Biochimie ( 1991 ) 73,235-244 © Soci6t6 fran~gaise de biochimie et biologie mol6culaire / Elsevier, Paris

235

Cloning of the recA gene of Bordetella pertussis and characterization of its product D Favre, SJ Cryz Jr, JF Viret Swiss Serum and Vaccine Institute, PO Box 2707, CHo3001 Berne, Switzerland

(Received 16 November 1990; accepted 10 December 1990)

Summary - - A recA gene of Bordetella pertussis was identified in a plasmid library by complementation of a recA mutation in E coli and subcloned as a 2. l-kb Sph I DNA fragment. Southern hybridization experiments showed no similarity to the E coli recA gene. but very strong similarity to other Bordetella species. E coli recA mutant cells containing the B pertussis recA gene at high gene dosage were resistant to DNA-damaging agents such as methyl methane sulfonate or 4-nitroquinoline-N-oxide, displayed induction of SOS functions, and were able to promote DNA recombination, but not induction of phage ~.. The latter phenotype distinguishes the B pertussis recA gene product from the corresponding proteins from most other Gram-negative organisms. Amino acid sequence comparisons revealed a high degree of structural conservation between prokaryotic RecA proteins. recA / ~. prophage induction / SOS induction / DNA recombination / DNA repair

Introduction Whopping cough, an acute respiratory disease, is caused by infection with the Gram-negative bacterium Bordetella pertussis. The action of pertussis toxin (PT) elaborated by B pertussis appears to mediate many of the symptoms characteristic of the disease [ 1]. A recent field trial in Swedish infants showed that immunization with ~ pertussis toxoid provided highly significant protection against disease [2]. However, concerns over the safety of the vaccine rules out its widespread use as a public health tool [3]. Several pertussis toxoids, derived either through recombinant DNA technology [4] or by chemical detoxification of PT [5] have been described and are undergoing clinical evaluation. One problem faced with the manufacturing of such vaccines is the low PT yield. Although the structural genes for PT have been cloned and sequenced, construction of a hypertoxinogenic strain has proven difficult. Several lines of evidence indicate that only members of the genus Bordetella may be suitable for the large-scale production of PT [4, 6]. One problem encountered in strain cons,ruction is the instability of plasmids introduced into B pertussis [7]. In an attempt to overcome this problem, we have initiated the construction of recombination-deficient mutants of B pertussis analogous t':J the E coli recA mutants. In E coli the RecA protein is involved in various cellular functions such as DNA homologous recombination, DNA repair, and induction of SOS response(s)

which include mutagenesis and cleavage of LexA and phage lambda repressors [8, 9]. The genes coding for RecA-like proteins have been cloned from a number of prokaryotic organisms often on the basis of complementation of E coli recA mutants. RecA-like proteins usually display a range of activities similar to the RecA protein of E coli [ 10-17]. In the present study, we have cloned and characterized the recA gene of B pertussis and compared the predicted amino acid sequence of its product [ 18] with those of RecA proteins from various prokaryotic organisms. Materials and Methods Bacterial strains and plasmids

Genomic DNA isolated from Bordetella pertussis 165 was used in the construction of a plasmid bank in vector pUC19 [19], and for later comparison with strains BP536. B bronchiseptica 932, and B parapertussis 207, 229, and 501. via Southern hybridization. Escherichia coli strains DH5ot. HB 101, C600 [20], and OT99 [21] have been described. Strain GY4533 is a fecAl strain used as a recipient for pOU61, a plasmid which replicates at parity with the chromosome at 37°C, but can be amplified by a temperature shift to 42°C followed by growth at 40°C or less [22]. E coli GY7313 thr-I leuB6 A (gpt-proA) his-4 argE3 i/v(Ts) galK2 aral4 xvl-5 mtl1 rpsL31 supE44 sfiA211 Alac ksfiA::lacZ ArecA306 srl::Tn 10 tsx33 was used to measure mitomycin C (MMC, Fluka. Buchs, Switzerland)-induced sfiA activity. Induction of k prophage and of recombination was determined using strains GY5360 (k) metB thi pyre lacMS286 080dlllacBKl ArecA306 and GY7066

236,

D Favre et al

metB thi pyrE lacMS286 ~'OdlilacBKl ,sfiB114 [231 respectively. Plasmid pSSVII9 is a pUCI9 derivative containing a i5.6-kb EcoRl fragment of chromosomal DNA bearing the recA gene ot B pertussis BPI65 (this study), pSSVI25-7 and psSV125-8 c,'u'ry the latter gene in opposite orientations on a 2.i-kb Sphl fi'agment cloned into pUCI9, pSSVI28 is a pOU61 defvative carrying the recA gene of B pertussis on a 9-kb BamHl fragment, pOU61 :RECA corresponds to pOU61 containing the recA gene of E coli (R Devoret, persona4 communication)

Induction ¢~'recomhination

Media and growth conditions

Polyacrylamide gel electrophoresis [25] with or without SDS, and Western blotting [26] were as described. For identification of RecA proteins blotted onto nitrocellulose, a polyclonal antiserum directed against the E coil RecA protein was preadsorbed to a crude lysate of GY4533 (recA1) cells before use.

