Gene, 129 (1993) 107-l 11

0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-I 119/93/$06.00

GENE 07141

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Analysis of tandem, multiple genes encoding 30-kDa membrane proteins in Pas teurella haemoly tica A 1

(Lipoprotein; transcription; gene duplication; Escherichia coli and Huemophilus injluenzae homology)

George L. Murphy and Lisa C. Whitworth

Oklahoma State University, Department of Veterinary Pathology, Stillwater, OK 74078, USA

Received by G. Wilcox: 28 December 1992; Revised/Accepted: 5 February/l2 February 1993; Received at publishers: 1 March 1993

SUMMARY

A number of outer membrane proteins (OMPs), including a 30-kDa protein, may be important in eliciting immunity to Pasteurella huemolyticu Al, the causative agent of bovine pneumonic pasteurellosis. To better understand the nature of the 30-kDa antigen, several genes encoding this protein were sequenced. Sequence analysis revealed that three separate genes encoding similar, yet distinct, versions of the 30-kDa protein are tandemly arranged on the P. huemolyticu Al chromosome. The genes appear to be transcribed from a single promoter. The deduced amino acid sequences of the proteins encoded by these genes are similar to a 28-kDa inner membrane lipoprotein of Escherichiu coli and a 28-kDa membrane protein which may contribute to the virulence of Huemophilus injluenzue type b strains.

INTRODUCTION

Pasteurella huemolyticu serotype Al is the bacterium most commonly responsible for bovine pneumonic pas- teurellosis (shipping fever pneumonia) in feedlot cattle. Immunological analyses indicate that high antibody responses to several saline-extractable surface antigens, including a 30-kDa protein, correlate statistically with resistance to pneumonic pasteurellosis (Mosier et al., 1989), and vaccination of cattle with P. huemolyticu

OMPs enhances their resistance to experimental chal- lenge (Morton et al., 1990).

A gene encoding one of these immunogenic OMPs, the 30-kDa protein, was cloned and expressed in E. coli

(Craven et al., 1989). Radioiodination of P. huemolyticu

Correspondence to: Dr. G.L. Murphy, Oklahoma State University,

College of Veterinary Medicine, Department of Veterinary Pathology,

Stillwater, OK 74078, USA. Tel. (405)744-4518; Fax (405)74&5275.

Abbreviations: aa, amino acid(s); BHI, brain heart infusion; bp, base

pair(s); H., Haemophilus; kb, kilobase or 1000 bp; Lpp, lipoprotein(s);

nt, nucleotide(s) (number of sequence); OMP, outer membrane protein;

ORF, open reading frame; P., Pasteurella; PCR, polymerase chain reac-

tion; RBS, ribosome-binding site(s); SDS, sodium dodecyl sulfate.

surface proteins and agglutination of whole P. huemolyt-

icu, with antiserum directed against this protein, verified that it is indeed a surface antigen (Craven et al., 1989).

Although the 30-kDa OMP has been shown to be an important one in immunity to pneumonic pasteurellosis, the exact nature of the antigen is unknown. To obtain more information about this protein and to facilitate fur- ther studies on its role in pathogenesis, we have deter- mined the DNA sequence of the previously cloned gene. We have also identified, cloned, and sequenced two other genes which encode similar proteins. Here we report the DNA sequences of these genes, examine their transcrip- tion, and discuss the similarities of the proteins encoded by these genes with membrane proteins of E. coli and Huemophilus injluenzue type b.

EXPERIMENTAL AND DISCUSSION

(a) DNA sequencing of three tandem ORFs encoding 30-

kDa proteins

The recombinant plasmid pRC12 consists of an 8-kb Suu3AI fragment of P. huemolyticu chromosomal DNA

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cloned into the vector pUC19 (Craven et al., 1989). A 2.8- kb fragment from this insert (Fig. IA) was previously shown to encode a 30-kDa P. haemolytica OMP (Craven et al., 1989). To determine the nature of the 30-kDa pro- tein, we sequenced this region of DNA. Two large ORFs were identified (Fig. 1A). ORFl (831 bp) encodes a pro- tein of approximately 30 kDa. 0RF2 is located 3’ of ORF 1 and represents a partial copy of a gene very similar to ORFl. Restriction enzyme mapping revealed that the 5.6-kb DNA insert, present in the recombinant plasmid pRC9 (Fig. lB), overlaps and extends beyond the 3’ end of the 2.8-kb insert in pGEB2830. Further DNA sequen- cing with pRC9 revealed that three complete genes are present, in tandem, at this locus (Fig. 1B).

