Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome

Matthew J. Nicholson, Michael K. Theodorou and Jayne L. Brookman. (2005). Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome. Microbiology, 151 (1), 121-133. Sponsorship: BBSRC RAE2008

Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

Investigation of growth and stress tolerance characteristics of Cronobacter spp.

Cronobacter spp. are opportunistic pathogens which can be isolated from a wide variety of foods and environments. They are Gram negative, motile, non-spore forming, peritrichous rods of the Enterobacteriaceae family. This food-borne pathogen is associated with the ingestion of contaminated infant milk formula (IMF), causing necrotizing enterocolitis, sepsis and meningitis in neonatal infants. The work presented in this thesis involved the investigation and characterisation of a bank of Cronobacter strains for their ability to tolerate physiologically relevant stress conditions that are commonly encountered in the gastrointestinal tract. While all strains were able to endure the suboptimal conditions tested, noteworthy variations were observed between strains. A collection of these strains were Lux-tagged to determine if their growth could be tracked in IMF by measuring bioluminescence. The resulting strains could be easily and reproducibly monitored in real time by measuring light emission. Following this a transposon mutagenesis library was created in one of the Lux-tagged strains of Cronobacter sakazakii. This library was screened for mutants with affected growth in milk. The majority of mutants identified were associated with amino acid metabolism. The final section of this thesis identified genes involved in the tolerance of C. sakazakii to the milk derived antimicrobial peptide, Lactoferricin B (Lfcin B). This was achieved by creating a transposon mutagenesis library in C. sakazakii and screening for mutants with increased susceptibility to Lfcin B. Overall this thesis demonstrates the variation between Cronobacter strains. It also identifies genes required for growth of the bacteria in milk, as well as genes needed for antimicrobial peptide tolerance.

CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo.

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.

Environmental and genetic determinants of colony morphology in yeast.

Nutrient stresses trigger a variety of developmental switches in the budding yeast Saccharomyces cerevisiae. One of the least understood of such responses is the development of complex colony morphology, characterized by intricate, organized, and strain-specific patterns of colony growth and architecture. The genetic bases of this phenotype and the key environmental signals involved in its induction have heretofore remained poorly understood. By surveying multiple strain backgrounds and a large number of growth conditions, we show that limitation for fermentable carbon sources coupled with a rich nitrogen source is the primary trigger for the colony morphology response in budding yeast. Using knockout mutants and transposon-mediated mutagenesis, we demonstrate that two key signaling networks regulating this response are the filamentous growth MAP kinase cascade and the Ras-cAMP-PKA pathway. We further show synergistic epistasis between Rim15, a kinase involved in integration of nutrient signals, and other genes in these pathways. Ploidy, mating-type, and genotype-by-environment interactions also appear to play a role in the controlling colony morphology. Our study highlights the high degree of network reuse in this model eukaryote; yeast use the same core signaling pathways in multiple contexts to integrate information about environmental and physiological states and generate diverse developmental outputs.

Design and Evaluation of a Retrofittable Electric Snow Melting System for Pavements

Gemstone Team SnowMelt

Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation.

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10-7) and correlated with stronger p53RE sequences (p<10-110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.

Laminar Free Convection from a Vertically-Oriented Concentrated Photovoltaic Cell with Uniform Surface Heat Flux

This research project uses field measurements to investigate the cooling of a triple-junction, photovoltaic cell under natural convection when subjected to various amounts of insolation. The team built an experimental apparatus consisting of a mirror and Fresnel lens to concentrate light onto a triple-junction photovoltaic cell, mounted vertically on a copper heat sink. Measurements were taken year-round to provide a wide range of ambient conditions. A surface was then generated, in MATLAB, using Sparrow’s model for natural convection on a vertical plate under constant heat flux. This surface can be used to find the expected operating temperature of a cell at any location, given the ambient temperature and insolation. This research is an important contribution to the industry because it utilizes field data that represents how a cell would react under normal operation. It also extends the use of a well-known model from a one-sun environment to a multi-sun one.

