRVAIGRTLVAEPSVFLLDZPL8NLDAALLENV-M--FGPRVRG ANKEE R V--GAE EM VAbVK MFPT A LLDNT-A--FG-MELAGINAEERREKALDALRV--GLENYAHSYPDEISGGMRQRVGLIRULAINPDILLMDIAFBALDPLI

DNIALT--RPGASMHEVVAAARLAGAHEFICQLPEQYDTMLGENGVGLSGGQRQRIGIARALIHRPRVLILDIAT8ALDYES.*..*. * *....**. ..**

NB2

KEKLLKQLQQINQLKTAPTLIYISRHPDVITDIFTHLLLLREGKVIQSGKKENLLNEKILTDFYQRHLIWERLRSLLA-R-GKTILLTTRIMEEAERLCDRLCVLEAGRKIAEGRPHALIEEQIGCPVIERVQMRIEISRLHK-RLGRTMIYVTEDQVEAMTLADKIVVLDAGRVAQVGKPLAV-PLSGRPFCRRRHEVLKVMQDL-A-EEGMTMVIVTEEIGFAEKVASRLIFIDKGRIAEDGNPQVLIKNPPSQRLQERTEMQDELVKLQA-KHQRTIVFISEDLDEAMRIGDRIAIMQNGEVVQVGTPDEILNNPANDYVRT

(259)(237)(228)(240)(400)

HlyB 641 EHVIMRNMKICK---GRTVIIIAERLS-TVKNADRIIVMEKGKIVEQGKHKELLSEPESLYSYL (707)CyaB 644 EHIIQRNMDICD---GRTVIIIAHRLS-AVRCADRIVVMEGGEVAECGSHETLLAAGG-LYARL (712)

** * *. ..-* * .-.- * *

FIG. 3. Alignment of six ABC proteins with L. lactis ORF2. Software of Needleman and Wunsch was used (40). Nucleotide bindingdomains (23) are indicated by NB1 and NB2; a short sequence shared by all members of the ATP-dependent transport family is highlighted(#). Other notation: *, same amino acid in all proteins; ., conservative substitutions; bold characters, same amino acid in at least five proteins.Numbers at the left correspond to amino acid positions; numbers in parentheses indicate protein size in amino acids. The functions of thevarious proteins are as follows: 1, nodulation in Rhizobium leguminosarum (13); 2, maltose transport in E. coli (19); 3, glutamine transport inE. coli (43); 4, glycine, betaine, and proline transport in E. coli (21); 5, hemolysin secretion in E. coli (15); 6, cyclolysin secretion in Bordetellapetussis (20).

not (Fig. 4B). In addition, the transcript carries a 51-bpmessage, starting with an ATG and ending with a TAGcodon, specifying a leader peptide of 16 amino acids, 4 ofwhich are consecutive leucines and isoleucines. Ribosomestalling at Leu and Ile codons is expected to prevent forma-tion of the termination signal and lead to transcription of thedownstream leu genes. Rho-independent transcription ter-minators were not found between the two gene clusters or

downstream of the ilvA gene.

DISCUSSION

Organization of the ilv operon. The BCAA genes in L.lactis subsp. lactis are organized in a large cluster which isdivided into two units, grouping leu and ilv genes. Both unitsare necessary for leucine biosynthesis, while only the secondis required for the synthesis of isoleucine and valine. Atransaminase, which carries out the last step of BCAAbiosynthesis (the ilvE gene product in E. coli), is not encodedwithin the cluster, which suggests that this reaction isperformed by a nonspecific transaminase or that the corre-sponding L. lactis subsp. lactis gene maps elsewhere on thechromosome. Both the leu and ilv gene clusters are precededby a putative promoter. However, they are not separated bya rho-independent transcription terminator, which suggeststhat they might form a single operon. The putative operonextends past the last biosynthetic gene, ilvA.

