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Characterization of the pasteurella multocida skp and firA genes
Gene, 161 (1995) 39 43 ©1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
39
GENE 08972
Characterization of the Pasteurella multocida skp and firA genes (DNA sequencing; N-terminal amino-acid sequence; amino-acid homology; lipid synthesis; porin)
C h r i s t i a n D e l a m a r c h e a, F a b r i c e M a n o h a a, G h i s l a i n e B 6 h a r b, R6mi H o u l g a t t e b, U l f H e l l m a n c a n d Henri Wr6blewski a a Dkpartement "Membranes et Osmorbgulation", Centre National de la Recherche Scientifique, Unit~ de Recherche AssociOe No. 256, Rennes, France; b Centre National de la Recherche Scientifique, UnitO Propre de Recherche no. 420, Villejuif, France. Tel. (33-1) 4958-1132; and ~ Ludwig Institute for Cancer Research, Uppsala, Sweden. Tel. (46-18) 550-188
Received by A.J. Podhajska: 18 December 1994; Accepted: 16 March 1995; Received at publishers: 7 April 1995
SUMMARY
A 2.9-kb fragment of the Pasteurella multocida (Pro) genome encoding proteins p25 (25 kDa) and p28 (28 kDa) has previously been cloned and expressed in Escherichia coli (Ec). In the present paper, the nucleotide (nt) sequence of a 1.8-kb subfragment encoding the two proteins is described. The cloned fragment contains three open reading frames (ORFs). ORF1 is incomplete. ORF2 is homologous to the skp gene of Ec. ORF3 overlaps ORF2 and is highly homologous to the firA gene of Ec. The skp and firA genes are part of an operon governing the first steps of lipid A synthesis. Comparing the nt sequence with the N-terminal sequences of p25 and p28 revealed that the two proteins are encoded by ORF2 (skp). The preprotein p28 is converted into p25 by cleavage of a 23-amino-acid leader peptide. Though it serologically cross-reacts with porin H of Pro, p25 is not related to known bacterial porins.
INTRODUCTION
Gram- eubacteria are surrounded by an outer membrane composed of lipopolysaccharides (LPS), phospholipids and proteins as major components. LPS and porins are the main immunogens of this membrane. Furhermore, owing to the toxicity of its lipid A moiety, LPS is also a Correspondence to: Dr. H. Wr6blewski, D6partement "Membranes et Osmor6gulation", Centre National de la Recherche Scientifique, Unit6 de Recherche Associ6e No. 256, Universit6 de Rennes 1, Campus de Beaulieu, F-35042 Rennes cedex, France. Tel. (33-99) 286-138; Fax (33-99) 286-700.
Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); Ec, Escherichia coli; FirA, UDP-3-O-(R-3-hydroxymyristoyl)-GlcN Nacyltransferase; firA, gene encoding FirA; GCG, Genetics Computer Group (Madison, WI, USA); IPTG, isopropyl-13-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; LPS, lipopolysaccharide; nt, nucleotide(s); ompH, gene encoding OmpH; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; Pro, Pasteurella multocida; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; Skp, a 25-kDa protein of P. multocida; skp, gene encoding Skp; St, Salmonella typhimurium; X, any aa. SSDI 0378-1119(95)00254-5
virulence factor. Therefore, obtaining data on the structure and the synthesis of porins and LPS should prove useful to the development of new therapeutic and prophylactic methods against pathogenic Gram- eubacteria. Pasteurella multocida (Pro), a widespread Grameubacterium, alone or in association with other pathogens, is responsible for severe diseases in mammals (including man) and birds (Chanter and Rutter, 1989). For example, the principal diseases of pig in which Pm is involved are atrophic rhinitis and pneumonia, which are both of economical importance. In atrophic rhinitis, Pm is often asssociated with Bordetella bronchiseptica, whereas in pneumonia it seems associated with Mycoplasma spp., B. bronchiseptica, Haemophilus pleuropneumoniae, Salmonella cholerae-suis or viruses. In an attempt to obtain genetic information on Pm cell surface antigens, a genomic library was screened with Ab directed against envelope proteins of this bacterium, including porin H (Chevalier et al., 1993). We have recently reported the cloning in Escherichia coli (Ec) of a 2.9-kb chromosomal fragment of Pm encoding the synthesis of
40 two polypeptides, p25 (25kDa) and p28 (28kDa) ( M a n o h a et al., 1994). Protein p25 was mainly found in the periplasm of Ec whereas p28 was principally detected in the cell envelope. In the present paper, we report the nt sequences of two genes localized in the cloned fragment of the Pm genome. Comparison of these genes with their homologues in Ec and in Salmonella typhimurium (St) and the determination of N-terminal aa sequences allowed the identification of p25 and p28.
A 2
B 3
2
kDa
9467-
43EXPERIMENTAL AND DISCUSSION
(a) Expression of p25 and p28 in Ec The p U C A plasmid containing a 2.9-kb TaqI fragment of the Pm chromosome (strain 9222, serotype D2, porcine isolate) was digested with HindIII + PvuII and the resulting 1.8-kb fragment was ligated into pUC19-digested with H i n d I I I + S m a I ( = p U C 2 ) or into pUC18 also digested with HindIII + Sinai (= pUC2 reverse), pUC2 and pUC2 reverse differ in the orientation of the insert with respect to the lac promoter (Manoha et al., 1994). The transformed cells (Ec JM109) were grown in LB broth supplemented with 100 ~g of ampicillin per ml and with 0.5 m M I P T G . The proteins of the envelope fraction of Pm strain 9222 were resolved by SDS-PAGE, electroblotted onto nitrocellulose membranes, and treated with different Ab. A major band of 37 k D a and two minor bands of 13 and 17 k D a were immunolabelled with a rabbit polyclonal antiporin H serum (Fig. 1A), whereas several major bands corresponding to apparent molecular masses of 17, 28, 31, 37, 40, 54 and 64 k D a were revealed with a rabbit anti-Pro envelope serum (Fig. 1B1). A pattern similar to the latter was obtained when using sera from pigs infected with Pm (data not shown). In a lysate of Ec transformed with pUC2 reverse, both p25 and p28 were immunolabelled by antienvelope sera containing Ab raised against 6 distinct strains of Pm (one example is illustrated in Fig. 1B2). In contrast, only p25 was labelled by the antiporin H serum (Fig. 1A2). Thus, p25 appeared as the sole protein of the Ec strain transformed with the pUC2 reverse plasmid displaying an immunological crossreactivity with porin H of Pro.
