Reflections on the Teaching Symposium at the Microbiology Society Annual Conference 2023.

The Microbiology Society Education and Outreach Network (EON) recently hosted the Teaching Symposium at the Microbiology Society Annual Conference, sponsored by Access Microbiology. The presence of the Symposium as an established parallel session within the wider Annual Conference reflects the importance of high-quality, contemporary microbiology education and outreach delivered in an enthusiastic and inclusive manner. At the 2023 Symposium, a variety of pedagogical research projects in higher education learning, teaching and assessment, as well as public engagement projects, were showcased through invited talks, offered talks, flash talks and posters. The event was attended by up to 70 delegates. Several themes were noted throughout the day: engaging with Gen Z (Generation Z, those born between 1996 and 2010), active learning, art in science and engaging with non-higher education (HE) audiences. Inclusivity was a key driver in the organization of the Symposium; the room was set up to encourage discussion and participants could ask questions using an online platform as well as speaking in the room. We now encourage all speakers to consider publishing their work as a peer-reviewed article for further dissemination and impact.

Developing research skills in medical students online using an active research study.

BACKGROUND: Developing research skills and scholarship are key components of medical education. The COVID-19 pandemic necessitated that all teaching be delivered online. We introduced an approach to small group teaching in the academic year 2020-2021 online which involved students in an active (ongoing) research study to develop their research skills. METHODS: We acquired student feedback to evaluate their perspectives quantitatively on development of research and scholarship skills, teaching content and format, and tutor performance using this teaching approach. In addition, we captured free text responses from both students and tutors on the positives and negatives of our course, and their suggested improvements. We also compared summative assessment marks for the online/active research course (2020-2021) with those obtained from previous (2017-2019) and subsequent (2021-2023) teaching sessions. RESULTS: Students were largely positive about most aspects of the online course utilising an active research study (n = 13). Students agreed that they were able to acquire research skills, particularly related to data analysis, transferable skills, and giving scientific presentations. A one-way ANOVA revealed no significant difference for assessment marks across all five teaching years (two years prior and two years following the online/active research course), indicating that the course achieved the learning outcomes. Students enjoyed the convenience of online teaching and the availability of course resources, but least liked the lack of in-person interaction and laboratory training. Tutors enjoyed the collaborative aspects of online teaching, but least liked the lack of face-to-face interactions with students. CONCLUSIONS: Our study demonstrates that delivering online teaching which involves students in active research engages and motivates them to develop their research and scholarship skills. We recommend that educators consider incorporating a current research study in their undergraduate courses as this can enhance the student learning experience as well as the research project itself.

Dynamics of a starvation-to-surfeit shift: a transcriptomic and modelling analysis of the bacterial response to zinc reveals transient behaviour of the Fur and SoxS regulators.

We describe a hybrid transcriptomic and modelling analysis of the dynamics of a bacterial response to stress, namely the addition of 200 µM Zn to Escherichia coli growing in severely Zn-depleted medium and of cells growing at different Zn concentrations at steady state. Genes that changed significantly in response to the transition were those reported previously to be associated with zinc deficiency (zinT, znuA, ykgM) or excess (basR, cpxP, cusF). Cellular Zn levels were confirmed by ICP-AES to be 14- to 28-fold greater after Zn addition but there was also 6- to 8-fold more cellular Fe 30 min after Zn addition. Statistical modelling of the transcriptomic data generated from the Zn shift focused on the role of ten key regulators; ArsR, BaeR, CpxR, CusR, Fur, OxyR, SoxS, ZntR, ZraR and Zur. The data and modelling reveal a transient change in the activity of the iron regulator Fur and of the oxidative stress regulator SoxS, neither of which is evident from the steady-state transcriptomic analyses. We hypothesize a competitive binding mechanism that combines these observations and existing data on the physiology of Zn and Fe uptake. Formalizing the mechanism in a differential equation model shows that it can reproduce qualitatively the behaviour seen in the data. This gives new insights into the interplay of these two fundamental metal ions in gene regulation and bacterial physiology, as well as highlighting the importance of dynamic studies to reverse-engineer systems behaviour.

Modelling complex biological systems using an agent-based approach.

Many of the complex systems found in biology are comprised of numerous components, where interactions between individual agents result in the emergence of structures and function, typically in a highly dynamic manner. Often these entities have limited lifetimes but their interactions both with each other and their environment can have profound biological consequences. We will demonstrate how modelling these entities, and their interactions, can lead to a new approach to experimental biology bringing new insights and a deeper understanding of biological systems.

Transcript profiling and inference of Escherichia coli K-12 ArcA activity across the range of physiologically relevant oxygen concentrations.

Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo' and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.

A novel method for exploring elemental composition of microbial communities: laser ablation-inductively coupled plasma-mass spectrometry of intact bacterial colonies.

Bacterial colonies are spatially complex structures whose physiology is profoundly dependent on interactions between cells and with the underlying semi-solid substratum. Here, we use bacterial colonies as a model of a microbial community to evaluate the potential of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to delineate elemental distributions within colonies with minimal pre-treatment. To reduce water content of the colony and limit undesirable absorption of laser energy, we compared methods of preparing 24h-old colonies of Escherichia coli TG1 on agar for laser ablation. Colonies on excised agar segments dried on chromatography paper were superior to colonies dried in a dessicator or by prolonged incubation, with respect to signal magnitude, signal:noise ratio and background signal. Having optimised laser scan speed (10 microm s(-1)) and laser beam diameter (100 microm), further improvements were achieved by growing colonies on nylon membranes over agar, which were then transferred to the ablation chamber without further treatment. Repeated line rasters across individual membrane-supported colonies yielded three-dimensional elemental maps of colonies, revealing a convex morphology consistent with visual inspection. By normalising isotope counts for P, Mn, Zn, Fe and Ca against Mg, the most abundant cellular divalent cation, we sought elemental heterogeneity within the colony. The normalised concentration of Mn in the perimeter was higher than in the colony interior, whereas the converse was true for Ca. LA-ICP-MS is a novel and powerful method for probing elemental composition and organisation within microbial communities and should find numerous applications in, for example, biofilm studies.

