BACKGROUND
erwinia amylovora is the causative agent of fire blight, a destructive, contagious disease of apple, pear, and other rosaceous plants  <cit> .
all aerial parts of the hosts can be infected by the pathogen.
e. amylovora enters its host plants through natural openings  and wounds  <cit> .
upon entry, the fire blight pathogen moves through intercellular spaces towards the xylem  <cit> .
typical symptoms include flower necrosis, immature fruit rot, shoot curvature , bacterial ooze secretion, and cankers on woody tissues  <cit> .
the most effective method to treat infected plants is pruning to remove all infected tissue.
however, fire blight can infect entire orchards within a single growing season leading to devastating economic losses  <cit> .
presently, there are no effective therapeutics available to cure fire blight and therefore prevention is considered the best solution to manage this plant disease.
current control efforts are rather rare and rely primarily on antibiotic applications  to protect flowers.
however, the use of antibiotics for the management of fire blight is highly controversial due to the potential risk of promoting the emergence and spread of antibiotic resistance  <cit> .
gram-negative bacteria often possess multidrug efflux transporters within the cytoplasmic membrane, which have been found to recognize and expel a broad range of structurally unrelated compounds from the cell  <cit> .
among the multidrug efflux pumps, members of the resistance-nodulation-cell division  family appear to be the most effective efflux systems in gram-negative bacteria.
rnd transporters form a tripartite complex, consisting of an inner membrane pump that recognizes and captures the substrates, a periplasmic membrane fusion protein  and an outer membrane channel  <cit> .
acrab is the major multidrug efflux pump in e. coli and shows high conservation among gram-negative bacteria  <cit> .
acrd, a close homolog of acrb, is an rnd-type efflux pump characterized as a transporter of aminoglycosides, a highly hydrophilic class of molecules, and as a transporter of several amphiphilic compounds  <cit> .
typically, the inner membrane pump and the periplasmic mfp are co-transcribed in tandem on polycistronic mrna molecules  <cit> .
interestingly, this is not the case for acrd, which appears as a single gene and seemingly functions in concert with acra, a mfp co-transcribed with acrb  <cit> .
both acrab and acrd efflux systems are also present in the plant pathogen e. amylovora.
acrab has already been characterized as an efflux system required for virulence of e. amylovora in resistance towards apple phytoalexins and for successful colonization of the host plant  <cit> .
acrab of e. amylovora showed a similar substrate spectrum as acrab of e. coli <cit> .
in this study, the substrate specificity of acrd from e. amylovora was characterized and its contribution to virulence in apple and pear analyzed.
as it was found that acrd is expressed only at low levels under in vitro conditions, we were interested in investigating whether the expression of the acrd transporter in e. amylovora is induced in planta.
multidrug transporters are often expressed under control of local, as well as, global transcriptional regulators  <cit> .
current data show that expression of acrd in e. coli can be induced by the two-component regulatory system baesr  <cit> .
two-component systems  play an important role in the regulation of physiological processes in response to environmental or cellular parameters and enable bacterial cells to adapt to changing environmental conditions.
tcss typically consist of a membrane-bound histidine protein kinase  whose autokinase activity is dependent upon sensing a specific environmental stimulus   <cit> .
the second protein of a tcs is a response regulator, onto which a phosphoryl group is transferred from the phosphorylated hpk, and which functions as a phosphorylation-activated switch that regulates output responses within the cell causing changes in the expression of target genes  <cit> .
baesr is a tcs that responds cell envelope damages in e. coli <cit> .
the small core regulon of baesr includes the rnd-type transporters acrd and mdtabc and the periplasmic chaperone spy  <cit> .
the presence of a homologous baesr system in e. amylovora, prompted us to analyze the impact of the response regulator baer on the expression levels of acrd.
herein, we report that overexpression of the rnd pump acrd in an acrb-deficient mutant leads to increased resistance to two substrates, clotrimazole and luteolin, previously not described as substrates of acrd in other enterobacteria.
in order to determine the promoter activity in vitro, we utilized a transcriptional fusion of the promoter regions of acrab and acrd, respectively, with the reporter gene egfp.
we demonstrate that the response regulator baer is able to bind to the upstream region of acrd in e. amylovora ea <dig> and to induce acrd expression.
furthermore, we show that the inactivation of the rnd pump acrd did not result in reduction of virulence of e. amylovora on host plants.
RESULTS
identification of an acrd homologue in e. amylovora ea1189
a search with the blastp program  using the amino acid sequence of acrd from e. coli k- <dig> as the query  identified a homologous sequence in the genome of e. amylovora cfbp <dig> .
the annotated protein eamy_ <dig> is  <dig> amino acids shorter at the n-terminus than the acrd protein of e. coli.
comparison of the acrd gene sequences from e. coli and e. amylovora suggests that the eamy_ <dig> gene of e. amylovora cfbp <dig> has been annotated with a wrong start codon.
sequence analysis revealed an alternative atg start codon 54 bp upstream of the annotated eamy_ <dig> gene.
the  <dig> amino acids, encoded by the  <dig> additional nucleotides, are 100% identical to the n-terminal region of acrd from e. coli.
we used the genome sequence of e. amylovora cfbp <dig> to design primers to pcr amplify the respective region from the genomic dna of our model strain e. amylovora ea <dig> whose genome sequence is not yet available.
the nucleotide sequence of acrd and its upstream region from e. amylovora ea <dig> show 100% identity to eamy_ <dig> and its upstream region from e. amylovora cfbp <dig> based on our sequencing results.
acrd is a member of the rnd superfamily of transporters belonging to the hydrophobe/amphiphile efflux- <dig>  family .
a blastp search  of the deduced acrd sequence from e. amylovora ea <dig> as query identified homologous multidrug efflux transporters with high degree of sequence identity  in several members of the enterobacteriaceae family .
a sequence alignment between acrd from e. amylovora ea <dig> and acrd from e. coli k- <dig> showed that the proteins share 79% identity and 89% similarity with each other .
substituted amino acids were distributed throughout the sequence, but they were at least 40% conserved   <cit>  and no insertion or deletion was observed.
analysis of the up- and downstream regions flanking the acrd homologues from e. amylovora, e. coli and s. enterica revealed several differences  including the two-component system narqp located upstream of acrd in e. amylovora.
this system is involved in the regulation of anaerobic nitrate/nitrite respiration, and consists of the sensor kinase narq and the response regulator narp.
in e. coli and s. enterica, only the sensor kinase narq is present upstream of acrd.
the response regulator narp is situated at different positions in the genomes of e. coli and s. enterica.
moreover, the sizes of the narq homologues are also disparate.
