JOURNUA OF BACTERIOLOGY, OCt. 1992, p. 6563-6570
Vol. 174, No. 20
0021-9193/92/206563-08$02.00/0 Copyright © 1992, American Society for Microbiology
Tryptophan Biosynthesis Genes in Lactococcus lactis subsp. lactis JACEK BARDOWSKI,* S. DUSKO EHRLICH, AND ALAIN CHOPIN Laboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France Received 21 April 1992/Accepted 12 August 1992
The Lactococcus lactis chromosomal region containing the seven structural genes required for tryptophan biosynthesis was characterized by cloning and sequencing. All of the thp genes were identified by the homology of their products with known Trp proteins from other organisms. The identification was confirmed for five genes by their ability to complement tip mutations in Escherichia coli. The seven structural genes are present in the order trpEGDCFBA and span a 7,968-bp segment. Each gene is preceded by a putative ribosome binding site complementary to the 3' end of the L. lactis 16S rRNA. Three pairs of genes (trpG-trpD, trpC-trpF, and trpB-trpA) overlap, and there is intercistronic spacing of 124, 46, and 585 bp between the trpE-trpG, trpD-trpC, and trpF-trpB gene pairs, respectively. No gene fusion was found. Upstream of the tip genes, a 457-bp noncoding DNA segment contains several regions fitting the consensus for gram-positive promoters and one region strongly resembling a transcription terminator. However, it seems unlikely that an attenuation mechanism similar to the one found in E. coli regulates tryptophan biosynthesis in L. lacti&, since no potential leader peptide was detected. We propose that a mechanisms resembling that described in BaciUls spp. can regulate tip genes expression in L. lactis.
Lactococcus lactis has recently become a focus of an increasing number of genetic studies, mostly because of its involvement in milk fermentation. A number of genes important for industrial use of lactococci have therefore been characterized. Examples include genes required for efficient growth in milk, such as those for utilization of lactose or degradation of caseins (16, 30, 57), genes required for synthesis of antimicrobial agents such as nisin and bacteriocins (27, 55), and genes which specify resistance to bacteriophages (8, 25, 26). However, most of these genes are carried on extrachromosomal elements (plasmids or transposons). As a consequence, only a few L. lactis chromosomal genes have been studied; of 39 sequenced genes present in the data bases, only 9 are chromosomal. Six of these, rplO, rpmJ, rpsM, infA, secY, and adk, encoding three ribosomal proteins (L15, B, and S13), initiation factor 1, secretion protein SecY, and adenylate kinase (28, 29), respectively, are clustered. Three others, pabB, pepXP, and usp-45, which encode para-aminobenzoate synthase B, X-prolyl dipeptidyl aminopeptidase (39, 41), and the major unidentified secreted protein (54), respectively, have been isolated. The total length of the known chromosomal sequence is well below 10 kb, and from such scarce data it is difficult to draw conclusions about gene organization and expression in L. lactis. We therefore undertook an analysis of three biosynthetic pathways, i.e., for tryptophan, histidine, and branched-chain amino acids (leucine, isoleucine, and valine), which have been previously studied in numerous other organisms. The main conclusions, based on analysis of over 30 kb of sequence, reported in this and the two accompanying papers (14, 21) are that (i) most if not all of the genes are clustered and are probably organized in operons, (ii) the operons might contain only genes coding for proteins highly homologous to the enzymes required for the biosynthesis of a given amino acid or up to 30% of genes not *
obviously required for this biosynthesis, and (iii) the operons seem compact, and a number of genes overlap. This might be a consequence of the relatively small size of the L. lactis genome (34) and represent a general mode of organization of biosynthetic genes in this organism. In addition, our analysis eliminates certain modes of control of gene expression and strongly suggests the existence of others (for instance, transcriptional attenuation does not control tip or his genes but appears to control leu genes) and therefore provides guidelines for future studies of gene regulation in L. lactis. The thp genes and their products have been characterized for many prokaryotes (12) and represent a paradigm for the study of gene organization and regulation. The tryptophanspecific pathway is catalyzed by seven enzymatic activities encoded by five to seven genes, localized either in one cluster as in the enterobacteriaceae (11, 60), Bacillus subtilis (24, 49), or Brevibacterium lactofermentum (38) or in several clusters as in fluorescent pseudomonads (5, 10). Study of the regulation of the expression of the tip genes has revealed a variety of mechanisms, including repression (59), activation (6), transcriptional attenuation (59), and a novel type of attenuation mediated by a regulatory protein (45). We report here the structure of the L. lactis subsp. lactis chromosomal region which contains the seven structural genes for Trp biosynthesis. Similar studies of genes required for histidine and branched-chain amino acid synthesis are described in the accompanying reports (14, 21). MATERIALS AND METHODS Bacterial strains, plasmids, and media. The bacterial strains and plasmids used are described in Table 1. L. lactis subsp. lactis IL1403 was grown at 30°C in M17 medium (52) in which lactose was replaced by glucose. Escherichia coli was grown in Luria-Bertani medium or in minimal medium M9 (36) at 37°C. B. subtilis was grown at 37°C in LuriaBertani medium or in Spizizen salt minimal medium (1) supplemented with the appropriate amino acid(s). When
Corresponding author. 6563
BARDOWSKI ET AL.
