Gene, 108 (1991) 39-45 0
1991 Elsevier
GENE
Science
Publishers
B.V. All rights reserved.
39
0378-l 119/91/$03.50
06185
Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes (Recombinant
DNA;
Gram-positive
bacteria;
mutagenesis;
xylE
reporter
gene; operator;
transcription
start points)
D. Speck”, 1. Ohsawa b, R. Gloeckler a, M. Zinsiusa, S. Bernard”, C. Ledouxa, T. Kisou b, K. Kamogawa b and Y. Lemoine”* ” Transghe S.A., 67082 Strasbourg Cedex (Francej; and h Biological Science Institute, R&D Center, Nippon Zeon Co. Ltd., Kawasaki 210 (Japan) Tel. (al-044)276-3744 Received by J.-P. Lecocq: 30 May 1991 Revised/Accepted: 20 August/23 August 199 I Received at publishers: 20 September 1991
SUMMARY
The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-701. Sequence analysis revealed that they are organized into two gene clusters, designated bioXWF and bioDAYB. The 5’-noncoding region of the bioD locus fused to the xylE reporter gene was inserted into the Gram-positive PUB 110 replicon and the resulting plasmid was introduced into B. sphaericus IF03525. Transformants expressed the xylE gene only if the biotin concentration in the growth medium remained below 50 ng/ml. After mutagenesis, colonies were screened for their ability to express the chromogenic marker in the presence of an excess of biotin. Most of these mutants escaped biotin repression of xylE gene expression. Classical genetic analysis showed they formed two main categories: chromosomal mutations, pleiotropically acting in tram on both bioXWF and bioDAYB 5’-noncoding regions, in which a 15-bp region common to both promoters represented a hot-spot for the second class of plasmid-associated mutations. These results, completed by the identification of transcription start points for the bioD and bioX genes, strongly suggest that this 15-bp sequence overlaps the site of a biotin-mediated negative regulation circuit controlling the transcription of the bio genes.
INTRODUCTION
Biotin represents an essential cofactor for carboxylase binding to carbon dioxide and activation (Moss et al., 1971). In addition to its crucial role in the cell metabolism, Correspondence
IO: Dr. D. Speck, Transgene
67082 Strasbourg
1I rue de Molsheim,
M&SO,.
Cedex (France)
Tel. (33-88)27.91.00; * Present
S.A.,
67085 Strasbourg
L.G.M.E.,
Faculte
11 rue Humann,
dine; acrylamide-gel
Bacihs;
BSA,
bio, gene(s)
bovine
dethiobiotin; Casamino
serum GP. acids
acid;
r-DHB,
of the biotin synthesis albumin;
in 1 liter: (vitamin-free
C230,
pathway;
catechol
20 g glycerol/30 Difco)/l
r-dehydrobiotin;
g
B.,
bp, base pair(s);
2,3-dioxygenase; g proteose K,HPO,/O.S
DTB,
peptone/ g
FeSO,
7H,0/0.01
7.0; h, hour(s);
g
KCljO.5 g
transcription x~IE, gene carrier
electrophoresis;
state.
M MgC1,/35
site;
ng
LB, Luria-Bertani
NTG; NN’-nitrosoguanipromoter; PAGE, poly-
Pm, pimelate; SMMP,
4-6H,0/20
7-keto-8-aminopelargonic
or 1000 bp; Km, kanamycin;
ribosome-binding
maleatej0.04
MnSO,
g
7-KAP,
mu, milliunit(s); nt, nucleotide(s); P, oligo, oligodeoxyribonucleotide;
RBS, actithiazic
Ig
(medium);
Cedex (France)
ACM,
HCI pH
acid; kb, kilobase de Medecine,
Tel. (33-88)37.12.55. Abbreviations:
7H,O/O.O
thiamine
Fax (33-88)22.58.07.