narq of e. amylovora ea <dig> is a protein consisting of  <dig> amino acids, whereas the narq homologues of e. coli and s. enterica consist of  <dig> amino acids.
the downstream region of acrd of e. amylovora ea <dig> contains an insertion of about  <dig>  kb encoding several small hypothetical proteins.
transmembrane organization of acrb and acrd in e. amylovora
in a previous study, the transmembrane organization of acrb and acrd from e. coli was analyzed in silico, with  <dig> transmembrane-spanning domains  and  <dig> large periplasmic loops predicted in both proteins  <cit> .
a similar approach was accomplished with acrb and acrd from e. amylovora ea <dig> using the online tool topcons  <cit> .
topology analysis predicted the typical  <dig> tmds and  <dig> periplasmic loops between tmd <dig> and  <dig> and tmd  <dig> and  <dig> for the rnd-type efflux pumps acrb and acrd from e. amylovora ea <dig> .
phenotypic characterization of the acrd mutant
to evaluate the role of acrd in antibiotic resistance and to identify substrates of this rnd-type efflux pump, susceptibility tests of the wild type and the acrd mutant to a variety of antimicrobial agents were performed.
deletion of acrd resulted in no significant changes in sensitivity to tested aminoglycosides, dyes or detergents.
however, the acrd mutant was 2-fold more sensitive to nitrofurantoin, erythromycin, silver nitrate and sodium tungstate in comparison to the wild type .
the differences in sensitivity were minor but reproducible.
complementation of the acrd mutant with plasmid pblueks.acrd, which carried the acrd gene of ea <dig> under control of the p
lac
, restored resistance to all tested antimicrobials .
e. amylovora
wild-type strain,
acrd
mutant,
acrb
mutant complemented with acrd-overexpression plasmids pbluesk.acrd  and pblueks.acrd-ext  and
acrb
mutant complemented with control plasmid pbluesk.acrd
a
lac
< <
acrd
lac
> >
acrd
lac
, p
acrd
> >
acrd
a
mic values were determined by the 2-fold dilution assay in three or more independent experiments with similar results.
boldface numbers indicate a higher or lower mic.
nd, not determined.
expression of acrd in an acrb-deficient mutant of e. amylovora
to investigate the substrate specificity of acrd from ea <dig>  overexpression of the corresponding gene from a high-copy plasmid was achieved in e. amylovora mutant ea1189- <dig>  which is hypersensitive to many drugs due to a deficiency of the major multidrug efflux pump acrb  <cit> .
three overexpression plasmids were generated: pblueks.acrd, expressing acrd under control of the lac promoter , pbluesk.acrd-ext, expressing acrd under control of its native promoter  and pblueks.acrd-ext, expressing acrd under control of both promoters plac and pacrd.
as a control, a promoterless acrd gene was cloned in the opposite direction of plac.
these plasmids were mobilized into the acrb-deficient mutant ea1189- <dig> and the sensitivity of the transformants to various substrates were determined .
ea1189- <dig>  expressing acrd under control of plac, exhibited elevated resistance to clotrimazole , fusidic acid , novobiocin , hygromycin b , cadmium acetate , zinc sulfate , bile salt , deoxycholate , and sds .
the expression of acrd under control of its native promoter in ea1189- <dig> showed an increase in resistance similar to that of plac-controlled acrd expression .
when acrd was under control of both promoters, plac and pacrd, it conferred elevated resistance.
compared to the control, ea1189- <dig> displayed increased resistance to clotrimazole , fusidic acid , novobiocin , hygromycin b , cadmium acetate , zinc sulfate , bile salt , deoxycholate , sds , luteolin  and ethidium bromide  .
rnd-type efflux pump expression during cellular growth
to monitor the expression levels of the rnd-type efflux pumps acrab and acrd at different growth states, total rna was isolated at distinct optical densities and expression levels analyzed by quantitative rt-pcr.
the expression values were normalized to the highest expression of the acra and acrd transcript, respectively .
while the expression levels of acra changed during the cell cycle, indicating a growth phase-dependent transcription with the highest expression in the early exponential phase, acrd showed constant expression during growth.
additionally, the constant expression of acrd was also connected to a low expression level as determined by ct values .
furthermore, we studied the effect of temperature on activation of the rnd-type efflux pump acrd using qrt-pcr.
bacteria were cultured in lb broth at 18°c and 28°c, respectively, where 28°c represents the optimal growth temperature and 18°c represents the temperature at which several genes involved in pathogenicity showed induction in e. amylovora <cit> .
however, no temperature dependence of the acrd expression was observed in vitro .
promoter activity of acrab and acrd in vitro
in order to monitor promoter activities of the rnd-type efflux pumps acrab and acrd in e. amylovora, transcriptional fusions of the acra upstream region  and acrd upstream region , respectively, to the enhanced green fluorescence protein-encoding gene  were constructed.
to determine whether bacterial growth influenced the promoter activity, fluorescence measurements at several optical densities were performed .
our data indicated that the promoter activities of both acrab and acrd were constant throughout the growth phases in lb broth.
furthermore, the activity of the acrd promoter was  <dig> to 5-fold lower than the activity of the acrab promoter throughout growth.
effect of substrate exposure on acrd expression
the expression of genes encoding multidrug efflux systems can be influenced by substrates, which interact with regulatory proteins and therefore increase gene transcription  <cit> .
above results prompted us to investigate whether antimicrobials affect the expression of the acrd gene in e. amylovora.
therefore, we utilized a transcriptional fusion between the promoter region of acrd and egfp .
in order to determine the promoter activity of acrd, we developed a screening assay in a 96-well-plate format.
antimicrobial compounds were added to the plasmid-harboring cells by the 2-fold dilution method and egfp fluorescence was determined after  <dig> hours.
only fluorescence values from substrate concentrations that did not inhibit bacterial growth were plotted versus optical density on a scatter plot .
outliers, showing higher fluorescence than the remaining dataset, thus potential inducers of acrd expression, were identified as deoxycholate, naringenin, tetracycline and zinc sulfate.
in the next step, the effect on the activity of the acrd promoter was evaluated in batch cultures.
we included novobiocin and fusidic acid since they were identified as substrates of acrd in e. coli <cit> .
additionally, we tested tannin because it displayed a 2-fold induction of acrd in qrt-pcr analysis .
after  <dig> hours incubation, the fluorescence signal was measured and normalized to an od <dig> of  <dig>  .
the tested substrates were able to induce the acrd promoter by approximately  <dig> to 3-fold.
among the tested substrates, deoxycholate and zinc, showed significant differences in comparison to the control .
contribution of acrd to virulence of e. amylovora on apple rootstocks
to study the impact of acrd on virulence of e. amylovora ea <dig>  apple rootstocks mm  <dig> were infected and the development of disease symptoms was monitored.
