Population genetics of a transformable bacterium: The influence of horizontal genetic exchange on the biology of Neisseria meningitidis

Abstract Information on the biochemistry and genetics of bacterial species, usually obtained by the study of single isolates, is enhanced by studies of populations of bacteria. Recent advances in molecular technology, particularly polymerase chain reaction-based nucleotide sequence analysis, provide powerful for the study of population genetics. Data obtained by such techniques indicate that, while some bacterial species have a clonal population structure, others are non-clonal or panmictic. Clonal populations are a consequence of asexual reproduction by binary fission; panmictic population structures results from 'horizontal' exchange of genetic material between clones. A consequence of horizontal genetic exchange is mosaic gene structures, recognisable by comparisons of nucleotide sequences. In transformable bacteria, for example the human pathogen Neisseria meningitidis , several different genes, including the gene encoding the class 1 outer membrane protein, a major surface antigen, are mosaics. This genetic process has implications both for vaccine design and in the interpretation of epidemiological data.     

Microevolution and epidemic spread of serogroup A Neisseria meningitidis – a review

An extensive and representative strain collection of serogroup A Neisseria meningitidis was established. These bacteria were obtained from different endemic and epidemic/pandemic sources and include strains from diseased patients and healthy carriers. The genetic relationships of the bacteria were defined by multi-locus enzyme electrophoresis and sequence polymorphisms of genetically variable antigens have been analyzed in closely-related groupings. The results are interpreted as reflecting a balance of recombination events, which disrupt clonal relationships, and sequential bottlenecks, which purify the bacterial population of genetic variants during epidemic spread.     

Estimating the minimum rate of genetic transformation in bacteria

Extensive data from multilocus electrophoresis are available for many bacterial populations. In some cases, for example Neisseria gonorrhoeae, these data are consistent with the population being in linkage equilibrium. This raises the following question. What frequency of transformation, or other means of genetic recombination, is needed, relative to mutation, to produce apparent panmixis? Simulation of a finite-population model suggests that, if transformation is at least twenty times as frequent as mutation, the population structure will be indistinguishable from a panmictic one, using the best available data sets. That is, relatively infrequent transformation is sufficient to produce approximate linkage equilibrium.     

Sequence Diversity, Predicted Two-Dimensional Protein Structure, and Epitope Mapping of Neisserial Opa Proteins

The sequence diversity of 45 Opa outer membrane proteins from Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria sicca, and Neisseria flava indicates that horizontal genetic exchange of opa alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane β-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.     

Genetic engineering: possibilities and prospects for its application in industrial microbiology

A wide range of techniques is now available for the construction of hybrid DNA molecules comprising components from disparate species. Transfer of segments of DNA from other organisms, and especially eukaryotes, to Escherichia coli permits their preparation in quantities sufficient for detailed analysis of their structure and mechanism of expression. This information could be exploited to enhance the quantity or quality of polypeptide products from bacterial cells. Greatly increased yields of bacterial enzymes have been obtained in this way in several instances. The approaches that have been pioneered with bacteria are currently being applied to higher organisms. Much work is in progress with yeasts, in which transformation has been successfully demonstrated, with animal viruses and cells in culture and with some plant systems and offers the promise of wider application of genetic engineering in the not too distant future.     

Analysis of genetic structure in soil populations of Rhizobium leguminosarum recovered from the USA and the UK

Although many studies have shown that animal-associated bacterial species exhibit linkage disequilibrium at chromosomal loci, recent studies indicate that both animal-associated and soil-borne bacterial species can display a nonclonal genetic structure in which alleles at chromosomal loci are in linkage equilibrium. To examine the situation in soil-borne species further, we compared genetic structure in two soil populations of Rhizobium leguminosarum bv. trifolii and two populations of R. leguminosarum bv. viciae from two sites in Oregon, with genetic structure in R. leguminosarum bv. viciae populations recovered from peas grown at a site in Washington, USA, and at a site in Norfolk, UK. A total of 234 chromosomal types (ET) were identified among 682 strains analysed for allelic variation at 13 enzyme-encoding chromosomal loci by multilocus enzyme electrophoresis (MLEE). Chi-square tests for heterogeneity of allele frequencies showed that the populations were not genetically uniform. A comparison of the genetic diversity within combined and individual populations confirmed that the Washington population was the primary cause of genetic differentiation between the populations. Each individual population exhibited linkage disequilibrium, with the magnitude of the disequilibrium being greatest in the Washington population and least in the UK population of R. leguminosarum bv. viciae. Linkage disequilibrium in the UK population was created between two clusters of 9 and 23 ETs, which, individually, were in linkage equilibrium. Strong linkage disequilibrium between the two major clusters of 8 and 12 ETs in the Washington population was caused by the low genetic diversity of the ETs within each cluster relative to the inter-cluster genetic distance. Because neither the magnitude of genetic diversity nor of linkage disequilibrium increased as hierarchical combinations of the six local populations were analysed, we conclude that the populations have not been isolated from each other for sufficient time, nor have they been exposed to enough selective pressure to develop unique multilocus genetic structure.     

