See also:     Microbial Biodegradation: Genomics and Molecular Biology

Interest in the microbial biodegradation of pollutants has intensified in recent years as mankind strives to find sustainable ways to cleanup contaminated environments. These bioremediation and biotransformation methods endeavour to harness the astonishing, naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons (e.g. oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), pharmaceutical substances, radionuclides and metals. Major methodological breakthroughs in recent years have enabled detailed genomic, metagenomic, proteomic, bioinformatic and other high-throughput analyses of environmentally relevant microorganisms providing unprecedented insights into key biodegradative pathways and the ability of organisms to adapt to changing environmental conditions.

The elimination of a wide range of pollutants and wastes from the environment is an absolute requirement to promote a sustainable development of our society with low environmental impact. Biological processes play a major role in the removal of contaminants and they take advantage of the astonishing catabolic versatility of microorganisms to degrade/convert such compounds. New methodological breakthroughs in sequencing, genomics, proteomics, bioinformatics and imaging are producing vast amounts of information. In the field of Environmental Microbiology, genome-based global studies open a new era providing unprecedented in silico views of metabolic and regulatory networks, as well as clues to the evolution of degradation pathways and to the molecular adaptation strategies to changing environmental conditions. Functional genomic and metagenomic approaches are increasing our understanding of the relative importance of different pathways and regulatory networks to carbon flux in particular environments and for particular compounds and they will certainly accelerate the development of bioremediation technologies and biotransformation processes.

Aerobic Biodegradation of Organic Pollutants
The burgeoning amount of bacterial genomic data provides unparalleled opportunities for understanding the genetic and molecular bases of the degradation of organic pollutants. Aromatic compounds are among the most recalcitrant of these pollutants and lessons can be learned from the recent genomic studies of Burkholderia xenovorans LB400 and Rhodococcus sp. strain RHA1, two of the largest bacterial genomes completely sequenced to date. These studies have helped expand our understanding of bacterial catabolism, non-catabolic physiological adaptation to organic compounds, and the evolution of large bacterial genomes. First, the metabolic pathways from phylogenetically diverse isolates are very similar with respect to overall organization. Thus, as originally noted in pseudomonads, a large number of "peripheral aromatic" pathways funnel a range of natural and xenobiotic compounds into a restricted number of "central aromatic" pathways. Nevertheless, these pathways are genetically organized in genus-specific fashions, as exemplified by the b-ketoadipate and Paa pathways. Comparative genomic studies further reveal that some pathways are more widespread than initially thought. Thus, the Box and Paa pathways illustrate the prevalence of non-oxygenolytic ring-cleavage strategies in aerobic aromatic degradation processes. Functional genomic studies have been useful in establishing that even organisms harboring high numbers of homologous enzymes seem to contain few examples of true redundancy. For example, the multiplicity of ring-cleaving dioxygenases in certain rhodococcal isolates may be attributed to the cryptic aromatic catabolism of different terpenoids and steroids. Finally, analyses have indicated that recent genetic flux appears to have played a more significant role in the evolution of some large genomes, such as LB400's, than others. However, the emerging trend is that the large gene repertoires of potent pollutant degraders such as LB400 and RHA1 have evolved principally through more ancient processes. That this is true in such phylogenetically diverse species is remarkable and further suggests the ancient origin of this catabolic capacity.

Anaerobic Biodegradation of Organic Pollutants
Anaerobic microbial mineralization of recalcitrant organic pollutants is of great environmental significance and involves intriguing novel biochemical reactions. In particular, hydrocarbons and halogenated compounds have long been doubted to be degradable in the absence of oxygen, but the isolation of hitherto unknown anaerobic hydrocarbon-degrading and reductively dehalogenating bacteria during the last decades provided ultimate proof for these processes in nature. Many novel biochemical reactions were discovered enabling the respective metabolic pathways, but progress in the molecular understanding of these bacteria was rather slow, since genetic systems are not readily applicable for most of them. However, with the increasing application of genomics in the field of environmental microbiology, a new and promising perspective is now at hand to obtain molecular insights into these new metabolic properties. Several complete genome sequences were determined during the last few years from bacteria capable of anaerobic organic pollutant degradation. The ~4.7 Mb genome of the facultative denitrifying "Aromatoleum aromaticum" strain EbN1 was the first to be determined for an anaerobic hydrocarbon degrader (using toluene or ethylbenzene as substrates). The genome sequence revealed about two dozen gene clusters (including several paralogs) coding for a complex catabolic network for anaerobic and aerobic degradation of aromatic compounds. The genome sequence forms the basis for current detailed studies on regulation of pathways and enzyme structures. Further genomes of anaerobic hydrocarbon degrading bacteria were recently completed for the iron-reducing species Geobacter metallireducens (accession nr. NC_007517) and the perchlorate-reducing "Dechloromonas aromatica" (accession nr. NC_007298), but these are not yet evaluated in formal publications. Complete genomes were also determined for bacteria capable of anaerobic degradation of halogenated hydrocarbons by halorespiration: the ~1.4 Mb genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1 and the ~5.7 Mb genome of Desulfitobacterium hafniense strain Y51. Characteristic for all these bacteria is the presence of multiple paralogous genes for reductive dehalogenases, implicating a wider dehalogenating spectrum of the organisms than previously known. Moreover, genome sequences provided unprecedented insights into the evolution of reductive dehalogenation and differing strategies for niche adaptation.

Detecting Bacteria Involved in Biodegradation
Traditional molecular analyses have led to great understanding of the microbial diversity in natural systems. These approaches can tell the presence of a particular group of bacteria, but does not address the activity. Molecular methods, including microautoradiography, mRNA analysis, growth assays, and incorporation of stable isotopes, can be used to determine which bacteria are involved in biodegradation of chemical pollutants. This information leads to a greater understanding of the role of microbial community structure and function with respect to bioremediation.

Bioremediation with Extracellular Electron Transfer
Geobacter species are often the predominant organisms when extracellular electron transfer is an important bioremediation process in subsurface environments. Therefore, a systems biology approach to understanding and optimizing bioremediation with Geobacter species has been initiated with the ultimate goal of developing in silico models that can predict the growth and metabolism of Geobacter species under a diversity of subsurface conditions. To date, these studies have included sequencing the genomes of multiple Geobacter species and detailed functional genomic/physiological studies on one species, Geobacter sulfurreducens. Genome-based models of several Geobacter species that are able to predict physiological responses under different environmental conditions are now available. Quantitative analysis of gene transcript levels during in situ uranium bioremediation has demonstrated that it is possible to track in situ rates of metabolism and the in situ metabolic state of Geobacter in the subsurface. Initial attempts to link in silico Geobacter models with existing subsurface hydrological and geochemical models are underway. It is expected that this systems approach to bioremediation with Geobacter will provide the opportunity to evaluate multiple Geobacter-catalyzed bioremediation strategies in silico prior to field implementation, thus providing substantial savings when initiating large-scale in situ bioremediation projects for groundwater polluted with uranium and/or organic contaminants.

Signalling Networks and Pollutant Biosensors
The two elements needed for an efficient utilization of aromatic compounds by bacteria are the enzymes responsible for their degradation and the regulatory elements that control the expression of the catabolic operons to ensure the more efficient output depending on the presence/absence of the aromatic compounds or alternative environmental signals. Transcriptional regulation seems to be the more common and/or most studied mechanism of expression of catabolic clusters, although post-transcriptional control also plays an important role. Transcription is dependent on specific regulators that channel the information between specific signals and the target gene(s). A more complex network of signals connects the metabolic and the energetic status of the cell to the expression of particular catabolic clusters, overimposing the specific transcriptional regulatory control. In general, the regulatory networks that control the operons involved in the catabolism of aromatic compounds are endowed with an extraordinary degree of plasticity and adaptability. Elucidating such regulatory networks pave the way for a better understanding of the regulatory intricacies that control microbial biodegradation of aromatic compounds, which are key issues for the rational design of more efficient recombinant biodegraders, bacterial biosensors, and biocatalysts.

