Gene, 87 (1990) 63-70 Elsevier

63

GENE03~8

Cloning and characterization of the Bacillus sphacricus genes controlling the bioeonversion of pimelate into dethiobiotia (Recombinant DNA; biotin synthesis; gone clusters; regulatory sequences; pimelate-7-keto-8-aminopelargonic acid bioconversion; pimelate cell permeation)

R. Gloeelder', I. Ohsawa b D. Speck', C. Ledoux', S. Bernard; M. Zinsius % D. Villeval", T. Kisoa b, K. Kamogawa b and Y. Lemoine '~* ° Transg~ne S.A., 67082 Swasbourg Codex (France) and b Biological Science Institute, R&D Center, N~ppon Zeon Co. Ltd., Kawasaki 210 (Japan) Tel. 044(266)6521 Received by J.-P. Lecocq and AJ. Podhajska: 9 June 1989 Revised: 1 September 1989 Accepted: 15 September 1989

SUMMARY

Using 8.8 kb of genetic information from Bacillus sphae6cus, it was possible to confer to Eschev~chia coli bio- swains, including AbioA-D, bloC-, bioH-, the ability to convert exogenous pimelate into biotin. The b/o genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of H/ndlll-digested B. sphae6cus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphae6cus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa etal., Gone 80 (1989) 39-48]. These genes are clustered in the order bioDA YB. The 4.5-kb fragment contains genetic information for three different proteins, the products ofbioX, bioW and bioF. Complementation studies using an E. coil bioF mutant and a B. subtgis bio 112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gone order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators. The presence, upstream from the coding sequences in both inserts, of an identical 15-bp sequence, located within sequences exhibiting a center of imperfect symmetry, strongly suggests the existence in B. sphaer/cus of a common regulatory mechanism acting on these regions.

INTRODUCTION

The addition of pimelic acid to the culture medium of several different microbes has been shown to enhance the Correspondenceto: Dr. R. Gloeckler, Transg~ne S.A., I I rue de Molsheim, 67082 Strasbour8 Codex (France) Tel. 88.22.24.90; Fax 88.22.58.07. * Present address: L.G.M.E., Facuit6 de M6decine, l l rue Humann, 67085 Strasbour8 Codex (France). Abbreviations: aa, amino acid(s); Ap, ampicillin;B., Bacillus;bio,gone(s) of the biotin synthesis pathway; bp, base pair(s); flu, colony-forming unit(s); Cm, chloramphenicol; CoA-SH, coenzyme A; A, deletion; 0378-1119/90/$03.50© 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)

biosynthesis of biotin and its intermediate vitamers (lzumi etal., 1980). This specific effect was studied in detail with B. sphaericus, the only microorganism in which the enzymes involved in biotin synthesis from pimelic acid (described in DAPA, 7,8-diaminopelargonic acid; DTB, dethiobiotin; E., F_.scher/ch/a; 7-KAP, 7-keto-8-aminopelargonic acid; kb, 1000bp; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; p, promoter; PAGE, polyacrylamide.gel electrophoresis; PLP, pyridoxal $'-phosphate; R resistance/resistant; RBS, ribosomebinding site; SAM, S-adenosyl-L-methionine; SDS, sodium dodecyl sulfate; Tc, tetracycline; u, unit(s); wt, wild type.

