Niilo Kaldalu | University of Tartu (original) (raw)

Papers by Niilo Kaldalu

Many bacterial pathogens can permanently colonize their host and establish either chronic or recu... more Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube. SUMMARY Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which surviv...

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bac... more Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to oflo...

The mqsRA locus of Escherichia coli K-12 codes for a translation-independent GCU site-specific en... more The mqsRA locus of Escherichia coli K-12 codes for a translation-independent GCU site-specific endoribonuclease MqsR and an antitoxin MqsA, which has an additional function as a transcriptional regulator of other genes. Besides binding to the regulatory region of its own promoter, the antitoxin MqsA binds to the promoter regions of cspD, rpoS, spy, and mcbR. By repressing these target genes, MqsRA participates in regulation of the general stress response and biofilm formation. Structural analyses have shown that MqsR belongs to the RelE superfamily and MqsR is thus the first known ribosome-independent mRNase belonging to this toxin family.

The majority of cells transferred from stationary-phase culture into fresh medium resume growth q... more The majority of cells transferred from stationary-phase culture into fresh medium resume growth quickly, while a few remain in a nongrowing state for longer. These temporarily nonproliferating bacteria are tolerant of several bactericidal antibiotics and constitute a main source of persisters. Several genes have been shown to influence the frequency of persisters in Escherichia coli, although the exact mechanism underlying persister formation is unknown. This study demonstrates that the frequency of persisters is highly dependent on the age of the inoculum and the medium in which it has been grown. The hipA7 mutant had 1,000 times more persisters than the wild type when inocula were sampled from younger stationary-phase cultures. When started after a long stationary phase, the two displayed equal and elevated persister frequencies. The lower persister frequencies of glpD, dnaJ, and surA knockout strains were increased to the level of the wild type when inocula aged. The mqsR and pho...

Microbiology and Molecular Biology Reviews

Many bacterial pathogens can permanently colonize their host and establish either chronic or recu... more Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube.

Smart Materials and Structures

Smart and soft electroactive polymer actuators have many beneficial properties, making them attra... more Smart and soft electroactive polymer actuators have many beneficial properties, making them attractive for biomimetic and biomedical applications. However, the selection of components to fabricate biofriendly composites has been limited. Although biofriendly options for electrodes and membranes are available, the conventional ionic liquids (ILs) often used as the electrolytes in the actuators have been considered toxic in varying degrees. Here we present a smart electroactive composite with carefully designed and selected components that have shown low toxicity and a biofriendly nature. In the present study, polypyrrole-PVdF trilayer actuators using six different choline ILs were prepared and characterized. Choline ILs have shown promise in applications where low environmental and biological impact is critical. Despite this, the anions in ILs have a strong impact on toxicity. To evaluate how the anions effect the bioactivity of the ILs used to prepare the actuators, the ILs were tested on different microbial cultures (Escherichia coli, Staphylococcus aureus, Shewanella oneidensis MR-1) and HeLa cells. All of the selected choline ILs showed minimal toxic effects even at high concentrations. Electro-chemomechanical characterization of the actuators indicated that polypyrrole-PVdF actuators with choline ILs are viable candidates for soft robotic applications. From the tested ILs, choline acetate showed the highest strain difference and outperformed the reference system containing an imidazolium-based IL.

Science Signaling

Bacterial persisters survive antibiotic treatment and can cause chronic infections. In this issue... more Bacterial persisters survive antibiotic treatment and can cause chronic infections. In this issue of Science Signaling, Pontes and Groisman suggest that there is no specific molecular pathway responsible for persister formation in Salmonella and that slow growth is the decisive factor.