6564
J. BACTERIOL.
TABLE 1. Strains and plasmids Strain or plasmid
Strains E. coli HB101
TG1 SURE
IL2699 IL3012 IL3013 IL3014 IL3015 B. subtilis MT119 L. lactis subsp. lactis IL1403 Plasmids pBluescript pIL253 pIL320
pIL341 pIL356 pIL357 pIL258
pIL499
Reference source or
Characteristics
hsdS recA ara-14 supE lacY galK proA xyl-5 mtl-l rpsL supE thi A(lac-proAB) hsd (F'+ traD proAB lacIqZ M15) recB recJ sbcC uvrC umuC::TnS mcrA mcrB mrr lac A(hsdRMS) endA gyrA thi relA supE (F'+ proAB lac]qZ M15 TnlO) W3110 trpA33 W3110 trpB9578 tnaA2 Smr W3110 trpC9941 W3110 trpD9923 W3110 trpE10220 leuB6 trpC2 r- mPlasmid free, Trp+
Apr, M13 on, pBR322 on Emr, high copy number 12.7-kb L. lactis DNA segment cloned into pIL253 pIL320 with pBR322 inserted into its PstI site pIL341 lacking a 9.8-kb EcoRV segment pIL341 lacking a 6.0-kb EcoRV segment pIL341 lacking a 3.8-kb EcoRV segment 3.6-kb XbaI-XhoI L. lactis DNA segment cloned into pBluescript
36 43
Stratagene
C. C. C. C. C. 50 7
Yanofsky Yanofsky Yanofsky Yanofsky Yanofsky
Stratagene 47 This work
This work This work This work This work
This work
necessary, erythromycin (5 p,g/ml for L. lactis subsp. lactis for E. coli), or 0.3 ,ug/ml for B. subtilis), ampicillin (50 or tetracycline (10 ,ug/ml for E. coli) was added to the
jig/ml
medium. Molecular cloning and DNA manipulations. Plasmid and chromosomal DNAs were prepared as previously described (35, 36, 48, 51). E. coli cells were transformed either by the CaCl2 standard procedure (36) or by electroporation (17). B. subtilis cells were induced for competence and transformed as described by Anagnostopoulos and Spizizen (1), with some modifications (4). Southern blotting and DNA hybridization were done as described by Maniatis et al. (36). DNA probes were prepared by using [cc-32P]dCTP (Amersham) and a nick translation kit (Boehringer Mannheim) according to the supplier's recommendations. Other molecular techniques were used as described by Maniatis and al. (36). DNA sequence analysis. E. coli clones for sequencing were obtained by subcloning specific DNA fragments in the pBluescript plasmids family and by using exonuclease III and mung bean nuclease (Stratagene) to generate a series of clones containing overlapping DNA fragments. Nucleotide sequence was determined as described elsewhere (41). The reported sequence was determined at least twice on both strands. The sequence of a part of the tip region was also determined on a polymerase chain reaction (PCR)-amplified
DNA segment. Synthetic oligonucleotides 5'-GCCTACGGTGTGGATGTT-3' and 5'-AAGGGTCTCCACAC`TGC-3' were used as primers. The PCR mixture was incubated on a Pharmacia-LKB ATAQ Controller apparatus for 30 cycles (94°C for 1 min, 50°C for 1 min, and 72°C for 2 min). The PCR-amplified DNA was subsequently sequenced by using a Taq DyeDeoxy Terminator Cycle sequencing kit and 370A sequencer, both from Applied Biosystems, Foster City, Calif. The DNA and protein sequences were analyzed with BISANCE and University of Wisconsin GCG software packages, implemented at the Centre InterUniversitaire d'Informatique a Orientation Biomedicale (Paris, France). Protein sequences were aligned with MULTALIN software (9). Nucleotide sequence accession number. The GenBank, EMBL, and DDBJ nucleotide sequence accession number for the sequence shown in Fig. 2 is M87483.
RESULTS Cloning of the thp region. The trp region was cloned by preparing a shotgun library of L. lactis subsp. lactis IL1403 total DNA in plasmid vector pIL253 (47) and transforming B. subtilis MT119 to tryptophan prototrophy. Sixteen micrograms of chromosomal DNA was partially digested with endonuclease Sau3A to obtain a majority of DNA fragments larger than 5 kb, ligated with 5 ,ug of BamHI-cleaved vector, and used for transformation. Only one Trp+ transformant was isolated among 14,000 Emr clones. It contained a plasmid named pIL320, consisting of pIL253 carrying a 12.7-kb insert. Southern blot analysis of XbaI-cleaved chromosomal DNA of L. lactis subsp. lactis IL1403 by using pIL320 as a probe revealed that the DNA insert in pIL320 consisted of two noncontiguous Sau3A segments, which were joined during ligation (not shown). Analysis of the insert (see below) revealed that one of the segments encodes all of the genes required for tryptophan biosynthesis except trpE, the 5' end of which was missing. The other Sau3A segment, which carried no tip genes, was not studied further. Southern analysis indicated that the complete trpE gene might be present on a 3.6-kb XbaI-XhoI segment. This segment was cloned in E. coli SURE in the pBluescript vector and detected by colony hybridization, using as a probe a segment internal to trpE gene (Fig. 1). One positive clone was analyzed and found to contain a plasmid of the expected size, which was named pIL499 (Fig. 1). This plasmid was used for further analysis. Complementation experiments. To identify the thp genes present on the cloned segments, pIL320 and pIL499 were made replicative in E. coli by inserting plasmid pBR322 at their unique PstI sites and were used to complement various E. coli Trp- mutants. The pIL320 insert complemented mutations in trpA, trpB, trpC, and t-pD but not in trpE, whereas the pIL499 insert complemented only a trpE mutation. Further complementation analysis, using pIL320 derivatives deleted of various EcoRV fragments, established the approximate locations of the tip genes on the insert (Fig. 1). Nucleotide sequence of the trp region. The nucleotide sequence of the 8,656-bp region was determined (Fig. 2). Open reading frames (ORFs) were searched for by the TestCode software (19), which measures the nonrandomness of nucleotide composition at every third base and therefore does not require knowledge of the codon preference, which has not been established previously for L. lactis subsp. lactis chromosomally carried genes. Eight probable coding regions were detected by this analysis. Eight ORFs were subsequently delineated by searching for translation initiation and
TRYPTOPHAN BIOSYNTHESIS GENES OF L. LACTIS
VOL. 174, 1992
A
6565
Complementation of E. coli trpE trpD trpC trpB trpA
Xhol
pIL341
pIL356 -[ pIL357 -[
XholJ
r
i
pIL358-
-
L
probe
Xbal
Xhol
pIL499 _ _
C
ti
t2
trpB
trpE _m-p3 .p1
p4
p
FIG. 1. (A) Structure of the DNA region carrying the L. lactis trp genes. The thick line indicates the two overlapping DNA fragments containing the entire trp region. The thin line corresponds to DNA that is simultaneously cloned with the tip region but is not contiguous in the L. lactis chromosome. The DNA probe used to identify the clone containing pILA99 is indicated. Plasmids pIL356 to pIL358 were obtained by cleavage of pL;341 with EcoRV and religation and were used in complementation experiments with various E. coli tip mutants. (B) Results of complementation. Growth (+) or lack of growth (-) on a medium without tiyptophan is indicated. (C) Expansion showing the organization of the tip region. pl to p4, putative transcription promoters; tl to t3, putative transcription terminators. ORF1 has no homology with the known thp genes.
termination codons, as well as the putative ribosome binding sites, defined as sequences complementary to the 3' end of the L. lactis subsp. lactis 16S rRNA (15) localized 5 to 7 bp upstream of the translation initiation codon. Assignment of the ORFs. Comparison of the protein sequences deduced from the eight ORFs with the sequences in the GenBank and NBRF data bases revealed that seven ORFs encode polypeptides highly homologous to the known Trp proteins (Table 2). Conserved residues are distributed throughout the sequences of all polypeptides, but the homology is higher in the regions of the Trp proteins which are highly conserved among microorganisms (not shown). Deviations from this general conclusion were found for TrpE, in which the C-terminal region of the polypeptide is better conserved than the N-terminal region, and TrpF, which is
about 130 amino acids longer than TrpF proteins from other organisms and is homologous to their amino-terminal parts only (data not shown). To exclude the possibility that the lactococcal trpF gene was rearranged during or after cloning in E. coli, the appropriate fragment of the chromosome was amplified by PCR and sequenced. Cloned and PCR-amplified segments had identical sequences, which suggests that the lactococcal TrpF protein might be fused to another polypeptide. The function of this polypeptide is not known, since the carboxy-terminal part of the TrpF protein is not homologous with any protein in the data bases. The protein sequence deduced from the remaining ORF, localized upstream of the tip genes, showed no significant homology with any protein in the data bases and was designated ORF1. The assignment of the ORFs in the tip region is summarized in Fig. 1.
TABLE 2. Conservation of proteins involved in tryptophan biosynthesis between L. lactis subsp. lactis and various organisms Organism compared
Bacillus subtilis LactobaciUlus casei Pseudononas aeruginosa Escherichia coli Brevibacterum lactofermentwn Haloferax volcanii Methanococcus voltae Methanobacterium thermoautotrophicum Saccharomyces cerevisiae
% Identical amino acids"
TrpE
TrpG
TrpD
TrpC
TrpF
TrpB
TrpA
40 b 43 36 33
44
40
35
38 38 36 33 32 25 33
24 29
38 36 34
32 38 36 35 35
61 56 59 54 50 49 60 55
36 32 30 28 25 30 31 28
36
26 25 20
Reference(s)
3, 24 42
18, 23 60 38 33 46 40
33 55 20, 53, 61, 62 a Calculated from the multiple alignments obtained by the method of Corpet (9) as the ratio of the number of perfect matches x 100 to the shorter protein length.
b
_, Sequence not available.
