Cloning of the trp Gene Cluster from a Tryptophan-Hyperproducing Strain of Corynebacterium glutamicum: Identification of a Mutation in the tip ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1993, p. 791-799 Vol. 59, No. 3
0099-2240/93/030791-09$02.00/0
Copyright © 1993, American Society for Microbiology
Cloning of the trp Gene Cluster from a Tryptophan-
Hyperproducing Strain of Corynebacterium glutamicum:
Identification of a Mutation in the tip Leader Sequence
D. M. HEERYt AND L. K. DUNICAN*
Department of Microbiology, University College, Galway, Ireland
Received 29 June 1992/Accepted 6 December 1992
Corynebacterium glutamicum ATCC 21850 produces up to 5 g of extracellular L-tryptophan per liter in broth
culture and displays resistance to several synthetic analogs of aromatic amino acids. Here we report the cloning
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
of the tryptophan biosynthesis (trp) gene cluster of this strain on a 14.5-kb BamHI fragment. Subcloning and
complementation of Escherichia coli trp auxotrophs revealed that as in Brevibacterium lactofermentum, the C.
glutamicum trp genes are clustered in an operon in the order trpE, trpD, trpC, trpB, trpA. The cloned fragment
also confers increased resistance to the analogs 5-methyltryptophan and 6-fluorotryptophan on E. coli. The
sequence of the ATCC 21850 tbpE gene revealed no significant changes when compared to the trpE sequence of
a wild-type strain reported previously. However, analysis of the promoter-regulatory region revealed a
nonsense (TGG-to-TGA) mutation in the third of three tandem Trp codons present within a tip leader gene.
Polymerase chain reaction amplification and sequencing of the corresponding region confirmed the absence of
this mutation in the wild-type strain. RNA secondary-structure predictions and sequence similarities to the E.
coli trp attenuator suggest that this mutation results in a constitutive antitermination response.
Escherichia coli and related enteric bacteria use a com- acid and nucleoside production. Hyperproducing strains are
bination of tryptophan-dependent repression mediated generally selected as mutants resistant to normally lethal
through the TrpR protein (11) and transcriptional attenuation concentrations of amino acid analogs, which can indicate
(17, 37) to control expression of the genes involved in the elevated expression of certain genes or deregulated activity
biosynthesis of L-tryptophan. In addition, other factors of enzymes involved in amino acid biosynthesis (36). For
involved in the control of the common aromatic amino acid example, a 5-fluorotryptophan-resistant strain of Brevibac-
pathway contribute to regulation of the tip genes, e.g., the terium lactofermentum was found to contain a missense
tyrR gene product modulates the expression of a number of mutation in the trpE gene which rendered the encoded
key genes in this pathway (13, 25, 31). Alternative regulatory anthranilate synthase insensitive to feedback inhibition (24).
mechanisms have been discovered in several nonenteric Similarly, a missense mutation or a frameshift mutation in
species. For example, the gram-positive organism Bacillus the 3' end of the C. glutamicum hom gene gave rise to a
subtilis requires the product of its mtrB gene, which is feedback-resistant mutant form of the encoded product,
related to the regulatory RNA-binding protein RegA of homoserine dehydrogenase (1, 29). The tryptophan-hyper-
bacteriophage T4, for transcriptional attenuation of the tip producing strain used in this study, C glutamicum ATCC
operon (2, 9, 34). The tipI gene of Pseudomonas aeruginosa 21850, was obtained by stepwise selection of clones resistant
encodes a transcriptional activator (TrpI) which induces to 5-methyltryptophan (5-MT), 6-fluorotryptophan (6-FT),
expression of the trpBA genes in the presence of indole and four other analogs of aromatic amino acids, in addition
glycerol phosphate (5, 19). It has been demonstrated in vitro to the introduction of aaxotrophy-inducing mutations in side
that TrpI can activate hrpBA while simultaneously autore- pathways to block the production of phenylalanine and
pressing the trpI promoter, which divergently overlaps the tyrosine (12). However, the nature of the mutations that
trpBA promoter (8). In contrast to the above, Rhizobium result in the above phenotypes in ATCC 21850 and related
meliloti appears to control expression of its tip genes solely tryptophan-hyperproducing strains was not determined.
by tryptophan-dependent attenuation (3). These examples Knowledge of which control systems are used by coryne-
illustrate the versatility of prokaryotic regulation of a con- form bacteria to regulate the energetically expensive trypto-
served biosynthetic pathway. phan biosynthesis pathway may facilitate the generation of
Corynebacterium glutamicum belongs to a group of bac- more efficient tryptophan-hyperproducing strains by a mo-
teria which were initially characterized by their natural lecular genetic approach.
ability to excrete large quantities of glutamic acid in broth This report describes the cloning and expression in E. coli
culture. Classical mutagenesis techniques have been em- of the tip gene cluster of a tryptophan-hyperproducing
ployed to broaden the range of amino acids overproduced by mutant of C. glutamicum. We demonstrate that resistances
these bacteria, which are now used commercially for amino to two tryptophan analogs exhibited by this strain are
associated with the trp gene cluster and that these resis-
tances are expressed in E. coli. The deduced amino acid
*
Corresponding author. sequence encoded by the trpE gene is compared with those
t Present address: Laboratoire de Genetique Moleculaire des encoded by trpE genes of other bacteria, and a model for
Eucaxyotes du Centre National de la Recherche Scientifique, Unite
184 de l'Institut National de la Sante et de la Recherche Medicale, regulation of the C. glutamicum trp operon expression by
Institut de Chimie Biologique, Faculte de Medecine, 67085 Stras- attenuation is proposed which is based on similarity to the E.