after one week of incubation all infected shoots showed typical disease symptoms including the shepherd’s crook-like bending of the shoot tip, tissue necrosis and ooze formation surrounding the infection site.
furthermore, bacterial populations were counted  <dig> and  <dig> day post inoculation, respectively.
however, no significant differences between the populations of the wild type and the mutant were observed .
a
a
bacteria were inoculated by prick technique in the shoot tips with an inoculum of  <dig> × 106 cfu/shoot.
establishment of a population of erwinia amylovora ea <dig> and acrd mutant  was determined  <dig> and  <dig> days post inoculation , respectively.
additionally, immature pear fruits were infected with the wild type and the acrd-deficient mutant and disease symptoms were monitored by means of the diameter of necrotic tissue surrounding the infection site .
after  <dig> days of incubation, when the pear fruit was almost completely necrotic, no significant differences between the wild type and the mutant were observed.
transcriptional analysis of acra and acrd of e. amylovora in planta
in order to analyze the acra and acrd promoter activities in planta, ea <dig> was infected into shoot tips of apple rootstocks mm  <dig> as well as into immature pear fruits.
several hours  and days , respectively, after inoculation bacteria were re-isolated by macerating infected plant areas.
total rna was isolated from recovered cells and transcript abundances of acra and acrd were determined by quantitative rt-pcr.
rt-pcr signals of recovered bacteria were compared with rt-pcr signals of ea <dig> cells grown in lb broth to an od <dig> of  <dig> .
for immature pear infections, we first determined the expression of the sigma factor hrpl, which coordinates the transcription of genes of the hypersensitive response and pathogenicity  type iii secretion system in e. amylovora, to identify the time of maximal expression of plant-inducible hrp genes.
compared to growth in lb broth, hrpl expression was 9-fold higher 6 h post inoculation into immature pears; after 12 h, expression was greatest , and after 18 h, hrpl expression decreased again .
hence, we investigated the expression of acra and acrd genes with ea <dig> cells recovered from infected immature pear fruits 12 h after inoculation and compared them with cells grown in lb broth to an od <dig> of  <dig>  .
our results indicated that neither acra nor acrd are induced in the early infection phase of immature pear fruits.
acra
and
acrd
after inoculation of
erwinia amylovora
ea <dig> on apple rootstocks mm <dig> and immature pear fruit slices, respectively
a
b
c
a
total rna was isolated from bacterial cells recovered from infected plant tissues.
transcript abundance of acra and acrd was determined by quantitative rt-pcr and was compared to rt-pcr signal from cells grown in lb broth to an od <dig> of  <dig> .
b
bacteria were re-isolated from infected shoots of apple rootstock  <dig>   <dig> and  <dig> days post inoculation .
c
bacteria were re-isolated from infected immature pears  <dig> hours post inoculation .
for apple rootstock infections, bacteria were re-isolated  <dig>   <dig> and  <dig> days after inoculation, respectively, and compared the abundance of acra and acrd transcripts with cells grown in lb broth .
due to the high activity of the acra promoter in lb broth, expression analysis by quantitative rt-pcr revealed a down regulation of this gene in planta.
on the other hand, since acrd was only expressed at a low level during cellular growth in lb broth, it showed a more than 3-fold induction in planta.
regulation of the rnd-type multidrug efflux pump acrd in e. amylovora
in other enterobacteria, e.g., e. coli and s. enterica, baer is involved in the regulation of the rnd-type efflux pumps mdtabc and acrd  <cit> .
baer is the response regulator of the two-component system baesr, which controls a small set of adaptive factors involved in a unique envelope stress response in e. coli <cit> .
a blastp search using the amino acid sequence of baer from e. coli k <dig> as the query identified a homologous sequence in the genome of e. amylovora cfbp <dig> .
these homologues share 74% amino acid sequence identity with each other.
in order to test whether baer plays a role in the regulation of the acrd promoter in e. amylovora, we analyzed whether the published baer-binding site sequence motif from e. coli  is present in the plant pathogen  <cit> .
indeed we identified a similar motif resembling the baer binding box located at position - <dig> to -148 bp upstream of the coding sequence of acrd in ea1189: 5′-ttcttcacgattactggc-3′ .
to confirm the binding of baer to the acrd promoter in vitro, an electrophoretic mobility shift assay  was performed.
dna fragments used in the emsa were the cy5-labeled upstream region of acrd , and as controls, the upstream regions of acrab  and tolc .
the dna fragments were incubated with increasing amounts of purified baer protein in presence of nonspecific competitor dna  .
the purified baer protein showed binding to the upstream region of acrd with increasing concentrations, which was detected as a smear .
a slight interaction between the acrab promoter and baer was detected at the highest protein concentration , which could suggest an unspecific binding .
no interactions between the fragment of the promoter region of tolc and baer were detected .
these results show that baer binds to the acrd regulatory region and is probably involved in its regulation.
induction of acrd through overexpression of baer
owing to the interaction between the acrd promoter region and baer observed during emsa assays, we investigated whether overexpression of baer may induce the expression of acrd.
therefore, we cloned baer under an arabinose-inducible promoter  and performed qrt-pcr analysis  <dig> hour after induction.
relative fold-changes in mrna transcripts of acra , acrd  and tolc  were determined.
the obtained data values correlate with the observed interaction of baer during emsa indicating a specific binding of baer to the regulatory region of acrd.
discussion
bacteria have evolved energy-dependent multidrug efflux pumps in order to prevent intracellular accumulation of toxic compounds, including antimicrobials, antibiotics, dyes and detergents  <cit> .
in several enterobacteria, including the human pathogen e. coli and the plant pathogen e. amylovora, the rnd transporter acrab-tolc has been described as the major multidrug efflux system providing resistance towards lipophilic and amphiphilic substrates but not towards hydrophilic compounds  <cit> .
another member of the rnd family, acrd, has been shown to efficiently efflux highly hydrophilic aminoglycosides from e. coli cells  <cit> .
here, we identified a gene encoding acrd in e. amylovora ea <dig>  which shows significant sequence homology to the cognate aminoglycoside efflux pump of e. coli and investigated the role of this transporter in the fire blight pathogen.
due to the high level of homology shared by acrd from e. coli and e. amylovora, it was not surprising to find similar substrate specificities.
previous studies of acrd in e. coli have characterized it as an efflux transporter which provides resistance to hydrophilic aminoglycosides as well as to some amphiphilic compounds such as bile acids, novobiocin and fusidic acid  <cit> .
it has also been reported that deletion of acrd does not cause hypersusceptibility to amphiphilic drugs  <cit> , which may be due to low expression levels during cellular growth  <cit> .
