Hana Sanderova

• PostDoc Position at The Czech Academy of Sciences

Bacterial transcription regulation is critical for adaptation and survival. CarD is an essential transcription factor in mycobacteria involved in regulation of gene expression. We searched for CarD interaction partners in the model organism Mycobacterium smegmatis and identified two proteins: ApeB (MSMEG_5828) and an uncharacterized protein, which...

HelD protein, also named HelR (encoded by MSMEG_2174 in Mycobacterium smegmatis), interacts with mycobacterial RNA polymerase (RNAP) and affects rifampicin resistance in Mycobacterium abscessus. Here, we provide data on rifampicin resistance and helD presence in the genomes of other clinically relevant nontuberculous mycobacteria. We show that helD...

In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of...

Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis f...

During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric septation is linked to the membrane where FtsZ and SpoIIE initiate the formation of the Z-ring and the E-ring, respectively. These rings then serve as a scaffold for the other cell division...

σ $$ \sigma $$ factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative σ $$ \sigma $$ factors occupies the primary channel of RNAP and also prevents binding of the σ $$ \sigma $$ factor to promoter DNA alone. Here, we show that domain 1.1 of Ba...

Rifampicin is a clinically important antibiotic that binds to, and blocks the DNA/RNA channel of bacterial RNA polymerase (RNAP). Stalled, nonfunctional RNAPs can be removed from DNA by HelD proteins; this is important for maintenance of genome integrity. Recently, it was reported that HelD proteins from high G + C Actinobacteria, called HelR, are...

The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-L...

Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorpor...

Active wound dressings are attracting extensive attention in soft tissue repair and regeneration, including bacteria-infected skin wound healing. As the wide use of antibiotics leads to drug resistance we present here a new concept of wound dressings based on the polycaprolactone nanofiber scaffold (NANO) releasing second generation lipophosphonoxi...

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate th...

The exponential increase in the number of conducted studies combined with the development of sequencing methods have led to an enormous accumulation of partially processed experimental data in the past two decades. Here, we present an approach using literature-mined data complemented with gene expression kinetic modeling and promoter sequence analy...

The expression of rRNA is one of the most energetically demanding cellular processes and, as such, it must be stringently controlled. Here, we report that DNA topology, i.e., the level of DNA supercoiling, plays a role in the regulation of Bacillus subtilis σA-dependent rRNA promoters in a growth phase-dependent manner. The more negative DNA superc...

RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The c...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the c...

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA p...

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate th...

It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate t...

Successful surgeries involving orthopedic implants depend on the avoidance of biofilm development on the implant surface during the early postoperative period. Here, we investigate the potential of novel antibacterial compounds—second-generation lipophosphonoxins (LPPOs II)—as additives to surgical bone cements. We demonstrate (i) excellent thermos...

RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1...

Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs present significant experimental challenges. Here we investigate the co...

HelD is a helicase‐like protein binding to Bacillus subtilis RNA polymerase (RNAP), stimulating transcription in an ATP‐dependent manner. Here, our small angle X‐ray scattering data bring the first insights into the HelD structure: HelD is compact in shape and undergoes a conformational change upon substrate analog binding. Furthermore, the HelD do...

Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two so far unreported forms of Mycobacteium smegmatis RNAP: core, and holoenzyme containing σ A but no other factors. Each form was detected by cryo-EM in two major conform...

Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non‐rRNA transcript in stationary phase in Mycobac...

σ I from Bacillus subtilis is a σ factor associating with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here we provide a comprehensive characterization of this transcriptional regulator. By RNA-seq of wt and σ I -null strains at 37°C and 52°C we identified ∼130 genes affected by the absence...

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction in the major groove of DNA containing 5-ethynyluracil (UE ) with azides was used for turning off sequence-specific protein-DNA interactions. The concept was first demonstrated on switching off cleavage of short modified DNA by restriction endonuclease BamHI-HF. Finally, DNA templ...

DNA templates containing 5-hydroxymethyluracil or 5-hydroxymethylcytosine were used in an in vitro transcription with RNA polymerase from Escherichia coli. A strong enhancement of transcription was observed from DNA containing the...

The increase in the number of bacterial strains resistant to known antibiotics is alarming. In this study we report the synthesis of novel compounds termed Lipophosphonoxins II (LPPO II). We show that LPPO II display excellent activities against Gram positive and -negative bacteria, including pathogens and multiresistant strains. We describe their...

Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies RNAP's DNA-binding channel. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNA...

