Vol. 174, No. 24

JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 8036-8042

0021-9193/92/248036-07$02.00/0 Copyright © 1992, American Society for Microbiology

Cloning and Characterization of the Brucella ovis Heat Shock Protein DnaK Functionally Expressed in Eschenichia coli M. F. M. CELLIER,t* J. TEYSSIER, M. NICOLAS, J. P. LIAUTARD, J. MARTI, AND J. SRI WIDADA Laboratoire de Biologie Cellulaire, De6partement Biologie-Sante, Institut National de la Sante6 et de la Recherche Medicale U-65, C.P. 100, Universite Montpellier II, F-34095 Montpellier Cedex 5, France Received 11 May 1992/Accepted 13 October 1992

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaj genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50°C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated BruceUla strain, suggesting that BruceUla hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42°C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.

and two others hsps, products of dnaJ and grpE genes, jointly stimulate in vitro the rate of ATP hydrolysis and could regulate DnaK activity in vivo (21). In this paper, we describe the cloning and sequencing of the hsp70 gene of Brucella ovis. Identification of the protein was based on sequence analysis, immunological reactivity of the polypeptide chain, and functional complementation of an E. coli dnaK mutant.

Brucella organisms are facultative intracellular bacterial parasites which cause infectious diseases in mammals. In humans, Brucella species owe much of their virulence to the ability to survive within phagocytic cells such as macrophages (38). Recently, major heat shock proteins (hsps) of intracellular parasites have attracted attention because of their unusual immunogenicity (37). Studies of these molecuIles have primarily focused on their structure in relation to their potential to elicit autoimmune responses (17). However, little is known about the regulation of their expression and their function in intracellular parasites. The major hsps have been divided into families according to their molecular weights (23). They are ubiquitous proteins, abundant in many cell types under normal conditions. hsps are thought to contribute to protein homeostasis by mediating synthesis, folding, and translocation of other proteins (16). The level of their synthesis is characteristically up-regulated in response to temperature elevation (22). In Escherichia coli, at least two minor transcription factors, sigma 32 and sigma E, activated upon cellular stress govern two heat shock gene regulons characterized by distinctive responsive sequences (10-12, 35). mRNA coding for hsps appear to be preferentially translated, while synthesis of non-hsps is suppressed (15). The heat shock response is a universal phenomenon by which cells adapt to thermal stress and to a variety of other environmental stresses (22). The accumulation of hsps is thought to preserve cellular function (28). Members of the hsp70 family have been highly conserved during evolution (23). Their ubiquitous distribution and the conservation of their sequences suggest that they are in-

MATERIALS AND METHODS Bacterial strains, plasmids, and media. E. coli AB1157 (wild type) and WG4813 (the dnaK52 mutant of AB1157), originally described by Paek and Walker (29), were a kind gift of L. D. Simon. The E. coli XL1 Blue strain was purchased from Stratagene. B. ovis 63/290T (ATCC 25840T) was obtained from the National Reference Center of Brucellosis, Montpellier, France. Working cultures of E. coli strains were grown overnight at 37°C, in Luria broth medium (2) with required antibiotics (per ml, 100 p,g of ampicillin, 25 p,g of tetracycline, and 25 p,g of chloramphenicol). Stock cultures were stored in LB medium supplemented with 50% glycerol at -20°C. The B. ovis strain was grown on Brucella agar plates (Difco Laboratories) for 48 h at 37°C. Stock cultures were stored in skim milk at -20°C. Plasmid p132E3, containing the Drosophila hsp70 gene, was a kind gift of A. P. Arrigo and has been described previously by Artavanis-Tsakonas et al. (1). Plasmid pBSII was purchased from Stratagene (pBluescriptII Exo/Mung Kit). DNA isolation and manipulation. High-molecular-weight B. ovis DNA was extracted and purified according to the method described previously for gram-negative bacteria (2). Plasmid and bacteriophage DNA was isolated and purified by standard methods and treated by standard recombinant DNA techniques (2). DNA-modifying enzymes were purchased from Boehringer Mannheim, Promega, New England Biolabs, and U.S. Biochemical Corp. and used as recommended by the manufacturers. Partial genomic libraries ofB. ovis DNA were constructed in pBSIIKS+ vector. B. ovis

volved in fundamental functions in all living cells. However, less conserved parts of the molecule could determine species-specific interactions necessary for hsp70 activity (13). The molecular chaperone function of hsp7O seems to be based on ATPase activity (8). DnaK is the hsp70 of E. coli (3) * Corresponding author.

t Present address: McGill Centre for the Study of Host Resis-

tance, Montreal General Hospital, 1650 Cedar Avenue, Room 7113, Montreal, Quebec H3G 1A4, Canada.