bourg Cedex, France. coli attenuator and mRNA folding predictions. Finally, we
791792 HEERY AND DUNICAN APPL. ENVIRON. MICROBIOL.
report a mutation in the t-p operon control region of the TABLE 1. Bacterial strains, plasmids, and phages used in
hyperproducing strain and suggest a role for its contribution this study
to the deregulation of the tip operon. Strain, plasmid, or
phage
Relevant characte.stic(S)a Source or
reference
MATERIALS AND METHODS E. coli strains
Bacterial strains and plasmids. The bacterial strains, plas- DH1 recAl thi-I hsdRl7 r- m- D. Hanahan
mids, and phage vectors used in this study are listed in Table Q359 r- m+ (P2 lysogen) Stratagene
1. W3110 (TrpA-) AtonB trpA905 C. Yanofsky
W3110 tnaA2 AtonB trpAB7 C. Yanofsky
Media and growth conditions. Coryneforms were grown on (TrpAB-)
Luria broth or Trypticase soya broth at 30°C. E. coli strains W3110 (TrpC-) tnaA2 AtrpClO-16 C. Yanofsky
were grown routinely on Luria broth or Luria broth supple- W3110 tnaA2 AtrpED102 C. Yanofsky
mented with 10 mM MgSO4- 7H20 and 2% maltose for (TrpED-)
propagation of A phage. M13 phage were propagated in E. W3110 (TrpE-) tnaA2 AtrpE5 C. Yanofsky
coli grown on 2x TY liquid medium (1.6% tryptone, 1% CGSC 4892 pyrD34 trpC45 his-68 CGSCb
tyrA2
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
yeast extract, 0.5% NaCl). Complementation and analog
resistance tests were performed on M9 minimal medium. CGSC 3248 aroG365 aroH367 aroF363 CGSC
hisG4 ilvC7 argE3
Tryptophan production medium M38 supplemented with C. glutamicum
phenylalanine and tyrosine was prepared as described by strains
Shiio et al. (33). Liquid media were solidified by addition of ATCC 13032 Wild type ATCCC
1.2 or 0.7% agar for overlays and supplemented as required ASO19 Rif (derivative of ATCC 40
with 0.02% 5-bromo-4-chloro-3-indolyl-,-D-galactopyrano- 13059)
side (X-Gal), 1 mM isopropylthio-p-D-galactopyranoside ATCC 21850 4-MTr 5-MTr 6-FTr 4-Apr ATCC
(IPTG), 100 pug of ampicillin per ml, and 10 ,ug of tetracycline 4-FPr TyrHxr Phe-
per ml. Tyr
Amino acid analog resistance. Resistance of E. coli strains ATCC 21851 6-FTr 4-APr 4-FPr TyrHxr ATCC
to amino acid analogs was assessed by spreading a lawn of PheHxr Phe- Tyr-
B. lactofermentum Wild type ATCC
cells on selective M9 plates containing appropriate supple- ATCC 13869
ments. A Whatman 3M paper disk (radius, 3 mm) was then C. lilium NRRL Wild type NRRL (USDA)d
placed in the center of the seeded plate, and 250 ptg (in 10 pAl B-2243
of solvent) of the appropriate analog was carefully applied to C. callunae NRRL Wild type NRRL(USDA)
the disk and allowed to dry. The relative degree of resistance B-2244
was then determined by measuring the radius of the zone of
inhibition of cell growth after 3 and 6 days at 37°C. The Plasmids
pBR322 Ampr Tcr F. Bolivar
solvent alone (usually ethanol) had no observable effect on pULT61 B. lactofermentum 6
growth. trpDCB in pBR322
L-Tryptophan production assay. Coryneform strains were pDH121(+) C. glutamicum trpEDCBA This study
cultured in 50 ml of medium M38 (33) supplemented with 150 in pBR322
,ug of L-tyrosine per ml and 300 ,ug of L-phenylalanine per ml pDH122(-) C. glutamicum trpEDCBA This study
in 500-ml baffled flasks for 5 days at 30°C. The level of in pBR322 (opposite
extracellular L-tryptophan in the medium was assayed spec- orientation)
trophotometrically as described by Skogman and Sjostrom
(35). Phages
Manipulation of DNA. High-molecular-weight chromo- XEMBLIII Amersham
somal DNA was prepared from coryneform strains as fol- International
M13mpl8 lacIZ' fl replicon Amersham
lows. Late-log-phase cells (100 ml) were harvested and International
washed in 20 ml of 10 mM Tris (pH 8.0). The pellet was then M13mpl9 lacI'Z' fl replicon Amersham
resuspended in ice-cold acetone, incubated at -20°C for 10 International
min, and centrifuged at 12,000 x g and 4°C for 15 min. The a Abbreviations: r, resistant; Rif, rifampin; Amp, ampicillin; Tc, tetracy-
acetone was decanted, and the pellet was air dried prior to cline; 4-MT, 4-methyltryptophan; 4-AP, 4-aminophenylalanine; 4-FP, 4-fluo-
resuspension in 5 ml of buffer containing 0.5 M sucrose, 10 rophenylalanine; TyrHx, tyrosine hydroxymate; PheHx, phenylalanine hy-
mM Tris (pH 8.0), 0.5% sodium dodecyl sulfate and protease droxymate; Phe-, phenylalanine auxotroph; Tyr-, tyrosine auxotroph.
K (1 mg/ml). After gentle mixing for 2 h at 37°C, lysis was b CGSC, E. coli Genetic Stock Center.
c ATCC, American Type Culture Collection.
achieved by addition of 4 ml of 5% sodium dodecyl sulfate d NRRL (USDA), Northern Regional Research Laboratories, U.S. Depart-
and two freeze-thaw cycles. The lysate was extracted with ment of Agriculture.
phenol and then chloroform and precipitated with 2 volumes
of ethanol. The DNA pellet was then resuspended in 1 ml of
sterile H20.