64 Fig. 1) have all been clearly identified (Izumi et al., 1981). As compared to wt E. coil strains, B. sphaericus IF03525 and NCIB9370 strains produce much higher amounts of total vitamers. Indeed, a difference of about 1000-fold has been observed between the respective basal levels ofvitamer concentrations in supernatants of cultures containing pimelate (Izumi et al., 1980). Interestingly, no increase in biotin excretion can be detected after the addition of pimelate to E. coli cultures (Pai and Lichstein, 1965). The permeability of E. coli cells to this metabolite had already been questioned (Pal and McLaughlin, 1969). Strikingly, 8-(2,4-dichloro-sulfanilido)-caproic acid, a toxic analog of pimelate, was reported not to inhibit the growth of E. coli, unlike the other biotin prototrophic microbes tested (Woolley, 1950). In E. coli the most recent genetic study of biotin auxotrophic mutants confirmed that six structural genes are specifically involved in the biotin synthetic pathway (Barker and Campbell, 1980). Establishment of a physical map of the E. coli hie operon confirmed the order of the bio and adjacent genes (Szybalski and Szybalski, 1982). The gene products of the bioF, bioA, bioD and bioB cistrons were clearly linked to the pathway of bioconversion from, pimelyl-CoA to biotin (Rolfe and Eisenberg, 1968; Cleary and Campbell, 1972). Two other genes, bioC and bioH, are still tentatively identified as encoding the enzyme(s), involved in pimelyl-CoA synthesis. Here, we describe the characterization of the genes from B. sphaericus which catalyze the bioconversion of pimelate into DTB. Our findings enabled us to discuss a number of interesting features related to the complementation of E. coil bioH- , bloC- and AbioA-D mutants using B. sphaericus bio genes and to clarify some puzzling questions concerning the biosynthesis of pimelyl-CoA in E. coil and the permeability of these cells to pimelic acid.

MATERIALS AND METHODS

(a) Nucleic acid biochemistry Genomic DNA from B. sphaericus IF03525 was prepared according to Saito and Miura (1963). Conventional recombinant DNA techniques were those described in Maniatis et al. (1982). (b) Cell transformation Strains are described in Table I. All the E. coil strains were made competent and transformed as described by Dagert and Ehrlich (1979). Non-replicating plasmids were introduced into B. subtilis strains by the competent cell-transformation techniques developed by Boylan etal. (1972). Successful integrative transformation ofthe JKB3112 and bio112TG3 strains re-

quired the use of the protoplast method of Chang and Cohen (1979). (c) Genomic bank Purified chromosomal DNA (20 #g) from B. sphaericus IF03525 was completely digested with HindIll (3 u/itg of DNA). pBR322 was treated with alkaline phosphatase (after complete HindIll digestion). Ligation was performed as recommended by the suppliers. Aliquots of the ligation mixture were then used to transform E. coli strain C600 by selecting the transformants for their resistance to Ap (100 pg/ml)on LB medium. Four different pools of plasmid DNA were then extracted, each corresponding to about 104 individual clones on the transformation dishes. (d) Measurement of 7-KAP in culture supernatants E. coli recombinant strains were cultured at 37-C for 24 h in GP medium with the addition of (per liter): 20 g glycerol; 30 g proteose peptone; 5 g vitamin-free Casamino acids; 1 g K2HPO4; 0.5g KCI; 0.5g MgSO4"TH20; 0.01 g FeSO4" 7H20; 0.001 g MnSO4'4H20 pH 6.8-7; 20#g thiamine. HCl plus 100#g Ap/ml and 0.3mg pimelate (pH 7.5)/ml. After chromatographic separation (Ogata et al., 1965), 7-KAP present in the supernatant was measured quantitatively in the biological assay with Saccharomyces cerevisiae ATCC7754 (Snell et al., 1940).

RESULTS AND DISCUSSION

(a) Cloning of Bacillussphaericus bioA and bioD genes: evidence for their clusteringwith bioY and bioB (1) Complementation of Escherichia coli and Bacillus subtilis strains E. coil mutants C268 (AbioA) and C173 (dbioD) were transformed with four DNA pools (see MATERIALSAND METHODS, section e) from the B. sphaericus IF03525 gene bank and the transformants were selected either in the presence of Ap on LB medium or for simultaneous antibiotic resistance and biotin prototrophy. Plasmids were isolated from clones growing on Ap plus avidin. The four plasmids analyzed (two originating from strain C268 and two from strain C173) contained a 4.3-kb Hindlll insert with an identical restriction pattern. By selecting for resistance to Ap and prototrophy for biotin, it was possible with one of these plasmids, designated pTG1400, to retransform strains C268 (AbioA), C173 (AbioD), R877 (bioDl9) and C162 (bioB) at the same frequency as that obtained for the selection on Ap alone (more than 103 cfu/#g of DNA). No complementation of the biotin auxotrophy of strains R878 (bloC18), R874

65

(bioFl2), PAS05MA,dI08 (~bioH) and R901 (AbioA-D) could be obtained using pTG1400. In a second step, plasmid pTG1400 was used to transform B. subtilis strains bioATG 1, bioBTG2 and bio 112TG3. Biotin prototrophic transformants were isolated at a very high frequency from strains bioATG 1 and bioBTG2 but not from strain bio 112TG3 (see legend to Fig. 2). Southern blots were used to detect the HindIII fragment ofpTG 1400 in the genomic DNA orB. sphaericus IF03525. Under stringent hybridization conditions, a single 4.3-kb HindIII band could be seen (not shown), while pBR322 did not cross-react with the genomic DNA.