34
36
25
36
34
AAGCTTCGTTTAACAAGAAGTTTGTCCAGTTTGATTTGCAATGGTAAGCGTTGAGAAGTTAGAATTGTCGCAGAATCTGAAAATCACACTTTAA
1202
AAAAGOACAAGAATTGACCTTTAACATTATCTGCGCTTTGTTGCTTGGATTTTCTCATTTGTTTTAAGACTTTCTACAGCCAAAACAACAGCGT
2404
RBS
L T I orf 1 -4
L
T
I
I
S
L
L
L
L
A
V
E
F
F
Y
I
H
L
Y
E
T
A
F
S
T
S
T
K
S
T
R
V
GTTTATATGGAAAGAGAGCAGAAGTTCTCAGTCAACGCTTTTAAAACAAGGATTATACGGTTGATGGTTAGGTTAATTATCAAATTCTTTCTCA F
N
G
M
E
E
K
E
L
S
R
S
Q
V
T
L
F
K
Q
N
G
Y
I
G
N
L
G
I
G
L
I
L
A
Y
F
I
S
S
F
ACAATTGAATTGTAGATACACTTTTTAATTATTTGTGGTTTGATGGTCTTGACAGTATAAAAGATATTTCACCAAGTGGTTAGCATTTAGCCTA Q
L
E
I
V
L
R
L
L
I
Y
I
I
V
L
A
Y
L
S
G
L
S
T
N
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K
I
L
I
Q
T
G
G
L
I
A
A
L
L
A I
35
3606
75
4808
115
6000
TTCTCATTTTTTAAAAATATTTTAATCTTGAAAGCTCAAAAAGTTTACATTATTTCACATCGAAAGTACTCTTATCGAATTTTCAGGAGTCGCG S SF F pl.-lO pl.-35 TGTGGAGCGATAATTCTCAGGCGATGGCTG.TTTGAAGTGAGACGATTGTTTTCCAATTGATTGAACAAAAAATCACGATCTCCGATACAAGGAC GTTGTAGAGAGGAAAGAGATTTTCTAGATTTTTTATACTTTAGGACCTTAATTCGGTGCACGCGTACAACGCCGTAAGTATTGTACTTACGGCT p2.-35
119
-------
7202
8404
-------> tl < .--
antiterminator TTTGTATTTTTTTGAAAGGATTTCTAAAATGAACCAACGATGGCGAAGCTTAAATTTTAACTAATCAGTATCAATGGAAAAGACTACCTCAAACCTATAGATAGAGAGAAAACATGAGAA ~~~~~M R t rpE p3.-10 p3.-35 p2.-10
~~~~~~~~~RBS
-----------
AAATAAAAAATTCACAGAACCTGACCCGTTTCGTTATCTCGCTGAAGGGAAATAAATGATTTAAATCATTCCAAGAAATGCCAACACTTTTC
10808
TTATGCGTGAACCTTGAACACTCAATTTCTGAGGATTTTAGCTAAAGACAAACATTGCGTGAAATCAATGAATTCTGAAAATTATTTTCAGC
12000
K
I
L
A
P
N
T
D
K
V
T
M
H
I
P
F
K
S
I
T
V
G
D
L
Y
I
R
S
L
G
K
L
V
K
D
N
N
K
I
E
V
I
S
I
D
S
E
L
E
N
I
P
P
R
F
E
M
E
N
L
Q
D
E
S
L
K
R
Q
C
V
$
F
42
82
P
13202
CAGAATGTTTTATGTATACAATAGCTTAATTTTACCAAAAGAGAAAACGATTTTATGAAACATACTACATCAGGTTCGAAAAGATTACAAAGC
14404
TGTAAGAATATGAGTATTACTTACTGCAGAGCAAAAAGAATGACGAATAAGAAACGAATTCGCAGCATAGTCTAAGACTTTTGAGAAAAGTGC
15606
AAGCAAGGACTATCAAAATGTGCATGTCCAGTTTTTTTCAAACGTTAAAGCGATTTACTACACCCTTTATTATACAGAAGTTCGGTTGAAACC P
16808 242
CATCTATAGTATTTAGGAATCGTAATTTCAGTCTTGGAGCCACCGAGCTTTGTTGAGTTATGAAATAAGTTCACCAACCATTGCGGAAAAGAA
18000
GCGGCAAATGATTTGAGACAAGTCTTTTGAGATTAGAAGTATCCAAAGAGTGCTGACACAAAGCTTTTGTTTAGACGAATATATGGTAAATTC
19202 322
S
T
D
G
L
L
D
F
E
A
L
Q
F
E
V
H
H
D
A
G
E
L
S
R
I
D
V
T
N
G
H
N
Q
N
L
N
P
R
I
Q
E
E
G
202
A
N
282
K
R
T
162
A
V
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122
T
N
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V
A
G
I
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E
F
K
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L
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E
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Y
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L
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M
F
P
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S
N
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N
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F
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Y
F
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D
L
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S
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tL
M
L
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N
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Y
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D
M
Y
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S
A
S
I
AAAGACTACGAAATTAGATTGCTTTACAGTGGGCGTTGGTATCTGGTTTATACTATGTATTTTAAGCATTAACTGAATGAAGATCGATGGAC
E
L
E
K
II
P
9602
G0T E F EDQ0AL I ED LE SD PK EV AE HK M LV DL GR ND I GK I S AATAGGTCAATGAATTCCGTTTCATAAATTGAAAAATCGTACTCATCATTTAAAGTAAGGACCGCGGCTTGTCTGAATTAAGCTTGGTGCCT
K YG
E VP
S I
K YR YVNHH
V F MK VE
I TS
T ANMD
EF
L RP
EV T GE
20404
362
A L
GCGCACATGCCGCGGGACCTACAGGGCACGAACATGAGCTACAAAGATTATGATTTAAAGCAAAACAGGATTTTGGTGAGTATCGTTTCTAC R AT L P A GT L S G AP K HR A YQ0RI Y E F E TQ0K RG I Y G GAI G Y L T AAAAGGAATTGGATTTGCATTGCATTGGACATGTCTTAAAACAAAAAGACAGTTCAGCAGAGCGGATTGTTACATTCGTACCGACATGATACA
21606
AAACTTAACAAGCCAAGGTTATGAAGTTGTCATAATAGACCAAAAGTGTGAGTAAAAATTAACTCACAGAAATTTGTAGTATTTTTATGACG *
24000
G
N
K
C
N
D
A
F
A
I
I
K A QG LL
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T
R
K
V
M
L
D
K
K
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H
A
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G
A
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402
22808
442
Q
Y
-------->456
VGQ0
Vol. 174, No. 20
0021-9193/92/206563-08$02.00/0 Copyright © 1992, American Society for Microbiology
Tryptophan Biosynthesis Genes in Lactococcus lactis subsp. lactis JACEK BARDOWSKI,* S. DUSKO EHRLICH, AND ALAIN CHOPIN Laboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France Received 21 April 1992/Accepted 12 August 1992
The Lactococcus lactis chromosomal region containing the seven structural genes required for tryptophan biosynthesis was characterized by cloning and sequencing. All of the thp genes were identified by the homology of their products with known Trp proteins from other organisms. The identification was confirmed for five genes by their ability to complement tip mutations in Escherichia coli. The seven structural genes are present in the order trpEGDCFBA and span a 7,968-bp segment. Each gene is preceded by a putative ribosome binding site complementary to the 3' end of the L. lactis 16S rRNA. Three pairs of genes (trpG-trpD, trpC-trpF, and trpB-trpA) overlap, and there is intercistronic spacing of 124, 46, and 585 bp between the trpE-trpG, trpD-trpC, and trpF-trpB gene pairs, respectively. No gene fusion was found. Upstream of the tip genes, a 457-bp noncoding DNA segment contains several regions fitting the consensus for gram-positive promoters and one region strongly resembling a transcription terminator. However, it seems unlikely that an attenuation mechanism similar to the one found in E. coli regulates tryptophan biosynthesis in L. lacti&, since no potential leader peptide was detected. We propose that a mechanisms resembling that described in BaciUls spp. can regulate tip genes expression in L. lactis.
