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,

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The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-70]. Sequence analysi...
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