The nt sequence (Fig. 2) reveals that potential - 35 and -10 promoter regions, with correct spacing, are present 5’ of the start codon for ORFl but do not appear to be present 5’ of ORF2 or 0RF3. A potential RBS precedes the putative start codon of each gene. Regions of dyad symmetry, capable of forming hairpins and acting as tran- scription terminators, are also present 3’ of each ORF. The nt sequences of the three genes are approximately 60% identical. The mol% G + C for each gene is 37-39%, normal for P. haemolytica DNA (Mannheim, 1984). The calculated sizes of the proteins encoded by ORFl, 0RF2, and 0RF3 are 30 kDa, 30.15 kDa, and 29.1 kDa, respectively.

(b) ORF transcriptional analysis To examine the transcription of these genes, we probed

total RNA isolated from P. haemolyticu Al with gene fragments specific for each individual ORF. The ORFl probe hybridizes with three transcripts of 0.9 kb, 1.8 kb, and 2.6 kb , the ORF2 probe with transcripts of 1.8 kb and 2.6 kb, and the 0RF3 probe with a single 2.6 kb transcript (Fig. 3). The transcript sizes correspond to

A. pGEB2830

those expected from the transcription of one (ORFl), two (ORFs 1 and 2), or all three genes (ORFs 1, 2, and 3) from the single putative promoter 5’ of ORFl. These data are also consistent with the presence of a transcription terminator 3’ of each gene as suggested by the DNA sequence.

(c) Alignment of aa sequences with similar proteins A search of the GenBank DNA and NBRF protein

sequence databases revealed similarities among the deduced aa sequences from ORFl, 0RF2, and 0RF3, an E. coli 28-kDa Lpp (Lpp-28) (Yu et al., 1986), and a 28- kDa membrane protein of I-Z. inJIuenzue b strains (Chanyangam et al., 1991). The aa identity among the proteins varies from 51% to 78% (Table I).

The optimal alignment of the deduced aa sequences of the five proteins is shown in Fig. 4. A consensus Lpp processing site, preceded by a presumptive signal peptide, is present in each aa sequence. At this time, lipid modifi- cation has only been demonstrated for the E. coli Lpp- 28. While lipid modification of the P. haemolyticu and H. injluenzue proteins has not yet been demonstrated, these data suggest that they are likely to be Lpp. The calculated sizes of the putative mature forms of the P. huemolyticu proteins encoded by ORFl, 0RF2, and 0RF3 are 28.1, 28.2, and 27.1 kDa, respectively. Those for the H. i&en-

zue b and E. coli mature proteins are 27.2 and 27 kDa, respectively.

The E. coli Lpp-28 is an inner membrane protein (Yu et al., 1986). Mutants lacking this protein remain viable (Yamaguchi and Inouye, 1988). The H. injluenzue protein is located in the total membrane fraction of that organ- ism. Analyses of mutants lacking that protein suggested that it may aid the transepithelial invasion of H. injluen- zue type b strains (Chanyangam et al., 1991). Our prelimi- nary studies indicate that one or more of the P.

Pst I Sau3A I

1 kb

B. pRC9

Pst I SaudA I

Fig. 1. Restriction maps of plasmid inserts showing ORFs which encode P. haemolytica 30-kDa proteins. (A) The 2.8-kb EcoRI-BamHI fragment from pRCI2 (Craven et al., 1989) was subcloned into pGEM4Z (Promega, Madison, WI, USA) creating pGEB2830. (b) The recombinant pRC9 (Craven et al., 1989) carries a 5.6-kb Sau3AI fragment from the P. haemolytica chromosome.