(b) Nucleotide sequence determination The nt sequence of the 1836-bp D N A of the pUC2 insert was determined. Three O R F s were found (Fig. 2). ORF1, containing 120-bp starts at the 5' end of the sequence and stops with two consecutive TAA triplets, ORF2, which contains 582 bp starting with an ATG start codon and ending with a TAA stop codon. The deduced aa sequence corresponds to a protein of 193 aa. O R F 2
30-
if" p28 if" p25
0
m
14. 4-
Fig. 1. Immunodetection of Pm antigens with anti-porin H (A) or antiPm envelope Ab (B). Samples were envelopes from Pm (lane 1, 10 gg protein), whole-cell lysates of E. coli transformed with pUC2 reverse (lane 2, 20 gg protein) or pUC18 (lane 3, 20 gg proteint. Size standards are shown on the left. Methods: Proteins were resolved by 0.1% SDS-12% PAGE according to Laemmli (1970) and electrotransferred onto nitrocellulose membranes for immunodetection (Towbin et al., 1979). Anti-porin H Ab were raised in rabbits with porin H purified by size exclusion chromatography (Chevalier et al., 1993). Sera were used at a dilution of 1:200. Secondary Ab (dilution, 1:500) were goat immunoglobulins directed against rabbit IgGs and conjugated with horseradisch peroxidase. 4-chloro-l-naphtol and H202 were used as enzyme substrates. A control blot without primary Ab was blank (data not shown).
is preceded by a putative Shine-Dalgarno sequence and a putative promoter region. The sequences T T G G A A ( - 3 5 region) and T A G T C T ( - 1 0 region) have 4- and 3-nt conservations, respectively, with respect to the two consensus hexamers of Ec promoters. The space between the two hexamers is of 16 nt. Downstream O R F 2 and with one nt overlapping its termination codon lies the 1017-nt ORF3 encoding a protein of 339 aa. Though ORF3 does not contain a stop codon at the 5' end, comparison with the related sequences suggests that the nt sequence is almost complete in pUC2 (see section d, below).
41
CAGCTGGGGT C.GCGCTTCAATGGCAATCGCCAATTGGACCGTTAGTG•TCTCTTATGCGAAACCT•TTAAAAAATACCAAGGCGATGAAATTGAGCAGTTCCAATTCAGCA•TGGTGGGACGTTCTAATAAGCGATTGGAA•TTATGTAA A G V A L Q W Q S P I G P L V F S Y A K P L K K Y Q G D E I E Q F Q F S I G G T F t *
150 (41)
-IQ RD --~ A A A A T T G T A G T C T A A A C A G C C A T C T G T G C A G T C A G C G T T C G G C A C A G T A T T T A T TCATATC~TTATTGAAA~AGGAAAAGAAACCAATGAAAAAAGCAGTCAAAGT~ACCC.CACTTTCTTTAGCATTAC.CcT~TAC~TCGTCTTTAGC~A 300 M M K A V K V T A L S L A L A F T S S L A M (22) TGGCCACAGAAAATAT TGCATTTATCAGT GGGGATTATTTATTTCAAAATCACCCAGATCGTAAAATG•TGGCAGAAAAATTAGAGTCAGAGTTTAAAGCCAGAGTAGAAAAACTCACTGCAAATAAAAAAAC.CATTGATGAAAAAATTG 450 A T E N I A F I S G D Y L F Q N H P D R }f M V A E K L E S E F K A R V £ K L T A N K K S I D E K I A {72) C C GC T TC T C A A A A G A A A G T T G A A GC C A A A GTTGC T C42ATTACAAAAAGAT GCC CC G A A A T T A C GCAGT G C A G A TAT TAAAAAAC GT G A A G A A G A A A T C A A T A A A C TC GC-CAATAGC GAGCAAGAAGC G A T C A A T A A A C T C G T T A C T G C A C 600 A S Q K K V E A K v A A L Q K D A P K L R S A D I ~[ K R £ E E I N K L G N S E Q E A I N K L V T A H (122)
EcaX A T G A T G A A G A A G T C A G T A A A T A T C A A G A C GATTATC-CAAAAC G T G A A C G T G A A G A A A C C G C A A A A T T A G T G G A T A G C A T • C A A A A C G C G G T A A A T A C G G T T G C A A G A G A G A A A A A C T A T A C A T T A G T A C T G A A T G A A G G T G C A G T C G T G T 750 D E E V S K Y Q D D Y A K R E R £ E T A K L V D S I Q N A V N T V A R E K N Y T L V L N E G A V V F (172) PIZX -~ TTGC T C.CAGAT G C A A A A A A T A T T A C T G A A G A T G T T • T A A A A G T C A T T C C T G C G A C A C A G G C G A A A T A A T G C A A G T T T A T T C C C T C C A A G A A T T A G C A C A A C A G A T C A A D A K N I T £ D V L K V I P A T Q A K * M Q v Y s L Q E L A Q Q I
GGCGCTACCATTCGTGGTAACGCCGAT GTTGTTGTAGAAAGTAT G
A
T
I
R
G
N
A
D
V
V
V
£
S
I
900 {193) (28}
TGCGCCTT T A G A T A A G C - C G A C A G A A A A A C A G C T T A C C T T T A T T T C T A A T C C T A A G T T C C G T T C C T T A C T G G C A C A A T C T C A T G C C G G G A T T TT G G T G G T G T C A G A A G C G G A T G T G G C G T T T T G T G C A G A G C A A A G T A A T T T A T T G A T T G T A P L D K A T E K Q L T F I S N P K F R S L L A Q s H A G I L V V S £ A D V A F C A E Q s N L L I V
1050 (78)
A A A A G A T C C GTAC G T G G C T T A T G C A G T T C T A G C A C A A T A T A T G G A T A G C A C G C C G A A A G C T G C G A G C G G C A T T G C C GCCAGYGCGGT GGTATC C - G C T A G T G C T G T G A T A G G C A A A A A T G T C T C A A T T G G T G C C A A T ~ T G A T T G ~ G A K D P Y V A Y A V L A Q Y M D S T P K A A S G I A A S A V V S A S A V I G K N V S I G A N A V I E D
1200 {128)
CGGGGTAACGCTTGGTGATCATGTAGTCATTGGTGCTAACTGCTTTGT~GGCAAAAATAGCAAAATTGGTGCTTATA~CCAGCTATGGGCAAATGTCAGTGTTTATCA~GAGG~GAAATAGGC~AACATTGTTTAATT~AATCTGGTGC
1350 (178)
G
V
T
L
G
D
H
V
V
I
G
A
N
C
F
V
