MicrobiologyBytes

There are many descriptions of bacterial genes that have been found within nematode and arthropod chromosomes. In this article in Microbiology Today, Julie Dunning Hotopp and Jason Rasgon explain how they got there:

The arthropod-infecting Wolbachia exert unusual effects on host reproduction, including parthenogenesis, whereby infected virgin females produce infected female offspring, male killing, whereby infected male embryos fail to develop, feminization, whereby genetic males develop into reproductively capable females, and cytoplasmic incompatibility, the most common phenotype, whereby the offspring of uninfected females and infected males fail to develop. Wolbachia are maternally inherited, being transferred through the egg cytoplasm. Therefore, these reproductive phenotypes favouring Wolbachia-infected females increase the proliferation of Wolbachia-infected arthropods. Wolbachia are parasitic endosymbionts, since the interaction benefits Wolbachia while exerting a negative effect on the host by limiting genetic exchange. However, a mutualistic role benefiting both organisms cannot be excluded.

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• Wolbachia

discovermagazine.com

It was just a matter of time before someone discovered that Madagascar is a museum for viruses. The discovery came when a team of American and English scientists perused the genome of the gray mouse lemur. Nestled among its genes were segments of DNA that bore a remarkable resemblance to HIV. How on Earth could a deadly virus’s genes become part of a primate’s own genome? Some kinds of viruses, known as retroviruses, replicate by inserting their DNA into host cells, where their DNA can guide the production of new viruses. But many studies indicate that sometimes these viruses infect the cells that will give rise to sperm and eggs. The virus ends up in a fertilized egg and gets passed down to ever cell in the developing embryo–including its own sex cells. Now the virus gets passed down through the generations. It may still retain the ability to infect other cells for a while, but mutations typically knock out that ability. Instead, the virus can only insert copies of its DNA back into its own host cell’s genome. Over millions of years, this viral DNA spreads through the host genome. Our own DNA contains 98,000 stretches of this virus DNA, plus 150,000 tiny viral fragments, making up about 8% of our genome – about five times more DNA than the DNA that encodes proteins.

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• Phoenix from the ashes: The 5 million year old virus

Bioremediation can be defined as a process that uses microorganisms, fungi, green plants or their enzymes to return the natural environment altered by contaminants to its original condition. A major advantage of bioremediation is its reduced cost compared to conventional cleanup techniques – the cost of remediation for all contaminated sites in the USA alone is estimated to be $1.7 trillion (Molecular approaches in bioremediation. Curr Opin Biotechnol. Nov 12 2008). In addition, bioremediation is often a permanent solution providing complete transformation of the pollutant to its molecular constituents like carbon dioxide and water rather than a partial method that transfers wastes from one phase to another. Unfortunately, there are many man-made compounds that lack good biological catalysts, and many instances where good biocatalysts fail to transform pollutants in the environment.

Bacteria have enormous potential for cleaning up wastes; however, the interactions between bacteria and pollutants are complex and suitable outcomes do not always take place. Hence, molecular approaches are being applied to enhance bioremediation. One advance in bioremediation to improve the stability of the biocatalyst is to create a system where degradation occurs in the area near the roots of plants known as the rhizosphere. In rhizoremediation, the bacteria degrade the pollutants while the plant roots provide a niche for the microorganism and key nutrients. The advantages of rhizoremediation include the ability of plant roots to provide a large surface area for bacterial propagation and biofilm formation, the roots transport the bacteria through the contaminated soil, the roots provide a niche for the bacteria by providing nutrients, and the roots facilitate oxygen exchange. Successful rhizoremediation systems have been established for pollutants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls (PCBs), fuels, metals, and pesticides such as parathion.

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Directed evolution or DNA shuffling is a powerful mutagenesis technique that mimics the natural molecular evolution of genes in order to efficiently re-design them. Its power lies in the fact that it can introduce multiple mutations into a gene in order to create new enzymatic activity, which can be discovered by a suitable method of selection (bioassays). Family shuffling applies DNA shuffling to groups of related genes to combine them in a manner that accelerates directed evolution. Genome shuffling recombines the chromosomes of several bacteria to enhance the activity of the whole organism.

