Molecular Microbiology (1992) 6(16), 2253-2265

Surface protein-CAT reporter fusions demonstrate differential gene expression in the wrregulon of Streptococcus pyogenes A. Podbielski,' J. A. Peterson^ and P. Cleary^* Unstitut fur Medische Mikrobiologie, Klinikum RWTH, Pauwelstrasse 30, 5100 Aachen, Germany. ^Department of Microbiology, University of Minnesota, Box 196 UHMC, 1460 Mayo Building, 420 Delaware Street. SE Minneapolis, Minnesota 55455-0312, USA. Summary Streptococcus pyogenes expresses at least two virulence factors, the anti-phagocytic M protein and an inhibitor of chemotaxis, the C5a peptidase (ScpA), under control of the virR locus. To facilitate studies of this regulatory unit, we constructed a new shuttle vector with a staphylococcal chloramphenicol acetyl transferase (CAT) reporter box which replicates in S. pyogenes. We cloned polymerase chain reaction (PCR)-derived potential promoter regions of the virR, M protein {emm12), and ScpA {scpA) genes from an M type 12 5. pyogenes, strain CS24. Promoter activity was assessed by measurements of specific mRNAs, transacetylase activity, and minimum inhibitory concentrations (MICs) for chloramphenicol resistance. We demonstrated that VirR is a necessary but not always sufficient positive frans-acting regulator of emm12 and scpA expression; however, virR is not autoreguiated. A potential wr/7-bindlng consensus sequence is postulated for emm12, scpA and other M-like protein genes. Promoter activity of the structural genes was found to be dramatically influenced by growth conditions such as anaerobiosis. Levels of control, over and above the requirement for virR, are realized. The virR and scpA promoters were mapped for the first time using primer extension analysis. The observed mRNA start sites did not completely agree within the sequence predicted start sites. Data suggest that scpA could be subject to transcription attenuation. Introduction

{Miller ef a/., 1989). Considerably less is known about the regulatory mechanisms of pathogenic streptococci. Genes which encode M proteins {emm) and the C5a peptidase (scpA) were shown to be linked and co-ordinately regulated (Haanes and Cleary, 1989; Simpson etai., 1990). Recent data indicates that immunoglobulin receptor genes forlgG(fcr>l, Heath and Cleary, 1989; protein H, Gomi et al., 1990) and for IgA iarp4. Fritz et al., 1989; emmL2.2, Bessen and Fischetti, 1992) are genetically linked to emm genes and may be coregulated (Cleary ef al., 1991). Transcription of emm12 and scpA genes is dependent on an adjacent upstream locus we termed virR (Robbins ef al., 1987; Simpson ef al., 1990), a designation used for regulatory genes which co-ordinate expression of multiple virulence factors in other bacteria (Stachel and Nester, 1986; Stibitz ef al., 1988). We postulated that phase variation in expression of emm12 and scpA, a characteristic of Ml 2 strains of streptococci, is also co-ordinated by the virR gene product. Transcription of the emm6 gene in another serotype of Streptococcus pyogenes also requires an upstream locus, the mry gene (Caparon and Scott, 1987). mry has been suggested to encode a frans-acting DNA-binding protein (Perez-Casa! ef a/., 1991). Comparison of the virR and mry nucleotide sequences showed that they are highly homologous (Chen, 1990). To further define and quantify the physiologic impact of the regulatory circuit which controls virulence in S. pyogenes we constructed a shuttle vector using the staphylococcal chloramphenicol transacetylase (CAT) gene as a reporter (Hudson and Stewart, 1986). Putative virR, emm12, and scpA promoters were inserted next to the promoterless CAT {cat) gene and then reintroduced into steptococci. In this study we assessed the strength of each promoter, defined the transcription start sites, and showed that VirR is a frans-acting positive regulator of emm12 and scpA expression. Moreover, data suggest that transcription of emm12 and scpA are regulated independently at another level, superimposed on the requirement for virR.

Expression of vinjience factors by pathogenic bacteria is controlled in several species by complex regulatory circuits which are sensitive to environmental changes

Results CAT-promoter constructs

Received 17 December, 1991; revised 15 April, 1992; accepted 22 April, 1992. 'For correspondence. Tel, (612) 624 6190; Fax (612) 626 0623.

The predicted promoter sequences of virR {p-virR), emm12 {p-emm12), and sopA {p-scpA) were amplified by

2254 A. Podbielski, J. A. Peterson and P. Cleary Table 1, Comparison of promoter strength ol different promoters ot the Vir regulon fused to a cat reporter box.

