We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.

The mercurial-resistance determinant from Staphylococcus aureus plasmid pI2S8 is located on a 6.4-kilobase-pairBgl H fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA sequence of the Bgl II fragment was determined. The mer DNAsequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and mern (organomerculrial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage identity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mor sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons.The mercury-resistance determinants from Gram-negative bacteria consist of a regulatory gene (merR), an operator/ promoter region, and at least three structural genes (1-7). The three structural genes merT, merP, and merA code for a membrane transport protein, a periplasmic Hg2+-binding protein, and the mercuric reductase enzyme subunit, respectively. The reductase functions to detoxify mercury by reducing Hg2+ and Hg+ ions to elemental Hg0 (8). Some plasmid mercurial-resistance determinants also contain an additional gene, merB, whose product is the enzyme organomercurial lyage. The lyase cleaves the carbon-mercury bond of organomercurials such as phenylmercuric acetate (9). One product is Hg2+, which is subsequently detoxified by the mercuric reductase. Mercurial-resistance determinants that contain the organomercurial Iyase gene are designated broad-spectrum determinants; those without the Iyase gene are designated narrow-spectrum determinants (6, 10).Less is known about mercurial resistance in Gram-positive bacteria. The most thoroughly studied determinant from a Gram-positive bacterium is the broad-spectrum mer operon of the Staphylococcus aureus plasmid p1258. This mer operon was mapped by both deletion mutants (11, 12) and transposon (Tn) insertion mutants (13). These studies established the gene order merR merA merB.Mercury resistance is widely distributed among genera of Gram-negative and -positive bacteria (6, 7, 10). The mercuryresistance determinants from most Gram-negative bacteria are highly homologous (2-4, 6, 14). Conversely, the determinants from Gram-negative sources are less homologous with those from Gram-positive organisms (11,14).The DNA of two narrow-spectrum mercurial-resistance determinants from Gram-negative bacteria, those from transposons Tn2J and Tn501, have been s...

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 × 108 transformants per μg of plasmid pSK265 DNA. This represents a 530‐fold improvement over the previously reported optimum efficiency of 7.5 × 105 transformants per μg of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at −80°C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1‐cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500‐fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 × 105 and 5 × 103 transformants per μg of pSK265 DNA, respectively.

Cd21 and Mn21 accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 ,uM '09Cd2+ or 54Mn2+, both strains accumnulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 ,uM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 p.M '09Cd2+ or 54Mn2+. 54Mn2+ and '09Cd2+ uptake was thergy dependent and temperature sensitive, but '09Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decreast in temperature. 109Cd2+ uptake in the sensitive strain followed MichaelisOIenten kinetics with a Km of 1.8 ,uM Cd2+ and a V..x of 1.5 ,imol/min. g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of "09Cd2+ uptake by the Cd2-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the V., for the sensitive strain. The Km and Vi.. for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of M4Mn2+ uptake had an apparent Ki of 3.4 and 21.5 ,uM Cd2+ for the sensitive and Cd2-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 ,uM Mn2+ for Inhibition of '09Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+. Cd2+ is toxic to bacteria (13). A mechanism of resistance to Cd2" has been studied in Staphylococcus aureus. S. aureus is normally sensitive to concentrations of Cd2" above about 2 ,uM, but if a plasmid which confers resistance to Cd2+ is introduced into the organism, it is resistant to up to 200 ,uM Cd2+ (14). The plasmid codes for a Cd2+ efflux (antiporter) transport system which rapidly transports Cd2+ out of the cells (9, 11). Recently, a Cd2+-resistant mutant of Bacillus subtilis 168 was isolated (10). Both the parental and mutant strains are plasmidless. The mutant strain was obtained by successive transfers of the parental strain into medium containing up to 90 ,.M Cd2+. Under steady-state conditions, the Cd2+sensitive parental strain accumulated 10 times the level of Cd2+ that the Cd2+-resistant mutant strain accumulated. This Cd2+-resistant strain appears to be the first chromosomally determined Cd2+-resistant mnutant isolated. Cd2+ resistance in this strain is expressed constitutively (10).

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.