Substantive changes in gene expression, metabolism, and the proteome are manifested in overall ch... more Substantive changes in gene expression, metabolism, and the proteome are manifested in overall changes in microbial population growth. Quantifying how microbes grow is therefore fundamental to areas such as genetics, bioengineering, and food safety. Traditional parametric growth curve models capture the population growth behavior through a set of summarizing parameters. However, estimation of these parameters from data is confounded by random effects such as experimental variability, batch effects or differences in experimental material. A systematic statistical method to identify and correct for such confounding effects in population growth data is not currently available. Further, our previous work has demonstrated that parametric models are insufficient to explain and predict microbial response under non-standard growth conditions. Here we develop a hierarchical Bayesian non-parametric model of population growth that identifies the latent growth behavior and response to perturbation, while simultaneously correcting for random effects in the data. This model enables more accurate estimates of the biological effect of interest, while better accounting for the uncertainty due to technical variation. Additionally, modeling hierarchical variation provides estimates of the relative impact of various confounding effects on measured population growth.
Mechanisms of plasma induced microbial inactivation have commonly been studied with physico-chemi... more Mechanisms of plasma induced microbial inactivation have commonly been studied with physico-chemical techniques. In this letter, Escherichia coli K-12 and its ΔrecA, ΔrpoS and ΔsoxS mutants are employed to discriminate effects of UV photons, OH radicals, and reactive oxygen species produced in atmospheric discharges. This microbiological approach exploits the fact that these E. coli mutants are defective in their resistance against various external stresses. By interplaying bacterial inactivation kinetics with optical emission spectroscopy, oxygen atoms are identified as a major contributor in plasma inactivation with minor contribution from UV photons, OH radicals, singlet oxygen metastables, and nitric oxide.
Escherichia coli has been the leading model organism for many decades. It is a fundamental player... more Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K-12. However, the antecedents of today's laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits. We wished to determine whether these genetic lesions altered the physiology of E. coli such that observations made for E. coli K-12 were not reflective of the true physiology of the species. Here we have repaired the wbbL locus, restoring the ability of E. coli K-12 strain MG1655 to express the O antigen on its cell surface. We demonstrate that O-antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, the presence of the O antigen enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans nematode worm and to kill C. elegans at rates similar to pathogenic bacterial species. We show that killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. We demonstrate C. elegans killing is a feature of other commensal E. coli and that killing occurs at the same rate as known pathogens. These results suggest C. elegans is not an effective model of human infectious disease.
Transposon-directed insertion site sequencing (TraDIS) is a highthroughput method coupling transp... more Transposon-directed insertion site sequencing (TraDIS) is a highthroughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.
Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed int... more Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed interest in the use of traditional anti-microbial treatments, including honey. However, understanding the underlying mechanism of the anti-microbial action of honey has been hampered due to the complexity of its composition. High throughput genetic tools could assist in understanding this mechanism. In this study, the anti-bacterial mechanism of a model honey, made of sugars, hydrogen peroxide, and gluconic acid, was investigated using genome-wide transposon mutagenesis combined with high-throughput sequencing (TraDIS), with the strain Escherichia coli K-12 MG1655 as the target organism. We identified a number of genes which when mutated caused a severe loss of fitness when cells were exposed to the model honey. These genes encode membrane proteins including those involved in uptake of essential molecules, and components of the electron transport chain. They are enriched for pathways involv...
Honey has been valued as a powerful antimicrobial since ancient times. However, the understanding... more Honey has been valued as a powerful antimicrobial since ancient times. However, the understanding of the underlying antibacterial mechanism is incomplete. The complexity and variability of honey composition represent a challenge to this scope. In this study, a simple model system was used to investigate the antibacterial effect of, and possible synergies between, the three main stressors present in honey: sugars, gluconic acid, and hydrogen peroxide (H2O2), which result from the enzymatic conversion of glucose on honey dilution. Our results demonstrated that the synergy of H2O2 and gluconic acid is essential for the antibacterial activity of honey. This synergy caused membrane depolarization, destruction of the cell wall, and eventually growth inhibition of E. coli K-12. The presence of H2O2 stimulated the generation of other long-lived ROS in a dose-dependent manner. Sugars caused osmosis-related morphological changes, however, decreased the toxicity of the H2O2/gluconic acid. The ...
