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Kaare M Nielsen

Publications and source records attributed to Kaare M Nielsen.

12 recordsLinked to original sources
PubMed2006

Modeling suggests frequency estimates are not informative for predicting the long-term effect of horizontal gene transfer in bacteria.

Horizontal gene transfer (HGT) is an important mechanism by which bacteria recombine and acquire novel genes and functions. Risk scenarios where novel plant transgenes transfer horizontally into bacteria have been addressed in numerical theoretical assessments and experimental studies. A key outcome of these studies has been that the frequencies of such inter-domain transfer are very low, if occurring at all, suggesting that such transfers would not occur at a level that is biologically significant. The relationship between transfer frequencies and the subsequent selection or genetic drift of transgene carrying bacteria often remains unresolved in these studies and assessments. Here we present a stochastic model to better understand the initial establishment and population dynamics of rare bacterial transformants carrying horizontally acquired (trans)genes. The following key parameters are considered: initial transformant numbers, strength of selection, bacterial population size and bacterial generations (time). We find that the initial number of transformants is important for the subsequent persistence of transformants only in the range of 1 to approximately 50 independent HGT events. Our simulations show that transformant populations under a wide range of HGT rates and selection coefficients undergo stochastic developments where they persist at low frequencies for up to several years (at frequencies that are below detection using available field sampling methodology), after which they eventually may go to fixation. Stochastic variability may thus play a crucial but disregarded role in the design of field monitoring strategies e.g. in biosafety assessments. We also estimate the time required for transformants to reach 0.0002% prevalence in a bacterial population, a threshold that allows experimental detection of transgene carrying bacteria through sampling of the larger bacterial populations.

Bacteria↗
PubMed2006

Population structure and gene evolution in Saccharomyces cerevisiae.

The fully sequenced genomes of four species within the Saccharomyces sensu stricto complex provide a wealth of information for molecular-evolutionary inference. Yet virtually nothing is known about population-genetic variation within these species, including the molecular-biological and genetic-model organism S. cerevisiae. Here we investigate the population-genetic variation and population structure of S. cerevisiae by sequencing the four loci CDC19, PHD1, FZF1 and SSU1 in 27 strains. Sequence analysis demonstrates a distinct population structure in S. cerevisiae, distinguishing strains collected from a Pennsylvanian oak forest and strains collected from vineyards, perhaps due to ecological rather than geographic factors. The low level of conflict observed between the gene trees estimated for each locus implies moderate recombination in nature. High polymorphism in the gene SSU1 provides evidence of diversifying selection on its protein product, a sulfite exporter, perhaps associated with the use of sulfur-based fungicides in vineyards. FZF1, encoding a transcription factor regulating the expression level of SSU1, displays even greater polymorphism. This, the first multilocus sequence study of population structure in natural isolates of S. cerevisiae, is the first study to demonstrate population structure within S. cerevisiae, and the first study to detect historical selection on a locus important to the natural history of wine yeast.

Cloning, Molecular↗
PubMed2005

Nucleic acid isolation from ecological samples--vertebrate gut flora.

The utility of DNA molecules in identifying and characterizing intestinal microorganisms depends on methods that facilitate access to DNA of sufficient purity, quantity, and integrity. An efficient and unbiased extraction of DNA is thus critical to the validity of the subsequent analysis of the prevalence and diversity of the DNA sources in the sample. The highly heterogeneous composition of the diet of vertebrates makes DNA isolation challenging for this environment. Here, we consider the key steps involved in DNA isolation from vertebrate gut microflora including sample homogenization, lysis of bacterial cells, and extraction and precipitation of DNA. A detailed protocol for DNA isolation of the microbial contents of intestine and feces is also provided. In addition, we refer to commercially available methods for DNA extraction from the vertebrate gut flora.

Animals↗
PubMed2005

Experimental methods for assaying natural transformation and inferring horizontal gene transfer.

The observation of frequent lateral acquisitions of genes in sequenced bacterial genomes has spurred experimental investigations to elucidate the factors governing ongoing gene transfer processes in bacteria. The uptake of naked DNA by natural transformation is known to occur in a wide range of bacterial species and in some archaea. We describe a series of protocols designed to dissect the natural genetic transformability of individual bacterial strains under conditions that progress from standard in vitro conditions to purely in situ, or natural, conditions. One of the most important factors in ensuring the success of any transformation assay system is the use of a sensitive, effective, and distinguishable selection regimen. Detailed template protocols for assaying bacterial transformation in vitro are presented using the naturally competent bacterium Acinetobacter baylyi strain BD413 as a model. Factors increasing the complexity of the assay systems are included in the following section describing the incorporation of components of natural systems to the in vitro models, such as in soil and water microcosm experiments. We then present template protocols for the transformation of bacteria in modified natural systems, such as in the presence of host tissues and extracts or in the greenhouse. Clear and ecologically meaningful demonstrations of in situ natural transformation are most desirable but are also the most complex and challenging. Because of the highly variable nature of these experiments, we include a discussion of important factors that should be considered when designing such experiments. Some advantages and disadvantages of the experimental systems with regard to resolving the hypotheses tested are included in each section.