address:
this molecule is the subject of increasing industrial interest due to its broad range of uses, e.g., animal foodstuffs, pharmaceutical and cosmetic applications, and glutamate fermentation. Biotin biosynthesis and regulation have been extensively
0.5 M
g per liter of antibiotic
R, resistance/resistant; sucrose/O.04 medium
M
Na’
3 (Difco); fsp,
start point(s); TVA, 5-(2-thienyl)-valeric acid; wt, wild type; encoding catechol 2,3_dioxygenase; [ 1, denotes plasmid-
40 studied in E. coli (reviewed by Eisenberg, 1985). Five genes coding for biosynthetic enzymes are organized in a divergent operon structure, bioABFCD (Guha et al., 1971). The repressor encoded by the birA locus is a bifunctional molecule carrying the holoenzyme synthetase activity (Barker and Campbell; 1981). This protein converts biotin into a biotin-adenylate form before transferring it to the apocarboxylase enzyme. This activated form of biotin also acts as the corepressor in the regulation of the transcription of the bioABFCD operon. E. coli mutants derepressed for biotin synthesis have been obtained either by cross-feeding experiments with biotin auxatrophic strains (Pai, 1972) or by selection for resistance to a biotin analog, r-DHB (Pai, 1974). Such clones are characterized increase in their level of biotin secretion.
by a
lOOO-fold
When grown in the presence of pimelic acid. Gram-positive B. sphuericw can accumulate high amounts of vitamers, the intermediary metabolites in biotin biosynthesis (Ogata et al., 1965; lzumi et al., 1973). Analysis of culture supernatants identified the main vitamer as DTB, the direct precursor of biotin. However, under these conditions, biotin accumulation -to the extent obtained in the E. coli derepressed strain - remained low for all the tested B. sphuerims strains. A strong biotin-mediated decrease in the level of the biotin biosynthetic enzymes was indeed demonstrated for 7-KAP synthetase (Izumi et al., 1973) and for biotin synthetase (Ogata and Izumi, 1974). It was thus of great interest to isolate B. ,~phcteuicus mutants which were no longer subjected to this control. The classical approach of studying resistance to vitamer structural analogs, such as TVA or ACM, has already been reported but no conclusive data were obtained concerning the nature ofthe isolated mutants (Yamada et al., 1983). Gent fusion, a useful tool for the study of gene regulation (Casadaban, 1976) was applied to B. ~~~huer~~~~.s, after isolation and characterization of genes involved in biotin synthesis, as described by Gloeckler et al. (1990). Seven open reading frames, organized in two gene clusters, were totally sequenced, leading to the identification of a common 15bp segment located in the 5’-noncoding region of each cluster, upstream from each first translation initiation site. These two promoter regions were fused precisely to the xy/E chromogenic marker (Zukowski et al., 1983) at the level of the hioD and bid initiation codons. These constructions were inserted into a plasmid able to replicate in Grampositive bacteria, and subseque~~tly introduced into B. sphutericus; .uq’lE expression was easily detected by the color of plated colonies. The corresponding activity was measured in crude extracts of transformants grown in the presence of different biotin concentrations. After mutagenesis. colonies were screened for x$E gene expression in the presence of exogenous biotin. The isolation and preliminary characterization ofthese mutants arc
described in this paper, which provides evidence for a transcriptional negative regulation exerted on B. .~ph~~~~j~~.~ hio genes.
RESULTS
AND DISCUSSION
(a) Isolation of ~aciliu~ ~~~a~~ic~~ mutants After transformation of I?. .~~~~~~jc~s strain
IF03525
with pTG2414 (Fig. 1 and Table I), single colonies were spotted on GP medium plates (Ohsawa et al., 1989) containing a range of biotin concentrations up to 100 ng/ml. These plates were incubated for 18 h at 37”C, before spraying with a 0.5 M catechol solution to nionitor the expression
of the marker gene (yellow and white colonies).
Our results showed that 50 ng/ml of exogenous biotin were sufficient to severely reduce the level of synthesis of the ,.uv(E gene product (C230) as seen by the total absence of color of the colonies under these experimental conditions. This was confirmed by measuring the C230 activity in crude extracts prepared from cells grown in liquid GP medium, with different biotin concentrations (Fig. 2). Because of the close correlation between the visual test and the C230 activity assay, the color of whole cells was used in the screening of mutants characterized by an increased synthesis of the .I$,!!? gene product on a medium containing excess biotin. The frequency of mutant clones was improved by mutagenesis of IF03525[pTG2414] with NTG. Cells were subsequently plated on GP medium supplemented with IO pg/mi biotin and 10’ colonies were screened for _qiE activity as described above. Fifteen candidates were yellow in this screening and were subcloncd for further analysis.
