Cloning and genetic analysis of serotype 5 M protein determinant of group A streptococci: evidence for multiple copies of the M5 determinant in the Streptococcus pyogenes genome

Abstract

A gene bank of group A Streptococcus strain Manfredo (M protein serotype 5) was constructed with a bacteriophage lambda vector-Escherichia coli K-12 host system and screened by immunoblotting hybrid phage plaques with antisera raised to purified pep M5 (serotype 5 M protein fragment released from the streptococcal cell surface by pepsin). Hybrid phage expressing M5 antigen (lambda M5) were detected in the gene bank at an unexpectedly high frequency. The cloned streptococcal DNA sequences from one lambda M5 phage were subcloned into an E. coli plasmid vector. The M5 gene (smp5) was mapped, and its transcriptional orientation was determined by isolating and characterizing deletion and transposon insertion mutants of the M5+ hybrid plasmid pMK207. This analysis indicated that the intact smp5 gene had been cloned. Anti-pep M5 sera reacted with five pMK207-encoded polypeptides having relative molecular sizes of 64,000, 56,000, 55,500, 52,500, and 50,000. All of these polypeptides were encoded by the same DNA sequences, and all reacted with antisera raised to a synthetic peptide corresponding to the amino-terminal end of pep M5, suggesting that proteolytic cleavage at the carboxy-terminal end of the smp5 gene product generates at least some of the lower relative molecular size forms. Southern blotting experiments with smp5 gene sequences as probes identified multiple copies of DNA sequences sharing homology with the smp5 gene in the type 5 group A streptococcal genome.

Cloning and expression in Escherichia coli of the Ibc protein genes of group B streptococci: binding of human immunoglobulin A to the beta antigen

“…It can be seen that the anti-aand anti-p-specific antibodies reacted with several polypeptides produced by E. coli JM83 harboring pPHC10 (lane c) and pPHC8 and pPHC33 (lanes d and e), which are not present in the absence of the cloned streptococcal DNA (lanes a and g). The Ibc proteins expressed in E. coli from cloned genes, therefore, appear to be processed or degraded in a similar fashion to that occurring in streptococci (2,12,21,27). The 130-kilodalton (kDa) p protein (lanes d and e) was identical to that present in crude preparations from the cell surface of a group B streptococcus, which was extracted as described previously (2) (data not shown).…”

“…The Aa+ phage contained 12.1 kb of inserted streptococcal DNA and the A,B+ phage contained 11.6 kb. Although instability problems of streptococcal DNA in E. coli have been encountered previously (12,13), attempts were made to subclone DNA from the recombinant phage into the high-copy-number E. coli plasmid pUC18 (29) by using EcoRI or HindIII endonucleases. Products from these digestions were ligated into the unique EcoRI or HindlIl sites in the pUC18 polylinker and were used to transform competent E. coli JM83 cells and colorless colonies selected on medium containing 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside and ampicillin.…”

“…This study would suggest that a similar processing occurs with Ibc proteins made in E. coli. Work on group A streptococci has shown a similar degradation of M proteins in streptococci and E. coli (12). Although there are similarities between the a and ,B protein patterns seen in streptococci and E. coli, they are not identical.…”

“…Recombinant phage suspensions were diluted, incubated with cells of E. coli Q359, and plated in soft agar on AM3 medium so that approximately 200 plaques per plate were obtained. Plates were overlaid with nitrocellulose membrane filters (82 mm; Schleicher & Schuell, Inc., Keene, N.H.) to absorb proteins, as described by Kehoe et al (12), and then stored at 4°C overnight. Proteins from SDS-polyacrylamide gels were electroblotted onto Gene-Screen paper (New England Nuclear Research Products, Boston, Mass.)…”

Cloning of a surface protein antigen gene from serotype c Streptococcus mutans

“…However, a similar multiple banding pattern has been observed for the group A streptococcal M protein produced in E coli (Scott and Fischetti. 1983;Kehoe et al, 1985). Genes encoding several other surface proteins of mutans streptococci have been cloned by others.…”