mBio

I n a recent paper, Nigam and colleagues analyzed the stress-related effects of the endoribonucle... more I n a recent paper, Nigam and colleagues analyzed the stress-related effects of the endoribonuclease toxin MazF on the Escherichia coli proteome (1). The authors from the lab of Hanna Engelberg-Kulka-the discoverer of the mazEF toxin-antitoxin system (2)-claim that MazF creates a unique stress-induced translation machinery (STM). The STM hypothesis states that the toxin cleaves selected mRNAs within 5=-leader sequences to produce a pool of leaderless transcripts that are, in turn, translated by special stress ribosomes (3, 4). The latter are formed when the toxin cleaves off an anti-Shine-Dalgarno sequence-containing fragment from the 3= end of 16S rRNA in mature ribosomes (3). Thus, MazF is postulated to reshape translation in stressed E. coli similarly to how the S factor reshapes transcription. Independent studies failed to support these findings. Transcriptome-wide mapping of the cleavage sites indicated that MazF cleaves most transcripts within their coding regions and produces very few full-length, leaderless mRNAs (5, 6). Contradicting the STM model, MazF does not cleave rRNA in mature, fully assembled ribosomes but instead targets rRNA precursors (5, 7). Finally, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics revealed that MazF generally inhibits protein synthesis and no proteins are selectively synthesized in response to the toxin (6). This result is at odds with the paper of Nigam and coworkers (1), who also used SILAC proteomics and report a group of 42 MazF-mediated, stress-induced E. coli proteins. Here we reanalyze their data and highlight several technical issues. The setup of the proteomics experiment and the lack of statistical analysis make it impossible to determine whether the reported differences in proteomes were caused by MazF or random fluctuations. The authors aimed to test which proteins are synthesized in the ΔmazEF mutant and its wild-type (wt) parent strain upon treatment with the quinolone antibiotic nalidixic acid (NA). To do that, Nigam et al. (1) grew bacteria in the light medium, added NA to the culture, and after 10 min, added heavy lysine and arginine in order to label the new proteins. The relative amounts of newly synthetized proteins were estimated based on heavy and light isotope ratio (H/L ratio) after an additional 5-min incubation. The experiment was repeated three times. The short length of pulse labeling resulted in low H/L values, which could, possibly, account for the high variability of results (see below). While the authors state that they "checked several time points and deduced that 5 min is the optimal time point to figure out which are the differential new proteins," they do not present the relevant supporting data. The authors state that the differences between the wt and ΔmazEF proteomes are specifically induced by stress but do not provide an essential control, i.e., proteomic analysis of these strains without NA treatment. Nigam and colleagues admit the lack of statistical analysis and, instead, chose all the proteins "that were induced more in the WT than in the mazEF mutants in all the repeats" as differentially expressed. They Citation Kaldalu N, Maiväli Ü, Hauryliuk V, Tenson T. 2019. Reanalysis of proteomics results fails to detect MazF-mediated stress proteins. mBio 10:e00949-19.

mBio

W e thank David W. Holden and Jeff Errington for their comments (1). We agree that scientific res... more W e thank David W. Holden and Jeff Errington for their comments (1). We agree that scientific research is inherently error-prone. Therefore, validation, reassessment, and reinterpretation of one's own and others' results is part of the scientific approach, regardless of whether the conclusions are affirming or critical. We also agree that further work is needed to establish whether, and in which settings, each individual toxin-antitoxin (TA) system contributes to persister formation in Escherichia coli K-12 and/or in other bacterial species. In the following paragraphs, we express our opinion on the topics on which Holden and Errington saw some overstatements and factual inaccuracies in our paper (2). The reason we tested the persistence phenotype of the newly constructed Δ10TA strain of E. coli K-12 at mid-exponential growth phase is because the original study by Maisonneuve et al. proposing the link between the TA systems on persister formation was performed under these conditions (3). We cannot therefore draw conclusions about the role of TA systems in E. coli K-12 under other experimental conditions. However, the key outcome of our study and the paper published by Harms and colleagues (4) is a call for setting up all necessary controls, cautiously double-checking new results, and critically reevaluating previously published findings that link TA systems and persister formation. Holden and Errington use several publications as evidence for TA's role in persistence. For example, an influential and much-cited study by Harrison et al. reported that deletion of the yafQ gene, encoding 1 of the 10 toxin genes deleted in the Δ10TA strain, caused a drop in persister levels surviving cefazolin (a cephalosporin) and tobramycin (an aminoglycoside antibiotic) in E. coli grown as a biofilm (5). To the best of our knowledge, no follow-up or independent study confirming this result has been published. Therefore, we draw attention to several crucial control experiments lacking in the original paper. First, given that the YafQ-inhibiting antitoxin DinJ itself has been reported to affect the general stress response by decreasing RpoS levels (6), it is necessary to assess whether deletion of the full dinJ-yafQ operon has the same effect as deletion of the sole yafQ gene. Additionally, deletion of yafQ had no effect on persistence to doxycycline or rifampin (5), indicating that YafQ does not play a general role in persistence. Second, restoration of the original persister level by complementation of the dinJ-yafQ knockout from a plasmid or reinsertion of the operon into the chromosome is essential for drawing reliable conclusions from the phenotypes observed with the knockout strain. We have previously encouraged complementation for validation of the gene knockout effects that change persister levels (7). Third, it

mBio, Jan 12, 2018

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bac... more Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicill...

