Molecular organization of plasmid R906 (Inc P-1)

Genetic and restriction (for enzymes EcoRI, BamHI and HindIII) maps of the relatively broad host range plasmid R906 are constructed. There are two non-essential regions on the R906 DNA which can be deleted and cloned. Non-essential regions confer a resistance to different agents and restriction sites are clustered in these regions. Essential and conjugativity genes are located in two other DNA regions approximately at 0-23 and 29-44 kb of the R906 map. These large regions share a high level of homology with Inc-1 group plasmids R751 and RP4 according to Southern-blot hybridization and heteroduplex analyses. A transposon-like structure is found on the R751 DNA among R751/R906 heteroduplex molecules. This transposon of total length 5.1 kb has 1.4 kb inverted repeats at the ends. Bla genes of R906 and RP4 plasmids do not have homologous sequences. Data evidence that IncP-1 group plasmids irrespective to their original bacterial source and range of coded antibiotic resistance have very similar molecular organization. The role of possible factors which are responsible for the broad host range property of the IncP-1 group plasmids is discussed.     

Molecular clocks: when times are a-changin'

The molecular clock has proved to be extremely valuable in placing timescales on evolutionary events that would otherwise be difficult to date. However, debate has arisen about the considerable disparities between molecular and palaeontological or archaeological dates, and about the remarkably high mutation rates inferred in pedigree studies. We argue that these debates can be largely resolved by reference to the "time dependency of molecular rates", a recent hypothesis positing that short-term mutation rates and long-term substitution rates are related by a monotonic decline from the former to the latter. Accordingly, the extrapolation of rates across different timescales will result in invalid date estimates. We examine the impact of this hypothesis with respect to various fields, including human evolution, animal domestication and conservation genetics. We conclude that many studies involving recent divergence events will need to be reconsidered.     

Molecular clocks: four decades of evolution

During the past four decades, the molecular-clock hypothesis has provided an invaluable tool for building evolutionary timescales, and has served as a null model for testing evolutionary and mutation rates in different species. Molecular clocks have also influenced the development of theories of molecular evolution. As DNA-sequencing technologies have progressed, the use of molecular clocks has increased, with a profound effect on our understanding of the temporal diversification of species and genomes.     

Biological clocks help reduce the physiological conflicts in avian migrants

We present evidence from experiments on overwintering populations of two Palearctic-Indian latitudinal migratory birds, the black-headed bunting (Emberiza melanocephala) and the red-headed bunting (E. bruniceps), that the bird’s clock in interaction with day length regulates seasonal rhythms of migration and reproduction such that physiological conflict between them is reduced. Initiation and termination of the body mass and testicular cycles are separately regulated photoperiodic events. For example, under stimulatory photoperiods the response curve of body mass does not overlap with that of the testicular growth. A response-specific photoperiodism is adaptive, since gain in body mass, critical to spring migration, precedes gonadal recrudescence. Finally, migration as indicated by the night-time migratory restlessness under experimental situations (e.g., intense locomotion under caged condition-called Zugunruhe) appears to be regulated by a separate circadian oscillator.     

Molecular clocks do not support the Cambrian explosion

The fossil record has long supported the view that most animal phyla originated during a brief period approximately 520 MYA known as the Cambrian explosion. However, molecular data analyses over the past 3 decades have found deeper divergences among animals (approximately 800 to 1,200 MYA), with and without the assumption of a global molecular clock. Recently, two studies have instead reported time estimates apparently consistent with the fossil record. Here, we demonstrate that methodological problems in these studies cast doubt on the accuracy and interpretations of the results obtained. In the study by Peterson et al., young time estimates were obtained because fossil calibrations were used as maximum limits rather than as minimum limits, and not because invertebrate calibrations were used. In the study by Aris-Brosou and Yang, young time estimates were obtained because of problems with rate models and other methods specific to the study, and not because Bayesian methods were used. This also led to many anomalous findings in their study, including a primate-rodent divergence at 320 MYA. With these results aside, molecular clocks continue to support a long period of animal evolution before the Cambrian explosion of fossils.     

Replication of plasmid R1: Meselson-Stahl density shift experiments revisited

Meselson-Stahl density shift experiments have been used extensively to study selection and timing of plasmid replication. Experiments with plasmid R1 were previously performed and the conclusion was that this plasmid replicates one copy at a time and that there is an eclipse period after each replication during which no further replications can take place in the cell (Nordström et al., Plasmid1, 187–203 (1978)). However, this interpretation is in conflict with other data, mainly with those obtained in copy number shift experiments (Gustafsson and Nordström, J. Bacteriol.141, 106–110 (1980)). However, the density shift experiments have now been reinterpreted such that there no longer is any conflict with the copy number shift experiments. There does not seem to be any such eclipse period, but newly replicated plasmid molecules are not available for a second replication for about 20% of a generation time.     

Gene and cell survival: lessons from prokaryotic plasmid R1

Plasmids are units of extrachromosomal genetic inheritance found in all kingdoms of life. They replicate autonomously and undergo stable propagation in their hosts. Despite their small size, plasmid replication and gene expression constitute a metabolic burden that compromises their stable maintenance in host cells. This pressure has driven the evolution of strategies to increase plasmid stability--a process accelerated by the ability of plasmids to transfer horizontally between cells and to exchange genetic material with their host and other resident episomal DNAs. These abilities drive the adaptability and diversity of plasmids and their host cells. Indeed, survival functions found in plasmids have chromosomal homologues that have an essential role in cellular responses to stress. An analysis of these functions in the prokaryotic plasmid R1, and of their intricate interrelationships, reveals remarkable overall similarities with other gene- and cell-survival strategies found within and beyond the prokaryotic world.     