B-sBhaeri_Qus

B. c o l i

Pimelic acid ( ~ )

CoA~

Unidentif£ed precursors

.~

! b,oC.b,oH

(~co~)

T-seo~ CHa(C~)4COOH /

~Ala~

1

PLP

QH2 ( OH2) 4COOH

H3C / 8~

bioF

PLP

~

bio~

pelargon£c a©£d (D&P&)

(2) Nucleotide sequence of the HindIll fragment of pTGl400 The nt sequence has been submitted to the GENBANK database (accession No. M29291) and is also available on request to the authors. Computer analysis of the sequence revealed that the fragment has four ORFs. Based on the most fikelystart codon, these four ORFs have a coding capacity for proteins of about 27, 47, 23 and 37 kDa, respectively. A palindromic region at the 3' end ofthe sequence was previously reported to be a putative transcription terminatiov site (Ohsawa et al., 1989). (3) Mapping of bio genes carried on the Hindlll fragment of pTGI400 Detailed complementation analysis (Fig. 2) showed that the first ORF region (with two possible start codons) corresponds to the bioD gene. Maxicell experiments showed that this region codes for an approx. 25-kDa polypeptide (not shown). The second ORF, comprising two possible start codons, corresponds to the bioA gene. Autoradiographic analysis of maxiceli experiments revealed an approx. 43-kDa polypeptide which could correspond to the product of the bioA gene (not shown). The function of the third ORF sequence, referred to as the bioY gene, could not be determined. Interestingly, its predicted gene product presents four hydrophobic regions with the appropriately sized transmembrane domains and a probable signal sequence (not shown) suggesting that this ORF could encode a membrane-anchored, 21-kDa protein. The fact that the bio Y gene belongs to a cluster with other bio genes indicates that it codes for a still unknown function involved in biotin metabolism. The fourth ORF corresponds to the region encoding the 37-kDa biotin synthetase protein (Ohsawa et al., 1989). Thus, the bioA, bioB, bioD and bio Y genes are clustered.

H3C OH2(CH2)4COOH I

H¢03--

g~p,,e+

bio~

¢

/\

dethiobiotin (DTS)

I

HC

I

I

H30

CH2(CH2)4COOH

suZfu~

I

donor

bioB

0

aII /\

HN

I

I

H2C

\/

L'H

I

o-blokin

I

CH(eH2 ) 4C00H

5 Fig. 1. Biotin biosynthesis pathway in B. sphaericus and E. ¢oli.

(b) Cloning of the Bacillus sphaericus bioF gene: evidence for its clustering with two other bio genes (1) Complementationof Escherichia coli bioF and of Bacillus subtifis bio112 strains E. coil bioF12 strain was transformed using the HindIIIdigested B. ~F~aericus IF03525 genomic DNA library and clones growing without biotin were isolated. Analysis of their plasmid contents showed that they all carried identical 4.5-kb HindIII inserts; some also bore an extra 0.6-kb HindIII piece of DNA, probably resulting from incomplete DNA digestion. One ofthese plasmids, pTG 1418, was used to transform the E. coil bioFl2 strata: the same number of transformants was obtained either after selection for Ap resistance solely, or for both Ap resistance and biotin prototrophy. Experiments evaluating 7-KAP production showed that the 4.5-kb HindIII fragment adequately provided the

Hindlll Xual

Hlndlll Ooill

Doll

I

i t

COmnlementation E,coli B.eubtili8 D'A* B "

(A)

Xlndlll I

Xaul Xmnl Pvull Pvull Clnl I I I I I

HIndill I

ulox

A ° El'

bloW --

, |

!