Regulation of the dv operon. Sequence analysis stronglysuggests that the operon is regulated by an attenuationmechanism, mediated by a leucine-rich leader peptide (Fig.4). This peptide is very similar to the leader peptides of theE. coli and S. typhimurium leu operons (18, 66) but differsfrom those of the E. coli ilvBN (16) and ilvGMEDA (35)operons.The current model proposes that the strength of attenua-

tion depends on the availability of charged tRNAItU duringtranslation of the leader peptide. The presence of rarecodons increases the response to leucine deprivation byincreasing the duration of ribosome stalling. In E. coli, thecodon specifying the four leucine residues present in theleader peptide is CUA, which corresponds to only 2% ofleucine codons used for the proteins of this organism (51). Incontrast, the leucine codon UUG, found three times in the L.lactis BCAA leader transcript, corresponds to 24% of theleucine codons in proteins of L. lactis (61). The isoleucinecodon is also present within the L. lactis leader peptide,closely following the Leu codons, which is not the case in E.coli and might affect the response of the operon to BCAAstarvation. Further studies are needed to determine whetherthe model proposed for regulation in E. coli can be directlyapplied to L. lactis.ORF2. ORF2 is not a BCAA biosynthetic gene, since it

specifies a product which is a member of the ABC proteinsuperfamily. Proteins from this family are found in prokar-yotes and eukaryotes and are similar in basic organization(24, 25). In prokaryotes, most members of this superfamilyare components of transport systems which involve periplas-mic binding proteins. These genes are generally cotrans-cribed with other carrier protein genes (25) and have neverbeen found within biosynthetic operons. Further work isrequired to establish the function and role of ORF2, but it istempting to speculate that it might be involved in thetransport of BCAA or in the regulation of BCAA genes, byanalogy with another ABC protein, MalK, which regulatesnegatively the expression of the mal regulon in E. coli (33).

Isoleucine, leucine, and valine residues constitute at least20% of the amino acid content of L. lactis (58), whiletryptophan and histidine, the two other amino acids forwhich biosynthetic pathway genes were studied in L. lactis(5, 10), correspond to less than 2%. This finding suggests that

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BRANCHED-CHAIN AMINO ACID GENES OF L. LACTIS 6587

uuuu uU GC-GG-CA-UU-AAAU AA-UAUC-G

U G (A)C-GC-GA-UG-CG-Cu uG GG UA-U C AA-U U CC-G C-G

(1) U G (2) (5) C-G (6)A U GG-C G-CU G C-GU-A A-UG U G-C

CA U-A GA GAGA-UU UUGU- GUCAUUUCGAG CC UUUUUU AACA CAGUAAAGUUC GGUU C A AA AGAAAAAAAACAAUUA

A UCA

AUGACAUAACACAUUCUUGUUGAUCAAGGUGGACCUACAUAGCUUUUUUGAM T Y T Q F S L L L I K V D L H *

(B)

GGAA CG-C

(3) C-G (4) UAAUAUU AUG--->leuAU-A GU G GU-Al AA I GC-G AU-A UG-C 5 A

AUA II AIA GI AIGC U A

G-C AG-C A

Iu u uIU C A

U-A AG-C U

21 U G I6 UC-G CC-G -W U

3GCUAAAAUAUGUGGGU CGUCUUUUUUGUU

AGuUCAAGAGAAAAAAAACAAUUAFIG. 4. Secondary structures of the leader transcript which might mediate transcriptional attenuation. (A) Termination configuration.

Repeats 1 and 2 are annealed, and repeats 5 and 6 form a transcription terminator. (B) Antitermination configuration. Repeats 3 and 4 areannealed, which sequesters repeats 5 and 6. Bold characters indicate the sequence encoding the leader peptide; bold italics indicate theribosome binding sites of the leader peptide and the leuA gene. Ribosome stalling at the successive Leu and Ile residues might sequester repeat1 and favor this configuration.

a fine regulation of BCAA gene expression might be re-quired. Furthermore, the three BCAA genes use a commonpathway, which also requires a special regulation. In E. coli,in which the BCAA genes are scattered, the regulation iscomplex and not as yet fully understood. In L. lactis, inwhich the BCAA genes are clustered, the regulation isprobably different. The coordination of expression of thesegenes is presently being studied.

ACKNOWLEDGMENTSWe are grateful to P. Renault for helpful comments and to C.