Acinetobacter↗
PubMed2005

Mechanisms of, and barriers to, horizontal gene transfer between bacteria.

Bacteria evolve rapidly not only by mutation and rapid multiplication, but also by transfer of DNA, which can result in strains with beneficial mutations from more than one parent. Transformation involves the release of naked DNA followed by uptake and recombination. Homologous recombination and DNA-repair processes normally limit this to DNA from similar bacteria. However, if a gene moves onto a broad-host-range plasmid it might be able to spread without the need for recombination. There are barriers to both these processes but they reduce, rather than prevent, gene acquisition.

Bacteria↗
PubMed2004

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.

Ecosystem↗
PubMed2003

Gene conversion as a source of nucleotide diversity in Plasmodium falciparum.

Examination of polymorphisms in the Plasmodium falciparum gene for falcipain 2 revealed that this gene is one of two paralogs separated by 10.8 kb in chromosome 11. We designate the annotated gene denoted chr11.gen_424 as encoding falcipain 2A and the annotated gene denoted chr11.gen_427 as encoding falcipain 2B. The paralogs are 96% identical at the nucleotide level and 93% identical at the amino acid level. The consensus sequences differ in 31/309 synonymous sites and 45/1140 nonsynonymous sites, including three amino acid replacements (V393I, A400P, and Q414E) that are near the catalytic site and that may affect substrate affinity or specificity. In six reference isolates, among 36 synonymous sites and 46 nonsynonymous sites that are polymorphic in the gene for falcipain 2A, falcipain 2B, or both, significant spatial clustering is observed. All but one of the polymorphisms appear to result from gene conversion between the paralogs. The estimated rate of gene conversion between the paralogs may be as many as 1,400 to 1,700 times greater than the rate of mutation. Owing to gene conversion, one of the falcipain 2A alleles is more similar to the falcipain 2B alleles than it is to other falcipain 2A alleles. Divergence among the synonymous sites suggests that the paralogous genes last shared a common ancestor 15.2 MYA, with a range of 8.8 to 20.6 MYA. During this period, the paralogs have acquired 0.10 synonymous substitutions per synonymous site in the coding region. The 5' and 3' flanking regions differ in 47.7% and 39.8% of the nucleotide sites, respectively. Hence synonymous sites and flanking regions are not conserved in sequence in spite of their high AT content and T skew.

Animals↗
PubMed2003

Horizontal acquisition of divergent chromosomal DNA in bacteria: effects of mutator phenotypes.

We examine the potential beneficial effects of the expanded access to environmental DNA offered by mutators on the adaptive potential of bacterial populations. Using parameters from published studies of recombination in E. coli, we find that the presence of mutators has the potential to greatly enhance bacterial population adaptation when compared to populations without mutators. In one specific example, for which three specific amino acid substitutions are required for adaptation to occur in a 300-amino-acid protein, we found a 3500-fold increase in the rate of adaptation. The probability of a beneficial acquisition decreased if more amino acid changes, or integration of longer DNA fragments, were required for adaptation. The model also predicts that mutators are more likely than nonmutator phenotypes to acquire genetic variability from a more diverged set of donor bacteria. Bacterial populations harboring mutators in a sequence heterogeneous environment are predicted to acquire most of their DNA conferring adaptation in the range of 13-30% divergence, whereas nonmutator phenotypes become adapted after recombining with more homogeneous sequences of 7-21% divergence. We conclude that mutators can accelerate bacterial adaptation when desired genetic variability is present within DNA fragments of up to approximately 30% divergence.

Data Interpretation, Statistical↗
PubMed2002

Excess polymorphisms in genes for membrane proteins in Plasmodium falciparum.

The detection of single-nucleotide polymorphisms in pathogenic microorganisms has normally been carried out by trial and error. Here we show that DNA hybridization with high-density oligonucleotide arrays provides rapid and convenient detection of single-nucleotide polymorphisms in Plasmodium falciparum, despite its exceptionally high adenine-thymine (AT) content (82%). A disproportionate number of polymorphisms are found in genes encoding proteins associated with the cell membrane. These genes are targets for only 22% of the oligonucleotide probes but account for 69% of the polymorphisms. Genetic variation is also enriched in subtelomeric regions, which account for 22% of the chromosome but 76% of the polymorphisms.

Animals↗
PubMed2002

The paradoxical population genetics of Plasmodium falciparum.

Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies.

Animals↗