For many years, studies on protein folding were done by biochemists and biophysicists using purif... more For many years, studies on protein folding were done by biochemists and biophysicists using purified components and defined conditions. More recently, attention has shifted to thinking about protein folding in the messier internal environment of the cell. Here, proteins are faced with many hazards not encountered in the test tube: other proteins are present at high concentrations, the cell is full of membranes that need to be crossed, and conditions that can have a large effect on protein folding may not be constant. Proteins are often not well suited for these vagaries of cellular life, and a host of accessory proteins need to be present to assist the process of protein folding. These accessory proteins are referred to as molecular chaperones, and they use various mechanisms to make sure that their client proteins stay on the straight and narrow path to the folded active state.
Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 1993
Molecular chaperones belonging to heat shock protein families have been identified as prominent a... more Molecular chaperones belonging to heat shock protein families have been identified as prominent antigens in the immune response to a wide variety of infections. Recognition of such highly conserved antigens may contribute to protective immunity but, in some circumstances, may also have pathological autoimmune consequences. Recognition of chaperones may be an inherent feature of the immune system. Peptide mapping experiments revealed an overlap between hsp 70-binding sites and immunodominant regions of three protein antigens, consistent with a possible functional activity for molecular chaperones in the processing and presentation of peptides during class II-restricted T lymphocyte responses. A functional role for molecular chaperones in antigen processing may be a factor which contributes to their immunogenicity.
В работе показано, что ген groEL S. meliloti, кодирующий субъединицы шаперонина с молекулярной ма... more В работе показано, что ген groEL S. meliloti, кодирующий субъединицы шаперонина с молекулярной массой 60 кДа, комплементирует делецию гена groEL Е. coli, осуществленную путем замены groEL на nptll ген с помощью трансдукции фагом РЇ При этом для экспрессии гена groEL из S. meliloti в клетках Е. сой использована плазм ида с промотором арабинозного оперона Е. coli, что позволило тонко регулировать синтез шаперонинов содержанием арабинозы и глюкозы в среде выращивания. Метод замен хромосомного гена groEL на гетерологичный groEL, примененный в данной работе, позволяет изучать свойства гомологичных шаперонинов в клетке, не несущей дополнительно температурочувствительных шаперонинов и, таким образом, избежать нежела тельного взаимодействия шаперонинов различного происхождения и включения в один шаперонин гетерологичных субъединиц.
Proteins that are translocated across the cytoplasmic membrane by Sec machinery must be in an unf... more Proteins that are translocated across the cytoplasmic membrane by Sec machinery must be in an unfolded conformation in order to pass through the protein- .
The version in the Kent Academic Repository may differ from the final published version. Users ar... more The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record.
(2020) Optimizing the antimicrobial performance of metallic glass composites through surface text... more (2020) Optimizing the antimicrobial performance of metallic glass composites through surface texturing. Materials Today Communications, 23. p. 101074.
The ability of chaperonins to buffer mutations that affect protein folding pathways suggests that... more The ability of chaperonins to buffer mutations that affect protein folding pathways suggests that their abundance should be evolutionarily advantageous. Here, we investigate the effect of chaperonin overproduction on cellular fitness inEscherichia coli. We demonstrate that chaperonin abundance confers (a) an ability to tolerate higher temperatures, (b) improved cellular fitness and (c) enhanced folding of metabolic enzymes, which is expected to lead to enhanced energy harvesting potential.
