Housekeeping Loci Research Articles

Multilocus sequence typing system for Campylobacter jejuni.

The gram-negative bacterium Campylobacter jejuni has extensive reservoirs in livestock and the environment and is a frequent cause of gastroenteritis in humans. To date, the lack of (i) methods suitable for population genetic analysis and (ii) a universally accepted nomenclature has hindered studies of the epidemiology and population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in seven housekeeping loci to determine the genetic relationships among isolates. The MLST system was established using 194 C. jejuni isolates of diverse origins, from humans, animals, and the environment. The allelic profiles, or sequence types (STs), of these isolates were deposited on the Internet (http://mlst.zoo.ox.ac.uk), forming a virtual isolate collection which could be continually expanded. These data indicated that C. jejuni is genetically diverse, with a weakly clonal population structure, and that intra- and interspecies horizontal genetic exchange was common. Of the 155 STs observed, 51 (26% of the isolate collection) were unique, with the remainder of the collection being categorized into 11 lineages or clonal complexes of related STs with between 2 and 56 members. In some cases membership in a given lineage or ST correlated with the possession of a particular Penner HS serotype. Application of this approach to further isolate collections will enable an integrated global picture of C. jejuni epidemiology to be established and will permit more detailed studies of the population genetics of this organism.

Identification of the major Spanish clones of penicillin-resistant pneumococci via the Internet using multilocus sequence typing.

Multilocus sequence typing was used to characterize isolates of the major Spanish clones of penicillin-resistant and multiple-antibiotic-resistant Streptococcus pneumoniae. Isolates of the multidrug-resistant Spanish serotype 23F clone and serotype variants of this clone either had identical allelic profiles or their allelic profiles differed from this typical allelic profile at only one of the seven housekeeping loci. Similarly, isolates of the Spanish serotype 6B and 14 clones and the penicillin-resistant serotype 9V clone (and serotype variants of this clone) each had the same allelic profiles or profiles that differed at a single locus. Multilocus sequence typing therefore allows resistant pneumococci to be assigned to the Spanish clones if they have the typical allelic profile of the clone or if their profiles differ from that profile at a single locus. A few resistant isolates that had allelic profiles typical of that of a Spanish clone or whose profiles differed from that of the typical profile at only a single locus possessed penicillin-binding protein pbp1a, pbp2b, or pbp2x genes that differed from those that are characteristic of the clone. In most cases these isolates could be assigned as variant members of the clone. Since almost all serotype 9V isolates have very similar genotypes, independently emerging penicillin-resistant clones of this serotype will inevitably appear to be similar by molecular typing procedures. Analysis of the pbp genes, in addition to multilocus sequence typing (or any other molecular typing procedure), is therefore required to assign isolates unambiguously to the penicillin-resistant Spanish serotype 9V clone.

The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis.

Multilocus sequence typing (MLST) is a recently developed nucleotide sequence-based method for the definitive assignment of isolates within bacterial populations to specific clones. MLST uses the same principles as multilocus enzyme electrophoresis and provides data that can be used to investigate aspects of the population genetics and evolution of bacterial species. We used an MLST data set consisting of the sequences of approximately 450-bp fragments from seven housekeeping loci from a large strain collection of Neisseria meningitidis to estimate the relative impact of recombination compared with point mutation in the diversification of N. meningitidis clonal complexes. 126 meningococcal isolates were assigned to 10 clonal complexes, 9 of which contained minor clonal variants. The allelic variation within each complex was classified as a recombinational exchange or a putative point mutation through a comparison of the sequences of each variant allele with that of the allele typically found in the clonal complex. The nine clonal complexes contained a total of 23 allelic variants, and analysis of the sequences of these variant alleles revealed that a single nucleotide site in a meningococcal housekeeping gene is at least 80-fold more likely to change as a result of recombination than as a result of mutation. This value is estimated to be 10-50-fold for Escherichia coli and approximately 50-fold for Streptococcus pneumoniae.

The Evolutionary Ecology of Dominance-Recessivity

An “adaptive dynamics” modelling approach to the evolution of dominance-recessivity is presented. In this approach, fitness derives from an explicit ecological scenario, and both evolutionary attractivity and invasibility of resident populations are examined.The ecology consists of a within-individual part representing a locus with regulated activity and a between-individual part that is a two-patch soft selection model. Evolutionary freedom is allowed at a single locus. The evolutionary analysis considers directed random walks on trait space, generated by repeated invasions of mutants.The phenotype of an individual is determined by allelic parameters. Mutations can have two effects: they either affect the affinity of the promoter sequence for transcription factors, or they affect the gene product. The dominance interaction between alleles derives from their promoter affinities.Additive genetics is evolutionarily unstable when selection and evolution maintain two alleles in the population. In such a situation, dominance interactions can become stationary and close to additive genetics or they continue to evolve at a very slow pace towards dominance-recessivity. The probability that a specific dominance interaction will evolve depends on the relative mutation rate of promoter compared to gene product and the distribution of mutational effect sizes. Either allele in the dimorphism can become dominant, and dominance-recessivity is always most likely to evolve. Evolution then approaches a population state where every phenotype has maximum viability in one of the two patches.When the within-individual part is replaced by a housekeeping locus that codes for a metabolic enzyme, evolution favours a population of two alleles under the same conditions as for a regulated locus. In the case of a housekeeping gene, however, the evolutionary dynamical system approaches a population state where the heterozygote and only one homozygote phenotype are equivalent to the optimum phenotypes in the two patches.