we have been able to detect similar low expression levels of acrd in e. amylovora ea <dig> during growth in lb broth .
moreover, we were unable to detect hypersusceptibility to any of the tested antimicrobial compounds in an acrd-deficient mutant .
as noted for other bacteria, the overproduction of acrd in an acrb-deficient host led to increased resistance towards detergents, novobiocin and fusidic acid  <cit> .
overproduction of acrd in an acrb-deficient mutant of e. amylovora ea <dig> increased the resistance to several antimicrobial compounds and heavy metals.
it is noteworthy that expression of acrd under control of the lac promoter displayed only a minor effect on the resistance level compared to acrd expression driven by a combination of the lac promoter and the native promoter .
it has previously been reported that strong overproduction of acrd may interfere with normal activity of the pump  <cit> .
in this study, we identified two new substrates, clotrimazole and luteolin, which increased the substrate spectrum of acrd in enterobacteria.
clotrimazole is a derivative of imidazole, commonly used in the treatment of fungal infections, and acts primarily by inhibiting the activity of cytochrome p <dig> mono-oxygenase  <cit> .
luteolin is one of the most common flavonoids present in many plant families.
one of the functions of flavonoids in plants is their protective role against microbial invasion.
luteolin was shown to inhibit bacterial n-acetyltransferase activity  <cit> .
since acrd conferred resistance to aminoglycosides in e. coli <cit> , we hypothesized that acrd of e. amylovora would display a similar substrate spectrum.
however, overexpression of acrd in e. amylovora ea1189- <dig> did not increase the mics of the aminoglycosides amikacin, gentamicin, streptomycin, and tobramycin.
although it is important to note that we observed occasional, but not reproducible, 2-fold differences between the aminoglycoside mics for different experiments .
while this result is contradictory to previous findings for e. coli <cit> , it may reflect a possible adaptation of the acrd transporter to a particular physiological function during growth in the plant environment.
to elucidate the role of acrd in the plant environment, we analyzed whether this rnd-type efflux pump is involved in pathogenesis of the plant pathogen.
previously, we have observed that disruption of the acrb efflux pump in e. amylovora significantly reduced virulence on apple rootstock  <cit> .
this prompted us to evaluate the effect of acrd on the virulence of the fire blight pathogen by studying development of disease symptoms.
however, one week after inoculation, the wild type as well as the acrd-deficient mutant conferred typical symptoms on all inoculated shoots, indicating that the disruption of the efflux pump had negligible impact on virulence on apple rootstock.
as the host range of e. amylovora also includes pear trees, we further investigated the virulence of the wild type and its acrd-deficient mutant on immature pear fruits  with the conclusion that acrd is not involved in the interaction of the fire blight pathogen with this host.
additionally, we studied the expression levels of the acrab and acrd efflux pumps in vitro and in planta, respectively.
the activity of the acra promoter was lower in planta than in lb medium .
however, it is possible that growth of the bacteria in lb broth may increase expression of the acrab pump.
a similar induction of the rnd-type efflux system mexab-oprm in pseudomonas syringae was observed during growth in complex king’s b medium  <cit> .
specific components of the complex media might induce the expression of these rnd efflux systems.
alternatively, the efflux pumps may play a role in the secretion of metabolites during exponential growth of bacteria in complex medium.
the level of acrd expression was low during growth in lb medium , whereas it was slightly induced in planta  indicating that plant-derived compounds are able to induce the acrd pump.
the nature of these compounds remains to be elucidated.
several multidrug transporters are induced in response to the presence of toxic substances  <cit> .
we identified the substrates deoxycholate, naringenin, tetracycline, novobiocin, fusidic acid, tannin and zinc as inducers of acrd in e. amylovora.
in prokaryotes, the expression of drug transporter genes is frequently mediated by transcriptional regulatory proteins, whose genes are often located adjacent to those encoding the transport system.
however, no local transcriptional regulator was identified flanking the acrd gene in e. amylovora, suggesting that expression of acrd may be subject to regulation at the global level.
the acrd gene belongs to the regulon of the envelope stress response, two-component system baesr in e. coli and salmonella enterica.
a baesr-deficient mutant of e. amylovora ea <dig> has previously been evaluated for virulence on immature pears, and exhibit full-virulence, as that of wild type, on immature pear fruits  <cit> .
the core regulon of baesr consists of spy, encoding a protein chaperon, and the rnd efflux pump genes acrd and mdtabc <cit> .
interestingly, we identified a partial overlap between the compounds inducing expression of acrd in e. amylovora and baer in e. coli, e.g., flavonoids , zinc, and tannin  <cit> .
accordingly, the contribution of the two-component system baesr to regulation of the acrd gene in e. amylovora became of particular interest to us.
in e. coli and s. enterica, baer, upon activation by phosphorylation through baes, binds to the upstream promoter region of mdta and acrd <cit> .
our results showed that baer of e. amylovora is able to bind the promoter region of acrd in e. amylovora, but not to the promoter regions of acra or tolc .
additional investigation of the regulatory networks controlling expression of acrd in growth cultures and in natural environments, such as within host plants, will need to be conducted in order to provide further insights into the role of this multidrug transporter in the physiology of the cell.
in summary, we have identified a homologue of the rnd-type multidrug efflux pump acrd in e. amylovora ea <dig>  despite the fact that acrd of ea <dig> was unable to efflux aminoglycosides, we detected a similar substrate spectrum compared to homologues of acrd from other enterobacteria.
finally, we identified two substrates, clotrimazole and luteolin, hitherto unreported as substrates of acrd in e. coli and s. enterica.
CONCLUSIONS
the aim of the present study was the characterization of acrd, a rnd-type multidrug efflux pump from the plant pathogen e. amylovora, causing fire blight on apple and pear.
our results demonstrated that acrd plays a role in drug resistance to a limited number of amphiphilic compounds.
we showed that the substrate specificity of acrd from e. amylovora and of acrd from e. coli is partly overlapping.
however, in contrast to acrd from e. coli, acrd from e. amylovora cannot provide resistance towards aminoglycosides.
the expression of acrd was up-regulated by the addition of several substrates and was found to be regulated by the envelope stress two-component regulatory system baesr.
an acrd mutant showed full virulence on apple rootstock and immature pear fruits.