Metabolic engineering involves redirecting a cell’s metabolism to achieve a particular goal using recombinant engineering. This technique has been used to create bacterial strains that degrade chlorinated ethenes through the addition of several cloned enzymes to the cell. Metabolic engineering has also been used successfully to handle difficult mixtures of pollutants.

Whole-transcriptome profiling using DNA microarrays has the advantage that the relative amount of transcripts from the whole genome may be easily determined compared to such techniques like proteomics. To understand the metabolism of bacteria in the rhizosphere, researchers have begun to utilize whole-genome profiling.

Although a tremendous amount of work remains to be performed, significant advances have been made through protein engineering and through metabolic engineering for the purposes of bioremediation. However, even though whole-transcriptome profiling and proteomics are utilized routinely in some disciplines, they remain to be utilized extensively in bioremediation. Furthermore, it is important to ensure engineered strains for field use are competitive; rhizoremediation can provide a niche for these engineered bacteria. Chromosomal integration of introduced genes can limit horizontal gene transfer to other species, but this should also be empirically verified to ensure that no adverse environmental effects occur.

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Huge numbers and variety of bacteriophages exist on our planet. In this article in Microbiology Today, Graham Hatfull describes this massive reservoir of unidentified genetic information:

You may well be under the impression that the largest number of undiscovered species – and the greatest pool of unknown genes – lie within the considerable biodiversity of the tropical rain forests. Not so. A compelling argument can be made that the biggest reservoir of unidentified genetic information is all around us, in the global population of bacteriophages.

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• MicrobiologyBytes: Bacteriophages
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One of the main problems in fighting malaria is the speed with which Plasmodium falciparum, the causative agent of human malaria, is able to vary its genetic makeup. This allows antigenic variation, which makes the creation of effective vaccines very difficult. Antigenic variability also gives P. falciparum the ability to persist in the face of an immune reaction and to reinfect people who have been previously exposed to the disease. Effective immunity to malaria requires repeated infections and is slow to develop, so children under ten years of age are most susceptible to illness. The entry of malaria parasites into red blood cells during the replication cycle creates two opportunities to evade host immunity. First, infected red blood cells do not induce a CTL response due to their lack of MHC I expression. Second, malaria antigens exposed on the surface of the cell are highly variable. The P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor which is expressed on the surface of infected erythrocytes and causes the blood cells to stick to the walls of small blood vessels, preventing infected cells from going through the general circulation and to the spleen (see: Giving malaria the slip).

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Red blood cells infected with Plasmodium display immunodominant parasite antigens on their surface. The reason for this is not clear, but it may be to modify the physical properties of the host cells so that they are not trapped and destroyed in the spleen. The expression of the immunodominant surface protein PfEMP1 is also linked to suppression of host interferon-gamma in the early immune response to the parasite, and low interferon-gamma levels may improve parasite survival.

PfEMP1 is in fact a family of cell surface molecules, encoded by approximately 60 var genes. Antigenic variation is controlled by epigenetic factors including monoallelic var transcription in separate domains at the nuclear periphery, differential histones on otherwise identical var genes, and var gene silencing mediated by telomeres (Antigenic variation in Plasmodium falciparum. Annu Rev Microbiol. 2008 62: 445-470).

Targeting the mechanisms responsible for antigen switching could be a promising approach to tackle the malaria parasite without having to deal with phenotypic variation of the surface molecule. The development of specific biological assays that target antigenic variation could uncover crucial mechanisms required for export to the cell surface, repression of the var gene family, or switching to new variants and would allow the screening of drugs which block these essential processes. Plasmodium’s trump card could yet prove to be its undoing.

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The lion (Panthera leo) is one of the world’s most charismatic carnivores. In a new article published in PLoS Genetics, an international team of researchers provides insights into the genetic structure and history of lion populations. Their work refutes the hypothesis that African lions consist of a single, randomly breeding (panmictic) population. It also indicates the importance of preserving populations in decline as opposed to prioritizing larger-scale conservation efforts.