8036

VOL. 174, 1992

MOLECULAR CHARACTERIZATION OF BRUCELLA OVIS DnaK

DNA was digested to completion with suitable enzymes, electrophoresed on agarose gels, and revealed with ethidium bromide. A DNA ladder (GIBCO BRL) was used to accurately localize the molecular weight range of the fragments of interest. After excision, DNA was recovered from lowmelting-point agarose (Bio-Rad) with a Quiagen column (Diagen) or from standard agarose (GIBCO BRL) with Geneclean (Bio 101) according to the manufacturer's instructions. After ligation to pBSIIKS+ plasmid, E. coli cells were transfected by electroporation with a Cellject system (Eurogentec), and the cells were spread on LB plates containing, per ml, 100 pg of ampicillin, 25 ,ug of tetracycline, 2 mM isopropylthiogalactoside, and 400 p,M 5-bromo-4-chloro-3indolyl-p-D-galactopyranoside and grown overnight at 37°C. White colonies were transfected to 96-well plates (Falcon), grown overnight in LB selective nutrient, and stored in 50% glycerol. Probes were labelled with digoxigenin-modified nucleotides by plasmid-directed RNA transcription (Stratagene) or by random priming of purified DNA fragments (DNA labelling and nonradioactive detection kit; Boehringer Mannheim). Southern and colony blots were carried out as recommended by the supplier, utilizing GeneScreen Plus nylon membranes (New England Biolabs). Stringency conditions of the Drosophila probe hybridization to B. ovis DNA were 5x SSPE (lx SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA [pH 7.7]) (2), 40% formamide, and 1% blocking reagent (Boehringer Mannheim) overnight at 37°C. Washing conditions were O.1x SSPE-0.5% sodium dodecyl sulfate (SDS) at 37°C. DNA sequencing. DNA sequencing was performed by the procedure of Sanger et al. (33). Sequencing kits were purchased from Amersham (Klenow DNA polymerase fragment utilized with [32P]ATP), U.S. Biochemical Corp. (Sequenase utilized with 35S-ATP), and Applied Biosystems (Taq polymerase utilized with fluorescence primers). Sequence determinations by fluorescence analysis were carried out by using a 373A DNA sequencing system. Various restriction fragments were subcloned at the M13mpl8 SmaI site. Fragment orientation was assessed by hybridization to single-stranded RNA probes. Sequences determined in the same orientation were confirmed by independent clones. DNA sequence analysis was carried out with the aid of the MacroBioSoft software package (Prolabo). Protein expression and characterization. After growth on agar plates, B. ovis cells were resuspended in one volume of phosphate-buffered saline. Total cellular proteins were precipitated by the addition of one volume of 10% trichloroacetic acid. Samples were kept on ice for 15 min and centrifuged for 10 min at 4°C and 16,000 x g. The pellet was washed with cold ethanol and diethyl ether before resuspension in an equal volume of 50 mM Tris-10 mM EDTA (pH 8) and 1 volume of 2x Laemmli buffer (19). For [35S]methionine pulse-labelling experiments (specific activity of 10 mCi/ml), E. coli cells were cultivated in LB medium at 30°C until the mid-log phase and resuspended in M9 minimal medium (2) supplemented with 10 p,M thiamine and 20 mM glucose and were then aliquoted (500 pl in 2-ml tubes). After 5 min of incubation at 30°C, aliquots were labelled either immediately at 30°C or after 4 or 20 min of preincubation at 50°C. Pulse-labelling, performed for 4 min with the addition of 500 pl of prewarmed medium containing 10 ,Ci of [35S]methionine, was followed by the addition of 50 pl of prewarmed unlabelled methionine (0.22 M) before precipitation with 1 volume of ice-cold 10% trichloroacetic acid. Samples were

8037

kept for 1 min at - 10°C and then for 15 min on ice before centrifugation (16,000 x g, +4°C, 10 min). Samples were processed as described above. Samples containing equal counts per lane were run on 10% polyacrylamide gel in denaturing conditions. The proteins were transferred to nylon membranes (Immobilon; Millipore) by using a Milliblot system (Millipore). Total proteins were revealed by Coomassie blue staining, and radiolabelled proteins were revealed by exposure to X-Omat AR films (Kodak). Western blot (immunoblot) analysis was performed with rabbit hyperimmune serum raised against E. coli DnaK (kind gift of A. Malki) and with infected and Brucella melitensis Revl-vaccinated sheep sera (tested by Rose Bengal and kindly provided by B. Andral). Revelation was achieved by using anti-rabbit and anti-sheep immunoglobulin G-alkaline phosphatase conjugates (Sigma). Plasmid construction and complementation test. pJT226 contains a 3.0-kb PstI fragment which includes the region encoding the NH2 terminus of the B. ovis DnaK. pMClH1 contains a 3.5-kb ClaI fragment which includes the region encoding the COOH terminus of the B. ovis DnaK and the entire DnaJ coding region (see Results). pJT227 was obtained by excision of the pJT226 insert, religation, and selection by hybridization to a single-stranded RNA probe so as to obtain the putative B. ovis gene in inverted orientation relative to the pBSIIKS+ ,-galactosidase promoter. pJH was obtained by digestion of pJT227 at the BglIl site of the insert and the Sall site of pBSIIKS+ and ligation to the 3.4-kb fragment of pMClH1 obtained by cutting at BglII and SafI sites (see Fig. 1). E. coli AB1157 and WG4813 were transfected with control plasmid pBSIIKS+, and strain WG4813 was transfected with the recombinant plasmid pJH. After cultivation at 30°C overnight in selective LB medium, clones were spread on selective LB plates in duplicate. The plates were incubated for 48 h at either 30 or 42°C. The plates incubated at 42°C were further incubated at 30°C in order to determine whether bacteria had been killed by exposure at 42°C. Nucleotide sequence accession number. The nucleotide sequence data described here have been deposited in GenBank under accession number M95799. RESULTS Isolation of the gene encoding B. ovis hsp7O. A PstI 1.0-kb fragment encoding the most conserved part of the Drosophila stress-inducible hsp7o molecule was prepared from plasmid p132E3 and labelled for hybridization experiments. Southern blot analysis revealed a 3.0-kb fragment among the products of digestion byPstI of B. ovis DNA. A recombinant clone (pJT226) hybridizing to the Drosophila probe was selected from a partial B. ovis genomic library constructed with the pool of PstI fragments around 3.0 kb. The nucleotide sequence at both ends of the cloned fragment revealed that the coding part corresponding to only the NH2 extremity of the protein had been obtained. A second partial library was constructed with electrophoretically purified B. ovis DNA ClaI fragments around 3.5 kb. With the coding part of pJT226 as a probe, the clone pMClH1 containing the rest of the gene was obtained. A 6-kb fragment of B. ovis DNA was reconstituted in a novel plasmid (pJH) by the ligation of the two overlapping clones at the BglII site. The restriction map of this fragment is shown in Fig. 1. DNA sequence analysis and comparison with related sequences. A sequence of 3,979 nucleotides was determined from the first NcoI site, starting from the left of the 6-kb

8038

CELLIER ET AL.

J. BACTERIOL.

J T 226 _~~~~~~~~~~~~~~~~~~~~~~~~~~

M C lHl

I P Pv

PEv

H

l

I

I

E

Ev

N

N

B

N

H

K

[

If l1I

1 1

.

Pv Ev Ev S ..

.

.

I

I

0

0

1,0

2,0

3,0

40

dna K

.

