A second gene in the Staphylococcus aureus cadA cadmium resistance determinant of plasmid pI258.

Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.     

The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+ resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA).

A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.     

Effects of multicopy LeuO on the expression of the acid-inducible lysine decarboxylase gene in Escherichia coli.

We previously reported that mutations in hns, the structural gene for the histone-like protein H-NS, cause derepressed expression of cadA, which encodes the acid-inducible lysine decarboxylase at noninducing pH (pH 8.0). This study reports the characterization of a plasmid isolated from an Escherichia coli library that suppresses the effect of an hns mutation on cadA expression. A previously sequenced open reading frame, leuO, proves to be the gene that causes the hns-complementing phenotype. The mechanism for this phenotype appears to be overexpression of leuO from a multicopy plasmid, which drastically reduces production of CadC, the essential activator for cadA induction. These results show an in vivo regulatory phenotype for leuO, consistent with its proposed protein sequence.     

Genetic Studies on Plasmid-Linked Cadmium Resistance in Staphylococcus aureus

The genetic basis of cadmium resistance conferred by three penicillinase plasmids, PI(524), PI(258), and PII(147), of Staphylococcus aureus was examined by mutation, recombination, and deletion analysis. Three separate loci were identified: cadA, responsible for high-level resistance; cadB, giving a low-level resistance, nonadditive to cadA; and mad, a locus marginally decreasing the cadmium resistance of plasmid-positive staphylococci. The loci cadA and mad were present on all three plasmids, but cadB was only found on PII(147). Spontaneous deletions of mad involved up to three-fourths of the plasmid genome, which allowed derivation of a partial deletion map of PII(147), a plasmid with a contour length of 10.9 mum, corresponding to a molecular weight of 20.4 x 10(6).     

Cloning and Expression of cadD, a New Cadmium Resistance Gene of Staphylococcus aureus

A cadmium resistance gene, designated cadD, has been identified in and cloned from the Staphylococcus aureus plasmid pRW001. The gene is part of a two-component operon which contains the resistance gene cadD and an inactive regulatory gene, cadX*. A high degree of sequence similarity was observed between cadD and the cadB-like gene from S. lugdunensis, but no significant similarity was found with either cadA or cadB from the S. aureus plasmids pI258 and pII147. The positive regulatory gene cadX* is identical to cadX from pLUG10 over a stretch of 78 codons beginning at the N terminus, but it is truncated at this point and inactive. Sequence analysis showed that the cadmium resistance operon resides on a 3,972-bp element that is flanked by direct repeats of IS257. The expression of cadD in S. aureus and Bacillus subtilis resulted in low-level resistance to cadmium; in contrast, cadA and cadB from S. aureus induced higher level resistance. However, when the truncated version of cadX contained in pRW001 is complemented in trans with cadX from plasmid pLUG10, resistance increased approximately 10-fold suggesting that the cadmium resistance operons from pRW001 and pLUG10 are evolutionarily related. Moreover, the truncated version of cadX contained in pRW001 is nonfunctional and may have been generated by deletion during recombination to acquire the cadmium resistance element.     

Regulation of the Escherichia coli cad operon: location of a site required for acid induction.

The cad operon encodes lysine decarboxylase and a protein homologous to amino acid antiporters. These two genes are induced under conditions of low pH, anaerobiosis, and excess lysine. The upstream regulatory region of the cad operon has been cloned into lacZ expression vectors for analysis of the sequences involved in these responses. Deletion analysis of the upstream region and cloning of various fragments to make cadA::lacZ or cadB::lacZ protein fusions or operon fusions showed that cadA was translated more efficiently than cadB and localized the pH-responsive site to a region near an upstream EcoRV site. Construction of defined end points by polymerase chain reaction further localized the left end of the regulatory site. The presence of short fragments bearing the regulatory region on high-copy-number plasmids greatly reduced expression from the chromosomal cad operon, suggesting that titration of an essential activator protein was occurring. With nonoptimal polymerase chain reaction conditions, a set of single point mutants were made in the upstream regulatory region. Certain of these altered regulatory regions were unable to compete for the regulatory factor in vivo. The locations of these essential bases indicate that a sequence near the EcoRV site is very important for the activator-DNA interaction. In vivo methylation experiments were conducted with cells grown at pH 5.5 or at pH 8, and a difference in protection was observed at specific G residues in and around the region defined as important in pH regulation by the mutation studies. This work defines essential sequences for acid induction of this system involved in neutralization of extracellular acid.     

