Biology subjects

Malyshev, D.

Publications and source records attributed to Malyshev, D..

8 recordsLinked to original sources

bioRxiv2026

Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

biochemistry↗

bioRxiv2025

Endospore Appendages Enhance Bacillus cereus Spore Adhesion to Industrial Surfaces, Modulated by Physicochemical Factors

Spores of species belonging to the Bacillus cereus group (B. cereus spp.) are common contaminants in food processing environments due to their ability to adhere to surfaces and resist cleaning procedures. These spores are equipped with pilus-like endospore appendages (ENAs), which are believed to promote surface adhesion. We have investigated the role of ENAs in spore adhesion to abiotic surfaces using wild-type (WT) B. cereus spp. strains and isogenic mutants lacking ENAs or an intact exosporium. WT spores expressing both short and long ENAs (S+L+) adhered significantly more to stainless steel (SS) and polypropylene (PP) compared to bald spores (S-L-) and spores of an exosporium-deficient mutant ({Delta}exsY), while adhesion to polystyrene (PS) and glass was not significantly affected by the presence of ENAs. The {Delta}exsY mutant showed the lowest adhesion across all tested surfaces, a pattern also observed for vegetative cells. The individual roles of S-ENA and L-ENA were also assessed, where the strongest adhesion occurred when both fibers were present on PP. A trend also emerged on PP; WT remained adhered for an hour, while bald spores tended to detach within that time. For saline conditions and pH, bald spores adhered strongly to SS, while for non-ionic surfactant and concentrated protein solutions, WT spores exhibited the strongest adhesion. These results highlight the crucial role of ENAs in B. cereus spp. spore adhesion to industrial relevant surfaces, providing mechanistic insight into spore persistence. This understanding supports the design of surface treatments to prevent contamination, spoilage, and foodborne illness. ImportanceBacteria belonging to the Bacillus cereus group represent a persistent challenge in food production due to their highly resilient endospores (spores), which resist cleaning and disinfection as well as food processing. Understanding the adhesion properties of these spores is essential for designing effective surface treatments that reduce chemical use, enhance food safety and quality, and minimize environmental impact. This study underscores the important role of endospore appendages (ENAs) in spore adhesion to common materials in food processing and laboratory environments. Wild-type spores expressing both S-ENA and L-ENA adhered significantly more than mutants lacking ENAs or the exosporium, highlighting ENAs as potential targets for disrupting spore adhesion. Time-dependent adhesion tests on polypropylene revealed strong, sustained attachment by wild-type spores, contrasting with weaker, transient adhesion by ENA-depleted mutants. These findings offer valuable insights into B. cereus spore adhesion dynamics, guiding the development of tailored cleaning protocols to improve contamination control and sustainability.

microbiology↗

bioRxiv2024

The role of endospore appendages in spore-spore contacts in pathogenic bacilli

Species within the spore-forming Bacillus cereus sensu lato group are recognized for their role in food spoilage and food poisoning. B. cereus spores are decorated with numerous pilus-like appendages, called S-ENAs and L-ENAs. These appendages are believed to play crucial roles in self-aggregation, adhesion, and biofilm formation. By using both bulk and single-cell approaches, we investigate the role of S-and L-ENAs as well as the impact of different environmental factors in spore-to-spore contacts and in the interaction between spores and vegetative cells. Our findings reveal that ENAs, and particularly their tip fibrilla, play an essential role in spore self-aggregation but not in the adhesion of spores to vegetative cells. The absence of L-BclA, which builds the L-ENA tip fibrillum, reduced both S-and L-ENA mediated spore aggregation, emphasizing the interconnected roles of S-and L-ENAs. Increased salt concentrations in the liquid environment significantly reduced spore aggregation, implying a charge dependency of spore-spore interactions. By elucidating these complex interactions, our study provides valuable insights into spore dynamics. This knowledge can guide future studies on spore behavior in environmental settings and aids in developing strategies to manage bacterial aggregation for beneficial purposes, like controlling biofilms in food production equipment.