The influence of recombination on the population structure and evolution of the human pathogen Neisseria meningitidis.

The extent to which recombination disrupts the bifurcating treelike phylogeny and clonal structure imposed by binary fission on bacterial populations remains contentious. Here, we address this question with a study of nucleotide sequence data from 107 isolates of the human pathogen Neisseria meningitidis. Gene fragments from 12 house-keeping loci distributed around the meningococcal chromosome were analyzed, showing that (1) identical alleles are disseminated among genetically diverse isolates, with no evidence for linkage disequilibrium; (2) different loci give distinct and incongruent phylogenetic trees; and (3) allele sequences are incompatible with a bifurcating treelike phylogeny at all loci. These observations are consistent with the hypothesis that meningococcal populations comprise organisms assembled from a common gene pool, with alleles and allele fragments spreading independently, together with the occasional importation of genetic material from other species. Further, they support the view that recombination is an important genetic mechanism in the generation new meningococcal clones and alleles. Consequently, for anything other than the short-term evolution of this species, a bifurcating treelike phylogeny is not an appropriate model.

Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the Internet

Multilocus sequence typing is a development of multilocus enzyme electrophoresis in which the alleles at multiple house-keeping loci are assigned directly by nucleotide sequencing, rather than indirectly from the electrophoretic mobilities of their gene products. A major advantage of this approach is that sequence data are unambiguous and electronically portable, allowing molecular typing of bacterial pathogens (or other infectious agents) via the Internet.

Bacterial population genetics, evolution and epidemiology.

Asexual bacterial populations inevitably consist of an assemblage of distinct clonal lineages. However, bacterial populations are not entirely asexual since recombinational exchanges occur, mobilizing small genome segments among lineages and species. The relative contribution of recombination, as opposed to de novo mutation, in the generation of new bacterial genotypes varies among bacterial populations and, as this contribution increases, the clonality of a given population decreases. In consequence, a spectrum of possible population structures exists, with few bacterial species occupying the extremes of highly clonal and completely non-clonal, most containing both clonal and non-clonal elements. The analysis of collections of bacterial isolates, which accurately represent the natural population, by nucleotide sequence determination of multiple housekeeping loci provides data that can be used both to investigate the population structure of bacterial pathogens and for the molecular characterization of bacterial isolates. Understanding the population structure of a given pathogen is important since it impacts on the questions that can be addressed by, and the methods and samples required for, effective molecular epidemiological studies.

Comparison of the evolutionary dynamics of symbiotic and housekeeping loci: a case for the genetic coherence of rhizobial lineages.

In prokaryotes, lateral gene transfer across chromosomal lineages may be mediated by plasmids, phages, transposable elements, and other accessory DNA elements. However, the importance of such transfer and the evolutionary forces that may restrict gene exchange remain largely unexplored in native settings. In this study, tests of phylogenetic congruence are employed to explore the range of horizontal transfer of symbiotic (sym) loci among distinct chromosomal lineages of native rhizobia, the nitrogen-fixing symbiont of legumes. Rhizobial strains isolated from nodules of several host plant genera were sequenced at three loci: symbiotic nodulation genes (nodB and nodC), the chromosomal housekeeping locus glutamine synthetase II (GSII), and a portion of the 16S rRNA gene. Molecular phylogenetic analysis shows that each locus generally subdivides strains into the same major groups, which correspond to the genera Rhizobium, Sinorhizobium, and Mesorhizobium. This broad phylogenetic congruence indicates a lack of lateral transfer across major chromosomal subdivisions, and it contrasts with previous studies of agricultural populations showing broad transfer of sym loci across divergent chromosomal lineages. A general correspondence of the three rhizobial genera with major legume groups suggests that host plant associations may be important in the differentiation of rhizobial nod and chromosomal loci and may restrict lateral transfer among strains. The second major result is a significant incongruence of nod and GSII phylogenies within rhizobial subdivisions, which strongly suggests horizontal transfer of nod genes among congenerics. This combined evidence for lateral gene transfer within, but not between, genetic subdivisions supports the view that rhizobial genera are "reproductively isolated" and diverge independently. Differences across rhizobial genera in the specificity of host associations imply that the evolutionary dynamics of the symbiosis vary considerably across lineages in native settings.

A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease.