LB broth, LB agar, McConkey agar, and soft agar used for ~. plating have been described previously [241. B pertussis cultures were grown on Bordet-Gengou (BG) agar (Difco) supplemented with 15% horse blood. Liquid cultures were ~ w n in modified Stainer-Sckolte medium (Gibco). Antibiotic resistance plasmids were selected by addition of 50 or 100 .uglml ampicillin (Ap) to the growth medium, as indicated. Rec + strains were selected on plates covered with 150 ~1 of 2% methyl methane sulfonate (MMS, Fluka) solution.

GY7066 transformants were plated onto McConkey agar plates containing 50 tag/ml Ap and either 0, 1.5, or 3 mM MMS. Alternatively, individual transformant colonies were grown overnight in LB broth and similar cell densities were plated out. In both cases, the plates were incubated for 2 days at 37°C followed by 36 h at 20°C. Colonies which exhibited red papillae were scored as lac +.

PAGE and Western blotting

Analysis of protein sequences Alignment of predicted amino acid sequences was carried out using the ALIGN program from the computer software package of the University of Wisconsin Genetics Computer Group in a VAX computer [27].

Recombinant DNA techniques General molecular techniques, including pl,smid sequential deletions and Southern hybridization, were performed according to standard procedures [201.

Resistam'e to MMS and 4-NQO Cultures were ~own at 37°C in LB broth to an absorbance at 600 nm (Aroo) of 0.3 (corresponding to --- 108 colony-formingunits (CFU)/ml), then shifted to 42°C for 60 min. 20 mM MMS or 0.2 mM 4-nitroquinoline-N-oxide (4-NQO, Sigma) (final concentrations) were then added and the cultures grown at 37°C. Samples (0.5 ml) were taken at various times, washed by centrifugation, and resuspended in an equal volume of phosphate-buffered saline. Dilutions of the cell susoensions were plated on LB agar and the plates incubated at 37°(2. SfiA induction Cultures of GY7313 derivatives were grown in LB broth at 37°C to an Ar00 of 0.2 and treated for 10 min with 20 lag/ml MMC, The drug was then removed by filtration and the cells resuspended in fresh prewarmed medium. Incubation was continued at 37°C and l-ml samples were collected at various times. Cell suspensions were sonicated and the lysates assayed for B-galactosidase activity and total protein content [24].

Prophage imhwtion GY5360 derivatives were grown in LB broth at 37°C to an Ar(~) of 0.2, diluted in the same medium to an A~0 of 0.1, temperature-shifted to 42°C and incubated until the A6(•) reached 0.2. Cultures were then split 2 ways. One portion was treated with 20 l.t~ml MMC for 10 min, the cells filtered, washed, resuspended into fresh prewarmed medium, and allowed to ~ o w at 37°C for 120 min. The other portion was grown for 120 rain without MMC treatment. 500 lLtl aliquots of the cultures were then mixed with 50 lal of chloroform to lyse the cells, and the cellular debris was pelleted by centrifugation. Phages "~,ere then titrated in the supematants by plating on lawns of E coli LE392 [20].

Results Cloning o f the r e c A gene o f B pertussis An EcoRI genomic bank of B pertussis was constructed in the h i g h - c o p y - n u m b e r - p l a s m i d p U C 19. Rec+ clones w e r e selected by plating H B 1 0 1 (recAl3) transf o r m a n t s directly onto M M S - c o n t a i n i n g LB-Ap (100 lag/ml) a g a r plates ( L B - A p - M M S plates). D N A isolated f r o m several Apt, M M S r colonies w a s transf o r m e d in parallel into f e c A l (DH50c, O T 9 9 ) and recA13 ( H B I0 I) recipients. All clones tested c o m p l e m e n t e d both recA m u t a t i o n s and were a s s u m e d to c a r r y a recA gene. O n e o f these, p S S V I 1 9 , w a s further a n a l y z e d . Restriction analysis defined a 15.6-kb EcoRI f r a g m e n t c a r r y i n g the gene. S u c c e s s i v e subcloning steps allowed the isolation o f the recA gene on a 2.1-kb Sphl f r a g m e n t cloned in both orientations into p U C 1 9 ( p S S V I 2 5 - 7 and p S S V I 2 5 - 8 ) . N e s t e d deletions o f the insert in both clones were g e n e r a t e d using a c o m b i n a t i o n o f e x o n u c l e a s e III a n d S1 nuclease treatment. Precise localization o f the gene w a s then d e t e r m i n e d by plating out cells c o n t a i n i n g p l a s m i d s with increasing deletions onto L B - A p M M S plates. S e q u e n c e analysis [18] s h o w e d that the g e n e w a s transcribed f r o m left to right on p l a s m i d p S S V I 2 5 - 7 (fig 1).

Relatedeness o f r e c A loci in Bordetella species U s i n g the SphI f r a g m e n t o f p S S V I 2 5 - 7 as a radiolabeled probe, we a s s a y e d the extent of similarity b e t w e e n the recA gene o f B pertussis and the corresp o n d i n g genes in B bronchiseptica, B parapertussis, and E coli by Southern hybridization. G e n o m i c D N A

ret'A gent of Bmde te//a pertussis

X PSaSaP

ES l

i

i

I

Resistance to DNA-damaging agents

SaSmSmSa

I

i

0

t

I

I

PSH I

i

1

I

2

kb

Fig 1. Physical map of the pSSVI25-7 insert (vector sequences not shown). Thick line: recA-like gene. Arrow: direction of transcription. Restriction endonucleases (RE): E, EcoRI; H, HindlIl; P, Pstl; S, Sphl; Sa, Sail: Srn, Smal; X, XhoI. Underlined RE abbreviations depict salient sites in the polylinker of the vector (other sites are not shown).

from B pertussis 165 and 536, B bronchiseptica 932, B parapertussis 207, 229, 501, and from E coli C600 were cut with either BamHI or XhoI and blotted onto nitrocellulose. Results showed no apparent homology to the E coli gene, even in conditions of low stringency hybridization. In contrast, hybridization to DNA from the various Bordetella strains was of equal strength and presented identical banding patterns, indicating a high degree of DNA similarity at the recA locus (results not shown). This is compatible with the previous reports of Musser et al [28] and Arico et al [29], who demonstrated a close genetic relatedness between all 3 Bordetella species.

