MicrobiologyBytes

Engineering of synthetic genetic circuits holds the promise to revolutionize medical treatment and industrial production from microbes. Yet progress over the last decade has been hindered by a lack of rational design principles to guide the interfacing of engineered components with the host organism. Recent work demonstrates that even constitutive protein expression, and much more so genetic circuits, can be strongly affected by the cell’s physiological state. Thus, it appears difficult to insulate synthetic circuitry from the growth state of the host. Despite the inherent crosstalk between synthetic and endogenous elements, some of the resultant global effects have been shown to obey simple mathematical relations referred to as “growth laws”. These quantitative relations may provide a framework for the design of robust synthetic systems, opening up new directions in bioengineering and biotechnology.

Bacterial growth laws and their applications. Curr Opin Biotechnol. May 16 2011
Quantitative empirical relationships between cell composition and growth rate played an important role in the early days of microbiology. Gradually, the focus of the field began to shift from growth physiology to the ever more elaborate molecular mechanisms of regulation employed by the organisms. Advances in systems biology and biotechnology have renewed interest in the physiology of the cell as a whole. Furthermore, gene expression is known to be intimately coupled to the growth state of the cell. Here, we review recent efforts in characterizing such couplings, particularly the quantitative phenomenological approaches exploiting bacterial ‘growth laws.’ These approaches point toward underlying design principles that can guide the predictive manipulation of cell behavior in the absence of molecular details.

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The most amazing thing about the “common cold”, or at least, the proportion of this spectrum of diseases which is caused by rhinoviruses (as opposed to adenoviruses, coronaviruses, or something else), is how little we still know about it. A few years ago I was involved in a proposal for a large project which would look at some of the issues adressed by a new research paper which has just appeared.  That proposal eventually collapsed under its own weight, so it’s fascinating to read these new results and see what we might have found.

The new study describes a phylogenetic analysis to compare the relative distribution of HRV species or serotypes according to the respiratory site (upper respiratory tract (URT) versus lower respiratory tract (LRT) ) and in protracted infection in hospital patients and immunosuppressed lung transplant recipients. In one case, rhinovirus genome variation was followed in the URT and LRT over a period of 27 months using both classical and ultra-deep sequencing methods.

Based on phylogenetic analysis, the frequency distribution of strains infecting the URT and LRT did not reveal any apparent correlation between a given HRV serotype or species and their ability to infect the LRT. Five lung transplant recipients were chronically infected with HRV during periods of time ranging from three to 27 months. Mutation mapping along the HRV genome showed that synonymous changes were roughly spread along the entire ORF, whereas non-synonymous changes clustered mostly in the capsid VP2, VP3, and VP1 genes. The capsid genes are also the most variable during acute infections in immunocompetent hosts.

As expected, the data suggests that immunocompromised patients cannot clear virus infections as well as immunocompetent individuals, and represent a potential reservoir for the emergence of new variants and inter-host transmission due to chronic virus infection. In addition, these patients may be co-infected by two viruses, thus opening the door to recombination, another putative driving force of rhinovirus evolution. With the emergence of new therapies and progress in transplantation, the population of immunocompromised patients is constantly increasing. Our results suggest that this could accelerate the ability of viruses to adapt to the host, evolve, and propagate and may favor a more rapid emergence of new viral variants.

Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections. (2011) PLoS ONE 6(6): e21163. doi:10.1371/journal.pone.0021163
Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5′ UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

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At the edge of a fungal colony, leading hyphae grow into new territory in search of food. Behind the colony edge, the hyphae interconnect to form a three-dimensional network that is optimized to extract nutrients from the surrounding medium in order to fuel continued exploration. Colony growth can be fast (about 10–100 μm min⁻¹, depending on the organism, nutrient availability and temperature) and involves the continuous synthesis of all the cellular constituents that are necessary for rapid cell expansion. A major driving force for cell expansion is pressure.

Pressure is a thermodynamic state property that affects the life of all organisms. In cells that lack a cell wall, excessive pressure can result in cell lysis and death. In cells that do have a wall (most bacteria, algae, fungi and plants), an internal hydrostatic pressure (turgor) provides both mechanical support for free-standing structures and a force that drives cellular expansion, substrate penetration and other processes. Extreme examples from fungi are the projectile release of spores at >100,000 × g (g is the acceleration due to gravity at the Earth’s surface) in ascomycetes and zygomycetes.

This review describes the roles of turgor and pressure in fungal growth. How is turgor regulated? How does it affect tip growth? Do intra-hyphal pressure gradients play a part in fungal growth (such as in the transport of new materials to the growing tip)? These areas of active research are revealing the mechanisms of hyphal growth in filamentous fungi and are relevant to applied research on pathogenicity and the control of fungal diseases.

