Vol. 174, No. 4

Cloning and Sequence Analysis of the Chlamydia trachomatis spc

Ribosomal Protein Gene ClusterRAVI KAUL,* GARY J. GRAY, NIELS R. KOEHNCKE, AND LIJIE GU

Division of Infectious Diseases, Department of Pediatrics, University ofAlberta, Edmonton T6G 2R7, Canada

Received 26 August 1991/Accepted 9 December 1991

We identified and sequenced a segment of Chlamydia trachomatis chromosomal DNA that shows homologyto the Escherichia coli spc and distal region of the S10 ribosomal protein (r-protein) operons. Its sequence

revealed a high degree of nucleotide and operon context conservation with the E. coli r-protein genes. The C.trachomatis spc operon contains the r-protein genes for L14, L24, L5, S8, L6, L18, S5, L15, and Sec Y alongwith the genes for r-proteins L16, L29, and S17 of the S10 operon. The two operons are separated by a 16-bpintragenic region which contains no transcription signals. However, a putative promoter for the transcriptionof the spc operon was found 162 nucleotides upstream of the CtrL14e start site; it revealed significant homologyto the E. coli consensus promoter sequences. Interestingly, our results indicate the absence of any structureresembling an EcoS8 regulatory target site on C. trachomatis spc mRNA in spite of significant amino acididentity between E. coli and C. trachomatis r-proteins. Also, the intrinsic aminoglycoside resistance in C.trachomatis is unlikely to be mediated by CtrL6e since E. coli expressing CtrL6e remained susceptible togentamicin (MIC < 0.5 ,ug/ml).

Chlamydia trachomatis infections represent major publichealth problems in both developing and industrialized coun-

tries (30). Chlamydia species have evolved a complex andunique developmental cycle which involves two distinctforms: the small (0.2 to 0.3 ,um) extracellular, rigid elemen-tary bodies and the large (1 ,um) intracellular, fragile reticu-late bodies (44). The genetics of chlamydial regulation islargely undefined, mainly because of the lack of any conve-nient system for gene transfer and also because of thepaucity of information about the signals and machinery thatgovern gene expression.Ribosomes constitute the protein-synthesizing machinery

of both prokaryotes and eukaryotes. The entire prokaryoticribosome comprises three rRNAs with sedimentation coef-ficients of 23S, 16S, and 5S and approximately 52 ribosomalproteins (r-proteins) that are organized into 19 differentoperons (26, 33). Earlier attempts at characterizing therRNA from Chlamydia species have met with some success.Tamura and Iwanaga (42) identified 21S, 16S, and 4S rRNAfractions in Chlamydia psittaci; of these, 21S and 16S weremore predominant forms in reticulate bodies, whereas 4Spredominated in the elementary bodies. Sarov and Beckerfound similar rRNA species in C. trachomatis (39). On thebasis of their 16S rRNA gene sequence, chlamydiae havebeen identified as eubacterial in origin, related peripherallyto planctomyces (45). Although a few other ribosomal deter-minants have been identified, r-protein gene organizationand expression have not been well characterized (7-10, 20,21). Interestingly, the organization and transcriptional regu-lation of r-protein operons appear to be well conservedamong eubacteria; however, their regulation among evolu-tionary distant species remains unelucidated (15).

Recently, we reported the cloning and sequencing of ther-protein CtrL6e from C. trachomatis, which is structurallyand functionally homologous to Escherichia coli r-proteinEcoL6 (13). The sequence flanking CtrL6e revealed homol-ogy to EcoS8 and EcoL18, suggesting that CtrL6e is in the

* Corresponding author.

same operon context as the E. coli spc operon. The spcoperon in E. coli contains genes encoding r-proteinsEcoL14, EcoL24, EcoL5, EcoS14, EcoS8, EcoL6, EcoL18,EcoS5, EcoL30, and EcoL15 as well as the Sec Y and Xproteins (5, 43). The operon is autogenously regulated by atranslation coupling mechanism whereby the EcoS8 geneproduct binds to the target site on the EcoL5 mRNA, leadingto repression of EcoL5 along with downstream translation(6, 31). In Bacillus subtilis and Thennus aquaticus, however,no structure resembling an S8 translational repressor hasbeen identified, suggesting some different mechanism oftranscriptional regulation (15, 18). Immediately upstream ofthe spc operon in E. coli lies the S10 operon, which sequen-tially encodes EcoS10, EcoL3, EcoL4, EcoL23, EcoL22,EcoS19, EcoS3, EcoL16, EcoL29, and EcoS17 (33). A162-bp spacer between these two operons contains thetranscription termination and initiation sequences. How-ever, in Mycoplasma capricolum, the spacer is only 15 bpand does not include transcription termination signals (34).Also, there is no space to accommodate intragenic promot-ers in T. aquaticus (1) and Methanococcus vannielii (2).

In this article, we report the cloning and sequence analysisof the C. trachomatis spc operon along with the distal regionof the S10 operon.

MATERIALS AND METHODS

Bacterial strains. C. trachomatis serovar L2 (L2/434/Bu)was grown in HeLa 229 cells as described previously (22).Elementary bodies were harvested at 48 h by centrifugationand purified as reported earlier (13).

E. coli DH5otF' (46) was used as a host for M13mpl8 andM13mpl9 (47). NM522 (12), and BNN103 (17) were used as

hosts for pUC (47) plasmids. These cells were made compe-tent for transformation or transfection essentially accordingto the method of Hanahan (14).DNA manipulations. Chromosomal DNA was isolated

from purified elementary bodies (13). Plasmid and bacterio-phage replicative-form DNA isolation, ligation, and transfor-mations of E. coli strains were done as described previously

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(13). Chromosomal DNA was analyzed by Southern hybrid-ization by the standard technique (40). Overlapping DNAfragments were generated with various restriction endonu-cleases. Large DNA fragments were purified on agarosegels, while smaller fragments were purified on polyacryl-amide gels. Subclone libraries were then constructed byinserting the restricted DNA at corresponding restrictionsites in pUC18 or M13mp18 pretreated with alkaline phos-phatase. Positive colonies and plaques were identified byhybridization to appropriate end-labeled fragments.DNA sequencing and sequence analysis. All DNA sequenc-

ing was performed by using the dideoxy chain terminationmethod (37) with [at-32P]dATP and the Sequenase sequenc-ing kit (United States Biochemical Corp., Cleveland, Ohio).Sequence reactions were primed with either universal prim-ers (Boehringer Mannheim Canada Ltd., Laval, Quebec,Canada) or synthetic oligonucleotides (DNA Synthesis Fa-cility, Department of Microbiology, University of Alberta,Edmonton, Canada) when necessary in order to continuesequencing. The sequence was either confirmed by analysisof complementary strand or determined by two independentreactions from different cloned fragments. Sequence compi-lation, open reading frame (ORF) identification and transla-tion, and restriction map construction were all performedwith DNA software for the MacIntosh computer (29). Tocompare the predicted products of ORFs with those ofNBRF library, we used the FASTP program on the MacIn-tosh SE computer (27).

