Analysis of mercuric reductase (merA) gene diversity in an anaerobic mercury-contaminated sediment enrichment

The reduction of ionic mercury to elemental mercury by the mercuric reductase (MerA) enzyme plays an important role in the biogeochemical cycling of mercury in contaminated environments by partitioning mercury to the atmosphere. This activity, common in aerobic environments, has rarely been examined in anoxic sediments where production of highly toxic methylmercury occurs. Novel degenerate PCR primers were developed which span the known diversity of merA genes in Gram-negative bacteria and amplify a 285 bp fragment at the 3' end of merA. These primers were used to create a clone library and to analyse merA diversity in an anaerobic sediment enrichment collected from a mercury-contaminated site in the Meadowlands, New Jersey. A total of 174 sequences were analysed, representing 71 merA phylotypes and four novel MerA clades. This first examination of merA diversity in anoxic environments suggests an untapped resource for novel merA sequences.     

Evolution of mercuric reductase (<Emphasis Type="Italic">merA</Emphasis>) gene: A case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA gene is key gene in mer operon and has been used for this swtudy. Phylogenetic analysis of aligned merA gene sequences shows broad similarities to the established 16S rRNA gene phylogeny. But there is no separation of bacterial merA gene from archael merA gene which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA gene and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.     

Evolution of gene fusions: horizontal transfer versus independent events

Background  

Gene fusions can be used as tools for functional prediction and also as evolutionary markers. Fused genes often show a scattered phyletic distribution, which suggests a role for processes other than vertical inheritance in their evolution.     

Evolution by acquisition: the case for horizontal gene transfers

One of the most debated questions in the field of molecular evolution is the possible role of horizontal transfer in evolution. Of all the claims that have been made over the years, those reporting transfers between eukaryotes and prokaryotes are the most controversial. Here we present the cases for and against several such possible gene acquisitions.     

Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria

Background  

The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 and nad5 genes. Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera).     

Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer

The isopenicillin N synthase genes from three fungal species, three Gram-positive species, and one Gram-negative bacterial species share an unusually high sequence similarity. A phylogenetic analysis was carried out to determine which type of evolutionary scenario best accounts for this similarity. The most plausible scenario is one in which a horizontal gene-transfer event, from the prokaryotes to the eukaryotes, occurred at a time close to the divergence between the Gram-positive and the Gram-negative bacteria.     

Evolution of acetoclastic methanogenesis in Methanosarcina via horizontal gene transfer from cellulolytic Clostridia

Phylogenetic analysis confirmed that two genes required for acetoclastic methanogenesis, ackA and pta, were horizontally transferred to the ancestor of Methanosarcina from a derived cellulolytic organism in the class Clostridia. This event likely occurred within the last 475 million years, causing profound changes in planetary methane biogeochemistry.     

Towards a quantitative understanding of horizontal gene transfer: a kinetic model

In this work, we present a simple kinetic model of horizontal gene transfer. It describes the processes of gene duplication, mutation, gene transfer and the regulation of the total size of the genome for genetically homogeneous prokaryotic species or strains. The emerging nonlinear system of first-order differential equations can be linearized at the stationary point. For selected models, we give an analytical solution for the number of foreign and native genes within a species. We identify a regime characterized by a fast gene transfer rate and species with a mixed genome, a slow gene transfer regime with pure organisms, and a crossover region. The data are compared to experiments, and the biological implications of our model are discussed.     

DarkHorse: a method for genome-wide prediction of horizontal gene transfer

A new approach to rapid, genome-wide identification and ranking of horizontal transfer candidate proteins is presented. The method is quantitative, reproducible, and computationally undemanding. It can be combined with genomic signature and/or phylogenetic tree-building procedures to improve accuracy and efficiency. The method is also useful for retrospective assessments of horizontal transfer prediction reliability, recognizing orthologous sequences that may have been previously overlooked or unavailable. These features are demonstrated in bacterial, archaeal, and eukaryotic examples.     