How does a hypha grow? The biophysics of pressurized growth in fungi. (2011) Nature Reviews Microbiology 9, 509-518 doi:10.1038/nrmicro2591
The mechanisms underlying the growth of fungal hyphae are rooted in the physical property of cell pressure. Internal hydrostatic pressure (turgor) is one of the major forces driving the localized expansion at the hyphal tip which causes the characteristic filamentous shape of the hypha. Calcium gradients regulate tip growth, and secretory vesicles that contribute to this process are actively transported to the growing tip by molecular motors that move along cytoskeletal structures. Turgor is controlled by an osmotic mitogen-activated protein kinase cascade that causes de novo synthesis of osmolytes and uptake of ions from the external medium. However, as discussed in this Review, turgor and pressure have additional roles in hyphal growth, such as causing the mass flow of cytoplasm from the basal mycelial network towards the expanding hyphal tips at the colony edge.

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Plant viruses are often accompanied by small parasitic RNAs termed satellite RNAs. Satellite RNAs range in size from ~220 to 1400 nucleotides (nt) in length and depend on their associated viruses (known as the helper virus) for replication, encapsidation, movement and transmission, but share little or no sequence homology to the helper virus itself. Most satellite RNAs do not encode functional proteins, yet can induce disease symptoms which range from chlorosis and necrosis, to total death of the infected plant. How such non-protein-coding RNA pathogens induce disease symptoms has been a longstanding question.

Early studies showed that the pathogenicity of a satellite RNA is determined at the nucleotide level, with one to several nucleotide changes dramatically altering both the virulence and host specificity of disease induction. Subsequent studies demonstrated that satellite RNA replication is associated with the accumulation of high levels of satellite RNA-derived small interfering RNAs (siRNA). These findings led to the suggestion that pathogenic satellite-derived siRNAs might have sequence complementarity to a physiologically important host gene, and that the observed disease symptoms are in fact due to satellite siRNA-directed silencing of the targeted host gene. However, to date, no such host gene has been identified, leaving the satellite RNA-induced disease mechanism unsolved.

This paper explores the sRNA-mediated disease mechanism using the Y-satellite of Cucumber mosaic virus (CMV Y-Sat). The CMV Y-Sat consists of a 369-nt single-stranded RNA genome and induces distinct yellowing symptoms in a number of Nicotiana species including N. tabacum (tobacco). Y-Sat-induced yellowing symptoms result from Y-Sat siRNA-directed silencing of the host chlorophyll biosynthetic gene, CHLI, and Y-Sat-induced symptoms can be prevented by transforming tobacco with a silencing-resistant version of CHLI. The observed species specificity of Y-Sat-induced disease symptoms is due to natural sequence variation within the targeted region of the CHLI transcript.

Viral Small Interfering RNAs Target Host Genes to Mediate Disease Symptoms in Plants. (2011) PLoS Pathog 7(5): e1002022. doi:10.1371/journal.ppat.1002022
The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5′ RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.

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Human parvovirus B19 (B19V) is the etiological agent of fifth disease seen in children, aplastic crisis in sickle cell disease patients, chronic anemia in immunocompromised patients, and hydrops fetalis in pregnant women. 35 years after its discovery, it was still not possible to propagate B19V in vitro in a productive and sustainable manner, which delayed progress in the study of B19V pathogenesis, and consequently finding ways to treat patients infected with B19V.

Researchers cultured human erythroid progenitor cells under hypoxic conditions by mimicking the natural niches of human bone marrow. This work demonstrates, for the first time, a long-term B19V infection of ex vivo expanded erythroid progenitor cells. This finding will largely facilitate the study of the mechanisms underlying B19V infection and more importantly, identification of approaches to treat B19V infection. Identification of the cellular signaling pathways in regulating B19V replication sheds light on the virus-host interaction and will nominate potential candidates for anti-virus drug targeting.