 erwinia amylovora is the causative agent of fire blight, a destructive, contagious disease of apple, pear, and other rosaceous plants. all aerial parts of the hosts can be infected by the pathogen. e. amylovora enters its host plants through natural openings and wounds. upon entry, the fire blight pathogen moves through intercellular spaces towards the xylem. typical symptoms include flower necrosis, immature fruit rot, shoot curvature, bacterial ooze secretion, and cankers on woody tissues. the most effective method to treat infected plants is pruning to remove all infected tissue. however, fire blight can infect entire orchards within a single growing season leading to devastating economic losses. presently, there are no effective therapeutics available to cure fire blight and therefore prevention is considered the best solution to manage this plant disease. current control efforts are rather rare and rely primarily on antibiotic applications to protect flowers. however, the use of antibiotics for the management of fire blight is highly controversial due to the potential risk of promoting the emergence and spread of antibiotic resistance. gram-negative bacteria often possess multidrug efflux transporters within the cytoplasmic membrane, which have been found to recognize and expel a broad range of structurally unrelated compounds from the cell. among the multidrug efflux pumps, members of the resistance-nodulation-cell division family appear to be the most effective efflux systems in gram-negative bacteria. rnd transporters form a tripartite complex, consisting of an inner membrane pump that recognizes and captures the substrates, a periplasmic membrane fusion protein and an outer membrane channel. acrab is the major multidrug efflux pump in e. coli and shows high conservation among gram-negative bacteria. acrd, a close homolog of acrb, is an rnd-type efflux pump characterized as a transporter of aminoglycosides, a highly hydrophilic class of molecules, and as a transporter of several amphiphilic compounds. typically, the inner membrane pump and the periplasmic mfp are co-transcribed in tandem on polycistronic mrna molecules. interestingly, this is not the case for acrd, which appears as a single gene and seemingly functions in concert with acra, a mfp co-transcribed with acrb. both acrab and acrd efflux systems are also present in the plant pathogen e. amylovora. acrab has already been characterized as an efflux system required for virulence of e. amylovora in resistance towards apple phytoalexins and for successful colonization of the host plant. acrab of e. amylovora showed a similar substrate spectrum as acrab of e. coli. in this study, the substrate specificity of acrd from e. amylovora was characterized and its contribution to virulence in apple and pear analyzed. as it was found that acrd is expressed only at low levels under in vitro conditions, we were interested in investigating whether the expression of the acrd transporter in e. amylovora is induced in planta. multidrug transporters are often expressed under control of local, as well as, global transcriptional regulators. current data show that expression of acrd in e. coli can be induced by the two-component regulatory system baesr. two-component systems play an important role in the regulation of physiological processes in response to environmental or cellular parameters and enable bacterial cells to adapt to changing environmental conditions. tcss typically consist of a membrane-bound histidine protein kinase whose autokinase activity is dependent upon sensing a specific environmental stimulus. the second protein of a tcs is a response regulator, onto which a phosphoryl group is transferred from the phosphorylated hpk, and which functions as a phosphorylation-activated switch that regulates output responses within the cell causing changes in the expression of target genes. baesr is a tcs that responds cell envelope damages in e. coli. the small core regulon of baesr includes the rnd-type transporters acrd and mdtabc and the periplasmic chaperone spy. the presence of a homologous baesr system in e. amylovora, prompted us to analyze the impact of the response regulator baer on the expression levels of acrd. herein, we report that overexpression of the rnd pump acrd in an acrb-deficient mutant leads to increased resistance to two substrates, clotrimazole and luteolin, previously not described as substrates of acrd in other enterobacteria. in order to determine the promoter activity in vitro, we utilized a transcriptional fusion of the promoter regions of acrab and acrd, respectively, with the reporter gene egfp. we demonstrate that the response regulator baer is able to bind to the upstream region of acrd in e. amylovora ea and to induce acrd expression. furthermore, we show that the inactivation of the rnd pump acrd did not result in reduction of virulence of e. amylovora on host plants.  identification of an acrd homologue in e. amylovora ea1189 a search with the blastp program using the amino acid sequence of acrd from e. coli k- as the query identified a homologous sequence in the genome of e. amylovora cfbp. the annotated protein eamy_ is amino acids shorter at the n-terminus than the acrd protein of e. coli. comparison of the acrd gene sequences from e. coli and e. amylovora suggests that the eamy_ gene of e. amylovora cfbp has been annotated with a wrong start codon. sequence analysis revealed an alternative atg start codon 54 bp upstream of the annotated eamy_ gene. the amino acids, encoded by the additional nucleotides, are 100% identical to the n-terminal region of acrd from e. coli. we used the genome sequence of e. amylovora cfbp to design primers to pcr amplify the respective region from the genomic dna of our model strain e. amylovora ea whose genome sequence is not yet available. the nucleotide sequence of acrd and its upstream region from e. amylovora ea show 100% identity to eamy_ and its upstream region from e. amylovora cfbp based on our sequencing results. acrd is a member of the rnd superfamily of transporters belonging to the hydrophobe/amphiphile efflux- family. a blastp search of the deduced acrd sequence from e. amylovora ea as query identified homologous multidrug efflux transporters with high degree of sequence identity in several members of the enterobacteriaceae family. a sequence alignment between acrd from e. amylovora ea and acrd from e. coli k- showed that the proteins share 79% identity and 89% similarity with each other. substituted amino acids were distributed throughout the sequence, but they were at least 40% conserved and no insertion or deletion was observed. analysis of the up- and downstream regions flanking the acrd homologues from e. amylovora, e. coli and s. enterica revealed several differences including the two-component system narqp located upstream of acrd in e. amylovora. this system is involved in the regulation of anaerobic nitrate/nitrite respiration, and consists of the sensor kinase narq and the response regulator narp. in e. coli and s. enterica, only the sensor kinase narq is present upstream of acrd. the response regulator narp is situated at different positions in the genomes of e. coli and s. enterica. moreover, the sizes of the narq homologues are also disparate. narq of e. amylovora ea is a protein consisting of amino acids, whereas the narq homologues of e. coli and s. enterica consist of amino acids. the downstream region of acrd of e. amylovora ea contains an insertion of about  kb encoding several small hypothetical proteins. transmembrane organization of acrb and acrd in e. amylovora in a previous study, the transmembrane organization of acrb and acrd from e. coli was analyzed in silico, with transmembrane-spanning domains and large periplasmic loops predicted in both proteins. a similar approach was accomplished with acrb and acrd from e. amylovora ea using the online tool topcons. topology analysis predicted the typical tmds and periplasmic loops between tmd and and tmd and for the rnd-type efflux pumps