MicrobiologyBytes

Regulons are the basic units of cellular response systems in bacterial cells, and represent a most basic concept in bacterial studies. A bacterial regulon is a group of operons that are transcriptionally co-regulated by the same regulatory machinery, consisting of trans regulators (transcription factors or simply TFs) and cis regulatory binding elements in the promoters of the operons they regulate. Operationally, a regulon contains operons regulated by one same transcription factor. Since the term regulon was first proposed in 1964, 173 regulons have been fully or partially identified in E. coli K12 and many more in other bacteria e.g. B. subtilis.

Loosely speaking, regulons can be categorized into two classes: local and global regulons, with the former corresponding to regulons consisting of only a few component operons and the latter having a relatively large number of operons. While the functionalities of the known regulons have been well studied, very little is known about how regulons are organized in a bacterial genome.

This paper examines the organizational principles of regulons in bacterial genomes, looking at E. coli K12 and Bacillus subtilis. The key findings are (1) operons of each regulon tend to form a few closely located clusters along with genome; (2) TFs are under stronger evolutionary constraints than their TGs; and (3) the global arrangement of the component operons of all the (known) regulons in a genome tend to minimize a simple scoring function.

Genomic Arrangement of Regulons in Bacterial Genomes. (2012) PLoS ONE 7(1): e29496. doi:10.1371/journal.pone.0029496
Regulons, as groups of transcriptionally co-regulated operons, are the basic units of cellular response systems in bacterial cells. While the concept has been long and widely used in bacterial studies since it was first proposed in 1964, very little is known about how its component operons are arranged in a bacterial genome. We present a computational study to elucidate of the organizational principles of regulons in a bacterial genome, based on the experimentally validated regulons of E. coli and B. subtilis. Our results indicate that (1) genomic locations of transcriptional factors (TFs) are under stronger evolutionary constraints than those of the operons they regulate so changing a TF’s genomic location will have larger impact to the bacterium than changing the genomic position of any of its target operons; (2) operons of regulons are generally not uniformly distributed in the genome but tend to form a few closely located clusters, which generally consist of genes working in the same metabolic pathways; and (3) the global arrangement of the component operons of all the regulons in a genome tends to minimize a simple scoring function, indicating that the global arrangement of regulons follows simple organizational principles. Tweet

Traditional approaches to phage therapy rely on the ability of viruses to kill their bacterial prey. However, the narrow host range or most bacteriophages and the ability of bacteria to become resistant to infection mean that in practice, using phage to simply replace antibiotics is not feasible. We need smarter approaches, which is where a recent paper comes in. Using phages to engineer sensitivity to antibiotics is a promising approach, but whether this proof-of-principle experiment ever makes it to the clinic is another matter.

Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. (2011)Appl. Environ. Microbiol. 23 Nov 2011 doi: 10.1128/AEM.05741-11
Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that will resensitize pathogens to approved antibiotics therefore holds key advantages. We present a proof-of-principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages encoding the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene’s sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage’s ability to kill pathogens in the infected host, but instead, to deliver genetic constructs into the bacteria, and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phages will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

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Cystic fibrosis is caused by mutations in the CFTR gene leading to a disrupted chloride channel. It is well established that the greatest contributor to patient morbidity and mortality is chronic lung disease, caused by a constant cycle of infection and inflammation throughout the patient’s life. The CFTR mutation leads to defective regulation of chloride and sodium, resulting in increased water absorption, depletion of airway surface liquid and dehydrated mucous. Consequently, the purulent sputum and mucus plugs together with an ineffective inflammatory response, all contribute to the chronic infections that are central to CF lung disease. From early childhood, CF patients experience recurrent pulmonary infections from a range of pathogens.

In spite of intensive antibiotic therapy, certain organisms persist, leading to pulmonary exacerbations, hospitalizations and patient death. These include Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and Achromobacter xylosoxidans, with Bcc being the most problematic. It was recently demonstrated that chronic colonisation by Bcc resulted in a greater lung function decline than by the other two pathogens. CF patients are also susceptible to colonisation by other pathogens, including Staphylococcus aureus (both methicillin-resistant and sensitive), genus Pandoraea, Stenotrophomonas maltophilia and non-tuberculous Mycobacteria, although the role of these latter four pathogens in CF lung disease is unclear. Furthermore, the identification of high levels of anaerobic organisms in CF sputum has added to the complex microbial population in the CF lung. These CF-associated anaerobes were not susceptible to antibiotics with known efficacy against anaerobes and the clinical significance of anaerobes in CF is not yet fully understood.

Bacterial host interactions in cystic fibrosis. Curr Opin Microbiol. Dec 1 2011
Chronic infection is a hallmark of cystic fibrosis (CF) and the main contributor to morbidity. Microbial infection in CF is complex, due to the number of different species that colonise the CF lung. Their colonisation is facilitated by a host response that is impaired or compromised by highly viscous mucous, zones of hypoxia and the lack of the cystic fibrosis transmembrane regulator (CFTR). Successful dominant CF pathogens combine an effective arsenal to establish infection and counter-attack the host response, together with an ability to adapt readily to an unfavourable environment. Hypermutability is common among CF pathogens facilitating adaptation and as the host response persists, progressive destruction of the normal architecture of lung tissue ensues with catastrophic consequences for the host.

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DNA fragments encoding enzymes, transcriptional regulators and virulence factors are fluxing through bacterial taxonomic walls by horizontal gene transfer. These elements often endow environmental and clinical strains of bacteria with new properties, including an enhanced virulence. Lateral genetic exchange, particularly of drug tolerance genes has been recognized for a long time; however, our understanding of this phenomenon is limited. Ontology and phylogeny of laterally transferred genetic elements are difficult to investigate, let alone the predictions of their insertion sites in hosts chromosomes.

An outbreak of the lethal Escherichia coli in Europe in 2011 highlighted the shortcoming of our knowledge on the basic principles of evolutionary trends of new pathogens. The outbreak first occurred in Germany in May 2011 where a rare enterohemorrhagic strain Escherichia coli O104:H4 caused haemolytic-uremic syndrome. The infection spread fast through many other European countries and sickened thousands of people. The level of lethality associated with the production of Shiga toxin by the strain and its resistance against many antibiotics was significant. A number of isolates from this outbreak have been sequenced and annotated. Based on the unique combination of genomic features these strains were suggested to represent a new pathotype Entero-Aggregative-Haemorrhagic E. coli (EAHEC).

Mainstreams of Horizontal Gene Exchange in Enterobacteria: Consideration of the Outbreak of Enterohemorrhagic E. coli O104:H4 in Germany in 2011. (2011) PLoS ONE 6(10): e25702. doi:10.1371/journal.pone.0025702
Escherichia coli O104:H4 caused a severe outbreak in Europe in 2011. The strain TY-2482 sequenced from this outbreak allowed the discovery of its closest relatives but failed to resolve ways in which it originated and evolved. On account of the previous statement, may we expect similar upcoming outbreaks to occur recurrently or spontaneously in the future? The inability to answer these questions shows limitations of the current comparative and evolutionary genomics methods. The study revealed oscillations of gene exchange in enterobacteria, which originated from marine γ-Proteobacteria. These mobile genetic elements have become recombination hotspots and effective ‘vehicles’ ensuring a wide distribution of successful combinations of fitness and virulence genes among enterobacteria. Two remarkable peculiarities of the strain TY-2482 and its relatives were observed: i) retaining the genetic primitiveness by these strains as they somehow avoided the main fluxes of horizontal gene transfer which effectively penetrated other enetrobacteria; ii) acquisition of antibiotic resistance genes in a plasmid genomic island of β-Proteobacteria origin which ontologically is unrelated to the predominant genomic islands of enterobacteria. Oscillations of horizontal gene exchange activity were reported which result from a counterbalance between the acquired resistance of bacteria towards existing mobile vectors and the generation of new vectors in the environmental microflora. We hypothesized that TY-2482 may originate from a genetically primitive lineage of E. coli that has evolved in confined geographical areas and brought by human migration or cattle trade onto an intersection of several independent streams of horizontal gene exchange. Development of a system for monitoring the new and most active gene exchange events was proposed. Tweet

As a new paper confirms the presence of thousands of “unknown” viruses in raw sewage, it brings to mind something we already knew – that although there are a lot of “unknown” viruses out there, most of them are tailed bacteriophages, blown apart, reshuffled and reassembled:

Raw Sewage Harbors Diverse Viral Populations. mBio 2 (5) e00180-11 4 October 2011 doi: 10.1128/​mBio.00180-11
At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity.

Analysis of the virus population present in equine faeces indicates the presence of hundreds of uncharacterized virus genomes. (2005) Virus Genes. 30(2): 151-156
Virus DNA was isolated from horse faeces and cloned in a sequence-independent fashion. 268 clones were sequenced and 178140 nucleotides of sequence obtained. Statistical analysis suggests the library contains 17560 distinct clones derived from up to 233 different virus genomes. TBLASTX analysis showed that 32% of the clones had significant identity to GenBank entries. Of these 63% were viral; 20% bacterial; 7% archaeal; 6% eukarya; and 5% were related to mobile genetic elements. Fifty-two percent of the virus identities were with Siphoviridae; 26% unclassified phages; 17% Myoviridae; 4% Podoviridae; and one clone (2%) was a vertebrate Orthopoxvirus. Genes coding for predicted virus structural proteins, proteases, glycosidases and nucleic acid-binding proteins were common.

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The advent of genetic engineering – the ability to edit and insert DNA into living organisms – in the latter half of the 20th century created visions of a new era of synthetic biology, where novel biological functions could be designed and implemented for useful purposes. We are witnessing an exciting revolution of scale, wherein technical progresses allow for the manipulation of genetic material at the whole genome level. This will enable the manufacture of increasingly complex genetic designs to solve pressing challenges in health, energy and the environment-if and when such designs can be specified.

This paper argues that the organized development of key common application organisms, engineered for engineerability, and attendant libraries of parts, pathways and standardized manufacturing are necessary for this genome-scale technology to realize its promise.

Toward rational design of bacterial genomes. Curr Opin Microbiol. Aug 22 2011

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Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection. These bacteria live as soil saprotrophs on decaying plant matter but also as intracellular parasites, using the cell cytosol as a replication niche. PrfA, a regulatory protein, integrates a number of environmental cues that signal the transition between these two contrasting lifestyles, activating a set of key virulence factors during host infection. While a number of details concerning the general mode of action of this virulence master switch have been elucidated, others remain unsolved. Recent work has revealed additional mechanisms that contribute to L. monocytogenes virulence modulation, often via cross-talk with PrfA, or by regulating new genes involved in host colonization.

Regulation of Listeria virulence: PrfA master and commander. Curr Opin Microbiol. Mar 7 2011

In this article in Microbiology Today Ed Feil describes how we must brace ourselves for the next wave of data as new sequencing techniques become available to determine and compare many sequences at once. The enormous amount of data soon to be generated will bring exciting new insights into how micro-organisms within communities evolve and interact:

Regardless of the species in question, announcements of completed genome sequencing projects in the mainstream media almost invariably make reference to ‘cracking a code’ or ‘deciphering a genetic blueprint’. For bacteria, these over-used analogies spectacularly fail to give a true sense of the fluidity of genome evolution. The doe-eyed assumption in the mid-1990s that a single genome sequence can safely be considered as a prescriptive ‘solution’ for a given bacterial species has been dramatically falsified. By the late 1990s, multiple genome sequences for Escherichia coli revealed extensive differences in gene content between strains, and it rapidly became clear that, for many taxa, an individual genome is most usefully considered as one of many possible combinations of genes drawn from a vast pool known as the pangenome. When faced with such a maelstrom, our natural inclination (as good cladists) is to try and tidy it up, and catalogue strains into pockets of relatedness. Fortunately, phylogenetic analyses are possible, even for very variable species like E. coli, because one can readily identify genes which are universally present in all strains. These essential ‘core’ genes can be thought of as representing the operating system of a given species. In contrast, the specialist software is provided by ‘non-core’ or ‘accessory’ genes which are variably present or absent, are commonly acquired by horizontal transfer, and tend to be restricted to hypervariable regions called genomic islands. These two sets of genes present a fundamental duality in bacterial genomics. Whilst core genes can satisfy our requirements for molecular phylogeny (i.e. what a strain is), accessory genes often play a significant role in adaptation and phenotypic differences (i.e. what a strain does). Conflicts between these two can go a long way to explaining the mystery behind the muddle that is bacterial systematics.

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While single cell studies have focused on bacteria and cyanobacteria, single virions have yet to be isolated and genomically described using similar mechanisms. Viruses are ubiquitous and the most numerous and diverse biological entities on our planet. Nearly all aspects of our lives are influenced by viruses through shaping the environments that surround us, our immune responses and even our genomes. The field of environmental viral metagenomics has gained momentum over the past several years; however, sequencing of individual environmental viral genomes is currently dependent on the establishment of cultivable virus-host systems. With this in mind, if less than one percent of microbial populations can be cultured using standard microbiological techniques due to incongruencies in direct counts versus cultivatable microbes, then only a very small number of viruses have the likelihood of being genomically described. Currently, viral genomic sequences are lacking in public databases, with the exception of human viruses and those of agricultural and industrial significance (e.g. Lactococcal phages). Clearly, a better understanding of virus diversity and evolution will not be achieved until the genomes of a broad range of viruses are available.

This paper introduces an approach for isolating and characterizing the genomes of viruses called “Single Virus Genomics” (SVG). The benefits of SVG will be far-reaching, enabling novel virus discovery in a variety of clinical and environmental settings, altering our understanding of virus evolution, adaptation and ecology and facilitating the interpretation of viral genomic and metagenomic data by providing suitable reference genomes.

Single Virus Genomics: A New Tool for Virus Discovery. 2011 PLoS ONE 6(3): e17722. doi:10.1371/journal.pone.0017722
Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called ‘Single Virus Genomics’, which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.