Promoter construct p-virRt-cat p-virRlt-cat p-emm 12-cat p-scpA-cat None

E. coli MIC 20-80

20-60 >200 120-160 1-4

S. pyogenes MIC^

S. pyogenes CAT activity"

2.5 2.5 40 2.5-10 1.25

0.5" 1.5° 669 60

S. pyogenes CAT specific mRNA= 10 10 0.6 1.25-2.5 n.d.

a. MIC is the minimum inhibitory concentration ior chloramphenicol in mg I V MIC values were obtained from either £, coli strains harbouring pMH109 recombinants or Vir" S. pyogenes. strain CS24, harbouring pNZ123 recombinants. b. CAT activity values are taken from crude extracts ot mid-exponential phase cells grown anaerobically. Activity is shown as milli-units per mg protein of the cell extract, 1 unit = 1 nmole of CM acetylated per minute. c. Minimum amount of total RNA (>igl required for the detection ot CAT-specific mRNA by hybridization with the Dig-dUTP labelled CAT universal REV oligonucleotide {Table 2) as a probe. mRNA was prepared from mid-exponential phase cells grown anaerobically. d. Activity was detectable, but not significantly different from cells without cat-fusions. •• n.d. = not detectable.

polymerase chain reaction (PCR) from genomic DNA of a serotype Ml 2 S. pyogenes strain, CS24. The amplified PCR products were cloned in front of a promoterless CAT reporter gene carried by the shuttle vector pMH109, a plasmid known to replicate in Streptococcus mutans and other streptococcal species (Hudson and Curtiss, 1990) (see Fig. 1 and the Experimental procedures for a detailed description of procedures used to construct the reporter fusions). With the exception of one p-scpA clone, ApscpA-cat, all constructs proved to have the expected sequence integrity and were shown to express CAT activity in Escherichia coll by MIC measurement (Table 1). The ip-scpA-cat construct harboured a deletion in the scpA sequence at position 721-832 bp, including the downstream PCR primer (Fig. 5c). The pMH109 plasmid and its reccmbinant could not be established in S. pyogenes, strains CS24 or Ti ,30/50, for unknown reasons. To circumvent this problem each promoter-caf reporter fusion was subcloned into pNZerm, a modified form of the shuttle vector pNZ123 (Fig. 1) (De Vos, 1987). The caf gene of pNZ123 was replaced by the promoter-caf reporter constructs from pMHI 09 constructs (see the Experimental procedures). Establishment and characterization of pNZerm cat fusion plasmids in streptocooci To compare promoter activities and their dependence on VirR. plasmids carrying the promoter fusions were transformed into S. pyogenes by electroporation. Transformants were initially selected on THB agar containing 5 mg r ^ Er and thereafter passaged on the same medium.

Transformants were determined to be the same phenotype as recipient cells by colony morphology on Islam medium, an indirect indicator of M protein phenotype (Simpson ef a/., 1987). Because plasmids with reporter genes had never been introduced into S. pyogenes we were obliged to assess their genetic stability, molecular integrity and extrachromosomal state. In the absence of selection these reporter plasmids were very unstable. After single passage of Er-resistant transformants in non-selective broth, only 25-60% of the colony forming units, depending on the culture, retained the plasmid. H/ndlll digests of total DNA from the various transformants revealed fragments of the expected size when probed with plasmid (pNZerm) fragments or CAT universal REV oligonucleotide (Fig. 2) specific probes. In all cases the plasmid specific bands could be visualized (data not shown). The presence of the correct promoter insert in recombinant plasmids was also evaluated by PCR. Several primer pairs were used in separate reactions. The primers erm REV and CAT REV amplified recombinant plasmid DNA irrespective of the streptococcal insert. Specific insert FOR and CAT univeral REV or appropriate insert REV and erm REV primer pairs (Fig. 2) expanded only specific promoter-plasmid recombinant DNAs. The correct nature of the resulting PCR products were confirmed by size, the location of restriction sites (Acc\ for virR, Sph\ for emm12, Nde\ for scpAr-promoier constructs) and hybridization to appropriate promoter probes. The equality of plasmid copy number of each reporter fusion construct in streptococci was assessed by target DNA dilution PCR assays using plasmid specific primers (erm REV and CAT universal REV) (see the Experimental procedures) (Kellogg ef al.. 1990). The yields of PCR products en different promoter-CAT constructs from CS24 were approximately equal. The limiting concentration of total DNA was 1 pg m\ ^ for each recombinant plasmid. The limiting concentration of total DNA for plasmids pAP211 and pAP251 (Table 2) in DNA from host strains A l , A7, and Tn30/50 was also 1 pg ml '. To ensure that promoter-caf fusions were extrachromosomal rather than integrated via homologous recombination, we analysed DNA from each streptococcal partial diploid for linkage between the erm gene and chromosomal DNA by PCR. Plasmids pAP230 and pAP251 residing in strains CS24, A l , A7, and T, ,30/50 were analysed with specific primer pairs, erm REV~emm FOR probe or erm REV-scp for primers, respectively (Fig. 2). Specific PCR products consistent with an integrated form of the recombinant plasmid were observed in total DNA preparations from the CS214, A l , and A7 clones but not in the T, ,30/50 clones. However, limiting total DNA concentration for these products was 100 ng ml ' in all cases. As mentioned above, the limiting total DNA concentration using primers

cat promoter fusions to study virR in Streptococcus pyogenes

2255

2256 A. Podbielski, J. A. Peterson and P. Cleary Sad

a. emm FOR

5'

CCC

CCC GAG CTC pos. 1028-1048

ACT TTC (emm)