acrb and acrd from e. amylovora ea. phenotypic characterization of the acrd mutant to evaluate the role of acrd in antibiotic resistance and to identify substrates of this rnd-type efflux pump, susceptibility tests of the wild type and the acrd mutant to a variety of antimicrobial agents were performed. deletion of acrd resulted in no significant changes in sensitivity to tested aminoglycosides, dyes or detergents. however, the acrd mutant was 2-fold more sensitive to nitrofurantoin, erythromycin, silver nitrate and sodium tungstate in comparison to the wild type. the differences in sensitivity were minor but reproducible. complementation of the acrd mutant with plasmid pblueks.acrd, which carried the acrd gene of ea under control of the p lac, restored resistance to all tested antimicrobials. e. amylovora wild-type strain, acrd mutant, acrb mutant complemented with acrd-overexpression plasmids pbluesk.acrd and pblueks.acrd-ext and acrb mutant complemented with control plasmid pbluesk.acrd a lac  < < acrd lac > >  acrd lac, p acrd > >  acrd a mic values were determined by the 2-fold dilution assay in three or more independent experiments with similar results. boldface numbers indicate a higher or lower mic. nd, not determined. expression of acrd in an acrb-deficient mutant of e. amylovora to investigate the substrate specificity of acrd from ea overexpression of the corresponding gene from a high-copy plasmid was achieved in e. amylovora mutant ea1189- which is hypersensitive to many drugs due to a deficiency of the major multidrug efflux pump acrb. three overexpression plasmids were generated: pblueks.acrd, expressing acrd under control of the lac promoter, pbluesk.acrd-ext, expressing acrd under control of its native promoter and pblueks.acrd-ext, expressing acrd under control of both promoters plac and pacrd. as a control, a promoterless acrd gene was cloned in the opposite direction of plac. these plasmids were mobilized into the acrb-deficient mutant ea1189- and the sensitivity of the transformants to various substrates were determined. ea1189- expressing acrd under control of plac, exhibited elevated resistance to clotrimazole, fusidic acid, novobiocin, hygromycin b, cadmium acetate, zinc sulfate, bile salt, deoxycholate, and sds. the expression of acrd under control of its native promoter in ea1189- showed an increase in resistance similar to that of plac-controlled acrd expression. when acrd was under control of both promoters, plac and pacrd, it conferred elevated resistance. compared to the control, ea1189- displayed increased resistance to clotrimazole, fusidic acid, novobiocin, hygromycin b, cadmium acetate, zinc sulfate, bile salt, deoxycholate, sds, luteolin and ethidium bromide. rnd-type efflux pump expression during cellular growth to monitor the expression levels of the rnd-type efflux pumps acrab and acrd at different growth states, total rna was isolated at distinct optical densities and expression levels analyzed by quantitative rt-pcr. the expression values were normalized to the highest expression of the acra and acrd transcript, respectively. while the expression levels of acra changed during the cell cycle, indicating a growth phase-dependent transcription with the highest expression in the early exponential phase, acrd showed constant expression during growth. additionally, the constant expression of acrd was also connected to a low expression level as determined by ct values. furthermore, we studied the effect of temperature on activation of the rnd-type efflux pump acrd using qrt-pcr. bacteria were cultured in lb broth at 18°c and 28°c, respectively, where 28°c represents the optimal growth temperature and 18°c represents the temperature at which several genes involved in pathogenicity showed induction in e. amylovora. however, no temperature dependence of the acrd expression was observed in vitro. promoter activity of acrab and acrd in vitro in order to monitor promoter activities of the rnd-type efflux pumps acrab and acrd in e. amylovora, transcriptional fusions of the acra upstream region and acrd upstream region, respectively, to the enhanced green fluorescence protein-encoding gene were constructed. to determine whether bacterial growth influenced the promoter activity, fluorescence measurements at several optical densities were performed. our data indicated that the promoter activities of both acrab and acrd were constant throughout the growth phases in lb broth. furthermore, the activity of the acrd promoter was to 5-fold lower than the activity of the acrab promoter throughout growth. effect of substrate exposure on acrd expression the expression of genes encoding multidrug efflux systems can be influenced by substrates, which interact with regulatory proteins and therefore increase gene transcription. above results prompted us to investigate whether antimicrobials affect the expression of the acrd gene in e. amylovora. therefore, we utilized a transcriptional fusion between the promoter region of acrd and egfp. in order to determine the promoter activity of acrd, we developed a screening assay in a 96-well-plate format. antimicrobial compounds were added to the plasmid-harboring cells by the 2-fold dilution method and egfp fluorescence was determined after hours. only fluorescence values from substrate concentrations that did not inhibit bacterial growth were plotted versus optical density on a scatter plot. outliers, showing higher fluorescence than the remaining dataset, thus potential inducers of acrd expression, were identified as deoxycholate, naringenin, tetracycline and zinc sulfate. in the next step, the effect on the activity of the acrd promoter was evaluated in batch cultures. we included novobiocin and fusidic acid since they were identified as substrates of acrd in e. coli. additionally, we tested tannin because it displayed a 2-fold induction of acrd in qrt-pcr analysis. after hours incubation, the fluorescence signal was measured and normalized to an od of. the tested substrates were able to induce the acrd promoter by approximately to 3-fold. among the tested substrates, deoxycholate and zinc, showed significant differences in comparison to the control. contribution of acrd to virulence of e. amylovora on apple rootstocks to study the impact of acrd on virulence of e. amylovora ea apple rootstocks mm were infected and the development of disease symptoms was monitored. after one week of incubation all infected shoots showed typical disease symptoms including the shepherd’s crook-like bending of the shoot tip, tissue necrosis and ooze formation surrounding the infection site. furthermore, bacterial populations were counted and day post inoculation, respectively. however, no significant differences between the populations of the wild type and the mutant were observed. a a bacteria were inoculated by prick technique in the shoot tips with an inoculum of × 106 cfu/shoot. establishment of a population of erwinia amylovora ea and acrd mutant was determined and days post inoculation, respectively. additionally, immature pear fruits were infected with the wild type and the acrd-deficient mutant and disease symptoms were monitored by means of the diameter of necrotic tissue surrounding the infection site. after days of incubation, when the pear fruit was almost completely necrotic, no significant differences between the wild type and the mutant were observed. transcriptional analysis of acra and acrd of e. amylovora in planta in order to analyze the acra and acrd promoter activities in planta, ea was infected into shoot tips of apple rootstocks mm as well as into immature pear fruits. several hours and days, respectively, after inoculation bacteria were re-isolated by macerating infected plant areas. total rna was isolated from recovered cells and transcript abundances of acra and acrd were determined by quantitative rt-pcr. rt-pcr signals of recovered bacteria were compared with rt-pcr signals of ea cells grown in lb broth to an od of. for immature pear infections, we first determined the