Expression and production of recombinant proteins in E.coli. E. coli NM522 harboring different plasmids was grownin an overnight culture. Bacteria from the overnight cultureswere diluted 1:100 in Luria broth containing 100 ,ug ofampicillin per ml and grown for 2 h at 37°C with adequateaeration. After the addition of isopropyl-o-D-thiogalactopy-ranoside (IPTG; 10 mM final concentration), incubation wascontinued for 4 h. Aliquots of approximately 40 ,ul each werepelleted for 1 min at 4°C in a microcentrifuge. The resultingpellet was solubilized in sample buffer, and proteins wereresolved by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) on 12.5% acrylamide (23).RNA isolation and Northern (RNA) blotting. Total RNA

was isolated from C. trachomatis-infected HeLa cells at 21and 36 h postinfection by using the guanidine isothiocya-nate-hot phenol method as described earlier (28), except thatcell extracts were treated with RNase-free DNase (PromegaCorp., Madison, Wis.) prior to proteinase K treatment.Isolated RNA was fractionated on 1% formaldehyde-agarosegels. A ladder of RNA species was used to provide markersfor size calibration. Northern blotting on DBM-paper wascarried out as described earlier (19). Primer extension assaywas carried out as described by Calzone et al. (4). Essen-tially, a 19-mer oligonucleotide primer (5' TCACATCACCGACCGTTGC 3') complementary to the coding strand(comprising the region from nucleotides 750 to 732 of the4.91-kb DNA sequence) was end labeled at the 5' end with[-y-32P]ATP by using T4 polynucleotide kinase. Approxi-mately 20 to 40 ,ug of total RNA was hybridized to the primerin annealing buffer by allowing the heated mixture to coolslowly to 42°C, and then the primer extension was performedwith a reaction solution containing 0.45 U of avian myelo-blastosis virus reverse transcriptase (20,000 U/ml; Pharma-cia LKB Corp.) per ,ul. The samples were analyzed on 6%polyacrylamide-urea gels.

Nucleotide sequence accession number. The nucleotidesequence data reported in this article have been submitted to

GenBank and have been assigned accession numberM80325.

RESULTS

Cloning of the C. trachomatis spc operon DNA. We previ-ously isolated a 3.2-kb Sacl fragment designated pCTJS1from a C. trachomatis gene library that encoded the r-pro-tein CtrL6e (13). The DNA sequence of an internal 1.2-kbXbaI-HindIII fragment designated pCTJS8 revealed thepresence of genes for proteins that are homologous to EcoS8and EcoL18 flanking the CtrL6e. This is reminiscent of thespc operon genes in E. coli. Consequently, we determinedthe sequence of the whole 3.2-kb Sacl fragment of pCTJS1.DNA sequence analysis of the HindIII-SacI fragment adja-cent to the previously sequenced HindIII site (from plasmidpCTJS8) revealed that this region corresponded to the car-boxy terminus of CtrL18e (13). In order to extend thesequence to cover the entire spc region, overlapping DNAfragments were generated through successive subcloningand hybridizations by using appropriate DNA fragments asprobes. A restriction map sequencing strategy and geneorganization of this C. trachomatis r-protein gene cluster areshown in Fig. 1.

Sequence analysis and gene organization. The DNA se-quence as determined on both strands of the entire 5-kbregion is shown in Fig. 2, together with the predicted aminoacid sequences of the encoded proteins. The protein-codingregions were identified by comparison with E. coli r-proteingene sequences by using the FASTP program (27). Allputative r-protein genes in this cluster initiated with ATGcodons and terminated with either TAG or TAA terminationcodons. Also, the intragenic regions in this gene clusterdiffer from one another in both length and sequence.The organization of r-protein genes in C. trachomatis

appears similar to that of the S10 and spc operons of E. coliexcept for the absence of r-proteins CtrS14e and CtrL30e inC. trachomatis. The gene order in the cluster was found tobe CtrL16e, CtrL29e, CtrS17e, CtrL14e, CtrL24e, CtrL5e,CtrL8, CtrL6e, CtrL18e, CtrSSe, and CtrLl5e. Furthersequencing downstream from the CtrLl5e gene revealed anORF whose partial amino acid sequence was similar to thatof the Sec Y protein involved in export pathway. The firstthree genes at the 5' end in this cluster are very similar to thelast three genes of the S10 operon. A 16-bp intragenic regionbetween the end of the CtrS17e coding sequence (SlOoperon) and the beginning of the CtrL14e coding sequence(spc operon) was found and did not include any apparenttranscription termination sequences. Two of the intragenicregions between genes for CtrL29e and CtrS17e and CtrSSeand CtrLl5e have overlapping translational stop and startcodons.

Analysis of the deduced amino acid sequence of r-proteins.Alignments of the amino acid sequences of the 11 r-proteinsof the S10 operon and the spc gene cluster from C. tracho-matis and E. coli, respectively, are shown in Fig. 3. Thepercent sequence identity varies from 57% for CtrL14e to28% for CtrL29e and CtrL24e. Consideration of conserva-tive substitution raises the homology to 77% for CtrL18e andapproximately 50% for CtrS8e. In the case of CtrL18e, thehomology was more marked toward its carboxy-terminalregion. Table 1 shows a comparison of the total numbers ofamino acid residues of each protein from C. trachomatis andE. coli. With the exception of CtrL5e, which is 24 amino acidresidues short, no significant differences were observedamong other r-proteins.

