Vadim Valerevich Fomin

The bioluminescence of entomopathogenic bacteria of the genus Photorhabdus was considered to be constitutive. However, it has recently been shown that under heat shock conditions, lux-operon is activated in Photorhabdus temperata cells. In the present study, bioluminescence thermoactivation was investigated in two isolated strains, Photorhabdus hainanensis FV2401 and P. temperata FV2402. The luminescence of the more thermotolerant P. hainanensis cells was activated at higher temperatures than that of P. temperata. At the maximum induction temperatures of 37 °C for FV2401 and 34 °C for FV2402, higher bioluminescence activation was observed in FV2401, whereas the increase in lux-genes mRNA levels was approximately the same in both strains. In P. hainanensis, elevated temperatures also affected the expression of the hexA, madB, and csrB genes, which are indirectly involved in luminescence in Photorhabdus. The heat-activated lux-operon promoter in P. hainanensis and P. temperata was determined. This is a σ⁷⁰-dependent promoter that differs in its distance from the luxC start codon. When promoter variants with or without the 5′-UTR mRNA were transferred to the heterologous Escherichia coli system, heat activation of luminescence was retained in constructs containing only the promoter and was independent of σ³² and σ^E, whereas constructs with the 5′-UTR exhibited repression of luminescence following thermoactivation. The obtained data on the temperature-induced promoter of the lux-operon can broaden understanding of the ecological and biological functions of Photorhabdus bioluminescence under heat shock conditions and provide a basis for the development of novel biosensors.

The fluorescent dyes 9-aminoacridine (9-AA) and acridine orange (AO) are known mutagens that induce frameshift mutations in cells by intercalating between DNA bases. However, these chemicals can also affect other cellular components, such as proteins. In this study, we tested the ability of 9-AA and AO to induce heat shock in bacteria using the following methods: lux-biosensors based on Escherichia coli cells with the luxCDABE genes transcriptionally fused to heat shock-specific inducible promoters, RT-qPCR, and nanoDSF. We demonstrated that acridine dyes not only induce mutagenesis but also cause heat shock in bacterial cells. AO significantly reduced the melting temperature of proteins and strongly activated σ^E- and σ³²-dependent promoters, but not PluxC, which is activated by elevated temperatures via a different mechanism. In contrast, 9-AA weakly denatured the proteins and induced the σ^E-dependent promoter; however, it activated the σ³²-dependent promoters and PluxC, supporting the hypothesis that the σ³² heat shock response system is activated via hairpin RNA denaturation by 9-AA. The study on the application of lux-biosensors was hampered by the high general toxicity and luminescence shielding effect of AO, and RT-qPCR’s sensitivity was insufficient for detection of the response to 9-AA. Thus, methodologically, it is justified to conduct a comprehensive study of substances that cause heat shock or affect bioluminescence by both RT-qPCR and lux-biosensors.

Ferritin FtnA is the main scavenger of Fe²⁺ and storage of Fe³⁺ in bacterial cells, together with Dps and Bfr it prevents the Fenton reaction. To study the regulation of Escherichia coli ftnA expression under oxidative stress conditions, we used P_ftnA‐luxCDABE transcriptional fusion. It was shown that P_ftnA is induced after the addition of oxidative stress inducers. This activation was independent of the presence of functional oxyR and soxR genes in the cell, but was completely abolished in the absence of fur. The response is amplified in the ftnA mutant and is diminished in the FtnA‐overproducing strain, which indicates that iron sequestration by apoferritin blocks the response and helps to cope with stress consequences. Comparison of the activation kinetics of the P_fecA and P_ftnA promoters, responsible for iron uptake and storage regulation, showed that the addition of H₂O₂ initially leads to the inactivation of Fur, causing derepression of iron uptake and, as a consequence, an increase in intracellular iron. As the redox balance in the cell is restored, Fur is reactivated, which leads to the induction of ftnA expression. Thus, oxidative stress leads to P_ftnA activation, which is mediated by Fur and time‐delayed in comparison with OxyR‐response.

The ability of aquatic mesofauna representatives involved in trophic chains to sorb and accumulate toxicants is important for understanding the functioning of aquatic ecosystems and for fishing industry. This study investigated the capacity of marine amphipod Gammarus oceanicus and freshwater amphipods Eulimnogammarus vittatus and Gammarus lacustris to absorb the DNA-alkylating agent methyl methanesulfonate (MMS). The presence of alkylating agents in the environment and in the tissues of the amphipods was determined using whole-cell lux-biosensor Escherichia coli MG1655 pAlkA-lux, in which the luxCDABE genes from Photorhabdus luminescens, enabling the luminescence of the cell culture, are controlled by the P_alkA promoter of DNA glycosylase. It was shown that within one day of incubation in water containing MMS at a concentration above 10 μM, the amphipods absorbed the toxicant and their tissues produce more alkylation damage to biosensor cells than the surrounding water. Concentrations of MMS above 1 mM in the environment caused the death of the amphipods before the toxicant could be significantly concentrated in their tissues. The sensitivity and the capacity to absorb MMS were found to be approximately the same for the marine amphipod G. oceanicus and the freshwater amphipods E. vittatus and G. lacustris.

The L. fermentum U-21 strain, known for secreting chaperones into the extracellular milieu, emerges as a promising candidate for the development of novel therapeutics termed disaggregases for Parkinson’s disease. Our study focuses on characterizing the secreted protein encoded by the C0965_000195 locus in the genome of this strain. Through sequence analysis and structural predictions, the protein encoded by C0965_000195 is identified as ClpL, homologs of which are known for their chaperone functions. The chaperone activity of ClpL from L. fermentum U-21 is investigated in vivo by assessing the refolding of luciferases with varying thermostabilities from Aliivibrio fischeri and Photorhabdus luminescens within Escherichia coli cells. The results indicate that the clpL gene from L. fermentum U-21 can compensate for the absence of the clpB gene, enhancing the refolding capacity of thermodenatured proteins in clpB-deficient cells. In vitro experiments demonstrate that both spent culture medium containing proteins secreted by L. fermentum U-21 cells, including ClpL, and purified heterologically expressed ClpL partially prevent the thermodenaturation of luciferases. The findings suggest that the ClpL protein from L. fermentum U-21, exhibiting disaggregase properties against aggregating proteins, may represent a key component contributing to the pharmabiotic attributes of this strain.

Strain L. fermentum U-21 secretes chaperones into the medium and it is considered as the candidate for the development of drugs, so-called disaggregases, for Parkinson’s disease therapy. This study is devoted to characterization of secreted protein encoded by C0965_000195 locus. Analysis of sequence and predicted structure of the protein encoded by C0965_000195 allows us to attribute it to the ClpL, homologs of which are known to be chaperones. The chaperone activity of ClpL L. fermentum U-21 was evaluated in vivo by refolding of differing in thermostability luciferases from Aliivibrio fischeri and Photorhabdus luminescens in Escherichia coli cells. It was observed that clpL from L. fermentum U-21 can compensate for the deficiency in the clpB gene and enhance the ability to refold thermodenatured proteins in clpB- cells. In vitro experiments have demonstrated the ability of a spent culture medium containing proteins secreted by L. fermentum U-21 cells, including ClpL, to prevent thermodenaturation of luciferases partially.We suggest that the ClpL protein from L. fermentum U-21 strain, which exhibits disaggregase properties against aggregating proteins, may be one of the clue components that play a role in the pharmabiotic properties of this strain.

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescensluxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxRA. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30–100 pM).