Naal t

pTG1418

| DTG1404 pl'Q1412

pTG1413

O*^ " B-

&'B"

D'A" B"

A'B"

D ' A * B"

A'B"

D÷A * B "

AOB-

D'A" B*

A'B*

D'A* B-

^°e"

D "^'B"

A-B"

> pTG1422 .~_. . . .

•

pTG1420 pTG1486

DTG1414 pTG|403 pTG1408 pTG1409

pTG143? pTG1435

(B) oomplementation

1-kb

Pla8mid

Fig. 2. Schematic representation of complementationmapping studies performed on the H/ndlll insert of pTGI400. A series of overlapping DNA fragments (pTG1412 to pTGI415) was obtained by the cyclone system used for sequencing experiments. Various E. coli and B. subtilis bio- strains were transformedby these different plasmids. The transformed £. coli cells were spread on LB medium containing avidin (0.2 u/ml) and 100 #g Ap/ml.This selection allowed the recovery of true biotin prototrophicclones, after incubation at 37°C for 16-24 h. Clones were furtherchecked for growth on CA synthetic medium,treated with charcoal to removethe traces of biotin and solidifiedusing 1.5% agarose, as previouslydescribed(Ohsawaetal., 1989).B. subtilishie mutants were complemented as follows. First, plasmid pTG475 was integrated into different B. subfflisbio mutants. It contains the xy/E gene encoding the enzyme C 2,3-oxygenase (Zukowski etal., 1983) which may be used as a chromogenic(yellow)markerand whose expressionis controlledby the inducible promoter of the B. subtilis levansucrase gene (sacB). This plasmid also contains a cat gene conferringresistance to Cm; the cat and xy/E genes were inserted into pBR322, pTG475 was integrated into the chromosome of the JKB3173, BGSCIA92 and JKB3112 B.subtilis strains. Transformedclones were selected on TBAB (tryptose blood agar base; Difco, Detroit, MI; plus 3 pg Cm/mi), Southernblots showed that bioA-, bloB- and bioll2 strains had integrated pTG475 via a single recombination event in the promoter region of sacB. The transformed strains orB. subtl/lsare referredto as bioATG i, bloBTG2and hieI 12TG3. Since pTG475 introducedstable pBR322 sequences into the genome of B. subtile,it becamepossible to integrate,by homologousrecombination, any foreign plasmid containing these same sequences into the abovementioned strains. To check integration of the B. sphaericusb/o genes, complementationwas monitoredon LB mediumcontaining 10 IzgCm/ml and avidin (0.2-0.5 u/mi).

7-KAP synthetase function responsible for the complementation of the bioFl2 mutation of E. coli strain R874. Southern-blot experiments identified the 4.5-kb Hindlll fragment in the genomic D N A of B. sphaericus IF03525 (not shown). No cross-reaction was observed between pTGI400 or the left and right SphI-Sphl flanking regions of the corresponding insert and pTGI418 (not shown). These findings demonstrated that, unlike E. coil, the bioF gene is not linked to the bioA, bioD and bioB genes, in B. sphaericus IF03525. B. subtilis mutant bioll2 was identified by nutritional tests as being specifically affected in the biosynthesis of

Corresponding In8ert

pTG1418

4 . 3 - k b Hlndlll

pTG1422

2 . 4 - k b Clal

pTG1420

2 . 0 - k b ClaI-Hindlll

pTG1436

1 . 3 - k b XmnI-Ncol

pTG1437

1.3-kb NcoI-Xmnl

pTG1435

0.6-kb

B. 8ubtlll8 • I

bio112TG3

]

E. c o i l b

R874

I~JI-Pvull

Integrative traneformutlon Performed on LB * atvldln (0.Gulml) b

* 10~9 (:m/1111

Pert~rmed on LB * atvldln (O.blml) * ~

Ap Iml

Fig. 3. Schematic representationofcomplementation studies performed on the HindlI! insert of pTGI418. (A)Gene localization and partial restriction map. SubclonedDNA fragmentsand correspondingplasmids are shown. Arrows indicate the orientation of the correspondinginsert after subcloning in plasmid pBR322. (B) Complementationstudies performed with these plasmids. For details see legend to Fig. 2.