The candidates were first tested for their ability to accumulate biotin in the presence of its precursor, pimelic acid (unpublished data). For one mutant strain, biotin accumulation in the culture supernatant increased by up to a factor of ten, as compared to the wt. This can be explained by the fact that, in strain IF03525, biotin gene product synthesis were strongly reduced as soon as the biotin concentration reached 50 ngjml. This best biotin producer, strain TK502-2[pTG2414] was chosen for further analysis. Plasmid curing experiments were undertaken in order to confirm the localization of the mutation. Cells were incubated for 18 h in LB at 37°C and l/l00 (v : v) of the preculture was inoculated into freshly prepared LB supplemented with O-10 ;&g/ml of novobiocin (Sigma). After 18 h of agitation, cultures showing slightly impaired growth were plated on GP solid medium and incubated for 18 h at 37°C. Cured strains lost their
41 TABLE
I
List of plasmids
and recombinant
M 13 used
Description”
Name
Reference
Plasmids
Bglll
bioDAYB gene cluster
pTG1400
in-
Gloeckler
et al. (1990) et al. (1990)
serted into pBR322 pTGl418
bioXWF gene cluster inserted into pBR322
Gloeckler
pUBll0
Staphylococcus aweus plas-
Gryczan
et al. (1978)
mid pTG445
xylE gene inserted PC194
pTG2414
PbioD-xylE fusion inserted into PUB 110
This study
pTG2416
PbioX-xylE fusion inserted into pUB 110
This study
pTG498
bioB gene inserted pUBll0
into
Ohsawa
et al. (1989)
pBHB5022
bioB gene inserted
into
Ohsawa
et al. (1989)
into
This study
BamH!” AG 7-E MC AM GGT GTA ATG CGA.:. TGA Met Asn Lys Gly Val Met Arg Stop
AGG GGG AGGTAC
XylE
RBS bioD
Zukowski
in
et al. (1983)
pUBll0 xylE gene inserted pUBll0
pTG2403
Recombinant Ml3 Derivative Ml3tg131
of M 13mp70 1
Kieny et al. (1983)
PbioD region on Ml3tg131
This study
Ml3tg432
PbioX region on Ml3tg131
This study
Ml3tg433
xylE gene downstream from PbioD
This study
M13tg436
xyIE gene fused to PbioD
This study
M 13tg44 1
xylE gene downstream from PbioX
This study
M 13tg442
xylE gene fused to PbioX
This study
M 13tg43 1
Barn;: d P is a promoter TGGG AGG AA+A GGA GG ATG MC AAA GGT GTA ATG CGA: Met Asn Lys Gly Val Met Arg I,
TGA Stop
RBS bioX Fig. 1. Construction
ofpTG2414
tation of pTG2414.
and pTG2416.
The 799-bp NsiI fragment
region of the bioDAYB cluster (Gloeckler pTGl400
and inserted
EcoRI fragment (Zukowski promoter
(A) Schematic containing
et al., 1990), was isolated
in the PstI site ofphage
containing
Ml3tgl31.
the xylE gene, isolated
et al., 1984), was inserted in the BarnHI-EcoRI
from the PbioDAYB
Ml3tg431.
By mutagenesis,
the xl&Y-coding region was previously
fused to the putative
of bioD and the Bg/II-EcoRI
containing
quently Inserted into the BumHI-EcoRI
from
The BamHI-
from vector pTG445
downstream
sites ofphage fragment
represen-
the noncoding
start codon
this fusion was subseB. subtilisstrain
sites of pUBll0.
BGSClA92 (bioB aroC932 sacA321) was used as a host for the construction of recombinant vectors. B. subtilis was transformed using the protoplast method formed
(Chang
as described
tation ofpTG2416. taneously digested containing
and Cohen, by Ohsawa
the noncoding
isopropanol
region of the bioXWF cluster was inserted
sites of Ml3tgl31.
EcoRl and SmaI restriction of pTG445.
EcoRI-generated
represen-
‘yellow’ phenotype (cells which are not synthesizing the C230) and their Km-resistance, and did not contain any detectable plasmid DNA. One of these, TK502-2-C5 was transformed with either pTG24 14 or pTG24 16 (Fig. 1) and these recombinant strains expressed the xy/E gene in the presence of 10 pg/ml biotin, as evidenced by their yellow color. These results demonstrated that the initial phenotype was linked to modification(s) at the chromosomal DNA level. As can be seen in Table II ,uylE gene expression in this strain escaped biotin-exerted regulation: in the presence of excess biotin, C230-specific activities were always superior for the recombinant strains TK502-2-C5[ pTG24 141 and TK502-2-C5[pTG2416], as compared to that measured for IF03525.