Bioavailability, Chemotaxis, and Transport of Organic Pollutants
Bacterial pathways for the degradation of organic pollutants have been the subject of intense study for decades. However, important physiological events that precede the catabolism of these compounds have recently been receiving significant scientific attention. Bioavailability, or the amount of a substance that is physiochemically accessible to microorganisms is a key factor in the efficient biodegradation of pollutants. Chemotaxis, or the directed movement of motile organisms towards or away from chemicals in the environment is an important physiological response that may contribute to effective catabolism of molecules in the environment. In addition, mechanisms for the intracellular accumulation of aromatic molecules via various transport mechanisms are also important.

Solvent Tolerance and Pumps That Extrude Toxic Chemicals
Organic solvents are toxic for microorganisms because they dissolve in the cytoplasmic membranes, a process that alters the membrane's physical structure and renders the cell unable to synthesize ATP. The degree of toxicity varies depending on the chemical and the strain involved, and chemical toxicity correlates with the partition coefficient of the compound in a mixture of octanol and water. Microbial tolerance to solvents can be mediated by physical and biochemical barriers. Physical barriers are usually based on increased membrane rigidity through the alteration of the cis/trans unsaturated fatty acid ratio, the increase in the saturated:unsaturated fatty acid ratio, or alteration in the phopholipd head groups. Although these barriers counteract the effect of initial toxicity, long-term resistance is based on the active extrusion of solvents, which is mainly mediated by extrusion pumps. Genomic analyses in Gram-negative bacteria have revealed that the resistance-nodulation-cell division (RND) family of efflux pumps is the main group involved in the removal of solvents from the cell. These pumps are made up of three components that span the membranes and extrude solvents from the inner membrane or cytoplasm to the outer medium. The level of expression of these extrusion pumps is finely modulated by transcriptional regulators belonging to different families, but which act in a similar fashion. Some regulators act as repressors that prevent access of the RNA polymerase to the promoter region of the cognate operon. These regulators recognize multiple drugs through a series of overlapping binding pockets, and upon the binding of an effector, transmit a signal to the DNA binding region so that the regulator is released and the genes encoding the efflux pumps are transcribed.

Evolution of Catabolic Pathways and Bacterial Adaptation to Xenobiotic Compounds
Bacteria adapt and become quite rapidly selected to xenobiotic compounds introduced into the environment, mainly via the usage of the compound as carbon, energy or nitrogen source. Important examples include chlorobenzenes, the herbicide 2,4-dichlorophenoxyacetic acid, chloroalkanes, lindane, atrazine and nitroaromatic compounds. At the genomic level, such bacteria show evidence for genetic rearrangements mediated by transposable elements or general recombination, the result being most often an expansion of existing catabolic properties with additional gene modules from outside sources. DNA from outside sources appears to have been trapped and mobilized via conjugative plasmids and genomic islands. Genomic evidence shows that most bacterial genomes contain considerable numbers of insertion elements, integrases, prophages and/or plasmids, which can contribute to their adaptation capacities.

Oil Biodegradation in Marine Systems
Petroleum oil is toxic for most life forms and episodic and chronic pollution of the environment by oil causes major ecological perturbations. Marine environments are especially vulnerable since oil spills of coastal regions and the open sea are poorly containable and mitigation is difficult. In addition to pollution through human activities, millions of tons of petroleum enter the marine environment every year from natural seepages. Despite its toxicity, a considerable fraction of petroleum oil entering marine systems is eliminated by the hydrocarbon-degrading activities of microbial communities, in particular by a remarkable recently discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB). Alcanivorax borkumensis, a paradigm of HCB and probably the most important global oil degrader, was the first to be subjected to a functional genomic analysis. This analysis has yielded important new insights into its capacity for (i) n-alkane degradation including metabolism, biosurfactant production and biofilm formation, (ii) scavenging of nutrients and cofactors in the oligotrophic marine environment, as well as (iii) coping with various habitat-specific stresses. The understanding thereby gained constitutes a significant advance in efforts towards the design of new knowledge-based strategies for the mitigation of ecological damage caused by oil pollution of marine habitats. HCB also have potential biotechnological applications in the areas of bioplastics and biocatalysis.

Emerging Technologies to Analyze Natural Attenuation and Bioremediation
Natural attenuation is one of several cost-saving options for the treatment of polluted environment, in which microorganisms contribute to pollutant degradation. For risk assessments and endpoint forecasting, natural attenuation sites should be carefully monitored (monitored natural attenuation). When site assessments require rapid removal of pollutants, bioremediation, categorized into biostimulation (introduction of nutrients and chemicals to stimulate indigenous microorganisms) and bioaugmentation (inoculation with exogenous microorganisms), can be applied. In such a case, special attention should be paid to its influences on indigenous biota and the dispersal and outbreak of inoculated organisms. Recent advances in microbial ecology have provided molecular technologies, e.g., detection of degradative genes, community fingerprinting and metagenomics, which are applicable to the analysis and monitoring of indigenous and inoculated microorganisms in polluted sites. Scientists have started to use some of these technologies for the assessment of natural attenuation and bioremediation in order to increase their effectiveness and reliability.

Analysis of Waste Biotreatment in Confined Environments
Sustainable development requires the promotion of environmental management and a constant search for new technologies to treat vast quantities of wastes generated by increasing anthropogenic activities. Biotreatment, the processing of wastes using living organisms, is an environmentally friendly, relatively simple and cost-effective alternative to physico-chemical clean-up options. Confined environments, such as bioreactors, have been engineered to overcome the physical, chemical and biological limiting factors of biotreatment processes in highly controlled systems. The great versatility in the design of confined environments allows the treatment of a wide range of wastes under optimized conditions. To perform a correct assessment, it is necessary to consider various microorganisms having a variety of genomes and expressed transcripts and proteins. A great number of analyses are often required. Using traditional genomic techniques, such assessments are limited and time-consuming. However, several high-throughput techniques originally developed for medical studies can be applied to assess biotreatment in confined environments.

Metabolic Engineering and Biocatalytic Applications of the Pollutant Degradation Machinery
The study of the fate of persistent organic chemicals in the environment has revealed a large reservoir of enzymatic reactions with a large potential in preparative organic synthesis, which has already been exploited for a number of oxygenases on pilot and even on industrial scale. Novel catalysts can be obtained from metagenomic libraries and DNA-sequence based approaches. Our increasing capabilities in adapting the catalysts to specific reactions and process requirements by rational and random mutagenesis broadens the scope for application in the fine chemical industry, but also in the field of biodegradation. In many cases, these catalysts need to be exploited in whole cell bioconversions or in fermentations, calling for system-wide approaches to understanding strain physiology and metabolism and rational approaches to the engineering of whole cells as they are increasingly put forward in the area of systems biotechnology and synthetic biology.

More Information:     Microbial Biodegradation: Genomics and Molecular Biology

See also:     Pseudomonas: Genomics and Molecular Biology

The Taxonomy of Pseudomonas
The studies on the taxonomy of this complicated genus groped their way in the dark while following the classical procedures developed for the description and identification of the organisms involved in sanitary bacteriology during the first decades of the twentieth century. This situation sharply changed with the proposal to introduce as the central criterion the similarities in the composition and sequences of macromolecules components of the ribosomal RNA. The new methodology clearly showed that the genus Pseudomonas, as classical defined, consisted in fact of a conglomerate of genera that could clearly be separated into five so-called rRNA homology groups. Moreover, the taxonomic studies suggested an approach that might proved useful in taxonomic studies of all other prokaryotic groups. A few decades after the proposal of the new genus Pseudomonas by Migula in 1894, the accumulation of species names assigned to the genus reached alarming proportions. At the present moment, the number of species in the current list has contracted more than ten-fold. In fact, this approximated reduction may be even more dramatic if one considers that the present list contains many new names, i.e., relatively few names of the original list survived in the process. The new methodology and the inclusion of approaches based on the studies of conservative macromolecules other than rRNA components, constitutes an effective prescription that helped to reduce Pseudomonas nomenclatural hypertrophy to a manageable size.