RNA Biology, 2016

The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and pr... more The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNAsequencing to map the toxin-cleaved 5 0-and 3 0-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF-or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

Scientific Reports, 2016

2-Bromo-5-(2-bromo-2-nitrovinyl)furan (G1 or Furvina) is an antimicrobial with a direct reactivit... more 2-Bromo-5-(2-bromo-2-nitrovinyl)furan (G1 or Furvina) is an antimicrobial with a direct reactivity against thiol groups. It is active against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi. By reacting with thiol groups it causes direct damage to proteins but, as a result, is very short-living and interconverts into an array of reaction products. Our aim was to characterize thiol reactivity of G1 and its conversion products and establish how much of antimicrobial and cytotoxic effects are due to the primary activity of G1 and how much can be attributed to its reaction products. Stability of G1 in growth media as well as its conversion in the presence of thiols was characterized. The structures of G1 decomposition products were determined using NMR and mass-spectroscopy. Concentration-and time-dependent killing curves showed that G1 is bacteriostatic for Escherichia coli at the concentration of 16 μg/ml and bactericidal at 32 μg/ml. However, G1 is inefficient against nongrowing E. coli. Addition of cysteine to medium reduces the antimicrobial potency of G1. Nevertheless, the reaction products of G1 and cysteine enabled prolonged antimicrobial action of the drug. Therefore, the activity of 2-bromo-5-(2-bromo-2-nitrovinyl)furan is a sum of its immediate reactivity and the antibacterial effects of the conversion products. 2-Bromo-5-(2-bromo-2-nitrovinyl)furan (G1 or Furvina) belongs to the group of 2-vinylfuran-based antimicrobials 1. It is an efficient antimicrobial with broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi 2-4. G1 is currently in medical use in Cuba. It is marketed as Dermofural ointment for treatment of human skin and nail infections and as Furvinol for veterinary use. In Europe and USA, G1 is not in clinical use but patents for its synthesis and application as an anti-infective have been granted internationally 5-7. The exact details of action of G1 are a matter of debate. Just like other vinylfurans (2-furylethylenes), it is a thiolreactive compound and reacts with functional thiol groups in living cells, including those of vital enzymes 4,8-10. The reaction center of vinylfurans in these addition reactions is the electrophilic exocyclic double bond 9,11. For example, G1 has been shown to react with the cysteine residues of MurA, a protein that is crucial to peptidoglycan synthesis and is the target of phosphomycin 4. Modification of cysteines is irreversible, supposedly rather nonselective and bactericidal, if it is extensive 4. Due to the thiol reactivity, G1 must be short-lived both in a patient and during the standard tests of antimicrobial activity because such tests are carried out in cysteine-containing complex media. Besides adduct formation, G1 induces oxidation of thiol groups and formation of non-native disulfide bonds in a redox reaction. That leads to formation of a thiol-reactive de-bromo derivative of G1, which possesses antibacterial activity acting by the same mechanism as G1 4. Furthermore, G1 is unstable in aqueous media even in the lack of thiols 4. It breaks up into 5-bromo-2-furaldehyde, which does not have any antimicrobial activity, and bromonitromethane, that has been patented as a biocide while its target and the mechanism of action are unknown 4,12,13. Volatility of G1 evokes a question, how much of the reported antibacterial and cytotoxic effects are attributed to the compound itself and how much are due to its reaction-and breakdown products. Standard measurements of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and cytotoxicity do

Applied microbiology and biotechnology, Jan 4, 2016

Persisters-a drug-tolerant sub-population in an isogenic bacterial culture-have been featured thr... more Persisters-a drug-tolerant sub-population in an isogenic bacterial culture-have been featured throughout the last decade due to their important role in recurrent bacterial infections. Numerous investigations detail the mechanisms responsible for the formation of persisters and suggest exciting strategies for their eradication. In this review, we argue that the very term "persistence" is currently used to describe a large and heterogeneous set of physiological phenomena that are functions of bacterial species, strains, growth conditions, and antibiotics used in the experiments. We caution against the oversimplification of the mechanisms of persistence and urge for a more rigorous validation of the applicability of these mechanisms in each case.

Methods in Molecular Biology, 2016

Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bacte... more Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bactericidal antibiotics. These cells are suggested to be involved in the establishment of chronic infections. Persister levels depend on growth conditions. Here, we discuss the parameters that have to be considered when measuring persister levels and provide a sample protocol to do it.

Biofilms, Infection, and Antimicrobial Therapy, 2005

BioTechniques, 2000

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epit... more We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprintin...

