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PubMed2003

Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes.

Ralstonia metallidurans, formerly known as Alcaligenes eutrophus and thereafter as Ralstonia eutropha, is a beta-Proteobacterium colonizing industrial sediments, soils or wastes with a high content of heavy metals. The type strain CH34 carries two large plasmids (pMOL28 and pMOL30) bearing a variety of genes for metal resistance. A chronological overview describes the progress made in the knowledge of the plasmid-borne metal resistance mechanisms, the genetics of R. metallidurans CH34 and its taxonomy, and the applications of this strain in the fields of environmental remediation and microbial ecology. Recently, the sequence draft of the genome of R. metallidurans has become available. This allowed a comparison of these preliminary data with the published genome data of the plant pathogen Ralstonia solanacearum, which harbors a megaplasmid (of 2.1 Mb) carrying some metal resistance genes that are similar to those found in R. metallidurans CH34. In addition, a first inventory of metal resistance genes and operons across these two organisms could be made. This inventory, which partly relied on the use of proteomic approaches, revealed the presence of numerous loci not only on the large plasmids pMOL28 and pMOL30 but also on the chromosome. It suggests that metal-resistant Ralstonia, through evolution, are particularly well adapted to the harsh environments typically created by extreme anthropogenic situations or biotopes.

Adenosine Triphosphatases↗
PubMed2002

Phylogenetic analysis shows that the OXA beta-lactamase genes have been on plasmids for millions of years.

The OXA genes encode a class of beta-lactamases that confer resistance to a wide range of beta-lactam antibiotics. To determine whether the diversity of the OXA beta-lactamases is the result of recent or ancient events, and to determine whether mobilization of the OXA genes from chromosomes to plasmids occurred recently or long ago, we have constructed a Bayesian phylogeny of the OXA beta-lactamase genes. Analysis of that phylogeny shows that much of the diversity is the result of ancient events and that the OXA genes were mobilized from chromosomes to plasmids on at least two independent occasions that occurred millions of years ago. That observation contradicts the commonly held impression that mobilization of antibiotic resistance genes is strictly the result of modern use of antibiotics.

Bayes Theorem↗
PubMed2003

Mitochondrial genome diversity: evolution of the molecular architecture and replication strategy.

Mitochondrial genomes in organisms from diverse phylogenetic groups vary in both size and molecular form. Although the types of mitochondrial genome appear very dissimilar, several lines of evidence argue that they do not differ radically. This would imply that interconversion between different types of mitochondrial genome might have occurred via relatively simple mechanisms. We exemplify this scenario on patterns accompanying evolution of mitochondrial telomeres. We propose that mitochondrial telomeres are derived from mobile elements (transposons or plasmids) that invaded mitochondria, integrated into circular or polydisperse linear mitochondrial DNAs (mtDNAs) and subsequently enabled precise resolution of the linear genophore. Simply, the selfish elements generated a problem - how to maintain the ends of a linear DNA - and, at the same time, made themselves essential by providing its solution. This scenario implies that insertion or deletion of such resolution elements may represent relatively simple routes for interconversion between different forms of the mitochondrial genome.

DNA Replication↗
PubMed1992

Mutagenesis induced by bacterial UmuDC proteins and their plasmid homologues.

The popular image of a world full of pollutants mutating DNA is only partly true since there are relatively few agents which can subtly and directly change base coding; for example, some alkylating agents alter guanine so that it pairs like adenine. Many more mutagens are less subtle and simply destroy coding altogether rather than changing it. Such mutagens include ultraviolet light, X-rays, DNA cross-linkers and other agents which make DNA breaks or large adducts. In Escherichia coli, mutagenesis by these agents occurs during a DNA repair process which increases cell survival but with an inherent possibility of changing the original sequence. Such mutagenic DNA repair is, in part, encoded by the E. coli umuDC operon. This article reviews the structure, function, regulation and evolution of the umuDC operon and similar genes found both in other species and on naturally occurring plasmids.