Understanding the broader aspects of the evolutionary history of the lion has been hindered by a lack of comprehensive sampling and appropriately informative genetic markers. Nevertheless, the unique social ecology of lions and the well-documented infectious diseases they have experienced, including lion-specific feline immunodeficiency virus (FIVPle), provides the opportunity to study lion evolutionary history using both host and virus genetic information. In total, a comprehensive sample of 357 individuals from most of the major lion populations in Africa and Asia were studied. The authors compared the large multigenic dataset from lions with patterns of genetic variation of FIVPle to characterize the genomic legacy of lion populations. The research reveals evidence of unsuspected genetic diversity even in the well-studied lion population of the Serengeti ecosystem, which consists of recently admixed animals derived from three distinct genetic groups.

The Evolutionary Dynamics of the Lion Panthera leo Revealed by Host and Viral Population Genomics. PLoS Genet 4(11): e1000251
The lion Panthera leo is one of the world’s most charismatic carnivores and is one of Africa’s key predators. Here, we used a large dataset from 357 lions comprehending 1.13 megabases of sequence data and genotypes from 22 microsatellite loci to characterize its recent evolutionary history. Patterns of molecular genetic variation in multiple maternal (mtDNA), paternal (Y-chromosome), and biparental nuclear (nDNA) genetic markers were compared with patterns of sequence and subtype variation of the lion feline immunodeficiency virus (FIVPle), a lentivirus analogous to human immunodeficiency virus (HIV). In spite of the ability of lions to disperse long distances, patterns of lion genetic diversity suggest substantial population subdivision and reduced gene flow, which, along with large differences in seroprevalence of six distinct FIVPle subtypes among lion populations, refute the hypothesis that African lions consist of a single panmictic population. Our results suggest that extant lion populations derive from several Pleistocene refugia in East and Southern Africa (324,000–169,000 years ago), which expanded during the Late Pleistocene (100,000 years ago) into Central and North Africa and into Asia. During the Pleistocene/Holocene transition (14,000–7,000 years), another expansion occurred from southern refugia northwards towards East Africa, causing population interbreeding. In particular, lion and FIVPle variation affirms that the large, well-studied lion population occupying the greater Serengeti Ecosystem is derived from three distinct populations that admixed recently.

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• Infectious diseases and extinction risk in wild mammals
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Emerging evidence indicates that the global organization of the bacterial chromosome is defined by its physical map. This architectural understanding has been gained mainly by observing the localization and dynamics of specific chromosomal loci. However, the spatial and temporal organization of the entire mass of newly synthesized DNA remains elusive. To visualize replicated DNA within living cells, we developed an experimental system in the bacterium Bacillus subtilis whereby fluorescently labeled nucleotides are incorporated into the chromosome as it is being replicated. Here, we present the first visualization of replication morphologies exhibited by the bacterial chromosome. At the start of replication, newly synthesized DNA is translocated via a helical structure from midcell toward the poles, where it accumulates. Next, additionally synthesized DNA forms a second, visually distinct helix that interweaves with the original one. In the final stage of replication, the space between the two helices is filled up with the very last synthesized DNA. This striking geometry provides insight into the three-dimensional conformation of the replicating chromosome.

Spatial organization of a replicating bacterial chromosome. 2008 PNAS 105: 14136–14140

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Listeria monocytogenes is a pathogen transmitted through contaminated food and is responsible for severe infections, including meningitis and abortion in animals and humans. It is known that many distinct strains of this pathogen exist, and that they differ in their virulence and epidemic potential. Unfortunately, there is currently no standard definition of strains and no comprehensive overview of their evolution. To tackle these serious limitations to the control of listeriosis and to improve knowledge of how virulence evolves, a new paper characterizes a large collection of isolates with sequence-based genotyping methods. The authors were able to identify precisely the most prevalent clones of L. monocytogenes, i.e., groups of isolates that descend from a single ancestral bacterium, which can now be characterized further for diagnostic purposes and determination of their precise ecology and virulence potential. They also determined how these clones evolved from their common ancestor and the evolutionary history by which they acquired their phenotypic characteristics, such as antigenic structures. Finally, they show that some particular strains tend to lose a virulence factor that plays a crucial role in infection in humans. This is a rare example of evolution towards reduced virulence of pathogens, and the discovery of the selective forces behind this phenomenon may have important epidemiological and biological implications.