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C. TRACHOMATIS spc r-PROTEIN GENE CLUSTER 1207

0

Sc

2

SR D R

3 4 5

E Ss E XP Xh R E D HScI, i I I .,

H Ha BD H

B HSc R R BII I I I

0.-4-a

------4-9

kb

PCTJSIPCTJHI

PCTJB1

4-4- 4-- 4 - 4-*~ ~ ~~~----.

4-.-4-

0-* 4- -* 4-- -* -. 4--

TqFL-2R --IF-4--- 4

L16iES1L 14 L24 L5 I S L6 18 L5 115 [_ SEC_Y_- s Io--Ii SPo

PCTRKIPCTRK2PCTJS8i

i I PCTRK3

FIG. 1. Restriction map, sequencing scheme, and subcloning strategy of a 4,913-bp C. trachomatis S10-spc gene cluster. Abbreviationsfor the restriction sites are as follows: Sc, SacI; S, SalI; R, RsaI; D, DraI; E, EcoRI; Bc, BclI; Ss, SspI; X, XbaI; Xh, XhoI; P, PstI; H,HindIII; Ha, HaeIII; B, BamHI. The extent and direction of different sequencing runs from the M13-specific universal primers are indicatedby simple arrows, while arrows with solid circles indicate runs from primers synthesized from the sequence determined in the earliersequencing runs. The ORFs identified as r-protein coding regions are boxed, and their directions of initiation of translation are indicated bylarge arrows. Regions corresponding to the S10 and spc operons are labeled on the bottom. The clones named at the far right representplasmid-derived recombinants for either expression or sequence extension studies.

Transcription and promoter studies. To study the temporalregulation of the spc and S10 operon gene transcripts, totalcellular RNA was isolated from C. trachomatis-infectedHeLa cells at 21 and 36 h postinfection and Northern blottedby using a 32P-labeled 0.8-kb HindIII probe. This fragmentencodes the genes for r-protein CtrS5e and part of CtrL18eand CtrLl5e. Only one transcript, approximately 5 kb, wasobserved at both time points (data not shown). To preciselylocate the transcription start site of the spc operon, we

analyzed the product of primer extension experiments byusing a 19-mer synthetic oligonucleotide. The transcriptinitiated at a cytosine residue 162 nucleotides upstream ofthe CtrL14e start site, the first gene of the spc operon (Fig.4). This cytosine residue is designated as + 1 in the sequence.We arbitrarily chose the cytosine instead of thymidine as theprobable start site since there appears to be no difference inthe intensity of bands corresponding to two initiation sites.Inspection of the sequence upstream of the transcriptionstart site identified -10 (TATACT) and -35 (CTGTTG)sequences within r-protein CtrS17e, which represents theend of S10 operon.

Expression and analysis of recombinant proteins. Recom-binants containing fragments of interest from this C. tracho-matis r-protein gene cluster were constructed by subcloningin the vector pUC18; construction was followed by analysisof their protein profiles. The recombinants designated pC-TRK1, pCTRK2, pCTJS8 (13), and pCTRK3, which encodegenes for r-proteins CtrS8e, CtrL5e, CtrL6e and CtrS5e,respectively, were transformed into E. coli NM522. Thecells were harvested, and extracted proteins were resolvedby SDS-15% PAGE. Figure 5 shows the expression ofCtrL6e and CtrL5e gene products after induction with IPTG.

The molecular masses of these proteins as determined bySDS-PAGE were calculated to be 23,000 and 17,000 Da,respectively. Surprisingly, no toxicity of heterologous geneproducts was observed in E. coli. In contrast, cells harboringpCTRK1 and pCTRK3 did not grow well and no additionalgene products were visualized compared with E. coli cellsharboring vector alone. Subsequently, cells harboring therecombinant plasmid pCTJS8 encoding 23-kDa CtrL6e weretested for susceptibility to gentamicin, an aminoglycosideantibiotic. Both the recombinant strain and E. coli contain-ing only pUC18 were highly susceptible to gentamicin; theMICs for each were <0.5 ,ug/ml.

TABLE 1. Comparison of r-proteins of C. trachomatis andE. coli

No. of amino acid residuesProtein

E. coli C. trachomatis

L29 63 72S17 84 83L14 123 122L24 104 111L5 179 155S8 130 133L6 177 183L18 117 123S5 167 165L15 144 144

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GAGCTCCTGATCACTGGGTAGCTGTTGTCCGTCCCGGACGTATTTTATTCGAAGTGGCAAA P D H W V A V V R P G R I L F E V A

ACGTTTCGAAAGAAGATGCTCAGGATGCTTTGAGAAGAGCTGCTGCAAAGTTAGGAATTAN V S K E D A Q D A L R R A A A K L G I

GAACACGATTTGTTAAGCGTGTGGAAAGGGTATAGTATGGGAGCAAAAAAGAATTTATTAR T R F V K R V E R V - M G A K K N L L

end Ctr L16e start Ctr L29eGCGGAGCTTAGAGAGAAGAGTTCTGAAGAGTTGGATGAGTTTATTCGTGATAATAAAAAAA E L R E K S S E E L D E F I R D N K K