3! .

237

.

.

.

.

.

.

.

Thc ability of the B pertussia recA gene to confer resistance to DNA-damaging agents was measured by the survival of GY4533 cells containing pSSVI28 after treatment with either 20 mM or 0.2 mM 4-nitroquinoline-N-oxidc (4-NQO/, a compound resulting in DNA lesions repaired by the same mechanisms as UV light-induced pyrimidine dimers [30]. When cells containing pSSVI28 were grown at 37°C, no MMS or 4-NQO resistance was detectable (data not shown). However, when plasmid copy-number was amplified = 25-fold via incubation at 42°C for 1 h, the cells became resistant to both MMS and 4-NQO. Negative controls were provided by the host E coli recA mutants alone or harbouring the plasmid vector. Positive control was pOU61 carrying the wild-type recA gene of E coli (pOU61 :RECA) (fig 2). Induction of SOS response sfiA induction One of the responses of E coli cells to SOS-inducing agents is filamentation [9]. When exposed to MMC the cells continue to elongate but do not form septa, as a result of the derepression of the sfiA gene through RecA-induced cleavage of the LexA repressor. To test the SOS-inducing proficiency of the B pertussis RecA protein, plasmids pSSVI28, pSSVI25-7, and control

.

i i

m

E

Q.

._I

-6 _'

0

I

I

I

I

I

I

80

40

60

80

100

120

Time in MMS (min)

0

I

I

I

I

10

20

30

40

Time in NQO (rain)

Fig 2, Survival of GY4533 derivatives after exposure to MMS or 4-NQO. Panel A: MMS treatment. Panel B: 4-NQO treatment. (e), no plasmid, (m), pOU61, (r~), pSSVI28, ( o ), pOU61 :RECA.

D Favre et al

..~

plasmids were transformed into the lac, recA mutant strain GY7313, which contains a sfiA::lacZ translational fusion in the chromosome (R Devoret, personal communication). In GY7313, derepression of sfiA translates into concomitant synthesis of B-galactosidase providing an easy assay for the induction of SOS functions. Following a 10-min exposure to 20 la~mi MMC, sfiA expression increases up to 8-fold in pOU61:RECA-bearing cells (fig 3). In contrast, the presence of pSSVI28 failed to stimulate any expression of sfiA even alter transient incubation at 42°C. However, the high-copy-number clone pSSVI25-7 provided an 18-fold activation, a phenotype indicating that the B pertussis recA gene product is actually able to catalyze the cleavage of the E coli LexA repressor.

Table I. Prophage induction in the ~, lysogenic recA strain GY5360. Induction experiments were performed as described in Materials and Methods. Plasmid

4

t~

'o ~3

I:: W c

PFU/ml from lvsates after treatment with." No MMC MMC

pOU61:RECA

+

9.4 104

6.5 106

pSSVI25-7

+

2.0 102

6.6 102

pSSVI28

+

0

3.0 101

pOU61

-

4.0 101

5.0 100

9.0 101

1.7 102

-

A Prophage induction RecA-induced cleavage of the phage lambda repressor is another component of the SOS response [9, 31]. Therefore we tested the ability of the B pertussis recA gene to induce the ~, prophage in strain GY5360, either spontaneously or in response to recA activation by MMC. After M M C induction of the positive control GY5360 (pOU61:RECA), phage titers were 60 times higher than those arising from spontaneous induction (table I). Titers for GY5360 (pSSVI28) were comparable to those of the negative controls. At higher gene dosage, such as provided in GY5360

recA genot3,pe

-

(pSSVI25-7), a low level of spontaneous phage induction occurred, which was of the same order of magnitude as in the absence of the cloned gene. Treatment with MMC, however, did not raise titers significantly. Induced recombination

In order to assay for the recombination-promoting capability of the pertussis recA gene, the latter was transformed into GY7066, an E coli recA strain which contains duplicated copies of partially deleted lac genes such that a recombinational event is required in order to reconstitute a functional operon [23]. Complementation of recombination deficiency gives rise to colonies containing red papillae when grown on McConkey agar. The transformed cells were directly plated onto McConkey agar with or without MMS to induce RecA functions. Alternatively, the transformed strains were grown overnight and similar cell densities were plated out. Both sets of experiments gave similar results. Data obtained by direct plating of transformation mixtures are presented in table II. In the absence of MMS, recombination Table II. Proficiency of induced and non-induced lac recombination in GY7066 transformants. Scoring of lac+ recombinants was as described in Materials and Methods. NG: no growth. Plasmid

t-

0

v 30

_~ 60

_-_ ~ 90

~120

Time (min) Fig 3. Induction of sfiA::lacZ fusi n expression in GY7313 derivatives. (e), no plasmid, (,.), pOU61, (A), pUC19, (~:), pSSVI28, (: k pSSVI25-7, (o), pOU61 :RECA.

pOU61 :RECA

Percentage of lac + colonies~plate no MMS 1.5 mM MMS 3 mM MMS

38

55 1.50

62

pSSVI25-7

0.006

0.75

pUCI98

0

NG

NG

pOU61

0

NG

NG

recA gene of Bordetella pertussis activity linked to the B pertussis recA clone was at least 3 orders of magnitude lower than for the E coli recA gene. When MMS was present in the plates, the percentage of red papillae obtained for GY7066 (pSSVI25-7) increased 250-fold amounting to 1-2% of the corresponding value for the E coli recA clone.