G
K
N
S
K
I
G
A
Y
T
Q
L
W
A
N
V
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AGTGATTGGTAGTGATGGTTTTGGTTACGCCAATGATCGTGG•CGTTGGATTAAAATTCCACAAGTTGGACAAGTCATTATTGGCAATCAT V I G $ D G F G Y A N D R G R W I K I P Q V G Q V I I G N H
GTC G A A A T T G G C G C T T G C A C T T G T A T T G A T C G A G G T G C G T T A G A T C C A A C G G T C A T T G A V E I G A C T C I D R G A L D P T V I E PBZX A G A T A A T G T G A T T A T T G A T A A T C T T T G C C A A A T T G C G C A T A A C G T C C A T A T T G G G A C C G G G A C T G C A G T T G C T G G T G G G G T G A T T A T G G C T G G T A G C TT GACCGTCGGACGCTATTGCTTAATTGGTGGTC-CCAGTGTGATCAATGGACA D N V I I D N L C Q I A H N V H I G T G T A V A G G V I M A G S L T V G R Y C L I G G A S V I N G H C A T G G A A A T C TGC G A T A A A G T G A C G A T T A C A G G C A T G G G G A T G G T G A T G C G T C C A A T T A C T G A A C C TGGCGTGTACTCT T C G G G T A T C C C A T T A C A A A C G A A T A A A G A A T G G C G T A A A A C C GCTGCATTGACGTTAGC-CATTGATGCGAT M E I C D K V T I T G M G M V M R P I T E P G V Y S S G I P L Q T N K E W R K T A A L T L G I D A M Ta~fX GAATAAACGGT TAAAAGCCTTAGA~GA~TTC N K P. L K A L E K K F
1500 (228) 1650 (278) 1800 (328)
GA 1836 (339)
Fig. 2. Nucleotide sequence of the 1836-bp Pm insert from pUC2 and deduced aa sequences. Possible -35 and -10 promoter regions and ShineDalgarno sequences are underlined. The stop codons are marked with asterisks. The ATG start codons for ORF2 and ORF3 are indicated by horizontal arrows above the nt sequence. The underlined aa sequences were determined by sequencing the N terminus of purified p25 and p28. This sequence was submitted to the EMBL data library and assigned accession No. X74357. Methods: Plasmid DNA from pUC2, pUC2 reverse, PvuIfSacl and PstI-PstI subclones was purified with the Magic Minipreps DNA purification system (Promega, Madison, WI, USA) and used for primer walking sequencing. The nt sequence was determined by dideoxysequencing (Sanger et al. 1977) on both strands using dye-labeled primers (forward and reverse primers) or dye-labeleddideoxynucleotides.The computer program used for the analyses of nt and aa were provided by the GCG package (Devereux et al., 1984). For N-terminal sequencing, the proteins resolved by one-dimensional SDS-PAGEwere electroblotted onto PVDF membranes (Towbin et al., 1979). The strips containing p25 or p28 were excised and the samples were analyzed in an Applied Biosystems Model 470A protein sequencer with an on-line PTH-analyzer Model 120A.
(e) Identification of ORF2 Comparison of the nt sequence of ORF2 with databases reveals significant homologies with the skp gene of Ec and the ompH gene of St (Holck and Kleppe, 1988; Koski et al., 1990). Fig. 3 shows the aligned sequences of four proteins of the Skp family. The Pm ORF2 displays 46 and 45% identity with the skp gene of Ec and the ompH gene of St, respectively, whereas there is 86% identity between the two latter genes. Therefore, we termed skp the gene corresponding to ORF2 and Skp the protein encoded by this gene. The N-terminal sequence of p28, determined over 18 aa, perfectly matches the 18-aa N-terminal sequence of the Skp polypeptide encoded by ORF2. The N-terminal sequence of p25, determined over 15 aa, is identical to the sequence running from aa 24 to 38 in Skp. Thus, Skp of Pm is synthesized as a precursor of 193 aa containing a leader peptide of 23 aa which is subsequently processed to yield a mature 170-aa protein starting with Thr. The leader peptide of Skp comprises an apolar stretch flanked by a short basic sequence (MKKAVK) on the N-terminal side and a cleavage site (AMA) on the C-terminal side. This arrangement is typical of bacterial signal peptides. The hydropathy plot of p28 reveals that
both ends are hydrophobic in contrast to the central region which is more hydrophilic (data not shown). This scheme is common to all Skp preproteins (Hirvas et al., 1991). Since in bacteria sequence signal-containing proteins are normally exported, the presence of a cleavable signal sequence in p28 is consistent with the localization of this polypeptide in the envelope (in principle in the plasma membrane) of the transformed Ec strain whereas p25 was found in the periplasmic fraction (Manoha et al., 1994). The same observations were made with the homologous proteins Skp in Ec (Thome and Mt~ller, 1991), O m p H in St (Koski et al., 1990) and O m p H in Yersinia (Hirvas et al., 1991). The function of these proteins has not yet been elucidated. But, since skp is localized in a complex operon governing the early steps of LPS synthesis, it is conceivable that Skp/OmpH is also a protein involved in the synthesis of LPS (Koski et al., 1989) or in the exportation of macromolecules through the bacterial envelope as recently demonstrated (Thome and MUller, 1991). Previous hypotheses considering these proteins as the possible components of a bacterial chromatine were grounded upon misleading data. Indeed, the binding of
