Skip to main content
  • ASM
    • 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 Special Collection
    • Archive
    • Minireviews
  • 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
    • 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 mBio
    • Editor in Chief
    • Board of Editors
    • AAM Fellows
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • ASM
    • 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
mBio
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Latest Articles
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • 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
    • 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 mBio
    • Editor in Chief
    • Board of Editors
    • AAM Fellows
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
Research Article | Molecular Biology and Physiology

Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters

Tyler C. Helmann, Caitlin L. Ongsarte, Jennifer Lam, Adam M. Deutschbauer, Steven E. Lindow
David S. Guttman, Editor
Tyler C. Helmann
aDepartment of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Caitlin L. Ongsarte
aDepartment of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jennifer Lam
aDepartment of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Adam M. Deutschbauer
aDepartment of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
bEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Steven E. Lindow
aDepartment of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Steven E. Lindow
David S. Guttman
University of Toronto
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/mBio.02614-19
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants.

IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.

INTRODUCTION

Bacteria, like all living organisms, must tolerate a variety of potentially harmful, chemically diverse molecules present in the environment. While many of these compounds can be degraded to prevent their accumulation to harmful levels within the cell, a common response to the presence of toxins is exportation of such toxins. Bacterial efflux transporters can function both to achieve stress tolerance and to contribute to virulence by secreting toxins or effectors of various kinds (1, 2). A given bacterial species commonly possesses a wide variety of efflux transporters, and it is presumed that they differ in the specificity of toxins that they export. Broad-specificity transporters actively remove toxins from the cell, and their substrates can include heavy metals, solvents, dyes, detergents, and antibiotics, as well as certain host-derived products (3–6). Promiscuous transporters in that category include the multidrug-resistant (MDR) efflux transporters that remove a wide range of structurally diverse chemical compounds from the cell interior (7). While genes encoding these exporters can be found on plasmids, pathogenic and nonpathogenic bacteria have comparable numbers of chromosomally encoded MDR systems (8). A high level of redundancy of transporters exists within a given strain, where they may share many of the same substrates (9, 10). These observations raise the issue of why so many transporters are present in a given bacterium, given the broad substrate range of these MDR pumps.

MDR efflux transporters are structurally diverse, being found in at least five distinct protein families: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, the multidrug and toxic compound extrusion (MATE) family, the ATP-binding cassette (ABC) superfamily, and the resistance-nodulation-division (RND) family (11, 12). Many of these transporter classes contain both substrate-specific transporters and less-specific MDR-type efflux pumps (8). For example, the characterized MFS transporters in Escherichia coli range from highly specific sugar-proton symporters such as LacY and XylE to multidrug efflux transporters such as EmrD (13). Our understanding of efflux-mediated resistance to toxins and antibiotics would benefit from identifying both the MDR transporters within the larger collection of genomic transporters for a given organism and the specific substrates for those transporters.

Substrate redundancy poses a challenge for the study of MDR transporters, as alternative transporters can mask mutations that disrupt the function of a given pump under investigation (9). The large number of potential MDR transporters in a given strain also makes their investigation by analysis of targeted gene deletion mutants laborious. An example of one of the few such intensive studies that have been performed is that of Sulavik et al. (14), who tested the susceptibility of E. coli strains with null mutations in seven known and nine predicted efflux genes against 35 toxicants. That study, and others (15, 16), identified the RND transporter AcrAB-TolC as the major determinant of intrinsic toxicant resistance in E. coli. Homologs of AcrAB-TolC are common in Gram-negative bacteria and have been shown to contribute to the virulence of plant pathogens as diverse as Erwinia amylovora, Pseudomonas syringae, Ralstonia solanacearum, and Xylella fastidiosa (17–20). Interestingly, inhibition of efflux pumps by known chemical inhibitors can increase the antimicrobial activity of compounds produced by plants (21–23) and might be considered a plant disease resistance trait.

P. syringae pv. syringae is a plant pathogen that is commonly found both in association with plants and in the water cycle (24). Those are habitats in which it might be expected to encounter a variety of toxic compounds. P. syringae pv. syringae strain B728a (B728a) was originally isolated from bean (Phaseolus vulgaris) (25) and is exposed to the antimicrobial compounds phaseollin and coumestrol, which accumulate in bean leaves in response to inoculation with both compatible and incompatible P. syringae strains (26). This species also can interact with many other plant species and thus is exposed to a variety of preformed inhibitors on plant surfaces as well as to induced phytoalexins and other toxic compounds found within diseased tissues. It also can be a transient soil colonist, being associated with dead plant material, and is likely exposed to the myriad of inhibitory compounds typically found in soils. MexAB-OprM is the best-characterized AcrAB-TolC homolog present in Pseudomonas species. While this protein complex has been proposed to secrete the iron-chelating molecule pyoverdine (27), it also contributes significantly to antibiotic resistance (11). In strain B728a, genes in this operon are generally expressed at much higher levels than genes encoding other RND transporters, both in culture and in cells on the leaf surface as well as in the apoplast (28). However, in P. aeruginosa, expression of mexAB-oprM is inversely correlated with expression of the related RND transporter operons mexEF-oprN and mexCD-oprJ (29). This suggests that regulation of the overall repertoire of efflux transporters is tightly controlled and that alterations in one or more can lead to compensatory changes in the others.

To examine the role of MexAB-OprM homologs and other potential MDR transporters present in strain B728a, we interrogated the fitness of a large library of randomly barcoded mariner transposon mutants in culture media containing diverse antimicrobial compounds. Random barcode-transposon insertion sequencing (RB-TnSeq) is a modification of transposon insertion sequencing where each transposon insertion is tagged with a unique 20-nucleotide barcode (30). As transposon insertions are mapped only once for a given library, this reduces the effort required for the use of that library for analysis of fitness contributions of genes under multiple conditions. Changes in relative barcode abundances over time are used as a proxy for the relative fitness contribution of a given gene under a given condition. This method can be used to associate bacterial genes with their importance to fitness under different growth conditions and has been used to improve genome annotations for diverse bacterial species (31). Of particular importance for this current study are the ease and scale associated with the use of this method to correlate genes encoding transporter proteins with their likely substrates. Here, we used RB-TnSeq to identify likely substrates for B728a efflux transporters, with a particular focus on complementary RND homologs of MexAB-OprM.

RESULTS

Creation of a barcoded transposon library in P. syringae B728a ΔmexB.To test the role of redundant RND efflux proteins in vitro, we created a barcoded mariner transposon library in an unmarked B728a ΔmexB mutant. Comparisons of gene fitness in the mutant library with that of the barcoded transposon library in wild-type (WT) strain B728a (32) allowed us to directly test the fitness contributions of B728a transporters in both genetic backgrounds, enabling the complementarity of other transporters with MexAB-OprM to be quantified. The ΔmexB transposon library contains 237,285 unique insertion strains at a median density of 16 central insertion strains per gene and insertion density and distribution levels similar to those of the WT library (see Table S1 in the supplemental material; see also Fig. S1 in the supplemental material). We hypothesized that insertional mutants in efflux genes in the ΔmexB genotype would be less fit than mutants in the WT genotype, particularly when exposed to toxic substrates of MexAB-OprM. The B728a genome encodes 668 predicted transport proteins (33); here, we primarily focused on the RND transporters but also investigated members of the other protein families that typically encode MDR efflux transporters. The B728a genome encodes 16 RND transporters, including MexAB-OprM (33).

