Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

Macrina, Francis L.; Evans, R P; Tobian, Janet; Hartley, Donna F.; Clewell, Don B.; Jones, Kathryn

doi:10.1016/0378-1119(83)90176-2

Cited by 207 publications

(123 citation statements)

References 14 publications

(12 reference statements)

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“…E. coli strain M2508 (melA metA lac) was kindly provided by Dr R. Schmitt (Schmitt, 1968). Plasmid pVA891 was kindly provided by Dr F. Macrina (Macrina et al, 1983). E. colistrains were grown with shaking in Luria Broth (Maniatis et al, 1982) with antibiotics as required: ampicillin (100 pg ml-I), erythromycin (400 pg ml-I).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and genetic analysis of Streptococcus mutans -galactosidase

Aduse-Opoku

Tao

Ferretti

et al. 1991

Journal of General Microbiology

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~ ~ ~~The aga gene coding for a-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage A. The gene was subcloned into plasmid vectors and shown to specify a novel protein of M, 8OOOO. Characterization of a-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the a-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted M, of 82022 and revealed regions of homology to agalactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all a-galactosidase activity and abolished the ability to ferment melibiose; aglucosidase activity was also lost, due to an indirect effect on the dexB gene.

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Section: Methodsmentioning

confidence: 99%

Biochemical and genetic analysis of Streptococcus mutans -galactosidase

Aduse-Opoku

Tao

Ferretti

et al. 1991

Journal of General Microbiology

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“…pVA891 is a derivative of E. coli plasmid pACYC184 (4) containing the pAM31 Emr gene which is expressed in lactococci (21). Several attempts to transform pVA891 into L. lactis LM0230 verified that this plasmid does not replicate in this host, as no Emr transformants were recovered.…”

Section: Resultsmentioning

confidence: 99%

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris

1991

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The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. Plasmid replication and stability have been extensively studied for several plasmids found in gram-negative species. These studies have led to identification of several plasmidencoded elements which mediate plasmid replication, control plasmid copy number, and stabilize plasmid inheritance (18,24,26,30 the reasons for the differing abilities of L. lactis and L. cremoris strains to maintain pSK11L, we attempted to identify the pSK11L replication origin and regions involved in plasmid maintenance since these regions would be most likely to interact with host plasmid replication and maintenance functions.We report here that a 14.8-kbp PvuII fragment of pSK11L confers the replication ability and the temperature-sensitive pSK11L maintenance phenotype of L. lactis LM0230. We also report identification of several regions on this fragment which appear to affect pSK11L maintenance. Several of these regions affect pSK11L maintenance differently in L. lactis LM0230 and L. cremoris EB5. MATERIALS AND METHODSBacterial strains and plasmids. The L. lactis strains used in this study included LM0230, a plasmid-free, prophage-free derivative of C2 (9), and JF3216, an LM0230 transformant containing pSK11L, a lactose plasmid from L. cremoris SK11 (10). The L. cremoris strain used was EB5, a plasmidfree derivative of KR1 (3). Strains LM0230 and EB5 were grown at 32°C in M17 broth (33) supplemented with 0.5% glucose (M17-G). Transformants derived from these strains were grown at 25°C in M17-G containing erythromycin (Em) (10 jig/ml). Strain JF3216 was grown at 25°C in M17 broth supplemented with 0.5% lactose. The Escherichia coli plasmid used to clone the replication region of pSK11L was pVA891, a derivative of pACYC184 (4) containing the pAM,1 Emr-encoding gene (21). pVA891 and its derivatives were maintained in E. coli XL1-Blue

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“…PCR amplification with primers tpxf2\tpxr2 (5h-CGTCCGATGAAGA CCGTTTC-3h, 5h-CGCTGTAACCATCAATGCGG-3h) of S. gordonii DL1 DNA template generated a 1190 bp fragment comprising the entire tpx gene (492 bp) and flanking sequences that was cloned into pGEM-T. A DNA fragment (1052 bp) containing the ermAM gene (GenBank accession no. AB057644) was PCR-amplified from plasmid pVA838 (Macrina et al, 1983) using primers ermf\ermr (5h-CCATAT CATAAAAATCGATACAGC-3h, 5h-CCTTATCGATACA AATTCCCCG-3h) that contained ClaI restriction sites. The PCR product was digested with ClaI and ligated into a unique ClaI site within the cloned tpx gene, thus generating plasmid pGEM-tpx.ermAM (Table 1).…”

Section: Methodsmentioning

confidence: 99%