A New Perspective on Listeria monocytogenes Evolution. 2008 PLoS Pathogens, 4 (9)
Listeria monocytogenes is a model organism for cellular microbiology and host–pathogen interaction studies and an important food-borne pathogen widespread in the environment, thus representing an attractive model to study the evolution of virulence. The phylogenetic structure of L. monocytogenes was determined by sequencing internal portions of seven housekeeping genes (3,288 nucleotides) in 360 representative isolates. Fifty-eight of the 126 disclosed sequence types were grouped into seven well-demarcated clonal complexes (clones) that comprised almost 75% of clinical isolates. Each clone had a unique or dominant serotype (4b for clones 1, 2 and 4, 1/2b for clones 3 and 5, 1/2a for clone 7, and 1/2c for clone 9), with no association of clones with clinical forms of human listeriosis. Homologous recombination was extremely limited, implying long-term genetic stability of multilocus genotypes over time. Bayesian analysis based on 438 SNPs recovered the three previously defined lineages, plus one unclassified isolate of mixed ancestry. The phylogenetic distribution of serotypes indicated that serotype 4b evolved once from 1/2b, the likely ancestral serotype of lineage I. Serotype 1/2c derived once from 1/2a, with reference strain EGDe (1/2a) likely representing an intermediate evolutionary state. In contrast to housekeeping genes, the virulence factor internalin (InlA) evolved by localized recombination resulting in a mosaic pattern, with convergent evolution indicative of natural selection towards a truncation of InlA protein. This work provides a reference evolutionary framework for future studies on L. monocytogenes epidemiology, ecology, and virulence.

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Penicillins and synthetic beta-lactam antibiotics have dramatically transformed health care and quality of life in the 80 years since Alexander Fleming’s discovery of Penicillium. Large-scale production of beta-lactam antibiotics is the result of sustained industrial strain improvement, representing numerous rounds of mutagenesis and selection. Penicillin titers and productivities have increased by at least three orders of magnitude in the past 60 years, representing an unprecedented success in industrial strain improvement.

Current industrial Penicillium strains are derived from a single natural isolate of P. chrysogenum obtained during WWII from an infected cantaloupe. Biochemical and genetic analysis of industrial strains led to the identification of several important mutations in high-producing strains, including amplification of penicillin biosynthesis genes. However, much of the molecular basis for improved productivity remains to be elucidated. A detailed understanding of the molecular biology of P. chrysogenum is not only relevant for natural penicillins, but by applying genetic engineering approaches, it has become possible to extend the range of fermentation products to include beta-lactam derivatives that could previously only be produced by chemical modification.

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To gain more insight into penicillin synthesis, researchers have recently sequenced the 32.19 Mbp genome of P. chrysogenum and identified numerous genes responsible for key steps in penicillin production (Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nature Biotechnology, 28 September 2008). DNA microarrays were used to compare the transcriptomes of the sequenced strain and a penicillinG high-producing strain. Transcription of genes involved in biosynthesis of valine, cysteine and alpha-aminoadipic acid – precursors for penicillin biosynthesis – as well as of genes encoding microbody proteins, was increased in the high-producing strain. Some gene products were shown to directly control beta-lactam output. Many key cellular transport processes involving penicillins and intermediates still remain to be characterized at the molecular level. Genes predicted to encode transporters were strongly overrepresented among the genes transcriptionally upregulated under conditions that stimulate penicillinG production, illustrating potential for future genomics-driven metabolic engineering.

Access to the full range of genomics techniques will be invaluable for further innovation in antibiotics production. Despite the massive improvements already achieved in classical strain improvement, further improvement of penicillin production remains a distinct possibility.

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