Ordered and ushered; the assembly and translocation of the adhesive Type I and P Pili

Type I and P pili are chaperone-usher pili of uropathogenic Escherichia coli, which allow bacteria to adhere to host cell receptors. Pilus formation and secretion are orchestrated by two accessory proteins, a chaperone, which catalyses pilus subunit folding and maintains them in a polymerization-competent state, and an outer membrane-spanning nanomachine, the usher, which choreographs their assembly into a pilus and drives their secretion through the membrane. In this review, recent structures and kinetic studies are combined to examine the mechanism of type I and P pili assembly, as it is currently known. We also investigate how the knowledge of pilus biogenesis mechanisms has been exploited to design selective inhibitors of the process

Allosteric signalling in the outer membrane translocation domain of PapC usher

PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around β12-β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability

The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS

The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species

Factors leading to negative attitudes towards learning of mathematics at secondary school in Kwekwe district

The performance in mathematics by students has persistently been poor. Students‘ attitudes towards mathematics have been a factor that is known to influence students‘ achievement in mathematics. The purpose of this study is to investigate the factors contributing to negative attitudes towards mathematics resulting in poor performance at O‘level and also establish the strategies that can be adopted to improve performance in mathematics by students in secondary schools in Kwekwe district in Midlands Province. The researcher used purposive sampling procedure in selecting the schools and the simple random selection was used for pupils. Data was collected through the use of questionnaires and interviews. The instruments were administered to [120] students, [12] secondary school teachers and [3] H.O.Ds for the schools used. Factors contributing to poor performance results from both teachers and students negative attitude, lack of motivation by teachers and the methods used for teaching. Parents‘ negative comments on mathematics have an effect in developing negative attitudes by pupils. The economic status of parents is also a contributory factor the school environment, influence of peers and adolescence all contribute in students‘ attitude towards mathematics. Improving these factors and sensitization of local community to discard practices which prohibit students‘ effective participation in learning maths could improve performance at O‘ level. It is anticipated that the findings of this study will give curriculum developers new insights in emerging issues on performance and influence the ministry of education on policy formulation. Students are also expected to benefit from the findings because improved mathematics performance will give them opportunity to pursue science related courses in higher institutions of learning

Clebsch-Gordan coefficients and electron-phonon scattering in the valence band of Ge

The abstract contains mathematical symbols and do not display correctly. It can be read in the document attached.Dissertation (MSc)--University of Pretoria, 2017.PhysicsMScUnrestricte

Pilus biogenesis in uropathogenic Escherichia coli : An electrophysiology study on the PapC usher

Uropathogenic Escherichia coli are the main causal agents of urinary tract infections. Pili are bacterial surface adhesive organelles that are critical virulence factors in the successful colonization of the urinary tract by uropathogenic E. coli. Pili are composite in structure and are formed from the polymerization of different subunits. The chaperone/usher pathway is a bi-component secretion system that is dedicated to the biogenesis and export of pili to the cell surface. It is comprised up of a periplasmic chaperone protein and an outer membrane usher protein that functions as the site of pilus assembly and simultaneous translocation. The usher exists as a dimer, where each monomer consists of a gated β-barrel pore with several domains. To shed insight into the mechanistic details behind pilus biogenesis we instituted the first electrophysiology study on the PapC usher channel. In this dissertation we present data on the pore properties of the PapC usher. Using planar lipid bilayer technique we found that PapC forms an ion conducting channel that is mostly in a closed state. However, the usher channel is dynamic and transiently opens to various conductance levels. A structure function relationship study was applied to investigate the roles of the α-helix, plug, N- and C-terminal domains on the usher channel behavior. Domain deletion mutants characterized using planar lipid bilayer showed the modulating effects of the α-helix, plug, N- and C-terminal domains on the usher channel behavior. In contrast to the wild type, domain deletion mutants displayed open channel behavior that was marked by frequent closing transitions. The ∆plug mutant formed a channel with an extremely large conductance indicating that the plug domain gates the usher closed and its removal creates a large pore. Single site substitution of the D234 residue resulted in an open channel as well, indicating the D234 residue plays a role in plug displacement. Following characterization of the usher, we investigated the modulatory effect of chaperone – subunit complexes on the usher channel behavior. We attained inconclusive results and found no clear-cut discernible effect on the usher channel behavior by the chaperone subunit complexes in the various assays reported in this dissertation. However, these results set the stage for more work on the modulation of the usher by substrates.Biology and Biochemistry, Department o

Components of a digital transformation strategy : a South African perspective

Most organisations have begun to take the phenomenon of digital transformation very seriously and in response, they have adopted a digital transformation strategy (DTS) to guide them on the journey to being digitally transformed. Despite the impetus to adopt a DTS, most organisations lack the understanding of what a DTS entails and the components of such a strategy. In an effort to bring better understanding on the components of a DTS, this study adopted a qualitative research approach and collected research data using an Internet-mediated questionnaire. Our research findings revealed that most organisations have recently adopted a DTS within the last 10 years with the exception of a few. Furthermore, our research findings reveal that a DTS must incorporate the following components, digitisation of the customer experience, digitisation of products and services, digitisation employee ways of working and digitisation of business processes. Our findings also revealed that a DTS leverages digital technologies enabling the organisation to compete, innovate, grow and achieve its business strategy. These results have implications for academics; our study adds to the digital transformation body of knowledge and specifically the components of a DTS. We also propose a definition of a DTS based on our findings of DTS components. For practitioners, managers formulating and refining their DTS can use the DTS components as a benchmark of what to incorporate in their DTS.http://www.springer.com/series/151792022-09-17hj2021Informatic