Plasmid-mediated horizontal gene transfer is a coevolutionary process

Conjugative plasmids are key agents of horizontal gene transfer (HGT) that accelerate bacterial adaptation by vectoring ecologically important traits between strains and species. However, although many conjugative plasmids carry beneficial traits, all plasmids exert physiological costs-of-carriage on bacteria. The existence of conjugative plasmids, therefore, presents a paradox because non-beneficial plasmids should be lost to purifying selection, whereas beneficial genes carried on plasmids should be integrated into the bacterial chromosome. Several ecological solutions to the paradox have been proposed, but none account for co-adaptation of bacteria and conjugative plasmids. Drawing upon evidence from experimental evolution, we argue that HGT via conjugation can only be fully understood in a coevolutionary framework.     

Phylogeny vs genome reshuffling: horizontal gene transfer

The evolutionary events in organisms can be tracked to the transfer of genetic material. The inheritance of genetic material among closely related organisms is a slow evolutionary process. On the other hand, the movement of genes among distantly related species can account for rapid evolution. The later process has been quite evident in the appearance of antibiotic resistance genes among human and animal pathogens. Phylogenetic trees based on such genes and those involved in metabolic activities reflect the incongruencies in comparison to the 16S rDNA gene, generally used for taxonomic relationships. Such discrepancies in gene inheritance have been termed as horizontal gene transfer (HGT) events. In the post-genomic era, the explosion of known sequences through large-scale sequencing projects has unraveled the weakness of traditional 16S rDNA gene tree based evolutionary model. Various methods to scrutinize HGT events include atypical composition, abnormal sequence similarity, anomalous phylogenetic distribution, unusual phyletic patterns, etc. Since HGT generates greater genetic diversity, it is likely to increase resource use and ecosystem resilience.     

Detecting highways of horizontal gene transfer

In a horizontal gene transfer (HGT) event, a gene is transferred between two species that do not have an ancestor-descendant relationship. Typically, no more than a few genes are horizontally transferred between any two species. However, several studies identified pairs of species between which many different genes were horizontally transferred. Such a pair is said to be linked by a highway of gene sharing. We present a method for inferring such highways. Our method is based on the fact that the evolutionary histories of horizontally transferred genes disagree with the corresponding species phylogeny. Specifically, given a set of gene trees and a trusted rooted species tree, each gene tree is first decomposed into its constituent quartet trees and the quartets that are inconsistent with the species tree are identified. Our method finds a pair of species such that a highway between them explains the largest (normalized) fraction of inconsistent quartets. For a problem on n species and m input quartet trees, we give an efficient O(m + n(2))-time algorithm for detecting highways, which is optimal with respect to the quartets input size. An application of our method to a dataset of 1128 genes from 11 cyanobacterial species, as well as to simulated datasets, illustrates the efficacy of our method.     

Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase

We have determined the nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 and have predicted the structure of the gene product, mercuric reductase. The DNA sequence predicts a polypeptide of Mr 58 660, the primary structure of which shows strong homologies to glutathione reductase and lipoamide dehydrogenase, but mercuric reductase contains as additional N-terminal region that may form a separate domain. The implications of these comparisons for the tertiary structure and mechanism of mercuric reductase are discussed. The DNA sequence presented here has an overall G+C content of 65.1 mol%, typical of the bulk DNA of Pseudomonas aeruginosa from which Tn501 was originally isolated. Analysis of the codon usage in the merA gene shows that codons with C or G at the third position are preferentially utilized.     

Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.     

Nucleotide sequence of the Thiobacillus ferrooxidans chromosomal gene encoding mercuric reductase

The nucleotide sequence of the Thiobacillus ferrooxidans chromosomal mercuric-reductase-encoding gene (merA) has been determined. The merA gene contains 1635 bp, and shares 78.2% and 76.6% sequence homology with the transposon, Tn501, and plasmid R100 merA genes, respectively. From the sequence, a 545-amino acid (aa) polypeptide was deduced, and comparison with those of Tn501 and R100 revealed 80.6% and 80.0% homology, respectively, at the aa sequence level. Divergence among the three merA aa sequences was clustered within a specific region (aa positions 41-87). By analysis of codon usage frequency, it is speculated that the T. ferrooxidans merA gene originated from Tn501, R100, or a common ancestral gene, but not from T. ferrooxidans itself.     