Molecular clocks and the incompleteness of the fossil record

Molecular clocks can be evaluated by comparing absolute rates of evolution and by performing relative-rate tests. Typically, calculations of absolute rates are based on earliest observed occurrences in the fossil record. Relative-rate tests, on the other hand, merely require an unambiguous outgroup. A major disadvantage of relative-rate tests is their insensitivity to concomitant and equal rate changes in all lineages. Apparent differences in absolute rates, in turn, may be artifacts that are attributable to an incomplete fossil record.Recently developed methods in quantitative biostratigraphy recognize the incompleteness of the fossil record and allow us to place confidence intervals on the endpoints of taxon ranges. These methods are applicable to taxa whose fossil records are of markedly different quality. When we extend these methods and integrate molecular and paleontologic data, we can test the null hypothesis that seemingly disparate rates of molecular evolution are in fact equal under the simplifying assumption that fossils are randomly and independently distributed over their temporal ranges and that fossils can be accurately placed in a phylogenetic context. We can also estimate the range of ticking rates, if any, that are compatible with known fossil data. Ultimately, more accurate rate estimates for widely divergent taxa should allow for more meaningful comparisons of evolutionary rates.DNA hybridization data for monotremes and marsupials suggest a 17-fold difference for 14 different rate calculations with a mean value of approximately 1% divergence per million years. Variation among marsupials is sevenfold. However, when we apply appropriate statistical tests and make additional allowances for fossils of uncertain taxonomic assignment, etc., all 14 rates are compatible with a molecular clock ticking at approximately 0.4% divergence per million years. In addition, this analysis brings relative- and absolute-rate tests into accord.     

Centromere parC of plasmid R1 is curved

The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.     

A cointegrate of the bacteriophage P1 genome and the conjugative R plasmid R100

Restriction cleavage analysis identified a P1CmSmSuTc plasmid isolated by Mise and Arber (1976) (Virology 69, 191–205) as a cointegrate between bacteriophage P1 and the R plasmid R100. Cointegration occurred by reciprocal recombination between the IS1 element of P1 and IS1b of R100. It involved neither gain nor loss of genetic material, so that the cointegrate carries three IS1 elements in the same orientation. The cointegrate propagates with relatively high stability as plasmid in Escherichia coli host bacteria. It displays the Tra⁺ functions of R100, incompatibility FII of R100, and incompatibility Y of P1, Res⁺ (P1), Mod⁺ (P1) functions of P1 and P1 immunity. Production of P1 phage particles is inducible as for wild type P1. However, because of the large genome size of 180 kb, progeny phage particles contain only a fraction (about 100 kb) of the cointegrate genome. Because of cyclic permutation all genome regions are equally represented in a population of the phage particles of an induced lysate. Occasionally, reciprocal recombination between IS1 elements allows the restoration of the P1 genome. These segregants are found as plaque formers at a rate of about 1 per 300 phage particles in induced lysates.     

Molecular evolution: Codes,clocks, genes and genomes

The discoveries, advancements and continuing controversies in the field of molecular evolution are reviewed. Topics summarized are (1) the evolution of the genetic code, (2) gene evolution including the demonstration of homology, estimation of sequence divergence, phylogenetic trees, the molecular clock and the origin of genes and gene families by various genetic mechanisms, and (3) eukaryotic genome evolution, including the highly repeated satellite sequences, the interspersed and potentially mobile repeated sequences and the unique sequence fraction of the genome.     

Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism

Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.     

Inferring clocks when lacking rocks: the variable rates of molecular evolution in bacteria

Background  

Because bacteria do not have a robust fossil record, attempts to infer the timing of events in their evolutionary history requires comparisons of molecular sequences. This use of molecular clocks is based on the assumptions that substitution rates for homologous genes or sites are fairly constant through time and across taxa. Violation of these conditions can lead to erroneous inferences and result in estimates that are off by orders of magnitude. In this study, we examine the consistency of substitution rates among a set of conserved genes in diverse bacterial lineages, and address the questions regarding the validity of molecular dating.     

TraM of plasmid R1 regulates its own expression

Regulation of the traM gene, which encodes a factor essential for conjugation of resistance plasmid R1, was studied in vivo using translational gene fusions. tram″lacZ fusion constructs were transferred to the chromosome via the recombinant phage λRZ5. The level of β-galactosidase expressed by the lysogens indicates that the traM promoters are very active. Expression of traM was diminished five- to sixfold when the single-copy plasmids R1 or R1-19 were present in trans. When recombinant plasmids carrying traM were present at higher copy numbers, traM expression was reduced as much as 45-fold. The negative effect of R1 plasmids on traM expression in trans, which we interpret as autoregulation, was observed regardless of whether the plasmids were conjugatjvely repressed or derepressed. Site-specific mutagenesis of the region encoding the N-terminus of the TraM protein eliminated the autoregulative effect indicating that the N-terminal amino acids of the protein are important to its DNA-binding function. The autoregulatory behaviour of TraM is allele specific. R1- or P307-encoded TraM molecules were found to recognize only the cognate DNA.