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80600.9053A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069
60510.9026Conservation and diversity of the IrrE/DdrO-controlled radiation response in radiation-resistant Deinococcus bacteria. The extreme radiation resistance of Deinococcus bacteria requires the radiation-stimulated cleavage of protein DdrO by a specific metalloprotease called IrrE. DdrO is the repressor of a predicted radiation/desiccation response (RDR) regulon, composed of radiation-induced genes having a conserved DNA motif (RDRM) in their promoter regions. Here, we showed that addition of zinc ions to purified apo-IrrE, and short exposure of Deinococcus cells to zinc ions, resulted in cleavage of DdrO in vitro and in vivo, respectively. Binding of IrrE to RDRM-containing DNA or interaction of IrrE with DNA-bound DdrO was not observed. The data are in line with IrrE being a zinc peptidase, and indicate that increased zinc availability, caused by oxidative stress, triggers the in vivo cleavage of DdrO unbound to DNA. Transcriptomics and proteomics of Deinococcus deserti confirmed the IrrE-dependent regulation of predicted RDR regulon genes and also revealed additional members of this regulon. Comparative analysis showed that the RDR regulon is largely well conserved in Deinococcus species, but also showed diversity in the regulon composition. Notably, several RDR genes with an important role in radiation resistance in Deinococcus radiodurans, for example pprA, are not conserved in some other radiation-resistant Deinococcus species.201728397370
64920.9022The VirAB ABC Transporter Is Required for VirR Regulation of Listeria monocytogenes Virulence and Resistance to Nisin. Listeria monocytogenes is a Gram-positive intracellular pathogen that causes a severe invasive disease. Upon infecting a host cell, L. monocytogenes upregulates the transcription of numerous factors necessary for productive infection. VirR is the response regulator component of a two-component regulatory system in L. monocytogenes In this report, we have identified the putative ABC transporter encoded by genes lmo1746-lmo1747 as necessary for VirR function. We have designated lmo1746-lmo1747 virAB We constructed an in-frame deletion of virAB and determined that the ΔvirAB mutant exhibited reduced transcription of VirR-regulated genes. The ΔvirAB mutant also showed defects in in vitro plaque formation and in vivo virulence that were similar to those of a ΔvirR deletion mutant. Since VirR is important for innate resistance to antimicrobial agents, we determined the MICs of nisin and bacitracin for ΔvirAB bacteria. We found that VirAB expression was necessary for nisin resistance but was dispensable for resistance to bacitracin. This result suggested a VirAB-independent mechanism of VirR regulation in response to bacitracin. Lastly, we found that the ΔvirR and ΔvirAB mutants had no deficiency in growth in broth culture, intracellular replication, or production of the ActA surface protein, which facilitates actin-based motility and cell-to-cell spread. However, the ΔvirR and ΔvirAB mutants produced shorter actin tails during intracellular infection, which suggested that these mutants have a reduced ability to move and spread via actin-based motility. These findings have demonstrated that L. monocytogenes VirAB functions in a pathway with VirR to regulate the expression of genes necessary for virulence and resistance to antimicrobial agents.201829263107
60830.9008Entamoeba histolytica Adaption to Auranofin: A Phenotypic and Multi-Omics Characterization. Auranofin (AF), an antirheumatic agent, targets mammalian thioredoxin reductase (TrxR), an important enzyme controlling redox homeostasis. AF is also highly effective against a diversity of pathogenic bacteria and protozoan parasites. Here, we report on the resistance of the parasite Entamoeba histolytica to 2 µM of AF that was acquired by gradual exposure of the parasite to an increasing amount of the drug. AF-adapted E. histolytica trophozoites (AFAT) have impaired growth and cytopathic activity, and are more sensitive to oxidative stress (OS), nitrosative stress (NS), and metronidazole (MNZ) than wild type (WT) trophozoites. Integrated transcriptomics and redoxomics analyses showed that many upregulated genes in AFAT, including genes encoding for dehydrogenase and cytoskeletal proteins, have their product oxidized in wild type trophozoites exposed to AF (acute AF trophozoites) but not in AFAT. We also showed that the level of reactive oxygen species (ROS) and oxidized proteins (OXs) in AFAT is lower than that in acute AF trophozoites. Overexpression of E. histolytica TrxR (EhTrxR) did not protect the parasite against AF, which suggests that EhTrxR is not central to the mechanism of adaptation to AF.202134439488
64240.9006Role of histone-like protein H-NS in multidrug resistance of Escherichia coli. The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulation of enteric bacteria. It is known that the expression of a variety of genes is repressed by H-NS, and mutations in hns result in various phenotypes, but the role of H-NS in the drug resistance of Escherichia coli has not been known. Here we present data showing that H-NS contributes to multidrug resistance by regulating the expression of multidrug exporter genes. Deletion of the hns gene from the DeltaacrAB mutant increased levels of resistance against antibiotics, antiseptics, dyes, and detergents. Decreased accumulation of ethidium bromide and rhodamine 6G in the hns mutant compared to that in the parental strain was observed, suggesting the increased expression of some drug exporter(s) in this mutant. The