42
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Di~i:KI AAS;i~iKi~!VE A~VAAi#I~[Ki~iA P[i~iLRii~iAi~iIKi~iRi~iiEE I .ii~iL G Ni!~iiEi~iE A I N K L Viii , 2 0
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St F A Q K A Q A F E :K: - i'~i R ~i R i~ S N:i~ii~i:R N~i!i~ ~ T R {i:~ii:i~T ~ii~ K K:~ ~ N D Q S I D~ i~ V D A N T ~ A Y N S S ~i V i~ D ~ 152 Ye F S T K A Q A F E ii~! - i,~!i N "R'Ri~ Q M:I~I~:ER N ~II':I::L: S R i!:~ii!,i~D~ ~ K S ~i~~ S K G G ~ D V ' ~ I D A N ~ ~ A Y ~ D P :S':- ii~D i i 15
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Fig. 3. Alignment of the deduced aa sequences (A), Preproteins encoded by skp (=ompH) genes from Ec (Ec, X75465), St (St, J0510l), Y. enterocolitica (Ye, M34854) and Pm (Pro, X74357; see Fig. 2). The first aa of mature proteins is indicated by the asterisk in the upper line. The gaps are marked with dashes. The aa identical to those of Pm Skp protein are shaded. (B) Sequences encoded by firA (=ssc) genes from Ec (Ec, X54797), St (St, M35193), Y. enterocolitica (Ye, Z25463) and Pm (Pro, X74357). The gaps are marked with dashes. The aa identical to those of Pm FirA protein are shaded. The aa conforming to the six-residue periodicity are boxed. Because the N-terminal Met is processed in the mature Ec Skp protein it was omitted in the other sequences. these proteins to D N A is p r o b a b l y unspecific a n d due to their richness in basic aa. T h o u g h p25 (the m a t u r e form of Skp of Pro) is not h o m o l o g o u s to k n o w n porins, it was labelled with a r a b b i t a n t i s e r u m c o n t a i n i n g A b raised against p o r i n H of Pro. This unexpected cross-reactivity m a y be paralleled with that o b s e r v e d between two o t h e r proteins: O m p A of Ec a n d O m p H of St ( V a a r a et al., 1990). Since L P S is usually tightly b o u n d to o u t e r m e m b r a n e proteins, these
cross-reactions m i g h t be artefactual a n d r a t h e r due to the presence of residual L P S in o u t e r m e m b r a n e p r o t e i n b a n d s resolved by S D S - P A G E . A n o t h e r e x p l a n a t i o n might be presence of trace a m o u n t s of S k p in p o r i n H p r e p a r a t i o n s which were used to elicit a n t i - p o r i n H Ab.
(d) Identification of ORF3 C o m p a r i s o n of O R F 3 with nt d a t a b a s e s reveals 6 3 % of i d e n t i t y with the f i r A gene of Ec ( D i c k e r a n d
43 Seetharam, 1991) and 65% of identity with the ssc gene of St (Hirvas et al., 1990). The deduced aa sequence of ORF3 displays about 66% of identity with FirA of Ec and Ssc of St. Fig. 4 shows the aligned sequences of four proteins of the FirA family. The terms firA gene of Pm and FirA for the encoded protein are thus proposed for ORF3. In Ec, FirA is an N-acylase involved in the synthesis of LPS (Kelly et al., 1993). We thus believe that FirA has the same function in Pm. Recent studies have shown that several acyl- and acetyltransferases including CysE, LacA, LpxA and FirA/Ssc are built from repetitive blocks of six aa according to the scheme [(I, L, V or M) XXXXX], (Vaara, 1992; Vuorio et al., 1991; 1994). Fig. 4 shows that the FirA sequence of Pm displays the same hexapeptide periodicity.
(e) Conclusions (I) We have isolated a Pm chromosome fragment containing three distinct ORFs. ORF1 was a fragment of an unidentified gene. ORF2 proved homologous to skp of Ec and to ompH of St. ORF3 appeared homologous to firA of Ec and ssc of St. (2) The number of known Pm genes was thus far limited to six: the gene for the toxin, the gene aroA encoding adenylate cyclase, the tetracycline resistance gene tetA and the repressor gene tetR both cloned from an avian strain and the 16S rRNA cistron. The data presented in this paper are the first description of Pm genes mapping to an operon involved in the first steps of lipid A biosynthesis. (3) The fact that a Skp protein exists in bacteria outside the Enterobacteriaceae family strengthens the idea that it is a generally important protein, even though its function is still unknown. The finding that Pm FirA shares the repeated hexapeptide theme with other FirAs thus far sequenced futher suggests that this theme is an essential feature of lipid A acyl transferases.
ACKNOWLEDGEMENTS
We are grateful to M. Kobisch (Centre National d'Etudes Vrtrrinaires et Alimentaires, Ploufragan, France) for providing us with Strain 9222 of Pro. We are thankful to Nathalie Durand for typing the manuscript, to Louis Communier for the illustrations, and to Muriel Durand (Genexpress, Grnrthon) for help in nt sequencing. This work was supported by the Rrgion Bretagne (Programme Britta) and the Langlois Foundation (Rennes).