Protein analysis S D S - P A G E analysis of crude lysates prepared from MMC-induced GY4533 cells bearing pOU61:RECA revealed high levels of the induced E coli 38-kDa RecA protein [32]. However, when plasmids pSSVI25-7, pSSVI25-8 or pSSVI28 were used in the same conditions the B pertussis gene product could not be detected. Attempts to detect the B pertussis protein, whether denatured or in its native conformation by Western blotting using a polyclonal antibody directed against the E coli RecA protein were equally unsuccessful (data not shown).

Amino acid sequence comparison of bacterial RecA proteins The amino acid sequence of the B pertussis RecA protein was deduced from the corresponding DNA sequence [18] and compared to its counterparts from various prokaryotes (fig 4). Numerous amino acid clusters with extensive similarity, and even total identity among all species, are observed, particularly in the middle part of the proteins. Many more similarity patterns can be drawn when pairwise comparisons are made. For example, the RecA proteins of the 2 Enterobacteriaceae species Serratia marcescens and Proteus mirabiiis, show = 85% (95% for the first 300 amino acids), respectively 75% identity to the E coli protein, whereas those of E coli and Synechococcus sp (a cyanobacterium) are only -- 55% identical. The B pertussis protein presents an intermediate situation with 63% identity to the E coli protein.

Discussion The recA gene product is a multifunctional protein involved in the promotion of DNA pairing in homologous recombination and recombination-dependent DNA repair, as well as in the derepression of SOS functions. Strains carrying a mutated recA gene therefore suffer from an impaired capability to repair their DNA after exposure to DNA-damaging agents. Taking advantage of this phenotype we have cloned a gene from B pertussis that complements recA defects in E coli and we have shown that this gene corresponds to the E coli recA homolog. First, the product of the cloned fragment is able to restore RecA-specific activities in E coli recA mutants, including DNA

239

repair, induction of filamentation, and homologous recombination. Second, the predicted amino acid sequence shows a high level of similarity to the recA gene product of E coli and other bacterial species. It is noteworthy that we were unable to detect any recA complementation when the B pertussis recA gene was present at parity with the host cell chromosome. A 25-fold amplification of plasmid copynumber was necessary to detect resistance to DNAdamaging agents, and the presence of the gene on a high-copy-number pUC plasmid was required for the induction of the sfiA gene and for homologous recombination. Although this may have resulted from functional differences, we favour the interpretation that the gene might be expressed at very low levels in E coli, as already reported for other B pertussis genes, such as those encoding PT [6]. This view is supported by the fact that the RecA protein could not be detected by PAGE or Western blotting, even under high-copynumber conditions. In contrast, the same antiserum used to probe the B pertussis RecA protein could recognize the RecE protein of B subtilis (JC Alonso, personal communication) in agreement with a previous report [11]. Likewise, the observed crossreactivity between RecA proteins of various Gramnegative species [10, 33-35] can be attributed to a high degree of structural conservation among RecA proteins. It is therefore unlikely that the B pertussis RecA protein encodes epitopes structurally unrelated to other recA gene products. In this context, it is probable that the B pertussis RecA protein is functionally as proficient as its E coli counterpart with respect to DNA repair, cleavage of the LexA repressor, and homologous recombination. in the latter case, the increment of recombination promoted by the DNA-damaging agent was much higher for pSSVI25-7 than for the E coli retA clone. This may stem from the very low frequency of recombination observed under non-induced conditions for the B pertussis recA clone compared to that of its E coli counterpart (38% of red papillae in the absence of MMS). Low recombinase activity may be due to the fact that this function requires high amounts of RecA protein interacting with the DNA substrate [36]. We then infer that, in spite of low gene expression, the recA gene product displays a measurable inducible recombinase activity. Efficient recombination was also demonstrated in pSSVI25-7-containing cells by their ability to rescue )~ red, gam mutants and by the quick accumulation of plasmid multimers after transformation with purified plasmid monomers (data not shown). In contrast to the above phenotypes, the B pertussis recA clone was unable to promote either spontaneous or SOS-induced cleavage of the )~ repressor, even when the gene was carried on a high-copy-number plasmid. Since the B pertussis RecA protein seems to

", ,,~

D Favre et a /

20

40

AIDENKQKALAAALGQIEKQ_F_GKGSIMRLGEDRSMDVETI

Sm

M M

Pm

MLHCPEGDNM

.....................

Pa

M

Tf

MDEQRSKG

Am

MDR

Bp

MDDKTSKAAAAE

Bs

MSDRQAA

Av

MAINTDTSG

.....

Ssp

MSAISNNPD

.E...NLV.N...RN

Ec

60 ALGAGGLPMGRIVE!

Ec

o

o

o

o

e

e

o

o

o

e

o

o

o

o

o

o

o

o

o

o

o

o

Pm

........

o

o

o

o

o

o

o

o

.,.I

....