B

K

H

hN1

EAM

.

mmmi

I

I

5,0

6,0 KIb

dna J

..., '. -----:- S .- -,: "..!. 1: 2 ,.' "... I..:...,. I ., .. ;...j p FIG. 1. Physical map of the 6-kb genomic fragment (pJH) of B. ovis. pJH results from ligation at the BglII site of the two overlapping clones pJT226 and pMClHl. The 3.0-kb PstI fragment of pJT226 contains the region encoding the NH2 terminus of the B. ovis DnaK protein. The 3.5-kb ClaI fragment of pMClHl contains almost the entire dnaK gene and the entire dnaJ gene of B. ovis. B, BamHI; Bg, BglII; E, EcoRI; Ev, EcoRV; H, HindIl; K, KpnI; N, NcoI; P, PstI; Pv, PvuI; S, SmaI. ..

fragment to the ClaI site located at the end of the fragment (Fig. 1). In the sequence shown in Fig. 2, two open reading frames, capable of encoding 638- and 375-residue polypeptide chains, respectively, were detected. The deduced amino acid sequence of the larger one was compared with hsp70 sequences of purple eubacteria. It shared 78% homology with the Caulobacter crescentus homolog (14) and 71% homology with the E. coli DnaK sequence (3). The grampositive Bacillus megaterium sequence (36) was found to have 65% similarity. Sequences of the intracellular parasites Chlamydia pneumoniae (18) and Mycobacterium leprae (26) exhibited residues that were 61 and 59% identical, respectively. From these results, we concluded that the B. ovis hsp70 gene-encoded protein belongs to the hsp7O protein family. Comparison of the overall nucleotide sequence determined in this study with the sequence of the E. coli operon which contains the dnaK gene linked to the dnaJ gene (4), the latter coding for the hsp40 homolog (5, 30), revealed a similar gene organization in B. ovis. However, the noncoding intergenic region was not conserved and was found to be larger in B. ovis (231 nucleotides) than in E. coli (88 nucleotides). A potential sigma 32-specific sequence was identified by comparison with the E. coli heat shock promoter -35 and -10 consensus sequences defined by Cowing et al. (10). This B. ovis DNA sequence also displayed significant homology with the E. coli clpB and htrC heat shock gene promoter sequences (35) around the -35 and 10 regions (Fig. 3). In Fig. 2, these regions are indicated by boxes around nucleotides 358 and 392, respectively. ShineDalgarno sequences (ribosome binding site [34]) were localized upstream from the putative translation initiation site of each open reading frame. Sequences considered to act as putative transcription attenuators, localized at the end of each open reading frame, are indicated by inverted arrows. Expression and temperature regulation of the B. ovis hsp7O in E. coli. In order to determine whether B. ovis hsp70 could be expressed in E. coli, the mutant dnaK52 lacking the DnaK protein was transfected either with plasmid pJH, containing the cloned fragment in an inverted orientation relative to the lacZ promoter of the vector, or with control plasmid pBSIIKS+. Pulse-label experiments (4 min) were performed with cells incubated at 30°C either directly or after being exposed -

-,

..

:

..

..

..

S...

to a temperature shift to 50°C for various periods. Figure 4A

(lanes 2, 6, and 10) shows a characteristic heat shock pattern of the wild-type E. coli strain featured by the prominent induction of proteins identified by others as DnaK and GroEL; meanwhile, the synthesis of various other proteins was suppressed. DnaK protein was absent from extracts of the dnaK52 mutant strain (Fig. 4A, lanes 3, 7, and 11). A novel protein with a size of ca. 70 kDa was hardly detected at 30°C in the dnaK52 strain transfected with pJH (Fig. 4A, lane 4); however, upon exposure to temperature upshift, synthesis of the recombinant protein became obvious (Fig. 4A, lanes 8 and 12). This indicated that the B. ovis hsp7O synthesis was up-regulated during stress conditions. These results demonstrate that not only transcription factors but also translation factors of E. coli determine successful expression of the B. ovis hsp70, especially during stress response

response.

Immunological characterization of B. ovis hsp7O. Immunological characterization of the B. ovis hsp7O was achieved by using a rabbit hyperimmune serum raised against E. coli DnaK. Figure 4B reveals the total proteins extracted from E. coli cells during pulse-label experiments as well as B. ovis proteins obtained after the cells were maintained at 24°C in the presence of 2% ethanol. Figure 4C shows an overexposed Western blot of the corresponding lanes. E. coli DnaK was detected in the wild-type strain (Fig. 4C, lanes 2, 6, and 10), whereas a probably truncated form of DnaK was revealed, together with bands of contaminants in the dnaK52 mutant strain (lanes 3, 7, 11). A novel band corresponding to the heat shock up-regulated recombinant B. ovis hsp70 was detected in the dnaK52 strain transfected with pJH (Fig. 4C, lanes 4, 8, and 12). Furthermore, this signal was found to colocalize with the unique native B. ovis protein revealed by the anti-E. coli DnaK antibodies (Fig. 4C, lanes 5 and 9), which was one of the major proteins found in stressed B. ovis cells (Fig. 4B, lanes 5 and 9). hsp70 of various intracellular parasites has been reported to be the major antigen during infection (9). We therefore analyzed B. ovis proteins by Western blot, with pooled sera from either infected sheep or sheep which had been vaccinated with the attenuated strain B. melitensis Revl. Figure 5