Plasmid isolations from E. coli were done by the alkaline ments in the size range of 15 to 24 kb were pooled and ligated
lysis method of Birnboim and Doly (4). A DNA and M13 to XEMBLIII BamHI vector arms. The ligation was pack-
single-stranded DNA were isolated by standard methods. E. aged in vitro and used to infect the P2 lysogen E. coli Q359,
coli was transformed with plasmid or M13 DNA by electro- which permits only recombinant clones to propagate.
poration as previously described (14). Polymerase chain reaction and DNA synthesis. Genomic
XEMBLIII gene library. Fifty micrograms of C. glu- DNA sequences were amplified by Taq polymerase by using
tamicum ATCC 21850 genomic DNA was partially digested 25 to 30 polymerase chain reaction cycles with a Perkin
with Sau3A and fractionated on sucrose gradients. Frag- Elmer Cetus DNA Thermal Cycler. The primer sequencesVOL. 59, 1993 C. GLUTAMICUM tip GENE CLUSTER 793
used in amplification of the AS019 trpE gene and regulatory hybridization. Both probes detected a single 14.5-kb BamHI
region were 5'-CATACTGTTGCGATGGTTGACGCGAAC fragment in the three strains of C. glutamicum examined but
GCA-3' and 5'-TGTTCCTCTGAGATTGGTGATGTCATC hybridized to a 9.6-kb BamHI fragment in B. lactofermen-
AT-3'. Oligonucleotides were synthesized on a Beckman tum and C. ilium. Furthermore, the trpB probe detected two
System 200-A DNA synthesizer by BioResearch Ireland. BamHI fragments (4.1 and 1.9 kb) of weaker intensity in C.
DNA labelling, hybridizations, and DNA sequencing. DNA callunae (data not shown). Southern hybridization analysis
probes were labelled with [a-32P]dCrP by random priming. with a panel of restriction enzymes revealed no obvious
Oligonucleotides were end end-labelled with [y-32P]ATP by rearrangements within the trp locus of hyperproducing strain
using the enzyme T4 polynucleotide kinase. Standard meth- ATCC 21850 compared with the isogenic strain C. glu-
ods were used for Southern transfers and hybridization. A tamicum ATCC 13032 or the related strain AS019 (data not
560-bp EcoRI fragment containing an internal portion of the shown).
B. lactofermentum trpB gene was isolated from plasmid Sau3a partial restriction fragments in the range of 12 to 20
pULT61 and used as a trpB probe. The trpE probe was an kb were cloned into the lambda vector EMBLIII to generate
oligonucleotide based on a sequence within the B. lactofer- a library of greater than 5 x 105 recombinant clones.
mentum trpE gene. The sequence used was 5'-CrACACAA Restriction analysis of DNAs prepared from 20 clones veri-
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
GAACCCAAAAATGATTAATAATTGA-3'. Single-strand- fied the presence of unique cloned fragments with an average
ed DNAs from recombinant M13 mp18 and mp19 clones were size of approximately 15 kb. The trpB probe was used to
sequenced with T7 DNA polymerase (Pharmacia LKB) in screen the library by in situ plaque hybridization in aliquots
accordance with the manufacturer's instructions. of 7,000 clones per filter. Clones that hybridized to this probe
Sequence analysis. Sequence data were analyzed with the were detected at a frequency of approximately 30 per 7,000
PCGENE (Intelligenetics, Inc.) and DNA Strider (20) pro- screened. Thirty-five trpB-positive clones detected on one
grams. Protein sequence alignments were generated by the filter were screened with the trpE oligonucleotide probe via
CLUSTAL program (16). Transcript secondary structure the plaque dot assay (28), and three clones which hybridized
was predicted by the RNAFOLD program of Zuker and to both probes were selected for miniprep analysis. Two of
Steigler (41). The EMBL, GenBank, and Swissprot se- the lambda clones contained a 14.5-kb BamHI fragment
quence data bases were accessed for homology searches. which hybridized to both trp probes (data not shown). This
fragment was subcloned into pBR322 in both orientations,
RESULTS and the resulting plasmids were designated pDH121(+) and
pDH122(-) to denote their orientations.
Hyperproduction of L-tryptophan by C. glutamicum ATCC Complementation of E. coli trp mutations. A series of E.
21850. C. glutamicum ATCC 21850 is a tryptophan-hyper- coli auxotrophic strains bearing mutations or deletions in
producing strain that exhibits multiple resistance to chemical one or more of the five tip biosynthesis genes (Table 1) were
analogs of L-tryptophan and other aromatic amino acids transformed with plasmids pDH121(+) and pDH122(-).
(Table 1). This strain was derived from C. glutamicum Plasmid pULT61 (a gift from J. F. Martin), containing the
ATCC 13032 by repeated chemical mutagenesis and screen- trpDCB genes of B. lactofermentum (6), and the parent
ing for increased extracellular production of L-tryptophan vector pBR322 were included in the experiment as positive
and was originally reported to produce up to 10 g of and negative controls, respectively. Several transformants
L-tryptophan per liter (12). Shiio et al. (33) reported that the for each E. coli auxotroph were examined for the ability to
strain produced up to 6 g of L-tryptophan per liter only upon grow on minimal medium (M9) lacking L-tryptophan. Trans-
inclusion of tyrosine and phenylalanine in the production formants containing pBR322 failed to grow on this medium,
medium. In our laboratory, the amounts of L-tryptophan while pULT61 complemented trpB, trpC, and trpD muta-
produced after 5 days of batch culture as described in tions as expected. However, plasmids pDH121(+) and
Materials and Methods varied between 2.5 and 5 g/liter in pDH122(-) complemented mutations in all five E. coli genes
four separate experiments. In contrast, two wild-type C. (trpE, trpD, trpC, trpB, and trpA), indicating that the 14.5-kb
glutamicum strains, AS019 (40) and ATCC 13032 (wild BamHI fragment of ATCC 21850 contains these genes. The
type), produced negligible amounts of extracellular L-tryp- growth rates of auxotrophs on selective medium were differ-
tophan (approximately 400 p,g/liter) in the same assays. ent for pDH121(+) and pDH122(-). Colonies containing
Thus, ATCC 21850 was considered a suitable candidate for pDH121(+) reached full size within 3 days, whereas
investigating the molecular nature of mutations which in- pDH122(-) colonies took up to 6 days to attain a similar
crease synthesis of L-tryptophan. size. This orientation dependence suggests that expression
Cloning of the C. glutamicum ATCC 21850 trp gene cluster. of the cloned tip genes in pDH122(+) may be facilitated by
The tip genes of the coryneform B. lactofennentum have a sequence within the vector, a likely candidate being the tet
previously been cloned (6, 23) and sequenced (22). How- gene promoter.