Genome Diversity of Pseudomonas aeruginosa
The G+C rich Pseudomonas aeruginosa chromosome consists of a conserved core and a variable accessory part. The core genomes of P. aeruginosa strains are largely collinear, exhibit a low rate of sequence polymorphism and contain few loci of high sequence diversity, notably the pyoverdine locus, the flagellar regulon, pilA and the O-antigen biosynthesis locus. Variable segments are scattered throughout the genome of which about one third are immediately adjacent to tRNA or tmRNA genes. The three known hot spots of genomic diversity are caused by the integration of genomic islands of the pKLC102 / PAGI-2 family into tRNA^Lys or tRNA^Gly genes. The individual islands differ in their repertoire of metabolic genes, but share a set of syntenic genes that confer their horizontal spread to other clones and species. Colonization of atypical disease habitats predisposes to deletions, genome rearrangements and accumulation of loss-of-function mutations in the P. aeruginosa chromosome. The P. aeruginosa population is characterized by a few dominant clones widespread in disease and environmental habitats. The genome is made up of clone-typical segments in core and accessory genome and of blocks in the core genome with unrestricted gene flow in the population.

Oligonucleotide Usage Signatures of the Pseudomonas putida KT2440 Genome
Di- to pentanucleotide usage and the list of the most abundant octa- to tetradecanucleotides are useful measures of the bacterial genomic signature. The Pseudomonas putida KT2440 chromosome is characterized by strand symmetry and intra-strand parity of complementary oligonucleotides. Each tetranucleotide occurs with similar frequency on the two strands. Tetranucleotide usage is biased by G+C content and physicochemical constraints such as base stacking energy, dinucleotide propeller twist angle or trinucleotide bendability. The 105 regions with atypical oligonucleotide composition can be differentiated by their patterns of oligonucleotide usage into categories of horizontally acquired gene islands, multidomain genes or ancient regions such as genes for ribosomal proteins and RNAs. A species-specific extragenic palindromic sequence is the most common repeat in the genome that can be exploited for the typing of P. putida strains. In the coding sequence of P. putida LLL is the most abundant tripeptide.

Genetic Tools for Pseudomonas
Genetic tools are required to take full advantage of the wealth of information generated by genome sequencing efforts, and ensuing global gene and protein expression analyses. Although the development of genetic tools has generally not kept up with the sequencing pace, substantial progress has been made in this arena. PCR- and recombination-based strategies allowed construction of whole genome expression and transposon insertion libraries. Similar strategies combined with improved transformation protocols facilitate high-throughput construction of deletion alleles and development of a broad-host-range mini-Tn7 chromosome integration system. While to date most of these tools and methods have been developed for and applied in P. aeruginosa, they will most likely also be applicable to other Pseudomonas with appropriate modifications.

Molecular Biology of Cell-Surface Polysaccharides in Pseudomonas aeruginosa: From Gene to Protein Function
Cell-surface polysaccharides play diverse roles in the bacterial "lifestyle". They serve as a barrier between the cell wall and the environment, mediate host-pathogen interactions, and form structural components of biofilms. These polysaccharides are synthesized from nucleotide-activated precursors and, in most cases, all the enzymes necessary for biosynthesis, assembly and transport of the completed polymer are encoded by genes organized in dedicated clusters within the genome of the organism. Lipopolysaccharide is one of the most important cell-surface polysaccharides, as it plays a key structural role in outer membrane integrity, as well as being an important mediator of host-pathogen interactions. The genetics for the biosynthesis of the so-called A-band (homopolymeric) and B-band (heteropolymeric) O antigens have been clearly defined, and a lot of progress has been made toward understanding the biochemical pathways of their biosynthesis. The exopolysaccharide alginate is a linear copolymer of ß-1,4-linked D-mannuronic acid and L-guluronic acid residues, and is responsible for the mucoid phenotype of late-stage cystic fibrosis disease. The pel and psl loci are two recently discovered gene clusters that also encode exopolysaccharides found to be important for biofilm formation. Rhamnolipid is a biosurfactant whose production is tightly regulated at the transcriptional level, but the precise role that it plays in disease is not well understood at present. Protein glycosylation, particularly of pilin and flagellin, is a recent focus of research by several groups and it has been shown to be important for adhesion and invasion during bacterial infection.

Pseudomonas aeruginosa Virulence and Pathogenesis Issues
Regulation of gene expression can occur through cell-cell communication or quorum sensing (QS) via the production of small molecules called autoinducers. QS is known to control expression of a number of virulence factors. Another form of gene regulation which allows the bacteria to rapidly adapt to surrounding changes is through environmental signaling. Recent studies have discovered that anaerobiosis can significantly impact the major regulatory circuit of QS. This important link between QS and anaerobiosis has a significant impact on production of virulence factors of this organism.

Pseudomonas aeruginosa Biofilms: Impact of Small Colony Variants on Chronic Persistent Infections
The achievements of medical care in industrialised societies are markedly impaired due to chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the ageing population. Chronic infections remain a major challenge for the medical profession and are of great economic relevance because traditional antibiotic therapy is usually not sufficient to eradicate these infections. One major reason for persistence seems to be the capability of the bacteria to grow within biofilms that protects them from adverse environmental factors. Pseudomonas aeruginosa is not only an important opportunistic pathogen and causative agent of emerging nosocomial infections but can also be considered a model organism for the study of diverse bacterial mechanisms that contribute to bacterial persistence. In this context the elucidation of the molecular mechanisms responsible for the switch from planctonic growth to a biofilm phenotype and the role of inter-bacterial communication in persistent disease should provide new insights in P. aeruginosa pathogenicity, contribute to a better clinical management of chronically infected patients and should lead to the identification of new drug targets for the development of alternative anti-infective treatment strategies.

Antibiotic Resistance in Pseudomonas
Pseudomonas aeruginosa is a highly relevant opportunistic pathogen. One of the most worrisome characteristics of P. aeruginosa consists in its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes and the low permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily develop acquired resistance either by mutation in chromosomally-encoded genes, either by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events that include acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown that phenotypic resistance associated to biofilm formation or to the emergence of small-colony-variants may be important in the response of P. aeruginosa populations to antibiotics treatment.

Iron uptake in Pseudomonas
Like all aerobic bacteria, pseudomonads need to take up iron via the secretion of siderophores which complex iron (III) with high affinity. Much progress has been made in the elucidation of siderophore-mediated high-affinity iron uptake by Pseudomonas, especially in the case of the opportunistic pathogen, P. aeruginosa. Fluorescent pseudomonads produce the high-affinity peptidic siderophore pyoverdine, but also, in many cases, a second siderophore of lesser affinity for iron. Some of the genes for the biosynthesis and uptake of these siderophores have been identified and the functions of the encoded proteins known. Iron uptake via siderophores is regulated at several levels, via the general iron-sensitive repressor Fur (Ferric Uptake Regulator), via extracytoplasmic sigma factors/anti-sigma factors or via other regulators. Since pseudomonads are ubiquitous microorganisms, it is not surprising to find in their genome a large number of genes encoding receptors for the uptake of heterologous ferrisiderophores or heme reflecting their great adaptability to diverse iron sources. Another exciting development is the recent evidence for a cross-talk between the iron regulon and other regulatory networks, including the diffusible signal molecule-mediated quorum sensing in P. aeruginosa.

More information:     Pseudomonas: Genomics and Molecular Biology

from: Roy, P. 2008 Molecular Dissection of Bluetongue Virus. Chapter 7 In: Animal Viruses Molecular Biology. Mettenleiter, T.C. and Sobrino, F. (Eds.) Caister Academic Press, UK

Bluetongue virus (BTV), a member of Orbivirus genus within the Reoviridae family causes serious disease in livestock (sheep, goat, cattle). Partly due to this BTV has been in the forefront of molecular studies for last three decades and now represents one of the best understood viruses at the molecular and structural levels. BTV, like the other members of the family is a complex non-enveloped virus with seven structural proteins and a RNA genome consisting of 10 double-stranded (ds) RNA segments of different sizes. Data obtained from studies over a number of years have defined the key players in BTV entry, replication, assembly and exit and have increasingly found roles for host proteins at each stage. Specifically, it has been possible to determine the complex nature of the virion through 3D structure reconstructions (diameter ~ 800 Å); the atomic structure of proteins and the internal capsid (~ 700 Å, the first large highly complex structure ever solved); the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell and the protein sequestration required for it; and the role of host proteins in virus entry and virus release. These areas are important for BTV replication but they also indicate the pathways that may be used by related viruses, which include viruses that are pathogenic to man and animals, thus providing the basis for developing strategies for intervention or prevention.