REFERENCES Chanter, N and Rutter, J.M.: Pasteurellosis in pigs and the determinants of virulence of toxigenic Pasteurella multocida. In: Adlam, C. and Rutter, J.T. (Eds.), Pasteurella and Pasteurellosis. Academic Press, London, 1989, pp. 161-195. Chevalier, G., Duclohier, H., Thomas, D., Shechter, E. and Wr6blewski, H.: Purification and characterization of protein H, the major porin of Pasteurella multocida. J. Bacteriol. 175 (1993) 266-270. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387 395. Dicker, I.B. and Seetharam, S.: Cloning and nucleotide sequence of the )irA gene and the firA200 (Ts) allele from Escherichia coll. J. Bacteriol. 173 (1991) 334-344. Hirvas, L., Koski, P. and Vaara, P.: Primary structure and expression of the SSC protein of Salmonella typhimurium. Biochem. Biophys. Res. Commun. 175 (1990) 53-59. Hirvas, L., Koski, P. and Vaara, M.: The ompH gene of Yersinia enterocolitica: cloning, sequencing, expression, and comparison with known enterobacterial ompH sequences. J. Bacteriol. 173 (1991) 1223 1229. Holck, A. and Kleppe, K.: Cloning and sequencing of the gene for the DNA-binding 17K protein of Escherichia coll. Gene 67 (1988) 117-124. Kelly, T.M., Stachula, S.A., Raetz, C.R.H. and Anderson, M.S.: ThefirA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)glucosamine N-acyltransferase. J. Biol. Chem. 268 (1993) 19866 19874. Koski, P., Hirvas, L. and Vaara, M.: Complete sequence of the ompH gene encoding the 16-kDa cationic outer membrane protein of Salmonella typhimurium. Gene 88 (1990) 117-120. Koski, P., Rhen, M., Kantele, J. and Vaara, M.: Isolation, cloning and primary structure of a cationic 16-kDa outer membrane protein of Salmonella typhimurium. J. Biol. Chem. 264 (1989) 18 973-18 980. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680 685. Manoha, F., Chevalier, G., Wr6blewski, H. and Delamarche, C.: Cloning and expression of two Pasteurella multoeida genes in Escherichia coll. Biochimie 76 (1994) 9-14. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Thome, B.M. and Mtiller, M.: Skp is a periplasmic Eseherichia coil protein requiring secA and sec Y for export. Mol. Microbiol. 5 (1991) 2815-2821.. Towbin, H., Staehelin, T. and Gordon, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4354. Vaara, M.: Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme. FEMS Microbiol. Lett. 97 (1992) 249-252. Vaara, M., Hirvas, L. and Koski, P.: The cationic 16-kDa outer membrane protein OmpH of enteric bacteria. In: Nowotny, A., Spitzer, J.J. and Ziegler, E.J. (Eds.), Cellular and Molecular Aspects of Endotoxin Reactions. Elsevier Biomedical Press, Amsterdam, 1990, pp. 197-204. Vuorio, R., Hirvas, L. and Vaara, M.: The Ssc protein of enteric bacteria has significant homology to the acyltransferase Lpxa of lipid A biosynthesis, and to three acetyltransferases. FEBS Lett. 292 (1991) 90-94. Vuorio, R., H~irkrnen, T., Tolvanen, M. and Vaara, M.: The novel hexapeptide motif found in the acyltransferase LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria. FEBS Lett. 337 (1994) 289 292.
39
GENE 08972
Characterization of the Pasteurella multocida skp and firA genes (DNA sequencing; N-terminal amino-acid sequence; amino-acid homology; lipid synthesis; porin)
C h r i s t i a n D e l a m a r c h e a, F a b r i c e M a n o h a a, G h i s l a i n e B 6 h a r b, R6mi H o u l g a t t e b, U l f H e l l m a n c a n d Henri Wr6blewski a a Dkpartement "Membranes et Osmorbgulation", Centre National de la Recherche Scientifique, Unit~ de Recherche AssociOe No. 256, Rennes, France; b Centre National de la Recherche Scientifique, UnitO Propre de Recherche no. 420, Villejuif, France. Tel. (33-1) 4958-1132; and ~ Ludwig Institute for Cancer Research, Uppsala, Sweden. Tel. (46-18) 550-188
Received by A.J. Podhajska: 18 December 1994; Accepted: 16 March 1995; Received at publishers: 7 April 1995
SUMMARY
A 2.9-kb fragment of the Pasteurella multocida (Pro) genome encoding proteins p25 (25 kDa) and p28 (28 kDa) has previously been cloned and expressed in Escherichia coli (Ec). In the present paper, the nucleotide (nt) sequence of a 1.8-kb subfragment encoding the two proteins is described. The cloned fragment contains three open reading frames (ORFs). ORF1 is incomplete. ORF2 is homologous to the skp gene of Ec. ORF3 overlaps ORF2 and is highly homologous to the firA gene of Ec. The skp and firA genes are part of an operon governing the first steps of lipid A synthesis. Comparing the nt sequence with the N-terminal sequences of p25 and p28 revealed that the two proteins are encoded by ORF2 (skp). The preprotein p28 is converted into p25 by cleavage of a 23-amino-acid leader peptide. Though it serologically cross-reacts with porin H of Pro, p25 is not related to known bacterial porins.
INTRODUCTION
Gram- eubacteria are surrounded by an outer membrane composed of lipopolysaccharides (LPS), phospholipids and proteins as major components. LPS and porins are the main immunogens of this membrane. Furhermore, owing to the toxicity of its lipid A moiety, LPS is also a Correspondence to: Dr. H. Wr6blewski, D6partement "Membranes et Osmor6gulation", Centre National de la Recherche Scientifique, Unit6 de Recherche Associ6e No. 256, Universit6 de Rennes 1, Campus de Beaulieu, F-35042 Rennes cedex, France. Tel. (33-99) 286-138; Fax (33-99) 286-700.
Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); Ec, Escherichia coli; FirA, UDP-3-O-(R-3-hydroxymyristoyl)-GlcN Nacyltransferase; firA, gene encoding FirA; GCG, Genetics Computer Group (Madison, WI, USA); IPTG, isopropyl-13-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; LPS, lipopolysaccharide; nt, nucleotide(s); ompH, gene encoding OmpH; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; Pro, Pasteurella multocida; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; Skp, a 25-kDa protein of P. multocida; skp, gene encoding Skp; St, Salmonella typhimurium; X, any aa. SSDI 0378-1119(95)00254-5
virulence factor. Therefore, obtaining data on the structure and the synthesis of porins and LPS should prove useful to the development of new therapeutic and prophylactic methods against pathogenic Gram- eubacteria. Pasteurella multocida (Pro), a widespread Grameubacterium, alone or in association with other pathogens, is responsible for severe diseases in mammals (including man) and birds (Chanter and Rutter, 1989). For example, the principal diseases of pig in which Pm is involved are atrophic rhinitis and pneumonia, which are both of economical importance. In atrophic rhinitis, Pm is often asssociated with Bordetella bronchiseptica, whereas in pneumonia it seems associated with Mycoplasma spp., B. bronchiseptica, Haemophilus pleuropneumoniae, Salmonella cholerae-suis or viruses. In an attempt to obtain genetic information on Pm cell surface antigens, a genomic library was screened with Ab directed against envelope proteins of this bacterium, including porin H (Chevalier et al., 1993). We have recently reported the cloning in Escherichia coli (Ec) of a 2.9-kb chromosomal fragment of Pm encoding the synthesis of
40 two polypeptides, p25 (25kDa) and p28 (28kDa) ( M a n o h a et al., 1994). Protein p25 was mainly found in the periplasm of Ec whereas p28 was principally detected in the cell envelope. In the present paper, we report the nt sequences of two genes localized in the cloned fragment of the Pm genome. Comparison of these genes with their homologues in Ec and in Salmonella typhimurium (St) and the determination of N-terminal aa sequences allowed the identification of p25 and p28.