ever, at the outset of this work, the extent of genetic Subcloning and further complementation analysis showed
similarity among the various glutamic acid-producing that the organization of the genes in the tip cluster of C.
coryneform species was unknown. Sequences internal to the glutamicum is very similar to that in E. coli and identical to
trpE and trpB genes of B. lactofermentum (see Materials and that in B. lactofermentum. Extensive mapping experiments
Methods) were used as probes to test the degree of similarity revealed a high degree of restriction site conservation be-
between several members of this group at the tip locus. tween the two coryneform species at this locus; however,
Genomic DNA from each of the coryneform strains listed in differences were encountered upstream of the tip operon. To
Table 1 was isolated as described in Materials and Methods. determine whether the cloned fragment contained other
Southern analysis revealed strong hybridization to unique genes involved in aromatic amino acid biosynthesis, we
restriction fragments of genomic DNAs from all of the transformed E. coli CGSC 4892 and CGCS 3257 (a tyrA
strains; however, the hybridization patterns indicated some mutant and an aroFGH triple mutant, respectively; Table 1)
genetic diversity among the species tested, as indicated by with pDH121(+) and pDH122(-). However, the transfor-
the sizes of fragments detected and the relative strengths of mants did not exhibit complementation of mutations in the794 HEERY AND DUNICAN APPL. ENvIRON. MICROBIOL.
aroF, aroG, or tyrA gene, as indicated by their inability to
grow on M9 minimal media containing appropriate supple-
ments (data not shown).
Tryptophan analog resistance. The prototrophic strain E.
coli DH.1 was found to be highly sensitive to the analogs B Bg Bg PE H HPP P S Bg SP E EH PBgB
5-MT and 6-Fl, which almost completely inhibited its ''U I II I III
... I. I, , II.. I. 1I I 11
growth in the qualitative assay described in Materials and lmk I
Methods. The sensitivities of various clones were deter-
mined on seeded plates by measuring the radius of the zone
of cell growth inhibition around a filter disk containing the trpE I trpD(G) I trpC I trpB| trpAI
test compound (Materials and Methods). To determine
whether the tryptophan analog resistance phenotypes exhib-
ited by C. glutamicum ATCC 21850 are associated with the
fragment containing the trp gene cluster, DH.1 was trans-
formed with pDH121(+), pDH122(-), pULT61, or parental ,BamHl 0.38
vector pBR322. Transformants containing pBR322 displayed
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
no increased resistance to 5-MT or 6-FT (radii of cleared
zone, >45 mm), even after 10 days of incubation. DH.1
containing pULT61 exhibited slightly increased resistance to
5-MT and 6-FT (radii of cleared zones, 28 and 34 mm,
respectively). In contrast, DH.1 transformed with pDH BamH 14.88
121(+) was strongly resistant to both 5-MT and 6-FT (radius
of cleared zone,VOL. 59, 1993
E.coli
C. glut
E. coli
C. glut
E. coli
C. glut
E. co1i
C. glut
E. co1i
C. glut
E .coli
C. glut
E. coli
C. glut
~. ~ .
MSTNPHVFSL --- DVRYHEDASALFAHLGGTTADDAALLESADITTKNGISSLAVLKSSV
....
RITALGDTVTIQALSGNGEALLALLDNALPAGVESEQSPNCRVLRFPPVSPLLDEDARLC
RITCTGNTVVTQPLTDSGRAVVARLTQQLGQYNTAENT ----- FSFPASDAV-DERERLT
SLSVFDAFRLLQNLLNVPKEEREAMFFSGLFSYDLVAGFEDLPQLSAE-NNCPDFCFYLA
APSTIEVLRKLQ--FESGYSDASLPLLMGGFAFDFLETFETLPAVEESVNTYPDYQFVLA
. ...
ETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNELRQQLTEAAPPLPVV- -SVPH---
EIVLDINHQDQTAKLTGVSNAPGE ----- LEAELNKLSLLIDAAL PATEHAYQTTPHDGD
.
-MRCECNQSDEEFGGVVRLLQKAIRAGEIFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPY
TLRVVADIPDAQFRTQINELKENIYNGDIYQVVPARTFTAPCPDAFAAYLQLRATNPSPY
MFFM----QDNDFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRDLDSRI
MFYIRGLNEGRSYELFGASPESNLKFTAANRELQLYPIAGTRPRGLNPDGSINDELDIRN
...... .. ...
ELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVADLTKVDRYSYVMHLVSRWGELR
ELDMRTDAKEIADDTMLVDLARNDLARVSVPASRRVADLLQVDRYSRVMHLVSRVTATLD
C. GLUTAMICUM trp GENE CLUSTER
downstream from the proposed -10 region, as determined
by Si mapping (20a).
A 17-codon open reading frame containing three tandem
Trp codons is located immediately downstream of the C.
glutamicum AS019 trp operon promoter and approximately
200 bp proximal to the trpE gene. The sequence of this
region is identical in B. lactofermentum (30), and the pro-
posed initiation codon of this open reading frame is a GUG
codon, which may also be used as an initiation codon in
795
prokaryotes. The position, length, and sequence of this open
reading frame strongly suggest its function as a leader
peptide gene, as found in other bacterial genes which are
regulated by attenuation. However, as shown in Fig. 3A,
ATCC 21850 contains a nonsense (TGG-to-TGA) mutation
in the third Trp codon in the leader gene.