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium.

pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences are similar to those of the cad operon of plasmid pI258 of S. aureus, known to prevent accumulation of Cd2+ in the bacteria by an ATPase efflux mechanism. The cadmium resistance determinant of L. monocytogenes does not confer zinc resistance, in contrast to the cadAC determinant of S. aureus, suggesting that the two resistance mechanisms are slightly different. Slot blot DNA-RNA hybridization analysis showed cadmium-inducible synthesis of L. monocytogenes cadAC RNA.     

Insertional inactivation of a gene which controls expression of vancomycin resistance on plasmid pHKK100

Expression of inducible high level vancomycin resistance (Vmr) in enterococci appears to require other plasmid-encoded genes in addition to the previously described structural genes vanA and vanH. Tn917 mutagenesis was used to identify such a region in the Vmr plasmid pHKK100. Insertional inactivation of a 693-bp open reading frame upstream from vanH resulted in complete loss of Vmr. This putative 26,642-Da protein has been designated VanR.     

Effect of Cd2+ on growth of the cadmium-resistant and -sensitive Staphylococcus aureus

The effect of Cd2+ on aerobic and anaerobic growth was studied in the Cd2+-resistant Staphylococcus aureus 17810R which harbours the cadA and cadB markers on a penicillinase plasmid pII17810. Also the effect of Cd2+ on growth of the plasmidless strain 17810S, sensitive to Cd2+ was investigated. The results indicate that under all growth conditions the Cd2+-resistant S. aureus 17810R is protected against Cd2+ toxicity up to 100 microM Cd2+ by the 2H+/Cd2+ antiporter, the product of the cadA gene. Energetics of growth of both strains under various conditions is also discussed.     

Integrated R2 sequence in mitochondria of fertile B37N maize encodes and expresses a 130 kD polypeptide similar to that encoded by the S2 episome of S-type male sterile plants.

Expression of a 130 kDa protein from open reading frame 1 of the integrated form of the R2 mitochondrial plasmid in normal mitochondria of B37 and other inbred lines is described. The protein appears identical to that synthesized by the closely related S2 episome found in cytoplasmic male sterile maize of the S type. Protein was detected using antisera raised against a beta-galactosidase:ORF1 fusion product containing the most antigenic region of the ORF1 product. Detection of this protein is in contrast to previous reports that mitochondria of normal, male-fertile lines either do not contain this protein, or that there are 11 in-frame stop codons in the reading frame. The integrated R2 of B37N was cloned and this region sequenced, confirming that a continuous open reading frame existed. These results are discussed in relation to the possible role of the S-type episomes in causing cytoplasmic male sterility.     

Cadmium and manganese transport in Staphylococcus aureus membrane vesicles.

The presence of plasmid gene cadB did not affect Cd2+ accumulation, whereas plasmid gene cadA reduced Cd2+ accumulation by whole cells but not by membrane vesicles. Membrane vesicle studies indicated that Cd2+ uptake occurred via the Mn2+ transport system which was energized by the membrane electrical potential. Mn2+ and Cd2+ were competitive inhibitors of each other's transport, with Km's of 0.95 microM Mn2+ and 0.2 microM Cd2+. The kinetic parameters were nearly identical with vesicles prepared from sensitive and resistant cells, indicating that the cadA-encoded Cd2+ efflux system was inoperative in membrane vesicle preparations. Experiments with energy-inhibited cells indicated that the cadB gene product may bind Cd2+.     

Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola.

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv. phaseolicola were generated by Tn5 transposon mutagenesis. A P. syringae pv. phaseolicola LR700 cosmid library was screened with Tn5-containing EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb sequenced region spanning the Tn5 insertion site contained four open reading frames. The transposon-inactivated gene, designated pilP, is 525 bp long, potentially encoding a 19.1-kDa protein precursor that contains a typical membrane lipoprotein leader sequence. Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expression of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa, in agreement with the sizes of the open reading frames. The three genes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature form was shown to be inhibited by globomycin, a specific inhibitor of signal peptidase II. The gene region identified shows a high degree of homology to a gene region reported to be required for Pseudomonas aeruginosa type IV pilus production.