Anagnostopoulos for critical reading of the manuscript.This work was supported in part by BRIDGE contract Biot-CT91-

0263 of the Commission of the European Communities.

REFERENCES1. Andreadis, A., Y.-P. Hsu, G. B. Kohihaw, and P. Schimmel.

1982. Nucleotide sequence of yeast leu2 shows 5'-noncodingregion has sequences cognate to leucine. Cell 31:319-325.

2. Armpriester, J. M., Jr., and P. S. Fink. 1990. Unpublished data.3. Bachmann, B. J. 1987. Linkage map of Escherichia coli K-12, p.

807-877. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B.Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Esche-richia coli and Salmonella typhimurium: cellular and molecularbiology, vol. 2. American Society for Microbiology, Washing-ton, D.C.

4. Barat, M., C. Anagnostopoulos, and A. M. Schneider. 1965.Linkage relationships of genes controlling isoleucine, valine,

and leucine biosynthesis in Bacillus subtilis. J. Bacteriol. 90:357-369.

5. Bardowsld, J., S. D. Ehrlich, and A. Chopin. 1992. Tryptophanbiosynthesis genes in Lactococcus lactis subsp. lactis. J. Bac-teriol. 174:6563-6570.

6. Beltzer, J. P., L.-F. L. Chang, A. E. Hinkkanen, and G. B.Kohlhaw. 1986. Structure of yeast leu4: the 5' flanking regioncontains features that predict two modes of control and twoproductive translation starts. J. Biol. Chem. 261:5160-5167.

7. Butlin, J. D., G. B. Cox, and F. Gibson. 1971. Oxydativephosphorylation in Escherichia coli K12. Biochem. J. 124:75-81.

8. Calvo, J. M. 1983. Leucine biosynthesis in prokaryotes, p.267-284. In K. M. Herrman and R. L. Somerville (ed.), Aminoacids, biosynthesis and genetic regulation. Addison-Wesley,Reading, Mass.

9. Cordes, C., L. Eggeling, and H. Sahm. 1990. Production ofL-isoleucine by recombinant Corynebacterium glutamicumstrains after cloning, analysis and amplification of genes in-volved in isoleucine biosynthesis, p. 339-351. In H. Heslot, J.Davies, J. Florent, L. Bobichon, G. Durand, and L. Penasse(ed.), 6th International Symposium on Genetics of IndustrialMicroorganisms. Societe Francaise de Microbiologie, Paris.

10. Delorme, C., S. D. Ehrlich, and P. Renault. 1992. Histidinebiosynthesis genes in Lactococcus lactis subsp. lactis. J. Bac-teriol. 174:6571-6579.

11. de Vos, W. M. 1987. Gene cloning and expression in lacticstreptococci. FEMS Microbiol. Rev. 46:281-295.

12. Driver, R. P., and R. P. Lawther. 1985. Restriction endonucle-ase analysis of the ilvGEDA operon of members of the family

VOL. 174, 1992

on May 25, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

6588 GODON ET AL.

Enterobacteriaceae. J. Bacteriol. 162:1317-1319.13. Evans, I. J., and J. A. Downie. 1986. The nodl gene product of

Rhizobium leguminosarum is closely related to ATP-bindingbacterial transport proteins; nucleotide sequence analysis of thenodI and nodJ genes. Gene 43:95-101.

14. Falco, S. C., K. S. Dumas, and K J. Livak. 1985. Nucleotidesequence of the yeast ilv2 gene which encodes acetolactatesynthase. Nucleic Acids Res. 13:11.

15. Felmlee, T., S. Pellett, and R. A. Welch. 1985. Nucleotidesequence of an Eschenichia coli chromosomal hemolysin. J.Bacteriol. 163:94-105.

16. Friden, P., T. Newman, and M. Freundlich. 1982. Nucleotidesequence of the ilvB promoter-regulatory region: a biosyntheticoperon controlled by attenuation and cyclic AMP. Proc. Natl.Acad. Sci. USA 79:6156-6160.

17. Friedberg, D., E. R. Rosenthal, J. W. Jones, and J. M. Calvo.1985. Characterization of the 3' end of the leucine operon ofSalmonella typhimunum. Mol. Gen. Genet. 199:486-494.