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
The amino acid sequences of the E. coli and coryneform
E. coli
C. glut
HDLDALHAYRACMNMGTLSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIVI leader peptides share very little homology. In contrast, the
PELDALDAYRACMNMGTLTGAPKLRAMELLRGVEKRRRGSYGGAVGYLRGNGDMDNCIVI
DNA sequences exhibit a much higher degree of conserva-
E. co1i RSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRAIATAHHAQETF-- tion (approximately 60%), supporting the hypothesis that it
C. glut RSAFVQDGVAAVQAGAGVVRDSNPQSEADETLHKAYAVLNAIALAAGSTLEVIR is the nucleotide sequence which is important in attenua-
FIG. 2. Alignment of the deduced amino acid sequences of the tional control (Fig. 3B).
trpE genes of C. glutamicum (C. glut) and E. coli. The alignment Predicted secondary structure of the attenuator region. The
was generated by the CLUSTAL program of Higgins and Sharp RNAFOLD program (41) was used to predict possible RNA
(16). Asterisks indicate matched residues, dots indicate conserva- secondary structures within the C. glutamicum attenuator
tive substitutions, and dashes are used to introduce gaps to facilitate region. It has been estimated that a ribosome will sterically
the alignment. The nucleotide sequences of the C. glutamicum trpL overlap 10 to 12 bp on either side of the codon at which it is
and trpE genes are available from the EMBL data library under positioned (37) and that this sequence will not be available
accession no. X55994.
for participation in transcript folding. We took this steric
effect into consideration by initiating folding from a position
10 bp downstream from hypothetical stall sites. Therefore,
however, that the A nucleotide at the fourth position within the structure predicted for the RNA sequence from +87 to
the proposed -35 hexamer sequence in the B. lactofernen- +300 represents positioning of the ribosome at any of the
tum promoter is replaced by a G in the C. glutamicum ATCC sensory Trp codons, whereas the sequence from +99 to
21850 (Fig. 3A) and AS019 (15) promoters. In B. lactofer- +300 represents positioning of the ribosome at the stop
mentum, transcription initiates from an A nucleotide 16 bp codon of the leader transcript. Figure 4 shows the alternative
structures predicted to form during conditions that favor
attenuation or readthrough. The position of the nonsense
mutation in the ATCC 21850 tip leader would therefore
(A) prevent formation of the termination structure and lead to a
constitutive antitermination response.
-35 -10 +1
GAGCCTGCGGAAACTACCAAGAACCCAAAAAATTAATAATGAG_CAA
DISCUSSION
GCTTCCCACTTATGTGATAAAGTCCCATTTT GTG AAT AAC TCT TGT
M N N S C Production of particular amino acids in coryneforms can
CTC AGT CAA AGC ACC CAG TGG TGG TGA CGC GCT AAC TAA
L S Q S T Q W W R A N
be improved by gene amplification (24, 26). This finding,
coupled with recent improvements in the frequency of
GCGACCTGACACCTCAAGTTGTTTTCACTTTGATGAATTTTTTAAGGCTC transformation of corynebacteria (7, 32), offers considerable
scope for increasing product yields by reintroducing dereg-
ulated genes into engineered strains. Thus, it is important to
(B)
understand the regulation of gene expression in coryne-
forms. In this study, it was shown that the genomic organi-
M N N S C L S Q S T Q W W W R A N
zation of trp genes and the sequences of the tipE genes and
GTGAATAACTCTTOGTTCAGTCAAAGCACCCAGTGGTGGTOGGCGCGCTAACTAA C. glu tip regulatory regions are highly conserved in C. glutamicum
ATGAA-AGCAAT?CGTACTGAAAG-------GTTGGTGCGCACTTCCTGA E coli
.
and B. lactofermentum. Southern hybridizations have also
M K A I F V L K G W W R T S indicated conservation of tip gene sequences in two other
FIG. 3. (A) Nucleotide sequence of the C. glutamicum ATCC coryneform species, C. lilium and C. callunae. Interestingly,
21850 (C. glu) tip operon promoter and tip leader gene. Potential the deduced amino acid sequences of coryneform trpE genes
-35 and -10 boxes are underlined. The -15 TG and a possible share greater homology with known sequences from enteric
transcription start site are also underlined. (Note that position + 1 is bacteria than with sequences from other species, including
an arbitrary designation.) The G nucleotide present in the -35 box gram-positive organisms. This is surprising, as coryneforms
of C. glutamicum ATCC 21850 and AS019 but not in B. lactofer- and enteric bacteria are generally considered to be phyloge-
mentum is in bold type. The position of the G-to-A nonsense
mutation in the tip leader gene is also in bold type. (B) Nucleotide netically distant.
sequence comparisons of the tip leader genes of C. glutamicum and The five genes in the C. glutamicum trp cluster can
E. coli. Dashes are used to introduce gaps for alignment, and an complement mutations in their counterparts in E. coli.
asterisk indicates identity. The deduced amino acid sequences are However, the efficiency of expression of the C. glutamicum
also shown. tip genes in E. coli was found to be dependent on their796
0S¢,¢+
N
CN C
,¢VOL. 59, 1993 C. GLUTAMICUM trp GENE CLUSTER 797
FIG. 4. Model for regulation of expression of the C. glutamicum trp operon by attenuation. Alternative secondary structures predicted for
the attenuator region of the C. glutamicum tip operon transcript in conditions that favor attenuation of transcription (A) or antitermination
(B) are shown. When charged tRNA is not limiting, ribosome stalling at the leader stop codon prevents formation of the 2:3 structure but
allows formation of the 3:4 terminator hairpin (A). Alternatively, when charged tryptophanyl tRNA is limiting, the ribosome may stall at one
of the three tandem sensory Trp codons, resulting in formation of the antiterminator structure (B) and transcriptional readthrough into the
tip operon. In tryptophan-hyperproducing C. glutamicum ATCC 21850, stalling of the ribosome at the stop codon created by the nonsense
mutation in the third sensory Trp codon would therefore cause constitutive antitermination. These data were generated by the RNAFOLD
program, which uses the algorithm of Zuker and Steigler (40). Nucleotides are numbered relative to the A designated +1 in Fig. 3A. The
sensory Trp codons, the leader gene stop codons, and the trpE start codons are boxed. The codon on which the ribosome is assumed to be
positioned (Ribosome Stall Site) is indicated by heavy underlining, and the boxed, shaded areas approximate the sequences estimated to be
sterically overlapped by the ribosome and thus prevented from participation in the secondary structure. The position of the G-to-A nonsense
mutation in the tip leader gene of ATCC 21850 is also indicated. The terminator hairpin (3:4) and antiterminator (2:3) loops are indicated. The
larger invariant hairpin structure which is located between the attenuator and the trpE start codon is also represented.