20

10

40

30

60

50

cASTDCCGcArCACCCATGACCATCCADCCAccATTcA0CCACCccATGa0Tc0TTT0C 70 C

GAT

120

110

100

90

60 CA

CAOAGCTTTGTcTOCTC

S

130

140

150

160

170

130

190

200

210

220

230

240

250

260

270

280

290

300

GAATCTGTTTTTGTCTGGCTCTTGTGACTTGCAGTDAAAAACTGCCTTTCTTATATACGC 310

320

330

340

370

380

390

400

430

490

520

510

530

V

C

M D G

V

A

550

K

A

N

I

S

610

630

620

V

C

K K

K

Q

R

V T

A

640

E G T

P

N

L

650 A

r

660

X

V

R

L

R

R

Y

P

D

D

730

V TKOD

M

0D

750

740

L

V

Y K

P

G D N

K

GD

W

A

V

780

770

760

600

790

V

E

H

610

Y

K

K

C 820

S

P

I

S

I

A

L

650

E T A

S K

K

Q

660

870

E

Y

S

360

L

T V

I

Q A V

P A

920

910

Y

690

N

r

930

Q

D A

D P

940

Q

P

A

950

D

G L

A

I

K

A

960

970

A A L A AYD AAAA Y G L

1030

F

D

1050

S V

V

1060

1110

1100

1090

Z I

L

T

P

E

1010

1060

1070 Z

r

V

1130

1120

S T N

D T

G

1150

r

L

G

r

G

D

L V

R

1210

1230

1220

K D A A

L

1270

C

A

K

1250

1240

1280

K T

1290

L

Z

S

S

Q

1350

1340

1410

1400

1450

S P

R

1540

1550

1560

1570

1580

1590

1600

1610

1620

1630

X

1640

D

V

L

L

1650

L

D

V

P

T

L

G

I

E T

L

1690

G

G

V

1700

L I

1710

E

R

S

Q

1750

T

F

S T A

1720

1760

E

D

G

G

V

F

Q

A

S

V

K

L

A A D

N

K

L

L

C

Q

V

G

I

P

P

A

P

R G V

R

Q

I

E

F

V

1740

N

G

I V

N

V

S A K

D GG

V

P

K

E

K

G

G

G

Q

T

Q

1800

D

2760

COAGGCSTACGAAACCCTGAAAGAC T L X D E A Y r

D

Y

R

F

G

7 N rZ

AA

H

2840

2850

2860

2890

2900

2910

2920

G

G

2620

2940

T

S

I

3020

3030

3040

T C

D Z C S C

S

3090

3080

3050 e

A

K

T

C

T

N

C

S

G

S

A

GCCC A Q

3100

G

R

3150

3140

C

30600 a s

P

3110

R

3160

G

31200 C F

R T C

P

G

C

N G

R

3210

3200

C Q

31800

3170

I

3220

I X

D

P

H

C

G

Q

G

R V

3260

D R

C

3230

R S

3270

3280

3330

3340

L

S

V

3240

N

I

3300

3290

3320

P A

G

D

L

Y

L L S V

I

3350

3380

3390

K

P

3400

H

Z

3360

r

F

A D L

Y C

X

V

P

I

3410

3420

S N T T A A

L

F

E

V

S

T

L

3500

D G

T

Q T

R

V

3520

3510

X

V

P

r

3530

G

N

X

Q

F

R L

X

G

X

3580

3570

3560

CN

P

C L

3540 R

Q

3590

3600 I

3630

3620

LLLE

3670

3680

F

£ X

3650

3640

3690

L S

W

Q Z N S

3660

3700

3710

P

K 3720

TCGCGGDCCCCTCTTTCCCCGGATGAAGGAGTTTTTGAAGGAATCGGCGAATAGATTATT F rZr G I G E * S A G L F s R M K ZE 3750 3740 3760 3730 3770 3760

