A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors.

TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families.

A method for targeting a specified segment of DNA to a bacterial microorganelle.

Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.

Discovery of a fragment hit compound targeting D-Ala:D-Ala ligase of bacterial peptidoglycan biosynthesis.

Bacterial resistance is an increasing threat to healthcare systems, highlighting the need for discovering new antibacterial agents. An established technique, fragment-based drug discovery, was used to target a bacterial enzyme Ddl involved in the biosynthesis of peptidoglycan. We assembled general and focused fragment libraries that were screened in a biochemical inhibition assay. Screening revealed a new fragment-hit inhibitor of DdlB with a Ki value of 20.7 ± 4.5 µM. Binding to the enzyme was confirmed by an orthogonal biophysical method, surface plasmon resonance, making the hit a promising starting point for fragment development.

The Small DdrR Protein Directly Interacts with the UmuDAb Regulator of the Mutagenic DNA Damage Response in Acinetobacter baumannii.

Acinetobacter baumannii poses a great threat in health care settings worldwide, with clinical isolates displaying an ever-evolving multidrug resistance. In strains of A. baumannii, expression of multiple error-prone polymerase genes are corepressed by UmuDAb, a member of the LexA superfamily, and a small protein, DdrR. It is currently unknown how DdrR establishes this repression. Here, we used surface plasmon resonance spectrometry to show that DdrR formed a stable complex with the UmuDAb regulator. Our results indicated that the carboxy-terminal dimerization domain of UmuDAb formed the interaction interface with DdrR. Our in vitro data also showed that RecA-mediated inactivation of UmuDAb was inhibited when this transcription factor was bound to its target DNA. In addition, we showed that DdrR interacted with a putative prophage repressor, homologous to LexA superfamily proteins. These data suggested that DdrR modulated DNA damage response and prophage induction in A. baumannii by binding to LexA-like regulators. IMPORTANCE We previously identified a 50-residue bacteriophage protein, gp7, which interacts with and modulates the function of the LexA transcription factor from Bacillus thuringiensis. Here, we present data that indicates that the small DdrR protein from A. baumannii likely coordinates the SOS response and prophage processes by also interacting with LexA superfamily members. We suggest that similar small proteins that interact with LexA-like proteins to coordinate DNA repair and bacteriophage functions may be common to many bacteria that mount the SOS response.

Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue.

The GIL01 bacteriophage is a temperate phage that infects the insect pathogen Bacillus thuringiensis. During the lytic cycle, phage gene transcription is initiated from three promoters: P1 and P2, which control the expression of the early phage genes involved in genome replication and P3, which controls the expression of the late genes responsible for virion maturation and host lysis. Unlike most temperate phages, GIL01 lysogeny is not maintained by a dedicated phage repressor but rather by the host's regulator of the SOS response, LexA. Previously we showed that the lytic cycle was induced by DNA damage and that LexA, in conjunction with phage-encoded protein gp7, repressed P1. Here we examine the lytic/lysogenic switch in more detail and show that P3 is also repressed by a LexA-gp7 complex, binding to tandem LexA boxes within the promoter. We also demonstrate that expression from P3 is considerably delayed after DNA damage, requiring the phage-encoded DNA binding protein, gp6. Surprisingly, gp6 is homologous to LexA itself and, thus, is a rare example of a LexA homologue directly activating transcription. We propose that the interplay between these two LexA family members, with opposing functions, ensures the timely expression of GIL01 phage late genes.

Aegerolysins: Lipid-binding proteins with versatile functions.

Proteins of the aegerolysin family span many kingdoms of life. They are relatively widely distributed in bacteria and fungi, but also appear in plants, protozoa and insects. Despite being produced in abundance in cells at specific developmental stages and present in secretomes, only a few aegerolysins have been studied in detail. In particular, their organism-specific physiological roles are intriguing. Here, we review published findings to date on the distribution, molecular interactions and biological activities of this family of structurally and functionally versatile proteins, the aegerolysins.

Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage.

The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription.

Antibiotic induced bacterial lysis provides a reservoir of persisters.

In a genetically uniform bacterial population a small subset of antibiotic-susceptible cells enter an antibiotic tolerant state and are hence referred to as persisters. These have been proposed to be rare phenotypic variants with several stochastically activated independent parallel processes. Here we show an overlooked phenomenon, bacterial tolerance of extraordinary high levels of ampicillin due to encasement of viable cells by an antibiotic induced network of cell debris. This matrix shields the entrapped cells from contact with the bacteriolytic ß-lactam antibiotic ampicillin and may be an underlying cause of notable variations in the level of ampicillin tolerant persisters as well as of considerable medical significance. Disruption of the matrix leads to the rapid elimination of hidden survivors, revealing their metabolically active state.

Structural insight into LexA-RecA* interaction.

