Skip to main content
  • ASM Journals
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Latest Articles
    • COVID-19 Research and News from ASM Journals
    • mSphere of Influence: Commentaries from Early Career Microbiologists
    • Archive
  • Topics
    • Applied and Environmental Science
    • Clinical Science and Epidemiology
    • Ecological and Evolutionary Science
    • Host-Microbe Biology
    • Molecular Biology and Physiology
    • Therapeutics and Prevention
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About mSphere
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • ASM Journals
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
mSphere
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Latest Articles
    • COVID-19 Research and News from ASM Journals
    • mSphere of Influence: Commentaries from Early Career Microbiologists
    • Archive
  • Topics
    • Applied and Environmental Science
    • Clinical Science and Epidemiology
    • Ecological and Evolutionary Science
    • Host-Microbe Biology
    • Molecular Biology and Physiology
    • Therapeutics and Prevention
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About mSphere
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
Research Article | Molecular Biology and Physiology

Genomewide Profiling of the Enterococcus faecalis Transcriptional Response to Teixobactin Reveals CroRS as an Essential Regulator of Antimicrobial Tolerance

Rachel L. Darnell, Melanie K. Knottenbelt, Francesca O. Todd Rose, Ian R. Monk, Timothy P. Stinear, Gregory M. Cook
Paul D. Fey, Editor
Rachel L. Darnell
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
bMaurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Melanie K. Knottenbelt
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Francesca O. Todd Rose
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ian R. Monk
cDepartment of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Timothy P. Stinear
cDepartment of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Timothy P. Stinear
Gregory M. Cook
aDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
bMaurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul D. Fey
University of Nebraska Medical Center
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/mSphere.00228-19
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Teixobactin is a new antimicrobial of significant interest. It is active against a number of multidrug-resistant pathogens, including Staphylococcus aureus and Enterococcus faecalis, with no reported mechanisms of teixobactin resistance. However, historically, mechanisms of resistance always exist and arise upon introduction of a new antimicrobial into a clinical setting. Therefore, for teixobactin to remain effective long term, we need to understand how mechanisms of resistance could develop. Here we demonstrate that E. faecalis shows a remarkable intrinsic tolerance to high concentrations of teixobactin. This is of critical importance, as antimicrobial tolerance has been shown to precede the development of antimicrobial resistance. To identify potential pathways responsible for this tolerance, we determined the genomewide expression profile of E. faecalis strain JH2-2 in response to teixobactin using RNA sequencing. A total of 573 genes were differentially expressed (2.0-fold log2 change in expression) in response to teixobactin, with genes involved in cell wall biogenesis and division and transport/binding being among those that were the most upregulated. Comparative analyses of E. faecalis cell wall-targeting antimicrobial transcriptomes identified CroRS, LiaRS, and YclRK to be important two-component regulators of antimicrobial-mediated stress. Further investigation of CroRS demonstrated that deletion of croRS abolished tolerance to teixobactin and to other cell wall-targeting antimicrobials. This highlights the crucial role of CroRS in controlling the molecular response to teixobactin.

IMPORTANCE Teixobactin is a new antimicrobial with no known mechanisms of resistance. Understanding how resistance could develop will be crucial to the success and longevity of teixobactin as a new potent antimicrobial. Antimicrobial tolerance has been shown to facilitate the development of resistance, and we show that E. faecalis is intrinsically tolerant to teixobactin at high concentrations. We subsequently chose E. faecalis as a model to elucidate the molecular mechanism underpinning teixobactin tolerance and how this may contribute to the development of teixobactin resistance.

INTRODUCTION

The emergence of multidrug-resistant pathogens has rendered standard antimicrobial treatments ineffective, allowing infections to persist and proliferate. This is compounded by our lack of understanding of how antimicrobial resistance develops and a severe deficiency of new antimicrobials to treat resistant infections. Teixobactin is the first new class of antimicrobial to be discovered in decades and has proven efficacy against multidrug-resistant pathogens, such as vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium tuberculosis (1). Teixobactin is a unique depsipeptide antimicrobial consisting of 11 amino acids, including methylphenylalanine, enduracididine, and four d-amino acids (1). It was isolated from a new species of Gram-negative betaproteobacteria, Eleftheria terrae, using the multichannel iChip device (1). The potency of teixobactin stems from its ability to dually target the essential pyrophosphate-saccharide (PP-sugar) moiety of the cell wall precursors lipid II and lipid III, inhibiting both peptidoglycan and cell wall teichoic acid biosynthesis (1). The bactericidal mode of action of teixobactin has been identified in S. aureus, with inhibition of cell wall teichoic acids leading to a dysregulation of cell wall autolysins, resulting in cell lysis and death (1, 2). However, this remains to be determined in other bacterial pathogens.

There are no reported mechanisms of teixobactin resistance, with the producer strain (and other Gram-negative bacteria) being innately resistant, likely due to the inability of teixobactin to penetrate the outer membrane (1). This is in contrast to other antimicrobial producer species, such as Bacillus licheniformis, a Gram-positive bacterium which requires a resistance cassette to provide protection against bacitracin production (3, 4). The absence of a known naturally occurring resistance cassette therefore makes teixobactin a promising new antimicrobial for the treatment of infections caused by multidrug-resistant organisms. However, historically, resistance mechanisms always exist and appear upon introduction of an antimicrobial into a clinical setting (5, 6). Therefore, for teixobactin to remain effective long term, we need to understand how mechanisms of resistance could develop.

