ABSTRACT
PREMISE OF THE STUDY: Microsatellite markers were developed for Spondias radlkoferi to assess the impact of primate seed dispersal on the genetic diversity and structure of this important tree species of Anacardiaceae. ⢠METHODS AND RESULTS: Fourteen polymorphic loci were isolated from S. radlkoferi through 454 GS-FLX Titanium pyrosequencing of genomic DNA. The number of alleles ranged from three to 12. The observed and expected heterozygosities ranged from 0.382 to 1.00 and from 0.353 to 0.733, respectively. The amplification was also successful in S. mombin and two genera of Anacardiaceae: Rhus aromatica and Toxicodendron radicans. ⢠CONCLUSIONS: These microsatellite loci will be useful to assess the genetic diversity and population structure of S. radlkoferi and related species, and will allow us to investigate the effects of seed dispersal by spider monkeys (Ateles geoffroyi) on the genetic structure and diversity of S. radlkoferi populations in a fragmented rainforest.
ABSTRACT
The Bacillus subtilis strain 168 genome contains the chr3N-chr3C genes encoding the Chr3N/Chr3C protein pair of the chromate ion transporter (CHR) superfamily. Chr3N/Chr3C confers chromate resistance in Escherichia coli only when both proteins are expressed. Upstream of chr3N is the chrS gene encoding ChrS, a protein with homology to the Lrp/AsnC family of transcriptional regulators. When the chrS-chr3N-chr3C gene cluster was transferred to E. coli, a diminished level of chromate resistance was observed, as compared with E. coli transformants bearing only the chromate resistance genes, which displayed full resistance. These data suggested that the chrS gene product acts as negative regulator. RT-PCR assays demonstrated that expression of chrS diminishes transcription of the chromate resistance genes in E. coli, and that this repression was overcome by chromate. Electrophoretic mobility shift assays showed that purified ChrS protein specifically binds to the 5' region of chrS. These results indicate that the chr gene cluster forms an operon regulated negatively by ChrS binding to its own gene's regulatory region, and positively by chromate ions. Sequence analysis revealed similar operons in many Bacillales strains, suggesting some adaptive advantage. This is the first example of a bacterial heavy-metal resistance system controlled by an Lrp-type transcriptional regulator.
Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Chromates/metabolism , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/biosynthesis , Repressor Proteins/genetics , Repressor Proteins/metabolism , Chromates/toxicity , DNA, Bacterial/metabolism , Drug Resistance, Bacterial , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Profiling , Leucine-Responsive Regulatory Protein/genetics , Multigene Family , Operon , Protein Binding , Real-Time Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
The cyanobacterium Synechococcus elongatus strain PCC 7942 possesses pANL, a plasmid rich in genes related to sulfur metabolism. One of these genes, srpC, encodes the SrpC protein, a homologue of the CHR chromate ion transporter superfamily. The srpC gene was cloned and expressed in Escherichia coli and its role in relation to sulfate and chromate was analyzed. srpC was unable to complement the growth of an E. coli cysA sulfate uptake mutant when sulfate was utilized as a sole sulfur source, suggesting that SrpC is not a sulfate transporter. Expression of srpC in E. coli conferred chromate resistance and caused diminished chromate uptake. These results suggest that the S. elongatus SrpC protein functions as a transporter that extrudes chromate ions from the cell's cytoplasm, and further demonstrate the close relationship between sulfate and chromate metabolism in this organism.
Subject(s)
Anti-Bacterial Agents/metabolism , Chromates/metabolism , Drug Resistance, Bacterial , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Synechococcus/genetics , Synechococcus/metabolism , Cloning, Molecular , Escherichia coli/genetics , Multigene Family , Plasmids , Sulfates/metabolismABSTRACT
Sulfur is an essential element for microorganisms and it can be obtained from varied compounds, sulfate being the preferred source. The first step for sulfate assimilation, sulfate uptake, has been studied in several bacterial species. This article reviews the properties of different bacterial (and archaeal) transporters for sulfate, molybdate, and related oxyanions. Sulfate uptake is carried out by sulfate permeases that belong to the SulT (CysPTWA), SulP, CysP/(PiT), and CysZ families. The oxyanions molybdate, tungstate, selenate and chromate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC system and tungstate by the TupABC and WtpABC systems. CysPTWA, ModABC, TupABC, and WtpABC are homologous ATP-binding cassette (ABC)-type transporters with similar organization and properties. Uptake of selenate and chromate oxyanions occurs mainly through sulfate permeases.
Subject(s)
Molybdenum/metabolism , Sulfates/metabolism , Anion Transport Proteins/metabolism , Anions/metabolism , Bacteria/metabolism , Biological Transport , Chromates/metabolism , Selenic Acid , Selenium Compounds/metabolism , Tungsten Compounds/metabolismABSTRACT
Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily (ywrB and ywrA) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C, respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli, suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacillus subtilis/metabolism , Chromates/metabolism , Drug Resistance, Bacterial , Escherichia coli/drug effects , Membrane Transport Proteins/metabolism , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromates/pharmacology , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Gene Order , Ions/metabolism , Ions/pharmacology , Membrane Transport Proteins/genetics , Operon , Transcription, GeneticABSTRACT
The plasmidic chromate resistance genes chrBAC from Shewanella sp. strain ANA-3 were transferred to Escherichia coli. Expression of chrA alone, on a high- or low-copy number plasmid, conferred increased chromate resistance. In contrast, expression of the complete operon chrBAC on a high-copy number plasmid did not result in a significant increase in resistance, although expression on a low-copy number plasmid made the cells up to 10-fold more resistant to chromate. The chrA gene also conferred increased chromate resistance when expressed in Pseudomonas aeruginosa. The chrR gene from the P. aeruginosa chromosome was necessary for full chromate resistance conferred by chrA. A diminished chromate uptake in cells expressing the chrA gene suggests that chromate resistance is due to chromate efflux.