RNA polymerase (RNAP) is the central enzyme of transcription of the genetic information from DNA into RNA. RNAP recognizes four main substrates: ATP, CTP, GTP and UTP. Experimental evidence from the past several years suggests that, besides these four NTPs, other molecules can be used to initiate transcription: (i) ribooligonucleotides (nanoRNAs) a...

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2′-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription...

The advantages offered by established antibiotics in the treatment of infectious diseases are endangered due to the increase in the number of antibiotic-resistant bacterial strains. This leads to a need for new antibacterial compounds. Recently, we discovered a series of compounds termed lipophosphonoxins (LPPOs) that exhibit selective cytotoxicity...

Search for a potential protein binding partner(s) of LPPOs B. subtilis cell lysate was incubated either with empty streptavidin beads (EB) or with the beads couples with biotinylated LPPO (DR5690). The proteins associating with EB and LPPO were then resolved on SDS—PAGE and stained with Commassie Blue. A labeled molecular weight ladder (M) is shown...

CF release curve fitting. Time course of liposome lysis induced by DR5026 (A). Solid lines show the fit of the functions: α(1-exp(-t/τ))n to the data. Leakage of liposomes prepared from the bacterial phospholipids exhibited a biphasic behavior—in this data set the parameters for the fit of the initial phase (f(x)) were α = 29.7, τ = 175.5 s, n = 0....

Concentration-dependent cytoplasmic membrane permeabilization induced by DR5026. The increase in fluorescence intensity reflected intercalation of PI into DNA and dsRNA that occurred after PI entry into the cell upon membrane permeabilization. (PDF)

Synthesis of DR5557, biotinylated LPPO DR5690, and fluorescently labeled LPPO DR5832. (PDF)

MD simulations. (PDF)

The induction of resistance of Enterococcus faecalis and Streptococcus agalactiae to compounds DR5047 and DR5026. Minimum inhibitory concentrations of the tested LPPOs after each induction step. The induction of resistance was carried out by repeated passages of E. faecalis and S. agalactiae strains with subinhibitory concentrations of the relevant...

Structures of potential metabolites of DR5047. In all HPLC-MS analyses only intact DR5026 was detected. In the case of DR5047 (blue rectangle) less than 5% of the compound with the exact mass of 761.42 was detected. It corresponds to a monoacetylated compound—highlighted by the green rectangle—the exact position of the acetyl group has not been det...

LogD calculation. (PDF)

Antibacterial activity of LPPOs DR5557, DR5690, and DR5823. (PDF)

MTD test in mouse model—a detailed description. (PDF)

Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulate...

The partially disordered δ subunit of RNA polymerase was studied by various NMR techniques. The structure of the well-folded N-terminal domain was determined based on inter-proton distances in NOESY spectra. The obtained structural model was compared to the previously determined structure of a truncated construct (lacking the C-terminal domain). On...

RNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the δ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of δ from Bacillus subtilis. We demonstrate that δ affects the regula...

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β'), a sigma factor and a promoter-carrying DNA templ...

Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The...

Novel compounds termed lipophosphonoxins were prepared using a simple and efficient synthetic approach. The general structure of lipophosphonoxins consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a hydrophobic module (lipophilic alkyl chain), and (iv) a phosphonate linker module that holds together modules i-iii....

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of (13)C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and...

In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decr...

A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assig...

Bacterial elongation factor Tu (EF-Tu) is a model monomeric G protein composed of three covalently linked domains. Previously, we evaluated the contributions of individual domains to the thermostability of EF-Tu from the thermophilic bacterium Bacillus stearothermophilus. We showed that domain 1 (G-domain) sets up the basal level of thermostability...

Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and pol...

We identify here a pattern in the transcription start sites (+1A or +1G) of sigma(A)-dependent promoters of genes that are up-/downregulated in response to amino acid starvation (stringent response) in Bacillus subtilis. Upregulated promoters initiate mostly with ATP and downregulated promoters with GTP. These promoters appear to be sensitive to ch...

Among the structurally diverse nucleoside phosphonic acids, several compounds possessing strong antiviral properties have been found. Our effort in this area was focused to the synthesis of novel compounds - pyrrolidine-based nucleoside phosphonic acids and their derivatives.

The effect of noncatalytic domains 2+3 on the intrinsic activity and thermostability of the EF-Tu GTPase center was evaluated in experiments with isolated domains 1 and six chimeric variants of mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) EF-Tus. The isolated catalytic domains 1 of both EF-Tus displayed simila...

Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF-Tus, their isolated G-domains, and six chimeric EF-Tus composed of domains of either EF-Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized. BstEF-Tu and BstG-domain bound GDP and GTP with a...