Amino Acid Sequence↗
PubMed1982

Structure and evolution of human alpha-fetoprotein deduced from partial sequence of cloned cDNA.

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human alpha-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% (56/128) amino acids and 54% (207/384) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.

Amino Acid Sequence↗
PubMed2002

[Molecular evolution of beta-glucuronidase in vitro: obtaining thermotolerant GUS gene].

The Escherichia coli beta-glucuronidase gene (gus) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited by some plants and plant-associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta-glucuronidase. Using plasmid pBI121 as template, a 1.8 kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves DNase I digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS3-3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3-3 enzyme can resistant high temperature up to 80 degrees C for 30 min. The nucleotide sequence analysis showed 99.2% homology between the GUS-ck gene from pBI121 and GUS3-3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3-3 is 80 degrees C and increased by 25 degrees C above GUS-ck (55 degrees C). The researches indicated the feasibility of the molecular evolution of beta-glucuronidase in vitro to improve enzymatic thermostability.

Amino Acid Sequence↗
PubMed1998

Witnessing the evolution of transcription in mitochondria: the mitochondrial genome of the primitive brown alga Pylaiella littoralis (L.) Kjellm. Encodes a T7-like RNA polymerase.

A region of the mitochondrial genome of the primitive brown alga Pylaiella littoralis containing a plasmid-like insert which contains a transcribed T7-phage-type RNA polymerase gene is described. This is a first report of a phage-type RNA polymerase gene integrated in a mitochondrial genome. As the mitochondrial genome of this alga also contains sigma-70 proteobacterial promoter regions, i.e. traces of the ancestral alpha2betabeta'sigma-70 proteobacterial RNA polymerase, this genome witnesses two types of RNA polymerases. As such the mitochondrial genome of P. littoralis represents a unique stage in the evolution of transcription in mitochondria, which contrasts with that of the primitive protist Reclinomonas americana, which still retains the ancestral alpha2betabeta'sigma-70 proteobacterial RNA polymerase genes, and with animals, land plants and fungi, which use phage-type polymerases.

Chromosome Mapping↗
PubMed1993

The specific growth rate of Pseudomonas putida PAW1 influences the conjugal transfer rate of the TOL plasmid.

The kinetics of the conjugal transfer of a TOL plasmid were investigated by using Pseudomonas putida PAW1 as the donor strain and P. aeruginosa PAO 1162 as the recipient strain. Short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. The experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. This method allowed analysis of the mating experiments by a single intrinsic kinetic parameter for conjugal plasmid transfer. Further results indicated that the specific growth rate of the donor strain antecedent to the mating experiment had a strong impact on the measured intrinsic plasmid transfer rate coefficient, which ranged from 1 x 10(-14) to 5 x 10(-13) ml per cell per min. Preliminary analysis suggested that the transfer rates of the TOL plasmid are large enough to maintain the TOL plasmid in a dense microbial community without selective pressures.

Conjugation, Genetic↗
PubMed2007

An H-NS-like stealth protein aids horizontal DNA transmission in bacteria.

The Sfh protein is encoded by self-transmissible plasmids involved in human typhoid and is closely related to the global regulator H-NS. We have found that Sfh provides a stealth function that allows the plasmids to be transmitted to new bacterial hosts with minimal effects on their fitness. Introducing the plasmid without the sfh gene imposes a mild H-NS(-) phenotype and a severe loss of fitness due to titration of the cellular pool of H-NS by the A+T-rich plasmid. This stealth strategy seems to be used widely to aid horizontal DNA transmission and has important implications for bacterial evolution.

Bacterial Proteins↗
PubMed1994

Chimeric evolution of the 2-microns genome in Saccharomyces cerevisiae.

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 microns) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-microns variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-microns plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection.

Base Sequence↗
PubMed1992

Characterization and nucleotide sequence of the oriT-traM-finP region of the IncFVII plasmid pSU233.