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
orientation in the vector, suggesting that the coryneform involved in the formation of the rho-independent terminator
promoter functions poorly in this organism. It is unlikely that (3:4) structure in E. coli. The similarities with leader gene
a sequence required for the function of the C. glutamicum (Fig. 3A) and attenuator sequences in other bacteria, cou-
trp promoter is absent in the cloned fragment, as there is pled with the finding that a mutation in the proposed termi-
approximately a 7-kb sequence upstream of the operon. It is nation loop sequence causes increased transcription of the
also unlikely that the cloned fragment is discontiguous, as an tip genes in B. lactofermentum (24), provide convincing
identically sized (14.5-kb) fragment was detected by South- evidence that this region constitutes the tip attenuator in C.
ern blot hybridization (data not shown). The C. glutamicum glutamicum and B. lactofermentum.
tip promoter region is almost identical to the B. lactofermen- The attenuator region of C glutamicum, although slightly
tum tip promoter, which has been localized to a 51-bp larger, is remarkably similar in overall structure to the E. coli
AluI-HindIII fragment which is functional as a promoter in attenuator. For analysis of secondary structure within the
E. coli. However, as shown in this study, the sequences of attenuator, an A nucleotide was arbitrarily designated + 1 on
the proposed -35 boxes of the trp promoters in these two the basis of its position relative to the -10 sequence (Fig.
species differ by 1 bp. It is possible that this difference 3A), and it has subsequently been shown that this is very
accounts for the apparently poor efficacy of the C. glu- close to the true transcription start site (20a). As shown in
tamicum promoter in E. coli, but this remains to be studied Fig. 4, alternative and mutually exclusive transcript confor-
in detail. mations are predicted, depending on the position of the
Increased resistance to the analogs 5-MT and 6-FT has stalled ribosome on the nascent transcript, an event which
been used in the selection of ATCC 21850 and other trypto- governs how much of the sequence is available for folding.
phan-hyperproducing strains of C. glutamicum (12). Such Folding of the transcript from the position arbitrarily desig-
resistance phenotypes are of great interest, as they can nated +1 (i.e., without consideration of ribosome protec-
provide positive selection for genes which may directly tion) predicts a 1:2:3:4 structure (not shown) identical to that
contribute to deregulation of tryptophan biosynthesis, e.g., reported by Sano and Matsui (30), who postulated that this
mutations which relieve feedback inhibition of enzymes. Our structure represents the transcription terminator in B. lac-
results show that genes responsible for resistance to 5-MT tofermentum. However, as practically all of the leader
and 6-FT in ATCC 21850 are tightly associated with the tip transcript sequence is involved in the formation of this
gene cluster and are expressed in E. coli. Expression of the potential structure, it seems likely that the primary loop
6-FT resistance phenotype, like that of the tip genes, is represents the transcription pause loop (or 1:2 loop), as
orientation dependent in E. coli, providing circumstantial observed in E. coli, whose function is to synchronize the
evidence that it is regulated by the tip promoter and associ- transcription and translation processes (37, 38). In fact, the
ated with one of the tip genes. However, in contrast to a position of the Trp codons in this structure is identical to
previous report concerning 5-fluorotryptophan resistance in their position in the pause loop of E. coli. When the
B. lactofennentum, we detected no mutation in the gene that sequence available for folding begins at position +101 (i.e.,
encodes component I of the anthranilate synthase complex. the leader stop codon plus 12 bp), which approximates the
Thus, the 6-FT resistance in strain ATCC 21850 may result situation resulting from ribosome stalling at the leader stop
from a mutation in a different gene in this pathway or may be codon, a structure reminiscent of the termination (3:4) loop
due to a mutation which enhances transcription of the tip (or of the E. coli attenuator is generated (Fig. 4A). This struc-
other) genes, resulting in elevated levels of their products. ture, formed by the sequence between +133 and +170,
Expression of the gene responsible for 5-MT resistance, on contains the CGGGC/GCCCG-like motif and is followed by
the other hand, is not dependent on orientation, and thus the a poly(U)-rich stretch, as found in most prokaryotic rho-
gene may be expressed from a distinct transcriptional unit. independent terminators (37). This position of the terminator
The sequence between the C. glutamicum tip leader gene hairpin in the transcript is consistent with those in other
and the trpE start codon contains extensive hyphenated operons which are regulated by attenuation, i.e., centered in
dyad symmetry. Analysis of this sequence (which is identical the region from + 130 to + 170 (17). In support of this, a
in B. lactofermentum) revealed that it contains short se- mutation in this region predicted to destabilize the hairpin
quences which are found in the tip attenuators of other structure resulted in enhanced expression of the tip operon
bacteria (37). These include the CGGGC motif (in this case, in a tryptophan-hyperproducing mutant of B. lactofermen-
CGAGC) which occurs 14 bp downstream of the leader Trp tum (24). A more extensive and invariant hairpin structure
codons and a complementary motif (GCTCG) further down- can be formed by the sequence from +170 to +290, but it is
stream, which are thought to be involved in stabilizing the not known whether this sequence is required for attenuation.