Productive Parvovirus B19 Infection of Primary Human Erythroid Progenitor Cells at Hypoxia Is Regulated by STAT5A and MEK Signaling but not HIFα. (2011) PLoS Pathog 7(6): e1002088. doi:10.1371/journal.ppat.1002088
Human parvovirus B19 (B19V) causes a variety of human diseases. Disease outcomes of bone marrow failure in patients with high turnover of red blood cells and immunocompromised conditions, and fetal hydrops in pregnant women are resulted from the targeting and destruction of specifically erythroid progenitors of the human bone marrow by B19V. Although the ex vivo expanded erythroid progenitor cells recently used for studies of B19V infection are highly permissive, they produce progeny viruses inefficiently. In the current study, we aimed to identify the mechanism that underlies productive B19V infection of erythroid progenitor cells cultured in a physiologically relevant environment. Here, we demonstrate an effective reverse genetic system of B19V, and that B19V infection of ex vivo expanded erythroid progenitor cells at 1% O2 (hypoxia) produces progeny viruses continuously and efficiently at a level of approximately 10 times higher than that seen in the context of normoxia. With regard to mechanism, we show that hypoxia promotes replication of the B19V genome within the nucleus, and that this is independent of the canonical PHD/HIFα pathway, but dependent on STAT5A and MEK/ERK signaling. We further show that simultaneous upregulation of STAT5A signaling and down-regulation of MEK/ERK signaling boosts the level of B19V infection in erythroid progenitor cells under normoxia to that in cells under hypoxia. We conclude that B19V infection of ex vivo expanded erythroid progenitor cells at hypoxia closely mimics native infection of erythroid progenitors in human bone marrow, maintains erythroid progenitors at a stage conducive to efficient production of progeny viruses, and is regulated by the STAT5A and MEK/ERK pathways.

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For more than a decade, the field of systems biology has advanced our knowledge of how networks of molecular processes enable cells to perceive their environment and trigger phenotypic changes in response. This view centers on the single cell as a computational unit. However, many biological processes are multicellular in nature and are the product of cell–cell interaction within populations. How do molecular networks at the single-cell level ultimately define collective cell behaviors via social interaction? Understanding how interaction among cells enables the spread of information and leads to dynamic population behaviors is a fundamental problem in biology. A closely related question is how adaptive social interactions evolved in spite of the conflicting selection pressures at the individual and the population levels.

Studies of social interaction in synthetic and natural microbial communities have produced important complementary insights into the social biology of microbes and cell populations in general. This article reviews key work in the emerging field of microbial social evolution and discusses how it contributes to our growing understanding of cell–cell interactions in all multicellular systems.

Social interaction in synthetic and natural microbial communities. (2011) Mol Syst Biol. 7: 483
Social interaction among cells is essential for multicellular complexity. But how do molecular networks within individual cells confer the ability to interact? And how do those same networks evolve from the evolutionary conflict between individual- and population-level interests? Recent studies have dissected social interaction at the molecular level by analyzing both synthetic and natural microbial populations. These studies shed new light on the role of population structure for the evolution of cooperative interactions and revealed novel molecular mechanisms that stabilize cooperation among cells. New understanding of populations is changing our view of microbial processes, such as pathogenesis and antibiotic resistance, and suggests new ways to fight infection by exploiting social interaction. The study of social interaction is also challenging established paradigms in cancer evolution and immune system dynamics. Finding similar patterns in such diverse systems suggests that the same ‘social interaction motifs’ may be general to many cell populations.

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The ability of bacteria to extend their sphere of metabolic influence long distances (microns) from the cell is key to their activity and survival, and is achieved by secretion of small molecules, such as acyl homoserine lactones, which can have broad, regulatory effects on the metabolism of neighboring bacteria, as well as macromolecules, namely enzymes and outer membrane vesicles (OMV), which transmit specific function(s). The latter are unique in that they can encompass a broad range of (macro)molecules, which mediate a variety of processes. For example, OMV can package small molecules for signaling and proteins that effect virulence. OMV-mediated DNA transfer has also been demonstrated. These vesicles are highly versatile as they can be designed for different functions by different organisms, and tasked for different activities by the same organism. Thus OMV are a type of bacterial “Swiss army knife” for projecting extracellular activities and, perhaps reflecting their utility, their production is widespread in proteobacteria. But, despite their prominence, the biology of OMV has been extensively studied only in pathogens, for which these are key vehicles for long distance transmission of virulence factors.

This report describes novel bacterial organelles termed “nanopods” that can project OMV long distances (≥6 µm) from the cell. Nanopod deployment of OMV is independent of diffusion, and thus represents a solution to constraints imposed by partial hydration, a new paradigm in the mechanisms of long distance interaction utilized by bacteria.

Nanopods: A New Bacterial Structure and Mechanism for Deployment of Outer Membrane Vesicles. (2011) PLoS ONE 6(6): e20725. doi:10.1371/journal.pone.0020725
Bacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most) bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ≤10 nm water film vs. 20 to >200 nm OMV;).
We have identified a new bacterial surface structure, termed a “nanopod”, that is a conduit for projecting OMV significant distances (e.g., ≥6 µm) from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA), and the interior structures identified as OMV. Specific metabolic function(s) for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods.
Nanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic sphere of influence in a manner previously unknown for these organisms.