Many bacterial pathogens can permanently colonize their host and establish either chronic or recu... more Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube. SUMMARY Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which surviv...

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bac... more Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to oflo...

The mqsRA locus of Escherichia coli K-12 codes for a translation-independent GCU site-specific en... more The mqsRA locus of Escherichia coli K-12 codes for a translation-independent GCU site-specific endoribonuclease MqsR and an antitoxin MqsA, which has an additional function as a transcriptional regulator of other genes. Besides binding to the regulatory region of its own promoter, the antitoxin MqsA binds to the promoter regions of cspD, rpoS, spy, and mcbR. By repressing these target genes, MqsRA participates in regulation of the general stress response and biofilm formation. Structural analyses have shown that MqsR belongs to the RelE superfamily and MqsR is thus the first known ribosome-independent mRNase belonging to this toxin family.

The majority of cells transferred from stationary-phase culture into fresh medium resume growth q... more The majority of cells transferred from stationary-phase culture into fresh medium resume growth quickly, while a few remain in a nongrowing state for longer. These temporarily nonproliferating bacteria are tolerant of several bactericidal antibiotics and constitute a main source of persisters. Several genes have been shown to influence the frequency of persisters in Escherichia coli, although the exact mechanism underlying persister formation is unknown. This study demonstrates that the frequency of persisters is highly dependent on the age of the inoculum and the medium in which it has been grown. The hipA7 mutant had 1,000 times more persisters than the wild type when inocula were sampled from younger stationary-phase cultures. When started after a long stationary phase, the two displayed equal and elevated persister frequencies. The lower persister frequencies of glpD, dnaJ, and surA knockout strains were increased to the level of the wild type when inocula aged. The mqsR and pho...

Microbiology and Molecular Biology Reviews

Many bacterial pathogens can permanently colonize their host and establish either chronic or recu... more Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube.

Smart Materials and Structures

Smart and soft electroactive polymer actuators have many beneficial properties, making them attra... more Smart and soft electroactive polymer actuators have many beneficial properties, making them attractive for biomimetic and biomedical applications. However, the selection of components to fabricate biofriendly composites has been limited. Although biofriendly options for electrodes and membranes are available, the conventional ionic liquids (ILs) often used as the electrolytes in the actuators have been considered toxic in varying degrees. Here we present a smart electroactive composite with carefully designed and selected components that have shown low toxicity and a biofriendly nature. In the present study, polypyrrole-PVdF trilayer actuators using six different choline ILs were prepared and characterized. Choline ILs have shown promise in applications where low environmental and biological impact is critical. Despite this, the anions in ILs have a strong impact on toxicity. To evaluate how the anions effect the bioactivity of the ILs used to prepare the actuators, the ILs were tested on different microbial cultures (Escherichia coli, Staphylococcus aureus, Shewanella oneidensis MR-1) and HeLa cells. All of the selected choline ILs showed minimal toxic effects even at high concentrations. Electro-chemomechanical characterization of the actuators indicated that polypyrrole-PVdF actuators with choline ILs are viable candidates for soft robotic applications. From the tested ILs, choline acetate showed the highest strain difference and outperformed the reference system containing an imidazolium-based IL.

Science Signaling

Bacterial persisters survive antibiotic treatment and can cause chronic infections. In this issue... more Bacterial persisters survive antibiotic treatment and can cause chronic infections. In this issue of Science Signaling, Pontes and Groisman suggest that there is no specific molecular pathway responsible for persister formation in Salmonella and that slow growth is the decisive factor.

