Anna A. Kudryavtseva

Antirestriction proteins protect mobile genetic elements from the host’s restriction-modification systems. Here, we investigated the ability of ArdA and ArdB antirestriction proteins to regulate gene expression in an engineered E. coli K-12 MG1655-based biosensor strain. This biosensor strain harbors a lux-based reporter system controlled by the AllR-repressed promoter. Although structurally similar, DNA-mimic ArdA proteins interact with AllR differently. Recently described small sArdC and well-known ArdA from the conjugative plasmid R64 appear to bind AllR and open the promoter, while the other tested antirestriction proteins (small sArdN protein and various full-sized ArdA proteins from different sources) have no effect on gene expression under AllR-controlled promoter. Direct binding between ArdA and AllR was experimentally confirmed using pull-down assays with His-tagged ArdA. Our study opens up prospects for the specific use of antirestriction proteins for the regulation of gene expression. Surprisingly, ArdB, a non-DNA-mimic antirestriction protein used initially as a control, was also able to open the promoter, apparently through nonspecific interaction with DNA. We verified this effect with a distant ArdB homolog from a rhizobacterium, which was also able to open the promoter.

Antirestriction proteins protect mobile genetic elements from the host’s 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, the sArdA family contains two subgroups, one of which is structurally similar to the N-terminal end of ArdA, whereas the other one matches the C-terminal end. Both the N-terminal and C-terminal domains of ArdA appear capable of independent expression. 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 in the angle between its two M-subunits while interacting with different ArdAs or DNA. Interestingly, both sArdN and sArdC triggered the same intermediate closed state of EcoKI, indicating 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 protected λ phage DNA from restriction by the type I RM system. However, they revealed specificities to different restriction-modification systems. Specifically, sArdC was more effective against EcoR124II, whereas sArdN was more potent against EcoKI. Furthermore, both genes demonstrated antimethylation activity against EcoKI. Our current findings suggest the idea that the binding specificity of DNA-mimicking proteins to their targets could also be achieved by very short proteins. IMPORTANCE: Our current findings suggest that the binding specificity of DNA-mimicking proteins to their targets could also be achieved by very short proteins. The ability of these DNA-mimicking proteins to specifically inhibit different DNA-binding proteins makes them a promising tool for regulating a range of intracellular processes, including gene expression.

Type I restriction-modification (RMI) systems play a crucial role in bacterial defense against mobile elements by distinguishing self and foreign DNA through sequence-specific methylation and cleavage. Here, we characterize BlihIA, a novel RMI system from Bacillus licheniformis DSM13 which features redundancy in its hsdS gene copies. Using ONT sequencing, we identify the bipartite recognition site of BlihIA as RTAC(N)₅GCT. We demonstrate the system’s activity both in vivo through efficiency of plaquing (EOP) assay and in vitro in a nuclease reaction with purified BlihIA complex. Notably, mutation of the recognition site abolished in vitro DNA cleavage, confirming sequence specificity. Furthermore, we show that the antirestriction protein ArdB from plasmid R64 effectively prevents DNA cleavage by BlihIA, suggesting a direct mechanism of inhibition. This study provides the first functional characterization of a novel RM system BlihIA, extending the diversity of RM systems in Bacillus species and suggesting potential applications for improving genetic transformation in industrial strains.

Antirestriction proteins are known to protect mobile genetic elements from the host’s 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 λ 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.

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.

ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA-mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimic might competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribed mechanism of action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site.

ArdB proteins are known to inhibit the activity of the type I restriction-modification (RM-I) system, in particular EcoKI (IA family). The mechanism of ArdB’s activity still remains unknown; the spectrum of targets inhibited has been poorly studied. In this work, it was shown that the presence of the ardB gene from the R64 plasmid could suppress the activity of EcoAI endonuclease (IB family) in Escherichia coli TG1 cells. Due to the absence of specificity of ArdB to a certain RM-I system (it inhibits both the IA- and IB-family), it can be assumed that the mechanism of the anti-restriction activity of this protein does not depend on the sequence DNA at the recognition site nor the structure of the restriction enzyme of the RM-I systems.

Anti-Restriction Activity of ArdB Protein against EcoAI Endonuclease Активность антирестрикционного белка ArdB в отношении эндонуклеазы EcoAI

Dicyclopropanated 5-vinyl-2-norbornene (dcpVNB) is a strained polycyclic hydrocarbon compound with a high energy content, which makes it promising for the development of propellant components based on it. In this work, the genotoxic properties of dcpVNB were studied using whole-cell lux-biosensors based on Escherichia coli and Bacillus subtilis. It was shown that the addition of dcpVNB to bacterial cells leads to the appearance of DNA damage inducing the SOS response and Dps expression with slight activation of the OxyR-mediated response to oxidative stress. The highest toxic effect of dcpVNB is detected by the following lux-biosensors: E. coli pColD-lux, E. coli pDps, B. subtilis pNK-DinC, and B. subtilis pNK-MrgA, in which the genes of bacterial luciferases are transcriptionally fused to the corresponding promoters: P_cda, P_dps, P_dinC, and P_mrgA. It was shown that lux-biosensors based on B. subtilis, and E. coli are almost equally sensitive to dcpVNB, which indicates the same permeability to this compound of cell wall of Gram-positive and Gram-negative bacteria. The activation of P_dps after dcpVNB addition maintains even in oxyR mutant E. coli strains, which means that the P_dps induction is only partially determined by the OxyR/S regulon. Comparison of specific stress effects caused by dcpVNB and 2-ethyl(bicyclo[2.2.1]heptane) (EBH), characterized by the absence of cyclopropanated groups, shows that structural changes in hydrocarbons could significantly change the mode of toxicity.