FIG S1

Insertion coverage of transposon mutant libraries. Data represent frequencies of mapped transposon insertions across the chromosome of P. syringae B728a WT (left) and P. syringae B728a ΔmexB (right). Download FIG S1, PDF file, 0.1 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

TABLE S1

Characteristics of the barcoded mariner transposon libraries in B728a WT and ΔmexB backgrounds. We defined genic strains as those that contain an insertion within the central 10% to 90% of a gene; only these insertions were used to calculate fitness. Total number of B728a genes, 5,216. Download Table S1, PDF file, 0.04 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

The mexB deletion completely eliminates the activity of MexAB-OprM, including inactivation of OprM. To determine if the deletion of mexB resulted in a polar mutation, we expressed mexAB and mexAB-oprM under the control of the native promoter in the ΔmexB mutant strain. While the ΔmexB strain containing the plasmid expressing the entire operon was able to tolerate acriflavine, berberine, and phloretin at or above WT concentrations, the ΔmexB strain expressing only mexAB was not (see Table 2; see also Fig. S2). This indicates that the ΔmexB transposon library does not produce a functional OprM.

FIG S2

Zone of growth inhibition assays to test antibiotic sensitivity of B728a ΔmexB complementation variants. Mean values are displayed as column heights. For each compound tested, means marked with the same letter do not differ at the P value of 0.01 (Tukey’s honestly significant difference [HSD] test). The lower limit of measurement is 6 mm (dotted line). Download FIG S2, PDF file, 0.1 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Testing the fitness of mutants in the WT and ΔmexB transposon libraries in vitro.Both mutant libraries were grown in the rich culture medium King’s B (KB) with different antimicrobial compounds. For the compounds where the MIC was known for the B728a WT and ΔmexB strains (19), we used ¼ the MIC. We successfully assayed fitness for 16 unique antimicrobial compounds (Table S2). Many of these compounds were most soluble in DMSO (dimethyl sulfoxide), and the controls contained the concentrations of DMSO used in these assays. For each gene, fitness was calculated as the log2 ratio of barcode abundance following growth in a given condition relative to barcode abundance measured initially at time 0. As expected, insertions in the majority of genes did not contribute to fitness as measured by relative barcode abundance in the population, and thus the fitness values for most genes were close to 0. A mutant with a fitness value of −1 is approximately 50% less abundant than the typical strain in the library under that experimental condition.

TABLE S2

Antimicrobial compounds used in growth assays of the barcoded mariner transposon libraries. For compounds with known (and different) MIC values for the WT and ΔmexB genotypes, 0.25 MIC values were used. Otherwise, the same concentration was used for both libraries. DMSO solvent controls were used at concentrations representative of the highest concentration used. Download Table S2, PDF file, 0.04 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Identification of MexAB-OprM substrates.For many substrates, the dominant activity of MexAB-OprM seen in other species was expected to mask any requirement for complementary transporters. We therefore hypothesized that the ΔmexB deletion library would unmask the contributions of such alternative transporters to tolerance of various toxins. The majority of antimicrobial compounds tested here were substrates of the MexAB-OprM transporter and have known MICs (19). Other previously uninvestigated compounds included capsaicin, flavone, rifampin, and rotenone.

For the majority of the antimicrobial compounds tested, disruption of any gene in the MexAB-OprM operon resulted in decreased fitness relative to insertions in the average gene in the WT strain (Fig. 1). No fitness change was observed for insertional strains in this operon when cells were exposed to capsaicin, rifampin, or rotenone. There was also no fitness change seen under conditions of exposure to ampicillin, a previously reported substrate (19). For erythromycin, only transposon insertions in oprM decreased mutant fitness. Flavone is a likely a substrate of this complex due to the large decreases in fitness comparable to that seen upon exposure to known substrates that accompanied disruptions across the mexAB-oprM operon. Using this method, we would expect insertional mutations in mexA and oprM to be neutral in the ΔmexB genetic background. All strains in the ΔmexB transposon library indeed were unable to assemble a complete MexAB-OprM complex, since insertions elsewhere in the mexAB-oprM operon did not alter fitness. For all antimicrobial compounds tested, mexA and oprM are dispensable in the ΔmexB library, with fitness values close to 0 (Fig. 1).

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

Fitness of transposon insertional mutants in the operon mexAB-oprM (Psyr_4007-9) both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.

Transporters homologous to MexAB contribute to resistance to diverse toxicants.To identify the efflux transporters that made the largest contribution to tolerance of various toxicants, we calculated the number of compounds in which genes encoding transporters or transporter components had a fitness value of less than −1. RND transporters generally made a larger contribution to toxin tolerance in the ΔmexB library than in the WT, but some were important in both backgrounds (Table 1). Due to our experimental design focusing on known MexAB-OprM substrates, mexA and mexB were required for competitive fitness in the presence of 10 compounds. The outer membrane transporter OprM is likely shared with additional efflux pumps and so is required for tolerance of an additional three compounds. In the ΔmexB deletion background, mexA is dispensable.

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

RND operons homologous to mexAB-oprM that likely contribute to multidrug resistancea

In addition to MexAB, four homologous RND transporters contributed substantially to tolerance of several toxicants (Table 1). The mexEF-oprN operon, however, was required for tolerance of these compounds only in the absence of MexB (Fig. 2). MexEF-OprN is likely redundant with MexAB-OprM for the substrates acridine orange, acriflavine, berberine, chloramphenicol, flavone, nalidixic acid, nitrofurantoin, and phloretin. The lack of a phenotype for mutants in the WT background is consistent with the likely subsidiary role of this transporter for these shared substrates and therefore with masking by the more highly expressed MexAB-OprM under these conditions.

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

Fitness of transposon insertional mutants in the operon mexEF-oprN (Psyr_2967-9) both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.

Psyr_2483-5 is likely redundant with MexAB-OprM with respect to the substrates acriflavine, berberine, carbenicillin, chloramphenicol, erythromycin, and phloretin, with decreased fitness for disruptions in the ΔmexB genetic background (Fig. 3). Psyr_2483-5 independently contributes to resistance to rifampin. This apparent operon encodes an unnamed RND transporter that is most similar to the MuxABC-OpmB complex in other pseudomonads, such as P. aeruginosa PAO1 (Table S3).

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

Fitness of transposon insertional mutants in the operon muxABC-opmB (Psyr_2482-5) both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.