Plasmid pTG1418 (Gloeckler et al., 1990) was simulby Hind111 and XmnI enzymes. The 1153-bp fragment
the HindHI-EcoRV fragment
1979). B. sphaericus cells were transet al. (1989). (B) Schematic
(see Fig. 1).
enzymes,
Phage
reaction
into with
polymerase
by the addition
treatment
BumHI termini. The DNA was precipitated under the same conditions as described above and incubated with T4 DNA ligase. Using in vitro mutagenesis,
at 15°C to ligate the
the DNA was precipitated
(50 : 50) before Klenow
digested
was ligated with the EcoRI-BarnHI
After an overnight
termini,
Ml3tg432,
of
to fill-in the
the fragment
containing
to the xylE gene. The fusion, carried M13tg442 pUBll0.
was finally inserted
the PbioXWF promoter on a BgllI-EcoRI
between
the BamHI
fragment
was fused of phage
and EcoRI sites of
42 TABLE
loo’
II
C230-specific
activity
Strain”
of Bacillus sphaericus strain Plasmids
TK502-2C5 -..
C230-specific
activity
(mu/mgY
‘i
////I
I/,2
10 Log &ok
(ng~ml)
Fig. 2. Effect of biotin concentration specific
activity.
(Ohsawa
B. sphaericus with lysozyme
before sonication.
by centrifu-
(2 mgjml for 15 min at 37 a C)
for 15 min at 4°C. Supernatants
according
Bradford’s
medium
Cellular debris was pelleted in 1.5 ml eppendorf
by centrifugation (1971). Protein
(Sigma)
to the method
concentrations
method,
reported
by the reagent
tubes
were tested for C230
by Sala-Trepat
of cellular extracts
as described
IF03525
pTG2414
85
2
pTG2414
480
340
IF03525
pTG2416
TK502-2-C5
pTG24 14
and Evans
were measured supplier
60
1
140
45
58
43
pTG2403
IF03525 a See RESULTS
C230-
in GP
et al., 1989) for 18 h at 37°C. Cells were collected
gation and treated
activity
incubated
( + biotin)
-
added to GP medium were
B
( -- biotin)
TK502-2X5
100
on the IF03525[pTG2414]
cells
J
A
using
(BioRad)
AND DISCUSSION,
b See Table I. Plasmid pTG2403 the control
of the ‘HpaII’ constitutive
’ To measure
the C230-specific
in GP medium.
by centrifugation
for 15 min at 37°C)
Supernatants Bradford’s
promoter
(Ohsawa
at 37°C
before
sonication.
Cellular
tubes, by centrifugation
were then tested (BioRad).
cells in the stationary
and treated with lysozyme (Sigma)
for C230
activity
and Evans (1971). Protein concentration method
under
et al., 1989).
activity, B. sphuericus cells were grown
pelleted in 1.5 ml in eppendorf Sala-Trepat
a and b.
the xylE gene expressed
After 18 h of incubation
phase were collected (2 mg/ml
sections
contains
A, without
debris
was
for 15 min at 4°C. using the method was evaluated
of
using
biotin; 8, with 10 pg/ml biotin.
::::::::: r-----3
_“35”
123
4
9
A
: : : : : :
PbioDAYB S-AGGCArrrACAkAAC~.~~~~,~~~~~~~~~A~
+l +l
_“I 0”
.:,., .,.. . ...::: / ,..,.,.. ..., .,.,..,.
TTGG’ITAACTAAAAGAGGGGGAGGTACAqTTG-3’
]
+I +1 _“I 0” .,:.,., .,...,_ .. ..,....,,.. .,..:,.:...‘.-:).::.:_‘_:_. .;.,,.,.,. ~‘_~TAAT~ACCITCCTAT~AT~~~.~~~,~~~~~~A~~G ,.: .;,,.,(,..,(( ..I ,; .. ,,..,I _“35”
pbio_
Fig. 3. The bioDA YB and bioXJW
promoter
regions.
Mapping
of the &-acting
mutations.
Base substitutions
(dashed
upward
arrows)
and the deletion
(d) are indicated; numbers correspond to the following mutants: 1, TK502-8; 2, TK222-5; 3, TK502-1; 4, TK212-4; + 1, indicates the fsp (first nt transcribed); the common 15 bp overlapping the operator region are shaded; overlines denote the putative -10 and -35 regions deduced from the localization of the tsp; the RBS is underlined twice and the potential start codon (TTG) is boxed.