Bacterial stress response systems can act as determinants of antimicrobial resistance (7). When bacteria are challenged with cell wall-acting antimicrobials, they encounter a number of cellular stresses, including oxidative stress, nutrient limitation, and cell envelope stress (8–11). These stresses elicit a variety of specific and highly regulated adaptive responses that not only protect the bacteria from the offending stress but also promote changes in the cell that can impact innate susceptibility to additional antimicrobials (7). Antimicrobial network biology involves bioinformatic approaches which use high-throughput genetic screening or gene expression profiling, i.e., RNA sequencing (RNA-seq), to explore the different response layers of bacteria to different antimicrobial treatments (12). These high-throughput methods allow monitoring of the global changes in gene expression and can provide important insights into how groups of genes interact in response to antimicrobial stress (12–14).

Enterococci are opportunistic pathogens localized to the gastrointestinal tract of humans and animals (15). They are one of the leading causes of hospital-acquired infection, and infections caused by these organisms are notoriously difficult to treat (16). Antimicrobial tolerance was recently described to be an essential precursor to the development of ampicillin resistance in Escherichia coli, and it has long been known that enterococci are intrinsically tolerant to cell wall-targeting antimicrobials (17, 18). Previous studies have identified SodA, a superoxide dismutase, to be a key component of antimicrobial tolerance in Enterococcus faecalis (19, 20). However, SodA is not differentially expressed in response to cell wall-targeting antimicrobials; therefore, we hypothesize that there may be more than one mechanism of antimicrobial tolerance in E. faecalis (10).

The aim of this study was to identify and characterize potential pathways of teixobactin tolerance in E. faecalis using RNA-seq analysis. We report on the teixobactin-induced transcriptome of E. faecalis and isolation of the cell wall stress response two-component system (TCS) CroRS as an essential regulator of teixobactin tolerance and a potential contributor to the development of teixobactin resistance.

RESULTS AND DISCUSSION

E. faecalis displays tolerance to teixobactin at high concentrations.Antimicrobial tolerance is the ability of an organism to survive for extended periods of time in the presence of high antimicrobial concentrations, even when growth is inhibited. Importantly, antimicrobial tolerance is a critical preliminary factor in the acquisition of antimicrobial resistance, and it has long been known that enterococci are intrinsically tolerant to cell wall-targeting antimicrobials (17, 18). A comparison of E. faecalis JH2-2 and S. aureus ATCC 6538 MICs and minimum bactericidal concentrations (MBCs) showed that while the growth of both species was inhibited at 2 μg ml−1 (MIC), E. faecalis showed remarkable tolerance to cell killing by teixobactin with an MBC of 16 μg ml−1, compared to an MBC of 2 μg ml−1 for S. aureus (Table 1). This tolerance is also consistent with that to other cell wall-targeting antimicrobials (Table 1). This was confirmed by time-dependent kill kinetic assays, which demonstrated that a culture of S. aureus (5 × 108 CFU ml−1) was effectively sterilized by teixobactin at 50× MIC with a 5-log reduction in the number of CFU ml−1 to the lowest detectable level (1 × 103 CFU ml−1) in 2 h, while E. faecalis remained tolerant at >24 h postchallenge at the same concentration (Fig. 1). Ling et al. were unable to generate resistance to teixobactin using spontaneous mutagenesis in S. aureus (1). As tolerance has been shown to precede the development of resistance, we hypothesize that a lack of tolerance to teixobactin in S. aureus may hinder the bacterium’s ability to acquire mutations that could potentially confer resistance (17).

FIG 1
  • Open in new tab
  • Download powerpoint
FIG 1

Time-dependent kill kinetic assay of S. aureus and E. faecalis upon teixobactin challenge. Strains were grown to mid-exponential phase (5 × 108 CFU ml−1) and untreated or challenged with 50× MIC of teixobactin. Cell survival (number of CFU ml−1) was measured at time zero and 1, 2, 3, 4, 6, and 24 h postchallenge. Results are the mean ± SD (data are for biological triplicates).

View this table:
  • View inline
  • View popup
TABLE 1

Teixobactin MICs and MBCs for S. aureus and E. faecalisa

SodA is a superoxide dismutase responsible for the conversion of superoxide (O2−) to hydrogen peroxide (H2O2) and has previously been associated with antimicrobial tolerance in enterococci (19). However, sodA gene expression does not appear to be significantly up- or downregulated in response to other cell wall-targeting antimicrobials; we therefore hypothesized that there could be additional pathways that regulate teixobactin tolerance in E. faecalis (10).

Global gene expression profiling of E. faecalis in response to teixobactin.To identify potential pathways of intrinsic teixobactin tolerance, we performed global gene expression profiling of E. faecalis in response to teixobactin. E. faecalis JH2-2 was grown microaerobically (130 rpm) to mid-exponential phase (optical density at 600 nm [OD600], 0.5) and challenged with 0.25× MIC of teixobactin (0.5 μg ml−1) for 1 h (see Fig. S1 in the supplemental material). Challenge at this concentration ensured a teixobactin-induced response while minimizing growth inhibition (30% inhibition), thereby reducing changes in growth rate-related gene expression. A total of 573 genes were differentially expressed (2.0-fold log2 change in expression) in response to teixobactin challenge, with 306 being upregulated and 268 being downregulated (Tables S1 and S2). These results were confirmed by quantitative real-time PCR (Fig. S2; Table S3 and S6). Genes were categorized into gene ontologies to achieve an overall view of pathways up- and downregulated at a global transcriptomic level (Fig. 2). Excluding genes of unknown function (25.2% upregulated, 12% downregulated), genes involved in cell wall biogenesis and division (18.6%) and transport/binding (17%) were the most frequently upregulated in response to teixobactin (Fig. 2), while genes involved in metabolism (22%) and transport/binding (27%)—specifically, phosphotransfer system (PTS) transporters (11.6%) involved in the uptake of carbon metabolites—were the most frequently downregulated in response to teixobactin (Fig. 2).

