P-type ATPase from the cyanobacterium Synechococcus 7942 related to the human Menkes and Wilson disease gene products.

Abstract

7).The number of identified P-type ATPases is growing rapidly. In the course of cloning other genes from Synechococcus 7942, we found one that appears to encode a P-type ATPase. During the preparation of this paper three other cyanobacterial P-type ATPase sequences were published (8, 9). The first of these new reports concerns two genes for P-type ATPases from the same Synechococcus strain used in this study (8). However, the sequences of these genes differ significantly from the one we describe here. In addition, biochemical evidence indicates that the Synechococcus plasma membrane contains a P-type ATPase (10).We (Pharmacia), was selected on plates containing kanamycin at 50 ug/ml. For selective growth of E. coli strain DH5a (GIBCO/BRL) carrying antibiotic-resistance plasmids, ampicillin at 100 pug/ml or tetracycline 12 pig/ml was used.Cloning and Sequence Determination. Standard molecular genetic techniques, including DNA isolation, restriction nuclease digestion and ligation, and Southern and Northern blotting, were as described by Sambrook et al. (13). Genomic DNA was prepared from cells of Synechococcus 7942 according to Brusslan (14). RNA isolation was as described by Schaefer and Golden (15). A fragment containing part of the ctaA gene was isolated from a two-step size-fractionated Synechococcus DNA library. Synechococcus genomic DNA was first digested with BamHI and the restriction fiagments were separated by electrophoresis in an agarose gel. Fragments of 3-6 kb were isolated and digested with HindIII. Fragments of 2-3 kb were isolated, cloned into plasmid pBR322, and transformed into E. coli strain DH5a. Clone pLP180 (containing a 2.6-kb HindIII-BamHI fiagment) was identified by screening individual plasmid DNAs with an E. coli fabE (16) fragment as probe. A 320-bp Pst I-BamHI fragment was isolated from the HindIII-BamHI insert of pLP180 and used as probe to screen a library of wild-type Synechococcus genomic DNA in the cosmid vector pWB79 (prepared by J. Brusslan, University of Chicago). A 7-kb HindIll fragment was identified and smaller restriction nuclease fragments from it were subcloned into pUC18 and pBluescript vectors (Stratagene).Subclones for sequencing were generated either by nested deletion using the Erase-a-base kit (Promega) or by digesting the original clones with appropriate restriction nucleases and religating. Sequencing was done on both strands by the dideoxy chain-termination method with Sequenase version 2.0 (United States Biochemical) with either M13 universal and reverse primers or synthetic oligonucleotide primers. Fig. 1 summarizes the physical map of the 7-kb HindIII fragment and the 4.5-kb HindIII-EcoRV region within it that was sequenced completely on both strands.Insertional Inactivation of the ctaA Gene. The kanamycinresistance cassette from pUC4K was inserted between the Bgl II and Pst I sites of pLP1.5 (a pUC18-derived plasmid

A P-type ATPase importer that discriminates between essential and toxic transition metals

“…Deletion of the chromosomal copy of the gene encoding HmtA resulted in a P. aeruginosa strain that was mildly more resistant to copper and zinc, again suggesting involvement of the gene in metal uptake. A similar increase in copper resistance was observed upon deletion of CtaA, a copper importer of Synechococcus (25). Robust metal uptake by HmtA could be detected at the nanomolar range, and a 50-to 100-fold gradient in total intracellular copper was generated within 10-15 min.…”

Mutagenesis of a Copper P-Type ATPase Encoding Genein Methylococcus capsulatus (Bath) Results inCopper-Resistance

“…This indicated that, unlike copper, the disruption of copA1 did not confer silver resistance or sensitivity. Such findings coincide with those obtained from copper-resistant Synechococcus ΔctaA mutant strain which showed similar response to silver compared to the wild-type [24]. However, such similarity in phenotype between the M. capsulatus ΔcopA and parent strain with respect to silver, does not necessarily mean that CopA does not transport silver.…”

Two Menkes-type ATPases Supply Copper for Photosynthesis inSynechocystis PCC 6803

“…Neither cell line grew in BG-11 medium supplemented with 2 M copper, whereas growth of both cell types was inhibited by 1 M copper to a similar extent. In contrast, disruption of ctaA from Synechococcus PCC 7942 resulted in increased tolerance to copper ions compared with wild type (16).…”

“…Similarity of the deduced products of sll1920 and slr1950 with PacS (15) and CtaA (16), respectively, from Synechococcus PCC 7942, encouraged a prediction that these polypeptides contribute to copper homeostasis. We report that this is correct and designate the Synechocystis genes pacS and ctaA.…”