RecA protein is a hallmark for the bacterial response to insults inflicted on DNA. It catalyzes the strand exchange step of homologous recombination and stimulates self-inactivation of the LexA transcriptional repressor. Importantly, by these activities, RecA contributes to the antibiotic resistance of bacteria. An original way to decrease the acquisition of antibiotic resistance would be to block RecA association with LexA. To engineer inhibitors of LexA-RecA complex formation, we have mapped the interaction area between LexA and active RecA-ssDNA filament (RecA*) and generated a three-dimensional model of the complex. The model revealed that one subunit of the LexA dimer wedges into a deep helical groove of RecA*, forming multiple interaction sites along seven consecutive RecA protomers. Based on the model, we predicted that LexA in its DNA-binding conformation also forms a complex with RecA* and that the operator DNA sterically precludes interaction with RecA*, which guides the induction of SOS gene expression. Moreover, the model shows that besides the catalytic C-terminal domain of LexA, its N-terminal DNA-binding domain also interacts with RecA*. Because all the model-based predictions have been confirmed experimentally, the presented model offers a validated insight into the critical step of the bacterial DNA damage response.

Fungal MACPF-like proteins and aegerolysins: bi-component pore-forming proteins?

Proteins with membrane-attack complex/perforin (MACPF) domains are found in almost all kingdoms of life, and they have a variety of biological roles, including defence and attack, organism development, and cell adhesion and signalling. The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with another group of proteins with unknown biological function, known as aegerolysins. These two protein groups coincide in only a few species, and they might operate in concert as cytolytic bi-component pore-forming agents. Representative proteins here include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These have been shown to act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin content. The aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerised pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type. The biological significance of these proteins for the fungal life-style is discussed.

The LexA regulated genes of the Clostridium difficile.

BACKGROUND: The SOS response including two main proteins LexA and RecA, maintains the integrity of bacterial genomes after DNA damage due to metabolic or environmental assaults. Additionally, derepression of LexA-regulated genes can result in mutations, genetic exchange and expression of virulence factors. Here we describe the first comprehensive description of the in silico LexA regulon in Clostridium difficile, an important human pathogen. RESULTS: We grouped thirty C. difficile strains from different ribotypes and toxinotypes into three clusters according to lexA gene/protein variability. We applied in silico analysis coupled to surface plasmon resonance spectroscopy (SPR) and determined 16 LexA binding sites in C. difficile. Our data indicate that strains within the cluster, as defined by LexA variability, harbour several specific LexA regulon genes. In addition to core SOS genes: lexA, recA, ruvCA and uvrBA, we identified a LexA binding site on the pathogenicity locus (PaLoc) and in the putative promoter region of several genes involved in housekeeping, sporulation and antibiotic resistance. CONCLUSIONS: Results presented here suggest that in C. difficile LexA is not merely a regulator of the DNA damage response genes but also controls the expression of dozen genes involved in various other biological functions. Our in vitro results indicate that in C. difficile inactivation of LexA repressor depends on repressor`s dissociation from the operators. We report that the repressor`s dissociation rates from operators differentiate, thus the determined LexA-DNA dissociation constants imply on the timing of SOS gene expression in C. difficile.

Double locking of an Escherichia coli promoter by two repressors prevents premature colicin expression and cell lysis.

The synthesis of Eschericha coli colicins is lethal to the producing cell and is repressed during normal growth by the LexA transcription factor, which is the master repressor of the SOS system for repair of DNA damage. Following DNA damage, LexA is inactivated and SOS repair genes are induced immediately, but colicin production is delayed and induced only in terminally damaged cells. The cause of this delay is unknown. Here we identify the global transcription repressor, IscR, as being directly responsible for the delay in colicin K expression during the SOS response, and identify the DNA target for IscR at the colicin K operon promoter. Our results suggest that, IscR stabilizes LexA at the cka promoter after DNA damage thus, preventing its cleavage and inactivation, and this cooperation ensures that suicidal colicin K production is switched on only as a last resort. A similar mechanism operates at the regulatory region of other colicins and, hence, we suggest that many promoters that control the expression of 'lethal' genes are double locked.

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response.

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations.

Unique relationships between phages and endospore-forming hosts.

As part of their survival strategy under harsh environmental conditions, endospore-forming bacteria can trigger a sporulation developmental program. Although the regulatory cascades that precisely control the transformation of vegetative bacteria into mother cells and resilient spores have been described in detail, less is known about how bacteriophages that prey on endospore-formers exploit sporulation. Herein, we argue that phages infecting these bacteria have evolved several specific molecular mechanisms, not yet known in other bacteria, that manifest from the phage-driven alliance to negative effects on the host. We anticipate that the relationships between phages and endospore-formers outlined here will inspire studies on phage ecology and evolution, and could facilitate important advances in the development of phage therapies against pathogenic spore-formers.