TABLE S3

Shared amino acid identity between P. syringae B728a Psyr_2482-5 and P. aeruginosa PAO1 MuxABC-OpmB. Amino acid sequence homology was calculated using NCBI blastp. “Positives” includes amino acids having similar chemical properties. Download Table S3, PDF file, 0.04 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

MexCD (Psyr_2282-3) was required for full competitive fitness in the presence of acriflavine, berberine, erythromycin, and nalidixic acid (Fig. S3). Importantly, these phenotypes could be seen in both the WT and ΔmexB backgrounds. The negative fitness values for insertional mutants in this operon were, however, greater in the WT background, although this may have been at least partially due to the higher toxicant concentrations used in testing the mutants in the WT background than were used with the ΔmexB library. This operon does not encode an outer membrane protein, and a gene for such a required component is likely located elsewhere in the genome.

FIG S3

Fitness of transposon insertional mutants in the operon Psyr_2282-Psyr_2283 both in a WT background and in cells in which mexAB-oprM has been disrupted. KB and DMSO, media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene. Download FIG S3, PDF file, 0.1 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Among the compounds tested here, Psyr_0344-6 contributed to tolerance of phloretin only in the ΔmexB background (Fig. 4). Interestingly, disruption of any gene in this operon resulted in a mutant strain that was more fit than mutants of other genes under conditions of exposure to acriflavine, berberine, or nalidixic acid, but only if mexB was also absent. This operon likely requires an unknown outer membrane protein located elsewhere in the genome.

FIG 4
  • Open in new tab
  • Download powerpoint
FIG 4

Fitness of transposon insertional mutants in the operon Psyr_0344-6 both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.

Inner membrane transporters appear more substrate specific.The same computational analysis was used to interrogate the MFS and other inner membrane transporters to identify those with fitness contributions in the presence of various toxic compounds in either or both genetic backgrounds. Among 68 MFS transporters examined, only Psyr_0228 contributed to tolerance of any tested compound. Psyr_0228 contributed to competitive fitness in both acriflavine and acridine orange (Fig. 5). Interestingly, this gene contributed to acriflavine resistance even in a WT background, indicating that its role was independent of mexB. In contrast, this gene contributed to acridine orange resistance only in the ΔmexB genotype. Disruption of the gene encoding the SMR transporter Psyr_0541 strongly decreased fitness in berberine and mildly decreased fitness in acriflavine, independently of the mexB genotype (Fig. 6). In the ΔmexB strain, disruption of Psyr_0541 resulted in mild susceptibility to carbenicillin (Fig. 6). For those substrates where fitness was dependent on the mexB genotype, it is not clear if these inner membrane transporters contribute to toxin tolerance requiring the entire MexAB-OprM complex or just the OprM outer membrane protein.

FIG 5
  • Open in new tab
  • Download powerpoint
FIG 5

Fitness of transposon insertional mutants in the major facilitator superfamily transporter gene Psyr_0228 both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene.

FIG 6
  • Open in new tab
  • Download powerpoint
FIG 6

Fitness of transposon insertional mutants in the SMR gene Psyr_0541 both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene.

Several ABC transporters contribute to growth in rich media.Most genes annotated as encoding ABC transporter subunits were putative amino acid or carbohydrate transporters. Under the conditions tested, several ABC transporters were required for competitive fitness in the rich medium controls. For example, insertions in Psyr_0917-8, encoding the polysaccharide permease ABC transporter RfbAB-2, strongly decreased fitness under all conditions, including the controls containing only KB medium, in both the WT and ΔmexB genotypes (Fig. S4a). RfbAB-2 appears nearly essential for growth in KB. Genes encoding a putative peptide ABC transporter, Psyr_1754-9, were required for competitive fitness under all conditions, including in the KB controls, but only in a ΔmexB mutant background (Fig. S4b).

FIG S4

Fitness of transposon insertional mutants in the operons Psyr_0917-Psyr_0918 (left) and Psyr_1754-Psyr_1759 (right) both in a WT background and in cells in which mexAB-oprM has been disrupted. KB and DMSO, media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene. Download FIG S4, PDF file, 0.1 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Overlapping substrate specificities between transporters.The MDR efflux transporters tested here displayed a range of apparent substrates, with various degrees of overlap of MexAB-OprM (Fig. 7). Compounds such as acriflavine, berberine, and phloretin are probable substrates of multiple RND transporters. MexCD was required for tolerance of fewer compounds tested than MexAB, MexEF, or MuxABC. As hypothesized, inner membrane transporters Psyr_0228, Psyr_4519, and Psyr_0541 contributed to resistance to only a few toxicants. However, for the compounds examined here, no other inner membrane transporters contributed to competitive fitness.

FIG 7
  • Open in new tab
  • Download powerpoint
FIG 7

Likely substrates of P. syringae B728a multidrug resistance RND transporters, MFS transporter Psyr_0228, and SMR pump Psyr_0541. Blue cells indicate the compound is an apparent substrate of that transporter, with insertional mutants of a given gene having decreased fitness (fitness value less than −1) in the WT and/or the ΔmexB genetic backgrounds. Red cells indicate increased fitness of insertional mutants in this operon.

Assays to test predicted mutant phenotypes.Using RB-TnSeq, the fitness contributions of the various transporter genes were examined at only one or two concentrations of a particular toxicant. The lower fitness values seen for various mutants suggested a hypersensitivity to that compound. To confirm that disruption of these transporters reduced the concentration of the toxicant at which any growth could occur, we constructed targeted deletion mutants of the substrate binding proteins MexF, MuxB, and MexK (Psyr_0346) in both the WT and ΔmexB genotypes. We used broth serial dilution tests and zone of inhibition (ZOI) assays to examine the antibiotic susceptibility profiles of these strains (Table 2; see also Fig. S5). The measured MIC values were generally higher in the KB rich medium than in the M9 minimal medium. However, as fitness assays for the transposon libraries were conducted in a rich medium we aimed to test susceptibility phenotypes under the same condition. The ΔmexB ΔmexF double mutant was more susceptible to berberine than the WT and either single-deletion strain (Table 2; see also Fig. S5a). Similarly, the ΔmexB ΔmuxB double mutant was more sensitive to berberine, phloretin, and rifampin than the WT and either single-deletion strain (Table 2; see also Fig. S5b). While double mutants in a ΔmexB mutant background were more susceptible to acriflavin than the ΔmexB mutant itself, as evidenced by a larger zone of inhibition in the more sensitive ZOI assay and as predicted by the RB-TnSeq data, the extreme sensitivity of the ΔmexB mutant to acriflavin apparently obscured differences in the sensitivities of the double mutants as assessed in MIC assays. As suggested by the positive fitness values observed in mixture studies, the ΔmexB ΔmexK double mutant exhibited decreased susceptibility to acriflavine and berberine in comparison to the ΔmexB mutant (Table 2; see also Fig. S5c). In most cases, there was a direct relationship between the change in the MIC of a particular compound for a mutant and the fitness value for that mutant exposed to that chemical (Fig. S6).