43 The xylE gene fused to the bioX WF promoter region was also constitutively expressed in TK502-2-C5. This suggests the existence of a common circuit regulating the expression of both biotin gene clusters. (2) Plasmid-linked mutations Mutation(s) associated with the pTG2414 were also identified by plasmid curing experiments. The 5’ region upstream from the xylE gene was subcloned from plasmids in M 13 vectors and sequenced. The elongation was initiated
is noteworthy
that the C230-specific
this mutant. (c) Identification of the tsp of the bioX and l&D genes Using primer-extension methodology (Fig. 4) two potential tsp were found for each gene cluster. These sites are
A
at an oligo hybridizing closely to the start codon of the xylE gene. This methodology enabled the reading of about 200 bp covering the bioDA YB promoter region. Several modifications were detected (Fig. 3); all were located inside the 15-bp region previously proposed by Gloeckler et al. (1990) as a potential site of the biotin control circuit acting in B. sphaericus. The C230-specific activities measured in three strains containing these mutated plasmids are shown in Table III. The xy/E gene is indeed constitutively expressed, demonstrating the crucial role of this region in the biotin regulation circuit. As reported for strain TK502-2-C5, these specific activities were higher in two mutant clones when compared to the reference strain, after harvesting the cells cultivated on GP medium. These results may reflect an incomplete elimination of the repression in the wt strain, under these experimental conditions. The 15-bp segment located within sequences exhibiting imperfect rotational symmetry (Heinzel et al., 1989) is the first example of a clearly identified regulatory region shared by two gene clusters in Gram-positive bacteria. Sequences corresponding to the biotin cisacting regulatory region in the Enterobacteriacea family, respectively E. coli, Citrobacter freundii and Salmonella typhimurium exhibit a high degree of conservation (Shiuan and Campbell, 1988). Comparison of the 15-bp segment and its flanking sequences identified in B. sphaericus to this consensus region did not reveal any significant similarity. It
AGCT
AGCT
a T
1234 B. sphaericus, grown
III
resuspended
C230-specific
activities
of Bacillus sphaericus strains
carrying
plasmid-
C230-specific
Mutation
activity
with lysozyme
reference
as described
by Robbins
TK212-4[pTG2414] TK222-5[pTG2414]
11 bp deleted transversion (G + T)
TK5028[pTG2414]
transversion
(T + A)
A
B
(Model
380B,
( - biotin)
( + biotin)
primers
OTC2358
2
202
54 91
47 827
extension
After primer
(lanes l-4).
containing described
cells were cultivated biotin
10 pg biotin/ml
in GP medium (B). C230
in the legend of Table II.
activity
(A) or GP mediumwas determined
as
the mixtures
positions sequence,
(panel
of the reaction.
of the primer-extension and correspond
to tsp.
oligo
to hybridization
with
growth
transcriptase
in
(Takara
(lane s). The dideoxy-
Sequenase (US Biochemicals, OTC2358 to Ml3TG431 (panel B) were used as M, standards
The letters above lanes l-4 indicate
used in the termination
(5’-TTCG-
during exponential
were electrophoresed
to Ml3TG432
OTC2358
OTC2359
using a DNA synthesizer
with reverse
products produced with OH, U.S.A.) after annealing
by
and RNA
of the radiolabeled
was subjected
GP medium.
A) or OTC2359 ’ Bacterial
and OTC2359
Each
RNA, extracted
terminated Cleveland,
3 See Fig. 3.
were prepared Biosystems).
B. sphaericus TK502-2-C5 Tokyo, Japan),
870
Applied
and
1 y0 mur-
recovered
et al. (1987). Oligos
(5’-GTTCCAACAACCCAAAAGTGTT)
85
1979) containing
(2 mg/ml) and N-acetyl
at 2600 x g at 4°C for 15 min, were disrupted
CATCCTCCTATTCCTCCC)
lF03525[pTG2414]
extraction, 1 A (measured
at 2600 x g for 10 min at 4°C. The pellet was
SC (0.2 mg/ml) at 37°C for 30 min. Protoplasts,
was isolated
(mU/mgY
RNA
to approx.
in 10 ml of SMMP (Chang and Cohen,
centrifugation Strain”
For
in 100 ml GP medium
(w/v) BSA before incubation amidase
linked mutations
1234
s experiments.