increased drug resistance and decreased drug accumulation caused by the hns deletion were completely suppressed by deletion of the multifunctional outer membrane channel gene tolC. At least eight drug exporter systems require TolC for their functions. Among these, increased expression of acrEF, mdtEF, and emrKY was observed in the Deltahns strain by quantitative real-time reverse transcription-PCR analysis. The Deltahns-mediated multidrug resistance pattern is quite similar to that caused by overproduction of the AcrEF exporter. Deletion of the acrEF gene greatly suppressed the level of Deltahns-mediated multidrug resistance. However, this strain still retained resistance to some compounds. The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of the MdtEF exporter. Double deletion of the mdtEF and acrEF genes completely suppressed Deltahns-mediated multidrug resistance, indicating that Deltahns-mediated multidrug resistance is due to derepression of the acrEF and mdtEF drug exporter genes.200414973023
72550.9002The Bacillus subtilis extracytoplasmic function σ factor σ(V) is induced by lysozyme and provides resistance to lysozyme. Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative σ factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative σ factors are the extracytoplasmic function (ECF) σ factors. In this paper, we demonstrate that the expression of the ECF σ factor σ(V) in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that σ(V) is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon σ(V). Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for σ(V)-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF σ factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF σ factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF σ factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.201121856855
33960.9001Multiple mechanisms of resistance to cisplatin toxicity in an Escherichia coli K12 mutant. The mechanisms underlying cellular resistance to the antitumor drug cis-diamminedichloro-platinum(II) (CDDP) were studied in Escherichia coli K12. A bacterial strain (MC4100/DDP) was selected from the MC4100 wild-type strain after growth for four cycles in CDDP. MC4100/DDP bacteria showed a high level of resistance and exhibited various modifications including (1) a decrease in drug uptake and platinum/DNA binding which only partly contributed to resistance, (2) an increase in glutathione content not involved in the resistant phenotype, (3) an increase in DNA repair capacity. Resistance was unmodified by introducing a uvrA mutation which neutralizes the excision-repair pathway. In contrast, it was abolished by deletion of the recA gene which abolishes recombination and SOS repair but also by a mutation in the recA gene leading to RecA co-protease minus (no SOS induction). RecA protein was unchanged in MC4100/DDP but the expression of RecA-dependent gene(s) was required for CDDP resistance. The regulation of genes belonging to the SOS regulon was analysed in MC4100/DDP by monitoring the expression of sfiA and recA::lacZ gene fusions after UV irradiation. These gene fusions were derepressed faster and the optimal expression was obtained for a lower number of UV lesions in MC4100/DDP, suggesting a role of RecA co-protease activity in the mechanism of resistance to CDDP in this E. coli strain.19947974517
12870.9001Identification of a novel system for boron transport: Atr1 is a main boron exporter in yeast. Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Delta mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Delta cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Delta cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.200919414602
60680.8996Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria. Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.202133923690
34590.8994Genetic redundancy, proximity, and functionality of lspA, the target of antibiotic TA, in the Myxococcus xanthus producer strain. We recently showed that type II signal peptidase (SPaseII) encoded by lspA is the target of an antibiotic called TA (myxovirescin), which is made by Myxococcus xanthus. SPaseII cleaves the signal peptide during bacterial lipoprotein processing. Bacteria typically contain one lspA gene; however, strikingly, the M. xanthus DK1622 genome contains four (lspA1 to lspA4). Since two of these genes, lspA3 and lspA4, are located in the giant TA biosynthetic gene cluster, we hypothesized they may play a role in TA resistance. To investigate the functions of the four M. xanthus lspA (lspA(Mx)) genes, we conducted sequence comparisons and found that they contained nearly all the conserved residues characteristic of SPaseII family members. Genetic studies found that an Escherichia coli ΔlspA mutation could be complemented by any of the lspA(Mx) genes in an lpp mutant background, but not in an E. coli lpp(+) background. Because Lpp is the most abundant E. coli lipoprotein, these results suggest the M. xanthus proteins do not function as efficiently as the host enzyme. In E. coli, overexpression of each of the LspA(Mx) proteins conferred TA and globomycin resistance, although LspA3 conferred the highest degree of resistance. In M. xanthus, each lspA(Mx) gene could be deleted and was therefore dispensable for growth. However, lspA3 or lspA4 deletion mutants each exhibited a tan phase variation bias, which likely accounts for their reduced-swarming and delayed-development phenotypes. In summary, we propose that all four LspA(Mx) proteins function as SPaseIIs and that LspA3 and LspA4 might also have roles in TA resistance and regulation, respectively.201424391051