Substantive changes in gene expression, metabolism, and the proteome are manifested in overall ch... more Substantive changes in gene expression, metabolism, and the proteome are manifested in overall changes in microbial population growth. Quantifying how microbes grow is therefore fundamental to areas such as genetics, bioengineering, and food safety. Traditional parametric growth curve models capture the population growth behavior through a set of summarizing parameters. However, estimation of these parameters from data is confounded by random effects such as experimental variability, batch effects or differences in experimental material. A systematic statistical method to identify and correct for such confounding effects in population growth data is not currently available. Further, our previous work has demonstrated that parametric models are insufficient to explain and predict microbial response under non-standard growth conditions. Here we develop a hierarchical Bayesian non-parametric model of population growth that identifies the latent growth behavior and response to perturbat...
Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in b... more Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial cochaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all cochaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial cochaperonin.
The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We... more The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We have investigated the modifications and the cellular location of ChiA when it is expressed in transgenic tobacco plants. Immunoblots on total leaf protein probed with antibody to ChiA showed that when the bacterial chitinase is expressed in plants, it migrates as a series of discrete bands with either the same or a slower mobility than the secreted bacterial protein. Analysis of the vacuum infiltrate of leaves expressing ChiA showed that the modified forms of the protein are enriched in the intercellular fluid. Media recovered from suspension cultures of cell lines expressing the chiA gene were also enriched for the modified forms of ChiA. Washed protoplasts, however, contained only the nonmodified form. The molecular weight of these polypeptides is reduced by treatment with glycopeptidase F but not with endoglycosidase H. Treatment of the suspension cultures with tunicamycin also leads to reduction in the molecular weight of the chitinase bands. We suggest that some of the ChiA protein is N-glycosylated and secreted when expressed in plants, and that the modifications are complex glycans. These results show that a bacterial signal sequence can function in plant cells, and that protein secretion from plant cells probably operates by a default pathway.
When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the pr... more When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the protein is glycosylated and secreted from the plant cells; however most of the protein remains inside the cells. We tested whether the efficiency of secretion could be improved by replacing the bacterial signal sequence with a plant signal sequence. We found the signal sequence and the first two amino acids of the PRlb protein attached to the ChiA mature protein directs complete glycosylation and secretion of the ChiA from plant cells. Glycosylation of this protein is not required for its efficient secretion from plant cells.
Mechanisms of plasma induced microbial inactivation have commonly been studied with physico-chemi... more Mechanisms of plasma induced microbial inactivation have commonly been studied with physico-chemical techniques. In this letter, Escherichia coli K-12 and its ΔrecA, ΔrpoS and ΔsoxS mutants are employed to discriminate effects of UV photons, OH radicals, and reactive oxygen species produced in atmospheric discharges. This microbiological approach exploits the fact that these E. coli mutants are defective in their resistance against various external stresses. By interplaying bacterial inactivation kinetics with optical emission spectroscopy, oxygen atoms are identified as a major contributor in plasma inactivation with minor contribution from UV photons, OH radicals, singlet oxygen metastables, and nitric oxide.
Chaperonins are required for correct folding of many proteins. They exist in two phylogenetic gro... more Chaperonins are required for correct folding of many proteins. They exist in two phylogenetic groups: Group I, found in bacteria and eukaryotic organelles, and Group II, found in archaea and eukaryotic cytoplasm. The two groups, while homologous, differ in significantly in structure and mechanism. Evolution of group II chaperonins has been proposed to have been key in enabling the expansion of the proteome required for eukaryotic evolution. In an archaeal species which expresses both groups of chaperonins, client selection is determined by structural and biochemical properties, rather than phylogenetic origin. It is thus predicted that Group II chaperonins will be poor at replacing Group I chaperonins. We have tested this hypothesis, and report here that the Group II chaperonin from M. maripaludis (Mm-cpn) can partially functionally replace GroEL, the Group I chaperonin of E. coli Furthermore, we identify and characterise two single point mutations in Mm-cpn that have an enhanced ab...
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