GCTCTCTTCGCTTTGCGTGCGGAAGCTGCTTTACAGAATAAAGTTGTGAAAACTCATCAGA L F A L R A E A A L Q N K V V K T H Q

TTTTCTCTGTATAAGAAAAGCATTGCTCGTGCTCTAATAATAAAACAAGAAAAAAAGGGTF S L Y K K S I A R A L I I K Q E K K G

AGAGTCCATGGCTAGTGATGTGAGAGGCCGTAGAAAGACCAAAATTGGTGTAGTAGTCTCR V H G - end Ctr L29e

M A S D V R G R R K T K I G V V V Sstart Ctr S17e

ATCAAAAATGGAAAAAACTGTTGTTGTTCGAGTCGAAAGGGTATACTCGCACCCTCAATAS K M E K T V V V R V E R V Y S H P Q Y

TGCTAAGGTGGTTAGGGATTCTAGCAAGTATTATGCGCATAATGAGTTGGATGTGAAAGAA K V V R D S S K Y Y A H N E L D V K E

AGGTGATACTGTTCGAATCCAAGAGACGCGTCCTTTGTCTAAAACGAAGAGATGGCGGGTG D T V R I Q E T R P L S K T K R W R V

TGTCGGACGTGTAAATTAGTAGTGGTTTAGCAATTATGATCCAGCAAGAAAGTCAGTTAAV G R V N - end Ctr S17e M I Q Q E S Q L

start Ctr L14eAAGTTGCCGATAATACAGGGGCTAAGAAAGTTAAGTGTTTCAAGGTTCTAGGCGGATCTCK V A D N T G A K K V K C F K V L G G S

GTCGACGTTATGCAACGGTCGGTGATGTGATTGTATGCTCTGTAAGAGATATTGAGCCTGR R R Y A T V G D V I V C S V R D I E P

ATAGTTCCGTAAAGAAGGGGGATGTTGTTAAGGCTGTAATCGTACGGACTCGAAACGATAD S S V K K G D V V K A V I V R T R N D

TCCATCGTAAAGATGGTTCTACACTAAGATTCGATACGAATAGTTGTGTAATCATCGATGI H R K D G S T L R F D T N S C V I I D

ATAAAGGCAATCCTAAAGGAACTAGAATTTTTGGGCCTGTAGCAAGGGAGATTCGAGACAD K G N P K G T R I F G P V A R E I R D

GAGGCTTTGTTAAGATTAGCTCTTTGGCTCCCGAGGTGATTTAAAGGTAAGATAGTATGAR G F V K I S S L A P E V I - M

end Ctr L14e startAGAGACGTAGTGTTTGTGTCGGTGACACTGTTTATGTGCTTGCTGGAAACGACAAAGGTAK R R S V C V G D T V Y V L A G N D K GCtr L24eAGCAAGGGAAAGTTTTACGTTGTTTGAAGGATAAGGTTGTTGTTGAAGGAATCAATGTCCK Q G K V L R C L K D K V V V E G I N V

GAGTAAAAAATATTAAACGCTCTCAAGAGAATCCTAAAGGGAAGCGCTTAATATTGAGGR V K N I K R S Q E N P K G K R I N I E

CTCCTCTCCTATCTAACGTACGTAAGTATCGATAATCAGCCTGCTAGACTGTTTGA P L H I S N V R L S I D N Q P A R L F

TCAAAGTTACAGAGAAAGGACGAGAGCTTTGGAATAAGCATTCCGATGGAAGTTCTTQTV K V T E K G R E L W N K H S D G S S S

TATACCGATTGGTAAGAGAGAGAAAGGGTTAATATGAGCAGGTTAAAAAAACTATATACTL Y R L V R E R K G - end Ctr L24e

GAAGAGATAAGAAAGACTCTTCAAGATAAGTTTCAGTATGAAAATGTAATGCAAATCCCTstart Ctr L5e M Q I P

GTTCTTAAGAAGATCGTAATAAGCATGGGGCTTGCAGAGGCTGCAAAGGATAAAAACCTTV L K K I V I S M G L A E A A K D K N L

TTCCAGGCTcATTTAGAGGAATTGGCGGTTATCTCTGGTCAAAAACCTTTGGTAACAAGAF Q A H L E E L A V I S G Q K P L V T R

GCTAAAAACTCTATCGCAGGCTTCAAGTTACGAGAGGGTCAGGGCATCGGAGCAAAAGTCA K N S I A G F K L R E G Q G I G A K V

ACTCTACGTGGAATCCGTATGTATGACTTTATGGACCGTTGCAAATATTGTCTCCCCAT L R G I R M Y D F M D R F C N I V S P

AGAATTCGAGACTTTAGAGGATTCTCTTGTAAAGGAGATGGACGAGGATGTTATTCCCTTR I R D F R G F S C K G D G R G C Y S L

GGTTTAGATGATCAGCAAATCTTTCCTGAAGTTGATTTAGATGCTGTTAAACGATCTCAGG L D D Q Q I F P E V D L D A V K R S Q

GGAATGAATATTACTTGGGTAACTACAGCACAAACCGATGCGGAGTGCCTTACCTTGTTAG M N I T W V T T A Q T D A E C L T L L

GAGTGTATGGGCTTGCGTTTCAAGAAGGCTCAATAAGGGAGATGTAGGTCGGTATGGGAAE C M G L R F K K A Q -end Ctr L5e M G

startTGACGAGTGATTCAATCGCAAATTTATTGACACGGATTCGAAATGCTTTGATGGCAGAGCM T S D S I A N L L T R I R N A L M A ECtr S8eATTTGTACATTGATATCGAGCATAGTAAATGCTTGAAGCAATAGTAAGAATTCTCAAGCH L Y I D I E H S K M L E A I V R I L K