antitermination (2:3) structure. The latter sequence is also In the event of reduced cellular levels of charged trypto-798 HEERY AND DUNICAN APPL. ENvIRON. MICROBIOL.
phanyl tRNA, the rate of translation of the sensory Trp mation of whole cells of amino acid producing coryneform
codons in the leader transcript is presumably decreased. bacteria using high voltage electroporation. Bio/Technology
Although there is generally only a short distance (12 to 18 bp) 7:1067-1070.
between the sensory codons and the leader stop codon, the 8. Gao, J., and G. N. Gussin. 1991. Activation of the trpBA
promoter of Pseudomonas aeruginosa by TrpI protein in vitro.
predicted effect on the transcript secondary structure is J. Bacteriol. 173:3763-3769.
dramatic. As shown in Fig. 4B, initiation of folding from 9. Gollnick, P., S. Ishino, M. I. Kuroda, D. J. Henner, and C.
positions +83 to +89 (i.e., Trp codon 1, 2, or 3 plus 12) Yanofsky. 1990. The mtr locus is a two-gene operon required for
predicts a very different structure. In this case, the sequence transcription attenuation of the trp operon of Bacillus subtilis.
from +89 to +100 is unmasked and can form a stable 2:3 Proc. Natl. Acad. Sci. USA 87:8726-8730.
loop involving the region from +90 to +148, preventing 10. Graves, M. C., and J. C. Rabinowitz. 1986. In vivo and in vitro
formation of the 3:4 termination structure. Interestingly, the transcription of the Clostridium pasteurianum ferroxidin gene:
structure of the large hairpin from positions + 170 to +290 is evidence for extended promoter elements in gram-positive or-
ganisms. J. Biol. Chem. 261:11409-11415.
unaltered. As in E. coli, a CGGGC-like sequence (CGAGC) 11. Gunsalus, R. P., and C. Yanofsky. 1980. Nucleotide sequence
located 14 bp downstream from the sensory Trp codons is and expression of Escherichia coli trpR, the structural gene
involved in the 2:3 structure. This sequence is likely to be of the tip aporepressor. Proc. Natl. Acad. Sci. USA 77:7117-
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
masked in conditions that favor termination, and its comple- 7121.
mentary sequence, GCUCG, would be involved in formation 12. Hagino, H., and K. Nakayama. 1975. L-Tryptophan production
of the 3:4 terminator hairpin (Fig. 4B). by analog-resistant mutants derived from a phenylalanine and
The tip leader sequence of hyperproducing strain ATCC tyrosine double auxotroph of Corynebacterium glutamicum.
21850 contains a G-to-A mutation which transforms the third Agric. Biol. Chem. 39:343-349.
sensory Trp codon to a TGA stop codon. This mutation 13. Heatwole, V. M., and R. L. Somerville. 1991. The tryptophan-
would be expected to result in ribosome pausing at the specific permease gene, mtr, is differentially regulated by the
tryptophan and tyrosine repressors in Escherichia coli K-12. J.
mutated Trp codon, regardless of the availability of charged Bacteriol. 173:3601-3604.
tryptophanyl tRNA. A previous study using the trp attenu- 14. Heery, D. M., and L. K. Dunican. 1989. Improved M13 cloning
ator of E. coli has shown that mutation of the second sensory using electroporation. Nucleic Acids Res. 17:8006.
TGG codon to TGA alters expression at this locus and 15. Heery, D. M., and L. K. Dunican. 1990. Nucleotide sequence of
results in almost complete readthrough into the gene coding the Corynebacterium glutamicum trpE gene. Nucleic Acids
sequences (18). Further work involving Northern (RNA) Res. 18:7138.
analysis, a suitable reporter gene, or a polymerase chain 16. Higgins, D. G., and P. M. Sharp. 1988. CLUSTAL: a package
reaction-based approach is required to determine whether for performing multiple sequence alignment on a microcom-
this mutation causes a constitutive antitermination response, puter. Gene 73:237-244.
17. Landick, R., and C. Yanofsky. 1987. Transcription attenuation,
resulting in increased readthrough of the tip attenuator. p. 1276-1301. In F. C. Neidhardt, J. L. Ingraham, K. B. Low,
B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.),
Escherichia coli and Salmonella typhimurium: Cellular and
ACKNOWLEDGMENTS molecular biology, vol. 1. American Society for Microbiology,
Washington, D.C.
We thank Charles Yanofsky, A. J. Sinskey, and Juan Martin for 18. Landick, R., C. Yanofsky, K. Choo, and L. Phung. 1990.
gifts of strains or plasmids and BioResearch Ireland at this Depart- Replacement of the Escherichia coli trp operon attenuation
ment for oligonucleotide synthesis. We also thank P. Moran for control codons alters operon expression. J. Mol. Biol. 216:25-
critical reading of the manuscript and R. Fitzpatrick for useful 37.
discussions. 19. Manch, J. N., and I. P. Crawford. 1982. Genetic evidence for a
This work was funded by the European Community BRIDGE positive regulatory factor mediating induction on the tryptophan
programme. pathway of Pseudomonas aeruginosa. J. Mol. Biol. 156:67-77.
20. Marck, C. 1988. "DNA Strider": a 'C' program for the fast
REFERENCES analysis of DNA and protein sequences on the Apple Macintosh
1. Archer, J. A. C., D. E. Solow-Cordero, and A. J. Sinskey. 1991. family of computers. Nucleic Acids Res. 16:1829-1836.
A C-terminal deletion in Corynebacterium glutamicum homo- 20a.Martin, J. F. Personal communication.
serine dehydrogenase abolishes allosteric inhibition by L-threo- 21. Matsui, K., M. Ishida, M. Tsuchiya, and K. Sano. 1988. Con-
nine. Gene 107:53-59. struction of tryptophan producing recombinant strains of Brevi-
2. Babitzke, P., P. Gollnick, and C. Yanofsky. 1992. The mtrAB bacterium lactofermentum using the engineered tip operons.
operon of Bacillus subtilis encodes GTP cyclohydrolase I Agric. Biol. Chem. 52:1863-1865.