mBio

I n a recent paper, Nigam and colleagues analyzed the stress-related effects of the endoribonucle... more I n a recent paper, Nigam and colleagues analyzed the stress-related effects of the endoribonuclease toxin MazF on the Escherichia coli proteome (1). The authors from the lab of Hanna Engelberg-Kulka-the discoverer of the mazEF toxin-antitoxin system (2)-claim that MazF creates a unique stress-induced translation machinery (STM). The STM hypothesis states that the toxin cleaves selected mRNAs within 5=-leader sequences to produce a pool of leaderless transcripts that are, in turn, translated by special stress ribosomes (3, 4). The latter are formed when the toxin cleaves off an anti-Shine-Dalgarno sequence-containing fragment from the 3= end of 16S rRNA in mature ribosomes (3). Thus, MazF is postulated to reshape translation in stressed E. coli similarly to how the S factor reshapes transcription. Independent studies failed to support these findings. Transcriptome-wide mapping of the cleavage sites indicated that MazF cleaves most transcripts within their coding regions and produces very few full-length, leaderless mRNAs (5, 6). Contradicting the STM model, MazF does not cleave rRNA in mature, fully assembled ribosomes but instead targets rRNA precursors (5, 7). Finally, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics revealed that MazF generally inhibits protein synthesis and no proteins are selectively synthesized in response to the toxin (6). This result is at odds with the paper of Nigam and coworkers (1), who also used SILAC proteomics and report a group of 42 MazF-mediated, stress-induced E. coli proteins. Here we reanalyze their data and highlight several technical issues. The setup of the proteomics experiment and the lack of statistical analysis make it impossible to determine whether the reported differences in proteomes were caused by MazF or random fluctuations. The authors aimed to test which proteins are synthesized in the ΔmazEF mutant and its wild-type (wt) parent strain upon treatment with the quinolone antibiotic nalidixic acid (NA). To do that, Nigam et al. (1) grew bacteria in the light medium, added NA to the culture, and after 10 min, added heavy lysine and arginine in order to label the new proteins. The relative amounts of newly synthetized proteins were estimated based on heavy and light isotope ratio (H/L ratio) after an additional 5-min incubation. The experiment was repeated three times. The short length of pulse labeling resulted in low H/L values, which could, possibly, account for the high variability of results (see below). While the authors state that they "checked several time points and deduced that 5 min is the optimal time point to figure out which are the differential new proteins," they do not present the relevant supporting data. The authors state that the differences between the wt and ΔmazEF proteomes are specifically induced by stress but do not provide an essential control, i.e., proteomic analysis of these strains without NA treatment. Nigam and colleagues admit the lack of statistical analysis and, instead, chose all the proteins "that were induced more in the WT than in the mazEF mutants in all the repeats" as differentially expressed. They Citation Kaldalu N, Maiväli Ü, Hauryliuk V, Tenson T. 2019. Reanalysis of proteomics results fails to detect MazF-mediated stress proteins. mBio 10:e00949-19.

mBio

W e thank David W. Holden and Jeff Errington for their comments (1). We agree that scientific res... more W e thank David W. Holden and Jeff Errington for their comments (1). We agree that scientific research is inherently error-prone. Therefore, validation, reassessment, and reinterpretation of one's own and others' results is part of the scientific approach, regardless of whether the conclusions are affirming or critical. We also agree that further work is needed to establish whether, and in which settings, each individual toxin-antitoxin (TA) system contributes to persister formation in Escherichia coli K-12 and/or in other bacterial species. In the following paragraphs, we express our opinion on the topics on which Holden and Errington saw some overstatements and factual inaccuracies in our paper (2). The reason we tested the persistence phenotype of the newly constructed Δ10TA strain of E. coli K-12 at mid-exponential growth phase is because the original study by Maisonneuve et al. proposing the link between the TA systems on persister formation was performed under these conditions (3). We cannot therefore draw conclusions about the role of TA systems in E. coli K-12 under other experimental conditions. However, the key outcome of our study and the paper published by Harms and colleagues (4) is a call for setting up all necessary controls, cautiously double-checking new results, and critically reevaluating previously published findings that link TA systems and persister formation. Holden and Errington use several publications as evidence for TA's role in persistence. For example, an influential and much-cited study by Harrison et al. reported that deletion of the yafQ gene, encoding 1 of the 10 toxin genes deleted in the Δ10TA strain, caused a drop in persister levels surviving cefazolin (a cephalosporin) and tobramycin (an aminoglycoside antibiotic) in E. coli grown as a biofilm (5). To the best of our knowledge, no follow-up or independent study confirming this result has been published. Therefore, we draw attention to several crucial control experiments lacking in the original paper. First, given that the YafQ-inhibiting antitoxin DinJ itself has been reported to affect the general stress response by decreasing RpoS levels (6), it is necessary to assess whether deletion of the full dinJ-yafQ operon has the same effect as deletion of the sole yafQ gene. Additionally, deletion of yafQ had no effect on persistence to doxycycline or rifampin (5), indicating that YafQ does not play a general role in persistence. Second, restoration of the original persister level by complementation of the dinJ-yafQ knockout from a plasmid or reinsertion of the operon into the chromosome is essential for drawing reliable conclusions from the phenotypes observed with the knockout strain. We have previously encouraged complementation for validation of the gene knockout effects that change persister levels (7). Third, it

mBio, Jan 12, 2018

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bac... more Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicill...