Electronic energy transfer and fluorescence quenching in the active sites of mercuric reductase

The FAD-containing enzyme mercuric reductase has been studied by means of steady-state and time-resolved fluorescence spectroscopy. The fluorescence relaxation of the excited state of the isoalloxazine ring of FAD can be described by a sum of two exponential functions. The two lifetimes are not due to a different lifetime of each of the two FAD molecules of mercuric reductase. The FAD molecules are quenched dynamically by a quencher that is not sensitive to the solvent viscosity. In vitro activation induces a dynamic quenching of fluorescence, while upon binding of NADP+ the FAD molecules are both statically and dynamically quenched. Time-resolved fluorescence anisotropy experiments of mercuric reductase in water show that the isoalloxazine ring probably undergoes a rapid and restricted vibrational motion of small amplitude. Electronic energy transfer occurs between the two FAD molecules at a rate of about 3.4 x 10(7) s-1. The angle between the emission transition dipole of the donor and the absorption transition dipole of the acceptor is 137 +/- 2 degrees (or 43 +/- 2 degrees). From previous X-ray data of glutathione reductase we find that the corresponding angle is 160 degrees. This suggests that the isoalloxazine rings of mercuric reductase and glutathione reductase are mutually tilted in slightly different ways.     

Monitoring and modeling horizontal gene transfer

Monitoring efforts have failed to identify horizontal gene transfer (HGT) events occurring from transgenic plants into bacterial communities in soil or intestinal environments. The lack of such observations is frequently cited in biosafety literature and by regulatory risk assessment. Our analysis of the sensitivity of current monitoring efforts shows that studies to date have examined potential HGT events occurring in less than 2 g of sample material, when combined. Moreover, a population genetic model predicts that rare bacterial transformants acquiring transgenes require years of growth to out-compete wild-type bacteria. Time of sampling is there-fore crucial to the useful implementation of monitoring. A population genetic approach is advocated for elucidating the necessary sample sizes and times of sampling for monitoring HGT into large bacterial populations. Major changes in current monitoring approaches are needed, including explicit consideration of the population size of exposed bacteria, the bacterial generation time, the strength of selection acting on the transgene-carrying bacteria, and the sample size necessary to verify or falsify the HGT hypotheses tested.     

Structural characterization of intramolecular Hg(2+) transfer between flexibly linked domains of mercuric ion reductase

The enzyme mercuric ion reductase MerA is the central component of bacterial mercury resistance encoded by the mer operon. Many MerA proteins possess metallochaperone-like N-terminal domains (NmerA) that can transfer Hg²⁺ to the catalytic core domain (Core) for reduction to Hg⁰. These domains are tethered to the homodimeric Core by ∼ 30-residue linkers that are susceptible to proteolysis, the latter of which has prevented characterization of the interactions of NmerA and the Core in the full-length protein. Here, we report purification of homogeneous full-length MerA from the Tn21 mer operon using a fusion protein construct and combine small-angle X-ray scattering and small-angle neutron scattering with molecular dynamics simulation to characterize the structures of full-length wild-type and mutant MerA proteins that mimic the system before and during handoff of Hg²⁺ from NmerA to the Core. The radii of gyration, distance distribution functions, and Kratky plots derived from the small-angle X-ray scattering data are consistent with full-length MerA adopting elongated conformations as a result of flexibility in the linkers to the NmerA domains. The scattering profiles are best reproduced using an ensemble of linker conformations. This flexible attachment of NmerA may facilitate fast and efficient removal of Hg²⁺ from diverse protein substrates. Using a specific mutant of MerA allowed the formation of a metal-mediated interaction between NmerA and the Core and the determination of the position and relative orientation of NmerA to the Core during Hg²⁺ handoff.