microbiology↗

bioRxiv2023

A hybrid CNN-Random Forest algorithm for bacterial spore segmentation and classification in TEM images

We present a new approach to segment and classify bacterial spore layers from Transmission Electron Microscopy (TEM) images using a hybrid Convolutional Neural Network (CNN) and Random Forest (RF) classifier algorithm. This approach utilizes deep learning, with the CNN extracting features from images, and the RF classifier using those features for classification. The proposed model achieved 73% accuracy, 64% precision, 46% sensitivity, and 47% F1-score with test data. Compared to other classifiers such as AdaBoost, XGBoost, and SVM, our proposed model demonstrates greater robustness and higher generalization ability for non-linear segmentation. Our model is also able to identify spores with a damaged core as verified using TEMs of chemically exposed spores. Therefore, the proposed method will be valuable for identifying and characterizing spore features in TEM images, reducing labor-intensive work as well as human bias.

microbiology↗

bioRxiv2022

Physico-chemical characterization of single bacteria and spores using optical tweezers

Spore-forming pathogenic bacteria are adapted for adhering to surfaces, and their endospores can tolerate strong chemicals making decontamination difficult. Understanding the physico-chemical properties of bacteria and spores is therefore essential in developing antiadhesive surfaces and disinfection techniques. However, measuring physico-chemical properties in bulk does not show the heterogeneity between cells. Characterizing bacteria on a single-cell level can provide mechanistic clues usually hidden in bulk measurements. This paper shows how optical tweezers can be applied to characterize single bacteria and spores, and how physico-chemical properties related to adhesion, fluid dynamics, biochemistry, and metabolic activity can be assessed.

biophysics↗

bioRxiv2022

Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores

Bacterial spores are highly resistant to heat, radiation, and various disinfection chemicals. These impacts on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15 % of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65 %. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.

biophysics↗

bioRxiv2020

Disinfection chemicals mode of action on the bacterial spore structure and their Raman spectra

Contamination of toxic spore-forming bacteria is problematic since spores can survive a plethora of disinfection chemicals. It is also problematic to rapidly detect if the disinfection chemical was active, leaving spores dead. Robust decontamination strategies, as well as reliable detection methods to identify dead from viable spores, are thus critical. Vibrational detection methods such as Raman spectroscopy has been suggested for rapid diagnostics and differentiation of live and dead spores. We investigate in this work, using laser tweezers Raman spectroscopy, the changes in Raman spectra of Bacillus thuringiensis spores treated with sporicidal agents such as chlorine dioxide, peracetic acid, and sodium hypochlorite. We also imaged treated spores using SEM and TEM to verify if any changes to the spore structure can be correlated to the Raman spectra. We found that chlorine dioxide did not change the Raman spectrum or the spore structure; peracetic acid shows a time-dependent decrease in the characteristic DNA/DPA peaks and [~]20 % of the spores were degraded and collapsed; spores treated with sodium hypochlorite show an abrupt drop in DNA and DPA peaks within 20 minutes all though the spore structure was overall intact, however, the exosporium layer was reduced. Structural changes appeared over several minutes, compared to the inactivation time of the spores, which is less than a minute. We conclude that vibrational spectroscopy provides powerful means to detect changes in spores but it might be problematic to identify if spores are live or dead after a decontamination procedure.

biophysics↗

bioRxiv2019

Electrically induced bacterial membrane potential dynamics correspond to cellular proliferation capacity

Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and inter- cellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cell proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely B. subtilis and E. coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5 sec electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+ and the deletion of the K+ channel suggested that the hyperpolarization response is caused by the K+ efflux through the channel. When cells are pre-exposed to UV-violet light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh-Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore or alcohol. Therefore, electrically induced membrane potential dynamics offer a novel and reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.

microbiology↗