AGCACGGGTTCATTGCTCACTTTTTAGTAAAAGAAGAAAATCGCAAAAGACTAATGAGAGQ H G F I A H F L V K E E N R K R L M R

TCTTTTTGCGGTACGGGGAAGATCGTAGACCTGTGATTCATGCTCTTAAGCGTGTGTCTAV F L R Y G E D R R P V I H A L K R V S

AACCTTCTAGAAGGGTTTATGTTTCTGCAGCAAAAATTCCTTATGTATTTGGAAATATGGK P S R R V Y V S A A K I P Y V F G N M

GTATTGCCGTTCTTTCGACTCCTCAAGGGGTTTTAGAAGGCTCTGTAGCAAGGGCTAAGAG I A V L S T P Q G V L E G S V A R A K

60 ATGTTGGCGGCGAATTGCTTTGTTTGGTTTGGTAGCAAATTAAAAGATTAGGACGGTAACN V. - - L I L VC1 -e--A er+- 0

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

1920

1980

2040

2100

2160

2220

2280

N V G G E; L L C L v w - end Ctr S*oe

GAATGTCTCGTAAAGCTCGAGACCCTATTGTGCTTCCTCAAGGCGTAGAGGTCTCTATTCM S R K A R D P I V L P Q G V E V S Istart Ctr L6e

AAAATGATGAAATCTCAGTAAAAGGTCCTAAAGGGTCTTTGACGCAGGTATTGGCTAAAGQ N D E I S V K G P K G S L T Q V L A K

AAGTTGAGATTGCCGTTAAAGGTAATGAGGTGTTTGTTGCTCCTGCGGCTCACGTTGTAGE V E I A V K G N E V F V A P A A H V V

ACAGACCTGGTCGTATGCAAGGGCTTTATTGGGCCTTAATAGCAAATATGGTCAAAGGTGD R P G R M 0 G L Y W A L I A N M V K G

TCCATACTGGATTTGAGAAGCGTTTAGAAATGATCGGAGTCGGCTTCAGAGCTGCAGTACV H T G F E K R L E M I G V G F R A A V

AAGGGTCCTTGTTAGATCTGTCAATAGGGGTTTCTCACCCTACAAAAATGCCTATTCCTAQ G S L L D L S I G V S H P T K M P I P

CGGGATTAGAAGTCTCTGTTGAGAAAAACACATTGATCTCCATTAAAGGTATCAATAAGCT G L E V S V E K N T L I S I K G I N K

AGTTAGTTGGAGAATTTGCGGCTTGTGTTCGTGCAAAACGCCCTCCAGAACCATACAAAGQ L V G E F A A C V R A K R P P E P Y K

GTAAAGGAATTCGTTACGAAAACGAATATGTTCGTCGTAAGGCTGGGAAAGCAGCGAAAAG K G I R Y E N E Y V R R K A G K A A K

CTGGTAAAAAATAGAGGGTAAAGTAGAGTCGAACTATGGAAAGCTCTTTATATAAGAAAAT G K K - end Ctr L6e M E S S L Y K K

start Ctr L18eCTTCGGGGAAAGCTCGTAGAGCTTTAAGAGTGCGGAAAGCCTTAAAGGGATGTTCTTTAAT S G K A R R A L R V R K A L K G C S L

AGCCCAGATTATCCGTTGTAAAGACAAATAAGCATGTTTATGTGCAGCTGATTGATGATGK P R L S V V K T N K H V Y V Q L I D D

TTGAAGGGAAAACTTTAGCATTTATTTCAACTTTGGCTAAGGTTGCAAAAACTTCTGGATV E G K T L A F I S T L A K V A K T S G

TAACTAGAAAAAATCAGGATAATGCCAAAGCTTTGGGAATAAAAATTGCTGAATTAGGGAL T R K N Q D N A K A L G I K I A E L G

AAGGCCTTCAAGTAGATCGAGTTGTTTTCGATCGAGGAGCTCATAAGTATCATGGTGTAGK G L Q V D R V V F D R G A H K Y H G V

TAGCTATGGTTGCTGATGGAGCCAGAGAGGGTGGATTAcAGTTTTAATGAAGGTTTAGATV A M V A D G A R E G G L Q F - end Ctr L18e

AATGACGCTATCAAGAAATTCTCATAAGGAAGATCAGCTGGAAGAGAAGGTTCTCGTCGTM T L S R N S H K E D Q L E E K V L V Vstart Ctr S5e

CAACCGTTGTTGTAAGGTTGTTAAAGGAGGCCGTAAGTTTAGTTTTTCTGCGCTTATTTTN R C C K V V K G G R K F S F S A L I L

V G D R K G R L G F G F A K A N E L T D

TGCCATCCGTAAAGGTGGGGATGCTGCTCGAAAAAATCTTGTCTCTATCAATTCTCTTGAA I R K G G D A A R K N L V S I N S L E

GGGAGGATCTATTCCTCATGAGGTTCTTGTCAATCATGATGGAGCAGAGCTTCTGTTAAAG G S I P H E V L V N H D G A E L L L K

ACCTGCTAAGCCAGGAACCGGAATCGTTGCAGGATCTCGTATTCGGTTGATTTTAGAGATP A K P G T G I V A G S R I R L I L E M

GGCCGGGGTAAAGGACATTGTAGCAAAGAGTTTAGGATCCAATAATCCTATGAATCAGGTA G V K D I V A K S L G S N N P M N Q V

TAAAGCGGCTTTTAAAGCTCTCCTGACACTCTCTTGTAAAGATGATATTATGAAAAGGAGK A A F K A L L T L S C K D D I M K R R

AGCCGTTATCAATGATTAAGTTAGAGTGTTTACAAGATCCTTCGCCTCGTAAGCGAAGAAA V I N D - end Ctr S5e

M I K L E C L Q D P S P R K R Rstart Ctr L15e

CGAAACTCTTGGGCCGAGGACCTTCTTCTGGTCACGGGAAAACAAGTGGTCGAGGACACAT K L L G R G P S S G H G K T S G R G H

AAGGGGACGGTAGCCGTTCTGGATACAAGAGACGTTTCGGATATGAAGGGGGAGGCGTACK G D G S R S G Y K R R F G Y E G G G V

CTTTATACAGAAGAGTTCCTACACGAGGATTTTCTCATAAACGCTTTGATAAATGTGTTGP L Y R R V P T R G F S H K R F D K C V

AAGAAATCACAACACAACGTTTGAATGAGATTTTTGACAATGGCGCAGAAGTATCTTTGGE E I T T Q R L N E I F D N G A E V S L

AAGCTTTAAAAGAAAGAAAAGTTATCCATAGAGAGACTTCTCGTGTTAAAGTAATCCTTAE A L K E R K V I H R E T S R V K V I L

AAGGAGCTCTGGATAAGAAATTAGTCTGGAAAGATGCTGCAATAGTGCTGTCAGAAGGAGK G A L D K K L V W K D A A I V L S E G

TAAAAAGTCTTATCGAGGCTGTTTAACTAGAACTTTTAGGTAAAGTTTATGGCTACATTGV K S L I E A V - end Ctr L15e M A T L

start Ctr Sec YeCGACAAGTGTTTTCGATTTCCGAACTGCGACAAAAAATATTTTTcAcATTTTCCTTGCTTR Q V F S I S E L R Q K I F F T F S L L

GCATTATGTAGAATCGGGGTGTTTATCCCTGTGCCTGGAATTAACGGAGACCGCGCCGTAA L C R I G V F I P V P G I N G D R A V

GCCTACTTTAACCAATTGCTGGGGTCTAGCCGGGGTTTGmCAGTTAGCTGACATTTTTA Y F N Q L L G S S R G L F Q L A D I F

TCTGGGGGAGCTTTTGCTCAAATGACGGTAATAGCTCTTGGAGTTGTTCCGTACATCTCGS G G A F A Q M T V I A L G V V P Y I S

GCTTCAATCATTGTACAGCTTCTTGTCGTCTTTATGCCGACTCTGCAAAGAGAAATGCGAA S I I V Q L L V V F M P T L Q R E M R

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C. TRACHOMATIS spc r-PROTEIN GENE CLUSTER 1209

E S P D Q G K R K L G R M T R L F T L V

L A C V Q S L L F A K F A L R M N L V V

CCAGGGATTGTTGCCAGCAATGTTGTCCTTAAAGCTGTTTGGGGTGCCTTGGGTATTT 4740P G I V L P A M L S L K L F G V P W V F

TATTTGACAACTGTTGTGrATGACAACAGGGACTCTrIACTTATGTGTGGTGAG 44800Y L T T V V V M T T G T L L L M W V G E

CAAATATCTGACAAAGGGATTGGTAATGGAATCAGTTTGATCATTACTCTCGGGATATTA 4860Q I S D K G I G N G I S L I I T L G I L

GCCTCTTTTCCTTCCGTAGGGTCTATATTTAACAAGTTAAATTTGGATCC 4913A S F P S V L G S I F N K L N L D

FIG. 2. The complete gene sequence of the C. trachomatisS10-spc operon region. The predicted amino acid sequence for eachgene is given in single-letter code below the DNA sequence, and theORF borders are indicated below the amino acid sequence. Thedashes represent the stop codons. The sequence for nucleotides2029 to 3222 was reported previously (13). The numbers to the rightof each row refer to nucleotide positions.

DISCUSSION

We have characterized a 5-kb region of the C. trachomatisgenome that is comparable to the promoter proximal regionof the E. coli spc r-protein operon (5). Our results indicate ahigh degree of conservation among r-protein structure andorganization despite the evolutionary distance between E.coli and C. trachomatis (32, 35, 45). The C. trachomatis spcoperon contains the r-protein genes for CtrL14e, CtrL24e,CtrL5e, CtrS8e, CtrL6e, CtrL18e, CtrS5e, and CtrL15e aswell as Sec Y, along with the adjacent genes for r-proteinsCtrL16e, CtrL29e, and CtrS17e. The 5' region of this genecluster is also similar to the last three genes of E. coli S10operon, which precedes the spc operon (49). This similaritysuggests that basic organization of the gene cluster wasestablished before divergence of C. trachomatis and E. coliin evolution. This hypothesis is consistent with the presenceof overlapping translational stop and start codons in two ofthe intragenic regions in C. trachomatis compared with threeoverlapping regions in E. coli (5, 49) and four in the M.capricolum spc r-protein gene cluster (34). Surprisingly,there are no stop or start codons in the archaebacterium M.vannielii despite strong conservation among spc r-proteingene clusters with respect to gene composition and organi-zation (2). In chlamydiae, however, the genes for CtrS14eand CtrL30e are absent from this gene cluster. Whetherthese two genes are located in a separate region of thechromosome or were lost completely during evolution is notclear from this study. On the basis of the evidence thatEcoS14 and Bacillus stearothermophilus BstS14e are essen-tial for in vitro reconstitution of functionally active 30Ssubunits (16), it is tempting to anticipate that the CtrS14egene is located distantly in the chromosome. However, thegene homolog for EcoL30 is also absent in M. capricolum(34). There is nearly 75% bias toward A and U in position 3of codons in the cluster. This codon usage pattern is similarto one reported earlier by us (13) and is different from thosein E. coli (49%) and M. capricolum (91%). The overallcalculated A+T contents of the genes in the cluster amongC. trachomatis, E. coli, and M. capricolum are 60, 48, and69%, respectively. These percents are consistent with thecalculated averages for the whole genomes.

In E. coli there is a 162-bp intragenic region betweenEcoS17, the last gene of S10 operon, and EcoL14, the firstgene of the spc operon (5). This space contains rho-indepen-dent termination signals for the upstream S10 operon as wellas promoters and initiation signals for the downstream spc

operon (24). In contrast, C. trachcmatis contains only a16-bp spacer between the two operons. No transcriptionsignals were found in this intragenic region, suggesting thatthe transcription of spc region may initiate from an upstreampromoter. Fusion of the S10 and spc operons into a singleoperon has been reported to occur in M. capricolum, whichcontains a 15-bp spacer. The transcription in T. aquaticusand the archaebacterium M. vannielii as well as Halobacte-rium marismortui begins at the S10 operon and most likelytraverses into the spc operon (1, 2, 18). Northern blotanalysis was used to evaluate whether chlamydial spc andS10 operons are transcribed as single or separate transcrip-tional units. While an -5-kb transcript hybridized to probesfrom spc operon genes, a different transcript size wasobtained when an identical blot was hybridized to S10operon genes (unpublished data), supporting independenttranscription of the spc and S10 regions. Further evidencefor the presence of an internal promoter regulating the spcoperon was obtained by analysis of the transcription initia-tion site by using the primer extension technique. Theapparent promoter shows some similarities to the E. coliconsensus promoter sequence recognizing the &0 subunit ofRNA polymerase (36). The -10 sequence shows identityamong 5 of 6 positions, while the -35 region reveals identityamong 2 of 6 positions and includes a highly conserved TTGsequence at its 5' end rather than the conventional 3' end ofthe -35 region. The lack of homology among the variouspromoter regions in chlamydiae has made it difficult to definea consensus promoterlike sequence in this organism and alsoaccounts for its failure to initiate any efficient transcription inE. coli (38, 41). The ability of the chlamydial spc operonpromoter, along with the promoters for other recently iden-tified genes, to transcribe in an E. coli system is currentlybeing studied.The synthesis of r-proteins in prokaryotes is tightly coor-

dinated and stoichiometrically balanced with the assembly ofmature ribosomes (25, 26, 33, 49). Such a coordinatedregulation is mediated by the autogenous feedback mecha-nism whereby certain r-proteins, when unbound by rRNA,prevent the translation of their own mRNA (11, 48). How-ever, the mechanism of transcriptional regulation differsamong E. coli r-protein operons (24-26). Recently, onegroup reported the regulation of spc operon r-genes bytranslational coupling in which the product of EcoS8 genebinds to the spc operon mRNA near the beginning of EcoL5,leading to repression of EcoL5 translation and subsequentlythe downstream spc operon (31). The same group (15),however, failed to observe similar structures in the B.subtilis gene cluster, prompting them to speculate that thetranslational feedback regulation system for control of r-pro-tein gene expression is fairly recent in evolution. Our ownsearches in and around CtrLSe protein-coding sequence ofthe mRNA that would form a structure similar to the EcoS8target site failed to identify one. These studies suggest theexistence of an alternate pathway for spc operon autoregu-lation in chlamydiae. Similar observations have been madefor T. aquaticus (18).

Overproduction of some r-proteins in E. coli can lead tohost toxicity or cell death because of the imbalance instoichiometry of ribosome synthesis or autogenous regula-tion. As discussed, the endogenous r-protein genes aresubject to feedback inhibition to minimize any reduction inribosome activity. Recently, we reported the substitution ofCtrL6e into E. coli ribosomes without any deleterious effectdespite its overproduction (13). To study the effect of otherchlamydial r-protein gene products on E. coli growth and

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Ctr S5e MTLSRNSHKEDQLEEKVLVVNRCCKVVKGGRKFSFSALILVGDRKGRLGFGFAKANELTD

Eco L16

Ctr L16e

Eco L16

ML0PKRTKFRKMHKGRNRGLAQGTDVSFGSFGLKAVGRGRLTARQIEAARRAMTRAVKRQ

GKIWIRVFPDKPITEKPLAVRMGKGKGNVEYWVALIQPGKVLYEMDGVPEELAREAFKLA

Ctr L16e AAKLGIRTRFVKRVERV

Eco L16 AAKLPIKTTFVTKTVM

Ctr L29e MGAKKNLLAELREKSSEELDEFIRDNKKALFALRAEAALQNKVVKTHQFSLYKKSIARAL

Eco L29 MKAKELREKSVEELNTELLNLLREQFNLRMQAA-SGQLQQSHLLKQVRRDVARVK

Ctr L29e IIKQEKKGRVHG

Eco L29 TLLNEKAGA

Ctr S17e MASDVRGRRKTKIGVVVSSKMEKTVVVRVERVYSHPQYAKVVRDSSKYYAHNELDVKE-G

Eco S17 TDKIRTLQGRVVSDKMEKSIVVAIERFVKHPIYGKFIKRTTKVHVHDENNECGIG

Ctr S17e DTV-RIQETRPLSKTKRWRVVGRVN

Eco S17 DVVCEIRECRPLSKTKSWTLVRVVEKAVL

Ctr L14e MIQQESQLKVADNTGAKKVKCFKVLGGSRRRYATVGDVIVCSVRDIEPDSSVKKGDVVKA

Eco L14 MIQEQTMLNVADNSGARRVMCIKVLGGSHRRYAGVGDIIKITIKEAIPRGKVKKGDVLKA

Ctr L14e VIVRTRNDIHRKDGSTLRFDTNSCVIIDDKG-NPKGTRIFGPVAREIRDRGFVKISSLAP

Eco L14 VVVRTKKGVRRPDGSVIRFDGNACVLLNNNSEQPIGTRIFGPVTRELRSEKFMKIISLAP

Ctr L14e EVI

Eco L14 EVL

Ctr L24e

Eco L24

Ctr L24e

Eco L24

Ctr L5e

Eco L5

Ctr L5e

MK8RRSVCVGDTVYVLAGNDKGKQGKVLRCL-KDKVVVEGINVRVKNIKRSQE-NPKGKRI

MAAKIRRDDEVIVLTGKDKGKRGKVKNVLSSGKVIVEGINLVKKHQKPVPALNQPGGIV

NIEAPLHISNVRLSIDNQPARLFVKVTEKGRELWNKHSDGSSSLYRLVRERKG

EKEAAIQVSNVAIFNAATGKADRVGFRFEDGKKVRFFKSNSETIK

MQIPVLKKIVISMGLAEAAKDKNLFQAHLEELAVI

MAKLHDYYKDEVVKKLMTEFNYNSVMQVPRVEKITLNMGVGEAIADKKLLDNAAADLAAI

SGQKPLVTRAKNSIAGFKLREGQGIGAKVTLRGIRMYDFMDRFCNIVSPRIRDFRGFSCK

Eco L5 SGQKPLITKARKSVAGFKIRQGYPIGCKVTLRGEREWEFFERLITIAVPRIRDFRGLSAK

Ctr L5e

Eco L5

Ctr L5e

Ctr S8e

Eco S8

G-DGRGCYSLGLDDQQIFPEVDLDAVKRSQGMNITWVTTAQTDAECLTLLECMGLRFKKA

SFDGRGNYSMGVREQIIFPEIDYDKVDRVRGLDITITTTAKSDEEGRALLAAFDFPFRK

MGGMTSDSIANLLTRIRNALMAEHLYIDIEHSKMLEAIVRILKQHGFIAHFLVKEENRKRL

MSMQDPIADMLTRIRNGQAANKAAVTMPSSKLKVAIANVLKEEGFIEDFKV-EGDTKPE

Eco S5 AHIEKQAGELQEKLIAVNRVSKTVKGGRIFSFTALTVVGDGNGRVGFGYGKAREVPA

Ctr S5e AIRKGGDAARKNLVSINSLEGGSIPHEVLVNHDGAELLLKPAKPGTGIVAGSRIRLILEMIdentity(%) ::

Eco S5 AIQKAMEKARRNMINV-ALNNGTLQHPVKGVHTGSRVFMQPASEGTGIIAGGAMRAVLEV38

Ctr S5e AGVKDIVAKSLGSNNPMNQVKAAFKALLTLSCKDDIIMKRRAVIND

Eco S5 AGVHNVLAKAYGSTNPINVVRATIDGLENMNSPEMVAAKRGKSVEEILGK

Ctr L15e

Eco L15

Ctr L15e RVPTRGFSHKRFDKCVEEITTQRLNEIFDNGAEVSLEALKERKVIHRETSRVKVILKGAL288

Eco L15 RLPKFGFTSRK-AAITAEIRLSDLAKV--EGGVVDLNTLKAANIIGIQIEFAKVILAGEV

Ctr L15e DKKLVWKDAAIVLSEGVKSLIEAV

Eco L15 TTPVT--VRGLRVTKGARAAIEAAGGKIEE

FIG. 3. Comparison of the deduced amino acid sequence of C.trachomatis (Ctr) S10-spc with that of the E. coli (Eco) r-protein

41 cluster. The single-letter amino acid code is used. Dashes

indicate gaps introduced to optimize alignment, while colons indi-cate identical amino acids. Numbers to the right refer to the percentamino acid identity between individual Ctr and Eco r-proteins.