FIG 2
  • Open in new tab
  • Download powerpoint
FIG 2

Pie charts showing the distribution of gene ontologies up- and downregulated in response to teixobactin. recomb., recombination.

TABLE S1

Genes upregulated in response to teixobactin in Enterococcus faecalis JH2-2. #, log2 fold change (2-fold log2 minimum threshold). Download Table S1, DOCX file, 0.07 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

TABLE S2

Genes downregulated in response to teixobactin in Enterococcus faecalis JH2-2. #, log2 fold change (2-fold log2 minimum threshold). Download Table S2, DOCX file, 0.06 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

TABLE S3

A comparison of the differential changes in gene expression for 10 genes in response to teixobactin using qRT-PCR and RNA-seq. #, not detected (ND; below the 2-fold log2 threshold in RNA-seq analysis). Download Table S3, DOCX file, 0.01 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

FIG S1

Optimization of teixobactin concentration for RNA sequencing. E. faecalis JH2-2 was grown to mid-exponential phase (OD600, 0.5) and challenged with 0, 0.2, 0.5, and 1 μg ml−1 of teixobactin or DMSO at time zero. Growth was measured by determination of the cell density (OD600) for 3 h postchallenge. RNA was extracted at 1 h postchallenge. Results are the mean ± SD (data are for technical duplicates). Download FIG S1, TIF file, 0.2 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

FIG S2

qRT-PCR of E. faecalis JH2-2 gene expression in response to teixobactin. Quantitative real-time-PCR was carried out for 10 genes using E. faecalis JH2-2 cDNA to validate the changes in gene expression observed in the RNA sequencing data. Fold change is represented as a ratio of the mean CT values normalized to the CT value for the constitutively expressed EF0013. Results are the mean ± SD (data are for technical triplicates). Download FIG S2, TIF file, 0.1 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

The 20 most upregulated genes included genes for three putative autolysins (EF1518 [9.5-fold], EF0443 [8.7-fold], and EF0737 [5.5-fold]), four efflux transporters (EF2986 [6.8-fold], EF2987 [6.3-fold], EF2050 [6.3-fold], and EF1198 [5.7-fold]), a penicillin binding protein (EF0680 [5.4-fold]), and a glutamate-5-kinase (EF0038 [6.2-fold]) involved in cell wall biosynthesis and amino acid metabolism (Table 2). A total of 11 genes with unknown function were also among the most upregulated, including EF1533 (7.9-fold), EF0932 (7.1-fold), and EF1258 (6.0-fold) (Table 2). EF1533 appears to have a role in bacitracin and vancomycin susceptibility, while EF0932 is also highly upregulated in response to the antiseptic chlorhexidine (10, 21). Previous attempts to create an EF1258 deletion mutant suggest that this protein has an essential function (10). Eighteen of the 20 most downregulated genes were involved in carbon metabolism, including 13 transporters (11 PTS transporters), 4 metabolic genes (EF3141 [−9.9-fold], EF3142 [−9.7-fold], EF3140 [−9.4-fold], and EF0413 [−9.7-fold]), and 1 regulator (EF2966 [−9.3-fold]) (Table 2). The remaining two genes, EF2582 (−9.0-fold) and EF2223 (−8.8-fold), encode a chlorohydrolase/aminohydrolase and an ABC transporter, respectively, both of which are of unknown function (Table 2). E. faecalis JH2-2 teixobactin MICs and MBCs were determined in M17 broth supplemented with 0.5% glucose to identify a role for the PTS system in teixobactin susceptibility. However, no significant difference in MICs or MBCs was observed (data not shown).

View this table:
  • View inline
  • View popup
TABLE 2

The 20 most up- and downregulated genes in response to teixobactin in E. faecalis V583 and JH2-2

Cell wall precursors as a target of teixobactin.Lipid II and lipid III are precursors of peptidoglycan and teichoic acid biosynthesis, and both share the PP-sugar target moiety of teixobactin. Inhibition of these biosynthetic pathways has previously been confirmed in S. aureus but has not been confirmed in other Gram-positive organisms (1, 2). In this study, genes involved in peptidoglycan, teichoic acid, and cell wall exopolysaccharide biosynthesis were upregulated in response to teixobactin (Table S1). Cell wall exopolysaccharides have a role in virulence during enterococcal infections and can increase cell wall density, which may protect against cell wall stress (22). In enterococci, UPP binds activated sugars to form the precursors UPP-MurNAc, UPP-GlcNAc, and UPP-ManNAc of peptidoglycan, teichoic acid, and cell wall exopolysaccharide biosynthesis, respectively (23). We therefore hypothesize that teixobactin targets not only peptidoglycan and teichoic acid biosynthesis but also cell wall exopolysaccharide biosynthesis to effectively inhibit enterococci.

Comparative analysis of the E. faecalis transcriptional responses to different cell wall-targeting antimicrobials.Stress response pathways are major contributors to intrinsic antimicrobial resistance (7). Despite this, the cell wall stress response network in enterococci is poorly understood. In order to identify potential pathways that could contribute to intrinsic teixobactin tolerance and, thus, the development of teixobactin resistance, we compared the teixobactin-induced transcriptomic response to previously published E. faecalis transcriptomes of the cell wall-targeting antimicrobials bacitracin, vancomycin, and ampicillin (Table S4) (10). Unsurprisingly, cell wall biogenesis and division, including the biosynthesis of isoprenoid (a precursor of UPP), were the most-upregulated pathways in response to cell wall-targeting antimicrobials (Table S4). Other important components appeared to be two putative autolysins (EF0443 and EF1518), a number of efflux transporters (five ABC transporters and one EmrB-QacA-like MFS transporter), and three TCSs, YclRK (EF1260-EF1261), LiaRS (EF2911-EF2912), and CroRS (EF3289-EF3290) (Table S4). We decided to investigate whether CroRS could have a potential role in teixobactin tolerance.