7-KAP (Pal, 1975). To characterize the mutation, strain bioll2TG3 was transformed by different plasmids described in Fig. 3 (pTGI418, 1420, 1422, 1435, 1436 and 1437) followed by selection on LB medium with 10 gg Cm/ml and avidin [0.5 u/ml (1 u of avidin is defined as the amount of protein which will bind 1.0/~g of d-biotin)]. Fig. 3 summarizes these results. Since this test performed on E. coil mutant R874 gave similar results, B. subtilis mutant hie 112 is likely to be affected in bioF. Complementation studies using subclones of the pTGI418 insert localized the bioF gene within a 1.3-kbXmnl-Ncol fragment (Fig. 3).

(2) Complete nucleotide sequence of the Hindlll fragment of pTGI418 (GENBANK accession No. M29292) The sequence contains three ORFs, bioX, bio W and bioF, which encode predicted proteins of 18, 28 and 42 kDa, based on sequence analysis. The last gene is localized in the region identified as bioF. Thus, the predicted size of this B. sphaen'cus protein is of the same order of magnitude as that of E. coil 7-KAP synthetase (45kDa; Eisenberg, 1973).

67 pbloD AATGT

TI--

TAA AACI~ TAGTI ~ A A A

AGAGGGGGAGGTACAG~ .... RB8

pbloX

Fig. 4. Alignmentof the sequences found upstream from the first ORF of each B. sphaericus biotin cluster. The blackened circles define possible centers of imperfect palindromic symmetryshown by the arrows and boxes. The common 15-bp sequences are doubly underlined. Putative RBS and start codons corresponding to the first gene of each cluster are underlined and circled, respectively.

The juxtaposition of these three ORFs might be indicative of an operon structure, as in the case of the plasmid pTG1400 insert. A 15-bp sequence can be identified upstream from the first gene and is also present upstream from the first gene (bioD) of the plasmid p T G 1400 insert. This common 15-bp sequence is located within sequences exhibiting a center of imperfect symmetry typical of control regions (Fig. 4). This important feature might indicate that the two groups of B. sphaericus biotin genes are subjected to at least one common regulation circuit (D. S., manuscript in preparation). The latter might correspond to the control

by biotin (or a compound derived from it), which has already been reported for 7-KAP synthetase (bioF) (Izumi et al., 1973) and for biotin synthetase (bioB) (Ogata and lzumi, 1974). A palindromic region at the 3' end of the sequence might act as transcription termination site.

(c) The bioW gene probably encodes pimelyl-CoA synthetase When competent cells of E. coli bioCl8 (R878) and AbioH (PA505MAAI08) mutants were transformed with plasmid p T G 1418 and then plated on Ap-selective medium

TABLE I Bacterial strains used in cloning and complementation experiments Strain

Relevant genotype"

Source or reference

B. sphaerieus IFO 3525

wt

Institute for Fermentation, Osaka

F- thi-I thr-I leuB6 lacYl tonA21 supE44 hsdR- hsdM ÷ ~F- ieu.6 purE42 thi.l ara-14 lacYl gaIK2 xyl.5 str-109 capli9 galU supE44 ~-

Appleyard (1954) Zehnbauer and Markovitz (1980) Izumi, Y.b Cleary and Campbell (1972) Cieary and Campbell (1972) Clear), and Campbell (1972) Cleary and Campbell (1972) Cleary and Campbell (1972) Cleary and Campbell (1972) Campbell et al. (1969) Hatfield et al. (1969)

E. ¢oli

C600 MC169 C162 C173 C268 R874 R877 R878 C261 R901 PAS05MAA108

bioB his ~bloD his Sma AbioA his SmR bioFl2 his bioD 19 his bioC18(23) his dbioFCD his AbioA-D Sma dmald dbioH Smn argH metA

B. sub61is

JKB3173 BGSCIA92 JKB3112 bioATGl bioBTG2 bio I 12TG3

bioA 173 aroG932 bioB l41 aroG932 sacA321 argA2 bio 112 aroG932 bioA 173 aroG932 (pTG475 chromosomally integrated) • . bioB 141 aroG932 sacA321 argA2 (pTG475 chromosomally integrated) bio112 aroG932 (pTG475 chromosomally integrated)

a dbioA-D means a complete deletion of the bioABFCD operon.

b Personal communication.