expression of the sigma factor hrpl, which coordinates the transcription of genes of the hypersensitive response and pathogenicity type iii secretion system in e. amylovora, to identify the time of maximal expression of plant-inducible hrp genes. compared to growth in lb broth, hrpl expression was 9-fold higher 6 h post inoculation into immature pears; after 12 h, expression was greatest, and after 18 h, hrpl expression decreased again. hence, we investigated the expression of acra and acrd genes with ea cells recovered from infected immature pear fruits 12 h after inoculation and compared them with cells grown in lb broth to an od of. our results indicated that neither acra nor acrd are induced in the early infection phase of immature pear fruits. acra and acrd after inoculation of erwinia amylovora ea on apple rootstocks mm and immature pear fruit slices, respectively a b c a total rna was isolated from bacterial cells recovered from infected plant tissues. transcript abundance of acra and acrd was determined by quantitative rt-pcr and was compared to rt-pcr signal from cells grown in lb broth to an od of. b bacteria were re-isolated from infected shoots of apple rootstock and days post inoculation. c bacteria were re-isolated from infected immature pears hours post inoculation. for apple rootstock infections, bacteria were re-isolated and days after inoculation, respectively, and compared the abundance of acra and acrd transcripts with cells grown in lb broth. due to the high activity of the acra promoter in lb broth, expression analysis by quantitative rt-pcr revealed a down regulation of this gene in planta. on the other hand, since acrd was only expressed at a low level during cellular growth in lb broth, it showed a more than 3-fold induction in planta. regulation of the rnd-type multidrug efflux pump acrd in e. amylovora in other enterobacteria, e.g., e. coli and s. enterica, baer is involved in the regulation of the rnd-type efflux pumps mdtabc and acrd. baer is the response regulator of the two-component system baesr, which controls a small set of adaptive factors involved in a unique envelope stress response in e. coli. a blastp search using the amino acid sequence of baer from e. coli k as the query identified a homologous sequence in the genome of e. amylovora cfbp. these homologues share 74% amino acid sequence identity with each other. in order to test whether baer plays a role in the regulation of the acrd promoter in e. amylovora, we analyzed whether the published baer-binding site sequence motif from e. coli is present in the plant pathogen. indeed we identified a similar motif resembling the baer binding box located at position - to -148 bp upstream of the coding sequence of acrd in ea1189: 5′-ttcttcacgattactggc-3′. to confirm the binding of baer to the acrd promoter in vitro, an electrophoretic mobility shift assay was performed. dna fragments used in the emsa were the cy5-labeled upstream region of acrd, and as controls, the upstream regions of acrab and tolc. the dna fragments were incubated with increasing amounts of purified baer protein in presence of nonspecific competitor dna. the purified baer protein showed binding to the upstream region of acrd with increasing concentrations, which was detected as a smear. a slight interaction between the acrab promoter and baer was detected at the highest protein concentration, which could suggest an unspecific binding. no interactions between the fragment of the promoter region of tolc and baer were detected. these results show that baer binds to the acrd regulatory region and is probably involved in its regulation. induction of acrd through overexpression of baer owing to the interaction between the acrd promoter region and baer observed during emsa assays, we investigated whether overexpression of baer may induce the expression of acrd. therefore, we cloned baer under an arabinose-inducible promoter and performed qrt-pcr analysis hour after induction. relative fold-changes in mrna transcripts of acra, acrd and tolc were determined. the obtained data values correlate with the observed interaction of baer during emsa indicating a specific binding of baer to the regulatory region of acrd. discussion bacteria have evolved energy-dependent multidrug efflux pumps in order to prevent intracellular accumulation of toxic compounds, including antimicrobials, antibiotics, dyes and detergents. in several enterobacteria, including the human pathogen e. coli and the plant pathogen e. amylovora, the rnd transporter acrab-tolc has been described as the major multidrug efflux system providing resistance towards lipophilic and amphiphilic substrates but not towards hydrophilic compounds. another member of the rnd family, acrd, has been shown to efficiently efflux highly hydrophilic aminoglycosides from e. coli cells. here, we identified a gene encoding acrd in e. amylovora ea which shows significant sequence homology to the cognate aminoglycoside efflux pump of e. coli and investigated the role of this transporter in the fire blight pathogen. due to the high level of homology shared by acrd from e. coli and e. amylovora, it was not surprising to find similar substrate specificities. previous studies of acrd in e. coli have characterized it as an efflux transporter which provides resistance to hydrophilic aminoglycosides as well as to some amphiphilic compounds such as bile acids, novobiocin and fusidic acid. it has also been reported that deletion of acrd does not cause hypersusceptibility to amphiphilic drugs, which may be due to low expression levels during cellular growth. we have been able to detect similar low expression levels of acrd in e. amylovora ea during growth in lb broth. moreover, we were unable to detect hypersusceptibility to any of the tested antimicrobial compounds in an acrd-deficient mutant. as noted for other bacteria, the overproduction of acrd in an acrb-deficient host led to increased resistance towards detergents, novobiocin and fusidic acid. overproduction of acrd in an acrb-deficient mutant of e. amylovora ea increased the resistance to several antimicrobial compounds and heavy metals. it is noteworthy that expression of acrd under control of the lac promoter displayed only a minor effect on the resistance level compared to acrd expression driven by a combination of the lac promoter and the native promoter. it has previously been reported that strong overproduction of acrd may interfere with normal activity of the pump. in this study, we identified two new substrates, clotrimazole and luteolin, which increased the substrate spectrum of acrd in enterobacteria. clotrimazole is a derivative of imidazole, commonly used in the treatment of fungal infections, and acts primarily by inhibiting the activity of cytochrome p mono-oxygenase. luteolin is one of the most common flavonoids present in many plant families. one of the functions of flavonoids in plants is their protective role against microbial invasion. luteolin was shown to inhibit bacterial n-acetyltransferase activity. since acrd conferred resistance to aminoglycosides in e. coli, we hypothesized that acrd of e. amylovora would display a similar substrate spectrum. however, overexpression of acrd in e. amylovora ea1189- did not increase the mics of the aminoglycosides amikacin, gentamicin, streptomycin, and tobramycin. although it is important to note that we observed occasional, but not reproducible, 2-fold differences between the aminoglycoside mics for different experiments. while this result is contradictory to previous findings for e. coli, it may reflect a possible adaptation of the acrd transporter to a particular physiological function during growth in the plant environment. to elucidate the role of acrd in the plant environment, we analyzed whether this rnd-type efflux pump is involved in pathogenesis of the plant pathogen. previously, we have observed that disruption of the acrb efflux pump in e. amylovora significantly reduced virulence on apple rootstock. this prompted us to evaluate the effect