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1 CGCCCCmAGGCMmAGCGATPACCPATGGCGAACACCGTAA TXGGTGATCA

12 1 ~CGlTACGA’ICATCG~ AlTTAAAAA~TATGAGTlTCAAGAAAAlTTTAGGCGTlYXATlGG TTl’CTGCATl’AGCATl’AACCGCTMAGMGAG

MSFKKILGVALVSALALTACKEE

480 CAATCAAAAGGTTl’AAACMCTTAGTGATlGTGGGTMCACCTlTGTTl’ATCCGTTAGCGGGCTACTCT AAAAAAGTGAAAAATGTGTAGTTAGCAGMGGTGCGGTAATTGCAGTA

QSKGLNNLVI VGNTFVYPLAGYSKKVKNVSELAEGAVIAV

60 0 CCAAATGATCCTPCAAA Cl-I’AGC’ICGTOCllTGATl’TpATTAGAAAAACAGGGGlTMTTAAA lTAAAAGATMTACCAATTTATTCTCAACTPCAGl?2GATATTA~GAAAATCCGAAA

PNDPSNLARALILLEKQGLI KLKDNTNLFSTSVDIIENPK

720 AACITAAAAA’ITAAAGAAG~ATAC~M~CC~~G~~AGA~ACGTAGA~G~~T~TMCAC~A~Cffi~A~TA~C~~TACCCMGA~A~T

NLKIKEVDTSIAAKALDDVDLAVVNNTYAGQVGLNTQDHG

840 GTATTTGTTGAGTCTAAAGATTCACCGTATGn;AATA~A~~C~GCC~GACMT~GA~C~CTM~TA~C~A~~~~AC~CCGMGMG~TACCM

VFVESKDSPYVNIIVARQDNKDAANVQNF IKSYQTEEVYQ

I I

960 GAAGCACRAAAACACmAAAGATCGTCTAGTAAAAGGTPGGGCCA ~~AMATATAGGA

EAQKHFKDGVVKGW’

1077 ?zmGcAAAcAATGAAcTrr AAAAAA~ATPAGG~TGGCGTPAGTATCTGCCTTAGCAC~AC~CG~T~GA~~GCAC~G~CC~CTA~CAGCT~C~~C

MNFKKLLGVALVSALALTACKDEKAQAPATTAKTEN

1195 AAAGCCCCATPAAAAGTGGGTGTGATGACCGGCCCTGAAGC

KAPLKVGVMTGPEAQMTEVAVKIAKEKYGLDVELVQFTEY

1315 ACTCAACCAAATGCCGCAC?CATITTAAAGAmAGATGCI

TQPNAALHSKDLDANAFQTVPYLEQEVKDRGYKLAI IGNT

1435 CTAGTATGGCCAATCGCGGC ‘RXAAAACAmCCGAGTTAAAAGACGGAGCGAC’XTltXGAlTCCAAACMTGCAAGTMTACTGC~TG~l-l’ATl’ATl’GCTr

LVWPIAAYSKKIKNISELKDGATVAIPNNASNT’ARALLLL

1555 CAAGCICACGGTTTAAAA GATCCGAAAAATG~II3TCTACffi~CGATA~A~G~CCCG~TA~~~GTA~ffi~TAC~C~~CCCGT

QAHGLLKLKDPKNVFATENDIIENPKNIKIVQADTSLLTR

16 7 5 An;TPRGATGATGTAGMCTPGCGGTAATCAACAACACTT

MLDDVELAVINNTYAGQAGLSPDKDGIIVESKDSPYVNLV

1795.GTMGTCGTGMGATMTAAAGATGACCCACGC~A~CTTlTG TGAAGPCA~CAAACCGAAGAAGTA~~GMG~~~A~C~~~~~~ffi~

VSREDNKDDPRLQTFVKSFQTEEVFQEALKLFNGGVVKGW

> -.