6189100.8992Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus. Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.201323894076
539110.8988A role of ygfZ in the Escherichia coli response to plumbagin challenge. Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation.201021059273
579120.8986Control of expression of a periplasmic nickel efflux pump by periplasmic nickel concentrations. There is accumulating evidence that transenvelope efflux pumps of the resistance, nodulation, cell division protein family (RND) are excreting toxic substances from the periplasm across the outer membrane directly to the outside. This would mean that resistance of Gram-negative bacteria to organic toxins and heavy metals is in fact a two-step process: one set of resistance factors control the concentration of a toxic substance in the periplasm, another one that in the cytoplasm. Efficient periplasmic detoxification requires periplasmic toxin sensing and transduction of this signal into the cytoplasm to control expression of the periplasmic detoxification system. Such a signal transduction system was analyzed using the Cnr nickel resistance system from Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34. Resistance is based on nickel efflux mediated by the CnrCBA efflux pump encoded by the cnrYHXCBAT metal resistance determinant. The products of the three genes cnrYXH transcriptionally regulate expression of cnr. CnrY and CnrX are membrane-bound proteins probably functioning as anti sigma factors while CnrH is a cnr-specific extracytoplasmic functions (ECF) sigma factors. Experimental data provided here indicate a signal transduction chain leading from nickel in the periplasm to transcription initiation at the cnr promoters cnrYp and cnrCp, which control synthesis of the nickel efflux pump CnrCBA.200516158236
331130.8984MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis. The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.201021062372
552140.8983Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment. Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild-type cells were not known. Using RNA-Seq and promoter-lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil.201930648305
626150.8982Enterococcus faecalis Adapts to Antimicrobial Conjugated Oligoelectrolytes by Lipid Rearrangement and Differential Expression of Membrane Stress Response Genes. Conjugated oligoelectrolytes (COEs) are emerging antimicrobials with broad spectrum activity against Gram positive and Gram negative bacteria as well as fungi. Our previous in vitro evolution studies using Enterococcus faecalis grown in the presence of two related COEs (COE1-3C and COE1-3Py) led to the emergence of mutants (changes in liaF and liaR) with a moderate 4- to16-fold increased resistance to COEs. The contribution of liaF and liaR mutations to COE resistance was confirmed by complementation of the mutants, which restored sensitivity to COEs. To better understand the cellular target of COEs, and the mechanism of resistance to COEs, transcriptional changes associated with resistance in the evolved mutants were investigated in this study. The differentially transcribed genes encoded membrane transporters, in addition to proteins associated with cell envelope synthesis and stress responses. Genes encoding membrane transport proteins from the ATP binding cassette superfamily were the most significantly induced or repressed in COE tolerant mutants compared to the wild type when exposed to COEs. Additionally, differences in the membrane localization of a lipophilic dye in E. faecalis exposed to COEs suggested that resistance was associated with lipid rearrangement in the cell membrane. The membrane adaptation to COEs in EFC3C and EFC3Py resulted in an improved tolerance to bile salt and sodium chloride stress. Overall, this study showed that bacterial cell membranes are the primary target of COEs and that E. faecalis adapts to membrane interacting COE molecules by both lipid rearrangement and changes in membrane transporter activity. The level of resistance to COEs suggests that E. faecalis does not have a specific response pathway to elicit resistance against these molecules and this is supported by the rather broad and diverse suite of genes that are induced upon COE exposure as well as cross-resistance to membrane perturbing stressors.202032117172
807160.8982Transcriptomic analysis of Saccharomyces cerevisiae upon honokiol treatment. Honokiol (HNK), one of the main medicinal components in Magnolia officinalis, possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5, a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae, particularly in inducing iron starvation to inhibit cell growth.201728499955
329170.8981Effect of NlpE overproduction on multidrug resistance in Escherichia coli. NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In this study, we report that overproduction of NlpE increases multidrug and copper resistance through activation of the genes encoding the AcrD and MdtABC multidrug efflux pumps in Escherichia coli.201020211889
558180.8981Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine. Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria.200516356850
9993190.8981Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases. Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile, σ(V) is required for lysozyme resistance, and σ(V) is activated in response to lysozyme. Once activated, σ(V), encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σ(V) regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find that pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficileIMPORTANCEClostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.202032868404