18. Gemmill, R. M., S. R. Wessler, E. B. Keller, and J. M. Calvo.1979. leu operon of Salmonella typhimunium is controlled by anattenuation mechanism. Proc. Natl. Acad. Sci. USA 76:4941-4945.

18a.Gibson, T. J. 1984. Ph.D. thesis. University of Cambridge,Cambridge, England.

19. Gilson, E., H. Nikaido, and M. Hofaung. 1982. Sequence of themalK gene in E. coli K12. Nucleic Acids Res. 10:7449-7458.

20. Glaser, P., H. Sakamoto, J. Bellalou, A. Ullmann, and A.Danchin. 1988. Secretion of cyclolysin, the calmodulin-sensitiveadenylate cyclase-haemolysin bifunctional protein of Bordetellapertussis. EMBO J. 7:3997-4004.

21. Gowrishankar, J. 1989. Nucleotide sequence of the osmoregu-latoryproU operon of Escherichia coli. J. Bacteriol. 171:1923-1931.

22. Gribskov, G., J. Devereux, and R. R. Burgess. 1984. The codonpreference plot: protein coding sequences and prediction ofgene expression. Nucleic Acids Res. 12:539-549.

23. HigAns, C. F., M. L. Gallagher, M. L. Mimmack, and S. R.Pearce. 1988. A family of closely related ATP-binding subunitsfrom prokaryotic and eukaryotic cells. BioEssays 8:111-116.

24. Higgins, C. F., I. D. Hiles, G. P. C. Salmond, D. R. Gill, J. A.Downie, I. J. Evans, I. B. Holland, L. Gray, S. D. Bucked, A. W.Bell, and M. A. Hermodson. 1986. A family of related ATP-binding subunits coupled to many distinct biological processesin bacteria. Nature (London) 323:448-450.

25. Higgins, C. F., I. D. Hiles, KL Whalley, and D. J. Jamieson. 1985.Nucleotide binding by membrane components of bacterial peri-plasmic binding protein-dependent transport systems. EMBO J.4:1133-1040.

26. Ikawa, S., T. Shibata, T. Ando, and H. Saito. 1980. Geneticstudies on site-specific endodeoxyribonucleases in Bacillus sub-tilis: multiple modification and restriction systems in transfor-mants of Bacillus subtilis 168. Mol. Gen. Genet. 177:359-368.

27. Imai, R., T. Sekiguchi, Y. Nosoh, and K Tsuda. 1987. Thenucleotide sequence of 3-isopropylmalate dehydrogenase genefrom Bacillus subtilis. Nucleic Acids Res. 15:4988.

28. Iturrliaga, E. A., J. M. Diaz-Minguez, E. P. Benito, M. I.Alvarez, and A. T. Eslava. 1990. Nucleotide sequence ofPhycomyces blakesleeanus leul gene. Nucleic Acids Res. 18:4612.

29. Kagawa, Y., H. Nojima, N. Nukdwa, M. Ishizuka, T. Nakajima,T. Yasuhara, T. Tanaka, and T. Oshima. 1984. High guanineplus cytosine content in the third letter of codons of an extremethermophile. J. Biol. Chem. 259:2956-2960.

30. Kielland-Brandt, M. C., S. Holmberg, J. G. L. Petersen, and T.Nilsson-Tlllgren. 1984. Nucleotide sequence of the gene forthreonine dehydratase ilv-1 of Saccharomyces cerevisiae. Carls-berg Res. Commun. 49:567-575.

31. Kohlhaw, G. B. 1983. Regulation of leucine biosynthesis inlower eukaryotes, p. 285-299. In K. M. Herrman and R. L.Somerville (ed.) Amino acids, biosynthesis and genetic regula-tion. Addison-Wesley, Reading, Mass.

32. Kolter, R., and C. Yanofsky. 1982. Attenuation in amino acidbiosynthetic operons. Annu. Rev. Genet. 16:113-134.

33. Kuhnau, S., M. Reyes, A. Sievertsen, H. A. Shuman, and W.Boos. 1991. The activities of the Escherichia coli MalK proteinin maltose transport, regulation, and inducer exclusion can beseparated by mutations. J. Bacteriol. 173:2180-2186.

