A novel gene encoding an SH3 domain protein is mutated in nephronophthisis type 1.

Juvenile nephronophthisis (NPH), an autosomal recessive cystic kidney disease, is the primary genetic cause of chronic renal failure in children. About two thirds of patients with NPH carry a large homozygous deletion at the gene locus NPH1 on 2q13. We here identify a novel gene. NPHP1, which extends over most of this common deletion. The 4.5-kb transcript encodes a protein with an SH3 domain, which is highly conserved throughout evolution. The 11-kb interval between the 3' end of NPHP1 and an inverted repeat containing the distal deletion breakpoint was found to contain the first exon of a second gene, MALL. In patients with a hemizygous deletion of the NPH1 region, additional point mutations were found in NPHP1 but not in MALL.

Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes.

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.

Genes involved in copper resistance influence survival of Pseudomonas aeruginosa on copper surfaces.

AIMS: To evaluate the killing of Pseudomonas aeruginosa PAO1 on copper cast alloys and the influence of genes on survival on copper containing medium and surfaces. METHODS AND RESULTS: Different strains of P. aeruginosa were inoculated on copper containing medium or different copper cast alloys and the survival rate determined. The survival rates were compared with rates on copper-free medium and stainless steel as control. In addition, the effect of temperature on survival was examined. CONCLUSIONS: Copper cast alloys had been previously shown to be bactericidal to various bacteria, but the mechanism of copper-mediated killing is still not known. In this report, we demonstrate that P. aeruginosa PAO1 is rapidly killed on different copper cast alloys and that genes involved in conferring copper resistance in copper-containing medium also influenced survival on copper cast alloys. We also show that the rate of killing is influenced by temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: To use copper surfaces more widely as bactericidal agents in various settings, it is important to understand how genes influence survival on these surfaces. Here we show that genes shown to be involved in copper resistance in P. aeruginosa PAO1 can have an impact on the length of survival time on copper cast alloys under certain conditions. This is an important first step for evaluation of future use of copper surfaces as bactericidal agents.

Microbial Virulence and Interactions With Metals.

Transition metals, such as iron, copper, zinc, and manganese play an important role in many bacterial biological processes that add to an overall evolutional fitness of bacteria. They are often involved in regulation of bacterial virulence as a mechanism of host invasion. However, the same transition metals are known to play an important role in host-defense mechanisms against bacteria through Fenton chemistry evoked toxicity as an example. Copper and zinc are used as a mechanism to poison bacteria whereas other metals, such as, iron and manganese are withheld by the predator to prevent reconstruction of Fe-S clusters and the use of Mn as a protectant against reactive oxygen species. Therefore, tight regulation of transition metal distribution in bacteria and hosts is a vital part of host-pathogen interactions.

A deletion distinct from the classical homologous recombination of juvenile nephronophthisis type 1 (NPH1) allows exact molecular definition of deletion breakpoints.

Juvenile nephronophthisis, an autosomal recessive cystic kidney disease, is the most common genetic cause of end-stage renal disease in children and young adults. We recently identified by positional cloning the causative gene, NPHP1. Its gene product nephrocystin may play a role in focal adhesion and adherens junction signaling. Approximately 80% of all patients with NPH1 carry large homozygous deletions, which contain the NPHP1 gene. These common deletions are positioned within a complex arrangement of large inverted and direct repeats, suggesting unequal recombination as a potential cause for their origin. In this study we have characterized the deletion breakpoints in a family with juvenile nephronophthisis that bears a unique maternal deletion of the NPHP1 gene, which is not the result of an event of homologous recombination. We molecularly characterized the centromeric and telomeric deletion breakpoints by extensive genomic sequencing, Southern blot analysis, and cloning and sequencing of the junction fragment. We were able to exactly localize the breakpoints at the position of two guanines. The centromeric breakpoint was positioned within intron 2 of the NPHP1 gene 360 bp downstream of the 5' end of a complete LINE-1 element. Multiple topoisomerase I and II consensus sequences were found at the breakpoint sites, suggesting the involvement of topoisomerase II in the deletion mechanism. These findings provide the first data on a potential mechanism for a deletion of the NPHP1 gene, that most likely is not the result of an event of homologous recombination and thereby distinct from the known common deletions.

Phytoremediation of heavy and transition metals aided by legume-rhizobia symbiosis.

Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.

Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids.

The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities.

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

Genes involved in copper homeostasis in Escherichia coli.

Recently, genes for two copper-responsive regulatory systems were identified in the Escherichia coli chromosome. In this report, data are presented that support a hypothesis that the putative multicopper oxidase CueO and the transenvelope transporter CusCFBA are involved in copper tolerance in E. coli.

CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli.

