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PubMed1996

Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1980

Persistence and spread of a chloramphenicol resistance-mediating plasmid in antigenic types of Escherichia coli, pathogenic for piglets.

All chloramphenicol-resistant Escherichia coli strains isolated from piglets in the State veterinary Serum Laboratory, Copenhagen, in 1974-1975 harbored plasmids of IncFII group with largely the same resistance markers. Two strains from 1978 carried plasmids with similar characters. Restriction enzyme analysis of DNA from these plasmids with restriction endonucleases EcoRI, BglII, and PstI shows that the Cm plasmids are extremely closely related; but the patterns obtained (particularly from PstI digests) enable the classification of the plasmids into groups. These bear a strong relation to time and place of isolation so that plasmids isolated on the same farm belong to the same group even when their host strains are of different antigenic types. It is concluded that these plasmids have evolved from a single plasmid.

Animals↗
PubMed2000

A pot-pourri of plasmid paradoxes: effects of a second copy.

Bacterial plasmids are exemplary subjects for study, being conveniently isolated, dissected, reassembled, and introduced into various hosts. Their versatility and power make them eminently worthy of our attention. In what follows I consider some consequences of simply doubling the dosage of particular plasmid genes or of forming a plasmid dimer. These consequences can be perverse, paradoxical, or informative. They bear on questions of cell viability, copy number limitation, clonal homogeneity, check-point control, and the recovery of mutants. They have relevance to biotechnology, evolution and medicine. In reviewing these effects, my motivation is largely to share my enthusiasm for certain kinds of biological narratives, the nature of which is best left for the reader to discern.

Bacteria↗
PubMed2006

Bacillus anthracis multiplication, persistence, and genetic exchange in the rhizosphere of grass plants.

Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Co-inoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species.

Bacillus anthracis↗
PubMed1994

Evidence for Darwinian selection of the 2-micron plasmid STB locus in Saccharomyces cerevisiae.

The 2-microns plasmid of industrial and laboratory strains of Saccharomyces cerevisiae exists as two main polymorphic forms designated type I and type II. Polymorphism is restricted to the 3200-bp right unique region where types I and II show approximately 10% nucleotide divergence in trans-acting REP1 and RAF loci and 30% divergence in the cis-acting STB locus. In addition, the cis-acting STB plasmid partition locus of type II plasmids varies in sequence and copy number of a 125-bp repeat. We devised chimeric and 2-microns plasmid stability experiments to evaluate the effect of STB polymorphism on plasmid fitness in amphiploid industrial and haploid laboratory strains. Reciprocal experiments of type-II STB chimeric plasmids in type-I bakers' yeast or a type-I chimeric plasmid in type-II distillers', wine, or haploid strains showed similar partition efficiencies. However, chimeric and 2-microns plasmids carrying a 250-bp STB from a type-II haploid strain had reduced fitness in a type-II industrial wine strain. These results in conjunction with molecular analyses of 2-microns-like and 2-microns plasmids indicates the coevolution of STB with trans-acting plasmid and host-cell factors.

Biological Evolution↗
PubMed2005

Changes in race-specific virulence in Pseudomonas syringae pv. phaseolicola are associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island.

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.

Amino Acid Sequence↗
PubMed1987

A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae.

We have developed an assay in S. cerevisiae in which clones of cells that contain intact dicentric minichromosomes are visually distinct from those that have rearranged to monocentric minichromosomes. We find that the instability of dicentric minichromosomes is apparently due to mitotic nondisjunction accompanied by occasional structural rearrangements. Monocentric minichromosomes arising by rearrangement of the plasmid are rapidly selected in the population since dicentric minichromosomes depress the rate of cell division. We show that the ability of one centromere to compete with another in dicentric minichromosomes requires the presence of both of the conserved structural elements, CDE II and CDE III. Dicentric minichromosomes can be stabilized if one of the centromeres on the molecule is functionally hypomorphic because of mutations in CDE II even though these mutant centromeres are highly efficient in monocentric molecules. Stable dicentric molecules can also be produced by decreasing the space between two wild-type centromeres on the same molecule. These results suggest plausible pathways for changes in chromosome number that accompany evolution.

Alleles↗
PubMed2001

Directed evolution of a recombinase for improved genomic integration at a native human sequence.

We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

Base Sequence↗
PubMed2006

Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.

Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.