TABLE S4

Comparison of differential gene expression in response to cell wall-targeting antimicrobials teixobactin (Teix), bacitracin (Bac), vancomycin (Van), and ampicillin (Amp) in Enterococcus faecalis. #, 2-fold log2 minimum threshold; †, differential gene expression in E. faecalis JH2-2 (this study); ‡, differential gene expression in E. faecalis O1GRF (10). Download Table S4, DOCX file, 0.04 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

The cell wall stress response two-component system CroRS is essential for teixobactin tolerance in E. faecalis.CroRS is a cell wall stress response TCS, and its role in cephalosporin resistance in enterococci has been well characterized (24, 25). More recently, the CroRS regulon of 219 genes, including an alanine racemase responsible for d-cycloserine resistance, was determined (26). Interestingly, EF0443 (LysM), the second-most-upregulated gene in response to teixobactin, was also characterized as a component of the CroRS regulon (Table 2) (26). To investigate the role of CroRS in teixobactin tolerance, we acquired an E. faecalis JH2-2 croRS deletion mutant and compared its MIC and MBC to those of the isogenic wild type (WT), E. faecalis JH2-2 (24). Interestingly, while there was no change in MIC, teixobactin tolerance was completely abolished in the ΔcroRS mutant, with a decrease in MBC from 16 μg ml−1 (WT) to 1 μg ml−1 (ΔcroRS mutant), the same concentration as the observed MIC (Table 3).

View this table:
  • View inline
  • View popup
TABLE 3

Cell antimicrobial MICs and MBCs for the E. faecalis JH2-2 wild type and ΔcroRS mutanta

To establish whether this was exclusive to teixobactin, we determined the MICs and MBCs for three other cell wall-targeting antimicrobials, vancomycin, bacitracin, and ampicillin, as well as the ribosome-targeting antimicrobial gentamicin, in the ΔcroRS mutant and compared these to the MICs and MBCs in the WT. We found that tolerance to vancomycin and bacitracin (but not ampicillin) was also completely abolished in the ΔcroRS mutant, with a decrease in the MBC from >128 μg ml−1 (WT) to 1 to 2 μg ml−1 (ΔcroRS mutant) and from 64 μg ml−1 (WT) to 16 μg ml−1 (ΔcroRS mutant), respectively (Table 3). Interestingly, the comparative transcriptomic analyses showed that CroRS was upregulated in response to teixobactin, vancomycin, and bacitracin, but not ampicillin (Table S4) (10). No change in the MIC was observed for vancomycin (1 μg ml−1) or ampicillin (0.5 μg ml−1), while a 2-fold decrease in the MIC (32 to 16 μg ml−1) was observed for bacitracin (Table 3). Time-dependent kill kinetics were determined for teixobactin and vancomycin in the ΔcroRS mutant and compared to those in the WT. These results definitively showed an increase in sensitivity to teixobactin- and vancomycin-induced cell killing in the ΔcroRS mutant (Fig. 3). The ΔcroRS mutant was effectively sterilized (5-log reduction) within 2 h of teixobactin challenge (25× MIC), whereas only a 2-log reduction of the WT was seen after 24 h (Fig. 3). For vancomycin, we observed an increase in the sensitivity of the ΔcroRS mutant with sterilization at 8 h after vancomycin challenge (50× MIC), whereas there was a 1-log reduction of the WT after 24 h (Fig. 3). Surprisingly, a 4-fold reduction in both the MIC and the MBC was observed for gentamicin (Table 3).

FIG 3
  • Open in new tab
  • Download powerpoint
FIG 3

Time-dependent kill kinetic assay of the E. faecalis JH2-2 wild type and ΔcroRS mutant. Strains were grown to mid-exponential phase (5 × 108 CFU ml−1) and untreated or challenged with 25× or 50× MIC of teixobactin (Tx) and vancomycin (Van), respectively. Cell survival (number of CFU ml−1) was measured at time zero and 1, 2, 3, 4, 6, 8, and 24 h postchallenge. Results are the mean ± SD (data are for biological triplicates).

Intrinsic low-level resistance to aminoglycosides, like gentamicin, is likely due to an inhibition of the cellular uptake of the antimicrobial (27). The addition of an agent that interferes with cell wall synthesis, such as a β-lactam (ampicillin) or a glycopeptide (vancomycin), greatly increases uptake of the aminoglycoside, enhancing effectivity (27, 28). We hypothesize that the cell wall of a ΔcroRS mutant is compromised, allowing for an increase in the uptake of gentamicin, resulting in a subsequent decrease in the gentamicin MIC and MBC. This is supported by an increase in sensitivity to glycine, a known destabilizer of the enterococcal cell wall, in the ΔcroRS mutant compared to the WT (Fig. S3) (29).

FIG S3

Glycine assay of the E. faecalis JH2-2 wild type (WT) and ΔcroRS mutant. SM17 broth (0.5 M sucrose plus M17 medium) containing a range of glycine concentrations was inoculated with an overnight culture of the E. faecalis JH2-2 WT or ΔcroRS mutant. Cultures were grown overnight at 37°C with no aeration, and growth was measured by determination of the OD600. Results are the mean ± SD (data are for biological triplicates). Download FIG S3, TIF file, 0.2 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Deletion of lysM (EF0443) is not sufficient for CroRS-mediated teixobactin tolerance in E. faecalis.LysM encodes a putative endopeptidase and was the second-most-upregulated gene in response to teixobactin (8.7-fold log2 change) (Table S1) (26). Cell wall autolytic enzymes, like endopeptidases, are responsible for regulating normal cell wall turnover; however, their dysregulation can lead to cell lysis (2, 30, 31). In fact, dysregulation of cell wall autolysins via a teixobactin-induced decrease in wall teichoic acids is believed to be the cause of teixobactin-induced cell death in S. aureus (1, 2). As CroRS appears to be important for teixobactin tolerance and is a known regulator of LysM, we sought to determine whether the CroRS-mediated regulation of LysM plays a role in teixobactin tolerance (26). We generated an E. faecalis JH2-2 ef0443 deletion mutant and subsequently determined the teixobactin MICs and MBCs and compared these to the MICs and MBCs for the WT (32). We observed no difference in the MIC or MBC for the Δef0443 mutant compared to that for the WT (Table S5). This suggests either that LysM does not play an essential role in CroRS-mediated teixobactin tolerance or that more than one factor is required to confer tolerance.