BTV is the type species of the genus Orbivirus within the family Reoviridae. The Reoviridae family is one of the largest families of viruses and includes major human pathogens (e.g., rotavirus) as well as other vertebrate, plant and insect pathogens. Like the other members of the family, Orbiviruses which encompass, besides BTV, the agents causing African horse sickness (AHSV) and epizootic hemorrhagic disease of deer (EHDV), have the characteristic double-stranded and segmented features of their RNA genomes. However, unlike the mammalian reoviruses, Orbiviruses comprising 14 serogroups, are vectored to a variety of vertebrates by arthropod species (e.g., gnats, mosquitoes and ticks) and replicate in both hosts. BTV, the etiological agent of Bluetongue disease of animals, is transmitted by Culicoides species. In sheep BTV causes an acute disease with high morbidity and mortality. BTV also infects goats, cattle and other domestic animals as well as wild ruminants (e.g., blesbuck, white-tailed deer, elk, pronghorn antelope, etc.). The disease was first described in the late 18th century and was believed for many decades to be confined to Africa. However, to date BTV has been isolated in many tropical, subtropical and temperate zones and 24 serotypes have been identified from different parts of the world. Due to its economic significance BTV has been the subject of extensive molecular, genetic and structural studies. As a consequence it now represents one of the best characterised viruses.

Unlike the reovirus and rotavirus particles, the mature BTV particle is relatively fragile and the infectivity of BTV is lost easily in mildly acidic conditions. BTV virions (550S) are architecturally complex structures composed of 7 discrete proteins that are organised into two concentric shells, the outer and inner capsids, and a genome of 10 dsRNA segments. The outer capsid, which is composed of two major structural proteins (VP2 and VP5), is involved in cell attachment and virus penetration during the initial stages of infection. Shortly after infection, BTV is uncoated, i.e. VP2 and VP5 are removed, to yield a transcriptionally active 470S core particle which is composed of two major proteins VP7 and VP3, and the three minor proteins VP1, VP4 and VP6 in addition to the dsRNA genome. There is no evidence that any trace of the outer capsid remains associated with these cores, as has been described for reovirus. The cores may be further uncoated to form 390S subcore particles that lack VP7, also in contrast to reovirus. Subviral particles are probably akin to cores derived in vitro from virions by physical or proteolytic treatments that remove the outer capsid and causes activation of the BTV transcriptase. In addition to the seven structural proteins, three non-structural (NS) proteins, NS1, NS2, NS3 (and a related NS3A) are synthesised in BTV-infected cells. Of these, NS3/NS3A is involved in the egress of the progeny virus. The two remaining non-structural proteins, NS1 and NS2, are produced at high levels in the cytoplasm and are believed to be involved in virus replication, assembly and morphogenesis.

More information: Roy, P. 2008 Molecular Dissection of Bluetongue Virus. Chapter 7 In: Animal Viruses Molecular Biology. Mettenleiter, T.C. and Sobrino, F. (Eds.) Caister Academic Press, UK

The Scientist is inviting nominations for the most popular science blogs. If you think that the Microbiology Blog deserves a mention please just leave a comment here.

Many thanks!

See also:     Bacteriophage: Genetics and Molecular Biology

The New Phage Biology: From Genomics to Applications
Bacterial viruses, or bacteriophages, are estimated to be the most widely distributed and diverse entities in the biosphere. From initial research defining the nature of viruses, to deciphering the fundamental principles of life, to the development of the science of molecular biology, phages have been 'model organisms' for probing the basic chemistry of life. With more recent advances in technology, most notably the ability to elucidate the genome sequences of phages and their bacterial hosts, there has been a resurgence of interest in phages as more information is generated regarding their biology, ecology and diverse nature. Phage research in more recent years has revealed not only their abundance and diversity of form, but also their dramatic impact on the ecology of our planet, their influence on the evolution of microbial populations, and their potential applications.

Bacteriophage Bioinformatics and Genomics
Bacteriophages have played and continue to play a key role in bacterial genetics and molecular biology. Historically, they were used to define gene structure and gene regulation. Also the first genome to be sequenced was a bacteriophage. However, bacteriophage research did not lead the genomics revolution, which is clearly dominated by bacterial genomics. Only very recently has the study of bacteriophage genomes become prominent, thereby enabling researchers to understand the mechanisms underlying phage evolution. Bacteriophage genome sequences can be obtained through direct sequencing of isolated bacteriophages, but can also be derived as part of microbial genomes. Analysis of bacterial genomes has shown that a substantial amount of microbial DNA consists of prophage sequences and prophage-like elements. A detailed database mining of these sequences offers insights into the role of prophages in shaping the bacterial genome.

Bacteriophage in the Environment
Some time ago it was detected that phages are much more abundant in the water column of freshwater and marine habitats than previously thought and that they can cause significant mortality of bacterioplankton. Methods in phage community ecology have been developed to assess phage-induced mortality of bacterioplankton and its role for food web process and biogeochemical cycles, to genetically fingerprint phage communities or populations and estimate viral biodiversity by metagenomics. The release of lysis products by phages converts organic carbon from particulate (cells) to dissolved forms (lysis products), which makes organic carbon more bio-available and thus acts as a catalyst of geochemical nutrient cycles. Phages are not only the most abundant biological entities but probably also the most diverse ones. The majority of the sequence data obtained from phage communities has no equivalent in data bases. These data and other detailed analyses indicate that phage-specific genes and ecological traits are much more frequent than previously thought. In order to reveal the meaning of this genetic and ecological versatility, studies have to be performed with communities and at spatiotemporal scales relevant for microorganisms.

Bacteriophages and Food Fermentations
A broad number of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various organic substrates. Because enormous amounts of bacteria are being cultivated each day in large fermentation vats, the risk that bacteriophage contamination rapidly brings fermentations to a halt and cause economical setbacks is a serious threat in these industries. The relationship between bacteriophages and their bacterial hosts is very important in the context of the food fermentation industry. Sources of phage contamination, measures to control their propagation and dissemination, and biotechnological defence strategies developed to restrain phages are of interest. The dairy fermentation industry has openly acknowledged the problem of phage and has been working with academia and starter culture companies to develop defence strategies and systems to curtail the propagation and evolution of phages for decades.

Bacteriophages in Medicine
Bacteriophages, or phages, are viruses of bacteria. Thus, by their very nature, they can be considered as potential antibacterial agents. Over the past decade or two, the idea of phage therapy, i.e. the use of lytic bacteriophages for both the prophylaxis and the treatment of bacterial infections, has gained special significance in view of a dramatic rise in the prevalence of highly antibiotic-resistant bacterial strains paralleled by the withdrawal of the pharmaceutical industry from research into new antibiotics. As an alternative to "classic" phage therapy, in which whole viable phage particles are used, one can also employ bacteriophage-encoded lysis-inducing proteins, either as recombinant proteins or as lead structures for the development of novel antibiotics. Two additional, rather less-recognized potential medical applications of phages are the treatment of viral infections and their use as immunizing agents in diagnosing and monitoring patients with immunodeficiences. Recent novel findings have demonstrated the immunomodulatory activity of bacteriophages, suggestive of a potential role of endogenous phages in maintaining the homeostasis of the immune system.