Fig. 4. Primer-extension at 660 nm) were centrifuged
TABLE
activity was higher in
the derepressed strain than in the wt suggesting that intracellular levels of biotin enzymes were strongly increased in
product
the dideoxynucleotides
The arrowheads
indicate
the
in the gel (lane s) and on the
44
123
M
adjacent or separated by one bp and are located between the 15bp regulatory sequence and the RBS (Fig. 3). The results are consistent with regulation of the biotin gene clusters at the transcriptional level.
kDa 200 97.4
(d) Amplification mutated strain
68
of the bioB gene in a chromosomally
Plasmids pTG498 and pBHB5022, containing the B. sphuericus bioB gene on the PUB 110 replicon (Ohsawa et al., 1989), were introduced into TK502-2-C5 cells in order to increase the biotin synthetase gene dosage. Polyacrylamide gel electrophoresis of crude extract proteins from these transformants (Fig. 5) revealed a substantial increase of the bioB gene product, corresponding to a new band of 37 000. These results were correlated with improved biotin production in the corresponding strains, reaching 30 pg/ml of biotin when pimelate was added at the onset of the culture (Table IV).
25.7
18 14
Fig. 5. Production
ofbiotin
TK502-2-C5[pTG498] medium containing in an Eppendorf
in B. sphaericus cells. B. sphaericus
synthetase
cells were incubated
for 18 h at 37°C
tube and the washed
described
by Peschke
SDS-13:~
polyacrylamide
pellet was subsequently
et al. (1985).
5 pl were
in 7.5% acetic acid/5%
TK502-2-CS[pUBl The arrow
TABLE
loaded
gel. After 5 h of migration
was stained in 7.5 % acetic acid/5 y0 methanol/O.25 destained
in GP
10 pg Km/ml. A 100 ~1 ofthe culture were centrifuged
methanol.
Lanes;
the band corresponding
as
a O.l”/;,
at 20 mA, the gel
7, Coomassie
IO]; 3, TK502-2-C5[pTG498];
indicates
treated
onto
blue and
1, TK502-2-C5; M, kDa
2,
standards.
to biotin synthetase.
IV
Biotin production
in Bacillus sphaericus strain TK502-2C5 Biotin excreted
Strain”
(pg/ml)h
A
B 0.5
. (5) Biotin synthetase expression was improved by amplifying the bioB gene on a multicopy plasmid (Ohsawa et al., 1989) and, combined with the use of TK502-2-C5 as the host strain, led to a 30-fold improvement in biotin secretion.
ACKNOWLEDGEMENTS
The support of A. Yoshioka throughout this study was much appreciated. We are grateful to Drs. A. Marquet and 0. Ploux for their active collaboration during different phases of this project. Thanks go to D. Villeval and Y. Cordier for DNA sequencing and oligo synthesis, B. Heller for artwork, and D. Heery for critical reading of the manuscript.
45 Nishimura,
REFERENCES
A. and Kikuchi,
microorganisms, Barker,
A.M.: The birA gene of Escherichia coli
D.F. and Campbell,
encodes
a biotin
holoenzyme
synthetase.
J. Mol. Biol. 146 (1981)
Casadaban, M.J.: Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104 (1976) 541-555. S. and Cohen,
subfilis protoplasts
by plasmid
DNA.
of Bacillus
transformation
Mol. Gen. Genet.
168 (1979)
Biotin
biosynthesis
Ann. NY Acad. Gloeckler,
of the biotin
and metabolism,
operon biotin
M., Villeval, D., Kisou, T., Kamogawa, of pimelate
T., Contente,
subtilis. J. Bacterial. A., Saturen,
scription
C., Bernard,
S., Zinsius,
K. and Lemoine,
Y.: Cloning
of the Bacillus sphaericus genes controlling into dethiobiotin.
S. and Dubnau,
coccus aureus plasmids Guha,
probe.
introduced
the
Gene 87 (1990) 63-70.
D.: Characterization
of Staphylo-
by transformation
into Bacillus
134 (1978) 318-329.
Y. and Szybalski,
orientation
of tran-
PI mutational
M. and Schuster,
analysis
H.: ban operon of bacteriophage
of the cl repressor-controlled
operator.