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

Antimicrobial susceptibility of P. syringae B728a wild-type, derivative mutant, and complementation strains

FIG S5

Zone of growth inhibition assays to test antibiotic sensitivity of B728a efflux pump deletion mutants (A) ΔmexB ΔmexF, (B) ΔmexB ΔmuxB, and (C) ΔmexB ΔmexK. Mean values are displayed as column heights. For each compound tested, means marked with the same letter do not differ at the P value of 0.01 (Tukey’s HSD test). The lower limit of measurement is 6 mm (dotted line). Download FIG S5, PDF file, 0.1 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

FIG S6

Relationship between the change in MIC and fitness value of mutants when exposed to a given compound. MIC fold change was calculated as the change in log2 dilution factor relative to the B728a WT strain for single mutants or relative to the ΔmexB mutant for double-deletion mutants. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene. All values were measured in kilobases. Download FIG S6, PDF file, 0.1 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

DISCUSSION

Bacteria encounter and must tolerate diverse and potentially toxic molecules in the various habitats that they occupy. While efflux transporters are generally thought to play a major role in the tolerance of toxins, it is still unclear why bacterial genomes encode such a large number of MDR transporters. With high expression levels and wide substrate ranges, RND efflux transporters such as AcrAB-TolC and MexAB-OprM are essential for environmental survival and colonization of eukaryotic hosts (34). However, for the same reasons, these transporters are thought to mask the contributions to resistance of homologous RND transporters (14). Prompting our investigation in P. syringae, studies have shown that expression of multiple alternative transporters is increased when these primary RND pumps are disrupted (29, 35). Our studies of these alternative and redundant pumps have benefited from the availability of a method that has previously been successfully employed that relies on the use of hypersusceptible pump mutants to unmask redundant transporters (36–38).

Strain B728a contains a diverse array of transporters, many with likely roles in multidrug resistance (33). The MexAB-OprM complex has been shown to be important for several P. syringae strains for tolerance of a variety of antibiotics and other toxins, as well as for virulence during plant colonization (19). However, it has remained a challenge to associate particular transporters with their substrates, especially for host-produced compounds. With few exceptions (17, 39), genes encoding efflux transporters in addition to AcrAB-TolC and MexAB-OprM have not been observed to contribute to fitness during plant colonization (32, 40, 41), likely due to their high level of redundancy. Here, we used a transposon library constructed in a ΔmexB strain to reveal the role of several partially redundant RND efflux pumps. While several of these pumps appeared to have been completely masked by the role of MexAB-OprM under the conditions used here, some were complementary to MexAB-OprM, contributing incrementally to tolerance of various toxicants.

Among those transporters that played an incremental role in tolerance were mexEF-oprN and muxABC-opmB. These transporters, however, were important for only a subset of the MexAB-OprM substrates tested. We expected MexEF-OprN to be important in the ΔmexB genotype for two main reasons. First, previous transcriptional measurements in P. syringae strain B728a showed that after mexAB-oprM, mexEF-oprN is generally the second most highly expressed RND transporter under a variety of conditions in culture and in planta (28). Second, overexpression of mexEF-oprN in P. aeruginosa strains lacking mexAB-oprM increased resistance to several antibiotics (10). MuxABC-OpmB is an unusual RND operon because it is expected to have two inner membrane components. This transporter was characterized in P. aeruginosa only recently (42) and is homologous to the E. coli complex MdtABCD (also annotated as YegMNOB) (36, 37). Rifampin was not previously known to be a substrate of any Mex pumps (10), but full resistance appears to require the MuxABC-OpmB complex.

Of the eight RND operons likely involved in multidrug resistance, four do not contain genes encoding outer membrane proteins. Transporters such as these likely require the presence of outer membrane proteins, encoded by genes located elsewhere in the genome. The archetypal outer membrane protein TolC and its homologs (such as OprM and OprN in Pseudomonas species) can couple to many different transporters and apparently do not play a role in substrate specificity (43). Since the construction of the unmarked ΔmexB strain also disrupted the oprM gene, this may have reduced the functionality of transporters MexCD, Psyr_0344-6, Psyr_2193-4, and Psyr_3130-2 in that mutant if they require OprM for maximum functionality. Combined with the minor roles of these other transporters relative to that of MexAB-OprM, this would potentially explain the lack of a fitness cost of their disruption in both the WT and ΔmexB genotypes. We should emphasize, however, that nearly all of the compounds tested here were known MexAB-OprM substrates. In such a scenario, it might be expected that MexAB-OprM would play a dominant role in tolerance of such compounds. Very few studies have addressed the breadth of the substrate range for MexAB-OprM beyond that of clinically important antibiotics, and, given the wide variety of other toxic natural products that a bacterial species such as P. syringae would encounter in its myriad of habitats, it seems likely that many would be found for which these other efflux transporters might play a dominant role. Indeed, evidence for such a role for Psyr_0228 in the tolerance of acriflavin was seen here. It should prove fruitful to test this hypothesis further by the testing of these transposon libraries for their tolerance of diverse additional compounds. Not only will this provide further evidence of the constraints of MexAB-OprM in the chemical ecology of P. syringae, but it will also provide contextual evidence for the retention of the myriad of other efflux pumps in this species that presumably remain under selection, suggesting its importance in at least some settings encountered by this cosmopolitan bacterium. While we observed redundancy in efflux transporter function, seen in the overlapping substrate ranges of several RND transporters, it may simply benefit the cell to have multiple mechanisms of resistance for a given toxicant.

In Gram-negative bacteria, inner membrane transporters such as those in the MFS can function cooperatively with outer membrane transporters (like RND transporters) (9). This has been proposed to synergistically increase overall toxin resistance for the cell (10). Together with the high level of substrate promiscuity for most MDR RND transporters, we reasoned that inner membrane transporters might contribute to substrate specificity. This would prevent unnecessary efflux, attenuating the proton motive force. If this were true, we would expect to observe that inner membrane transporters would each contribute strongly to resistance to only a few toxicants. For Psyr_0228, which contributes to the tolerance of only acridine orange and acriflavine, two molecules that are structurally similar, this appeared to be the case. (Technically, acridine orange and acriflavine represent three molecules, as acriflavine is typically sold as a mixture of the related molecules acriflavine and proflavine). SMR protein Psyr_0541 is homologous to the quaternary ammonium compound resistance protein QacE in P. fluorescens SBW25 and QacH in P. aeruginosa PA14 (44). Both acriflavine and berberine are quaternary ammonium compounds, and it is noteworthy that Psyr_0541 was found to be necessary for tolerance of both of these compounds. Insertional mutants at this locus were particularly susceptible to berberine. The inner membrane transporters Psyr_0228 and Psyr_0541 appear to share some substrates with MexAB-OprM. More information is required to determine to what extent these transporters function cooperatively, if at all. In addition, it is possible that these transporters interact with molecules that are not substrates of MexAB-OprM, potentially with the assistance of outer membrane transporters other than OprM. It would be interesting if these protein interactions differed among particular substrates.