A 2
B 3
2
kDa
9467-
43EXPERIMENTAL AND DISCUSSION
(a) Expression of p25 and p28 in Ec The p U C A plasmid containing a 2.9-kb TaqI fragment of the Pm chromosome (strain 9222, serotype D2, porcine isolate) was digested with HindIII + PvuII and the resulting 1.8-kb fragment was ligated into pUC19-digested with H i n d I I I + S m a I ( = p U C 2 ) or into pUC18 also digested with HindIII + Sinai (= pUC2 reverse), pUC2 and pUC2 reverse differ in the orientation of the insert with respect to the lac promoter (Manoha et al., 1994). The transformed cells (Ec JM109) were grown in LB broth supplemented with 100 ~g of ampicillin per ml and with 0.5 m M I P T G . The proteins of the envelope fraction of Pm strain 9222 were resolved by SDS-PAGE, electroblotted onto nitrocellulose membranes, and treated with different Ab. A major band of 37 k D a and two minor bands of 13 and 17 k D a were immunolabelled with a rabbit polyclonal antiporin H serum (Fig. 1A), whereas several major bands corresponding to apparent molecular masses of 17, 28, 31, 37, 40, 54 and 64 k D a were revealed with a rabbit anti-Pro envelope serum (Fig. 1B1). A pattern similar to the latter was obtained when using sera from pigs infected with Pm (data not shown). In a lysate of Ec transformed with pUC2 reverse, both p25 and p28 were immunolabelled by antienvelope sera containing Ab raised against 6 distinct strains of Pm (one example is illustrated in Fig. 1B2). In contrast, only p25 was labelled by the antiporin H serum (Fig. 1A2). Thus, p25 appeared as the sole protein of the Ec strain transformed with the pUC2 reverse plasmid displaying an immunological crossreactivity with porin H of Pro.
(b) Nucleotide sequence determination The nt sequence of the 1836-bp D N A of the pUC2 insert was determined. Three O R F s were found (Fig. 2). ORF1, containing 120-bp starts at the 5' end of the sequence and stops with two consecutive TAA triplets, ORF2, which contains 582 bp starting with an ATG start codon and ending with a TAA stop codon. The deduced aa sequence corresponds to a protein of 193 aa. O R F 2
30-
if" p28 if" p25
0
m
14. 4-
Fig. 1. Immunodetection of Pm antigens with anti-porin H (A) or antiPm envelope Ab (B). Samples were envelopes from Pm (lane 1, 10 gg protein), whole-cell lysates of E. coli transformed with pUC2 reverse (lane 2, 20 gg protein) or pUC18 (lane 3, 20 gg proteint. Size standards are shown on the left. Methods: Proteins were resolved by 0.1% SDS-12% PAGE according to Laemmli (1970) and electrotransferred onto nitrocellulose membranes for immunodetection (Towbin et al., 1979). Anti-porin H Ab were raised in rabbits with porin H purified by size exclusion chromatography (Chevalier et al., 1993). Sera were used at a dilution of 1:200. Secondary Ab (dilution, 1:500) were goat immunoglobulins directed against rabbit IgGs and conjugated with horseradisch peroxidase. 4-chloro-l-naphtol and H202 were used as enzyme substrates. A control blot without primary Ab was blank (data not shown).
is preceded by a putative Shine-Dalgarno sequence and a putative promoter region. The sequences T T G G A A ( - 3 5 region) and T A G T C T ( - 1 0 region) have 4- and 3-nt conservations, respectively, with respect to the two consensus hexamers of Ec promoters. The space between the two hexamers is of 16 nt. Downstream O R F 2 and with one nt overlapping its termination codon lies the 1017-nt ORF3 encoding a protein of 339 aa. Though ORF3 does not contain a stop codon at the 5' end, comparison with the related sequences suggests that the nt sequence is almost complete in pUC2 (see section d, below).
41
CAGCTGGGGT C.GCGCTTCAATGGCAATCGCCAATTGGACCGTTAGTG•TCTCTTATGCGAAACCT•TTAAAAAATACCAAGGCGATGAAATTGAGCAGTTCCAATTCAGCA•TGGTGGGACGTTCTAATAAGCGATTGGAA•TTATGTAA A G V A L Q W Q S P I G P L V F S Y A K P L K K Y Q G D E I E Q F Q F S I G G T F t *
150 (41)
-IQ RD --~ A A A A T T G T A G T C T A A A C A G C C A T C T G T G C A G T C A G C G T T C G G C A C A G T A T T T A T TCATATC~TTATTGAAA~AGGAAAAGAAACCAATGAAAAAAGCAGTCAAAGT~ACCC.CACTTTCTTTAGCATTAC.CcT~TAC~TCGTCTTTAGC~A 300 M M K A V K V T A L S L A L A F T S S L A M (22) TGGCCACAGAAAATAT TGCATTTATCAGT GGGGATTATTTATTTCAAAATCACCCAGATCGTAAAATG•TGGCAGAAAAATTAGAGTCAGAGTTTAAAGCCAGAGTAGAAAAACTCACTGCAAATAAAAAAAC.CATTGATGAAAAAATTG 450 A T E N I A F I S G D Y L F Q N H P D R }f M V A E K L E S E F K A R V £ K L T A N K K S I D E K I A {72) C C GC T TC T C A A A A G A A A G T T G A A GC C A A A GTTGC T C42ATTACAAAAAGAT GCC CC G A A A T T A C GCAGT G C A G A TAT TAAAAAAC GT G A A G A A G A A A T C A A T A A A C TC GC-CAATAGC GAGCAAGAAGC G A T C A A T A A A C T C G T T A C T G C A C 600 A S Q K K V E A K v A A L Q K D A P K L R S A D I ~[ K R £ E E I N K L G N S E Q E A I N K L V T A H (122)