J.
of 7-keto-7-ami-
sequencing
vectors
R. and Lecocq, based
J.-P.: New versatile
on bacteriophage
cloning
M13. Gene
of dehydrobiotin134 (1974)
345-357. Peschke,
U.. Beuck, V., Bujard,
H., Gentz.
R. and Le Grice, S.: Efficient
ofEscherichiu coli transcriptional
signals in Bacillus subtilis.
J. Mol. Biol. 186 (1985) 547-555. Robbins,
J.C., Spanier,
J.G., Jones, S.J., Simpson,
W.J. and Cleary, P.P.:
Streprococcuspyogenes type 12M protein gene regulation sequences. Sala-Trepat,
J. Bacterial.
D. and
W.C.: The meta cleavage
Eur. J. Biochem.
Campbell,
by upstream
169 (1987) 5633-5640.
J.M. and Evans,
Azotobacter species.
of catechol
by
20 (1971) 400-413.
A.: Transcriptional
regulation
and
gene
of Escherichia coli, Citrobacter .fieundii and Salmonella
arrangement
Gene 67 (1988) 203-21 I. with Lactobacillus
ofbiotin
arabinosus. Proc. Sot. Exp. Biol. Med. 56 (1944) 95-98.
Zukowski,
H., Osakai,
M., Tani, Y. and Izumi, Y.: Biotin overproduction
M.M., Gaffney,
Wisecup,
26 (1983)
regulatory
mutants
of Bacillus sphaericus. Agric. Biol.
D.G., Speck, D., Kauffmann,
A. and Lecocq, J.-P.: Chromogenic
M., Findeli, A.,
identification
signals in Bacillus subfilis based on expression
ofgenetic of a cloned
Pseudomonas gene. Proc. Natl. Acad. Sci. USA 80 (1983) 1101-l 105. enzymes.
Adv. Enzymol.
35
Zukowski,
M.M., Speck, D., Kaufmann,
genic detection
K. and Izumi, Y.: Metabolism
of biotin by microorganisms.
Rev.
Vitam. 48 (1974) 159-178. K., Tochikura,
characterization
of Escherichia coli. Mol. Gen. Genet.
mutants
and
(1971) 321-442.
Ogata,
and genetic
levels of the biotin
112 (1972) 1280-1287.
Chem. 47 (1983) 101 l-1016.
91-99. Moss, J. and Lane, D.: The biotin-dependant Ogata,
J. Bacterial.
of biotin analog-resistant
nopelargonic acid synthetase in bacteria and the control mechanism of the enzyme activity. Agric. Biol. Chem. 37 (1973) 1335-1340. M.P., Lathe,
M., Gloeckler,
in Escherichia coli and
ofEscherichiu coli with derepressed
enzymes.
Pai, C.H.: Biochemical
Yamada, K.: Distribution
K., Zinsius,
K.: Cloning of the biotin synthetase
Wright, L.D. and Skeggs, H.R.: Determination
Mol. Biol. 205 (1989) 127-135. Izumi, Y., Sato, K., Tani, Y. and Ogata,
Y. and Kamogawa,
typhimurium biotin operons.
from the biotin locus. J. Mol. Biol. 56 (1971) 53-62.
Heinzel, T., Velleman,
Kieny,
synthetic
Shiuan,
W.: Divergent
by
Bacilli. Gene 80 (1989) 39-48.
utilization
I., Speck, D., Ledoux,
and characterization Gryczan,
as a molecular
VI.
Sci. 447 (1985) 335-349.
R., Ohsawa,
bioconversion
in E. coli. Part.
of biotin by
accumulated
Agric. Biol. Chem. 29 (1965) 895-901.
gene from Bacillus sphaericus and expression
resistant M.A.: Regulation
of biotin-vitamers
I., Speck, D., Kisou, T., Hayakawa,
Pai, C.H.: Mutant
S.N.: High frequency
111-115. Eisenberg,
Ohsawa,
microorganisms.
R., Lemoine,
45 l-467.
Chang,
various
M.: Studies on biosynthesis
II. Identification
T., Iwahara,
In: Ganesan,
ofgenetic
K., Takasawa,
S.,
M. and Lecocq, J.-P.: Chromosignals cloned in Bacillus subrilis.
A.T. and Hoch, J.A. (Eds.), Genetics
of Bacilli. Academic S., Ikushima,
regulatory
Press, New York,
and Biotechnology
1984, pp. 309-319.