RNA Biology, 2016

The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and pr... more The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNAsequencing to map the toxin-cleaved 5 0-and 3 0-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF-or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

Scientific Reports, 2016

2-Bromo-5-(2-bromo-2-nitrovinyl)furan (G1 or Furvina) is an antimicrobial with a direct reactivit... more 2-Bromo-5-(2-bromo-2-nitrovinyl)furan (G1 or Furvina) is an antimicrobial with a direct reactivity against thiol groups. It is active against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi. By reacting with thiol groups it causes direct damage to proteins but, as a result, is very short-living and interconverts into an array of reaction products. Our aim was to characterize thiol reactivity of G1 and its conversion products and establish how much of antimicrobial and cytotoxic effects are due to the primary activity of G1 and how much can be attributed to its reaction products. Stability of G1 in growth media as well as its conversion in the presence of thiols was characterized. The structures of G1 decomposition products were determined using NMR and mass-spectroscopy. Concentration-and time-dependent killing curves showed that G1 is bacteriostatic for Escherichia coli at the concentration of 16 μg/ml and bactericidal at 32 μg/ml. However, G1 is inefficient against nongrowing E. coli. Addition of cysteine to medium reduces the antimicrobial potency of G1. Nevertheless, the reaction products of G1 and cysteine enabled prolonged antimicrobial action of the drug. Therefore, the activity of 2-bromo-5-(2-bromo-2-nitrovinyl)furan is a sum of its immediate reactivity and the antibacterial effects of the conversion products. 2-Bromo-5-(2-bromo-2-nitrovinyl)furan (G1 or Furvina) belongs to the group of 2-vinylfuran-based antimicrobials 1. It is an efficient antimicrobial with broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi 2-4. G1 is currently in medical use in Cuba. It is marketed as Dermofural ointment for treatment of human skin and nail infections and as Furvinol for veterinary use. In Europe and USA, G1 is not in clinical use but patents for its synthesis and application as an anti-infective have been granted internationally 5-7. The exact details of action of G1 are a matter of debate. Just like other vinylfurans (2-furylethylenes), it is a thiolreactive compound and reacts with functional thiol groups in living cells, including those of vital enzymes 4,8-10. The reaction center of vinylfurans in these addition reactions is the electrophilic exocyclic double bond 9,11. For example, G1 has been shown to react with the cysteine residues of MurA, a protein that is crucial to peptidoglycan synthesis and is the target of phosphomycin 4. Modification of cysteines is irreversible, supposedly rather nonselective and bactericidal, if it is extensive 4. Due to the thiol reactivity, G1 must be short-lived both in a patient and during the standard tests of antimicrobial activity because such tests are carried out in cysteine-containing complex media. Besides adduct formation, G1 induces oxidation of thiol groups and formation of non-native disulfide bonds in a redox reaction. That leads to formation of a thiol-reactive de-bromo derivative of G1, which possesses antibacterial activity acting by the same mechanism as G1 4. Furthermore, G1 is unstable in aqueous media even in the lack of thiols 4. It breaks up into 5-bromo-2-furaldehyde, which does not have any antimicrobial activity, and bromonitromethane, that has been patented as a biocide while its target and the mechanism of action are unknown 4,12,13. Volatility of G1 evokes a question, how much of the reported antibacterial and cytotoxic effects are attributed to the compound itself and how much are due to its reaction-and breakdown products. Standard measurements of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and cytotoxicity do

Applied microbiology and biotechnology, Jan 4, 2016

Persisters-a drug-tolerant sub-population in an isogenic bacterial culture-have been featured thr... more Persisters-a drug-tolerant sub-population in an isogenic bacterial culture-have been featured throughout the last decade due to their important role in recurrent bacterial infections. Numerous investigations detail the mechanisms responsible for the formation of persisters and suggest exciting strategies for their eradication. In this review, we argue that the very term "persistence" is currently used to describe a large and heterogeneous set of physiological phenomena that are functions of bacterial species, strains, growth conditions, and antibiotics used in the experiments. We caution against the oversimplification of the mechanisms of persistence and urge for a more rigorous validation of the applicability of these mechanisms in each case.

Methods in Molecular Biology, 2016

Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bacte... more Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bactericidal antibiotics. These cells are suggested to be involved in the establishment of chronic infections. Persister levels depend on growth conditions. Here, we discuss the parameters that have to be considered when measuring persister levels and provide a sample protocol to do it.

Biofilms, Infection, and Antimicrobial Therapy, 2005

BioTechniques, 2000

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epit... more We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprintin...