Microbial Pathogenesis 1996; 20: 297–307

Cloning, sequencing and expression of the CAMPfactor gene of Streptococcus uberis∗

Min Jiang,1 Lorne A. Babiuk1,2 and Andrew A. Potter2,†

1Department of Veterinary Microbiology and 2Veterinary Infectious DiseaseOrganization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N5E3

(Received September 1, 1995; accepted in revised form December 19, 1995)

Jiang, M. (Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon,Saskatchewan, Canada S7N 5E3), L. A. Babiuk and A. A. Potter. Cloning, sequencing andexpression of the CAMP factor gene of Streptococcus uberis. Microbial Pathogenesis 1996;20: 297–307.

The gene coding for the CAMP factor from a strain of Streptococcus uberis (ATCC 9927)was cloned in Escherichia coli. Chromosomal DNA from Streptococcus uberis was usedto construct a gene library in plasmid pTZ18R and six CAMP-reaction positive clones wereobtained from a total of 10 000 transformants. One clone, pJLD21, was subcloned and theCAMP factor gene was located in a 3.2 kb BamHI fragment. The nucleotide sequence ofStreptococcus uberis CAMP factor gene was determined and the deduced amino acidsequence is highly homologous to the corresponding Streptococcus agalactiae protein.Immunoblot analysis revealed that the recombinant strain pJLD21 expressed a proteinwith a molecular weight of 28 000. Antibodies raised against purified Streptococcus uberisCAMP factor cross-reacted with Streptococcus agalactiae protein B.

1996 Academic Press Limited

Key words: Streptococcus uberis; CAMP factor; gene cloning; gene sequencing; primerextension.

Introduction

Streptococcus uberis is an important cause of mastitis in dairy cattle and isresponsible for about 20% of all clinical cases.1–3 Since antimicrobial treatment isgenerally ineffective in treating S. uberis mastitis, the development of controlmeasures must be based on an understanding of virulence factors and protectiveantigens involved in invasion and protection of the mammary gland.4–6 It is knownthat some S. uberis strains can produce a hyaluronic acid capsule,7 hyaluroni-dase,8 R-like protein,9 and a chemolysin, the CAMP factor.10 However, verylittle is known of their roles in pathogenicity.

The effect of CAMP factor was first described by Christie et al. in 1944.11 Theseauthors found that group B streptococci (S. agalactiae) produced a distinct zoneof complete haemolysis when grown near the diffusion zone of the Staphylococcusaureus beta-toxin (sphingomyelinase). This phenomenon was called CAMP

† Author to whom correspondence should be addressed.∗ Published with the consent of the director of the Veterinary Infectious Disease Organization as

Journal Series no. 201.

0882–4010/96/050297+11 $18.00/0 1996 Academic Press Limited

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reaction and the compound for this reaction was characterized as the CAMPfactor, an extracellular protein with a molecular weight of 23 50012 and a pI of8.9.13 The amino acid sequence was determined by Ruhlmann et al.14

The mechanism of the CAMP reaction was disclosed by the work of Bernheimeret al.,12 Sterzik et al.,15,16 and Fehrenbach et al.17,18 The CAMP factor has lytic actionon a variety of target cells in addition to sheep and bovine erythrocytes andartificial membranes in which membrane phospholipids and sphingomyelin werepreviously hydrolysed by phospholipase or sphingomyelinase.

The role of CAMP factor in pathogenicity is unclear, but the followingobservations suggest that it is a virulence determinant. A partially purified CAMPfactor from S. agalactiae was lethal to rabbits when injected intravenously10;intraperitoneal injection of purified CAMP factor into mice significantly raised thepathogenicity of sublethal dose of group B streptococci17; and, like protein A ofS. aureus, CAMP factor can bind the Fc sites of immunoglobulins and was thereforedesigned protein B.19

In addition to group B streptococci and S. uberis, a few other bacteria, includingListeria monocytogenes and Listeria seeligeri,20 Aeromonas sp.,21 Rhodococcusequi,22 A. pleuropneumoniae23 and certain Vibro spp.24 produce reactions similarto the CAMP effect. The CAMP factor genes of group B streptococci and A.pleuropneumoniae have been cloned and expressed in Escherichia coli.25,26

To study the CAMP factor of S. uberis and compare it with those of S. agalactiaeand other bacteria, we cloned, sequenced and expressed the CAMP factor geneof S. uberis.

Results

Cloning and expression of the S. uberis CAMP factor gene

Chromosomal DNA of S. uberis (ATCC 9927) was partially digested with Sau3AIand size fractionated in a sucrose gradient; from this, 2 to 5 kb DNA fragmentswere recovered. The ends of these fragments were partially filled in with dGTPand dATP and ligated into pTZ18R which was cut with SalI and partially filled inwith dTTP and dCTP. Following transformation of E. coli JF1754 competent cells,clones expressing the CAMP factor gene were identified on blood plates withampicillin and beta-toxin on the surface. Six clones from a total of 10 000 werephenotypically haemolytic and each one mediated a distinct CAMP reaction. Oneof them, containing recombinant plasmid pJLD21, was selected for further studyand its CAMP reaction is shown in Fig. 1.

Plasmid pJLD21 contained a 5.2 kb DNA insert and the CAMP factor gene, cfu,was localized within a 3.2 kb BamHI fragment in the subclone pJLD21-2 (Fig. 2).

To study the expression of the recombinant CAMP factor, SDS-PAGE analysisof supernatant proteins from Cfu+ E. coli JF1754 (pJLD21) and host E. coliJF1754 (pTZ18R) was performed (Fig. 3A). Compared to the vector control, nodistinguishable band was observed in the lane containing supernatant from theCfu+ clone, indicating that either expression was at a very low level or the proteinwas not secreted efficiently. To identify the CAMP factor encoded by pJLD21,proteins separated by SDS-PAGE were transferred to a nitrocellulose membraneand immunoblotted using mouse antiserum against purified S. uberis CAMPfactor (Fig. 3B). The Cfu+ E. coli clone carrying pJLD21 expressed a protein withmolecular weight of 28 000 (lane 2), similar to the native CAMP factor of S. uberis(lane 1).

CAMP factor gene of Streptococcus uberis 299

Fig. 1. CAMP reaction of S. uberis and recombinant E. coli clones. CAMP reaction was done on abase no. 2 blood plate as described in Materials and methods. Vertical streak: S, Staphylococcusaureus. Horizontal streak: 1, S. uberis; 2, E. coli JF1754 (pTZ18R) (negative control); 3, E. coli JF1754(pJLD21) (CAMP-positive recombinant); 4, E. coli JF1754 (pJLD21-2) (CAMP-positive subclone).

Nucleotide sequence of S. uberis CAMP factor gene

To obtain the nucleotide sequence of S. uberis CAMP factor gene, each of theEcoRI, HindIII, HincII and SacI fragments of pJLD21-2 was individually cloned intopTZ18R. Fragments were sequenced in both orientations as shown in Fig. 2. Thecomplete DNA sequence is presented in Fig. 4. An open reading frame beginningwith an ATG codon located at positions 157 to 159 and terminating with the TAAstop codon at positions 925 to 927 was found which could encode a 256-residuepolypeptide with a calculated molecular weight of 28 363. Analysis of the N-terminus of the predicted sequence showed a secretory signal sequence, with apotential cleavage site between residue 28 and 29. A purine-rich sequenceAAGAGG, which serves as a ribosome binding site in E. coli27 was found in frontof the ATG start codon.

Primer extension analysis

To localize the start site of transcription and the promoter region of the CAMPfactor gene, primer extension analysis of RNA from S. uberis (ATCC 9927), E.coli JF1754(pJLD21) and E. coli JF1754(pTZ18R) was done by using a syntheticoligonucleotide complementary to the DNA sequence from position 201 to 184.A strong primer extension product corresponding to base 91 (‘T’) was identifiedfrom both S. uberis and E. coli JF1754(pJLD21), but not from E. coli JF1754(pTZ18R)(Fig. 5). This data indicates that there is a major transcript of S. uberis CAMPfactor gene initiated with an ‘A’ residue (+1 in Fig. 4). Both −10 and −35

M. Jiang et al.300

1 Kb

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PLASMID CAMPREACTION

pJLD21

pJLD21-1

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probe

Fig. 2 Restriction enzyme maps of recombinant plasmid pJLD21 and the subclones. Lines indicateS. uberis insert DNA, while boxes represent the multiple cloning sites of vector pTZ18R. The CAMPactivities of recombinant plasmid pJLD21 and its derived subclone pJLD21-1, pJLD21-2 are indicatedon the right (+, CAMP reaction positive; −, CAMP reaction negative). The small horizontal arrowsrepresent start points and directions of sequencing experiments. The probe fragment used for Southernblot experiments is indicated by the large arrow. The bar at the bottom indicates the location of theopen reading frame of CAMP factor gene of S. uberis (cfu).

Fig 3. SDS-PAGE and immunoblot analysis of the S. uberis and S. agalactiae CAMP factors. (A)Coomassie blue-stained 12% polyacrylamide-SDS gel. Lane 1, partially purified S. uberis CAMP factor;lane 2, supernatant of E. coli JF1754 (pJLD21) (CAMP positive); lane 3, supernatant of E. coli JF1754(pTZ18R) (CAMP negative); lane 4, supernatant of S. agalactiae. Prestained molecular weight standard(lane Mw). Numbers on the left of the figure indicate the positions of the molecular weight markers(in thousands). (B) Immunoblot of the gel presented in panel A. Samples were transferred to anitrocellulose membrane and reacted with mouse antiserum against purified S. uberis CAMP factor.

regions, characteristic of E. coli promoters,28 were identified at the upstream ofthe transcriptional start site (Fig. 4).

Comparison of the S. uberis CAMP factor with S. agalactiae CAMP factor

To compare the S. uberis CAMP factor with protein B of S. agalactiae, aconcentrated culture supernatant of S. agalactiae containing protein B13 was

CAMP factor gene of Streptococcus uberis 301

Fig. 4. Nucleotide sequence of S. uberis CAMP factor gene and its promoter region. The deducedamino acid sequence of S. uberis CAMP factor is shown in the single-letter code below the sequence.The start site of transcription is represented by +1, and the −10 and −35 regions of the promoterare also indicated. The putative Shine-Dalgarno sequence is marked as SD, and the secretory signalsequence is shown in italics.

separated by SDS-PAGE and analysed by immunoblotting with antibodies againstthe purified S. uberis CAMP factor. A 25 kDa protein band from the S. agalactiaesupernatant (Fig. 3A, lane 4) reacted in the immunoblot (Fig. 3B, lane 4). Thisdata indicated that monospecific antibodies raised against the S. uberis CAMPfactor could cross-react with S. agalactiae protein B. This is not surprising, since

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Fig. 5. Primer extension analysis of the promoter region of S. uberis CAMP factor gene. The primerextension reactions were done as described in Materials and methods. The cellular RNAs used wereobtained from S. uberis (lane 1), E. coli JF1754 (pJLD21) (lane 2), and E. coli JF1754 (pTZ1754 (pTZ18R)(lane 3). The dideoxy sequencing ladder was generated by using the same primer and is shown as T,G, C, A above each lane.

alignment of the 226-amino acid sequence of the S. agalactiae CAMP factor, whichwas determined from the protein directly,14 with the deduced 256 amino acids ofthe S. uberis CAMP factor showed 66.4% identical residues (Fig. 6). The N-terminaltail of S. uberis CAMP factor lacking matches with the protein B sequence of S.agalactiae was from the secretory signal sequence which would be cleaved justbefore residue N.

Distribution of CAMP factor genes in eight S. uberis strains. ChromosomalDNA prepared from eight S. uberis strains were digested with the restrictionendonuclease HindIII and separated on an agarose gel. Southern blot analysiswith the 576 bp HindIII-EcoRI fragment of pJLD21 as a probe (Fig. 2) showed thata fragment identical in size to the HindIII fragment (1.2 kb) in pJLD21 was presentin three S. uberis strains which were CAMP-reaction positive, while none of theCAMP reactions negative strains reacted with the probe (Fig. 7). Thus, the CAMP-negative strains do not contain the cfu gene.

Discussion

Streptococcus uberis is an increasingly important cause of bovine mastitis, andthere are no effective measures for its control due to a lack of understanding of

CAMP factor gene of Streptococcus uberis 303

Fig. 6. Homology between S. uberis CAMP factor (upper lines) and S. agalactiae CAMP factor (lowerlines). Amino acids are identified in the single-letter code. Identical amino acids are indicated bydouble dots (:). The aligned sequences show 66.4% identity. Spaces (indicated by dash) were introducedin the sequences to optimize alignment. The secretory signal sequence of S. uberis CAMP factor isshown in italics.

Fig. 7. Southern blot analysis of different S. uberis strains. The origin of the probe used is indicatedin Fig. 2; chromosomal DNA from each S. uberis strain was cut with HindIII. Lane 1, 2, 3 and 4 arethe DNA samples from ATCC strain 9927, 13386, 13387 and 19436, respectively; lane 5, 6, 7 and 8 arethe DNA samples from field isolates. The arrowhead on the right indicates the position and size ofthe hybridizing restriction enzyme fragments. CAMP reaction phenotype of each strain is indicated atthe bottom by “+” (CAMP reaction positive) and “−” (CAMP reaction negative).

the virulence determinants and pathogenesis of this organism. Some S. uberisstrains produce a CAMP factor, which has been gaining more attention as apotential virulence factor because of its cohemolytic and Fc-binding properties.

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We have cloned the gene coding for the CAMP factor, cfu, from S. uberis ATCC9927 based on functional activity in E. coli. Even though the CAMP activity ofclone pJLD21 and pJLD21-2 was observed on blood agar after 6 h incubation,and a 28000 MW protein reacting with anti-S. uberis antibodies was found in theculture supernatant, we do not know whether the CAMP factor was released bycell lysis or secreted by recombinant E. coli.

In order to localize the cfu gene, we established restriction enzyme maps of therecombinant plasmid pJLD21 and generated several subclones of this plasmidfor sequencing. An open reading frame encoding a 256-residue polypeptidewas identified. A putative signal sequence was found at the N-terminus of thispolypeptide. This observation predicts the fact that the CAMP factor is secretedin S. uberis. However, we have no experimental evidence that this Gram-positivesignal sequence can lead the secretion of the CAMP factor from recombinant E.coli. From primer extension experiments, we found that transcription of the CAMPfactor gene is initiated from an ‘A’ residue which is preceded by a −10 box and−35 region (Fig. 4).

We analysed the culture supernatant of S. agalactiae by immunoblotting withantibodies against the S. uberis CAMP factor to see whether the proteins fromthese two streptococcal species are related. We found that a 25 kDa protein bandfrom the culture supernatant of S. agalactiae reacted with antibodies raised againstthe S. uberis protein, indicating that the two CAMP factors are immunologicallysimilar. The similarity was subsequently demonstrated at the amino acid level.

Based on the mapped location of the cfu gene, a HindIII-EcoRI fragment waschosen as a DNA probe to confirm the identity of the cloned DNA and itsdistribution in other S. uberis strains. The Southern blot analysis demonstratesthat the S. uberis strains containing cfu genes can show positive CAMP reactionphenotype.

Materials and methods

Bacterial strains, plasmids and growth conditions. The Escherichia coli strain used wasJF1754 (hsdR lac gal metB leuB hisB),29 which is from the laboratory collection. CompetentE. coli JF1754 was made as described.30 E. coli cells were grown in Luria broth (DifcoLaboratories) or on Luria-agar (Difco Laboratories) plates. Ampicillin was used at 50 lg/mlfor the growth of E. coli strains containing recombinant plasmids. Four S. uberis strains,S. agalactiae and S. aureus were obtained from the American Type Culture Collection(ATCC nos. 9927, 13386, 13387, 19436, 27541 and 25923, respectively). Other S. uberisstrains are field isolates kindly provided by M. Chirino-Trejo, University of Saskatchewan.All streptococcal strains were grown in brain heart infusion broth (BHI, Difco Laboratories)or on base no. 2 blood agar plates containing 5% sheep blood (PML microbiologicals).

The cloning vector pTZ18R31 was obtained from Pharmacia Canada Ltd.

Preparation of S. aureus beta-toxin. S. aureus was cultured in BHI for 18 h at 37°C andthe supernatant obtained after centrifugation at 5000 g was sterilized by filtration througha 0.22 lm filter (Nalge company). This material is referred to as crude beta-toxin and wasstored at −20°C.

CAMP reaction. Bacteria were screened for CAMP activity as described.26 Briefly, strainswere streaked perpendicular to a streak of beta-toxin-producing S. aureus on blood agarplates and after 6–20 h incubation at 37°C, they were observed for haemolysis.

Purification of CAMP factor. CAMP factor was partially purified from the culturesupernatant of S. uberis (ATCC 9927) by Octyl-Sepharose CL-4B (Pharmacia)chromatography as described by Jurgens et al.11

CAMP factor gene of Streptococcus uberis 305

Polyclonal antibodies. To analyse the recombinant CAMP factor of S. uberis, polyclonalantibodies directed against the purified CAMP factor were obtained. Mice were immunizedby intraperitoneal injection of 20 lg of the purified CAMP protein with complete Freund’sadjuvant. This primary immunization was followed 3 weeks later by the secondintraperitoneal injection of the same amount of CAMP protein with incomplete Freund’sadjuvant and a further 3 weeks later by the third intravenous injection of 20 lg of CAMPprotein with incomplete Freund’s adjuvant. Serum samples were then taken 10 days later.

PAGE and immunoblotting. Protein samples of S. agalactiae and E. coli were obtainedfrom culture supernatants by trichloroacetic acid (TCA)-precipitation at a final concentrationof 10%. SDS-polyacrylamide gel electrophoresis (PAGE) of proteins was performed asdescribed by Laemmli.32 Proteins were electroblotted onto nitrocellulose membranes asrecommended by the supplier (Bio-Rad) and the blots were developed as describedelsewhere33 with the following differences. The first antiserum used was mouse polyclonalantiserum against partially-purified S. uberis CAMP protein, and it was absorbed withantigens of the E. coli host strain as described previously.25 The second antibody used inblotting procedure was the goat anti-mouse IgG coupled to alkaline phosphatase(Kirkegaard & Perry Laboratories, Inc.).

DNA manipulations. All molecular techniques were as recommended by the supplier(Pharmacia Canada Ltd.) or Maniatis et al.34

Chromosomal DNA of S. uberis was prepared from cells grown in 100 ml BHI plus 5%(w/v) glycine. Cells were pelleted and resuspended in 2.5 ml of TES buffer (30 mm Tris-HCl, 5 mm EDTA, 50 mm NaCl; pH 8.0) with 25% sucrose and 1.6 mg/ml lysozyme (Sigma).The suspension was incubated for 1 h at 37°C, followed by freezing at −70°C. The frozencells were thawed in a 65°C water bath. EDTA and proteinase K (Pharmacia) were addedto final concentrations of 20 mm and 1.2 mg/ml, respectively, before incubation at 65°C for30 min. To lyse cells completely, sarkosyl was added to 1% and incubated at 37°C for 1 h.Two ml of TE buffer (10 mm Tris-HCl, 1 mm EDTA; pH 8.9) was added prior to phenol:chloroform extraction. DNA was recovered by ethanol precipitation and was treated withRNase (Pharmacia Canada Ltd.).

Size-fractionated Sau3AI-digested chromosomal DNA fragments were isolated by sucrosedensity gradient centrifugation.34

DNA sequence was determined by the dideoxy-chain termination method of Sanger etal.35 on double-stranded plasmid templates by using a T7 Sequencing kit (Pharmacia CanadaLtd.).

RNA analyses. RNA from E. coli strains was isolated as described previously14 with anadditional RNase-free DNase I digestion.

RNA from S. uberis was prepared as follows. The cell pellet from a 10 ml culture (OD600=0.6) was resuspended in 250 ll of TE buffer (pH 8.0) containing 500 lg of mutanolysin(Sigma) and incubated at 37°C for 30 min. Lysis buffer (250 ll) (60 mm Tris-HCl pH 7.4,200 mm NaCl, 10 mm EDTA, 2% SDS) and 100 lg/ml (final concentration) of proteinase Kwas added and the incubation continued for 1 h. The sample was extracted once with 65°Cphenol (water saturated, pH 4.0) and twice with room temperature phenol. RNA wasrecovered by ethanol precipitation and treated with DNase I (Pharmacia Canada Ltd.).

Primer extension assay was performed as described by Miller et al.37 RNasin and moloneymurine leukemia virus reverse transcriptase were obtained from Pharmacia Canada Ltd.

Nucleotide sequence accession number. The nucleotide sequence reported in this paperhas been submitted to GenBank and has been assigned accession number U34322.

We would like to thank Dr M. Chirino-Trejo for providing S. uberis strains and to Dr C.Rioux and Reno Pontarollo for advice and discussion. We appreciate the technical assistanceof Gordon Crockford and Neil Rawlyk. This work was supported by grants from the NationalScience and Engineering Research Council of Canada, Agriculture Canada and the DairyProducers of Alberta and Manitoba.

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