Efflux transporters are typically easily detected and annotated computationally due to their sequence homology and transmembrane domains. Here, we show that, using a range of structurally diverse antimicrobial compounds, RB-TnSeq can be used to characterize the substrates of MDR efflux transporters. This method is sensitive because it measures competitive fitness, rather than simple inhibition of growth, a phenotype important to bacteria in complex environments where they face competition with other microbes. RB-TnSeq is also very cost-effective once transposon mutants are initially mapped, making it practical to readily test a large number of conditions and compounds. Characterizing MDR transporter redundancy is essential to our understanding of not just clinical antibiotic resistance but also the role of these abundant proteins in bacterial survival in diverse environments.

Using RB-TnSeq in a hypersusceptible mutant strain allowed us to examine the role of alternative transporters that might not otherwise be active or discernible. Furthermore, plant-produced compounds are often not as toxic to bacteria as common antibiotics produced by bacteria or fungi, and this has been hypothesized to be due to the activity of efflux transporters (21). There is some evidence that plants can produce MDR pump inhibitors in addition to antimicrobial compounds (45), and this reinforces the importance of multi-“drug” resistance transporters in tolerating diverse plant-produced and other naturally occurring antimicrobial molecules. Multiple-component outer membrane transporters are apparently highly redundant, while inner membrane transporters can be more substrate specific. This may help explain the abundance of partially redundant MDR efflux transporters found in many bacteria, especially those strains typically found in environments with exposure to diverse toxic chemicals.

MATERIALS AND METHODS

Bacterial strains and growth media.P. syringae pv. syringae B728a was originally isolated from a bean leaf (Phaseolus vulgaris) in Wisconsin (25). The complete genome sequence for B728a is available on NCBI GenBank under accession number CP000075.1 (46). B728a and derivative mutant strains were grown at 28°C on King’s B (KB) agar or in KB broth (47). E. coli strains S17-1, TOP10, and XL1-Blue were grown on LB agar or in LB broth at 37°C. When appropriate, the following antibiotics were used at the indicated concentrations: 100 μg/ml rifampin, 50 μg/ml kanamycin, 15 μg/ml tetracycline, 100 μg/ml spectinomycin, and 40 μg/ml nitrofurantoin. A full list of strains, plasmids, and primers used in this study is contained in Table S4 in the supplemental material.

TABLE S4

Strains, plasmids, and oligonucleotides used in this study. Download Table S4, PDF file, 0.1 MB.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Complementation of the mexB deletion strain.Expression constructs of mexAB and mexAB-OprM were constructed through PCR amplification of this partial or complete operon, as well as of 1,161 bp of upstream sequence that would include potential promoter regions. These PCR products were ligated into the XbaI and EcoRI restriction enzyme sites of the plasmid p519ngfp (48). The ligation mixture was subsequently transformed into chemically competent E. coli XL1-Blue. Plasmids were confirmed to contain the correct insertion sequences by Sanger sequencing and were then electroporated into E. coli donor strain S17-1. Plasmids were introduced into B728a WT and B728a ΔmexB strains by conjugation on KB overnight and were then selected for 3 days on KB containing kanamycin and nitrofurantoin (E. coli counterselection).

Construction of bar-coded transposon libraries.Construction of the B728a WT barcoded transposon insertion library was described previously (32). The ΔmexB library was constructed in a similar fashion. For the ΔmexB library, we first removed the kanamycin resistance cassette from the B728a ΔmexB deletion mutant. The pFLP2Ω plasmid, containing the Flp recombinase and a spectinomycin resistance cassette (49), was introduced into the ΔmexB mutant by conjugation. Exconjugants were selected on spectinomycin. Colonies were screened for the loss of kanamycin resistance by plating, and the loss of the kanamycin cassette in sensitive colonies was confirmed by PCR. The pFLP2Ω plasmid was cured from the deletion strain by several overnight passages in KB containing rifampin only, and spectinomycin sensitivity was confirmed by plating. The rifampin-resistant ΔmexB deletion mutant was used as the recipient for conjugation with the barcoded mariner transposon library using the same protocol.

In vitro growth of the library.Aliquots of the transposon libraries that had been stored at −80°C were removed from cold storage, thawed, and inoculated into 25 ml fresh KB with 100 μg/ml kanamycin and grown for approximately 7 h at 28°C with shaking until the culture reached mid-log phase (optical density at 600 nm [OD600] of 0.5 to 0.7). For time 0 samples, 1-ml aliquots were pelleted by centrifugation and the pellets were frozen at −20°C until DNA purification. All in vitro experiments were performed in 24-well plates containing 1 ml total volume per well. Each compound used was added to KB containing 100 μg/ml kanamycin to reach a final volume of 950 μl. Cell suspensions (50 μl) were added to each well last. Libraries were grown overnight (15 h) at 28°C with shaking. The cells from each well were then pelleted and frozen until DNA purification.

DNA isolation and library preparation.DNA from frozen pellets was isolated using a Qiagen DNeasy blood & tissue kit according to manufacturer’s instructions. Cell lysis was performed at 50°C for 10 min per manufacturer instructions. Purified genomic DNA was measured on a NanoDrop device, and 200 ng of total DNA was used as a template for DNA barcode amplification and adapter ligation as established previously (30). For each time 0 sample, two separately purified DNA samples were sequenced as technical replicates.

Sequencing and generation of fitness data.Barcode sequencing, mapping, and analysis to calculate the relative abundances of barcodes were performed using the RB-TnSeq methodology and computation pipeline developed by Wetmore et al. (30; code available at bitbucket.org/berkeleylab/feba/). Briefly, TnSeq was used to map the insertion sites and for association of the DNA barcodes with these insertions. Fitness values for each gene were calculated as the log2 ratio of relative barcode abundance following library growth under a given condition divided by the relative abundance in the time 0 sample. Barcode counts were summed between replicate time 0 samples. For analysis, genes were required to have adequate coverage in the time 0 sample, i.e., at least 3 reads per strain and 30 reads per gene (30). The fitness values were calculated based on the “central” transposon insertions only, i.e., those within the central 10% to 90% of a gene. Fitness values were normalized across the genome such that the median gene had a fitness value of 0. All experiments described here passed previously described quality control metrics (30). Experimental fitness values are publicly available at fit.genomics.lbl.gov.

Identification of efflux pumps and assessment of fitness.We focused our initial analysis on genes that are homologous to mexAB-oprM. In the Transport Database (membranetransport.org) (33), B728a is annotated as containing 16 RND transporters. We manually curated this list of genes to focus on those operons potentially involved in “multidrug resistance,” namely, known mex genes or operons containing an “acriflavin resistance” or “hydrophobe/amphiphile efflux-1” and HlyD secretion protein. We filtered out 2 pseudogenes and 6 genes encoding proteins with known or likely unrelated function: protein export proteins SecD and SecF (Psyr_1230-1), a syringomycin efflux protein (Psyr_2622), and heavy metal efflux pump CzcA (Psyr_4803). Psyr_1128 is adjacent to a heavy metal (Cu/Ag) two-component system. Psyr_1701 is a hypothetical protein in the syringolin A biosynthesis gene cluster.