34. Lawther, R. P. Unpublished data.35. Lawther, R. P., and G. W. Hatfield. 1980. Multivalent transla-

tional control of transcription termination at attenuator ofilvGEDA operon of Escherichia coli K-12. Proc. Natl. Acad.Sci. USA 77:1862-1866.

36. Lawther, R. P., R. C. Wek, J. M. Lopes, R. Pereira, B. E.Taillon, and G. W. Hatfield. 1987. The complete nucleotidesequence of the ilvGMEDA operon of Escherichia coli K-12.Nucleic Acids Res. 15:2137-2155.

37. Mackey, C. J., R. J. Warburg, H. 0. Halvorson, and S. A.Zahler. 1984. Genetic and physical analysis of the ilvBC-leuregion in Bacillus subtilis. Gene 32:49-56.

38. Mackey, C. J., and S. A. Zahler. 1982. Insertion of bacterio-phage SPP into the citF gene of Bacillus subtilis and special-ized transduction of the ilvBC-leu genes. J. Bacteriol. 151:1222-1229.

39. Marsch, N. J., and D. E. Duggan. 1972. Ordering of mutant sitesin the isoleucine-valine genes of Eschenichia ccli by use ofmerogenotes derived from F14: a new procedure for fine-structure mapping. J. Bacteriol. 109:730-740.

40. Needleman, S. B., and C. D. Wunsch. 1970. A general methodapplicable to the search for similarities in the amino acidsequence of two proteins. J. Mol. Biol. 48:443-453.

41. Niaudet, B., and S. D. Ehrlich. 1982. Insertional mutagenesis:use in cloning of Bacillus subtilis genes, p. 201-209. In M.Polsinelli and G. Mazza (ed.), Transformation-80. CotswoldPress, Oxford, England.

42. Niaudet, B., L. Janniere, and S. D. Ehrlich. 1985. Integration oflinear, heterologous DNA molecules into the Bacillus subtilischromosome: mechanism and use in induction of predictablerearrangements. J. Bacteriol. 163:111-120.

43. Nohno, T., T. Saito, and J.-S. Hong. 1986. Cloning and completenucleotide sequence of the Escherichia coli glutamine permeaseoperon (glnHPQ). Mol. Gen. Genet. 205:260-269.

44. Pattee, P. A. 1976. Genetic linkage of chromosomal tetracyclineresistance and pigmentation to a purine auxotrophic marker andthe isoleucine-valine-leucine structural genes in Staphylococcusaureus. J. Bacteriol. 127:1167-1172.

45. Petersen, J. G. L., and S. Holmberg. 1986. The ilvS gene ofSaccharomyces cerevisiae is highly expressed. Nucleic AcidsRes. 14:9631-9651.

46. Piggot, P. G., and J. A. Hoch. 1985. Revised genetic linkage mapof Bacillus subtilis. Microbiol. Rev. 49-158-179.

47. Ricca, E., and J. M. Calvo. 1990. The nucleotide sequence ofleuA from Salmonella typhimurium. Nucleic Acids Res. 18:1290.

48. Roncero, M. I. G., L. P. Jepsen, P. Stroman, and R. vanHeeswjck 1989. Characterization of a leuA gene and an ARSelement from Mucor circineloides. Gene 84:335-343.

49. Rosenthal, E. R., and J. M. Calvo. 1990. The nucleotide se-quence of leuC from Salmonella typhimurium. Nucleic AcidsRes. 18:3072.

50. Seklguchi, T., J. Ortega-Cesena, Y. Nosoh, S. Ohashi, K. Tsuda,and S. Kanaya. 1986. DNA and amino-acid sequences of 3-iso-propylmalate dehydrogenase of Bacillus coagulans. Compari-son with the enzymes of Saccharomyces cerevisiae and Ther-mus thermophilus. Biochim. Biophys. Acta 867:36-44.