...V

.... R..V

Am

...I..V.R

.......

o

o

•

TMV.N...RS

•

•

.

e,

•

•

•

•

•

•

•

•

.............

....... .M.

Ec

G_VDIDNLLCSOPDTGEQALEICDALARSGAVDV

•

•

EV ..........

R..V

.EI ..........

............

T.M.V..N ~ M V

T. . . . . . . . . . . . .

.

.

.

.

.

.

.

.

.

.

-

-

.

.

.

.

.

.

.

Bp

..NLTD..I

............

T...V...S..L

Bs

• .N.EE..L

............

AE..V

Av

.... Q...V

.......

Ssp

.... E...VA...N..S

EC

LAARMMSQ_AMEKLAGNLKQSNTLLIFINOIRMKIGVMFGN • .................NA...........~........ -

o

e

o

e

.......

o

o

e

o

o

o

o

o

o o o o

o

.....

o o

o o

o o

V

o G o o o

.G..L

....

.G..V .G.,.

....

.A..T

A .T. ,L .A .T..Q

.......

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

A.T.V.P

.... V ........ ........... .... V..:

.....

~

.

.....

~

e

.Q..L

.... L..IT..

.... V .......

o

o

N

e

o

o

e

I.NA.C.V

o

o

e

e

o

o

o

D

•

o G o

•

G

•

o H o

•

•

o S o

•

V • .Q.

•

.... T •. SA. T • SAA.

...........

.

d.

•

•

V ........ •

•

•

•

L ........ ..I

•

•

•

•

•

•

•

•

M. oA.V. ~ V

e&-ao

•

•

•

•

V.RG.L...M..N...

......

V ........

M...LP.

V ......

V .......

DM .... V.

I ......

V.R

.....

DM..A.V.

......

V.R

......

..I

M..VQV. 220

PETTTGGNALKFYASVRLD ................... o

o

e

o

o

o

o

e

o

o

o

e

o

e

o

o

o

o

e

o

o

e

o

o

o

o

o

o

...................................

Tf

.Q..L

.... L.N.

Am

.H..L

.... L...T.SVSK.K.IV

..............

Bp

•Q . . L

.... L...TATI.RT.CMV

..........

TANISR

•

. • . . . . . . . . . . . . . . . . . . . .

......

o

o K o

• oQ..

VQ

200

e

•

DSVAALTPKAEIEGEIGDSHM_G

.........

S .... V.Q.VP.A

•

S

...........

.I.

.

•

I . . . . . . . . . . . . .

160

.... A.Q.V..A...L

Pa

.... V .............

Bs

.Q..L

.... L...S.AINK.K.IA

.......

Av

.Q..L

.... L..IT..IGK.GCTV

.....

Ssp

•Q . . L . . K . L . . I . . . M G R . G C T V . . L . . L . Q . . . I S Y

F i g 4. See l e g e n d n e x t p a g e .

•

EM.KL.G

180

e

•

.... I ........ T. 7

• AL.E..I.E..A

o

•

IVV

Am

o

o

140

• .NV.D..V

.

•

S ......

VA.HA..EV.KA.GVA

Pa

o

o

VA.HA..EV.K..GIA..V...Q

................

• • . . . • . . . . • . . . • • • . . . • • . • . T o . . • . . .

'

•

VA.HA..EVTQQRTS

Sm Pm

o

•

A.HI..E..KK.G

120

Sm

o

I .... DAAQ.K.A..P.

S...E..KQ.A

EV ...........

Ssp

Pm

e

100

.EV .................

.

o

N ....

HA..SC.AA.G.A

Av

.

o

K...KTDTRIS.VP...

.....

................

....

.

o

.... A. .... DATR.R

. . . . . . . . . . . . . . . . .

.... AAV

.

o

80

....

.

S_TGSLSLDI

o

Y.DNEVEHDIQVV

............

...V

.

o

K..GKDQVVETEVV..RI

S .............

...I..Y.R

.

o

AV .... DHNAIKDIEVY

Bp

LW.

o

......

Bs

Tf

o

.S...S..D

K ......

..F.R

o

YGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY_ARKL o

Pa

o

AV..M.DHERQAIPA

R..M.,.

Tf

o

R .....

.A .......

o

o

.... KR ..........

.DM..K

o

o

. .............

.... E..VS...RA

Sm

o

o

....-

................... ................

A ..................

E.V

L.Q

Y.S

.... FRE

.... TY.S ....

.......

R .....

ME S ..... S .... E

................... V .... T ...........

240 260 280 IEIGAVEEGENVV GSETRVKWKNKIAAPJKQAEFQJLYGEGINFYGELV~GVKEKL EC Sm .... ..I...DE ........................ ..M......SR.........H .M ... ..S..N.DE .I .. ..A.......V...........M......T Pm .. ..I Pa . ..T......DE .. .......... ..VSP..R.........K..YRT..II::::,k .... ..I.KSDE .. .ND.........V.P..RE...A.Y.....SRLS........FD I Tf . ..V..I.DRDE .. .NQ.........L.P...WD.D.M.....SKM...I Am ... ..ANV .... ..I.K.DE .. .N..........V.P.......D.M..S..SRE..II....Q AN-V BP Bs V..AEQL.Q.ND .M .NK.KI......V.P..RT..VD.M.....SKE..II...TELD I .. ..QTL.K.TDE F .NRVK...A...V.P..RI...D.IF.K.VSTL.C....AEETG I Av V ... D.IF.K..SRV.CML..AEQTG SSP .. ..QTL.K.SEGEF.IRAK...A...V.P..RI EC Sm Pm Pa Tf Am Bp Bs Av Ssp