TABLE S5

Teixobactin MIC and MBC for E. faecalis JH2-2 Δef0443 compared to those for its isogenic wild type (WT). Mean MICs for three biological replicates are reported. †, MIC; minimum bactericidal concentration. Download Table S5, DOCX file, 0.01 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Conclusion.Antimicrobial tolerance is an important preliminary factor in the acquisition of antimicrobial resistance (17). Here we present the teixobactin-induced transcriptome of E. faecalis JH2-2 and isolated CroRS as an important cell wall stress response TCS upregulated by a number of cell wall-targeting antimicrobials. We show that CroRS is an essential regulator of antimicrobial tolerance in E. faecalis; however, the CroRS regulon composes 219 genes, and those responsible for conferring this tolerance are yet to be determined (26).

MATERIALS AND METHODS

Bacterial strains and growth conditions.Enterococcus faecalis strain JH2-2 and variants were routinely grown in brain heart infusion (BHI) broth and agar (1.5%, wt/vol) overnight at 37°C with no aeration unless otherwise stated. Staphylococcus aureus strain ATCC 6538 was routinely grown in tryptic soy broth (TSB) and agar overnight at 37°C with aeration (200 rpm) unless otherwise stated. Cultures for RNA-seq and optimization were grown at a ratio of two-thirds headspace to reduce aeration and minimize bias, as E. faecalis is a facultative anaerobe. Growth was measured as the optical density at a 600-nm wavelength (OD600). Teixobactin stocks were made with dimethyl sulfoxide (DMSO) and stored at −20°C.

Construction of the ef0443 gene deletion in E. faecalis JH2-2.An in-frame deletion of ef0443 was constructed in E. faecalis strain JH2-2 using the pIMAY-Z allelic exchange plasmid, as previously described (32, 33). Primers EF0443_AF, EF0443_BR, EF0443_CF, and EF0443_DR (see Table S6 in the supplemental material) were used to amplify the deletion construct by SLiCE (seamless ligation cloning extract) overlap extension PCR and cloned into the vector by SLiCE. The pIMAY-ZΔef0443 construct was electroporated into JH2-2 using the method of Cruz-Rodz and Gilmore (29).

TABLE S6

List of primer sequences used in this study. Download Table S6, DOCX file, 0.01 MB.
Copyright © 2019 Darnell et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

MIC and MBC assays.Strains were grown to mid-exponential phase (OD600, 0.5; 5 × 108 CFU ml−1) and were diluted to a final OD600 of 0.005 (0.5 McFarland standard). MICs were determined using the broth microdilution method with Muller-Hinton broth in 96-well flat-bottom microtiter plates in accordance with CLSI guidelines. Growth was measured by determination of the OD600 using a Varioskan plate reader after 20 h. The MIC was denoted as the lowest inhibitory concentration. Minimum bactericidal concentration (MBCs) were determined by sampling a range of concentrations from a completed MIC plate, followed by serial dilution in 1× phosphate-buffered saline (PBS) and spot plating on BHI or TSB agar (no antibiotic). The plates were incubated at 37°C for 24 h. The MBC was denoted as the lowest concentration that prevented growth on solid agar.

Time-dependent kill assays.Time-dependent kill assays were carried out to determine cell death kinetics over time. E. faecalis and S. aureus strains were grown to mid-exponential phase (OD600, 0.5; ∼5 × 108 CFU ml−1) in BHI and TSB, respectively, and challenged with and without teixobactin (25× MIC or 50× MIC) or vancomycin (50× MIC). Samples were taken at 0, 1, 2, 3, 4, 6, 8, and 24 h postchallenge, serially diluted in 1× PBS, and spot plated on BHI or TSB agar (no antibiotic). The plates were incubated at 37°C for 24 h, and the numbers of CFU ml−1 were determined.

Extraction and preparation of RNA samples for sequence analysis.To optimize the teixobactin concentration for RNA-seq analysis, E. faecalis JH2-2 cultures were grown in technical duplicate to an OD600 of 0.5 and challenged with a range of teixobactin concentrations (0, 0.2, 0.5, and 1 μg ml−1) (Fig. S1). DMSO was used as a negative control. The optimal concentration was 0.5 μg ml−1.

E. faecalis JH2-2 cultures were grown in technical triplicate to an OD600 of 0.5 (37°C, 130 rpm), where each replicate was subsequently split to produce two sets of technical triplicates. One set was challenged with 0.5 μg ml−1 of teixobactin, while the other one remained unchallenged. Total RNA was isolated using TRIzol-chloroform extraction as previously described (34). RNA samples were run through an RNeasy minikit (Qiagen) per the manufacturer’s instructions. An Agilent RNA 6000 Nano kit and an Agilent 2100 bioanalyzer (RNA integrity number, >8) were used to verify RNA quality per the manufacturer’s instructions, and the RNA concentration was determined using a NanoDrop ND-100 spectrophotometer.