By hybridizing the IncFVII haemolytic plasmid pSU233 with a probe containing the origin of transfer of the IncFII plasmid R1, we isolated a 1.9 kb BglII fragment containing at least the origin of transfer (oriT), and the genes traM and finP. Functional complementation analysis of deletion derivatives was used to map the origin of transfer. We also determined the nucleotide sequence of traM and finP. Comparison with similar regions of several plasmids, also belonging to the Rep-FIIA family, revelaed that pSU233 resembles the F plasmid by very close. The homology is not evenly distributed along this region, but clustered into homologous regions (TraZb-oriT, TraMb-oriT and traM separated by non-homologous regions (TraYb-oriT, finP). This organization resembles that reported for the replication region and also suggests evolution by exchange of modules. In addition, the nucleotide sequence of finP is different from those previously described for other IncF plasmids and constitutes a new allele, which we have denominated allele VI.

Amino Acid Sequence↗
PubMed1987

Structure and expression of dog apolipoprotein C-II and C-III mRNAs. Implications for the evolution and functional constraints of apolipoprotein structure.

The complete cDNAs for canine apolipoprotein (apo) C-II and C-III have been cloned in the plasmid vector, pUC-18, and the phage vector, lambda gt11. With 32P-labeled nick-translated cDNAs as hybridization probes, apoC-II and apoC-III mRNAs were found to be expressed in canine liver, jejunum, ileum, colon, pancreas, spleen, ovary, and bladder at the following relative concentrations: 100, 35, 30, 10, 15, 5, 30, and 2% for apoC-II, and 100, 35, 15, 2, 20, 5, 30, and 5% for apoC-III. Neither mRNA was detected in canine brain, kidney, lung, muscle, oviduct, uterus, or testis. Both mRNAs are approximately 700 nucleotides in length. Complete sequence analysis revealed that apoC-II mRNA codes for a protein of 101 amino acid residues including 22 residues in the signal peptide and 79 residues in the mature peptide region. ApoC-III mRNA codes for a protein of 100 amino acid residues, including 20 in the signal peptide and 80 residues in the mature peptide region. It contains a single cysteine residue in position 54. Secondary structural analysis shows the presence of two regions each of amphipathic helix for apoC-II (residues 14-33 and 44-63) and apoC-III (residues 16-33 and 50-69). Computer analysis of the amphipathic helical regions demonstrates the importance of variation in the dihedral angles of the side chains in the charge microenvironment of the helices. Statistical analysis of sequence similarities between human apoC-II, apoC-III, apoA-II, apoA-I, apoE, and apoA-IV, and dog apoC-II and apoC-III, and rat apoC-III, apoA-II, apoA-I, apoE, and apoA-IV indicates that all these proteins have evolved rapidly, especially in the rat in which apoC-III has evolved at three times the rate in man and dog. Further, the shorter apolipoproteins, apoA-II, apoC-II, and a-poC-III, have evolved more rapidly than the longer ones, apoA-I, apoE, and apoA-IV. Some regions in the individual apolipoproteins are better conserved than others. The rate of evolution of individual regions seems to be related to the stringency of their functional requirements.

Amino Acid Sequence↗
PubMed2000

Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda, and COS7 cells for recombinant gene expression. Application to a rabbit liver carboxylesterase.

Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells. Although, recombinant protein was observed in E. coli sonicates, little or no enzymatic activity was detected. Similarly, no activity was observed following expression in S. cerevisiae. In contrast, active protein was produced in P. pastoris, from S. frugiperda following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA. For the preparation of small amounts of protein for kinetic and biochemical studies, enzyme expressed in P. pastoris has proved sufficient. However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated expression of a secreted form of the protein in S. frugiperda was the most efficient. Using this system, we have generated and purified milligram quantities of essentially pure protein. These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase, and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification.