EC Sm Pm Pa Tf Am BP Bs Av Ssp

320 340 300 IE _KAA~YSYKGEKIG~~_ATAW~KDNPETAKEIEKKV~ELLLSNPNSTPDFSVDDS . . . . . . ..N...........CNF..E..AI...LD..L.D...HSGGELVAASG..F 6: . . . . . . ..N . . . . . . . . . . ..NY..EH..MYN.LNT.L..M..NHAGEFTSAADF IGSVL..TI.DQ..AKSGPVKADAEEVA V. .S......Q.S.........AKY.E.... V. .S . . . . ..Q.HR.....D..RQY.. VH..L.AN..QRI.AAAAGH.LAFAEEVESPQ .GAI.QNAGLISEALAAVPDL.G VK.S . ..F..NSTR . . ..RE..KQF.R...AM.A.. VD .S......S.NR.....D.VREY..EHK.M.I...N....NQGIVSRAATFPASEAE VQ .S.S....EE.RL...RE..KQF..E.KDIMLM.QEQI..HYGLDN.GWQQQAEET LLR. . . . . ..N.DN.S..RD..IKY.EEK..F.EQ.KQQ...K.DKGAWSANSVAKAN .TR. . . . . ..E.DN.A..RD..VKY.EE..DV.AIVTQ....N.DMSSMGFG.EHHTTE 352 EGVAETNEDF .DDEAETSEQF

(100%)

DAEAD RSAS TP... D QEEL.FEE .EDE.DVDLDEEE .E

( ( ( ( ( ( (

( 87%) ( 84%) 71%) 66%) 62%) 63%) 60%) 58%) 56%)

Fig4.Alignment of the RecA protein amino acid sequences from EC: E coli K-12 [32], Sm: Sewatia tnarcescens [41]. Pm: Proteus mirahilis [42], Pa: Pseudomonas aei-rrginosa [43], Tf: T~~ioha~illus.fer.r-oo.~idans [44], Am: Aquaspirillur?1 magnetotac*ticurn [45] Bp: Bordetella per-tussis [ 181, Bs: Bacillus suhtilis [46], Av: Auahaerla rTar.iahilis [35], Ssp: S_vjrechococ*crtssp [47]. Periods indicate identity with the E coli sequence. Total identity among species is denoted by underlined letters in the E coli sequence. When necessary, gaps (blank spaces) were introduced to optimize alignments. Percentages of identical amino acids with respect to E coli RecA protein are shown in parentheses at the end of each sequence.

242

D Favre et al

be activated by MMC treatment as revealed by its capability to cleave the LexA repressor with the ensuing induction of the sfiA gene, we conclude that it differs markedly from its E coli counterpart with respect to phage repressor cleavage. RecA proteins from other bacterial species differ also in their capability to induce 3-, as shown by either in vivo or in vio'o studies. Table III summarizes available in vivo data on cleavage of the 3- repressor by heterologous RecA proteins. It appears that the recovery of spontaneous induction levels comparable to that for the E coli K-12 recA gene occur for most of the genes tested, except for those isolated from the strict aerobes L pneumophila and B pertussis which are phylogenetically re]atively distant from E coli. In contrast, SOS-induced phage production correlates only loosely with the phylogenetic proximity of the various organisms. Owttrim and Coleman [35] have suggested that the failure of the cyanobacterium A variabilis recA gene to promote SOS induction may have resulted from the absence of binding of the E coli LexA repressor to the regulatory region of the cloned recA gene. A similar explanation for our data is not likely, since a typical LexA box was found in a putative promoter region of the B pertussis gene [ 18]. A phenotype similar to that observed for the B pertussis gene, namely the ability of promoting cleavage of the LexA repressor but not that of the 3repressor, was previously reported for the purified RecE protein of B subtilis in vio'o [11 ]. Inversely, in the ,~ subtilis system, the E coli recA gene product permits the induction of an SOS response through the putative inactivation of a LexA-like effector, but not cleavage of the lysogenic phage 0105 repressor [37]. On the other hand, in vitro cleavage of the Salmonella phage P22 repressor in the presence of the E coli RecA protein has been documented [38] whereas the P mirabilis RecA protein is able to catalyze the in vio-o inactivation of both E coli LexA and 3repressors [39]. Therefore, available data point to functional conservation of RecA-like proteins with respect to the induction of SOS functions and to greater oiversity with regard to the cleavage of phage repressors. While the ft~rmer phenotype may s'em from the necessary coevolution of a multicomponent system (eg, RecA/ LexA couple, multiple lexA boxes), the latter reflects a stronger divergence of phage repressors in the course of evolution. Comparison of RecA protein sequences among various prokaryotic genera revealed a high degree of similarity. This was suspected earlier on the grounds of the aforementioned studies showing immunological cross-reactivity and a large extent of functional conservation between heterologous RecA proteins with the exception of 3, repressor cleavage, which is the only clearly diverging phenotype. Dutreix et al