51. Sharp, P. M., E. Cowe, D. G. Higgins, D. C. Shieds, K. H.Wolfe, and F. Wright. 1988. Codon usage patterns in Esche-richia coli, BaciUus subtilis, Saccharomyces cerevisiae, Schizo-saccharomyces pombe, Drosophila melanogaster, and Homosapiens; a review of the considerable within-species diversity.Nucleic Acids Res. 16:8207-8211.

52. Simon, D., and A. Chopin. 1988. Construction of a vectorplasmid family for molecular cloning in Streptococcus lactis.Biochimie 70:559-566.

53. Skala, J., E. Capieaux, E. Balzi, W. Chen, and A. Goffeau. 1991.Complete sequence of the Saccharomyces cerevisiae leul geneencoding isopropylmalate isomerase. Yeast 7:281-285.

J. BAC-rERIOL.

on May 25, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

VOL. 174, 1992 BRANCHED-CHAIN AMINO ACID GENES OF L. LACTIS 6589

54. Somers, J. M., A. Amzallag, and R. B. Middleton. 1973. Geneticfine structure of the leucine operon of Escherichia coli K-12. J.Bacteriol. 113:1268-1272.

55. Squires, C. H., M. DeFelice, J. Devereux, and J. M. Calvo. 1983.Molecular structure of ilvIH and its evolutionary relationship toilvG in Escherichia coli. Nucleic Acids Res. 11:5299-5313.

56. Squires, C. H., M. DeFelice, S. R. Wessler, and J. M. Calvo.1981. Physical characterization of the ilvHI operon of Esche-richia coli K-12. J. Bacteriol. 147:797-804.

57. Tanaka, T. 1979. Restriction of plasmid-mediated transforma-tion in Bacillus subtilis 168. Mol. Gen. Genet. 175:235-237.

58. Thomas, T. D., and G. G. Prichard. 1987. Proteolytic enzymesof dairy starter cultures. FEMS Microbiol. Rev. 46:245-268.

59. Umbarger, H. E. 1983. The biosynthesis of isoleucine and valineand its regulation, p. 245-266. In K. M. Herrman and R. L.Somerville (ed.), Amino acids, biosynthesis and genetic regula-tion. Addison-Wesley, Reading, Mass.

60. Umbarger, H. E. 1987. Biosynthesis of the branched-chainamino acids, p. 353-367. In F. C. Neidhardt, J. L. Ingraham,K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger(ed.), Escherichia coli and Salmonella typhimuinum: cellularand molecular biology, vol. 2. American Society for Microbiol-ogy, Washington, D.C.

61. Van de Guchte, M., J. Kok, and G. Venema. 1992. Gene

expression in Lactococcus lactis. FEMS Microbiol. Rev. 88:73-92.

62. Vandeyar, M. A., C. J. Mackey, R. H. Lipsky, and S. A. Zahler.1986. The ilvBC-leu operon of Bacillus subtilis, p. 295-305. InA. T. Ganesan and J. A. Hoch (ed.), Bacillus molecular geneticsand biotechnology applications. Academic Press, Inc., NewYork.

63. Ward, J. B., and S. A. Zahler. 1973. Genetic studies of leucinebiosynthesis in Bacillus subtilis. J. Bacteriol. 116:719-726.

64. Wek, R. C., and G. W. Hatfield. 1986. Nucleotide sequence andin vivo expression of ilvY and ilvC genes in Escherichia coliK12. J. Biochem. Chem. 261:2441-2450.

65. Wek, R. C., C. A. Hauser, and G. W. Hatfield. 1985. Thenucleotide sequence of the ilvBN operon of Escherichia coli:sequence homologies of the acetohydroxy acid synthase iso-zymes. Nucleic Acids Res. 13:3995-4010.

66. Wessler, S. R, and J. M. Calvo. 1981. Control of leu operonexpression in Eschenichia coli by transcription attenuationmechanism. J. Mol. Biol. 149:579-597.

67. Wilbur, W. J., and D. UIpman. 1983. Rapid similarity searchesof nucleic acid and protein data banks. Proc. Natl. Acad. Sci.USA 80:726-730.

68. Zahler, S. A. Personal communication.

on May 25, 2021 by guest

http://jb.asm.org/

Dow

nloaded from