RNA sequencing and gene expression analysis.(i) cDNA library preparation and sequencing of the Enterococcus faecalis JH2-2 transcriptome. rRNA was removed from total RNA using a Ribo-Zero RNA removal kit, and cDNA libraries were created using an Illumina TruSeq stranded total RNA library preparation kit. Sequencing was completed using an Illumina MiSeq (v3) system, generating 150-bp single-end reads.

(ii) Analysis of RNA sequencing data. Adapter sequences were removed from raw fastq files using Flexbar software (35), and reads shorter than 50 bp were discarded. Sequence reads from each sample were independently mapped to each contig (GenBank accession numbers NZ_KI518257.1 and NZ_KI518256.1) of the E. faecalis JH2-2 genome using Bowtie 2 software (36) to produce a table of raw read counts for all of the JH2-2 genes in each sample. Statistical and principle-component analyses were performed using the Bioconductor DESeq2 package (37). Parameters considered during analysis were the fold change (>2.0-fold log2), the mean number of reads (>50), and the adjusted P value (Padj < 0.1). Genes were also annotated with the E. faecalis V583 (GenBank accession number NC_004668.1) gene homolog using the NCBI BLAST program for continuity. Gene function and ontology were assigned using public databases (NCBI, UniProt, and KEGG) and complemented with literature searches (38–40).

qRT-PCR of E. faecalis genes in response to teixobactin.RNA-seq data were validated by quantitative real-time PCR (qRT-PCR) with primers specific to 10 genes, 8 differentially expressed genes and 2 constitutively expressed genes. Gene-specific primers were designed using Primer 3 (v0.4.0) software (http://primer3.ut.ee/ ). Primer sequences can be found in Table S6, and primer concentrations were optimized prior to final validation. cDNA was synthesized using a SuperScript III reverse transcriptase kit (Invitrogen) per the manufacturer’s instructions. cDNA was purified by ethanol precipitation and stored at −20°C. qRT-PCR was carried out using a ViiA 6 real-time PCR system with SYBR green and carboxy-X-rhodamine (Thermo Fisher Scientific). Differential expression was determined using the change in threshold cycle (ΔCT) values and normalized using the constitutively expressed EF0013 (dnaB).

Glycine assay.Glycine assays of E. faecalis JH2-2 WT and the ΔcroRS mutant were carried out in M17 medium with 0.5 M sucrose broth. Cultures were challenged with 0, 3, 5, 6, 7, 8, and 9% glycine and incubated overnight at 37°C with no aeration. Inhibition of growth was measured by determination of the OD600 for each strain at each concentration.

Accession number(s).The data from this study may be found in ArrayExpress under accession number E-MTAB-6484.

ACKNOWLEDGMENTS

This research was funded by the Maurice Wilkins Centre for Molecular Biodiscovery. R.L.D. was supported by a University of Otago doctoral scholarship and a Todd Foundation scholarship.

Library preparation and RNA-seq were carried out by the Otago Genomics and Bioinformatic Facility (OGBF) and New Zealand Genomics Ltd. (NZGL).

We thank Dallas Hughes of NovoBiotic Pharmaceuticals for the kind gift of teixobactin and Michel Arthur and Jean-Emmanuel Hugonnet for their kind gift of the E. faecalis ΔcroRS mutant strain. We also thank Shaun Ferguson and Hannah Hampton for their help and guidance in the RNA-seq analyses.

We declare no conflicts of interest.

FOOTNOTES

    • Received March 27, 2019.
    • Accepted April 16, 2019.
  • Copyright © 2019 Darnell et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