The population biology of Streptococcus pneumoniae is poorly understood. Most of the important issues could be addressed by the molecular characterization of large, well sampled populations from carriage and from the different manifestations of pneumococcal disease. The authors have therefore developed a pneumococcal multilocus sequence typing scheme and database by sequencing approximately 450 bp fragments of seven housekeeping loci from 295 isolates. The combination of alleles at the seven loci provided an allelic profile, or sequence type (ST), and the relatedness between isolates was obtained by constructing a dendrogram from the matrix of pairwise differences between STs. The typing scheme was validated using pneumococci of known genetic relatedness and could resolve >6 billion STs. Among 274 isolates from recent cases of invasive pneumococcal disease in eight countries, 143 STs were resolved, but 12 STs contained at least five isolates (range 5-21 isolates). The repeated recovery of indistinguishable isolates from invasive disease in different countries implies that these STs define strains with an increased capacity to cause invasive disease. The relationship between STs and serotypes suggested that, in the longer term, capsular genes have been distributed horizontally within the pneumococcal population, but in the short term, expansion of clones occurs with only occasional changes of serotype. The multilocus sequence typing scheme provides a powerful new approach to the characterization of pneumococci, since it provides molecular typing data that are electronically portable between laboratories, and which can be used to probe aspects of the population and evolutionary biology of these organisms. A Web site for the molecular characterization of pneumococci by MLST is available (http ://mlst.zoo.ox.ac.uk).

Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells.

Salmonella species are facultative intracellular pathogens that invade epithelial cells and reside within lysosomal membrane glycoprotein (lgp)-containing vacuoles. Coincident with the onset of bacterial replication inside these vacuoles, Salmonella induce the formation of stable lgp-containing filamentous structures that connect with the Salmonella-containing vacuoles. Salmonella typhimurium SL1344::Tn l0dCm mutant strains unable to induce these structures were isolated. All contained insertions within a novel Salmonella induced filament gene A (sifA). sifA is present only in Salmonella species and encodes a protein with a predicted molecular mass of 38 kDa and an apparent molecular mass of 35 kDa. sifA is flanked by 300 base pairs, and sifA and its flanking DNA show no homology to sequences in DNA databases. sifA is located within the potABCD operon, a housekeeping locus involved in periplasmic transport of polyamines. Fourteen-base-pair direct repeats mark the probable site of integration of sifA and its flanking DNA have a significantly reduced G+C content (41%) when compared with the potABCD operon (51%) and the Salmonella genome (52-54%). Deletion mutant strains in sifA or in the downstream potC were constructed. Delta sifA does not produce Salmonella-induced filaments in epithelial cells, and is attenuated in mice. Delta potC produces Salmonella-induced filaments in epithelial cells, and was fully virulent. Collectively, these results suggest that sifA arose by horizontal gene transfer into Salmonella and its product is involved in a virulence-associated intracellular phenotype related to Salmonella-induced filament formation.

Determination of DNA replication kinetics in synchronized human cells using a PCR-based assay.

Studies on the temporal order of DNA replication are difficult due to the lack of sensitivity of methods available for replication kinetic analysis. To overcome problems associated with the current techniques, we propose a PCR-based assay to determine the replication time of any single-copy DNA sequence in complex genomes. Human cells labeled with 5-bromodeoxyuridine (BrdU) were flow sorted, according to their DNA content, at different times after synchronous release from the G1/S phase boundary. The selective removal of newly-replicated BrdU-substituted DNA was achieved by UV light irradiation followed by S1 nuclease treatment. The timing of replication of selected DNA sequences (housekeeping, tissue-specific, and non-coding loci) was determined by polymerase chain reaction (PCR) amplification using appropriate primers. DNA sequences localized in inactive replication units allowed amplification whereas those that have replicated will not be amplified by PCR. Using this sensitive and quantitative assay the replication kinetic analysis of a number of different DNA sequences can be performed from a single sorting experiment.

Genetic analysis of a vital mammalian housekeeping locus using CHO cells that express a transfected mutant allele

We describe a novel approach for the isolation of null mutations in a vital Chinese hamster ovary (CHO) cell housekeeping gene. Our experimental strategy required introduction of an expressible DNA clone encoding a recessive emetine-resistance allele of ribosomal protein S14 into wild-type CHO cells. Transgene heterozygote (TGH) cell lines, which harbor multiple emetine-resistance S14 transgenes, survive mutations that inactivate the CHO RPS14 locus by virtue of the transgenes' biological function. Null mutations in RPS14 yield TGH clones that display the transgene's drug-resistance phenotype. A large collection of emetine-resistant clones was isolated from one TGH cell line and shown to consist of three types of S14 mutations: (1) nonsense null mutations in the RPS14 protein coding sequence; (2) missense null mutations that affect S14 amino acid residues that have been conserved stringently during eukaryotic evolution; and (3) a recurrent missense mutation that results in a new, functional RPS14 emetine-resistance allele.