Table III. Induction of prophage ~. by heterologous RecA proteins. Ec, Av, and Bp. See legend to figure 4. Sf: Shigella flexneri [10], Va: $Tbrio anguillarum [16], Bf: Bacteroides fragilis [13], EcB/r: Escherichia coli B/r [10], Pv: Proteus vulgaris [10], Eca: Erwinia carotovora [10], Lp: Legicnella pneumophila [40]. Effect of heterologous recA clones on phage titers is: (+) comparable to that of the wild-type E coli recA gene; (-) insignificant. Effect of heterologous recA clones on phage titers after treatment with DNA-damaging agents is: (+) > 20-fold, (+/-) 5-20-fold, (-) < 5-fold stronger than that of the untreated control. Strain

Spontaneous induction

Ec Sf Va Bf EcB/r Pv Eca Av Lp Bp

+ + + + + + + + -

SOS induction

+ + + + +/+/_ _ _

[23] showed that a glycine to serine change at codon 204 of the E coli protein abolishes the proteolysis of the 3- repressor. Comparison of the amino acid sequence at, and around, codon 204 reveals that this residue is unchanged in all species except in B subtilis and is part of a highly conserved region. In B pertussis, the next 14 codons downstream and upstream from this site are identical to those of the E coli RecA protein, except for an alanine replacing a glycine at position 200 (E coli coordinates), a relatively conservative change. Although the latter substitution may account for the lack of 3- induction from the B pertussis RecA protein, it is most likely that additional changes within other distinct, possibly less conserved areas also affect the cleavage of the phage repressor. In B subtilis, codon 204 is replaced non-conservatively by a phenylalanine residue, followed downstrer_m by a further non-conservative change (asparagine ,~,q arginine) and a glutamic acid insertion. On the whole, this area of the B subtilis RecA protein has sufficiently diverged to possibly account for the observed lack of 3, repressor cleavage in vitro. By taking advantage of functional differences such as that described above, we believe that extensive amino acid sequence comparisons between RecA proteins from diversely related organisms should complement the genetical approach to the study of structure-function relationships. This general strategy should allow better definition of potential targets for future site-directed mutagenesis experiments and

recA gene of Bordetelht pertussis

t h e r e b y h e l p to u n r a v e l the i n t e r p l a y a n d c o n t r i b u t i o n s o f structural m o t i f s to the p h e n o t y p e s a s s o c i a t e d w i t h the R e c A p r o t e i n .

relative various 14 15

Acknowledgments We thank J Jelk for excellent technical assistance. We gratefully acknowledge R Devoret for the gift of plasmids and bacterial strains, and the communication of data presented in figure 4. We are also grateful to JC Alonso for the RecA antiserum and communication of unpublished data, and to AB Lang for performing the Western blots.

16 17 18

References 1 2

3 4

5

6 7

8 9

10

11 12

13

Weiss AA, Hewlett EL (1986) Virulence factors of Bordetella pertussis. Altttlt Rev Microbioi 40, 66 !-686 Ad Itoc Group for the Study of Pertussis Vaccines (1988) Placebo-controlled trial of two acellular pertussis vaccines in Sweden-Protective efficacy and adverse events. Lancet i, 955-960 Cameron J (1987) Safer aceilular and whole-cell pertussis vaccines. Lancet i, 1427 Pizza M, Covacci A, Bartoloni A, Perugini M, Nencioni L, De Magistris MT, Villa L, Nucci D, Manetti R, Bugnoli M, Giovannoni F. Olivieri F, Barbieri JT, Sato H, Rappuoli R (1989) Mutants of pertussis toxin suitable for vaccine development. Science 246, 497-500 Sekura RD, Zhang Y, Roberson R, Acton B, Troilfors B, Tolson N, Shiloach J, Bryla D, Muir-Nash J, Koeller D, Schneerson R, Robbins JB (1988) Clinical, metabolic, and antibody responses of adult volunteers to an investigationai vaccine composed of pertussis toxin inactivated by hydrogen peroxide. J Pediatr ! 13,806-813 Nicosia A, Bartoloni A, Perugini M, Rappuoli R (1987) Expression and immunological properttes of tile five subunits of pertussis toxin, h!/'ect lmmtol 55,963-967 Lee CK, Roberts A, Perrin S (1989) Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids, h~'ect Immun 57, 1413-1418 Radding CM (1989) Helical RecA nucleoprotein filaments mediate homologous pairing and strand exchange. Biochim Biophys Acre 1008, ! 3 i - 145 Walker GC (1987) The SOS response of Escherichia colt. In: Escherichia colt attd Salmonella typhin.urium. Cellular attd Molecular Biology (Neidhardt FC, Ingraham JL, Low KB, Magasacrik B, Schaechter M, Umbarger HE, eds) American Society for Microbiology, Washington, DC Keener SL, McNamee KP, McEntee K (1984) Cloning and characterization of recA genes from Proteus vulgaris, Erwinia carotovora, Shigeila flexneri, and Escherichia colt B/r. J Bacteriol 160, 153-160 Lovett CM, Roberts JW (1985) Purification of a RecA protein analogue from Bacillus subtilis. J Biol Chem 260, 3305-3313 Murphy RC, Bryant DA, Porter RD, Tandeau de Marsac N (1987) Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp strain PCC 7002. J Bacteriol 169, 2739-2747 Goodman HJK, Parker JR, Southern JA, Woods DR (1987) Cloning and expression in Escherichia colt of a