7 0

AAATAAAAAAGCCATGCGGCTCCSTaTA

0 1I

H

R AA

3610

R Q R

1660 1910 1920 1900 1890 GACGCCAACGGCATCGTCAACGTTTCGGCCAAGGATGGTGGGGTACCTAAGGAGCACCAG A

I D

r

N P 2750

2740

CGCCAGCGCGACCTTCTT?AGGAGTTTGAAAAGCTTTCCTDDCAGGAGAACAGCCCGAAA

CCGCCTTGCTT

3610 3620 3630 3600 3840 ATAAACTTCAATGCGGCATAGATTTGTCTAATAACGACAATGGGAGCAGCAAGAATGGCA 3790

1870

D

7 1

K

P D R

H

Y

2730

2830

3550

1610 1650 1860 1820 1830 1840 CTCGTTGGCATTCCGCCCGCACCGCGCGGTGTGCGGCAGATCGAAGTGACCTTCGACATC L

L A N

TCTGTTACC.CGCATCTCTATATACAGATCGACATCGAAACGCCGCAGAATCTCTCCAAG S V T G D L Y I Q ID II T P Q N L S K

CCCGTCTTCCAGGCGAGCGTGAAATTGGCAGCCGATAACAACCTGCCTGTGACAGTTCaAC R

K

2720

3490

I

V

1790

1780

1770

A

K

1680

1730

Q S

N

2640

GGCACGCAGAACGGCAAGCAGTTCCGCCTGAAAGGCAAGGATGCCGGGTCTGCGCCAG

CCGACCAAGAAGTCGCAGACCTTCTCCACCGCTGAGGACAACCAGTCGGCCGTGACGATC P T K K

2630

2620

2610

3450 3440 3460 3470 3460 3430 GGCGGCCAGTTCGAGGTTTCGACACTTGATGTACCCACACCCGCGTGAAGGTGCCGAA

Ir

NS

F R

3370

S

1670

1660

F T R

2600

CAGCGTGATDGCGCGDCCTTTATTGCAAGGTGCCGATCTCAATGACCACGGCGGCTCTG

CTCGGCATTGAAACGCTGGGCGGCGTGTTCACCCGCCTGATCGAACGCAACACCACTATC L

2590

3310

R

OCGCCCGTTTTGCAGGGCrACrTCAADDACGTGCTGCTGCTCGACGTGACCCCGCTTTCr V

2540

Q R D G

1530

D

2530

s V E

1500

1520

Q G

2520

CGCGGCGGACCGGCGGGTGATCTCTATATCCTTCTGTCGDTGAAGCCGCACGAATTCTTC

E I Q K C V K a r F G

1510

L

2510

3250

AAGGAACCGCACAAGGGCGTGAACCCGGATGAAGTCOTGGCCATGGGCGCGCGATCCAG K E P H K G V N P D E V V A M G a A I Q G G V

2500

CCCGCAGTATCGAGGACAGAACCCCGTATCCCTCTGGCTCGCTAAGGCGAGGCGGGCCTG P A V S R T E P R I R L A ZE G E A GDL

1440

1490

1480

1470

1460

M T

2490

3190

C

CTGGTCGGCGGCATGACCCGCATGCCGGAGATTCAGAAGTGCGTGAAGGCCTTCTTCGGC G

2480

TGCAAAAAGTGCCATCGCCACDCCCG TTGACCGACGACGSTCGCSSTCGGTCAATATT

CCGTGCAAGGCGGCGCTCAACGATGCCGGCCTCAAGGCTGGCGAAATTrGACGAATGGTT P C I A A L K D A D L K A E IID r VV L V G

T

1380

1430

1420

2470

3130

F

AAGCTGTCGCGCGCCAATGTTGAAAGCCTGGTCGATGATGCGGTGCATGCCACGGTCGAG SVI K L S R A N V I S L V D D A V H AT 1390

P

1320

1370

1360

2460

TTCTCGGTGGAACCTACCTGCCCAGGCTGTAACGCACGCGDCCAGATCATCAAGGATCCG

ACCGAAATCAACCTGCCGTTCATCACGGCTCGACCAGACTGGCCCGAAGCATCTGCGATC T E I N L P F I T A D Q T G P K H L A r 1330

2450

3070

Q

Q

1310

1300

P

1260

S

2440

CAOCCCACCACCTGTACCATGTDCTCCDOCTCCGGCCD..D.CGT

CTGCAGCGCCTCAAGCACGCTDccGAGCCAAAGCCGAACTGTCGTCCTCGCAGCAG Q R

V

1200

TATCTDGTTGCCGAGTTCAADAADDAAADTDGCATCGACCTGAAGAACDACAADCTTDCC Y L V A E * K K E S G I D L K N D K LA L

2430

3010 R

E

1190

1180

1170

1160

Ir

r

2420

a r. x n OORRRRIOA1ROA r7GSS a a R R R R n a A * R a A D

1140

AAGTCCACCAATGGCGACACGTTCCITTGCGGTGAArACTTCGATATTCGTCTCGTcGAA K

S*

2960 2990 2970 30000 2950 2960 Cr0c1CTAcAAtAT0GAA0TGAc0e1O0GA0GAA0CCTAT0cGGDcAA0ACcDCAAATC L R Y N N S V T L 9 I A Y A C K T A Q S

AG V

Q K

II

I

G D C

2230

2270

1

2410

E A E R

1020

IAVYDz a v Y D

D DIS K S Z OK a K T

1040

G G GT

N

1000

CTTGGAGGCGGTACTTTCGAA L

I

I

R

L

V

E

990

X L

2680 2870 GGCCGTGGCGGCAATGGCTrTGCCGGTCGGGCCt CrmTCCDATATrTTCGADGAT E D l G G F G N G F A D A D G F A D 1r7

960

ACCAAGGATGCCGGCAAGATCGCCGGTCTGGAAGTTCTGCCCATCATCAACGAGCCGACC T K

X

2620 2610 2790 2770 2780 2800 CCGCAAAAGCGCGCCGCTTATGATCGCTTCGGCCATI GCAGCCTTTGAAAATGGCGGTATC

900 R

S

V

2260

A

A Q

A

DATCCCGAAGCCGAACGCAAGTTCAAGGAAATCGGCC

ACCGTTACGCAGGCCGTCATCACGGTTCCGGCCTACTTCAACGACGCCCAGCGTCAGGCC T V T

S

2710

E

G

z

M Y

L K AA

T

940

CAGATCTCCGCCATGATCCTTCAGAAGATGAAGGAAACGGCTGAATCCTACCTTGGCAA Q

2250

2240

2220

2210

A I

L

ACGCTGAAGGCAGCGTTTCGCAAGCTCGCCATrUCAGI;TATCATCCGCACCGTAACCCDAT

S

330

Q A

rCTCACCAGAACCCCAGATGACAAA GAACTGATSAAGATCGACTATTACGAAGCACTDDD? V T R T A D D K A LD K I D Y YY 2700 2690 2660 2670 2650 2680

2

GTCAAGGGCGACAATGGCGATGCCTGGGTCGAGGTGCACGGCAAGAAATATTCTCCGTCG V

T

ADCGGCGlDTATACDATATCCATGAArGCATGAGGCGCGTTCCAGCCGCCGATAAC

L

720 710 700 690 670 660 GGCCGTCGCTATGACGATCCGATGGTCACCAAGGACAAGGATCTTGTTCCTTACAAGATC G

2200

2190

2180

CAOGCTGCATGAAACCGAAAATT4;CAACDCTDCCTOCTTDAOCATGTCDCCDGTOCDO

P

GCGAAGCGTCAGGCCGTCACCAACCCGGAAGGCACCCTTTSTGCCGTCAAGCGCCTGCTT A

2160

2150

GCAGGTICGCCGGACAGGCAAAAAwiADAArTTGTTT rci:AGCGGGCTTGCCGAAAGC ;G(;CGDDT

600

A G Q

2140

C A C A C E 2340 2330 2310 2320 2300 2290 ATTACGAAOAAATCGAC CQ Ca CAOGCCTCTTCGTCCAAGGACGAT TOTCDATOCCC ;ACAAC Q A 5 S S 1 D 0 V V D A D Y R 9 I D D N 2400 2390 2370 2360 2350 2380 TCAAM;CCCGWO=CCCA2 rr AAGAADTCGTCDTAATTCCTGACCICWDCDTGC

TGGGAW

Z

XA x N Q

V

X S

2130

x IA A K

z

2230

A

590

CACCGA

F T D G D

I A

G

Z

DA

C Q A

540

A

E N

S06

570

560

CGCACCACCCCTCGATCATCGCCTTCACGGAC R T T P

I

V

K

2040 2030 mAAOCCAaAAT?CAO

2020

R R

1970 1960 uu0asTO0AAGaC 1591vx 0

A SDTDCDGDTG=cA=GD IC.IGTGAA ANo DGTCAGCCCATCTACGAACCAGCCC CAGCCTGCCA A

TCCTGCGTTGCCGTCATGGACGGAAAGAACGCGAADGTCATTGAAAATGCAGAAGGTGCA S

2010

2120

2170

420

CTTGC

r a

D

GAAAcDCGADC.ATATCAAGDCGMA~AAACCCADDCC ccI17=CGDAAGTTTCCATS Vu

S A

500

X

2110

470 480 450 460 CTAAAD??ATTDGTA CDATC?GGGAACDACCAAC K V I G I D L G T SN

440

DTTTGCTGAATGQ2AGAAATA

X

D A

DcDCATGACAAAGcGCDATTGADGATG ATCGCAGCCCTCAAOACCTCrCTTDAAOT I A -A -L -x T S L 1 c AD DDK A I Z D

360

410

2000 1990 c C.AAAT0C00A0 4o A * A N a Z A D

I

350

ACrCTCGCT

s

2100 2090 2060 2070 2060 G CCC0AAAGCCTDGTCCATrT2rCCACCCAAAACTC.CTGCCC.AATAT rGc QGACAAODcmcD A C S L V H S T I XS L A Z Y a D K V S