57

survival, we transformed E. coli with plasmids containinggenes for CtrL5e, CtrL6e, CtrS5e, and CtrS8e. Only thegene products from CtrL5e and CtrL6e were visible in vitrowhen resolved by SDS-PAGE, suggesting an unstable natureof the CtrS5e and CtrS8e transcripts and/or their geneproducts in E. coli. B. stearothernophilus BstS5e also

28 appears to be toxic to E. coli in spite of the fact that

A , A T r 1 o

52 _

TAG

> T

R,.

T,.

TGC

-T

37

Ctr S8e MRVFLRYGEDRRPVIHALKRVSKPSRRVYVSAAKIPYVFGNMGIAVLSTPQGVLEGSVAR

Eco S8 LELTLKYFQG-KAVVESIQRVSRPGLRIYKRKDQLPKVMAGLGIAVVSTSKGVMTDRAAR

Ctr S8e AKNVGGELLCLVW

Eco S8 QAGLGGEIICYVA

Ctr L6e

Eco L6

Ctr L6e

Eco L6

Ctr L6e

Eco L6

Ctr L6e

Ctr L18e

Eco L18

Ctr L18e

Eco L18

BMSRVAKAPVVVPAGVDVKINGQVITIKGKNGELTRTLNDAVEVKHADNTLTFGPRDGYAD

RPGRMQGLYWALIANMVKGVHTGFEKRLEMIGVGFRAAVQGSLLDLSIGVSHPTKMPIPT

GWA-QAGTARALLNSMVIGVTEDFTKKLQLVGVGYRAAVKGNVINLSLGFSHPVDHQLPA

GLEVSVEKNTLISIKGINKQLVGEFAACVRAKRPPEPYKGKGIRYENEYVRRKAGKAAKT

GITAECPTQTEIVLKGADKQVIGQVAADLRAYRRPEPYKGKGVRYADEVVRTKEAKKK

GKK

MESSLYKKTSGKARRALRVRKALKGCSLKPRLSVVKTNKHVYVQLIDDVEGKTLAFISTL

MDKKSARIRRATRARRKLQELG-ATRLWHRTPRHIYAQVIAPNGSEVLVAASTV

AKVAKTSGLTRKNQDNAKALGIKIAELGKGLQVDRVVFDRGAHKYHGVVAMVADGAREGG

EKAIAEQLKYTGNKDAAAAVGKAVAERALEKGIKDVSFDRSGFQYHGRVQALADAAREAG

Ctr L18e LQF

Eco L18 LQF

420 -35 -10CATCRRRRRTGGRRRRRRCTGTTGTTGTTCGRGTCGRRRGGGTRTaCTCG

470 + 1

CRCCCTCRRTRTGCTRRGGTGGTTRGGGRTTCTRGCRRGTRTTRTGCGCR

FIG. 4. Identification of the transcription start site of the spcoperon by primer extension analysis. (A) The 5' end of the transcriptis a doublet located 162 nucleotides upstream of the CtrL14e

36 translation codon. A 19-mer synthetic oligonucleotide was annealedto total RNA from uninfected 36-h-old HeLa cells (lane 1) or

infected cells (lane 2). The DNA sequencing ladders derived fromthe same primer are shown with GAT and C reactions from left toright. A complementary sequence is written on the right side of thesequencing ladder. Two arrows indicate the start site. (B) Nucleo-tide sequence of the spc operon promoter region. The transcriptionstart site is indicated as +1. Both -10 and -35 promoter sequencesare labeled above the DNA sequence, and their nucleotides are

underlined.

1210 KAUL ET AL. J. BACTERIOL.

40

MIKLECLQDPSPRKRRTKLLGRGPSSGHGKTSGRGHKGDGSRSGYKRRFGYEGGGVPLYR

MRLNTLSPAEGSKKAGKRLGRGIGSGLGKTGGRGHKGQKSRSGGGVRRGFEGGQMPLYR

38

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C. TRACHOMA4TIS spc r-PROTEIN GENE CLUSTER 1211

M 1 2 3 4 5

94

67

43

30

20

14

FIG. 5. Identification and expression of C. trachomatis r-pro-teins in E. coli. The Coomassie blue-stained SDS-PAGE analysis ofpolypeptides synthesized by the vector pUC18 and constructedplasmids in the host NM522 cells is shown. Lanes 1 to 5 representpUC18, pCTRK2 harboring CtrLSe, pCTRK1 harboring CtrS8e,pCTJS8 harboring CtrL6e, and pCTRK3 harboring CTrSSe, respec-tively. Approximately equal amounts of protein were loaded on eachlane. Standard molecular weight markers are shown in lane M.

endogenous overproduction of EcoS5 has not led to anytoxicity (33). The product of CtrS8e could be lethal by virtueof its characteristic binding to the translational repressor inE. coli.

Unlike many gram-negative bacteria, chlamydiae are com-pletely resistant to the aminoglycoside gentamicin. In E.coli, alterations in EcoL6 have been reported to causegentamicin resistance, invoking it as a possible target site(3). These studies prompted us to look for such a situationwith CtrL6e, the chlamydial EcoL6 homolog. E. coli harbor-ing the recombinant pCTJS8 encoding CtrL6e were highlysusceptible to gentamicin, suggesting that the intrinsic ami-noglycoside-resistant locus in C. trachomatis is located atsome other place in the genome. Alternatively, the resis-tance could be ascribed simply to aminoglycoside imperme-ation of elementary bodies because of their highly disulfide-linked outer membrane and a protective intracellular hostenvironment which reticulate bodies inhabit.

Efforts to understand the mechanism of operon regulationin chlamydiae, especially its transcription and terminationsignals, are under way. We are currently studying the effectof overexpressed proteins in an in vitro transcription-trans-lation system in order to look for specific repressor mole-cules. These studies will be helpful in understanding themechanisms of ribosome stoichiometry.

ACKNOWLEDGMENTS

We thank W. M. Wenman for generous support and helpfuldiscussions, K L. Roy for advice on oligonucleotide primers, F.Cooper for manuscript preparation, and S. Vinh for photography.

This work was supported by a grant from the Canadian MedicalResearch Council (MA7951) and the Canadian Bacterial Diseases

Network. G.J.G. was a graduate student and N.R.K. was a summerstudent of the Alberta Heritage Foundation for Medical Research.

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