19

20 21 22 23

24 25 26

27 28 29 30

31 32

243

recA-like gene from Bacteroi~h's fra~ili.~. Gene 58. 265-27 [ Resnick D, Nelson DR (19881 Cloning and characterization of tile Aeromonas caviae recA gene and construction of an A caviae recA mutant. J Bacteriol ! 70. 48-55 Farrand KS. O'Morchoe SP. McCutchan J (1989) Construction of an Agrobacterium tlonefaciens C58 recA mutant. J Bacteriol ! 7 i, 5314-5321 Singer JT (1989) Molecular cloning of the recA analog from the marine fish pathogen Vibrio anguillarum 775. J Bacteriol 17 I, 6367-6371 Liao CL, Liu YT (1989) Cloning of the Serratia marcestens recA gene and construction of a Serratia marcexcens recA mutant. ,I Gen Microhiol 135, 3319-3327 Favre D, Viret JF (1990) Nucleotide sequence of the recA gene of Bordetella pertussis. Nucleic Acids Res 18.4243 Yanish-Perron C. Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M l 3 m p l 8 and pUCI9 vectors. Gene 33. 103-109 Sambrook J, Fritsch EF, Maniatis T (1989) Molecuhw Cloning: A Labm'atorv Manual. Cold Spring Harbor Laboratory Press, Cold ~;pring Harbor, NY Harayama S, Oguchi T, Tino T (1984) Does TnlO transpose via the cointegrate molecule? Mol Gen Genet 194. 4A.A A50 Larsen JEL, Gerdes K, Light J, Molin S (1984) Low-copynumber plasmid-cloning vectors amplifiable by derepression of an inserted foreign gene. Gene 28, 45-54 Dutreix M, Moreau PL, Bailone A, Galibert F. Battista JR. Walker GC, Devoret R (1989) New recA mutations that dissociate the various RecA protein activities in Escherichia colt provide evidence for an additional role for RecA protein in UV mutagenesis. J Bacteriol 17 I, 2415-2423 Miller JH (1972) E.weriments in Molectdar Genetics. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685 Burnette WN (1981) "Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, i 95-203 Devereux J. Haeberli P. Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395 Musser JM, Hewlett EL. Peppier MS. Selander RK (1986) Genetic diversity and relationships in populations of Bordetella spp. ,I Bacteriol 166, 230-237 Arico B, Gross R, Smida J, Rappuoli R (1987) Evolutionary relationships in the genus Bordetella. Mol Microhiol 1,30 !-308 Kondo S, Ichiwaka H. Iwo K, Kato T (1970) Base change mutagenesis and prophage induction in strains of Escherichia colt with different DNA repair capacities. Genetics 66, 187-217 Dutreix M, Bailone A, Devoret R (1985) Efficiency of induction of prophage lambda mutants as a function of recA alleles. J Bacteriol 161, ! 080- ! 085 Sancar A, Stachelek C, Konigsberg W, Rupp WD (1990) Sequences of the recA gene and protein. Proc Natl Acad Sci USA 77.2611-2615

244 33 34 35 36 37 38 39 40

D Favre et al Hamot~! AN, Pettis GS, Parker CD, Mclntosh MA (1986) Isolation and characterization of the Vibrio cholerae recA gene. J Bacteriol i 67, 375-378 Miller RV, Kokjohn TA (1988) Expression of the recA gene of Pseudomonas aeruginosa PAO is inducible by DNA-damaging agents. J Bacteriol 170, 2385-2387 Owttrim GW. Coleman JR (1989) Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene. J Bacteriol 171, 57 i 3-5719 West SC (1988) Protein-DNA interactions in genetic recombination. Trends Genet 4, 8-13 Love PE, Yasbin RE (1986) Induction of the Bacillus suhtilis SOS-like response by Escherichia coli RecA protein. Proc Nati Acad Sci USA 83, 5204-5208 Sauer RT, Ross MJ, "'Ptashne M (1982) Cleavage of the )~ and P22 repressors by recA protein. J Biol Chem 257, 4458--4462 West SC, Little JW (1984) P mirabilis RecA protein catalyses cleavage of E coli LexA protein and the ~. repressor in vitro. Mol Gen Genet 194, 111-1 i 3 Dreyfus LA (1989) Molecular cloning and expression in Escherichia coli of the recA gene of Legionella pneumophila. J Gen Microbiol 135, 3097-3107

41 42 43 44

45 46 47

Ball TK, Wasmuth CR, Braunagei SC, Benedik MJ (1990) Expression of Serratia marcescens extracellular proteins requires recA. J Bacteriol 172, 342-349 Akaboshi E, Yip MLR, Howard-Flanders P (1989) Nucleotide sequence of the recA gene of Proteus mirabilis. Nucleic Acids Rcs 17, 4390 Sano Y, Kageyama M (1987) The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO. Mol Gen Genet 208, 412--419 Ramesar RS, Abratt V, Woods DR, Rawlings DE (1989) Nucleotide sequence and expression of a cloned Thiobacillus ferooxidans recA gene in Escherichia coli. Gene 78, 1-8 Berson AE, Peters MR, Waleh NS (1990) Nucleotide sequence of recA gene of Aquaspirillum magnetotacticum. Nucleic Acids Res 18, 675 Stranathan MC, Bayles KW, Yasbin RE (1990) The nucleotide sequence of the recE+ gene of Bacillus subtilis. Nucleic Acids Res 18, 4249 Murphy AC, Gasparich GE, Bryant DA, Porter RD (1990) Nucleotide sequence and further characterization of the Svnechococcus sp strain PCC 7002 recA gene: complementation of a cyanobacteriai recA mutation by the Escherichia coli recA gene. J Bacteriol 172, 967-976