REFERENCES

  1. 1.↵
    1. Ling LL,
    2. Schneider T,
    3. Peoples AJ,
    4. Spoering AL,
    5. Engels I,
    6. Conlon BP,
    7. Mueller A,
    8. Schäberle TF,
    9. Hughes DE,
    10. Epstein S,
    11. Jones M,
    12. Lazarides L,
    13. Steadman VA,
    14. Cohen DR,
    15. Felix CR,
    16. Fetterman KA,
    17. Millett WP,
    18. Nitti AG,
    19. Zullo AM,
    20. Chen C,
    21. Lewis K
    . 2015. A new antibiotic kills pathogens without detectable resistance. Nature 517:455–459. doi:10.1038/nature14098.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    1. Homma T,
    2. Nuxoll A,
    3. Gandt AB,
    4. Ebner P,
    5. Engels I,
    6. Schneider T,
    7. Götz F,
    8. Lewis K,
    9. Conlon BP
    . 2016. Dual targeting of cell wall precursors by teixobactin leads to cell lysis. Antimicrob Agents Chemother 60:6510–6517. doi:10.1128/AAC.01050-16.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Podlesek Z,
    2. Comino A,
    3. Herzog-Velikonja B,
    4. Žgur-Bertok D,
    5. Komel R,
    6. Grabnar M
    . 1995. Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance. Mol Microbiol 16:969–976. doi:10.1111/j.1365-2958.1995.tb02322.x.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    1. Podlesek Z,
    2. Herzog B,
    3. Comino A
    . 2006. Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis. FEMS Microbiol Lett 157:201–205. doi:10.1111/j.1574-6968.1997.tb12774.x.
    OpenUrlCrossRef
  5. 5.↵
    1. Davies J,
    2. Davies D
    . 2010. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74:417–433. doi:10.1128/MMBR.00016-10.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. García-Solache M,
    2. Rice LB
    . 2019. The enterococcus: a model of adaptability to its environment. Clin Microbiol Rev 32:e00058-18. doi:10.1128/CMR.00058-18.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Poole K
    . 2012. Bacterial stress responses as determinants of antimicrobial resistance. J Antimicrob Chemother 67:2069–2089. doi:10.1093/jac/dks196.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    1. Utaida S,
    2. Dunman PM,
    3. Macapagal D,
    4. Murphy E,
    5. Projan SJ,
    6. Singh VK,
    7. Jayaswal RK,
    8. Wilkinson BJ
    . 2003. Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon. Microbiology 149:2719–2732. doi:10.1099/mic.0.26426-0.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Jordan S,
    2. Hutchings MI,
    3. Mascher T
    . 2008. Cell envelope stress response in Gram-positive bacteria. FEMS Microbiol Rev 32:107–146. doi:10.1111/j.1574-6976.2007.00091.x.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    1. Abranches J,
    2. Tijerina P,
    3. Avilés-Reyes A,
    4. Gaca AO,
    5. Kajfasz JK,
    6. Lemos JA
    . 2014. The cell wall-targeting antibiotic stimulon of Enterococcus faecalis. PLoS One 8:e64875. doi:10.1371/journal.pone.0064875.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Abranches J,
    2. Martinez AR,
    3. Kajfasz JK,
    4. Chávez V,
    5. Garsin DA,
    6. Lemos JA
    . 2009. The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance, and virulence in Enterococcus faecalis. J Bacteriol 191:2248–2256. doi:10.1128/JB.01726-08.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Kohanski MA,
    2. Dwyer DJ,
    3. Collins JJ
    . 2010. How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol 8:423–435. doi:10.1038/nrmicro2333.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    1. Brazas MD,
    2. Hancock R
    . 2005. Using microarray gene signatures to elucidate mechanisms of antibiotic action and resistance. Drug Discov Today 10:1245–1252. doi:10.1016/S1359-6446(05)03566-X.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    1. Dwyer DJ,
    2. Kohanski MA,
    3. Collins JJ
    . 2008. Networking opportunities for bacteria. Cell 135:1153–1156. doi:10.1016/j.cell.2008.12.016.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    1. Amyes SGB
    . 2007. Enterococci and streptococci. Int J Antimicrob Agents 29(Suppl 3):43–52.
    OpenUrl
  16. 16.↵
    1. Hollenbeck BL,
    2. Rice LB
    . 2012. Intrinsic and acquired resistance mechanisms in Enterococcus. Virulence 3:421–433. doi:10.4161/viru.21282.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    1. Levin-Reisman I,
    2. Ronin I,
    3. Gefen O,
    4. Braniss I,
    5. Shoresh N,
    6. Balaban NQ
    . 2017. Antibiotic tolerance facilitates the evolution of resistance. Science 355:826–830. doi:10.1126/science.aaj2191.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Krogstad DJ,
    2. Pargwette AR
    . 1980. Defective killing of enterococci: a common property of antimicrobial agents acting on the cell wall. Antimicrob Agents Chemother 17:965–968. doi:10.1128/AAC.17.6.965.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Bizzini A,
    2. Zhao C,
    3. Auffray Y,
    4. Hartke A
    . 2009. The Enterococcus faecalis superoxide dismutase is essential for its tolerance to vancomycin and penicillin. J Antimicrob Chemother 64:1196–1202. doi:10.1093/jac/dkp369.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    1. Ladjouzi R,
    2. Bizzini A,
    3. Lebreton F,
    4. Sauvageot N,
    5. Rincé A,
    6. Benachour A,
    7. Hartke A
    . 2013. Analysis of the tolerance of pathogenic enterococci and Staphylococcus aureus to cell wall active antibiotics. J Antimicrob Chemother 68:2083–2091. doi:10.1093/jac/dkt157.
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Bhardwaj P,
    2. Ziegler E,
    3. Palmer KL
    . 2016. Chlorhexidine induces VanA-type vancomycin resistance genes in enterococci. Antimicrob Agents Chemother 60:2209–2221. doi:10.1128/AAC.02595-15.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Teng F,
    2. Singh KV,
    3. Bourgogne A,
    4. Zeng J,
    5. Murray BE
    . 2009. Further characterization of the epa gene cluster and epa polysaccharides of Enterococcus faecalis. Infect Immun 77:3759–3767. doi:10.1128/IAI.00149-09.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Hancock LE,
    2. Murray BE,
    3. Sillanpää J
    . 2014. Enterococcal cell wall components and structures, p 1–35. In Gilmore MS, Clewell DB, Ike Y (ed), Enterococci: from commensals to leading causes of drug resistant infection. Massachusetts Eye and Ear Infirmary, Boston, MA.
  24. 24.↵
    1. Comenge Y,
    2. Quintiliani R,
    3. Li L,
    4. Dubost L,
    5. Brouard J-P,
    6. Hugonnet J-E,
