Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant

1991 ◽  
Vol 37 (8) ◽  
pp. 624-631 ◽  
Author(s):  
Kenneth Babich ◽  
Mike Engle ◽  
Jeffery S. Skinner ◽  
Richard A. Laddaga
Keyword(s):  
Staphylococcus Aureus ◽  
Ion Transport ◽  
Copy Number ◽  
Deletion Mutant ◽  
Resistance Determinant ◽  
Mercury Resistance ◽  
Mutant Analysis ◽  
Mer Operon ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid

Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid. Key words: mercury resistance, Staphylococcus aureus plasmid.

scholarly journals The phasmid as a tool for plasmid genetics: II. Isolation of point mutations that affect replication of a ColE1-related plasmid

1982 ◽  
Vol 40 (3) ◽  
pp. 233-247 ◽  
Author(s):  
Gianni Cesareni ◽  
Luisa Castagnoli ◽  
Sydney Brenner
Keyword(s):  
Copy Number ◽  
Point Mutations ◽  
Plasmid Replication ◽  
Single Base ◽  
Lambdoid Phage ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid ◽  
Single Base Pair ◽  
Related Plasmid ◽  
Single Base Pair Change

SUMMARYThe insertion of a high-copy-number plasmid into a lambdoid phage chromosome which lacks a functional repressor gene confers on the hybrid ‘phasmid’ the capacity to grow on an immune lysogen. This was found to be due to titration of repressor because of plasmid replication. We have exploited this property in order to isolate mutants that affect plasmid replication. These mutants have been mapped in a region that was previously characterized as necessary for plasmid replication and incompatibility properties. Some of the mutations could revert at frequencies characteristic of single-base-pair change mutations.

scholarly journals Nucleotide sequence of a high copy number plasmid fromHalobacteriumstrain GRB

1990 ◽  
Vol 18 (11) ◽  
pp. 3408-3408 ◽  
Author(s):  
Neil R. Hackett ◽  
Mark P. Krebs ◽  
Shiladitya DasSarma ◽  
Werner Goebel ◽  
Uttam L. RajBhandary ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Copy Number ◽  
High Copy Number ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid

scholarly journals Contribution of Aggregation-Promoting Factor to Maintenance of Cell Shape in Lactobacillusgasseri 4B2

2003 ◽  
Vol 185 (11) ◽  
pp. 3288-3296 ◽  
Author(s):  
Ivana Jankovic ◽  
Marco Ventura ◽  
Valerie Meylan ◽  
Martine Rouvet ◽  
Marina Elli ◽  
...  
Keyword(s):  
Cell Division ◽  
Copy Number ◽  
Promoter Region ◽  
Cell Shape ◽  
Down Regulation ◽  
Copy Number Plasmid ◽  
Recombinant Cells ◽  
High Copy Number Plasmid ◽  
Induced Changes ◽  
First Time

ABSTRACT Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of Lactobacillus gasseri 4B2, whose corresponding apf gene was cloned and sequenced. In this report, we identified and sequenced an additional apf gene located in the region upstream of the previously published one. Inactivation of both apf genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the apf2 promoter region on a high-copy-number plasmid, which recruited a putative apf activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for L. gasseri 4B2 cell shape maintenance.

scholarly journals Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

2004 ◽  
Vol 70 (4) ◽  
pp. 2508-2513 ◽  
Author(s):  
Satoshi Tomita ◽  
Kenji Tsuge ◽  
Yo Kikuchi ◽  
Mitsuhiro Itaya
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Positional Cloning ◽  
Deletion Mutant ◽  
Essential Gene ◽  
Transfer System ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Subtilis Genome

ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

Development of Small High-Copy-Number Plasmid Vectors for Gene Expression in Caulobacter crescentus

Plasmid ◽  
2001 ◽  
Vol 46 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Elizabeth Umelo-Njaka ◽  
John F. Nomellini ◽  
Harry Yim ◽  
John Smit
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Caulobacter Crescentus ◽  
High Copy Number ◽  
Plasmid Vectors ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid

scholarly journals Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(−)-Hydroxycarboxylic Acids

2003 ◽  
Vol 69 (6) ◽  
pp. 3421-3426 ◽  
Author(s):  
Sang Yup Lee ◽  
Young Lee
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Ralstonia Eutropha ◽  
Enantiomerically Pure ◽  
Pha Depolymerase ◽  
E Coli ◽  
Theoretical Yield ◽  
Hydroxycarboxylic Acids ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid

ABSTRACT A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.

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