Phage Therapy: The Western Perspective
Phage therapy has a long and colourful history. Phages have been explored as means to eliminate pathogens like Campylobacter in raw food and Listeria in fresh food or to reduce food spoilage bacteria. In agricultural practice phages were used to fight pathogens like Campylobacter , Escherichia and Salmonella in farm animals, Lactococcus and Vibrio pathogens in fish from aquaculture and Erwinia and Xanthomonas in plants of agricultural importance. The oldest use was, however, in human medicine. Phages were used against diarrheal diseases caused by E. coli, Shigella or Vibrio and against wound infections caused by facultative pathogens of the skin like staphylococci and streptococci. Phage therapy therefore looks like a platform technology. This impression is reinforced by recent extension of the phage therapy approach to systemic and even intracellular infections and the addition of non-replicating phage and isolated phage enzymes like lysins to the antimicrobial arsenal. However, despite some hope and hype in recent editorials on phage therapy, definitive proof for the efficiency of these phage approaches in the field or the hospital is only provided in a few cases.


Bacteriophage Host Interaction in Lactic Acid Bacteria
The first contact between an infecting phage and its bacterial host is the attachment of the phage to the host cell. This attachment is mediated by the phage's receptor binding protein (RBP), which recognizes and binds to a receptor on the bacterial surface. RBP's are also referred to as: host specificity protein, host determinant, and anti-receptor. For simplicity, the RBP term will be used here. A variety of molecules have been suggested to act as host receptors for bacteriophages infecting lactic acid bacteria (LAB); among those are polysaccharides, (lipo)teichoic acids as well as a single membrane protein. A number of RBPs of LAB phages have been identified by the generation of hybrid phages with altered host range. These studies, however, also found additional phage proteins to be important for successful a phage infection. Analysis of the crystal structure of several RBPs indicated that these proteins share a common tertiary folding as well as supporting previous indications of the saccharide nature of the host receptor. The Gram-positive LAB have a thick peptidoglycan layer, which must be traversed in order to inject the phage genome into the bacterial cytoplasm. Peptidoglycan-degrading enzymes are expected to facilitate this penetration and such enzymes have been found as structural elements of a number of LAB phages.

Transfer of DNA From Phage to Host
Phage DNA transport is atypical among membrane transport and thus poses a fascinating problem: transport is unidirectional; it concerns a unique molecule the size of which may represent 50 times that of the bacterium. The rate of DNA transport can reach values as high as 3 to 4 thousands base pairs / sec. This raises many questions. Is there a single mechanism of transport for all types of phages? How does the phage genome overcome the hydrophobic barrier of the host envelope? Is DNA transported as a free molecule or in association with proteins? Is such transport dependent on phage and / or host cell components? What is the driving force for transport?

Prophages and Their Contribution to Host Cell Phenotype
In many bacterial species, prophages figure prominently in the biology of these cells, often conferring key phenotypes that can convert a non-pathogenic strain into a pathogen. The source of these phenotypic changes can be through prophage-encoded toxins, bacterial cell surface alterations, or resistance to the human immune system. Further, prophage integration into the host genome can inactivate or alter the expression of host genes. In addition to these direct genetic alterations associated with the addition or inactivation of genes, prophages can also alter the phenotype of bacteria at the population level by facilitating the spread of favorable genes through transduction.

Prophage Induction of Phage λ
The gene regulatory circuitry of phage λ is among the best-understood circuits at the mechanistic level. This circuitry involves several interesting regulatory behaviors. An infected cell undergoes a decision between two alternative pathways, the lytic and lysogenic pathways. If the latter is followed, the lysogenic state is established and maintained. While this state is highly stable, it can switch to the lytic pathway in the process of prophage induction, which occurs when the host SOS response is triggered by DNA damage.

Phage Φ29: Membrane-associated DNA Replication and Mechanism of Alternative Infection Strategy
Continuous research, spanning a period of more than three decades, has made the Bacillus bacteriophage Φ29 a paradigm for the study of several molecular mechanisms of general biological processes, including DNA replication and regulation of transcription. The genome of Φ29 consists of a linear double-stranded (ds) DNA, which has a terminal protein covalently linked to its 5' ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail in vitro and is considered to be a model system that is also used by other linear genomes with a terminal protein linked to their DNA ends. Phage Φ29 has also been proven to be a versatile system to study in vitro transcription regulation in general and the switch from early to late phage transcription in particular. The detailed knowledge of in vitro phage Φ29 DNA replication and transcription regulation makes it an attractive model to study these processes in vivo. For many years it has been known that (i) phage Φ29 DNA replication, as well as that of other prokaryotic genomes, occurs at the cytosolic membrane, and (ii) the lytic Φ29 cycle is suppressed in early sporulating cells and under these conditions the infecting phage genome becomes trapped into the spore. The molecular mechanisms involved in these processes were largely unknown.

Release of Progeny Phages from Infected Cells
Progeny release from phage-infected cells can occur either by lysis of the host or by a singular secretion mechanism, which has been only documented so far for filamentous phages. All known double stranded DNA phages synthesize two lysis effectors, an endolysin and a holin, the first providing a muralytic function and the second a lysis timing device. Endolysins and holins from different phages can be structurally very diverse in spite of their functional similarities. In its export to the cell wall, the endolysin can either be dependent on holin-formed membrane lesions or use the general secretion pathway of the host. In several known cases an antiholin is also produced. This protein can be either soluble or membrane-bound. In T4, the anti-holin is crucial in the response to superinfecting phage, in a process known as lysis inhibition (LIN). Phage members of the Microviridae and Leviviridae families are also bacteriolytic but use a single gene lysis strategy to release their progeny. The mechanism employed relies on the production of murein synthesis inhibitors and thus lysis by such phages is akin to lysis mediated by antibiotics which target the cell wall. The Inoviridae, filamentous phages, do not lyse their hosts. They are assembled during export, using transmembrane channels formed by at least one inner membrane phage-encoded protein and an outer membrane secretin.

More information:     Bacteriophage: Genetics and Molecular Biology

More information: The Cyanobacteria: Molecular Biology, Genomics and Evolution

Cyanobacteria and Earth History
The biochemical capacity to use water as the source for electrons in photosynthesis evolved once, in a common ancestor of extant cyanobacteria. The geological record indicates that this transforming event took place early in our planet's history, at least 2450-2320 million years ago (Ma), and possibly much earlier. Geobiological interpretation of Archean (>2500 Ma) sedimentary rocks remains a challenge; available evidence indicates that life existed 3500 Ma, but the question of when oxygenic photosynthesis evolved continues to engender debate and research. A clear paleontological window on cyanobacterial evolution opened about 2000 Ma, revealing an already diverse biota of blue-greens. Cyanobacteria remained principal primary producers throughout the Proterozoic Eon (2500-543 Ma), in part because the redox structure of the oceans favored photautotrophs capable of nitrogen fixation. Green algae joined blue-greens as major primary producers on continental shelves near the end of the Proterozoic, but only with the Mesozoic (251-65 Ma) radiations of dinoflagellates, coccolithophorids, and diatoms did primary production in marine shelf waters take modern form. Cyanobacteria remain critical to marine ecosystems as primary producers in oceanic gyres, as agents of biological nitrogen fixation, and, in modified form, as the plastids of marine algae.

Insights into Cyanobacterial Evolution from Comparative Genomics
Recent high-throughput sequencing has provided DNA sequences at an unprecedented rate, posing considerable analytical challenges, but also offering insight into the genetic mechanisms of adaptation. Here we present a comparative genomics-based approach towards understanding the evolution of these mechanisms in cyanobacteria. Historically, systematic methods of defining morphological traits in cyanobacteria have posed a major barrier in reconstructing their true evolutionary history. The advent of protein, then DNA, sequencing - most notably the use of 16S rRNA as a molecular marker - helped circumvent this barrier and now forms the basis of our understanding of the history of life on Earth. However, these tools have proved insufficient for resolving relationships between closely related cyanobacterial species. The 24 cyanobacteria whose genomes have been compared occupy a wide variety of environmental niches and play major roles in global carbon and nitrogen cycles. By integrating phylogenetic data inferred for hundreds to nearly 1000 protein coding genes common to all or most cyanobacteria, we are able to reconstruct an evolutionary history of the entire phylum, establishing a framework for resolving how their metabolic and phenotypic diversity came about.

