Cloning of chromosomal genes in Streptococcus pneumoniae.

Abstract

A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the maiM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid, pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme.A 10-kilobase restriction fragment containing the sd-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment ofa recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtiis are considered and compared.Despite the fact that bacterial transformation was discovered in Streptococcus pneunwniae, this species has not until now been used to clone recombinant DNA. In the bacterium commonly used for cloning, Escherichia coli, the artificial method of transformation allows uptake of DNA without alteration, whereas the naturally evolved DNA uptake systems of S. pneumoniae and Bacillus subtilis process the incoming DNA (1). The cutting of DNA during its binding and entry and the necessity to reconstruct a plasmid from single-stranded fragments are impediments to cloning in these Gram-positive bacteria. Plasmid introduction in B. subtilis requires a multimeric donor molecule (2), although monomers have been shown to be active in S. pneumoniae (3, 4). Nevertheless, cloning ofchromosomal genes by introduction of a recombinant plasmid has been achieved in B. subtilis (5, 6). However, a more promising approach may be the transformation of an endogenous plasmid by recombinant DNA (7). The latter process can be called plasmid transformation, as opposed to the former process of plasmid transfer, in which a new plasmid replicon is established in the cell. The recent introduction of multicopy plasmids into S. pneumoniae (4, 8) has made cloning in this species practicable.Our approach to cloning in S. pneumoniae depended on previous analysis of the maltose-utilization system (9, 10). The mal region-a cluster of genes concerned with maltosaccharide utilization-includes the maiM gene, which specifies amylomaltase, and a regulatory gene. Restriction fragments, either unfractionated or enriched for the maiM gene, were ligated to the plasmid pMV158 (11) and used to transform recipient cells with and without endogenous plasmid.Successful cloning of the wild-type allele of the malM gene was achieved. A recom...

Biological role of the pneumococcal amidase

“…When crude sonicated extracts were obtained from the strains used in the experiments reported in Figs 3 and 4, we found ( Table 2) that the extracts obtained from M51 were about fivefold more active than those obtained from the tyt' strain when tested on purified, choline-labeled pneumococcal cell walls, whereas M31 and M32 (a frame-shift mutant of the lytA gene) completely lacked amidase activity [8]. The higher level of amidase activity found in M52 would be due to the presence of several copies of pRG2 per cell as previously found for the vector plasmid pLSl [16]. In fact we have found that M51 contains about 120 copies of pRG2 as compared to the 45 copies of pLSl found in M34.…”

Streptococcus pneumoniae DNA polymerase I lacks 3'-to-5' exonuclease activity: localization of the 5'-to-3' exonucleolytic domain

“…DNA fragments were separated by electrophoresis on agarose gels and purified by binding to glass milk, according to the specifications of the supplier of GeneClean (BIO-101, Inc.). The plasmid content of the clones was analyzed in alkaline lysates prepared by the method of Birnboim and Doly (3) for E. coli and by a modification of that method (37) for S. pneumoniae. Cultures of S. pneumoniae were grown and transformed as previously described (23).…”

Rambling and Scrambling in Bacterial Transformation— a Historical and Personal Memoir

“…Among the tools and methods developed in my laboratory, one of the most important was the pneumococcal cloning system using derivatives of the plasmid pMV158 (53). We were forced to turn to this system by our inability to clone many important pneumococcal genes, such as malM, endA, and hexA, in the available E. coli systems, apparently due to the strong promoters of S. pneumoniae and the toxicity of the gene products in E. coli (52).…”