ATC

GCA

AAG

AGG

TTA

3'

CTC

TAG AAT Xbai (emm)

CAT

TTT

TAG

GAA

AAA

ACC

CTC

AGA (vir)

AAT

AAA

TGC

GTG

ATC

G 3'

CTC

ATA (vir)

AAT

GGC

ATA

AAA

AAT

CG

GGG GAG CTC pos. 1039-1019

TTT (vir)

AAT

TTT

CTT

GAT

ITT

CTT G

3'

CTC

TAA (scp)

AAG

AAT

GAG

GAT

AAG

GAG G

3'

CTC

CTG tscp)

ATG

AGA

GAC

TTT

GTC

TTT A

3'

GAT (cat)

ATG

CCT

CC

3'

GCG TAG (ermi)

AAA

COT

AAA

AGA

AGT

TA

ATA CAA (enn2)

ATT

CCC

CGT

AGG

C

3'

Sad emm REV

5'

CCC

CCC

GAG

pos. 1261-1241

vir 1 FOR

5'

GGG

GGG GAG pos. 897-915

C 3'

Sad vir 11 FOR

5'

GGG

GGO GAG pos. 819-838

3'

Sad vir REV

5'

GGG

Sad scpFOR

5'

GGO

GGG GAG pos. 542-563 Sad

scpREV

5'

GGG

b.

GGG GAG pos. 832-811

Start

CAT universal REV

5'

AGT TCA TTT pos. 1266-1247 Sad

cm FOR

5'

CCC

CCC GAG pos. 236-258

CTC

3'

Xhol ermREV

5'

CCC

CCC CTC GAG pos. 1185-1167 HifuUIl

CAT FOR

5'

GGG GGG AAG CTT CCA TTC ATC TIT TTT AAC GGC anneals 60 to 40 nucleotides upstream to the 3' end of CAT universal REV on pMHlO9

3'

SaU CAT REV

5'

GGG

sq) probe

GGG (ca:)

GCA

GGT

TAG

TGA

CAT

TAG

TAT

GCT

CC

3'

BamHI

C. emm probe

GGG GTC GAC pos, 1970-1950

5'

CCC

CCC GGA TCC pos. 2844-2825

TTT (vir)

AGC

CAT

GTT

AAT

GGC AAG pos. 888-868

ATC (scp)

AAA

TGG

3'

3'

Fig. 2. Sequences of oliQonucteotides used in this study for PCR, DNA sequencing, hybridization and primer extension experiments. Restriction srtes or special features (SD' Shine-Dalgamo sequence: start: translation start codon) within a given oUgonucleotlde are shown above the region ot interest. The nucleotide position numbers refer to the toHowing articles: emm. Robbins ef a/. (1987); vir, Chen (1990): scp. Chen and Cleary (1990): C3t. Horinouchi and Weisblum (1982): ermi. Brehm etal. (1987); 0rm2, Marijn etal. (1987). (a) oligonucleotides used for PCR ampliiication of promoter regions of the vir reguion (b) oligonucleotides used for sequencing, hydridization, and PCR amplification of the genes for erythromycin resistance and chloramphenicol resistance (c) oligonucleotides used for a PCR based screening to demonstrate chromosomally integrated recombinant pAP plasmids

cat promoter fusions to study virR in Streptococcus pyogenes specific extrachromosomal plasmids was 1 pg ml \ indicating a ratio of autonomous to integrated recombinant plasmid of 10^:1. This suggests that the overwhelming majority of cells contain the recombinant plasmid in an extrachromosomal form. Comparison of promoter activities, dependence on virR and growth conditions The chloramphenicol (Cm) and erythromycin (Er) minimum inhibitory concentrations (MICs) of streptococcal recipients are 1.25ande 12 M protein gene regulation by upstream sequences. J Bacteriol 169: 5633-5640. Saiki. R.K.. Gelfand, D.H.. Stoffel, S., Scharf, S.J.. Higuohi. R.. Horn, G.T.. Mullis, KB., and Eriich. HA. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. Sambrook, J., Fritsch. E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Hartior Laboratory Press. Sanger. F., Nicklen, S.. and Coulson. A.R. (1977) DNA sequencing

cat promoter fusions to study virR in Streptococcus pyogenes with chain-tennlnating inhibitors. Proc NatI Acad Sci USA 74: 5463-5467. Shaw. W.V. (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol 43: 737755. Simpson, W.J., and Cleary. P.P. (1987) Expression of M type 12 protein by a group A streptococcus exhibits phase-like variation: evidence for coregulation of colony opacity determinants and M protein. Infect Immun 55: 2448-2455. Simpson, W.J., LaPenta, D., Chen, C , and Cleary, P.P. (1990) Coregulation of t^pe 12 M protein and streptococcal C5a

2265

peptidase genes in group A streptococci: evidence for a virulence regulon controlled by the virR locus. J Bacteriol 172: 696-700. Stachel. S.E.,and Nester, E.W. (1986) The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMB0J5: 1445-1454. Stibitz. S., Weiss, A.A., and Falkow, L. (1988) Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir. J Bacteriol 170: 2904-2913.