Lactococcus lactis has recently become a focus of an increasing number of genetic studies, mostly because of its involvement in milk fermentation. A number of genes important for industrial use of lactococci have therefore been characterized. Examples include genes required for efficient growth in milk, such as those for utilization of lactose or degradation of caseins (16, 30, 57), genes required for synthesis of antimicrobial agents such as nisin and bacteriocins (27, 55), and genes which specify resistance to bacteriophages (8, 25, 26). However, most of these genes are carried on extrachromosomal elements (plasmids or transposons). As a consequence, only a few L. lactis chromosomal genes have been studied; of 39 sequenced genes present in the data bases, only 9 are chromosomal. Six of these, rplO, rpmJ, rpsM, infA, secY, and adk, encoding three ribosomal proteins (L15, B, and S13), initiation factor 1, secretion protein SecY, and adenylate kinase (28, 29), respectively, are clustered. Three others, pabB, pepXP, and usp-45, which encode para-aminobenzoate synthase B, X-prolyl dipeptidyl aminopeptidase (39, 41), and the major unidentified secreted protein (54), respectively, have been isolated. The total length of the known chromosomal sequence is well below 10 kb, and from such scarce data it is difficult to draw conclusions about gene organization and expression in L. lactis. We therefore undertook an analysis of three biosynthetic pathways, i.e., for tryptophan, histidine, and branched-chain amino acids (leucine, isoleucine, and valine), which have been previously studied in numerous other organisms. The main conclusions, based on analysis of over 30 kb of sequence, reported in this and the two accompanying papers (14, 21) are that (i) most if not all of the genes are clustered and are probably organized in operons, (ii) the operons might contain only genes coding for proteins highly homologous to the enzymes required for the biosynthesis of a given amino acid or up to 30% of genes not *
obviously required for this biosynthesis, and (iii) the operons seem compact, and a number of genes overlap. This might be a consequence of the relatively small size of the L. lactis genome (34) and represent a general mode of organization of biosynthetic genes in this organism. In addition, our analysis eliminates certain modes of control of gene expression and strongly suggests the existence of others (for instance, transcriptional attenuation does not control tip or his genes but appears to control leu genes) and therefore provides guidelines for future studies of gene regulation in L. lactis. The thp genes and their products have been characterized for many prokaryotes (12) and represent a paradigm for the study of gene organization and regulation. The tryptophanspecific pathway is catalyzed by seven enzymatic activities encoded by five to seven genes, localized either in one cluster as in the enterobacteriaceae (11, 60), Bacillus subtilis (24, 49), or Brevibacterium lactofermentum (38) or in several clusters as in fluorescent pseudomonads (5, 10). Study of the regulation of the expression of the tip genes has revealed a variety of mechanisms, including repression (59), activation (6), transcriptional attenuation (59), and a novel type of attenuation mediated by a regulatory protein (45). We report here the structure of the L. lactis subsp. lactis chromosomal region which contains the seven structural genes for Trp biosynthesis. Similar studies of genes required for histidine and branched-chain amino acid synthesis are described in the accompanying reports (14, 21). MATERIALS AND METHODS Bacterial strains, plasmids, and media. The bacterial strains and plasmids used are described in Table 1. L. lactis subsp. lactis IL1403 was grown at 30°C in M17 medium (52) in which lactose was replaced by glucose. Escherichia coli was grown in Luria-Bertani medium or in minimal medium M9 (36) at 37°C. B. subtilis was grown at 37°C in LuriaBertani medium or in Spizizen salt minimal medium (1) supplemented with the appropriate amino acid(s). When
Corresponding author. 6563
BARDOWSKI ET AL.
