The involvement of the second pair of chlorophylls, termed A_-1A and A_-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A_-1B and A_-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700⁺–P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700⁺–P700) FTIR DS cannot be due to only the P_A and P_B pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A_-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A_-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A_-1A and A_-1B pigments are modified upon P700⁺ formation. Previously, we showed that the A_-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

Fourier-transform infrared (FTIR) spectroscopy can detect biomolecular changes in bacterial cells in response to drugs or other stimuli. Fully developing this area requires an understanding of IR spectral changes associated with the growth of unperturbed cells. Such an understanding is still lacking, however. To address this issue, attenuated total reflectance (ATR) FTIR spectroscopy has been used to probe changes in the composition of Staphylococcus aureus ATCC 6538 cells during exponential growth, with a 30 min time resolution. We find prominent spectral changes in proteins, nucleic acids, and carbohydrates evolving from the early (30–120 min) to the late (240–360 min) log phase of growth. Principal component analysis (PCA) shows that spectra obtained for cells during the early and late log phases of growth can be discriminated against with 100% accuracy. Protein-related spectral features are most significant in spectra collected at 30- and 90-min post-inoculation and provide a robust basis for temporal differentiation. Spectral changes that occur during the first 30 min after inoculation are shown to reverse over the next 30–120 min, indicating dynamic adaptations during cellular growth. Overall, we demonstrate a band assignment strategy based on time resolution, underscoring the utility of FTIR spectroscopy in dynamic studies of bacterial cells.

Fourier transform infrared (FTIR) spectroscopy can detect biomolecular changes in bacterial cells in response to drugs or other stimuli. To fully develop that area requires an understanding of IR spectral changes associated with the growth of unperturbed cells. Such an understanding is still lacking, however. To address this issue, attenuated total reflectance (ATR) FTIR spectroscopy has been used to probe changes in the composition of Staphylococcus aureus ATCC 6538 cells during exponential growth, with 30-minute time resolution. We find prominent spectral changes associated with proteins, nucleic acids and carbohydrates on evolving from the early (30-120 min) to the late (240-360 min) log phase of growth. Principal component analysis (PCA) shows that spectra obtained for cells during the early and late log phases of growth can be discriminated against with 100% accuracy. Protein-related spectral features are most significant in spectra collected at 30- and 90-minutes post-inoculation and provide a robust basis for temporal differentiation. Spectral changes that occur during the first 30 minutes after inoculation are shown to reverse over the next 30-120 minutes, indicating dynamic adaptations during cellular growth. Overall, we demonstrate a band assignment strategy based on time resolution, underscoring the utility of FTIR spectroscopy in dynamic studies of bacterial cells.

Global analysis of time-resolved spectra fits shared kinetic parameters across all spectral channels, but nonlinear fits can depend on starting values and stopping settings. We describe UFiτ (UFit), a reproducible two-stage protocol for time-resolved Fourier transform infrared (FTIR) difference spectra. Stage 1 fits the M channels with the largest peak-to-peak change to estimate shared lifetimes and stretching exponents; these estimates initialize Stage 2, a variable-projection fit of the full matrix. Using 6940 independently generated matrices with known parameters, we compared this top-M initialization with a direct fit from a fixed generic start, a singular value decomposition (SVD)-assisted prefit, uniform and random channel subsets, and best-of-ten multistart. Starting-value errors differed by up to about twentyfold between methods, yet final recovery was similar in most conditions. In a predeclared close-lifetime test, top-M and SVD initialization recovered all components in 118 and 113 of 200 matrices (p = 0.27); in exploratory comparisons, top-M recovered fewer matrices in two of twelve conditions, and ranking can omit a weak late component. Endpoint agreement depended mainly on stopping settings: with scaled residuals and tighter tolerances, direct, continuation, SVD-assisted and top-M fits of one measured photosystem I recording met a common numerical endpoint screen; uniformly spaced and random-subset prefits ended at a slightly lower objective with a different, undetermined slow component. We provide the protocol, a component-resolution checklist, and benchmark code and results.