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Noncovalent biological interactions are commonly subjected to mechanical force, particularly when they are involved in adhesion or cytoskeletal movements. While one might expect mechanical force to break these interactions, some of them form so-called catch bonds that lock on harder under force, like a nanoscale finger-trap. The adhesive protein FimH, which is located at the tip of E. coli fimbriae, allows bacteria to bind to urinary epithelial cells in a shear-dependent manner, binding at high but not at low flow. Isolated fimbrial tips, consisting of elongated protein complexes with FimH at the apex, reproduce this behavior in vitro. Models of the fimbrial tip structure show that FimH is shaped like a hook that is normally rigid but opens under force, causing structural changes that lead to firm anchoring of the bacteria on the surface. In contrast, the more distal adaptor proteins of the fimbrial tip create a flexible connection of FimH to the rigid fimbria, enhancing the ability of the adhesin to move into position and form bonds with mannose on the surface. The entire tip complex forms a hook-chain, ideal for rapid and stable anchoring in flow.

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor. 2011 PLoS Biol 9(5): e1000617. doi:10.1371/journal.pbio.1000617
There is increasing evidence that the catch bond mechanism, where binding becomes stronger under tensile force, is a common property among non-covalent interactions between biological molecules that are exposed to mechanical force in vivo. Here, by using the multi-protein tip complex of the mannose-binding type 1 fimbriae of Escherichia coli, we show how the entire quaternary structure of the adhesive organella is adapted to facilitate binding under mechanically dynamic conditions induced by flow. The fimbrial tip mediates shear-dependent adhesion of bacteria to uroepithelial cells and demonstrates force-enhanced interaction with mannose in single molecule force spectroscopy experiments. The mannose-binding, lectin domain of the apex-positioned adhesive protein FimH is docked to the anchoring pilin domain in a distinct hooked manner. The hooked conformation is highly stable in molecular dynamics simulations under no force conditions but permits an easy separation of the domains upon application of an external tensile force, allowing the lectin domain to switch from a low- to a high-affinity state. The conformation between the FimH pilin domain and the following FimG subunit of the tip is open and stable even when tensile force is applied, providing an extended lever arm for the hook unhinging under shear. Finally, the conformation between FimG and FimF subunits is highly flexible even in the absence of tensile force, conferring to the FimH adhesin an exploratory function and high binding rates. The fimbrial tip of type 1 Escherichia coli is optimized to have a dual functionality: flexible exploration and force sensing. Comparison to other structures suggests that this property is common in unrelated bacterial and eukaryotic adhesive complexes that must function in dynamic conditions.

There are numerous threats facing honey bee populations and the recent losses of honey bee colonies in the United States, Canada, and Europe is alarming. In the U.S., annual honey bee colony losses increased from 17–20% to 32% during the winter of 2006/07 with some operations losing 90% of their hives. Average annual losses have remained high, averaging 32% from 2007–2010. One factor contributing to increased losses is Colony Collapse Disorder (CCD), an unexplained loss of honey bee colonies fitting a defined set of criteria. While factors such as pesticide exposure, transportation stress, genetic diversity, and nutrition affect colony health, the most significant CCD-associated variable characterized to date is increased pathogen incidence. Although greater pathogen incidence correlates with CCD, the cause is unknown in part due to insufficient knowledge of the pathogenic and commensal organisms associated with honey bees.

To gain a more complete understanding of the spectrum of infectious agents and potential threats found in commercially managed migratory honey bee colonies, researchers conducted a 10-month investigation. Analysis incorporated a suite of molecular tools (custom microarray, polymerase chain reaction (PCR), quantitative PCR (qPCR) and deep sequencing) enabling rapid detection of the presence (or absence) of all previously identified honey bee pathogens as well as facilitating the detection of novel pathogens. This study provides a comprehensive temporal characterization of honey bee pathogens and offers a baseline for understanding current and emerging threats to this critical component of U.S. agriculture.

Discovery and characterization of four new viruses will facilitate future monitoring of bee colones. Temporal characterization of these and other microbes offers a more complete view of the possible microbe-microbe and microbe-environment interactions. Further studies examining any subtle or combinatorial effects of these novel microbes are required to understand their role in colony health.

Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia. (2011) PLoS ONE 6(6): e20656. doi:10.1371/journal.pone.0020656
Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (~10^11 viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January.

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