6564
J. BACTERIOL.
TABLE 1. Strains and plasmids Strain or plasmid
Strains E. coli HB101
TG1 SURE
IL2699 IL3012 IL3013 IL3014 IL3015 B. subtilis MT119 L. lactis subsp. lactis IL1403 Plasmids pBluescript pIL253 pIL320
pIL341 pIL356 pIL357 pIL258
pIL499
Reference source or
Characteristics
hsdS recA ara-14 supE lacY galK proA xyl-5 mtl-l rpsL supE thi A(lac-proAB) hsd (F'+ traD proAB lacIqZ M15) recB recJ sbcC uvrC umuC::TnS mcrA mcrB mrr lac A(hsdRMS) endA gyrA thi relA supE (F'+ proAB lac]qZ M15 TnlO) W3110 trpA33 W3110 trpB9578 tnaA2 Smr W3110 trpC9941 W3110 trpD9923 W3110 trpE10220 leuB6 trpC2 r- mPlasmid free, Trp+
Apr, M13 on, pBR322 on Emr, high copy number 12.7-kb L. lactis DNA segment cloned into pIL253 pIL320 with pBR322 inserted into its PstI site pIL341 lacking a 9.8-kb EcoRV segment pIL341 lacking a 6.0-kb EcoRV segment pIL341 lacking a 3.8-kb EcoRV segment 3.6-kb XbaI-XhoI L. lactis DNA segment cloned into pBluescript
36 43
Stratagene
C. C. C. C. C. 50 7
Yanofsky Yanofsky Yanofsky Yanofsky Yanofsky
Stratagene 47 This work
This work This work This work This work
This work
necessary, erythromycin (5 p,g/ml for L. lactis subsp. lactis for E. coli), or 0.3 ,ug/ml for B. subtilis), ampicillin (50 or tetracycline (10 ,ug/ml for E. coli) was added to the
jig/ml
medium. Molecular cloning and DNA manipulations. Plasmid and chromosomal DNAs were prepared as previously described (35, 36, 48, 51). E. coli cells were transformed either by the CaCl2 standard procedure (36) or by electroporation (17). B. subtilis cells were induced for competence and transformed as described by Anagnostopoulos and Spizizen (1), with some modifications (4). Southern blotting and DNA hybridization were done as described by Maniatis et al. (36). DNA probes were prepared by using [cc-32P]dCTP (Amersham) and a nick translation kit (Boehringer Mannheim) according to the supplier's recommendations. Other molecular techniques were used as described by Maniatis and al. (36). DNA sequence analysis. E. coli clones for sequencing were obtained by subcloning specific DNA fragments in the pBluescript plasmids family and by using exonuclease III and mung bean nuclease (Stratagene) to generate a series of clones containing overlapping DNA fragments. Nucleotide sequence was determined as described elsewhere (41). The reported sequence was determined at least twice on both strands. The sequence of a part of the tip region was also determined on a polymerase chain reaction (PCR)-amplified
DNA segment. Synthetic oligonucleotides 5'-GCCTACGGTGTGGATGTT-3' and 5'-AAGGGTCTCCACAC`TGC-3' were used as primers. The PCR mixture was incubated on a Pharmacia-LKB ATAQ Controller apparatus for 30 cycles (94°C for 1 min, 50°C for 1 min, and 72°C for 2 min). The PCR-amplified DNA was subsequently sequenced by using a Taq DyeDeoxy Terminator Cycle sequencing kit and 370A sequencer, both from Applied Biosystems, Foster City, Calif. The DNA and protein sequences were analyzed with BISANCE and University of Wisconsin GCG software packages, implemented at the Centre InterUniversitaire d'Informatique a Orientation Biomedicale (Paris, France). Protein sequences were aligned with MULTALIN software (9). Nucleotide sequence accession number. The GenBank, EMBL, and DDBJ nucleotide sequence accession number for the sequence shown in Fig. 2 is M87483.
RESULTS Cloning of the thp region. The trp region was cloned by preparing a shotgun library of L. lactis subsp. lactis IL1403 total DNA in plasmid vector pIL253 (47) and transforming B. subtilis MT119 to tryptophan prototrophy. Sixteen micrograms of chromosomal DNA was partially digested with endonuclease Sau3A to obtain a majority of DNA fragments larger than 5 kb, ligated with 5 ,ug of BamHI-cleaved vector, and used for transformation. Only one Trp+ transformant was isolated among 14,000 Emr clones. It contained a plasmid named pIL320, consisting of pIL253 carrying a 12.7-kb insert. Southern blot analysis of XbaI-cleaved chromosomal DNA of L. lactis subsp. lactis IL1403 by using pIL320 as a probe revealed that the DNA insert in pIL320 consisted of two noncontiguous Sau3A segments, which were joined during ligation (not shown). Analysis of the insert (see below) revealed that one of the segments encodes all of the genes required for tryptophan biosynthesis except trpE, the 5' end of which was missing. The other Sau3A segment, which carried no tip genes, was not studied further. Southern analysis indicated that the complete trpE gene might be present on a 3.6-kb XbaI-XhoI segment. This segment was cloned in E. coli SURE in the pBluescript vector and detected by colony hybridization, using as a probe a segment internal to trpE gene (Fig. 1). One positive clone was analyzed and found to contain a plasmid of the expected size, which was named pIL499 (Fig. 1). This plasmid was used for further analysis. Complementation experiments. To identify the thp genes present on the cloned segments, pIL320 and pIL499 were made replicative in E. coli by inserting plasmid pBR322 at their unique PstI sites and were used to complement various E. coli Trp- mutants. The pIL320 insert complemented mutations in trpA, trpB, trpC, and t-pD but not in trpE, whereas the pIL499 insert complemented only a trpE mutation. Further complementation analysis, using pIL320 derivatives deleted of various EcoRV fragments, established the approximate locations of the tip genes on the insert (Fig. 1). Nucleotide sequence of the trp region. The nucleotide sequence of the 8,656-bp region was determined (Fig. 2). Open reading frames (ORFs) were searched for by the TestCode software (19), which measures the nonrandomness of nucleotide composition at every third base and therefore does not require knowledge of the codon preference, which has not been established previously for L. lactis subsp. lactis chromosomally carried genes. Eight probable coding regions were detected by this analysis. Eight ORFs were subsequently delineated by searching for translation initiation and
TRYPTOPHAN BIOSYNTHESIS GENES OF L. LACTIS
VOL. 174, 1992
A
6565
Complementation of E. coli trpE trpD trpC trpB trpA
Xhol
pIL341
pIL356 -[ pIL357 -[
XholJ
r
i
pIL358-
-
L
probe
Xbal
Xhol
pIL499 _ _
C
ti
t2
trpB
trpE _m-p3 .p1
p4
p
FIG. 1. (A) Structure of the DNA region carrying the L. lactis trp genes. The thick line indicates the two overlapping DNA fragments containing the entire trp region. The thin line corresponds to DNA that is simultaneously cloned with the tip region but is not contiguous in the L. lactis chromosome. The DNA probe used to identify the clone containing pILA99 is indicated. Plasmids pIL356 to pIL358 were obtained by cleavage of pL;341 with EcoRV and religation and were used in complementation experiments with various E. coli tip mutants. (B) Results of complementation. Growth (+) or lack of growth (-) on a medium without tiyptophan is indicated. (C) Expansion showing the organization of the tip region. pl to p4, putative transcription promoters; tl to t3, putative transcription terminators. ORF1 has no homology with the known thp genes.