CTCGCACAGGGCTSTGATGACAGCCGCGAAACCGGTCCGCTTTCTGGAACCCAACCC

igCAGGCATTGTGAAAACCGATAGWCAA =pG

L

2060

GTGCCGCTCTTCTCGCTGGAATTCCAGGGCGAACCAGAAATTCATCDATGCDAmAT TCCGCGGGCAGAAGCGGGGTGAGCGAGACTGTGTACGCCTGTACCCAGTGCGGGCAAGA

1960

1950

1940 1930 A TCWATCAGOTTC go I R I Q a 2 a a

Q

3850

3660

3910

3920

3670

3930

3880

3890

3900

3940

3950

3960

TCTAGACAWCCAAGCACTCGGSCGATTl!CTCCCCACr.GTCCATCACW=CGCe 3970

CDATGGCGAGTCATCG

FIG. 2. B. ovis nucleotide sequence and deduced amino acid sequences from the two open reading frames extending from nucleotide 442 to nucleotide 2355 and from nucleotide 2587 to nucleotide 3714. Boxed regions around nucleotide positions 358 and 392 represent putative -35 and -10 elements analogous to the E. coli sigma 32-specific promoter (see Fig. 3). Ribosome binding sites are underlined. Putative transcription attenuators are indicated by inverted arrows. 8039

8040

CELLIER ET AL.

J. BACT1ERIOL. -35

1.

TCTGGTCAATAACCTTGAAT ---- AATTGAGGGATG----------ACCTCATTTAATCT *****

3. dnaK Brucella ovis

TCCCCAT

CCCTTGAAA

32 consensus

2. clpB

-10

****

*********

*

*

***

**

TCTGGAACCCAACCTTGAAGCAGGCATTGTGAAAACCGATAGGAACTGCCCATGGAACGC ***

4. htrc

*

****

*

****

*

**

***

TATCCTGAAACTGACTGAAC--- TAATTGAGTCA---------AACTCGGCAAGGATTCG

FIG. 3. Comparison of the B. ovis DNA sequence with E. coli sigma 32 consensus promoter sequences. The best matching scores to -35 and -10 along the 396-bp NcoI fragment of B. ovis DNA (nucleotides 1 to 396 in Fig. 2) are shown. Homology of the identified regions with aligned promoter sequences of E. coli clpB and htrC genes (35) is indicated by asterisks. Gaps, indicated by dashes, were introduced to obtain alignment of the sequences. consensus sequences

shows that a major antigen recognized by either infected or vaccinated sheep sera was found to colocalize with the B. ovis protein immunologically related to E. coli DnaK. Rescue of the E. coli dnaKS2 mutant with pJH. The above findings showed that B. ovis hsp70 was structurally and immunologically related to E. coli DnaK and could be expressed in E. coli under thermal stress conditions. These results prompted us to investigate the possibility for the B. ovis protein to exert a biological activity in E. coli. We therefore assayed a complementation test in E. coli dnaK52, a mutant which exhibits temperature-sensitive cell growth (29). This mutant is viable at 30°C, but a deletion of a part of the dnaK gene renders it unable to grow at 42°C. Phenotypic complementation was examined in the pJH- and pBSIIKS + transfected mutant strain in comparison with the pBSIIKS+-transfected wild-type strain. The dnaK52 mutant transfected with pBSIIKS+ was unable to grow at 42°C and even upon further incubation at 30°C, indicating that these cells had been killed, while the dnaK52 mutant transfected with pJH recovered ability to form colonies at 42°C (Fig. 6). -

It therefore appears that B. ovis hsp70 may be functional in preventing cell death of an E. coli dnaK mutant at a nonpermissive temperature. DISCUSSION We used a Drosophila hsp70 gene fragment as a probe to clone the B. ovis homologous gene. We attempted this approach since it has been established that the E. coli dnaK gene was homologous to a Drosophila hsp70 gene (3). A similar approach was used to clone the dnaK gene of C. crescentus (14). Comparison of the putative B. ovis hsp70 amino acid sequence with other bacterial hsp70 homolog sequences confirmed the identity of the newly isolated gene. This conclusion was corroborated by serological studies. The unique protein recognized by anti-E. coli DnaK serum in protein extracts of B. ovis exhibited the same electrophoretic mobility as that of the recombinant protein which was revealed as a novel band in extracts from E. coli transfected with pJH.

C

;9

10

1112

1

13

2

3

4

5 6 7

8 9

10 11 12 13

I '

FIG. 4. Characterization of B. ovis hsp70. (A) SDS-polyacrylamide gel electrophoresis analysis of [35S]methionine pulse-labelled proteins from E. coli AB1157 wild-type strain transfected with pBSIIKS+ (lanes 2, 6, and 10) and E. coli WG4813 (dnaK52 mutant strain of AB1157, thus lacking DnaK protein) transfected with pBSIIKS+ (lanes 3, 7, and 11) or with pJH (lanes 4, 8, and 12). Cells were labelled for 4 min, as described in Materials and Methods, at 30°C (lanes 2, 3, and 4) or after 4 (lanes 6, 7, and 8) or 20 (lanes 10, 11, and 12) min of preincubation at 50°C. An equal amount of radioactivity was loaded per well. (B) Coomassie blue staining of total protein extracts from E. coli strains corresponding to those described for panel A. Lanes: 5 and 9, B. ovis-extracted proteins after exposure of the cells to a stress induced by 2% ethanol for 30 min (note prominent band around 70 and 60 kDa); 1 and 13, molecular mass markers (94, 67, 43, and 14.4 kDa). (C) Western blot analysis developed with anti-DnaK serum raised against E. coli protein. Lanes are the same as those described for panels A and B. Closed arrowheads indicate the positions of E. coli and B. ovis DnaK proteins (upper and lower arrowheads, respectively); the open arrowhead indicates the position of the E. coli GroEL protein.