1915 TMTCAGTIGGACGGCATAT‘IGC~TCC~C~ CAGATMTGAAMTMTGMA~AGCCGGn;CAGTPGCAG mMCCGmTAATGAT l MKIHKLAGAVAIFSLFLTACND

20 3 5 AAAGCCGAAAAG~~GTCGDPGTGA’ITPCCGGCCCPG CAAAAATTGCAAAGGMAAA TATAACCG~Al-G~TGlGGTATlTACCGAT KAEKLKVGVISGPEHKVMEVAAKIAKEKYNRDVELVVFTD

215 5 TATGCCACGCi!TMTGCAGCPFl’AGATAAAGGCGATCITGATTXMl’XTlTCCAGCATAMCCTl’ATl-l’AGATMCCAAATl’CAGGAAAAAGG Cl’ATAAATPAClGCCGGTtXGCAAT

YATPNAALDKGDLDLNAFQHKPYLDNQIQEKGYKLVPVGN

2215 ACmTCTl’TATCCGATPGCGGCTPATn: CAAAAAAATPAAATCGCPGG~GAAACATGGTGA~~A~~~AG~C~M~A~CGA~~A~CC~~M~A

TFVYPIAAYSKKIKSLAELKDGDTIAVPNDPTNLARALIL

23 9 5 TlCGAAAAACAAGAl-lTMTl’MGCTGCGAGCAGAXCA GGCTPAAAAGCMCCAG~ATA~AT~GAAJACCCTCGTAAA’I=~W~~GATCCMGAAAT~~AAGCACCATTATPXCT

LEKQDLIKLRADAGLKATSVDI IENPRKLVIQEIE.A.PLLP

2635 A~~CCCG~~PGAAAATAACCAACATPCTGAAGCCC

IVARENNQHSEAVKDLVKAYQTEKVYNKANEEFKGAMIKG

--

2755 TGGTAATTGMGATMATMGGCATAGAT’ICCTA’IBCCTTACll-MTATGlTACAGACC?%TCCGGC’ICTTAAXCTIC GGCTTTGTCAGTTFMTC CCAAGGAAAAXXTTACGACCG

w l

Fig.2. The nt sequence of the region encoding the P. hnemolytica 30-kDa proteins. Recombinant plasmids were purified using Qiagen columns (Qiagen, Chatsworth, CA). Nested deletions were created with the Erase-a-Base kit (Promega, Madison, WI). Both strands of DNA were sequenced using the Sequenase kit (US Biochemical, Cleveland, OH). Potential -35 and -10 regions 5’ of ORF 1 are underlined. Putative RBS 5’ of each ORF are overlined. Regions of dyad symmetry, 3’ of each ORF, which may act as transcription terminators, are overlined with arrows. The regions used as gene-specific probes for Northern blots are for: ORFl, nt 272-835; 0RF2, nt 1185-1747; 0RF3, nt 2005-2590. Each region was amplified, by PCR from pRC9, using oligodeoxynucleotide primers designed to hybridize to their 5’ and 3’ ends, and cloned into the EcoRI + HindIII-digested pGEM4Z. The resulting recombinant plasmids were designated pGORF30-I, pGORF30-2, and pGORF30-3. The identities of the cloned fragments were verified by nt sequencing from each end using the universal and reverse primers. The aa sequences of the three proteins are numbered in Fig. 4, as mature proteins Phi, 2, and 3. The GenBank accession No. for this sequence is L11037.

110

kb

4.4

2.4

1.77 1.52 1.28

Fig. 3. Autoradiograph of total P. haemolyticn RNA probed with (lane A) the ORFl-specific probe, (lane B) the ORF2-specific probe, and (lane C) the ORF3-specific probe. P. hnemolytica Al was grown in BHI broth at 37°C on a rotary shaker at 175 rpm. Cells from 100 ml of culture were harvested in early log phase of growth, resuspended in TES at 4”C, and immediately lysed by adding an equal volume of TES- 1 %SDS and plunging into a 100°C water bath. Total RNA was isolated by repeated extractions of the aqueous phase with 65°C phenol pre- viously equilibrated with 50 mM Naacetate (pH 5.0). RNA was precipi- tated with two volumes of 100% ethanol. Total RNA (10 ug) was separated by electrophoresis through 1.1% agarose gels, as described (McMaster and Carmichael, 1977), and transferred to Genescreen (NEN Research Products, Boston, MA) using the Posiblot apparatus (Stratagene Cloning Systems, La Jolla, CA) according to the manufac- turer’s instructions. Hybridizations, in sealed plastic bags, and washes were performed according to the Genescreen instructions. TES is 10 mM Tris, 10 mM EDTA, 100 mM NaCl, pH 80.

haemolytica proteins are located in the cell membrane fraction (data not shown). Similarities between H. influenzae- and P. haemolytica-induced pneumonias sug- gest that the 30-kDa protein(s) in P. haemolytica may also play a role in virulence. Analyses of P. haemolytica mutants lacking the 30-kDa protein(s) are necessary for determining their roles in bovine pneumonic pasteurello- sis. Construction and analysis of P. haemolytica mutants, which lack these genes, are in progress in our laboratory.

TABLE I

Amino acid identity among mature forms of the 30-kDa proteins

Ph28.1b Ph28.2” Hi28d Ec28’

Ph27.1” Ph28.1b Ph28.2’ Hi28d

60% 55% 59% 54% (100%) 63% 78% 51%

(100%) 58% 48% - - (100%) 51%

“Encoded by 0RF3 (see Fig. 1). ‘Encoded by ORFl (see Fig. 1). ‘Encoded by ORF2 (see Fig. 1). dFrom Haemophilus in@enzae (Chanyangam et al., 1991). ‘From Escherichin coli (Yu et al., 1986).

Phl MSFKKIL---GVALV-SALALTA CKEEKKAESTAAPAAQ--APAKIKVGV 25 Ph2 MNFKKLL---GVALV-SALALTA CKDEKAQAPATTAKTENKAP--L*"* 25 Ph3 MKIMKL-A--GAVAIFS-LFLTA CND-K-AR_-______-_-_-KL*.** 12 Hi MKLKQLFAIT--AIA-SALS'LTG CKEDKKPE--AA-AA----PLKI**** 20 EC MKLTTHHLRTGAALLLAGILLAG (-DQS,$SDAm-_--_______-I*“’ 15

Phl MSGPEHTVAERAAQIAKEKYGLEVEFVLFNDYALPNTAVFQHKP 78 Ph2 MT*P*AQMT'V*VKI****'GLD'EL'Q'TEYTQ**A*LHSKD"A'AF'TV' 78 Ph3 IS*P*HKVM*V'AKI*'*'*NRD*EL'V*TDYAT**A*LDKGD'*L*AF*HK' 65 Hi MS*P*HQVA*I*AKV**"*GLD*QF*E*~YAL"E'VS*HK* 73 EC IN*A'QDVA*V*KKV**'**GLD*EL'G'SGSLL**D*T~GE*'A*VF*HR' 68

Phl YLDK-DSQSKGLNNLVIVGNTFVYPLAGYSKKVKNVSELAEGAVIAVPNDPSN 130 Ph2 Y*E-QEVKDRGYK-*AII**'L'W*I*AY***I*NISELKD*ATVAI**NAS' 129 Ph3 Y*DNQ-IQEKG~-*VPV***F'Y*I*AY***I*S~LKD*~IAV**DPT* 116 Hi Y*DE-DAKAKNLNN*VIV*'*F*Y*L'GT l **I'NVNELQD'AKVVV*'DPT* 125 EC F'E-QDNQAHGYK-*VAV*'*F*F*M*GY***I*~AQIKE*A~AI~*DPT' 119

Phl LARALILLEKQGLIKLKDNTNLFSTSVDIIENPKNLKIKEVDTSIAAKALDD- 182 Ph2 TA***L**QAHG'LK'KDPKNVFA'EN '*IE"KNIK'VQADTSLLTRM"'- 181 Ph3 LA*"I'*EKQD*IK'RADAGLKA*SV **IE**RKLV'QEIEAPLLPRT**'- 168 Hi RG"*I"EKQG*IK'KDANNLLS*VL**VE'*KKLN*TEVDTSVAA~*"*- 177 EC LG***L**QKEK*IT*KEGKGLLP'AL **TD*'RHLQ'MELEGAQLPRV'*'P 172

Phl Ph2 Ph3 Hi EC

-VDLAWNNTYAGQVGL~QDHGVFVESKDSPYVNIIVARQDNKDUNVQNFI 234 -'EIJ,VINN**AG'A'*SPDK"GIIV S D l ' l ++**LV*S*ED+KDDPRLQTFV 233 -'AFSIINT*'AG'N*'TPTK*GIFV D D l l l *'*'LI*A*EN'QHSEAVKDLV 220

l l -'DLAVVNN"AG*V*'NAQD'GVFV D D "**'II'S*TD'KDSKAVQDFV 229 K'DVAIIST'*IQ*T**SPVH*SVFI*D*N****'IL*A*ED*KNAENVKEFL 225

Phl KSYQTEEVYQEAQKHFKDGVVKGW 258 Ph2 KSF*TE"FQE*LKL'NGGWK'* 257 Ph3 KAY*TE"YNK'NEE'KGAMIK** 244 Hi KSY*TE*'YQE'QKH'KEGWK** 253 EC QSY*SP"AKA*ETI'NGGAVP** 249

Fig. 4. The aa alignment of the P. haemolytica Al (Ph), H. injluenzae (Hi) (Chanyangam et al., 1991), and E. coli (EC) (Yu et al., 1986) 28-30- kDa proteins. The aa residues identical in all three proteins are desig- nated by a asterisks (*). Dashes (-) have been inserted for absent residues to allow for optimal alignment. Numbering begins at the cysteine resi- due (+l), which is presumed to represent the N-terminal aa of the mature forms of these membrane proteins. The putative signal peptides are represented as negatively numbered residues. Phi, Ph2, and Ph3 were deduced from the nt sequence in Fig. 2.

(d) Conclusions

(I) Three genes encoding proteins of approximately 30 kDa and exhibiting 55-60% nt sequence identity are tandemly arranged on the P. haemolytica Al chromosome.

(2) Transcription appears to be driven by a single pro- moter 5’ of the gene cluster. Three transcripts correspond- ing to one, two, or all three genes are produced in P. haemolytica Al.

(3) The deduced aa sequences of the proteins display a

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high degree of identity with an E. coli inner membrane Lpp and an H. injuenzae membrane protein which may be associated with invasion.

ACKNOWLEDGEMENTS

This research was supported by the Oklahoma Agricultural Experiment Station, and by grant No. 92-03744 from the National Research Initiative Competitive Grants Program/USDA. This manuscript is submitted as No. 592-054.

REFERENCES

Chanyangam, M., Smith, A.L. , Moseley, S.L., Kuehn, M. and Jenny, P.: Contribution of a 28-kilodalton membrane protein to the viru- lence of Haemophilus inj7uenzae. Infect. Immun. 59 (1991) 600-608.

Craven, R.C., Confer, A.W. and Gentry, M.J.: Cloning and expression of a 30 kDa surface antigen of Pasteurella haemolytica. Vet. Microbial. 27 (1991) 63-78.

Mannheim, W.: Family III. Pasteurellaceae. In: Krieg, N.R. (Ed.), Bergey’s Manual of Systematic Bacteriology, Vol. 1. Williams and Wilkins, Baltimore, MD, 1984, pp. 550-558.

McMaster, G.K. and Carmichael, G.C.: Analysis of single- and double- stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74 (1977) 4835-4838.

Morton, R.J., Confer, A.W. and Panciera, R.J.: Vaccination of cattle with outer membrane protein enriched fractions of Pasteurella

haemolytica. Abstr. 71st Annu. Meet. Conf. Res. Workers in Anim. Dis. (1990) p. 51, CRWAD, Chicago, IL, USA.

Mosier, D.A., Simons, K.R., Confer, A.W., Panciera, R.J. and Clinkenbeard, K.D.: Pasteurella haemolytica antigen associated with resistance to pneumonic pasteurellosis. Infect. Immun. 57 (1989) 711-716.

Yamaguchi, K. and Inouye, M.: Lipoprotein 28, an inner membrane protein of Escherichia coli encoded by nlpA, is not essential for growth. J. Bacterial. 170 (1988) 3747-3749.

Yu, F., Inouye, S. and Inouye, M.: Lipoprotein-28, a cytoplasmic mem- brane lipoprotein from Escherichia coli. J. Biol. Chem. 261 (1986) 2284-2288.