Characterization of mal recombination plasmids cloned in Streptococcus pneumoniae

Conference · Thu Jan 01 04:00:00 EST 1981

OSTI ID:6123116

Stassi, D L; Lopez, P; Espinosa, M; Lacks, S A

The malM locus of Streptococcus pneumoniae was cloned into one of the two PstI sites of the multicopy S. pneumoniae plasmid pMV158. To eliminate chromosomal transformants in the simultaneous selection for tetracycline resistance (coded by pMV158) and maltose utilization, the host cells contained a chromosomal deletion of the mal gene cluster. Two clones were isolated; one with a 3.3 kb insert (pLS70) which behaved like wild type with respect to maltose utilization, and another with a 2.9 kb insert (pLS69) which behaved as though it contained a down promoter mutation. Preliminary mapping of these clones by restriction analysis placed the 0.4kb deletion on a HindIII fragment in the interior of the chromosomal insert. The recombinant plasmids were able to transform over 50% of a recipient population to Mal/sup +/. Enzyme measurements of the clones indicated an overproduction of amylomaltase, constituting up to 10% of the total cellular protein, and supported the theory that the deletion in the pLS69 is in the promoter region. Protein analysis by polyacrylamide gel electrophoresis confirmed that the amylomaltase polypeptide was produced in large amounts in induced cells containing the pLS70. Another polypeptide, possibly a fragment of the phosphorylase or X protein of the mal gene cluster, was also produced to a similar extent.

• Research Organization: Brookhaven National Lab., Upton, NY (USA)
• DOE Contract Number: AC02-76CH00016
• OSTI ID: 6123116
• Report Number(s): BNL-29853; CONF-810694-3; ON: DE81029322
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
CELL CONSTITUENTS
CLONING
DATA
EXPERIMENTAL DATA
GENE RECOMBINATION
GENES
INFORMATION
MICROORGANISMS
NUMERICAL DATA
PLASMIDS
STREPTOCOCCUS
TRANSFORMATIONS