For the remaining eight RND operons, we compared fitness values for all genes within each operon across all experiments that passed quality control in both libraries. We focused on pumps where multiple encoding genes in the same operon contributed strongly to competitive fitness (fitness values of less than −1). As a negative control, the eight genes with expected functions unrelated to drug resistance were confirmed to not be required under any conditions tested (fitness values of less than −0.5 in both libraries). We performed the same analysis using 68 predicted MFS genes (encoding a mixture of potential drug resistance transporters as well as diverse sugar transporters), 210 ABC transporter genes, SMR gene Psyr_0541, and norA (Psyr_0073). Heat maps were plotted in R (50) using the gplots package, version 3.0.1.1 (51).

Construction of targeted deletion mutants.Deletion strains were constructed using an overlap extension PCR protocol as described previously (52). Briefly, 1-kb DNA fragments upstream and downstream of the genes of interest were amplified along with a kanamycin resistance cassette from pKD13 (53). These three fragments were joined by overlap extension PCR. The resulting fragment was blunt end ligated into the SmaI site of pTsacB (54) and transformed into E. coli subcloning strain TOP10 or strain XL1-Blue and then E. coli conjugation donor strain S17-1. This suicide plasmid was conjugated into B728 on KB overnight and was then selected for 3 days on KB containing kanamycin and nitrofurantoin. Putative double-crossover colonies that were kanamycin resistant and tetracycline sensitive were selected for screening using external primers and further confirmed by PCR and Sanger sequencing.

Drug susceptibility tests.The MICs of drugs for P. syringae strains were determined by growth of cells in 2-fold dilutions of test compounds in 96-well plates containing KB or M9 minimal medium supplemented with 0.2% (vol/vol) glycerol to reach a total volume of 200 μl per well. Bacterial cultures were grown to mid-log phase, diluted to an OD600 of 0.3, and washed three times in M9 before inoculation of 20 μl into each well. A semipermeable sealing membrane (Breathe-Easy) was used to cover the plates, which were maintained at 28°C with shaking. Growth of bacteria was examined by visual inspection after 24 h in KB and 48 h in M9. Three or four replicates were measured for each strain, compound, and medium combination. For zone of inhibition assays, strains were grown overnight on KB agar containing rifampin, resuspended in 10 mM KPO4, and diluted to an OD600 of 0.1. A 200-μl volume of the cell suspension was spread on each plate. Paper discs (6-mm diameter) were used to absorb the following antibiotics (vehicle): acriflavin (20 μl of 3 mg/ml H2O), berberine (30 μl of 50 mg/ml DMSO), flavone (15 μl of 20 mg/ml DMSO), and phloretin (30 μl of 30 mg/ml DMSO). Three replicate discs were used per plate. Plates were incubated overnight at 28°C, and the diameter of the clearance zone was measured. Graphs were plotted in R using the ggplot2 package version 3.1.1 (55).

ACKNOWLEDGMENTS

We thank Morgan Price for assistance with RB-TnSeq sequence analysis, Jayashree Ray for mapping the insertion sites of the B728a WT and ΔmexB transposon libraries, and Olga Smelik for experimental assistance. Capsaicin, flavone, and rotenone were kindly provided by Cat Adams and Thomas Bruns at the University of California, Berkeley (UC Berkeley).

Funding for T.C.H. was partially provided by the Arnon Graduate Fellowship and the William Carroll Smith Fellowship. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH instrumentation grant S10 OD018174.

FOOTNOTES

    • Received 3 October 2019
    • Accepted 10 October 2019
    • Published 29 October 2019