Gene transfer to Cyanobacteria in the Laboratory and in Nature
Horizontal (lateral) gene transfer is a postulated mechanism influencing bacterial evolution. Known mechanisms of DNA transfer into cyanobacteria include genetic transformation and conjugation with Escherichia coli, which are widely used in the laboratory with several different cyanobacterial strains. Additionally, direct (likely conjugal) transfer of DNA between cyanobacterial strains has been demonstrated. These transfer mechanisms can represent the basis for genetic recombination in natural populations of cyanobacteria, for which several possible examples have been described, as well as for the horizontal transfer that is deduced for some genes in protein phylogeny studies, which have been made possible by the current availability of numerous complete cyanobacterial genomic sequences.

Molecular Ecology and Environmental Genomics of Cyanobacteria
The application of molecular biology and genomics to microbial ecology has been a transformative force, making possible the discovery of layer upon layer of complexity in natural communities of microbes. Diversity surveying, community fingerprinting, and functional interrogation of natural populations have become common, enabled by a battery of molecular and bioinformatics techniques, some specifically developed for the cyanobacteria. The ensuing effects on our views of cyanobacterial ecology have been perhaps less revolutionary, because of the special characteristics of cyanobacteria among microbes, but also significant. We have come to realize that the present taxonomic system is often phylogenetically incorrect; several new cyanobacteria or groups thereof have been discovered, and some established groups have been found to be constructs. Surveying efforts have covered many habitats and have demonstrated that cyanobacterial communities tend to be habitat-specific, and that plenty of undescribed genetic diversity is concealed among morphologically simple types. We can now select among isolates those that are good representatives of natural populations, enabling, among other objectives, ecologically motivated genome sequencing efforts. We have witnessed the first studies addressing population genetics and the blooming of functional studies based on detection of gene expression in Nature. We are entering an era of expansion of the polyphasic approaches that combine molecular, bioinformatics, physiological, and geochemical techniques to study natural communities

Comparative Genomics of Marine Cyanobacteria and their Phages
At present there are 24 cyanobacteria for which a total genome sequence is available. 15 of these cyanobacteria come from the marine environment. These are six Prochlorococcus strains, seven marine Synechococcus strains, Trichodesmium erythraeum IMS101 and Crocosphaera watsonii WH8501. Several studies have demonstrated how these sequences could be used very successfully to infer important ecological and physiological characteristics of marine cyanobacteria. However, there are many more genome projects currently in progress, amongst those there are further Prochlorococcus and marine Synechococcus isolates, Acaryochloris and Prochloron, the N₂-fixing filamentous cyanobacteria Nodularia spumigena, Lyngbya aestuarii and Lyngbya majuscula, as well as bacteriophages infecting marine cyanobaceria. Thus, the growing body of genome information can also be tapped in a more general way to address global problems by applying a comparative approach. Some new and exciting examples of progress in this field are the identification of genes for regulatory RNAs, insights into the evolutionary origin of photosynthesis, or estimation of the contribution of horizontal gene transfer to the genomes that have been analyzed.

Stress Responses in Synechocystis: Regulated Genes and Regulatory Systems
Genome-wide investigations of gene expression at the transcriptional level in cyanobacteria, using DNA microarrays, have allowed identification of genes whose expression is induced or repressed by various types of environmental stress and also of previously uncharacterized genes that appear to be involved in stress responses. Acclimation to stress begins with perception of stress and transduction of the stress signal. A combination of the systematic mutation of potential sensors and transducers and DNA microarray analysis has led to significant progress in understanding the mechanisms of perception of and reaction to environmental stress in cyanobacteria. Recent progress has been made in the identification of stress-inducible genes and of systems that regulate responses to stress that has been made using DNA microarrays in cyanobacteria and, in particular, in Synechocystis sp. PCC 6803.

Bioactive Compounds Produced by Cyanobacteria
Cyanobacteria produce a large variety of bioactive compounds, including substances with anti-cancer and anti-viral activity, UV protectants, specific inhibitors of enzymes, and potent hepatotoxins and neurotoxins. Only a few biosynthetic pathways have been elucidated. So far genes have been identified for several bioactive proteins, "ribosomal" and "non-ribosomal" peptides, and peptide-polyketide hybrid molecules. Potential functions of bioactive compounds for the producing cells and evolutionary aspects are discussed, and methods for the detection of cyanobacterial toxins and harmful cyanobacteria are described.

The Cyanobacterial Circadian Clock and the KaiC Phosphorylation Cycle
The circadian clock is an endogenous timing mechanism in living organisms that coordinates their lives with environmental changes. Cyanobacteria are the simplest organisms that are known to exhibit circadian rhythms, and they have become one of the most successful model organisms for circadian biology. Although the circadian clock in cyanobacteria has the same fundamental features as that in eukaryotes, its individual components that have been identified to date are unique. The molecular mechanism of the cyanobacterial clock is different from that described for the eukaryotic clock. The clock core in cyanobacteria is the KaiC phosphorylation cycle.

Molecular Structure of the Photosynthetic Apparatus
The process of conversion of light energy from the sun into chemical energy is catalyzed by oxygenic photosynthesis. It is the process that provides all higher life on earth with energy. All oxygen in the atmosphere is evolved by this process, which was invented 2.8 billion years ago by the ancestors of cyanobacteria. Cyanobacteria are even nowadays very important members of the global ecosystem, and contribute up to 30% of the yearly oxygen production on earth. The structure and function of the protein complexes that catalyze the first steps of the energy conversion have been described. Light is captured by antenna complexes and transferred to two large bio-solar systems, photosystem I and II, which catalyze the transmembrane charge separation. This drives the photosynthetic process and provides the energy for production of the high-energy substrate ATP and reduced hydrogen in the form of NADPH. The photosystems are functionally coupled by the cytochrome b₆f complex, the membrane intrinsic plastoquinone pool and lumenal electron carriers. The reactions of the electron transport chain lead to an electrochemical proton gradient, which drives synthesis of ATP by the molecular motor, the ATP synthase. The structures of the complexes have been described in respect to the function and evolution of the photosynthetic apparatus.

Membrane Systems in Cyanobacteria
Cyanobacteria are photosynthetic prokaryotes with highly differentiated membrane systems. In addition to a Gram-negative-type cell envelope with plasma membrane and outer membrane separated by a periplasmic space, cyanobacteria have an internal system of thylakoid membranes where the fully functional electron transfer chains of photosynthesis and respiration reside. The presence of different membrane systems lends these cells a unique complexity among bacteria. Cyanobacteria must be able to reorganize the membranes, synthesize new membrane lipids, and properly target proteins to the correct membrane system. The outer membrane, plasma membrane, and thylakoid membranes each have specialized roles in the cyanobacterial cell. Understanding the organization, functionality, protein composition and dynamics of the membrane systems remains a great challenge in cyanobacterial cell biology.

Biogenesis and Dynamics of Thylakoid Membranes and the Photosynthetic Apparatus
Thylakoid membranes are the site of the light-reactions of photosynthesis, and they are crucial to the photosynthetic lifestyle of cyanobacteria. Recent knowledge has been obtained regarding the structure, organisation and biogenesis of thylakoid membranes in cyanobacteria. In particular the dynamics of the membrane, and the roles that protein diffusion may play in membrane biogenesis, regulation of photosynthesis and the turnover and repair of the photosynthetic apparatus. Although we have detailed knowledge of many thylakoid membrane components and some thylakoid membrane processes, much remains to be learned about the large-scale organisation and biogenesis of thylakoid membranes.

Carbon Acquisition by Cyanobacteria: Mechanisms, Comparative Genomics and Evolution
Tthe mechanisms of inorganic carbon uptake, photorespiration, and the regulation between the metabolic fluxes involved in photoautotrophic, photomixotrophic and heterotrophic growth have been identified including the genes involved, their regulation and phylogeny.