Hoch, J.A.b; Pai (1975) Bacillus Genetic Stock Center

Hoch, J.A.b; PUi (1975) This study This study This study

68 without biotin, many small clones could be detected after 36 h ofincubation at 37 ° C. The frequency of appearance of the transformed clones on this medium was of the same order of magnitude as that measured on LB + Ap. Growth of the transformed clones, which was normal on LB + Ap, was retarded on biotin-depleted medium. However, complementation ofthe biotin auxotrophy of the bioC and bioH mutants was significant, considering the total lack of residual growth on biotin-depleted medium of the same mutants, transformed by plasmids derived from pBR322, including pTG1400, which did not contain the 4.5-kb pTG 1418 insert. A striking observation was that the addition of neutralized pimelic acid (concentrations of 0.5-500 pg/ml), which by itself was unable to correct the biotin requirement of the bloC and bioH mutants, completely restored their growth on selective biotin-free medium when they were transformed ~ith pTG 1418. With plasmids carrying either the bioXW or the bio WF genes fused to the E. coli lac promoter, the same complementation phenotype ofbioC and bioH cells was observed: slow growth on biotin-free medium evolving to normal growth in the presence of pimelate. The use of a plasmid containing only the blow gene, in serted down stream from the Tc R(pT~) promoter, revealed a weak complementation of the bloC and bioH mutants, but only on pimelate-supplemented medium. With the same strains containing bioX, expressed as a single unit from the Tc R promoter, biotin prototrophy was not restored, even in the presence of pimelate. On pimelate-contalning medium, full complementation ofbioC or bioHE, cog strains required a minimal level of expression of the bloW gene product (detected by SDS-PAGE when expressed by the plao-bioXW construction, our unpublished results); the simultaneous amplification of the bioF gene product compensated for its suboptimal expression (e.g., as in a pTc-bioXWF construction). Expression ofthe blow gene as a single unit was probably difficult due to its native structure in a tightly coupled polygenic association. An in vivo bioconversion test was used to further demonstrate that the bioWF sequence enables the cells to convert pimelate into 7-KAP. With plac-bioWF introduced into competent cells of E. cog MC169, 7-KAP (30 pg/ml) could be measured in the culture supernatant, after 24 h of incubation with pimelate (0.3 mg/ml), whereas MC169bearing pBR322 did not show detectable conversion of pimelate into 7-KAP. A similar level of 7-KAP production was obtained with the same plasmid introduced into AbioH or AbioA-D genetic backgrounds, proving that the E. cog gene products of bioC or bioH did not contribute to this pimelate-7-KAP conversion. That bioW and bioF were both involved in the observed bioconversion was further demonstrated by the fact that

precise, short 4-aa deletions introduced into each of them, at the level of a pTc-bioXWF plasmid structure, totally abolished 7-KAP production from pimelate. At this stage, we cannot completely attribute pimelylCoA synthesis from pimelate to the bloW gene product; in vitro enzymatic studies will help to further clarify this point (O. Ploux, P. Soularue, R.G., Y.L. and A. Marquet, manuscript in preparation). But we can exclude that blow encodes a pimelate transport function, because both the phenotype of slow growth on nonsupplemented biotin-free medium and its aa sequence hardly resemble those of a membrane protein. (d) Biotin prototrophy on pimelate-supplemented medium of £scherichia coli C261 (AbioFCD) and R901 (AbioA-D) cells transformed with both Bacillus sphaericus bid gene clusters E. coli C261 (AbioFCD) and R901 (AbioA-D) mutants were used as test strains in complementation experiments. An expression vector (pTG1440) containing both B. sphaericus biotin clusters was constructed by subcloning in pBR322 the inserts carried on pTG1400 and pTGI418. With pTG1440 introduced into C261 and R901 E. coli strains, the frequency of biotin prototrophs was equivalent to that of Ap R clones and these transformed clones exhibited a maximal growth rate on biotin-free medium upon addition of pimelate (2/zg/ml). Again, weak growth of these strains and of bioC and b/oH pTG1440-containing strains could be seen on biotin-depleted medium.

