Biology subjects

Manukhov, I. V.

Publications and source records attributed to Manukhov, I. V..

4 recordsLinked to original sources

bioRxiv2025

A new family of small ArdA proteins reveals an antirestriction activity.

Antirestriction proteins are known to protect mobile genetic elements from the hosts restriction-modification (RM) systems. In our study, we identified a new family of small proteins, which we named sArdA The sArdA proteins are homologous to DNA-mimicking ArdA proteins but differ in size, being approximately one-third the length of full ArdAs. Moreover, sArdA family contains two subgroups, one of which is structurally similar to the N-terminal end of ArdA, while the other one - to the C-terminal end. Phylogenetic analysis demonstrated that genes encoding these proteins evolved into evolutionarily stable subfamilies, named sArdN and sArdC, respectively. Alphafold structure prediction of sArdA interaction with RM systems revealed four states of EcoKI, which differ the angle between its two M-subunits while interacting with different agents. Interestingly, both sArdN and sArdC triggered the same intermediate closed state of EcoKI indicating the possible new interaction pathways of Ards with RM systems. For phenotypic studies in Escherichia coli cells, we cloned the sardN gene from the chromosome of Corynebacterium pilbarense and the sardC gene from Lactococcus cremoris. Both genes were shown to protect {lambda} phage DNA from restriction by the type I RM system. However, they revealed specificity to different restriction-modification systems. Specifically, sArdC was more effective against EcoR124II, whereas sArdN was more potent against EcoKI. Furthermore, both genes demonstrated high antimethylation activity against EcoKI. Our current findings suggest the idea that binding specificity of DNA-mimicking proteins to their targets could also be achieved by very short proteins.

molecular biology↗

bioRxiv2025

The capability of plant-bacteria consortia to reduce the genotoxicity of unsymmetrical dimethylhydrazine in the environment

Unsymmetrical dimethylhydrazine (UDMH) despite its proven high toxicity continues to be used in rocket technology and some other areas of human activity. In this work, the ability of plant-bacteria consortia to reduce the genotoxicity of UDMH incomplete oxidation products was investigated. Genotoxicity was assessed using a specific lux-biosensor Escherichia coli MG1655 pAlkA-lux sensitive to DNA alkylation in cell. For microbiological biodegradation, the Bacillus subtilis KK1112 strain was obtained by the isolation from soil with a subsequent selection for resistance to high UDMH concentrations (more than 5000 MAC). Its ability to biodegrade UDMH was shown by observing the reduction of DNA alkylation of the KK1112-treated UDMH. The ability of KK1112 cells to act in a bacterial-plant consortium with following fodder halo-phytes was studied: Bromus inermis Leyss, Medicago varia Mart. and Phleum pratense L. A synergistic reduction in the alkylating properties of UDMH oxidation products was observed under the combined use of bacteria and plant seedlings. The greatest effect was obtained when bacteria was used in combination with B. inermis. It was shown that KK1112 cells accelerate the seedlings development and mitigate the growth inhibition that occurs during incubation with UDMH. The obtained results indicate that it is optimal to introduce the bacterium KK1112 in combination with B. inermis plants for soils in arid climate zones during reclamation after UDMH exposure.

ecology↗

bioRxiv2025

Oxidative stress leads to Fur-mediated activation of ftnA in Escherichia coli independently of OxyR/SoxRS regulators

Ferritin FtnA is the main scavenger of Fe2+ and storage of Fe3+ in bacterial cells, together with Dps and Bfr preventing the Fenton reaction and thus protecting the cell from iron-induced oxidative stress. However, until now, it was not known how its expression is regulated under conditions of oxidative stress, and the available evidence was contradictory. To study the regulation of E. coli ftnA expression in response to oxidative stress, PftnA-luxCDABE transcriptional fusion in different strains was used. It has been shown that PftnA is induced after the addition of oxidative stress inducers. The maximum amplitude of this activation did not depend on the presence of functional genes oxyR and soxR in the cell, but completely disappeared 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 blocks the response. Exposure of a cell to H2O2 initially inactivates Fur and a number of iron-utilizing proteins, and derepresses iron uptake. This results in an increase in the cellular iron content with the consequent Fur reactivation, which leads to the induction of ftnA expression. Thus, oxidative stress leads to PftnA activation, which is mediated by Fur and time-delayed in comparison with OxyR-response.

molecular biology↗

bioRxiv2023

luxA gene from Enhygromyxa salina encodes a functional homodimeric luciferase

Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is catalyzed by the subunit LuxA, while LuxB is inactive. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product of the luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes both in vivo in Escherichia coli and in vitro. Overall, EsLuxA is less bright compared with luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 [A] reveals a classical monooxygenase fold, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a {beta}-hairpin or a coil in Vibrio harveyi LuxA, form -helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for discovery of new luciferases that have an advantage of being encoded by a single gene.

biochemistry↗