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

REFERENCES

  1. 1.↵
    1. Blanco P,
    2. Hernando-Amado S,
    3. Reales-Calderon J,
    4. Corona F,
    5. Lira F,
    6. Alcalde-Rico M,
    7. Bernardini A,
    8. Sanchez M,
    9. Martinez J
    . 2016. Bacterial multidrug efflux pumps: much more than antibiotic resistance determinants. Microorganisms 4:14. doi:10.3390/microorganisms4010014.
    OpenUrlCrossRef
  2. 2.↵
    1. Piddock LV
    . 2006. Multidrug-resistance efflux pumps—not just for resistance. Nat Rev Microbiol 4:629–636. doi:10.1038/nrmicro1464.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    1. Nies DH
    . 2003. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27:313–339. doi:10.1016/S0168-6445(03)00048-2.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    1. Fernandes P,
    2. Sommer Ferreira B,
    3. Sampaio Cabral JM
    . 2003. Solvent tolerance in bacteria: role of efflux pumps and cross-resistance with antibiotics. Int J Antimicrob Agents 22:211–216. doi:10.1016/S0924-8579(03)00209-7.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Nikaido H
    . 1996. Multidrug efflux pumps of gram-negative bacteria. J Bacteriol 178:5853–5859. doi:10.1128/jb.178.20.5853-5859.1996.
    OpenUrlFREE Full Text
  6. 6.↵
    1. Stermitz FR,
    2. Lorenz P,
    3. Tawara JN,
    4. Zenewicz LA,
    5. Lewis K
    . 2000. Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5’-methoxyhydnocarpin, a multidrug pump inhibitor. Proc Natl Acad Sci U S A 97:1433–1437. doi:10.1073/pnas.030540597.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Blair JM,
    2. Piddock LV
    . 2009. Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update. Curr Opin Microbiol 12:512–519. doi:10.1016/j.mib.2009.07.003.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    1. Saier MH,
    2. Paulsen IT,
    3. Sliwinski MK,
    4. Pao SS,
    5. Skurray RA,
    6. Nikaido H
    . 1998. Evolutionary origins of multidrug and drug-specific efflux pumps in bacteria. FASEB J 12:265–274. doi:10.1096/fasebj.12.3.265.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Tal N,
    2. Schuldiner S
    . 2009. A coordinated network of transporters with overlapping specificities provides a robust survival strategy. Proc Natl Acad Sci U S A 106:9051–9056. doi:10.1073/pnas.0902400106.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Lee A,
    2. Mao W,
    3. Warren MS,
    4. Mistry A,
    5. Hoshino K,
    6. Okumura R,
    7. Ishida H,
    8. Lomovskaya O
    . 2000. Interplay between efflux pumps may provide either additive or multiplicative effects on drug resistance. J Bacteriol 182:3142–3150. doi:10.1128/jb.182.11.3142-3150.2000.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Paulsen IT,
    2. Brown MH,
    3. Skurray RA
    . 1996. Proton-dependent multidrug efflux systems. Microbiol Rev 60:575–608.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Paulsen IT
    . 2003. Multidrug efflux pumps and resistance: regulation and evolution. Curr Opin Microbiol 6:446–451. doi:10.1016/j.mib.2003.08.005.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    1. Yan N
    . 2013. Structural advances for the major facilitator superfamily (MFS) transporters. Trends Biochem Sci 38:151–159. doi:10.1016/j.tibs.2013.01.003.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    1. Sulavik MC,
    2. Houseweart C,
    3. Cramer C,
    4. Jiwani N,
    5. Murgolo N,
    6. Greene J,
    7. DiDomenico B,
    8. Shaw KJ,
    9. Miller G,
    10. Hare R,
    11. Shimer G
    . 2001. Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob Agents Chemother 45:1126–1136. doi:10.1128/AAC.45.4.1126-1136.2001.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Nishino K,
    2. Yamaguchi A
    . 2001. Analysis of a complete library of putative drug transporter genes in Escherichia coli. J Bacteriol 183:5803–5812. doi:10.1128/JB.183.20.5803-5812.2001.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Okusu H,
    2. Ma D,
    3. Nikaido H
    . 1996. AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J Bacteriol 178:306–308. doi:10.1128/jb.178.1.306-308.1996.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Brown DG,
    2. Swanson JK,
    3. Allen C
    . 2007. Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence. Appl Environ Microbiol 73:2777–2786. doi:10.1128/AEM.00984-06.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Burse A,
    2. Weingart H,
    3. Ullrich MS
    . 2004. The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora. Mol Plant Microbe Interact 17:43–54. doi:10.1094/MPMI.2004.17.1.43.
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    1. Stoitsova SO,
    2. Braun Y,
    3. Ullrich MS,
    4. Weingart H
    . 2008. Characterization of the RND-type multidrug efflux pump MexAB-OprM of the plant pathogen Pseudomonas syringae. Appl Environ Microbiol 74:3387–3393. doi:10.1128/AEM.02866-07.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Reddy JD,
    2. Reddy SL,
    3. Hopkins DL,
    4. Gabriel DW
    . 2007. TolC is required for pathogenicity of Xylella fastidiosa in Vitis vinifera grapevines. Mol Plant Microbe Interact 20:403–410. doi:10.1094/MPMI-20-4-0403.
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    1. Tegos GP,
    2. Stermitz FR,
    3. Lomovskaya O,
    4. Lewis K
    . 2002. Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrob Agents Chemother 46:3133–3141. doi:10.1128/aac.46.10.3133-3141.2002.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Lewis K,
    2. Ausubel FM
    . 2006. Prospects for plant-derived antibacterials. Nat Biotechnol 24:1504–1507. doi:10.1038/nbt1206-1504.
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    1. Rempe CS,
    2. Burris KP,
    3. Lenaghan SC,
    4. Stewart CN
    . 2017. The potential of systems biology to discover antibacterial mechanisms of plant phenolics. Front Microbiol 8:422. doi:10.3389/fmicb.2017.00422.
    OpenUrlCrossRef
  24. 24.↵
    1. Morris CE,
    2. Sands DC,
    3. Vinatzer BA,
    4. Glaux C,
    5. Guilbaud C,
    6. Buffière A,
    7. Yan S,
    8. Dominguez H,
    9. Thompson BM
    . 2008. The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. ISME J 2:321–334. doi:10.1038/ismej.2007.113.
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    1. Loper JE,
    2. Lindow SE
    . 1987. Lack of evidence for in situ fluorescent pigment production by Pseudomonas syringae pv. syringae on bean leaf surfaces. Phytopathology 77:1449–1454. doi:10.1094/Phyto-77-1449.
    OpenUrlCrossRefWeb of Science
  26. 26.↵
    1. Lyon FM,
    2. Wood R
    . 1975. Production of phaseollin, coumestrol and related compounds in bean leaves inoculated with Pseudomonas spp. Physiol Plant Pathol 6:117–124. doi:10.1016/0048-4059(75)90039-9.
    OpenUrlCrossRef
  27. 27.↵
    1. Poole K,
    2. Heinrichs DE,
    3. Neshat S
    . 1993. Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine. Mol Microbiol 10:529–544. doi:10.1111/j.1365-2958.1993.tb00925.x.
    OpenUrlCrossRefPubMedWeb of Science
  28. 28.↵
    1. Yu X,
    2. Lund SP,
    3. Scott RA,
    4. Greenwald JW,
    5. Records AH,
    6. Nettleton D,
    7. Lindow SE,
    8. Gross DC,
    9. Beattie GA
    . 2013. Transcriptional responses of Pseudomonas syringae to growth in epiphytic versus apoplastic leaf sites. Proc Natl Acad Sci U S A 110:E425–E434. doi:10.1073/pnas.1221892110.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Li X-Z,
    2. Barre N,
    3. Poole K
    . 2000. Influence of the MexA-MexB-OprM multidrug efflux system on expression of the MexC-MexD-OprJ and MexE-MexF-OprN multidrug efflux systems in Pseudomonas aeruginosa. J Antimicrob Chemother 46:885–893. doi:10.1093/jac/46.6.885.
    OpenUrlCrossRefPubMedWeb of Science
  30. 30.↵
    1. Wetmore KM,
    2. Price MN,
    3. Waters RJ,
    4. Lamson JS,
    5. He J,
    6. Hoover CA,
    7. Blow MJ,
    8. Bristow J,
    9. Butland G,
    10. Arkin AP,
    11. Deutschbauer A
    . 2015. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 6:e00306-15. doi:10.1128/mBio.00306-15.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Price MN,