    7. Arthur M
    . 2003. The CroRS two-component regulatory system is required for intrinsic beta-lactam resistance in Enterococcus faecalis. J Bacteriol 185:7184–7192. doi:10.1128/JB.185.24.7184-7192.2003.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Kellogg SL,
    2. Little JL,
    3. Hoff JS,
    4. Kristich CJ
    . 2017. Requirement of the CroRS two-component system for resistance to cell wall-targeting antimicrobials in Enterococcus faecium. Antimicrob Agents Chemother 61:e02461-16. doi:10.1128/AAC.02461-16.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Muller C,
    2. Massier S,
    3. Le Breton Y,
    4. Rincé A
    . 2018. The role of the CroR response regulator in resistance of Enterococcus faecalis to d-cycloserine is defined using an inducible receiver domain. Mol Microbiol 107:416–427. doi:10.1111/mmi.13891.
    OpenUrlCrossRef
  27. 27.↵
    1. Chow JW
    . 2000. Aminoglycoside resistance in enterococci. Clin Infect Dis 31:586–589. doi:10.1086/313949.
    OpenUrlCrossRefPubMed
  28. 28.↵
    1. Moellering RC,
    2. Weinberg AN
    . 1971. Studies on antibiotic synergism against enterococci. II. Effect of various antibiotics on the uptake of 14C-labeled streptomycin by Enterococci. J Clin Invest 50:2580–2584. doi:10.1172/JCI106758.
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    1. Cruz-Rodz A,
    2. Gilmore M
    . 1990. High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation. Mol Gen Genet 224:152–154.
    OpenUrlCrossRefPubMedWeb of Science
  30. 30.↵
    1. Tomasz A,
    2. Albino A,
    3. Zanati E
    . 1970. Multiple antibiotic resistance in bacterium with suppressed autolytic system. Nature 227:138–140. doi:10.1038/227138a0.
    OpenUrlCrossRefPubMedWeb of Science
  31. 31.↵
    1. Park JT,
    2. Uehara T
    . 2008. How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiol Mol Biol Rev 72:211–227. doi:10.1128/MMBR.00027-07.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    1. Pidot SJ,
    2. Gao W,
    3. Buultjens AH,
    4. Monk IR,
    5. Guerillot R,
    6. Carter GP,
    7. Lee JYH,
    8. Lam MMC,
    9. Grayson ML,
    10. Ballard SA,
    11. Mahony AA,
    12. Grabsch EA,
    13. Kotsanas D,
    14. Korman TM,
    15. Coombs GW,
    16. Robinson JO,
    17. Gonçalves da Silva A,
    18. Seemann T,
    19. Howden BP,
    20. Johnson PDR,
    21. Stinear TP
    . 2018. Increasing tolerance of hospital Enterococcus faecium to handwash alcohols. Sci Transl Med 10:eaar6115. doi:10.1126/scitranslmed.aar6115.
    OpenUrlFREE Full Text
  33. 33.↵
    1. Monk IR,
    2. Shah IM,
    3. Xu M,
    4. Tan M-W,
    5. Foster TJ
    . 2012. Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. mBio 3:e00277-11. doi:10.1128/mBio.00277-11.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Petridis M,
    2. Benjak A,
    3. Cook GM
    . 2015. Defining the nitrogen regulated transcriptome of Mycobacterium smegmatis using continuous culture. BMC Genomics 16:821. doi:10.1186/s12864-015-2051-x.
    OpenUrlCrossRef
  35. 35.↵
    1. Dodt M,
    2. Roehr J,
    3. Ahmed R,
    4. Dieterich C
    . 2012. FLEXBAR—flexible barcode and adapter processing for next-generation sequencing platforms. Biology (Basel) 1:895–905. doi:10.3390/biology1030895.
    OpenUrlCrossRefPubMed
  36. 36.↵
    1. Langmead B,
    2. Salzberg SL
    . 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi:10.1038/nmeth.1923.
    OpenUrlCrossRefPubMedWeb of Science
  37. 37.↵
    1. Love MI,
    2. Huber W,
    3. Anders S
    . 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. doi:10.1186/s13059-014-0550-8.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Altschul SF,
    2. Gish W,
    3. Miller W,
    4. Myers EW,
    5. Lipman DJ
    . 1990. Basic Local Alignment Search Tool. J Mol Biol 215:403–410. doi:10.1016/S0022-2836(05)80360-2.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    1. Apweiler R,
    2. Bairoch A,
    3. Wu CH,
    4. Barker WC,
    5. Boeckmann B,
    6. Ferro S,
    7. Gasteiger E,
    8. Huang H,
    9. Lopez R,
    10. Magrane M,
    11. Martin MJ,
    12. Natale DA,
    13. O ’Donovan C,
    14. Redaschi N,
    15. Yeh L-SL
    . 2004. UniProt: the Universal Protein knowledgebase. Nucleic Acids Res 32(Database issue):D115–D119. doi:10.1093/nar/gkh131.
    OpenUrlCrossRefPubMedWeb of Science
  40. 40.↵
    1. Kanehisa M,
    2. Goto S
    . 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 28:27–30. doi:10.1093/nar/28.1.27.
    OpenUrlCrossRefPubMedWeb of Science
PreviousNext
Back to top
Download PDF
Citation Tools
Genomewide Profiling of the Enterococcus faecalis Transcriptional Response to Teixobactin Reveals CroRS as an Essential Regulator of Antimicrobial Tolerance
Rachel L. Darnell, Melanie K. Knottenbelt, Francesca O. Todd Rose, Ian R. Monk, Timothy P. Stinear, Gregory M. Cook
mSphere May 2019, 4 (3) e00228-19; DOI: 10.1128/mSphere.00228-19

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print
Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this mSphere article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Genomewide Profiling of the Enterococcus faecalis Transcriptional Response to Teixobactin Reveals CroRS as an Essential Regulator of Antimicrobial Tolerance
(Your Name) has forwarded a page to you from mSphere
(Your Name) thought you would be interested in this article in mSphere.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Genomewide Profiling of the Enterococcus faecalis Transcriptional Response to Teixobactin Reveals CroRS as an Essential Regulator of Antimicrobial Tolerance
Rachel L. Darnell, Melanie K. Knottenbelt, Francesca O. Todd Rose, Ian R. Monk, Timothy P. Stinear, Gregory M. Cook
mSphere May 2019, 4 (3) e00228-19; DOI: 10.1128/mSphere.00228-19
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS AND DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

CroRS
Enterococcus
RNA sequencing
teixobactin
antimicrobial resistance
antimicrobial tolerance
mechanisms of resistance

Related Articles

Cited By...

About

  • About mSphere
  • Board of Editors
  • Policies
  • For Reviewers
  • For the Media
  • Embargo Policy
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Author Warranty
  • Types of Articles
  • Getting Started
  • Ethics
  • Contact Us

Follow #mSphereJ

@ASMicrobiology

       

 

Website feedback

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Online ISSN: 2379-5042