Cary 50 uv visible spectrophotometer

Anaerobic UV-visible spectral changes of holo-OAM and variants upon binding with d-ornithine or DABA were followed in a Cary 50 UV-visible spectrophotometer (Varian Inc.) contained in an anaerobic glove box. The wild-type OAM or variant holoenzyme solutions contained 15 μm apoenzyme, 15 μm PLP, and 15 μm AdoCbl in a total volume of 1 ml in anaerobic buffer. Spectral changes for holo-OAM were recorded at 25 °C, at 0 s and 10 s and then at every 60 s up to 25 min following the addition of 2.5 mmd-ornithine or 2.5 mm DABA.
Aerobic UV-visible spectral changes of holo-OAM and variants upon binding with the inhibitor DAPA and subsequent changes on continuous photolysis were followed in a Cary 50 UV-visible spectrophotometer (Varian Inc.). The holoenzyme solution contained 30 μm apoenzyme (wild-type OAM or OAM variants), 30 μm PLP, and 30 μm AdoCbl in a total volume of 1 ml in aerobic buffer (100 mm NH₄-EPPS, pH 8.5), and the reaction was initiated by adding 5 mm DAPA. After 25 min of incubation, holoenzyme was subjected to continuous illumination from a Schott KL1500 electronic light source, which provided illumination at an intensity of 1000 μmol m⁻² s⁻¹, and spectral changes were recorded at 25 °C at 0 and 10 s and then at every 60 s up to 25 min. A red insert filter (<530-nm cutoff filter) was attached to the light path to avoid protein photo-degradation.

The MIC of rifampicin for the SL strain was determined using a micro-broth dilution method. Briefly, rifampicin was solubilised in dimethyl sulfoxide (DMSO) to 10 mg/ml and diluted to 8,000 μg/ml as the initial rifampicin concentration before serial 2-fold dilution in oleic acid-albumin-dextrose-catalase- (OADC-) enriched 7H9 media, with either Tween-20 (0.1%) or Tween-80 (0.05%), followed by the addition of mid-growth M. smegmatis diluted to the equivalent of A_600nm 0.006. No drug was added to the final row, and no bacteria were added to the first as positive and negative controls, respectively. Plates were sealed in zip lock bags and incubated at 37°C for 3–4 days before visual inspection for growth.
For the determination of the sublethal rifampicin drug concentration by treatment curve, duplicate flasks of 75 ml liquid cultures in Tween-20 (0.1%, see subsection Growth Conditions and Harvesting) in log phase growth (A_600nm 1.2) were treated with rifampicin solubilised in DMSO to a final concentration of 2.5 μg/ml, or with DMSO only as a control. Cell density, and hence bacterial growth, was inferred from A_600nm readings measured every 1.5–2.5 h from 2 h prior to treatment until 7 h post-treatment and then again 52 h post-treatment using a Varian Cary 50 UV-visible spectrophotometer.

The average chlorophyll specific optical absorption cross section (a*) of the cells was determined according to Kromkamp and Limbeek [34 (link)] from in vivo absorption spectra (range 400–750 nm) recorded on a Varian Cary50 UV–visible spectrophotometer. To minimize the impact of the light scattering effect from the cells’ surface on the absorbance measurement, the sample in the cuvette was positioned right next to the detector window.
The photosynthetic electron transport rate of PSII (ETR, μmol e⁻ mg chl⁻¹ s⁻¹) was estimated from the following relationship: $$\text{ETR}=\mathrm{\Delta }F/F_{\text{m}}^{\prime }\times \text{PAR}\times a^{\ast }\times 0.5$$ assuming that the photosystem I (PSI) to PSII (PSI/PSII) ratio of Synechocystis PCC6803 is 1 according to Fujimori et al. [22 (link)]. Thus, 50 % of the photons are absorbed by PSII [35 (link)], and a* (m² mg chl⁻¹) was the average chlorophyll optical cross section normalized to chlorophyll a. This equation assumes that no cyclic electron transport by PSI occurs.
The quantum yield of CO₂ assimilation, Φ_CO2, was calculated from the product of ΦPSII × fraction PSII × (1/4) [36 (link)] where Φ_PSII = ΔF/F_m^′.