Nitrogen Assimilation and C/N Balance Sensing
Cyanobacteria inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. Generally, cyanobacteria are able to utilize a variety of inorganic and organic sources of combined nitrogen, like nitrate, nitrite, ammonium, urea or some amino acids. Several cyanobacterial strains are also capable of diazotrophic growth. Genome sequencing has provided a large amount of information on the genetic basis of nitrogen metabolism and its control in different cyanobacteria. Comparative genomics, together with functional studies, has led to a significant advance in this field over the past years. 2-oxoglutarate has turned out to be the central signalling molecule reflecting the carbon/nitrogen balance of cyanobacteria. Central players of nitrogen control are the global transcriptional factor NtcA, which controls the expression of many genes involved in nitrogen metabolism, as well as the P_II signalling protein, which fine-tunes cellular activities in response to changing C/N conditions. These two proteins are sensors of the cellular 2-oxoglutarate level and have been conserved in all cyanobacteria. In contrast, the adaptation to nitrogen starvation involves heterogeneous responses in different strains.

Transcriptional and Developmental Responses by Anabaena to Deprivation of Fixed Nitrogen
Deprivation of fixed nitrogen, to which the formation of dinitrogen-fixing cells called heterocysts is a conspicuous response, appears to initiate a switch from reliance on photosynthesis for ATP, reductant, and carbon to reliance-in developing cells-on endogenous glycogen stores, heightened utilization of the oxidative pentose phosphate pathway, and-at least in mature heterocysts-photosystem I and influx from vegetative cells. Sugars are needed to produce heterocyst envelope layers of polysaccharide and glycolipid. Decreased transcription and translation of a few highly expressed genes may make possible the increased transcription and translation of many others. Although we interpret metabolic capabilities from microarray data, we stress the hazards of doing so, and emphasize that the interpretations remain to be evaluated. Little is known of the regulation of gene expression during heterocyst differentiation. NtcA is required for the expression of nrrA and many other genes, NrrA is required for the full induction of hetR, and HetR is required for the expression of many downstream genes. Numerous regulatory-family genes are significantly induced during heterocyst differentiation and may importantly regulate the process. Some genes of unknown function increase many-fold in expression during differentiation, leading one to wonder what their roles may be.

Cyanobacterial Nitrogen Fixation in the Ocean: Diversity, Regulation and Ecology
Nitrogen is an essential and major component of biomass. While virtually all life depends on combined forms of nitrogen that are usually limited in availability, some prokaryotes, including many groups of cyanobacteria, can use the ubiquitous atmospheric dinitrogen (N₂). As photoautotrophic bacteria they can easily meet the energy demand that is required by nitrogenase, the enzyme that reduces N₂to NH₃. However, nitrogenase is very sensitive to oxygen and the oxygenic cyanobacteria have evolved various strategies to cope with this paradox. Primary production in the ocean is generally considered to be limited by nitrogen. In recent years it has become clear that N₂-fixing cyanobacteria are important in the nitrogen budget of the surface oceans. Estimates of N₂ fixation indicate that approximately half of global N₂ fixation occurs in the sea. N₂ fixation is not distributed homogenously throughout the oceans. Pelagic diazotrophic cyanobacteria are only found in (sub)tropical oceans and are notably absent in temperate and colder seas. However, at lower salinities in estuaries and other brackish environments, N₂-fixing cyanobacteria can be abundant. N₂-fixing cyanobacteria are also abundant in benthic mats in coastal and aquatic environments all over the globe, including polar regions. This demonstrates that N₂-fixing cyanobacteria are not excluded from temperate and cold marine environments, even though they are only found in the water column of warm oceans.

Cyanobacterial-plant Symbioses: Signalling and Development
Cyanobacteria form stable nitrogen-fixing symbioses with diverse eukaryotes. With few exceptions the cyanobacteria belong to the terrestrial and widespread genus Nostoc. This genus has a notable morphological plasticity which may be in part responsible for its symbiotic competence. In contrast, the symbiotic host range is wide, from mosses to angiosperms. The plant symbioses range from less intimate interactions, such as in mosses, to highly intricate symbioses, such as the intracellular symbiosis with the angiosperm Gunnera. In Azolla spp. the relationship is perpetual and maintained between generations. Nostoc is also one of the most developmentally advanced prokaryotes and capable of differentiating several cell types with various functions. Individual vegetative cells of Nostoc may differentiate into nitrogen-fixing heterocysts, and filaments may fragment into hormogonia, a motile life-stage and a prerequisite for plant infection. On internalization, the hormogonia are turned into multi-heterocystous filaments. The high frequency of heterocysts is reflected in their high nitrogen-fixing activities, and in the transfer of the fixed nitrogen to the plant. A sequence of inter-organism communication events between the partners and cellular adaptations is therefore obvious.

More information: The Cyanobacteria: Molecular Biology, Genomics and Evolution

International Microbiology (2007) 10:227 has published a highly positive review of a recent book on Bacillus subtilis. Gemma Reguera (Michigan State University) writes "In addition to the depth of knowledge and detailed description of the latest research, the book chapters are well written and nicely organized so that the reader can follow the description of cell cycle events such as DNA replication, DNA repair, chromosome segregation and cell division and cell cycle regulatory mechanisms just as they would be timed in the cell cycle. Care also has been taken to blend writing styles and allow the flow from chapter to chapter, thus facilitating the reading. The book is also perfect as a reference for advanced undergraduate and graduate-level courses, as it presents the latest research in bacterial molecular biology, differentiation, gene/protein regulation and development in a clearly written and well-illustrated style. It provides a perfect balance of descriptive background information and detailed experimental methods, while giving a thorough account of the latest discoveries. A must read for anybody interested in just about any aspect of bacterial research."

Details of the book

A new case of foot-and-mouth disease has been confirmed today (September 12, 2007) in Surrey, UK. The last outbreak occurred in August 2007. A 10km control zone centred on the affected farm near Egham, Surrey has been put in place and cattle from the herd are being culled. A national movement ban is in effect to prevent spread of the disease. A report on the August outbreak blamed a leaking pipe at a research laboratory in Pirbright, about 10 miles away from the current outbreak. Foot-and-mouth disease is caused by the virus (FMDV) which is the prototypic member of the Aphthovirus genus in the Picornaviridae family.

Animal Viruses: Foot-and-Mouth Disease Virus

Coronaviruses: Molecular and Cellular Biology

Coronaviruses are positive-strand, enveloped RNA viruses that are important pathogens of mammals and birds. This group of viruses cause enteric or respiratory tract infections in a variety of animals including humans, livestock and pets. The important discovery in 2003 that the causative agent of severe acute respiratory syndrome (SARS) was a new, potentially lethal coronavirus named SARS-CoV provided major impetus to coronavirus research. SARS-CoV spread within months to more than 30 countries causing the first epidemic of the new millennium and becoming a public health nightmare in the countries affected.

Binding and Entry
Coronaviruses bind to host cells primarily through interactions between viral spike glycoproteins and specific host cell surface glycoproteins. Some coronaviruses also bind to sialic acids on glycoproteins and glycolipids via their spike and/or hemagglutinin esterase glycoproteins. The interactions between coronaviruses and host cell receptors are critical determinants of species-specificity, tissue tropism, and virulence.

Replication
Coronaviruses have single-stranded, positive-sense RNA genomes of about 30 kilobases, by far the largest non-segmented RNA virus genomes currently known. The key functions required for coronavirus RNA synthesis are encoded by the viral replicase gene. The gene comprises more than 20,000 nucleotides and encodes two replicase polyproteins, pp1a and pp1ab, that are proteolytically processed by viral proteases. Over the past years, it has become clear that the unique size of the coronavirus genome and the special mechanism that coronaviruses (and several other nidoviruses) have evolved to produce an extensive set of subgenome-length RNAs is linked to the production of a number of nonstructural proteins (nsps) that is unprecedented among RNA viruses. Many of these replicase cleavage products in fact are multidomain proteins themselves, thus further increasing the complexity of protein functions and interactions. Structural studies suggest that several nsps, following their release from larger precursor molecules, form dimers or even multimers. The various pp1a/pp1ab precursors and processing products are thought to assemble into large, membrane-associated complexes that, in a temporally coordinated manner, catalyze the reactions involved in RNA replication and transcription and, most probably, serve yet other functions in the viral life cycle.