termination codons, as well as the putative ribosome binding sites, defined as sequences complementary to the 3' end of the L. lactis subsp. lactis 16S rRNA (15) localized 5 to 7 bp upstream of the translation initiation codon. Assignment of the ORFs. Comparison of the protein sequences deduced from the eight ORFs with the sequences in the GenBank and NBRF data bases revealed that seven ORFs encode polypeptides highly homologous to the known Trp proteins (Table 2). Conserved residues are distributed throughout the sequences of all polypeptides, but the homology is higher in the regions of the Trp proteins which are highly conserved among microorganisms (not shown). Deviations from this general conclusion were found for TrpE, in which the C-terminal region of the polypeptide is better conserved than the N-terminal region, and TrpF, which is
about 130 amino acids longer than TrpF proteins from other organisms and is homologous to their amino-terminal parts only (data not shown). To exclude the possibility that the lactococcal trpF gene was rearranged during or after cloning in E. coli, the appropriate fragment of the chromosome was amplified by PCR and sequenced. Cloned and PCR-amplified segments had identical sequences, which suggests that the lactococcal TrpF protein might be fused to another polypeptide. The function of this polypeptide is not known, since the carboxy-terminal part of the TrpF protein is not homologous with any protein in the data bases. The protein sequence deduced from the remaining ORF, localized upstream of the tip genes, showed no significant homology with any protein in the data bases and was designated ORF1. The assignment of the ORFs in the tip region is summarized in Fig. 1.
TABLE 2. Conservation of proteins involved in tryptophan biosynthesis between L. lactis subsp. lactis and various organisms Organism compared
Bacillus subtilis LactobaciUlus casei Pseudononas aeruginosa Escherichia coli Brevibacterum lactofermentwn Haloferax volcanii Methanococcus voltae Methanobacterium thermoautotrophicum Saccharomyces cerevisiae
% Identical amino acids"
TrpE
TrpG
TrpD
TrpC
TrpF
TrpB
TrpA
40 b 43 36 33
44
40
35
38 38 36 33 32 25 33
24 29
38 36 34
32 38 36 35 35
61 56 59 54 50 49 60 55
36 32 30 28 25 30 31 28
36
26 25 20
Reference(s)
3, 24 42
18, 23 60 38 33 46 40
33 55 20, 53, 61, 62 a Calculated from the multiple alignments obtained by the method of Corpet (9) as the ratio of the number of perfect matches x 100 to the shorter protein length.
b
_, Sequence not available.
34
36
25
36
34
AAGCTTCGTTTAACAAGAAGTTTGTCCAGTTTGATTTGCAATGGTAAGCGTTGAGAAGTTAGAATTGTCGCAGAATCTGAAAATCACACTTTAA
1202
AAAAGOACAAGAATTGACCTTTAACATTATCTGCGCTTTGTTGCTTGGATTTTCTCATTTGTTTTAAGACTTTCTACAGCCAAAACAACAGCGT
2404
RBS
L T I orf 1 -4
L
T
I
I
S
L
L
L
L
A
V
E
F
F
Y
I
H
L
Y
E
T
A
F
S
T
S
T
K
S
T
R
V
GTTTATATGGAAAGAGAGCAGAAGTTCTCAGTCAACGCTTTTAAAACAAGGATTATACGGTTGATGGTTAGGTTAATTATCAAATTCTTTCTCA F
N
G
M
E
E