EcaX A T G A T G A A G A A G T C A G T A A A T A T C A A G A C GATTATC-CAAAAC G T G A A C G T G A A G A A A C C G C A A A A T T A G T G G A T A G C A T • C A A A A C G C G G T A A A T A C G G T T G C A A G A G A G A A A A A C T A T A C A T T A G T A C T G A A T G A A G G T G C A G T C G T G T 750 D E E V S K Y Q D D Y A K R E R £ E T A K L V D S I Q N A V N T V A R E K N Y T L V L N E G A V V F (172) PIZX -~ TTGC T C.CAGAT G C A A A A A A T A T T A C T G A A G A T G T T • T A A A A G T C A T T C C T G C G A C A C A G G C G A A A T A A T G C A A G T T T A T T C C C T C C A A G A A T T A G C A C A A C A G A T C A A D A K N I T £ D V L K V I P A T Q A K * M Q v Y s L Q E L A Q Q I
GGCGCTACCATTCGTGGTAACGCCGAT GTTGTTGTAGAAAGTAT G
A
T
I
R
G
N
A
D
V
V
V
£
S
I
900 {193) (28}
TGCGCCTT T A G A T A A G C - C G A C A G A A A A A C A G C T T A C C T T T A T T T C T A A T C C T A A G T T C C G T T C C T T A C T G G C A C A A T C T C A T G C C G G G A T T TT G G T G G T G T C A G A A G C G G A T G T G G C G T T T T G T G C A G A G C A A A G T A A T T T A T T G A T T G T A P L D K A T E K Q L T F I S N P K F R S L L A Q s H A G I L V V S £ A D V A F C A E Q s N L L I V
1050 (78)
A A A A G A T C C GTAC G T G G C T T A T G C A G T T C T A G C A C A A T A T A T G G A T A G C A C G C C G A A A G C T G C G A G C G G C A T T G C C GCCAGYGCGGT GGTATC C - G C T A G T G C T G T G A T A G G C A A A A A T G T C T C A A T T G G T G C C A A T ~ T G A T T G ~ G A K D P Y V A Y A V L A Q Y M D S T P K A A S G I A A S A V V S A S A V I G K N V S I G A N A V I E D
1200 {128)
CGGGGTAACGCTTGGTGATCATGTAGTCATTGGTGCTAACTGCTTTGT~GGCAAAAATAGCAAAATTGGTGCTTATA~CCAGCTATGGGCAAATGTCAGTGTTTATCA~GAGG~GAAATAGGC~AACATTGTTTAATT~AATCTGGTGC
1350 (178)
G
V
T
L
G
D
H
V
V
I
G
A
N
C
F
V
G
K
N
S
K
I
G
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T
Q
L
W
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V
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AGTGATTGGTAGTGATGGTTTTGGTTACGCCAATGATCGTGG•CGTTGGATTAAAATTCCACAAGTTGGACAAGTCATTATTGGCAATCAT V I G $ D G F G Y A N D R G R W I K I P Q V G Q V I I G N H
GTC G A A A T T G G C G C T T G C A C T T G T A T T G A T C G A G G T G C G T T A G A T C C A A C G G T C A T T G A V E I G A C T C I D R G A L D P T V I E PBZX A G A T A A T G T G A T T A T T G A T A A T C T T T G C C A A A T T G C G C A T A A C G T C C A T A T T G G G A C C G G G A C T G C A G T T G C T G G T G G G G T G A T T A T G G C T G G T A G C TT GACCGTCGGACGCTATTGCTTAATTGGTGGTC-CCAGTGTGATCAATGGACA D N V I I D N L C Q I A H N V H I G T G T A V A G G V I M A G S L T V G R Y C L I G G A S V I N G H C A T G G A A A T C TGC G A T A A A G T G A C G A T T A C A G G C A T G G G G A T G G T G A T G C G T C C A A T T A C T G A A C C TGGCGTGTACTCT T C G G G T A T C C C A T T A C A A A C G A A T A A A G A A T G G C G T A A A A C C GCTGCATTGACGTTAGC-CATTGATGCGAT M E I C D K V T I T G M G M V M R P I T E P G V Y S S G I P L Q T N K E W R K T A A L T L G I D A M Ta~fX GAATAAACGGT TAAAAGCCTTAGA~GA~TTC N K P. L K A L E K K F
1500 (228) 1650 (278) 1800 (328)
GA 1836 (339)
Fig. 2. Nucleotide sequence of the 1836-bp Pm insert from pUC2 and deduced aa sequences. Possible -35 and -10 promoter regions and ShineDalgarno sequences are underlined. The stop codons are marked with asterisks. The ATG start codons for ORF2 and ORF3 are indicated by horizontal arrows above the nt sequence. The underlined aa sequences were determined by sequencing the N terminus of purified p25 and p28. This sequence was submitted to the EMBL data library and assigned accession No. X74357. Methods: Plasmid DNA from pUC2, pUC2 reverse, PvuIfSacl and PstI-PstI subclones was purified with the Magic Minipreps DNA purification system (Promega, Madison, WI, USA) and used for primer walking sequencing. The nt sequence was determined by dideoxysequencing (Sanger et al. 1977) on both strands using dye-labeled primers (forward and reverse primers) or dye-labeleddideoxynucleotides.The computer program used for the analyses of nt and aa were provided by the GCG package (Devereux et al., 1984). For N-terminal sequencing, the proteins resolved by one-dimensional SDS-PAGEwere electroblotted onto PVDF membranes (Towbin et al., 1979). The strips containing p25 or p28 were excised and the samples were analyzed in an Applied Biosystems Model 470A protein sequencer with an on-line PTH-analyzer Model 120A.