MOLECULAR CHARACTERIZATION OF BRUCELLA OVIS DnaK

VOL. 174, 1992 1

2 3 4 _

..

*as

A

I1 i

I

I

tI L

FIG. 5. Western blot analysis of B. ovis proteins developed with control serum (lane 1), rabbit hyperimmune serum raised against E. coli DnaK (lane 2), pooled sera from infected sheep (lane 3), or pooled sera from B. melitensis Revl-vaccinated sheep. The arrowhead indicates the position of the B. ovis hsp7O protein.

We found that the B. ovis hsp7O gene is physically associated with a second gene homologous to the dnaJ heat shock gene. A similar gene organization has been described for E. coli (4), C. crescentus (14), and Mycobacterium tuberculosis (20) and could be a common feature in eubacteria. B. ovis hsp7O was expressed in E. coli either under normal conditions or during a thermal stress. Enhanced expression was also observed during ethanol treatment (data not shown). Expression of Brucella abortus genes in E. coli has been reported previously (25). However, E. coli transcription factor determining stress-regulated expression of B. ovis hsp70 gene remains to be identified. The occurrence of a sigma 32-related sequences 5' upstream the B. ovis dnaK

FIG. 6. Restoration of viability at 42°C of the temperaturesensitive dnaK52 mutant. Experiments were performed as described in Materials and Methods. (A) E. coli wild-type strain AB1157/ pBSIIKS+; (B) E. coli dnaK52 mutant strain WG4813/pBSIIKS+; (C) E. coli dnaK52 mutant strain WG4813/pJH. Plates were incubated either at 30'C for 72 h or at 42°C for 48 h and then at 30°C for 24 h.

8041

gene together with absence of sequences related to the sigma E-responsive elements (12) suggests that sigma 32 is responsible for the observed expression. Furthermore, the existence of a sigma 32-related transcription factor has been postulated for Agrobacterium tumefaciens (24), a bacterium closely related to Brucella species (39). We were able to perform phenotypical complementation of the E. coli dnaK52 mutant transfected with the B. oviscloned fragment. The inability of this strain to survive at 42°C has been clearly correlated with the lack of DnaK since, while DnaJ is still expressed in this mutant strain, normal growth can be restored only by the introduction of a plasmid carrying the autologous dnaK gene (7). The present results indicate that the B. ovis recombinant hsp7O may exert the same DnaK function. To our knowledge, this is the first report of heterologous complementation in a dnaK-deficient strain. Previous attempts with genes from M. tuberculosis (7, 27) and from B. megaterium (36) have been reported to be unsuccessful. This difference may be related to the extent of structural conservation since comparison of B. ovis and E. coli DnaK amino acid sequences revealed about 70% similarity, whereas B. megaterium and M. tuberculosis sequences exhibited 60 and 54% similarity, respectively, with the E. coli DnaK sequence. The functional complementation observed in this study is thus likely to be due to extensive conservation between the B. ovis and E. coli DnaK sequences. Homologous regions are unevenly distributed along the hsp7O polypeptide chain, and it was suggested that the lack of complementation observed in other studies could be due to the less conserved parts of the protein, which could be involved in interactions with other cell components (7, 27). In E. coli, DnaJ has been shown to interact with DnaK and to be necessary to stimulate its ATPase activity (21). This may imply that heterologous DnaK complementation of the dnaK52 mutant strain also requires the dnaJ gene from the same species. Thus, the simultaneous presence of B. ovis DnaK and DnaJ in the dnaK52 mutant could explain the observed complementation. hsps have been reported to be major antigens in a variety of infections (reviewed in reference 9). While it is possible that these proteins possess specific immunogenic properties, their abundance during infection is probably another factor contributing to this phenomenon. An increased synthesis of hsps may occur during a stress response of the infectious organism, triggered by the hostile environment encountered during host colonization (31, 40). Up-regulation of a pathogen's hsps has been described in vitro upon phagocytosis and was suggested to be associated with an increased ability of the pathogen to withstand exposure to macrophage antimicrobial armory (6). Our results indicated that the DnaK protein may be recognized by sera from sheep either infected by Brucella species or vaccinated with an attenuated living Brucella strain. Recently, it was reported that another member of the hsp family, the B. abortus hsp60, homologous to E. coli GroEL, was a major antigen during naturally acquired and experimentally induced brucellosis (32). These results may indicate that Brucella cells react by a stress response during their development in the host. The availability of molecular probes for the major Brucella hsps will help in analyzing this phenomenon.

ACKNOWLEDGMENTS We are indebted to A. P. Arrigo, who provided plasmid p132E3; L. D. Simon, who supplied E. coli AB1157 and WG4813; A. Malki, who purified E. coli DnaK and kindly supplied the anti-E. coli DnaK serum; B. Andral, who furnished sheep sera; and R. Alary (Institut

8042

CELLIER ET AL.

J. BAcrERIOL.

National de la Recherche Agronomique) for help in performing the automatic sequencing. This work was supported by funds from Recherche & Partage, Chimie-Ecologie, and Caisse Nationale d'Assurance Maladie.

21.

REFERENCES

22.

