Stina Lindberg

Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli

ABSTRACT Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.

Structure of FocB--a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli.

In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form homomeric or heteromeric complexes and exert both positive and negative effects on the transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 Å resolution. FocB is an all α‐helical protein with a helix‐turn‐helix motif. Interestingly, conserved residues important for DNA‐binding are located not in the postulated recognition helix of the motif, but in the preceding helix. Results from protein–DNA‐binding studies suggest that FocB interacts with the minor groove of its cognate DNA target, which is indicative of a DNA interaction that is unusual for this motif. FocB crystallizes in the form of dimers. Packing interactions in the crystals give two plausible dimerization interfaces. Conserved residues, known to be important for protein oligomerization, are present at both interfaces, suggesting that both sites could play a role in a functional FocB protein.

Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors

The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy)alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors – a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 – showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries.

A luminescence-based assay of UDP-sugar producing pyrophosphorylases†

A coupled luminescence assay was applied to monitor pyrophosphate (PPi) production by either purified barley UDP-glucose pyrophosphorylase (UGPase) or purified Leishmania UDP-sugar pyrophosphorylase (USPase). In the assay, the PPi produced by the pyrophosphorylases was converted to ATP by ATPsulfurylase, and the ATP produced was linked to luminescent light formation through the action of firefly luciferase. The assay allowed for a quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The activities were linear with time and proportional to the amount of the enzyme added, and were neither affected by Pi nor by DTT. For UGPase,Km values with UTP and Glc-1-P were 0.14 and 0.26 mM, respectively, whereas for USPase the respective Km values with UTP, Glc-1-P and Gal-1-P were 0.4, 2.9 and 3.9 mM. Possible applications of the luminescence-based assay for not only UDP-sugar producing pyrophosphorylases, but also other types of pyrophosphorylases are discussed.

Motivating students with authentic science experiences : Changes in motivation for school science

Abstract Background: Students’ motivation for science declines over the early teenage years, and students often find school science difficult and irrelevant to their everyday lives. This paper asks whether creating opportunities to connect school science to authentic science can have positive effects on student motivation. Purpose: To understand how authentic science experiences can affect students’ motivation for science and students’ goals, values, beliefs and attitudes towards science. Programme description: The Medicine Hunt brought scientists and students together to find bacteria that produce secondary metabolites with antibiotic effects. Scientists received help with collecting soil samples and teachers and students took an active role in research and worked with solving an authentic problem as a part of their ordinary school science over a course of six months. Sample: About 388 students from 18 lower-secondary school classes participating in the Medicine Hunt. Students were enrolled in grade seven to nine (13–15 years old). At this stage, science is compulsory, and all students follow the same science course. The classes represented different geographical regions of Sweden. Design and methods: Students filled in motivation questionnaires before and after the Medicine Hunt. Paired t-tests were used to evaluate how students’ intrinsic motivation, goals, values, beliefs and attitudes towards science changed over the project period. Results: Students’ intrinsic motivation for school science and plans for future participation in science remained unchanged during the period they participated in the Medicine Hunt, and students’ goals, values and attitudes followed the well-documented pattern of decline. Thus, the authentic experience can arrest the well-described decline for some motivation-related factors. Conclusions: The findings suggest that the authentic experience can arrest some aspects of the decline in motivation for science in the teenage years. The paper discusses the processes around students’ motivation in relation to authentic experiences.

Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli.

The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His(6)-tagged fusion protein was captured by Ni(2+)-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His(6) tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 A resolution was collected at 100 K using synchrotron radiation.

Increasing the permeability of Escherichia coli using MAC13243

The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.