Bacterial evolution: bacteria play pass the gene

DNA transfer between related bacterial species is enhanced by species-specific uptake sequences. These sequences have been used to identify genes that have been transferred from Haemophilus to Neisseria, providing a clear example of interspecific transfer of DNA in the evolution of the pathogenic Neisseria.     

Whole-genome sequence of the transformable Neisseria meningitidis serogroup A strain WUE2594

Serogroup A meningococci are a leading cause of bacterial meningitis in children and young adults worldwide. However, the genetic basis of serogroup A strains' virulence and their epidemiological properties remain poorly understood. Therefore, we sequenced the complete genome of the transformable Neisseria meningitidis serogroup A strain WUE2594.     

Sexuality in a natural population of bacteria–Bacillus subtilis challenges the clonal paradigm

Reproduction by binary fission necessarily establishes a clonal genotypic structure in bacterial populations unless a high rate of genetic recombination opposes it. Several genetic properties were examined for a wild population of Bacillus subtilis in the Sonoran Desert of Arizona to assess the extent of recombination in a natural population. These properties included allozyme variation revealed by multilocus enzyme electrophoresis, phage and antibiotic resistance, and restriction fragment length polymorphism with Southern hybridization. Evidence of extensive genetic recombination was found along with evidence of modest clonal structure. Recombination must be frequent relative to binary fission in this population. This mixed population structure provides broader options for bacterial evolution than would a purely clonal structure.     

Clinical Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Neisseria meningitidis in Cerebrospinal Fluid

BackgroundNeisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF).Conclusions/SignificanceCompared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.     

Genome plasticity in Neisseria gonorrhoeae

Abstract The pathogenic Neisseria have exploited the processes of horizontal DNA transfer and genetic recombination as mechanisms for the generation of extensive protein variation and modulation of gene expression. Localized recombinations have been well documented in members of multigene families as have alterations in short repetitive sequences. Here we report an analysis of the chromosomal structure of a defined lineage of Neisseria gonorrhoeae strain MS 11 pilin variants. This study reveals the occurrence of large rearrangements, including the amplification of a 26 kb region and an inversion involving more than a third of the chromosome. Additionally, a restriction site polymorphism that correlates with pilin expression has been observed. These findings highlight the flexibility of the gonococcal genome.     

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

Sufu (Suppressor of Fused), a two‐domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu‐like proteins have previously been identified based on sequence similarity to the N‐terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu‐like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu‐like protein. The structure revealed a striking similarity to the N‐terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ～15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu‐like proteins that are present in ～200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.     

The role of porins in neisserial pathogenesis and immunity

Neisseria meningitidis and Neisseria gonorrhoeae are Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia, and the sexually transmitted disease gonorrhea, respectively. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells (APCs). Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of APCs, an effect that is Toll-like receptor 2- and MyD88-dependent. The effect of neisserial porins on the immune system also involves interaction with components of the complement cascade. Furthermore, neisserial porins co-localize with mitochondria of target cells, where they appear to modulate apoptosis.     