(e) Analysis of the structural analogies between the Escherichia coil and Bacillus sphaericus biotin biosynthetic enzymes Sequence comparisons between the E. cog (Otsuka et al., 1988) and B. sphaericus biotin biosynthetic enzymes revealed many interesting features. Amino acid sequence analysis according to the rules of Needleman and Wunsch (1970) using the DNASTAR software (Madison, WI; data not shown) indicated broad similarities between the corresponding bioA, bioD and bioF gene products of 65, 52 and 65~o, respectively (allowing conservative replacements). Previously reported comparisons of bioB gene products (Ohsawa et al., 1989), indicated the same extent of similarity. However, no significant homology could be found between the K. coli bloc and B. sphaericus bioX, bloW or bioY gene products. Reciprocally, no homology was found between the putative BIOX, BIOW or BIOY proteins and the gene product of ORFI, located downstream from the E. coli bioA gene (Otsuka et al., 1988). This total absence of similarity between the bidW, bioXB, sphaericusgene product and the bioC or bioH (O'Regan et al., 1989), E. coli gene

69 products could underline essential differences in the cellular functions of these proteins.

(f) Conclusions and discussion Analysis of all the data communicated here, led us to believe that the bloC and bioH gene products were not catalyzing the specific transformation of pimelate into pimelyl-CoA. Essentially two different possibilities can be considered to specify their role.

(I) Pimelate synthesis from unknown precursors In this case, it remains unclear why, until now, none of the described complementation groups of E. coli bio mutants have included mutants solely affected in the pimelate-pimelyl-CoA metabolic step. In addition, the striking phenotypic effects of exogenous pimelate reported here do not include a release from the biotin auxotrophy of the bloC and bioH strains. This finding could, however, result from the combination of an inefficient pimelate cell permeation and an in vivo limiting rate of pimelyl-CoA synthesis. If this holds true, the following experiments should provide useful information: isolation of pimelatedependent bloC (bioH) mutant strains on biotin-depleted medium would confirm that increased cell permeability to pimelate can ensure their proper nutrition, as expected; shotgun cloning of E. coil genetic material into bioC (bioH) strains with a selection of clones strictly dependent upon pimelate for their biotin prototrophy would allow the isolation of the putative E. coli gene encoding pimelyl-CoA synthetase (ifa single subunit exists, as is probably the case for B. sphaericus ). (2) Catalysis of pimelyi-CoA synthesis from unknown precursors, other than pimelate A similar metabolic pathway has already been discussed by Eisenberg (1987), and is supported, in the case of Achromobacter, by results of isotopic labeling of biotin. The weak but significant complementation in the absence of pimelate of various E. coil bio- strains (including deletion mutants) reported here for several expression vectors containing B. sphaericus bio genes supports the existence of a small intracellular pimelate pool, most probably not linked to the physiological E. coil biotin biosynthetic pathway.

ACKNOWLEDGEMENTS

We are greatly indebted to A. Campbell, M. Schwartz and J.A. Hoch for generously providing us with E. coli and B. subtilb bio mutants. The support of J.-P. Lecocq and A. Yoshioka throughout this study was much appreciated. A. Marquet and O. Ploux are greatly acknowledged for their

active collaboration at the end of this project. Their results on the in vitro detection of enzymatic activities and on in vitro cross-complementation studies were of great help for us in the fmal analysis of in vivo complementation assays with the bioXWF gene cluster. Thanks go to J.F. Viret and M. O'Regan for suggestions to improve the manuscript, N. Poujol and E. Chambon for expert secretarial assistance and to E. Maetz for artwork.

REFERENCES Appleyard, R.K.: Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from E. coil K-12. Genetics 39 (1954) 440-450.

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NOTE ADDED IN PROOF

Careful analysis of the bioX aa sequence revealed an almost perfect match (at coordinates 1353-1382 of the nt sequence) with the consensus phosphopantetheine attachment site sequence: (L,I)-G-(A,L,I,V,M,F,Y)-D-S-(L,I)-X3-(D,E) This could suggest that one of the functions of the BIOX protein requires the binding of an acyl group.