    2. Wetmore KM,
    3. Waters RJ,
    4. Callaghan M,
    5. Ray J,
    6. Liu H,
    7. Kuehl JV,
    8. Melnyk RA,
    9. Lamson JS,
    10. Suh Y,
    11. Carlson HK,
    12. Esquivel Z,
    13. Sadeeshkumar H,
    14. Chakraborty R,
    15. Zane GM,
    16. Rubin BE,
    17. Wall JD,
    18. Visel A,
    19. Bristow J,
    20. Blow MJ,
    21. Arkin AP,
    22. Deutschbauer AM
    . 2018. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 557:503–509. doi:10.1038/s41586-018-0124-0.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Helmann TC,
    2. Deutschbauer AM,
    3. Lindow SE
    . 2019. Genome-wide identification of Pseudomonas syringae genes required for fitness during colonization of the leaf surface and apoplast. Proc Natl Acad Sci 116:18900–18910 doi:10.1073/pnas.1908858116.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    1. Elbourne LDH,
    2. Tetu SG,
    3. Hassan KA,
    4. Paulsen IT
    . 2017. TransportDB 2.0: a database for exploring membrane transporters in sequenced genomes from all domains of life. Nucleic Acids Res 45:D320–D324. doi:10.1093/nar/gkw1068.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Li XZ,
    2. Plésiat P,
    3. Nikaido H
    . 2015. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin Microbiol Rev 28:337–418. doi:10.1128/CMR.00117-14.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Blair JM,
    2. Smith HE,
    3. Ricci V,
    4. Lawler AJ,
    5. Thompson LJ,
    6. Piddock LV
    . 2015. Expression of homologous RND efflux pump genes is dependent upon AcrB expression: implications for efflux and virulence inhibitor design. J Antimicrob Chemother 70:424–431. doi:10.1093/jac/dku380.
    OpenUrlCrossRefPubMed
  36. 36.↵
    1. Baranova N,
    2. Nikaido H
    . 2002. The BaeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate. J Bacteriol 184:4168–4176. doi:10.1128/JB.184.15.4168-4176.2002.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Nagakubo S,
    2. Nishino K,
    3. Hirata T,
    4. Yamaguchi A
    . 2002. The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system, MdtABC. J Bacteriol 184:4161–4167. doi:10.1128/JB.184.15.4161-4167.2002.
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    1. Pletzer D,
    2. Weingart H
    . 2014. Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora. BMC Microbiol 14:185–115. doi:10.1186/1471-2180-14-185.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Santamaría-Hernando S,
    2. Senovilla M,
    3. González-Mula A,
    4. Martínez-García PM,
    5. Nebreda S,
    6. Rodríguez-Palenzuela P,
    7. López-Solanilla E,
    8. Rodríguez-Herva JJ
    . 2019. The Pseudomonas syringae pv. tomato DC3000 PSPTO_0820 multidrug transporter is involved in resistance to plant antimicrobials and bacterial survival during tomato plant infection. PLoS One 14:e0218815. doi:10.1371/journal.pone.0218815.
    OpenUrlCrossRef
  40. 40.↵
    1. Lindow SE,
    2. Andersen GL,
    3. Beattie GA
    . 1993. Characteristics of insertional mutants of Pseudomonas syringae with reduced epiphytic fitness. Appl Environ Microbiol 59:1593–1601.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Royet K,
    2. Parisot N,
    3. Rodrigue A,
    4. Gueguen E,
    5. Condemine G
    . 2019. Identification by Tn-seq of Dickeya dadantii genes required for survival in chicory plants. Mol Plant Pathol 20:287–306. doi:10.1111/mpp.12754.
    OpenUrlCrossRef
  42. 42.↵
    1. Mima T,
    2. Kohira N,
    3. Li Y,
    4. Sekiya H,
    5. Ogawa W,
    6. Kuroda T,
    7. Tsuchiya T
    . 2009. Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa. Microbiology 155:3509–3517. doi:10.1099/mic.0.031260-0.
    OpenUrlCrossRefPubMedWeb of Science
  43. 43.↵
    1. Higgins CF
    . 2007. Multiple molecular mechanisms for multidrug resistance transporters. Nature 446:749–757. doi:10.1038/nature05630.
    OpenUrlCrossRefPubMedWeb of Science
  44. 44.↵
    1. Winsor GL,
    2. Griffiths EJ,
    3. Lo R,
    4. Dhillon BK,
    5. Shay JA,
    6. Brinkman F
    . 2016. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res 44:D646–D653. doi:10.1093/nar/gkv1227.
    OpenUrlCrossRefPubMed
  45. 45.↵
    1. Belofsky G,
    2. Percivill D,
    3. Lewis K,
    4. Tegos GP,
    5. Ekart J
    . 2004. Phenolic metabolites of Dalea versicolor that enhance antibiotic activity against model pathogenic bacteria. J Nat Prod 67:481–484. doi:10.1021/np030409c.
    OpenUrlCrossRefPubMed
  46. 46.↵
    1. Feil H,
    2. Feil WS,
    3. Chain P,
    4. Larimer F,
    5. DiBartolo G,
    6. Copeland A,
    7. Lykidis A,
    8. Trong S,
    9. Nolan M,
    10. Goltsman E,
    11. Thiel J,
    12. Malfatti S,
    13. Loper JE,
    14. Lapidus A,
    15. Detter JC,
    16. Land M,
    17. Richardson PM,
    18. Kyrpides NC,
    19. Ivanova NN,
    20. Lindow SE
    . 2005. Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000. Proc Natl Acad Sci U S A 102:11064–11069. doi:10.1073/pnas.0504930102.
    OpenUrlAbstract/FREE Full Text
  47. 47.↵
    1. King EO,
    2. Ward MK,
    3. Raney DE
    . 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44:301–307.
    OpenUrlPubMedWeb of Science
  48. 48.↵
    1. Matthysse AG,
    2. Stretton S,
    3. Dandie C,
    4. McClure NC,
    5. Goodman AE
    . 1996. Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145:87–94. doi:10.1111/j.1574-6968.1996.tb08561.x.
    OpenUrlCrossRefPubMedWeb of Science
  49. 49.↵
    1. Hoang T,
    2. Karkhoff-Schweizer R,
    3. Kutchma A,
    4. Schweizer HP
    . 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86. doi:10.1016/s0378-1119(98)00130-9.
    OpenUrlCrossRefPubMedWeb of Science
  50. 50.↵
    R Core Team. 2017. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.
  51. 51.↵
    1. Warnes GR,
    2. Bolker B,
    3. Bonebakker L,
    4. Gentleman R,
    5. Huber W,
    6. Liaw A,
    7. Lumley T,
    8. Maechler M,
    9. Magnusson A,
    10. Moeller S,
    11. Schwartz M,
    12. Venables B
    . 2019. gplots: various R programming tools for plotting data. https://rdrr.io/cran/gplots/.
  52. 52.↵
    1. Hockett KL,
    2. Burch AY,
    3. Lindow SE
    . 2013. Thermo-regulation of genes mediating motility and plant interactions in Pseudomonas syringae. PLoS One 8:e59850. doi:10.1371/journal.pone.0059850.
    OpenUrlCrossRefPubMed
  53. 53.↵
    1. Datsenko KA,
    2. Wanner BL
    . 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645. doi:10.1073/pnas.120163297.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    1. Chen C,
    2. Malek AA,
    3. Wargo MJ,
    4. Hogan DA,
    5. Beattie GA
    . 2010. The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds. Mol Microbiol 75:29–45. doi:10.1111/j.1365-2958.2009.06962.x.
    OpenUrlCrossRefPubMed
  55. 55.↵
    1. Wickham H
    . 2016. ggplot2: elegant graphics for data analysis. Springer-Verlag, New York, NY.
PreviousNext
Back to top
Download PDF
Citation Tools
Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters
Tyler C. Helmann, Caitlin L. Ongsarte, Jennifer Lam, Adam M. Deutschbauer, Steven E. Lindow
mBio Oct 2019, 10 (5) e02614-19; DOI: 10.1128/mBio.02614-19

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 mBio 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.
Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters
(Your Name) has forwarded a page to you from mBio
(Your Name) thought you would be interested in this article in mBio.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters
Tyler C. Helmann, Caitlin L. Ongsarte, Jennifer Lam, Adam M. Deutschbauer, Steven E. Lindow
mBio Oct 2019, 10 (5) e02614-19; DOI: 10.1128/mBio.02614-19
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

endophytes
epiphytes
fitness

Related Articles

Cited By...

About

  • About mBio
  • Editor in Chief
  • Board of Editors
  • AAM Fellows
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Author Warranty
  • Article Types
  • Ethics
  • Contact Us

Follow #mBio

@ASMicrobiology

       

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: 2150-7511