(e) Identification of ORF2 Comparison of the nt sequence of ORF2 with databases reveals significant homologies with the skp gene of Ec and the ompH gene of St (Holck and Kleppe, 1988; Koski et al., 1990). Fig. 3 shows the aligned sequences of four proteins of the Skp family. The Pm ORF2 displays 46 and 45% identity with the skp gene of Ec and the ompH gene of St, respectively, whereas there is 86% identity between the two latter genes. Therefore, we termed skp the gene corresponding to ORF2 and Skp the protein encoded by this gene. The N-terminal sequence of p28, determined over 18 aa, perfectly matches the 18-aa N-terminal sequence of the Skp polypeptide encoded by ORF2. The N-terminal sequence of p25, determined over 15 aa, is identical to the sequence running from aa 24 to 38 in Skp. Thus, Skp of Pm is synthesized as a precursor of 193 aa containing a leader peptide of 23 aa which is subsequently processed to yield a mature 170-aa protein starting with Thr. The leader peptide of Skp comprises an apolar stretch flanked by a short basic sequence (MKKAVK) on the N-terminal side and a cleavage site (AMA) on the C-terminal side. This arrangement is typical of bacterial signal peptides. The hydropathy plot of p28 reveals that
both ends are hydrophobic in contrast to the central region which is more hydrophilic (data not shown). This scheme is common to all Skp preproteins (Hirvas et al., 1991). Since in bacteria sequence signal-containing proteins are normally exported, the presence of a cleavable signal sequence in p28 is consistent with the localization of this polypeptide in the envelope (in principle in the plasma membrane) of the transformed Ec strain whereas p25 was found in the periplasmic fraction (Manoha et al., 1994). The same observations were made with the homologous proteins Skp in Ec (Thome and Mt~ller, 1991), O m p H in St (Koski et al., 1990) and O m p H in Yersinia (Hirvas et al., 1991). The function of these proteins has not yet been elucidated. But, since skp is localized in a complex operon governing the early steps of LPS synthesis, it is conceivable that Skp/OmpH is also a protein involved in the synthesis of LPS (Koski et al., 1989) or in the exportation of macromolecules through the bacterial envelope as recently demonstrated (Thome and MUller, 1991). Previous hypotheses considering these proteins as the possible components of a bacterial chromatine were grounded upon misleading data. Indeed, the binding of
42
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St F A Q K A Q A F E :K: - i'~i R ~i R i~ S N:i~ii~i:R N~i!i~ ~ T R {i:~ii:i~T ~ii~ K K:~ ~ N D Q S I D~ i~ V D A N T ~ A Y N S S ~i V i~ D ~ 152 Ye F S T K A Q A F E ii~! - i,~!i N "R'Ri~ Q M:I~I~:ER N ~II':I::L: S R i!:~ii!,i~D~ ~ K S ~i~~ S K G G ~ D V ' ~ I D A N ~ ~ A Y ~ D P :S':- ii~D i i 15
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Fig. 3. Alignment of the deduced aa sequences (A), Preproteins encoded by skp (=ompH) genes from Ec (Ec, X75465), St (St, J0510l), Y. enterocolitica (Ye, M34854) and Pm (Pro, X74357; see Fig. 2). The first aa of mature proteins is indicated by the asterisk in the upper line. The gaps are marked with dashes. The aa identical to those of Pm Skp protein are shaded. (B) Sequences encoded by firA (=ssc) genes from Ec (Ec, X54797), St (St, M35193), Y. enterocolitica (Ye, Z25463) and Pm (Pro, X74357). The gaps are marked with dashes. The aa identical to those of Pm FirA protein are shaded. The aa conforming to the six-residue periodicity are boxed. Because the N-terminal Met is processed in the mature Ec Skp protein it was omitted in the other sequences. these proteins to D N A is p r o b a b l y unspecific a n d due to their richness in basic aa. T h o u g h p25 (the m a t u r e form of Skp of Pro) is not h o m o l o g o u s to k n o w n porins, it was labelled with a r a b b i t a n t i s e r u m c o n t a i n i n g A b raised against p o r i n H of Pro. This unexpected cross-reactivity m a y be paralleled with that o b s e r v e d between two o t h e r proteins: O m p A of Ec a n d O m p H of St ( V a a r a et al., 1990). Since L P S is usually tightly b o u n d to o u t e r m e m b r a n e proteins, these
cross-reactions m i g h t be artefactual a n d r a t h e r due to the presence of residual L P S in o u t e r m e m b r a n e p r o t e i n b a n d s resolved by S D S - P A G E . A n o t h e r e x p l a n a t i o n might be presence of trace a m o u n t s of S k p in p o r i n H p r e p a r a t i o n s which were used to elicit a n t i - p o r i n H Ab.
(d) Identification of ORF3 C o m p a r i s o n of O R F 3 with nt d a t a b a s e s reveals 6 3 % of i d e n t i t y with the f i r A gene of Ec ( D i c k e r a n d
43 Seetharam, 1991) and 65% of identity with the ssc gene of St (Hirvas et al., 1990). The deduced aa sequence of ORF3 displays about 66% of identity with FirA of Ec and Ssc of St. Fig. 4 shows the aligned sequences of four proteins of the FirA family. The terms firA gene of Pm and FirA for the encoded protein are thus proposed for ORF3. In Ec, FirA is an N-acylase involved in the synthesis of LPS (Kelly et al., 1993). We thus believe that FirA has the same function in Pm. Recent studies have shown that several acyl- and acetyltransferases including CysE, LacA, LpxA and FirA/Ssc are built from repetitive blocks of six aa according to the scheme [(I, L, V or M) XXXXX], (Vaara, 1992; Vuorio et al., 1991; 1994). Fig. 4 shows that the FirA sequence of Pm displays the same hexapeptide periodicity.
(e) Conclusions (I) We have isolated a Pm chromosome fragment containing three distinct ORFs. ORF1 was a fragment of an unidentified gene. ORF2 proved homologous to skp of Ec and to ompH of St. ORF3 appeared homologous to firA of Ec and ssc of St. (2) The number of known Pm genes was thus far limited to six: the gene for the toxin, the gene aroA encoding adenylate cyclase, the tetracycline resistance gene tetA and the repressor gene tetR both cloned from an avian strain and the 16S rRNA cistron. The data presented in this paper are the first description of Pm genes mapping to an operon involved in the first steps of lipid A biosynthesis. (3) The fact that a Skp protein exists in bacteria outside the Enterobacteriaceae family strengthens the idea that it is a generally important protein, even though its function is still unknown. The finding that Pm FirA shares the repeated hexapeptide theme with other FirAs thus far sequenced futher suggests that this theme is an essential feature of lipid A acyl transferases.
ACKNOWLEDGEMENTS
We are grateful to M. Kobisch (Centre National d'Etudes Vrtrrinaires et Alimentaires, Ploufragan, France) for providing us with Strain 9222 of Pro. We are thankful to Nathalie Durand for typing the manuscript, to Louis Communier for the illustrations, and to Muriel Durand (Genexpress, Grnrthon) for help in nt sequencing. This work was supported by the Rrgion Bretagne (Programme Britta) and the Langlois Foundation (Rennes).
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