1. Artavanis-Tsakonas, S., P. Schedl, R. Steward, W. J. Gehring, M. E. Mirault, L. Moran, M. Goldschmidt-Clermont, P. Arrigo, A. Tissieres, and J. Lis. 1979. Heat activated genes of Drosophila melanogaster, p. 72. In Specific eukaryotic genes, Alfred Benzon Symposium, Munksgaard, Denmark, July, 1979. 2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1988. Current protocols in molecular biology, vol. 1. Wiley Interscience, New York. 3. Bardweli, J. C. A., and E. A. Craig. 1984. Major heat shock gene of Drosophila and the E. coli heat inducible dnaK gene are homologous. Proc. Natl. Acad. Sci. USA 81:848-852. 4. Bardwell, J. C. A., K. Tilly, E. Craight, J. King, M. Zylicz, and C. Georgopoulos. 1986. The nucleotide sequence of the Escherichia coli K12 dnaJ gene. J. Biol. Chem. 261:1782-1785. 5. Blumberg, H., and P. A. Silver. 1991. A homologue of the bacterial heat shock gene dnaJ that alters protein sorting in yeast. Nature (London) 349:627-630. 6. Buchmeier, N. A., and F. Heffron. 1990. Induction of Salmonella stress proteins upon infection of macrophages. Science 248:730732. 7. Bukau, B., and G. C. Walker. 1989. Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism. J. Bacteriol. 171: 2337-2346. 8. Chappel, T. G., B. B. Karforti, S. L. Schmid, and J. E. Rothman. 1987. The ATPase core of a clathrin uncoating protein. J. Biol. Chem. 262:746-751. 9. Cohen, I. R., and D. B. Young. 1991. Autoimmunity, microbial immunity and the immunological homunculus. Immunol. Today 12:105-110. 10. Cowing, D. W., J. C. A. A. Bardwell, E. A. Craig, C. Woolford, R. W. Hendrix, and C. A. Gross. 1985. Consensus sequence for E. coli heat shock gene promoters. Proc. Natl. Acad. Sci. USA 82:2679-2683. 11. Craig, E. A., and C. A. Gross. 1991. Is hsp70 the cellular thermometer. Trends Biol. Sci. 16:135-140. 12. Erickson, J. W., and C. A. Gross. 1989. Identification of the sigmaE subunits of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high temperature gene expression. Genes Dev. 3:1462-1471. 13. Gething, M. J., and J. Sambrook 1992. Protein folding in the cell. Nature (London) 355:33-45. 14. Gomes, S. L., J. W. Gober, and L. Shapiro. 1990. Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures. J. Bacteriol. 172:3051-3059. 15. Henry, M. D., S. D. Yancey, and S. P. Kushner. 1992. Role of the heat shock response in stability of mRNA in Eschenichia coli K-12. J. Bacteriol. 174:743-748. 16. Hightower, L. E. 1991. Heat shock, stress protein, chaperones and proteotoxicity. Cell 66:191-197. 17. Hill Gaston, J. S. 1991. Heat shock proteins and autoimmunity. Semin. Immunol. 3:35-42. 18. Kornak, J. M., C.-C. Kuo, and L. A. Campbell. 1991. Sequence analysis of the gene encoding the Chlamydiapneumoniae DnaK protein homolog. Infect. Immun. 59:721-725. 19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680-685. 20. Lathigra, R. B., D. B. Young, D. Sweetser, and R. A. Young. 1988. A gene from Mycobacterium tuberculosis which is homol-

23.

24. 25.

26.

27.

ogous to the dnaJ heat shock protein of E. coli. Nucleic Acids Res. 16:636. Liberek, K., J. Marszalek, D. Ang, and C. Georgopoulos. 1991. E. coli dnaJ and grpE heat shock proteins jointly stimulate ATPase activity of dnaK. Proc. Natl. Acad. Sci. USA 88:28742878. Lindquist, S. 1986. The heat shock response. Annu. Rev. Biochem. 55:1151-1191. Lindquist, S., and E. A. Craig. 1988. The heat shock proteins. Annu. Rev. Genet. 22:631-677. Mantis, N. J., and S. C. Winans. 1992. Characterization of the Agrobacterinum tumefaciens heat shock response: evidence for a &2-like factor. J. Bacteriol. 174:991-997. Mayfield, J. E., B. J. Bricker, H. Godfrey, R. M. Crosby, D. J. Knight, S. M. Halling, D. Balintsky, and L. B. Tabatabai. 1988. The cloning, expression and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein. Gene 63:1-9. McKenzie, K. R., E. Adams, W. J. Britton, R. J. Garcia, and A. Basten. 1991. Sequence and immunogenicity of the 7OkDa heat shock protein of Mycobacterium leprae. J. Immunol. 147:312319. Mehlert, A., and D. B. Young. 1989. Biochemical and antigenic characterization of the Mycobacterium tuberculosis 71 kDa antigen, a member of the 70 kDa heat shock protein family. Mol.

Microbiol. 3:125-130. 28. Neidhardt, F. C., and R. A. VanBogelen. 1983. Heat shock response, p. 1334-1345. In J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. 29. Paek, K.-H., and G. C. Walker. 1987. Escherichia coli dnaK null mutants are inviable at high temperature. J. Bacteriol. 169:283290. 30. Raabe, T., and J. L. Manley. 1991. A human homologue of the E. coli dnaJ heat shock protein. Nucleic Acids Res. 19:6645. 31. Res, P. C. M., J. E. R. Thole, and R. R. P. deVries. 1991. Heat shock proteins in immunopathology. Curr. Opin. Immunol.

3:924-929. 32. Roop, R. M., M. L. Price, B. E. Dunn, S. E. Boyle, N. Sriranganathan, and G. G. Schurig. 1992. Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus hsp60 protein, BA60K. Microb. Pathog. 12:47-62. 33. Sanger, F., A. R. Coulson, B. G. Barrell, A. J. H. Smith, and B. A. Roe. 1980. Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143:161-178. 34. Shine, J., and L. Dalgarno. 1974. The 3' terminal sequence of E. coli 16S ribosomal RNA: complementary to non-sense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71: 1342-1346. 35. Squires, C. L., S. Pedersen, B. M. Ross, and G. Squires. ClpB is the Escherichia coli heat shock protein F84.1. J. Bacteriol.

173:4254-4262. 36. Sussman, M. D., and P. Setlow. 1987. Nucleotide sequence of a Bacillus megaterium gene homologous to the gene of E. coli. Nucleic Acids Res. 15:3923-3928. 37. Young, D., R. Lathigra, R. Hendrix, D. Sweetser, and R. A. Young. 1988. Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85:4267-4270. 38. Young, E. J. 1983. Human brucellosis. Rev. Infect. Dis. 5:821842. 39. Young, J. P. W., H. L. Downer, and B. D. Eardly. 1991. Phylogeny of the phototrophic rhizobium strain BTAil by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J. Bacteriol. 173:2271-2277. 40. Young, R. A., and T. J. Elliott. 1989. Stress proteins, infection and immune surveillance. Cell 59:5-8.