The putative multi-copper oxidase CueO had previously been implicated in intrinsic copper resistance in Escherichia coli. In this report we showed that the presence of CueO in the periplasm protected alkaline phosphatase from copper-induced damage. CueO contained four copper atoms per molecule and displayed spectroscopic properties typical of blue copper oxidases. CueO catalyzed the oxidation of p-phenylenediamine (pPD), 2,6-dimethoxyphenol (DMP) and exhibited ferroxidase activity in vitro.

The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli.

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.

New functions for the three subunits of the CzcCBA cation-proton antiporter.

The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport. The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit. The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation. Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter. Fusions of CzcC and CzcB with alkaline phosphatase and beta-galactosidase are in agreement with a periplasmic location of most parts of both proteins. Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein. Based on these data, a new working model for the function of the Czc system is discussed.

The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase.

The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA::kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.

Pb(II)-translocating P-type ATPases.

The cad operon of Staphylococcus aureus plasmid pI258, which confers cadmium resistance, encodes a transcriptional regulator, CadC, and CadA, an ATP-coupled Cd(II) pump that is a member of the superfamily of cation-translocating P-type ATPases. The Escherichia coli homologue of CadA, termed ZntA, is a Zn(II)/Cd(II) pump. The results described in this paper support the hypothesis that ZntA and CadA are Pb(II) pumps. First, CadC is a metal-responsive repressor that responds to soft metals in the order Pb>Cd>Zn. Second, both CadA and ZntA confer resistance to Pb(II). Third, transport of 65Zn(II) in everted membrane vesicles of E. coli catalyzed by either of these two P-type ATPase superfamily members is inhibited by Pb(II).

Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coli.

In a search for genes that produce hypersensitivity to cadmium salts in Escherichia coli, random transposon mutagenesis with TnphoA was used. One of the mutant strains obtained was sensitive to Cd2+ and Zn2+. Sequence analysis showed that the TnphoA insertion was located in the dsbA gene coding for a periplasmic protein required for disulfide bond formation.

A Zn(II)-translocating P-type ATPase from Proteus mirabilis.

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis gene was expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.

Escherichia coli CopA N-terminal Cys(X)(2)Cys motifs are not required for copper resistance or transport.

Escherichia coli CopA is a Cu(I)-translocating P-type ATPase that is involved in copper export and resistance. It is an orthologue of the human Menkes and Wilson disease-related proteins. Each of those two human copper pumps has six N-terminal Cys(X)(2)Cys sequences, but their function in transport is unclear. CopA has two N-terminal Cys(X)(2)Cys sequences, GLSC(14)GHC(17) and GMSC(110)ASC(113). The requirement of these cysteine motifs was investigated by mutagenesis of the codons for all four cysteine residues, singly and in combination. Cells of a copA deletion strain expressing genes for the mutant genes were nearly as resistant to copper as the wild type. In addition, everted membrane vesicles from cells expressing the mutant copA genes exhibited ATP-coupled accumulation of copper similar to that of the wild type. The results indicate that neither of two N-terminal Cys(X)(2)Cys motifs is required for either resistance or transport.

CopA: An Escherichia coli Cu(I)-translocating P-type ATPase.

The copA gene product, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in Escherichia coli. The copA gene was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain was more sensitive to copper salts but not to salts of other metals, suggesting a role in copper homeostasis. The copper-sensitive phenotype could be rescued by complementation by a plasmid carrying copA from E. coli or copB from Enterococcus hirae. Expression of copA was induced by salts of copper or silver but not zinc or cobalt. Everted membrane vesicles from cells expressing copA exhibited ATP-coupled accumulation of copper, presumably as Cu(I). The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases.

Children with ocular motor apraxia type Cogan carry deletions in the gene (NPHP1) for juvenile nephronophthisis.

Congenital ocular motor apraxia type Cogan is characterized by impairment of horizontal voluntary eye movements, ocular attraction movements, and optokinetic nystagmus. Two patients with congenital ocular motor apraxia type Cogan exhibited a newly recognized association with nephronophthisis type 1, an autosomal recessive kidney disease. Both patients possess large deletions of the NPHP1 gene. The deletion occurred on both chromosomes 2q13 in one patient and heterozygously in combination with a point mutation of the NPHP1 gene in the other. The findings will help to elucidate the pathogenetic processes involved.

Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli.

In a search for genes responsible for the accumulation of antimonite in Escherichia coli, TnphoA was used to create a pool of random insertional mutants, from which one antimonite-resistant mutant was isolated. Sequence analysis showed that the TnphoA insertion was located in the glpF gene, coding for the glycerol facilitator GlpF. The mutant was shown to be defective in polyol transport by GlpF. These results suggest that in solution Sb(III) is recognized as a polyol by the glycerol facilitator.