Impacts of the Oceans on Climate Change

The oceans play a key role in climate regulation especially in part buffering (neutralising) the effects of increasing levels of greenhouse gases in the atmosphere and rising global temperatures. This chapter examines how the regulatory processes performed by the oceans alter as a response to climate change and assesses the extent to which positive feedbacks from the ocean may exacerbate climate change. There is clear evidence for rapid change in the oceans. As the main heat store for the world there has been an accelerating change in sea temperatures over the last few decades, which has contributed to rising sea‐level. The oceans are also the main store of carbon dioxide (CO2), and are estimated to have taken up ∼40% of anthropogenic-sourced CO2 from the atmosphere since the beginning of the industrial revolution. A proportion of the carbon uptake is exported via the four ocean ‘carbon pumps’ (Solubility, Biological, Continental Shelf and Carbonate Counter) to the deep ocean reservoir. Increases in sea temperature and changing planktonic systems and ocean currents may lead to a reduction in the uptake of CO2 by the ocean; some evidence suggests a suppression of parts of the marine carbon sink is already underway. While the oceans have buffered climate change through the uptake of CO2 produced by fossil fuel burning this has already had an impact on ocean chemistry through ocean acidification and will continue to do so. Feedbacks to climate change from acidification may result from expected impacts on marine organisms (especially corals and calcareous plankton), ecosystems and biogeochemical cycles. The polar regions of the world are showing the most rapid responses to climate change. As a result of a strong ice–ocean influence, small changes in temperature, salinity and ice cover may trigger large and sudden changes in regional climate with potential downstream feedbacks to the climate of the rest of the world. A warming Arctic Ocean may lead to further releases of the potent greenhouse gas methane from hydrates and permafrost. The Southern Ocean plays a critical role in driving, modifying and regulating global climate change via the carbon cycle and through its impact on adjacent Antarctica. The Antarctic Peninsula has shown some of the most rapid rises in atmospheric and oceanic temperature in the world, with an associated retreat of the majority of glaciers. Parts of the West Antarctic ice sheet are deflating rapidly, very likely due to a change in the flux of oceanic heat to the undersides of the floating ice shelves. The final section on modelling feedbacks from the ocean to climate change identifies limitations and priorities for model development and associated observations. Considering the importance of the oceans to climate change and our limited understanding of climate-related ocean processes, our ability to measure the changes that are taking place are conspicuously inadequate. The chapter highlights the need for a comprehensive, adequately funded and globally extensive ocean observing system to be implemented and sustained as a high priority. Unless feedbacks from the oceans to climate change are adequately included in climate change models, it is possible that the mitigation actions needed to stabilise CO2 and limit temperature rise over the next century will be underestimated.

Thermal front variability along the North Atlantic Current observed using satellite microwave and infrared data

Thermal fronts detected using multiple satellite sensors have been integrated to provide new information on the spatial and seasonal distribution of oceanic fronts in the North Atlantic. The branching of the North Atlantic Current (NAC) as it encounters the Mid-Atlantic Ridge (MAR) is reflected in surface thermal fronts, which preferentially occur at the Charlie Gibbs Fracture Zone (CGFZ) and several smaller fracture zones. North of the CGFZ there are few thermal fronts, contrasting with the region to the south, where there are frequent surface thermal fronts that are persistent seasonally and interannually. The alignment of the fronts confirms that the shallower Reykjanes Ridge north of the CGFZ is more of a barrier to water movements than the ridge to the south. Comparison of front distributions with satellite altimetry data indicates that the MAR influence on deep ocean currents is also frequently exhibited in surface temperature. The improved spatial and temporal resolution of the front analysis has revealed consistent seasonality in the branching patterns. These results contribute to our understanding of the variability of the NAC, and the techniques for visualising oceanic fronts can be applied in other regions to reveal details of surface currents that cannot be resolved using satellite altimetry or in situ measurements.

Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron

Objectives: The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance.
Methods: The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays.
Results: A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose.
Conclusions: These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron

Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116, Two copies of IS2112 were found in R, rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the l-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp, HA1 or in several Rhodococcus strains which do not utilize haloalkanes, IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064 which harbours genes encoding utilization of l-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA), Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.

Identification of essential operons with a rhamnose-inducible promoter in Burkholderia cenocepacia

Scanning of bacterial genomes to identify essential genes is of biological interest, for understanding the basic functions required for life, and of practical interest, for the identification of novel targets for new antimicrobial therapies. In particular, the lack of efficacious antimicrobial treatments for infections caused by the Burkholderia cepacia complex is causing high morbidity and mortality of cystic fibrosis patients and of patients with nosocomial infections. Here, we present a method based on delivery of the tightly regulated rhamnose-inducible promoter P(rhaB) for identifying essential genes and operons in Burkholderia cenocepacia. We demonstrate that different levels of gene expression can be achieved by using two vectors that deliver P(rhaB) at two different distances from the site of insertion. One of these vectors places P(rhaB) at the site of transposon insertion, while the other incorporates the enhanced green fluorescent protein gene (e-gfp) downstream from P(rhaB). This system allows us to identify essential genes and operons in B. cenocepacia and provides a new tool for systematically identifying and functionally characterizing essential genes at the genomic level.