Genomic Cis-Acting Elements
In common with the genomes of all other RNA viruses, coronavirus genomes contain cis-acting RNA elements that ensure the specific replication of viral RNA by a virally encoded RNA-dependent RNA polymerase. The embedded cis-acting elements devoted to coronavirus replication constitute a surprisingly small fraction of the total genome, but this is probably a reflection of the fact that coronaviruses have the largest genomes of all RNA viruses. The boundaries of cis-acting elements essential to replication are fairly well-defined, and an increasingly well resolved picture of the RNA secondary structures of these regions is emerging. However, we are only in the early stages of understanding how these cis-acting structures and sequences interact with the viral replicase and host cell components, and much remains to be done before we understand the precise mechanistic roles of such elements in RNA synthesis

Genome Packaging
The assembly of infectious coronavirus particles requires the selection of viral genomic RNA from a cellular pool that contains an abundant excess of non-viral and viral RNAs. Among the seven to ten specific viral mRNAs synthesized in virus-infected cells, only the full length genomic RNA is packaged efficiently into coronavirus particles. Studies have revealed cis-acting elements and trans-acting viral factors involved in coronavirus genome encapsidation and packaging. Understanding the molecular mechanisms of genome selection and packaging is critical for developing antiviral strategies and viral expression vectors based on the coronavirus genome.

SARS
Human infection by SARS coronavirus appears to be limited to the respiratory tract where infection of susceptible cells leads to damage to the pneumocytes resulting in a histological picture of diffuse alveolar damage and a clinical picture of adult respiratory distress syndrome. Diarrhoea is also present but there is limited evidence of damage to the intestinal epithelium. The damage to the respiratory tree appears limited to the lower respiratory tract and there is evidence that the immune response plays a part in the outcome of patients with SARS.

Antiviral Research
Before the emergence of SARS-CoV, no efforts were put into the search for antivirals against coronaviruses. The rapid transmission and high mortality rate made SARS a global threat for which no efficacious therapy was available and empirical strategies had to be used to treat the patients. New insights into the field of the SARS-CoV genome structure and pathogenesis revealed novel potential anti-coronavirus targets. Several proteins encoded by the SARS-CoV could be considered as targets for therapeutic intervention: the spike protein, the main protease, the NTPase/helicase, the RNA dependent RNA polymerase and different other viral protein-mediated processes. Potential anti-SARS-CoV drugs are currently being developed in vivo. The development of effective drugs against SARS-CoV may also provide new strategies for the prevention or treatment of other coronavirus diseases in animals or humans.

Vaccine Development
The emergence and identification of several common and rare human coronaviruses that cause severe lower respiratory tract infection argues for the judicious development of robust coronavirus vaccines and vector platforms. Currently, limited information is available on the correlates of protection against SARS-CoV and other severe lower respiratory tract human coronavirus infections, a clear priority for future research. Passive immunization has been successful in establishing protection from SARS-CoV suggesting an important role for neutralizing antibodies. One important property of future vaccine candidates is the ability to confer protection against multiple variant strains of SARS-CoV, especially in senescent populations that are most at risk for severe disease. Many vaccine candidates are capable of inducing humoral and cellular responses. The development of infectious clones for coronaviruses has facilitated the identification of attenuating mutations, deletions and recombinations which could ultimately result in live attenuated vaccine candidates. Stable vaccine platforms should be developed that allow for rapid intervention strategies against any future emergence coronaviruses. Vaccine correlates that enhance disease after challenge should be thoroughly investigated and mechanisms devised to circumvent vaccine-associated complications.

Further Information

Coronaviruses: Molecular and Cellular Biology (Volker Thiel)

Animal Viruses: Molecular Biology (Thomas C. Mettenleiter and Francisco Sobrino)

Acinetobacter Molecular Biology

The genus Acinetobacter is a group of Gram-negative, non-motile and non-fermentative bacteria belonging to the family Moraxellaceae. They are important soil organisms where they contribute to the mineralisation of, for example, aromatic compounds. Acinetobacter are able to survive on various surfaces (both moist and dry) in the hospital environment, thereby being an important source of infection in debilitated patients. These bacteria are innately resistant to many classes of antibiotics. In addition, Acinetobacter is uniquely suited to exploitation for biotechnological purposes.

Taxonomy
The genus Acinetobacter comprises 17 validly named and 14 unnamed (genomic) species. Some unrelated (genomic) species have common designations, while some other species seem to be congruent but have different names. The knowledge of the biology or ecology of acinetobacters at species level is limited. This is due to the fact that identification of acinetobacters at species level is difficult. A phenotypic species identification system has been described and a variety of genotypic methods has been explored and applied to investigate the diversity or phylogeny in the genus. These methods include high resolution fingerprinting with AFLP, PCR-RFLP with digestion of PCR amplified sequences, and analysis of various DNA sequences. Of these, AFLP analysis and amplified 16SrDNA ribosomal DNA restriction analysis have been validated with large numbers of strains of all described species. Nucleotide sequence based methods are expected to be the standard for identification in the near future.

Clinical Significance
Several species persist in hospital environments and cause severe, life-threatening infections in compromised patients. The spectrum of antibiotic resistances of these organisms together with their survival capabilities make them a threat to hospitals as documented by recurring outbreaks both in highly developed countries and elsewhere. An important factor for their pathogenic potential is probably an efficient means of horizontal gene transfer even though such a mechanism has so far only been observed and analyzed in Acinetobacter baylyi, a species that lives in the soil and has never been associated with infections.

Phage Therapy
A phage directed against Acinetobacter showed a remarkable lytic activity both in vitro and in vivo: as few as 100 pfu of phage protected mice against Acinetobacter. Further information on phage therapy ...

Biotechnology
Many of the characteristics of Acinetobacter ecology, taxonomy, physiology and genetics point to the possibility of exploiting its unique features for future applications. Acinetobacter strains are often ubiquitous, exhibit metabolic versatility, are robust and some provide convenient systems for modern molecular genetic manipulation and subsequent product engineering. These characteristics are being exploited in various biotechnological applications including biodegradation and bioremediation, novel lipid and peptide production, enzyme engineering, biosurfactant and biopolymer production and engineering of novel derivatives of these products. It is anticipated that progress in these fields will broaden the range of applications of Acinetobacter for modern biotechnology.

Catabolism of Aromatic Compounds
Acinetobacter strains isolated from the environment are capable of the degradation of a wide range of aromatic compounds. However the predominant route for the final stages of assimilation to central metabolites is through catechol or protocatechuate (3,4-dihydroxybenzoate) and the beta-ketoadipate pathway, and the diversity within the genus lies in the channelling of growth substrates, most of which are natural products of plant origin, into this pathway.

Molecular Basis of Antibiotic Resistance in Acinetobacter spp.
Members of the genus Acinetobacter have the ability to develop resistance to new antibiotics extremely rapidly. Most multiresistant isolates of Acinetobacter spp. belong to the Acinetobacter baumannii complex, and many clinical isolates of A. baumannii are now resistant to all conventional antimicrobial agents, including carbapenems. Molecular studies have characterised most of the responsible genes and mechanisms of resistance to antibiotics found within the genus. Multidrug resistance typically results from the accumulation of multiple mutations and/or the acquisition of resistance genes from other bacterial genera, with the latter occurring by a variety of mechanisms, including the transfer of plasmids, transposons and integrons carrying clusters of genes encoding resistance to several unrelated families of antibiotics simultaneously. Whole-genome sequence analysis has identified the presence of resistance islands, apparently built through the successive insertion of broad host-range mobile genetic elements into an insertion hotspot on the A. baumannii chromosome. This ability to 'switch' its genetic structure may explain the unmatched speed at which A. baumannii captures resistance markers when under antibacterial selection pressure. Overall, the emergence of resistance among clinical isolates of A. baumannii appears to be a combined effect of gene acquisition, following lateral gene transfer, and clonal spread of multiresistant clones.

For further information see the newly published book Acinetobacter Molecular Biology edited by Ulrike Gerischer