Animals↗
PubMed2002

Characterisation and distribution of a cryptic Salmonella typhi plasmid pHCM2.

pHCM2 is a 106 kbp cryptic plasmid harboured by Salmonella typhi CT18, originally isolated from a typhoid patient in Vietnam. The genome of S. typhi CT18, including pHCM2, has recently been completely sequenced and annotated. Bioinformatic analysis revealed that 57% of the coding sequences (CDSs) encoded on pHCM2 display over 97% DNA sequence identity to the virulence-associated plasmid of Yersinia pestis, pFra. pHCM2 encodes no obvious virulence-associated determinants or antibiotic resistance genes but does encode a wide array of putative genes directly related to DNA metabolism and replication. PCR analysis of a series of S. typhi isolates from Vietnam detected pHCM2-related DNA sequences in some S. typhi isolated before, but not after, 1994. Similar pHCM2-related sequences were also detected in S. typhi isolated from other regions of South East Asia and Pakistan but not elsewhere in the world.

Base Sequence↗
PubMed1991

Elements in microbial evolution.

Spontaneous mutation, selection, and isolation are key elements in biological evolution. Molecular genetic approaches reveal a multitude of different mechanisms by which spontaneous mutants arise. Many of these mechanisms depend on enzymes, which often do not act fully at random on the DNA, although a large number of sites of action can be observed. Of particular interest in this respect are DNA rearrangement processes, e.g., by transposition and by site-specific recombination systems. The development of gene functions has thus to be seen as the result of both DNA rearrangement processes and sequence alterations brought about by nucleotide substitutions and small local deletions, insertions, and duplications. Prokaryotic microorganisms are particularly appropriate for studying the effects of spontaneous mutation and thus microbial evolution, as they have haploid genomes, so that genetic alterations become rapidly apparent phenotypically. In addition, bacteria and their viruses and plasmids have relatively small genomes and short generation times, which also facilitate research on evolutionary processes. Besides the strategy of development of gene functions in the vertical transmission of genomes from generation to generation, the acquisition of short DNA segments from other organisms appears to be an important strategy in microbial evolution. In this process of horizontal evolution natural vector DNA molecules are often involved. Because of acquisition barriers, the acquisition strategy works best for relatively small DNA segments, hence at the level of domains, single genes, or at most operons. Among the many enzymes and functional systems involved in vertical and horizontal microbial evolution, some may serve primarily for essential life functions in each individual and only secondarily contribute to evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1987

Evolutionary relationship between plasmids and phages: phasmid P4 as a model.

Phasmid (phage-plasmid) P4 is capable of propagating as a temperate phage, lytic helper-dependent phage, and as a plasmid (pP4vir1). Alteration of cellular growth conditions can be used to manipulate the copy number (n) of pP4vir1 from low (n = 1-2) to high (n = 30-40). The levels of gene expression of P4 in the plasmid and phage states are radically different. While there is high expression of P4 proteins in the phage state, low levels of gene expression are found in the plasmid mode of propagation thereby allowing pP4vir1 to remain intracellularly without perturbing host homeostasis. Possible evolutionary origins of phasmid P4 are discussed.

Bacteriophage P2↗
PubMed2005

Predicted hexameric structure of the Agrobacterium VirB4 C terminus suggests VirB4 acts as a docking site during type IV secretion.

The Agrobacterium T-DNA transporter belongs to a growing class of evolutionarily conserved transporters, called type IV secretion systems (T4SSs). VirB4, 789 aa, is the largest T4SS component, providing a rich source of possible structural domains. Here, we use a variety of bioinformatics methods to predict that the C-terminal domain of VirB4 (including the Walker A and B nucleotide-binding motifs) is related by divergent evolution to the cytoplasmic domain of TrwB, the coupling protein required for conjugative transfer of plasmid R388 from Escherichia coli. This prediction is supported by detailed sequence and structure analyses showing conservation of functionally and structurally important residues between VirB4 and TrwB. The availability of a solved crystal structure for TrwB enables the construction of a comparative model for VirB4 and the prediction that, like TrwB, VirB4 forms a hexamer. These results lead to a model in which VirB4 acts as a docking site at the entrance of the T4SS channel and acts in concert with VirD4 and VirB11 to transport substrates (T-strand linked to VirD2 or proteins such as VirE2, VirE3, or VirF) through the T4SS.

Amino Acid Sequence↗