K
E
L
S
R
S
Q
V
T
L
F
K
Q
N
G
Y
I
G
N
L
G
I
G
L
I
L
A
Y
F
I
S
S
F
ACAATTGAATTGTAGATACACTTTTTAATTATTTGTGGTTTGATGGTCTTGACAGTATAAAAGATATTTCACCAAGTGGTTAGCATTTAGCCTA Q
L
E
I
V
L
R
L
L
I
Y
I
I
V
L
A
Y
L
S
G
L
S
T
N
K
K
I
L
I
Q
T
G
G
L
I
A
A
L
L
A I
35
3606
75
4808
115
6000
TTCTCATTTTTTAAAAATATTTTAATCTTGAAAGCTCAAAAAGTTTACATTATTTCACATCGAAAGTACTCTTATCGAATTTTCAGGAGTCGCG S SF F pl.-lO pl.-35 TGTGGAGCGATAATTCTCAGGCGATGGCTG.TTTGAAGTGAGACGATTGTTTTCCAATTGATTGAACAAAAAATCACGATCTCCGATACAAGGAC GTTGTAGAGAGGAAAGAGATTTTCTAGATTTTTTATACTTTAGGACCTTAATTCGGTGCACGCGTACAACGCCGTAAGTATTGTACTTACGGCT p2.-35
119
-------
7202
8404
-------> tl < .--
antiterminator TTTGTATTTTTTTGAAAGGATTTCTAAAATGAACCAACGATGGCGAAGCTTAAATTTTAACTAATCAGTATCAATGGAAAAGACTACCTCAAACCTATAGATAGAGAGAAAACATGAGAA ~~~~~M R t rpE p3.-10 p3.-35 p2.-10
~~~~~~~~~RBS
-----------
AAATAAAAAATTCACAGAACCTGACCCGTTTCGTTATCTCGCTGAAGGGAAATAAATGATTTAAATCATTCCAAGAAATGCCAACACTTTTC
10808
TTATGCGTGAACCTTGAACACTCAATTTCTGAGGATTTTAGCTAAAGACAAACATTGCGTGAAATCAATGAATTCTGAAAATTATTTTCAGC
12000
K
I
L
A
P
N
T
D
K
V
T
M
H
I
P
F
K
S
I
T
V
G
D
L
Y
I
R
S
L
G
K
L
V
K
D
N
N
K
I
E
V
I
S
I
D
S
E
L
E
N
I
P
P
R
F
E
M
E
N
L
Q
D
E
S
L
K
R
Q
C
V
$
F
42
82
P
13202
CAGAATGTTTTATGTATACAATAGCTTAATTTTACCAAAAGAGAAAACGATTTTATGAAACATACTACATCAGGTTCGAAAAGATTACAAAGC
14404
TGTAAGAATATGAGTATTACTTACTGCAGAGCAAAAAGAATGACGAATAAGAAACGAATTCGCAGCATAGTCTAAGACTTTTGAGAAAAGTGC
15606
AAGCAAGGACTATCAAAATGTGCATGTCCAGTTTTTTTCAAACGTTAAAGCGATTTACTACACCCTTTATTATACAGAAGTTCGGTTGAAACC P
16808 242
CATCTATAGTATTTAGGAATCGTAATTTCAGTCTTGGAGCCACCGAGCTTTGTTGAGTTATGAAATAAGTTCACCAACCATTGCGGAAAAGAA
18000
GCGGCAAATGATTTGAGACAAGTCTTTTGAGATTAGAAGTATCCAAAGAGTGCTGACACAAAGCTTTTGTTTAGACGAATATATGGTAAATTC
19202 322
S
T
D
G
L
L
D
F
E
A
L
Q
F
E
V
H
H
D
A
G
E
L
S
R
I
D
V
T
N
G
H
N
Q
N
L
N
P
R
I
Q
E
E
G
202
A
N
282
K
R
T
162
A
V
E
122
T
N
K
V
A
G
I
L
E
F
K
S
D
K
L
R
T
K
E
E
Y
S
L
S
Q
Y
V
E
R
S
M
F
P
Q
S
N
P
Q
I
Y
S
V
D
G
E
N
F
T
A
F
D
N
F
V
L
I
E
L
K
A
G
Y
F
S
T
E
T
I
L
L
P
D
L
E
R
S
F
K
T
Q
S
G
A
R
E
L
I
Y
K
N
V
V
G
I
A
F
E
F
N
G
I
T
M
D
S
T
F
S
S
N
M
P
E
L
R
F
L
Y
S
I
Y
D
L
E
tL
M
L
M
I
E
Y
N
F
N
K
S
R
Y
R
A
E
D
M
Y
K
S
A
S
I
AAAGACTACGAAATTAGATTGCTTTACAGTGGGCGTTGGTATCTGGTTTATACTATGTATTTTAAGCATTAACTGAATGAAGATCGATGGAC
E
L
E
K
II
P
9602
G0T E F EDQ0AL I ED LE SD PK EV AE HK M LV DL GR ND I GK I S AATAGGTCAATGAATTCCGTTTCATAAATTGAAAAATCGTACTCATCATTTAAAGTAAGGACCGCGGCTTGTCTGAATTAAGCTTGGTGCCT
K YG
E VP
S I
K YR YVNHH
V F MK VE
I TS
T ANMD
EF
L RP
EV T GE
20404
362
A L
GCGCACATGCCGCGGGACCTACAGGGCACGAACATGAGCTACAAAGATTATGATTTAAAGCAAAACAGGATTTTGGTGAGTATCGTTTCTAC R AT L P A GT L S G AP K HR A YQ0RI Y E F E TQ0K RG I Y G GAI G Y L T AAAAGGAATTGGATTTGCATTGCATTGGACATGTCTTAAAACAAAAAGACAGTTCAGCAGAGCGGATTGTTACATTCGTACCGACATGATACA
21606
AAACTTAACAAGCCAAGGTTATGAAGTTGTCATAATAGACCAAAAGTGTGAGTAAAAATTAACTCACAGAAATTTGTAGTATTTTTATGACG *
24000
G
N
K
C
N
D
A
F
A
I
I
K A QG LL
E T LN
T
R
K
V
M
L
D
K
K
K
H
A
V
Q
G
A
G
A
V
I
Y
S
D
P
V
E
E
H
402
22808
442
Q
Y
-------->456
VGQ0
