Michaela Sikova

• Complex of Biomedical Institutes at Krč

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...

Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in...

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...

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...

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...

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)

Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with th...