Animals↗
PubMed2002

Identification of a chromosomal tra-like region in Agrobacterium tumefaciens.

The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.

Agrobacterium tumefaciens↗
PubMed2006

Detection of a novel variant bla(CTX-M-3) extended spectrum beta-lactamase gene in a community-acquired Escherichia coli isolate.

A highly cefotaxime- and cefepime-resistant but ceftazidime-sensitive Escherichia coli isolate was recovered from a community-acquired urinary infection of a Greek patient. Susceptibility testing, transfer assays, plasmid analysis as well as PCR and sequencing techniques were used to investigate the underlying mechanism of resistance. The isolate carried a new variant of the bla(CTX-M-3) gene that possessed a T instead of A at nt position 663. Cefotaxime resistance was transferable and carried on a 60 kb plasmid. The bla(CTX-M-3) variant was located downstream of an ISEcp1B element. The emergence of this new derivative indicates further evolution of the worldwide-distributed bla(CTX-M-3) gene.

Anti-Bacterial Agents↗
PubMed2001

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols.

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4'-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5' hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended.

Amino Acid Substitution↗
PubMed2007

Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.

Adaptation, Physiological↗
PubMed1983

Evolution of the influenza virus neuraminidase gene during drift of the N2 subtype.

The complete genetic information for the neuraminidase (NA) gene of influenza virus A/Bangkok/1/79 has been cloned by in vitro synthesis of dsDNA, insertion into pBR322 plasmid, and transformation of Escherichia coli. The nucleotide sequence of the NA gene has been determined by the Maxam and Gilbert method. It is 1466 nucleotides long and contains a single open reading frame with a coding capacity for 469 amino acids. When compared to the NA genes of the N2 strains A/Victoria/3/75, A/Udorn/72, A/NT/60/68, and A/RI/5-/57, 90% of the nucleotide positions and 87% of the amino acid positions remained invariant. Forty-two nucleotide changes and 14 amino acid changes accumulated in the period 1975-1979, but the general structure of the protein appeared to remain constant.

Amino Acid Sequence↗
PubMed1992

In vivo duplication of genetic elements by the formation of stem-loop DNA without an RNA intermediate.

Gene duplication through cDNA synthesis by reverse transcriptase is believed to have played an important role in the diversification of genomes during evolution. Here, we demonstrate that a genomic DNA sequence can be duplicated in vivo as a result of template switching. When an inverted repeat (IR) structure was inserted in a site downstream from a ColE1 plasmid origin of DNA replication, transformation of Escherichia coli cells with this plasmid resulted in the production of a new DNA fragment encompassing the region from the origin to the center of the IR structure. The structure of this DNA molecule is composed of a long stem-loop formed by a single-stranded DNA, in which the loop is formed by the IR structure. The DNA fragment is designated slDNA, for stem-loop DNA. The experiments in this study suggest that during DNA replication, template switching at the stem-loop structure formed by the IR structure gives rise to slDNA utilizing the nascent DNA strand or the parental strand as a template. The mechanistic implications of slDNA synthesis, and its possible roles in genome evolution, are discussed.

Base Sequence↗
PubMed1999

Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa.

We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.

Amino Acid Sequence↗
PubMed1996

cis-acting elements within an RNA coliphage genome: fold as you please, but fold you must!!

Using an in vivo complementation system, we conducted a mutational analysis of the bacteriophage Q beta readthrough cistron. In the Q beta cDNA-containing plasmid, pQ beta m100, we constructed six defined Q beta deletion cDNA genomes, each missing between 86 and 447 nucleotides from within the readthrough cistron. These deletion plasmids were introduced into host cells that are constitutively supplied with Q beta readthrough protein from the plasmid pQ beta RT. Under these conditions, all six deletion genomes spontaneously generated phage particles, each exhibiting a characteristic plaque phenotype and virus forming potential. Isolated readthrough-defective phage particles were subsequently used to infect host cells that carried helper readthrough protein. Passaged viruses yielded both larger plaques and higher titers, compared with those of the parent phages. Sequence analysis revealed that the genomes of the passaged viruses had deleted additional regions of readthrough RNA sequence. We discuss the possibilities that (1) the disruption of a well-defined structural domain in Q beta RNA was selectively disadvantageous to phage infection, and that (2) the evolved viral populations were selected by virtue of their ability to restore critical integrity of short and/or long-range nucleotide interactions within this region of Q beta RNA.

Base Sequence↗