of acrd on the virulence of the fire blight pathogen by studying development of disease symptoms. however, one week after inoculation, the wild type as well as the acrd-deficient mutant conferred typical symptoms on all inoculated shoots, indicating that the disruption of the efflux pump had negligible impact on virulence on apple rootstock. as the host range of e. amylovora also includes pear trees, we further investigated the virulence of the wild type and its acrd-deficient mutant on immature pear fruits with the conclusion that acrd is not involved in the interaction of the fire blight pathogen with this host. additionally, we studied the expression levels of the acrab and acrd efflux pumps in vitro and in planta, respectively. the activity of the acra promoter was lower in planta than in lb medium. however, it is possible that growth of the bacteria in lb broth may increase expression of the acrab pump. a similar induction of the rnd-type efflux system mexab-oprm in pseudomonas syringae was observed during growth in complex king’s b medium. specific components of the complex media might induce the expression of these rnd efflux systems. alternatively, the efflux pumps may play a role in the secretion of metabolites during exponential growth of bacteria in complex medium. the level of acrd expression was low during growth in lb medium, whereas it was slightly induced in planta indicating that plant-derived compounds are able to induce the acrd pump. the nature of these compounds remains to be elucidated. several multidrug transporters are induced in response to the presence of toxic substances. we identified the substrates deoxycholate, naringenin, tetracycline, novobiocin, fusidic acid, tannin and zinc as inducers of acrd in e. amylovora. in prokaryotes, the expression of drug transporter genes is frequently mediated by transcriptional regulatory proteins, whose genes are often located adjacent to those encoding the transport system. however, no local transcriptional regulator was identified flanking the acrd gene in e. amylovora, suggesting that expression of acrd may be subject to regulation at the global level. the acrd gene belongs to the regulon of the envelope stress response, two-component system baesr in e. coli and salmonella enterica. a baesr-deficient mutant of e. amylovora ea has previously been evaluated for virulence on immature pears, and exhibit full-virulence, as that of wild type, on immature pear fruits. the core regulon of baesr consists of spy, encoding a protein chaperon, and the rnd efflux pump genes acrd and mdtabc. interestingly, we identified a partial overlap between the compounds inducing expression of acrd in e. amylovora and baer in e. coli, e.g., flavonoids, zinc, and tannin. accordingly, the contribution of the two-component system baesr to regulation of the acrd gene in e. amylovora became of particular interest to us. in e. coli and s. enterica, baer, upon activation by phosphorylation through baes, binds to the upstream promoter region of mdta and acrd. our results showed that baer of e. amylovora is able to bind the promoter region of acrd in e. amylovora, but not to the promoter regions of acra or tolc. additional investigation of the regulatory networks controlling expression of acrd in growth cultures and in natural environments, such as within host plants, will need to be conducted in order to provide further insights into the role of this multidrug transporter in the physiology of the cell. in summary, we have identified a homologue of the rnd-type multidrug efflux pump acrd in e. amylovora ea despite the fact that acrd of ea was unable to efflux aminoglycosides, we detected a similar substrate spectrum compared to homologues of acrd from other enterobacteria. finally, we identified two substrates, clotrimazole and luteolin, hitherto unreported as substrates of acrd in e. coli and s. enterica.  the aim of the present study was the characterization of acrd, a rnd-type multidrug efflux pump from the plant pathogen e. amylovora, causing fire blight on apple and pear. our results demonstrated that acrd plays a role in drug resistance to a limited number of amphiphilic compounds. we showed that the substrate specificity of acrd from e. amylovora and of acrd from e. coli is partly overlapping. however, in contrast to acrd from e. coli, acrd from e. amylovora cannot provide resistance towards aminoglycosides. the expression of acrd was up-regulated by the addition of several substrates and was found to be regulated by the envelope stress two-component regulatory system baesr. an acrd mutant showed full virulence on apple rootstock and immature pear fruits. 
an acrd mutant exhibited full virulence on apple rootstock and immature pear fruits. multidrug efflux pumps are membrane translocases that have the ability to extrude a variety of structurally unrelated compounds from the cell. acrd, a resistance-nodulation-cell division transporter, was shown to be involved in efflux of highly hydrophilic aminoglycosides and a limited number of amphiphilic compounds in e. coli. the substrate specificity of acrd was studied by overexpression of the corresponding gene from a high-copy plasmid in e. amylovora ea1189- which is hypersensitive to many drugs due to a deficiency of the major multidrug pump acrb. moreover, a direct binding of baer, the response regulator of the two-component regulatory system baesr, to the acrd promoter was observed as has already been shown in other enterobacteria. acrd from e. amylovora is involved in resistance to a limited number of amphiphilic compounds, but in contrast to acrd of e. coli, it is not involved in resistance to aminoglycosides. here, a homologue of acrd in the plant pathogen and causal agent of fire blight disease erwinia amylovora was identified. plant pathogenfire blighterwinia amylovoramultidrug effluxrnd transporteracrd acrd mediated resistance to several amphiphilic compounds including clotrimazole and luteolin, two compounds hitherto not described as substrates of acrd in enterobacteria. the expression of acrd was up-regulated by addition of the substrates deoxycholate, naringenin, tetracycline and zinc. rt-pcr analysis revealed an induction of acrd expression in infected apple tissue but not on pear fruits.
 multidrug efflux pumps are membrane translocases that have the ability to extrude a variety of structurally unrelated compounds from the cell. acrd, a resistance-nodulation-cell division transporter, was shown to be involved in efflux of highly hydrophilic aminoglycosides and a limited number of amphiphilic compounds in e. coli. here, a homologue of acrd in the plant pathogen and causal agent of fire blight disease erwinia amylovora was identified.  the substrate specificity of acrd was studied by overexpression of the corresponding gene from a high-copy plasmid in e. amylovora ea1189- which is hypersensitive to many drugs due to a deficiency of the major multidrug pump acrb. acrd mediated resistance to several amphiphilic compounds including clotrimazole and luteolin, two compounds hitherto not described as substrates of acrd in enterobacteria. however, acrd was not able to expel aminoglycosides. an acrd mutant exhibited full virulence on apple rootstock and immature pear fruits. rt-pcr analysis revealed an induction of acrd expression in infected apple tissue but not on pear fruits. moreover, a direct binding of baer, the response regulator of the two-component regulatory system baesr, to the acrd promoter was observed as has already been shown in other enterobacteria.  acrd from e. amylovora is involved in resistance to a limited number of amphiphilic compounds, but in contrast to acrd of e. coli, it is not involved in resistance to aminoglycosides. the expression of acrd was up-regulated by addition of the substrates deoxycholate, naringenin, tetracycline and zinc. acrd appears to be regulated by the baesr two-component system, an envelope stress signal transduction pathway. plant pathogenfire blighterwinia amylovoramultidrug effluxrnd transporteracrd 