(MtrA), an enzyme involved in folic acid biosynthesis, and 22. Matsui, K., K. Miwa, and K. Sano. 1986. Complete nucleotide
MtrB, a regulator of tryptophan biosynthesis. J. Bacteriol. and deduced amino acid sequences of the Brevibacterium lact-
174:2059-2064. ofermentum tip operon. Nucleic Acids Res. 14:10113-10114.
3. Bae, Y. M., and I. P. Crawford. 1990. The Rhizobium meliloti 23. Matsui, K., K. Miwa, and K. Sano. 1987. Cloning of the
tzrpE(G) gene is regulated by attenuation, and its product, tryptophan genes of Brevibacterium lactofermentum, a glutamic
anthranilate synthase, is regulated by feedback inhibition. J. acid producing bacterium. Agric. Biol. Chem. 51:823-828.
Bacteriol. 172:3318-3327. 24. Matsui, K., K. Miwa, and K. Sano. 1987. Two single-base-pair
4. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction substitutions causing desensitization to tryptophan feedback
procedure for screening recombinant plasmid DNA. Nucleic inhibition of anthranilate synthase and enhanced expression of
Acids Res. 7:1513-1523. tryptophan genes of Brevibacterium lactofermentum. J. Bacte-
5. Crawford, I. P., and I. C. Gunsalus. 1966. Inducibility of riol. 109:5330-5332.
tryptophan synthase in Pseudomonasputida. Proc. Natl. Acad. 25. Muday, G. K., D. I. Johnson, R. L. Sommerville, and K. M.
Sci. USA 56:717-724. Hermann. 1991. The tyrosine repressor negatively regulates
6. Del Real, G., A. Aguilar, and J. F. Martin. 1985. Cloning and aroH expression in Escherichia coli. J. Bacteriol. 173:3930-
expression of tryptophan genes from Brevibacterium lactofer- 3932.
mentum in Escherichia coli. Biochem. Biophys. Res. Commun. 26. Ozaki, A., R. Katsumata, T. Oka, and A. Furuya. 1985. Cloning
133:1013-1019. of the genes concerned in phenylalanine biosynthesis in Coryne-
7. Dunican, L. K., and E. Shivnan. 1989. High frequency transfor- bacterium glutamicum and its application to breeding of aVOL. 59, 1993 C. GLUTAMICUM trp GENE CLUSTER 799
phenylalanine producing strain. Agric. Biol. Chem. 49:2925- 1986. Novel form of transcription attenuation regulates expres-
2930. sion of the Bacillus subtilis tryptophan operon. J. Bacteriol.
27. Peoples, 0. P., W. Liebi, M. Bodis, P. J. Maeng, M. T. Follettie, 166:461-471.
J. A. Archer, and A. J. Sinskey. 1988. Nucleotide sequence and 35. Skogman, G. S., and J. Sjostrom. 1984. Factors affecting the
fine structural analysis of the Corynebactenum glutamicum biosynthesis of L-tryptophan by genetically modified strains of
hom-thrB operon. Mol. Microbiol. 2:63-72. Escherichia coli. J. Gen. Microbiol. 130:3091-3100.
28. Powell, R., J. Neilan, and F. Gannon. 1986. Plaque dot assay. 36. Sommerville, R. L. 1983. Tryptophan: biosynthesis, regulation
Nucleic Acids Res. 14:1541. and large scale production, p. 351-378. In K. L. Herrmann and
29. Reinscheid, D. J., B. J. Eikmanns, and H. Sahm. 1991. Analysis R. L. Somerville (ed.), Amino acids: biosynthesis and genetic
of a Corynebacterium glutamicum hom gene coding for a regulation. Addison-Wesley Publishing Co., Inc., Reading,
feedback-resistant homoserine dehydrogenase. J. Bacteriol. Mass.
173:3228-3230. 37. Yanofsky, C. 1981. Attenuation in the control of expression of
30. Sano, K., and K. Matsui. 1987. Structure and function of the trp bacterial operons. Nature (London) 289:751-758.
operon control regions of Brevibactenum lactofermentum, a 38. Yanofsky, C. 1984. Comparison of regulatory and structural
glutamic acid-producing bacterium. Gene 53:191-200. regions of genes of tryptophan metabolism. Mol. Biol. Evol.
31. Sarsero, J. P., P. J. Wookey, and A. J. Pittard. 1991. Regulation 1:143-161.
of expression of the Escherichia coli K-12 mtr gene by the TyrR 39. Yanofsky, C. 1988. Transcription attenuation. J. Biol. Chem.
Downloaded from http://aem.asm.org/ on March 8, 2021 by guest
protein and Trp repressor. J. Bacteriol. 173:4133-4143. 263:609-612.
32. Schwarzer, A., and A. Puhler. 1991. Manipulation of Coryne- 40. Yoshihama, M., K. Higashiro, E. A. Rao, M. Akedo, W. G.
bacterium glutamicum by gene disruption and replacement. Shanabruch, M. T. Follettie, G. C. Walker, and A. J. Sinskey.
Bio/Technology 9:84-87. 1985. Cloning vector system for Corynebacterium glutamicum.
33. Shiio, I., S. Sugimoto, and K. Kawamura. 1984. Production of J. Bacteriol. 162:591-597.
L-tryptophan by sulphonamide resistant mutants. Agric. Biol. 41. Zuker, M., and P. Steigler. 1981. Optimal computer folding of
Chem. 48:2073-2080. large RNA sequences using thermodynamics and auxiliary
34. Shimotsu, H., M. I. Kuroda, C. Yanofsky, and D. J. Henner. information. Nucleic Acids Res. 9:133-148.You can also read