Severe zinc depletion of Escherichia coli: roles for high affinity zinc binding by ZinT, zinc transport and zinc-independent proteins.

Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn(2+) or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

Expression of the PitA phosphate/metal transporter of Escherichia coli is responsive to zinc and inorganic phosphate levels.

Escherichia coli possesses two major systems for inorganic phosphate (P(i)) uptake. The Pst system (pstSCAB) is inducible by low phosphate concentrations whereas the low-affinity transporter (pitA) has been described as constitutively expressed. PitA catalyses transport of metal [Mg(II), Ca(II)]-phosphate complexes, and mutations in pitA confer Zn(II) resistance. Here we report that pitA transcription is not constitutive; activity of a single-copy pitA-lacZ transcriptional fusion (monolysogen) was maximal at high extracellular Zn(II) (150 microM), in the absence of added P(i), and in a well-defined pitA mutant strain. Intracellular zinc levels were unaffected by adding Zn(II) to the medium for both the wild-type and mutant strains. However, in the wild-type strain, Mg levels (per gram of dry biomass) fell by eightfold in cells grown with added Zn(II) and by 20-fold when Zn(II) and P(i) were added to cultures. Mutation of pitA reduced the effects of external Zn(II) and phosphate levels on Mg pools, consistent with competition or inhibition by Zn(II) of PitA. The mechanism of pitA regulation by extracellular Zn(II) and P(i) is unknown but appears not to involve Fur or other well-characterized regulators.

NMR structural analysis of cadmium sensing by winged helix repressor CmtR.

CmtR from Mycobacterium tuberculosis is a winged helical DNA-binding repressor of the ArsR-SmtB metal-sensing family that senses cadmium and lead. Cadmium-CmtR is a dimer with the metal bound to Cys-102 from the C-terminal region of one subunit and two Cys associated with helix alphaR from the other subunit, forming a symmetrical pair of cadmium-binding sites. This is a significant novelty in the ArsR-SmtB family. The structure of the dimer could be solved at 312 K. The apoprotein at the same temperature is still a dimer, but it experiences a large conformational exchange at the dimer interface and within each monomer. This is monitored by an overall decrease of the number of nuclear Overhauser effects and by an increase of H(2)O-D(2)O exchange rates, especially at the dimeric interface, in the apo form with respect to the cadmium-bound state. The C-terminal tail region is completely unstructured in both apo and cadmium forms but becomes less mobile in the cadmium-bound protein due to the recruitment of Cys-102 as a metal-ligand. DNA binds to the apo dimer with a ratio 1:3 at millimolar concentration. Addition of cadmium to the apo-CmtR-DNA complex causes DNA detachment, restoring the NMR spectrum of free cadmium-CmtR. Cadmium binding across the dimer interface impairs DNA association by excluding the apo-conformers suited to bind DNA.

A cadmium-lead-sensing ArsR-SmtB repressor with novel sensory sites. Complementary metal discrimination by NmtR AND CmtR in a common cytosol.

We report a cadmium- and lead-detecting transcriptional repressor from Mycobacterium tuberculosis designated CmtR. Two genes were co-transcribed with cmtR, one encoding a deduced P1 type ATPase. Purified CmtR bound to the cmt operator-promoter, and repression of transcription was lost after introduction of a stop codon into cmtR. Assays of metal-dependent expression from cmt and nmt operator-promoters established that the metal specificity of CmtR in vivo was perfectly inverted relative to the nickel-cobalt sensor NmtR from the same organism, with CmtR totally insensitive to Co(II) or Ni(II) and NmtR totally insensitive to Cd(II) or Pb(II). Absorption spectroscopy of Cd(II)-, Co(II)-, and Ni(II)-substituted CmtR revealed S- to metal-charge-transfer which was absent in NmtR, providing diagnostic metal-difference spectra that discriminated between metal-binding to these two proteins. Ni(II)-binding isothermal titrations of CmtR are complex, with Kapp = 1.8 x 10(4) m(-1) for site1, three orders of magnitude weaker than KNi for NmtR. Mixing equimolar apo-NmtR and apo-CmtR with 0.9 equivalents of Cd(II) gave Cd(II)-dependent difference spectra almost identical to Cd(II)0.9-CmtR. Thus, Cd(II) bound to CmtR in preference to NmtR, whereas the converse was true for Ni(II); this correlates faithfully with and provides a simplistic basis for metal-sensing preferences. In contrast, CmtR and NmtR had similar affinities for Co(II), and alternative explanations for Co(II) sensitivities are invoked. ArsR-SmtB repressors detect metals through derivatives of one or both of two possible allosteric sites at either carboxyl-terminal alpha5 helices or helix alpha3 proximal to the DNA-binding site. Unexpectedly, neither site was required for inducer recognition by CmtR. The mutants in potential metal ligands in, or near, these regions, Cys4, Cys35, Asp79, His81, Asp97, Asp99, Glu105, Glu111, and Glu114, retained both repression and inducer recognition. Crucially, substitution of Cys57, Cys61, and Cys102 with Ser revealed that each of these three residues is obligatory for Cd(II) detection, and this defines completely new sensory sites.