Dissecting giant hailstones: A glimpse into the troposphere with its diverse bacterial communities and fibrous microplastics.

The formation of giant hailstones is a rare weather event that has devastating consequences in inhabited areas. This hazard has been occurring more frequently and with greater size of hailstones in recent years, and thus needs to be better understood. While the generally accepted mechanism is thought to be a process similar to the formation of smaller hailstones but with exceptional duration and stronger updrafts, recent evidence suggests that biotic and abiotic factors also influence the growth of these unusually large ice chunks. In this study, we improved these findings by determining the distribution of a wide variety of these factors throughout the hail volume and expanding the search to include new particles that are common in the environment and are of anthropogenic origin. We melted the concentric layers of several giant hailstones that fell to the ground over a small region in Slovenia in 2019. The samples, up to 13 cm in diameter, were analyzed for biotic and abiotic constituents that could have influenced their formation. Using 16S rRNA-based metagenomics approaches, we identified a highly diverse bacterial community, and by using scanning electron microscopy and Raman spectroscopy, we found natural and synthetic fibers concentrated in the cores of the giant hailstones. For the first time, we were able to detect the existence of microplastic fibers in giant hailstones and determine the changes in the distribution of sand within the volume of the samples. Our results suggest that changes in the composition of hail layers and their great diversity are important factors that should be considered in research. It also appears that anthropogenic microfiber pollutants were a significant factor in the formation of the giant hailstones analyzed in this study.

Inhibition of the SARS-CoV-2 3CLpro main protease by plant polyphenols.

The abundance of polyphenols in edible plants makes them an important component of human nutrition. Considering the ongoing COVID-19 pandemic, a number of studies have investigated polyphenols as bioactive constituents. We applied in-silico molecular docking as well as molecular dynamics supported by in-vitro assays to determine the inhibitory potential of various plant polyphenols against an important SARS-CoV-2 therapeutic target, the protease 3CLpro. Of the polyphenols in initial in-vitro screening, quercetin, ellagic acid, curcumin, epigallocatechin gallate and resveratrol showed IC50 values of 11.8 µM to 23.4 µM. In-silico molecular dynamics simulations indicated stable interactions with the 3CLpro active site over 100 ns production runs. Moreover, surface plasmon resonance spectroscopy was used to measure the binding of polyphenols to 3CLpro in real time. Therefore, we provide evidence for inhibition of SARS-CoV-2 3CLpro by natural plant polyphenols, and suggest further research into the development of these novel 3CLpro inhibitors or biochemical probes.

A small bacteriophage protein determines the hierarchy over co-residential jumbo phage in Bacillus thuringiensis serovar israelensis.

Bacillus thuringiensis serovar israelensis is the most widely used biopesticide against insects, including vectors of animal and human diseases. Among several extrachromosomal elements, this endospore-forming entomopathogen harbors two bacteriophages: a linear DNA replicon named GIL01 that does not integrate into the chromosome during lysogeny and a circular-jumbo prophage known as pBtic235. Here, we show that GIL01 hinders the induction of cohabiting prophage pBtic235. The GIL01-encoded small protein, gp7, which interacts with the host LexA repressor, is a global transcription regulator and represses the induction of pBtic235 after DNA damage to presumably allow GIL01 to multiply first. In a complex with host LexA in stressed cells, gp7 down-regulates the expression of more than 250 host and pBtic235 genes, many of which are involved in the cellular functions of genome maintenance, cell-wall transport, and membrane and protein stability. We show that gp7 homologs that are found exclusively in bacteriophages act in a similar fashion to enhance LexA's binding to DNA, while likely also affecting host gene expression. Our results provide evidence that GIL01 influences both its host and its co-resident bacteriophage.

DNA sampling: a method for probing protein binding at specific loci on bacterial chromosomes.

We describe a protocol, DNA sampling, for the rapid isolation of specific segments of DNA, together with bound proteins, from Escherichia coli K-12. The DNA to be sampled is generated as a discrete fragment within cells by the yeast I-SceI meganuclease, and is purified using FLAG-tagged LacI repressor and beads carrying anti-FLAG antibody. We illustrate the method by investigating the proteins bound to the colicin K gene regulatory region, either before or after induction of the colicin K gene promoter.

Surface plasmon resonance approach to study drug interactions with SARS-CoV-2 RNA-dependent RNA polymerase highlights treatment potential of suramin.

The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is essential for virus replication, therefore it is a promising drug target. Here we present a surface plasmon resonance approach to study the interaction of RdRp with drugs in real time. We monitored the effect of favipiravir, ribavirin, sofosbuvir triphosphate PSI-7409 and suramin on RdRp binding to RNA immobilized on the chip. Suramin precluded interaction of RdRp with RNA and even displaced RdRp from RNA.