Exploration of soil bacterial communities for their potential as bioresource

Soil is a repository of diverse microorganisms, which has frequently been used to isolate and exploit microbes for industrial, environmental and agricultural applications. Knowledge about the structure and dynamics of bacterial communities in soil has been limited as only a small fraction of bacterial diversity is accessible to culture methods. Traditional enrichment techniques and the pure culture approach for microbiological studies have offered only a narrow portal for examining the soil microbial flora due to their limited selectivity. Therefore, the morphological and nutritional criteria used to describe bacterial community failed to provide a natural taxonomic order according to evolutionary relationship. Molecular methods under an emerging discipline of biology "molecular microbial ecology" are now helping in getting these constraints removed to some extent. Nucleic acid extraction from soil is the first crucial step in the application of most of the molecular techniques, which have largely been dominated by diverse variations of PCR. Due to its rapidity, sensitivity and specificity, PCR-based finger printing techniques have proved extremely useful in assessing the changes in microbial community structure. Such techniques can yield complex community profiles and can also provide useful phylogenetic information. Fluorescent in situ hybridization (FISH) can be used to evaluate the distribution and function of bacterial population in situ. DNA microarray techniques have also been developed and being frequently used for the evaluation of ecological role and phylogenetic affiliations of bacterial populations in the soil.     

Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis

Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.     

Effects of two different application methods of Burkholderia ambifaria MCI 7 on plant growth and rhizospheric bacterial diversity

In order to acquire a better understanding of the effects of the different delivery modes of bacterial inoculants on plant growth and on the community structure of rhizosphere bacterial populations, Burkholderia ambifaria MCI 7 (formerly B. cepacia MCI 7) was inoculated into the rhizosphere of maize plants by either seed adhesion or incorporation into soil. Plant growth was evaluated at different inoculum concentrations. The community structure of rhizosphere bacterial populations was evaluated by analysing the restriction patterns of the DNA coding for 16S rRNA amplified by polymerase chain reaction (PCR) (ARDRA) of 745 bacterial isolates. A number of diversity indices (richness, Shannon diversity, evenness and mean genetic distance) were calculated for each bacterial population isolated from control and treated plants according to the concept of the r/K strategy. Moreover, the analysis of molecular variance (AMOVA) method was applied to estimate the genetic differences among the various bacterial populations. Our results showed that the method of application can be an essential element in determining the effects of the inoculant on plant growth. In fact, when applied as a maize seed treatment, B. ambifaria MCI 7 promoted plant growth significantly; on the contrary, when incorporated into soil, the same strain reduced plant growth markedly. As far as the bacterial community structure is concerned, B. ambifaria MCI 7 affected the indigenous microflora of treated plants according to the application method: seed treatment brought about an abrupt decrease in bacterial diversity, whereas incorporation into soil increased bacterial diversity. Moreover, changes in bacterial diversity were limited to r-strategist bacteria. In conclusion, B. ambifaria MCI 7 can act as both a plant growth-promoting rhizobacterium and a deleterious rhizobacterium depending on the inoculation method.     

Intercontinental spread of Neisseria meningitidis clones of the ET-5 complex

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 structural genes encoding enzymes in 688 isolates. Variation among strains in serogroup and serotype has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at more than half their genetic loci. Clones of one of these lineages, the ET-5 complex, have been identified as the causative agent of recent outbreaks and epidemics of meningococcal disease in Europe, South Africa, Latin America, and the United States. There is evidence that organisms of the ET-5 complex reached Florida via human immigrants from Cuba.     

Structure of a sialic acid-activating synthetase, CMP-acylneuraminate synthetase in the presence and absence of CDP

The x-ray crystallographic structure of selenomethionyl cytosine-5'-monophosphate-acylneuraminate synthetase (CMP-NeuAc synthetase) from Neisseria meningitidis has been determined at 2.0-A resolution using multiple-wavelength anomalous dispersion phasing, and a second structure, in the presence of the substrate analogue CDP, has been determined at 2.2-A resolution by molecular replacement. This work identifies the active site residues for this class of enzyme for the first time. The detailed interactions between the enzyme and CDP within the mononucleotide-binding pocket are directly observed, and the acylneuraminate-binding pocket has also been identified. A model of acylneuraminate bound to CMP-NeuAc synthetase has been constructed and provides a structural basis for understanding the mechanism of production of "activated" sialic acids. Sialic acids are key saccharide components on the surface of mammalian cells and can be virulence factors in a variety of bacterial species (e.g. Neisseria, Haemophilus, group B streptococci, etc.). As such, the identification of the bacterial CMP-NeuAc synthetase active site can serve as a starting point for rational drug design strategies.