Expression of the DisA amino acid decarboxylase from Proteus mirabilis

Expression of the DisA amino acid decarboxylase from Proteus mirabilis inhibits motility and class 2?agellar gene expression in Escherichia coli

Lindsay G.Stevenson a ,Bree A.Szostek a ,Katy M.Clemmer b ,Philip N.Rather a ,b ,*

Department of Microbiology and Immunology,Emory University School of Medicine,Atlanta,GA,USA

Veterans Affairs Medical Center,Decatur,GA 30322,USA

Received 20April 2012;accepted 31August 2012

Available online 11September 2012

Abstract

In Proteus mirabilis ,a putative phenylalanine decarboxylase (DisA)acts in a regulatory pathway to inhibit class 2?agellar gene expression and motility.In this study,we demonstrate that DisA expression in Escherichia coli blocked motility and resulted in a 50-fold decrease in the expression of class 2(?iA )and class 3(?iC )?agellar genes.However,the expression of ?hDC encoding the class 1activator of the ?agellar cascade was unchanged by DisA expression at both the transcriptional and translational levels.Phenethylamine,a decarboxylation product derived from phenylalanine,was able to mimic DisA overexpression and decrease both motility and class 2/3?agellar gene expression.In addition,both DisA overexpression and phenethylamine strongly inhibited bio?lm formation in E.coli .DisA overexpression and exogenous phenethylamine could also reduce motility in other enteric bacteria,but had no effect on motility in non-enteric Gram-negative bacteria.It is hypothesized that phenethylamine or a closely related compound formed by the DisA decarboxylation reaction inhibits the formation or activity of the FlhD 4C 2complex required for activation of class 2genes.Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Keywords:Motility;Decarboxylase;FlhDC

1.Introduction

Bacterial motility,mediated through the use of ?agella,plays an important role in locomotion,host colonization,adhesion,and bio?lm formation (Giron et al.,2002;Lane et al.,2005).Two types of ?agellar-mediated motility,swim-ming and swarming,have been described (reviewed in Harshey,2003;Jarrell and McBride,2008).In Escherichia coli ,swimming motility occurs in liquid medium where the predominant cell type is a short vegetative rod with few ?agella.Swarming motility also requires ?agella and occurs in Gram-negative bacteria such as Proteus mirabilis ,Serratia marcescens ,E.coli ,and Salmonella typhimurium when cells contact a solid surface (Allison et al.,1992;Kim and Surette,

2003;Rauprich et al.,1996).During a swarming state,cells become elongated,aseptate and multinucleate and an increase in ?agellar gene expression results in a hyper?agellated swarmer cell.Swarmer cells act in a coordinated manner that may involve cell e cell signaling to move across a solid surface (Sturgill and Rather,2004).In E.coli ,the analysis of de?ned mutants in the Keio collection has revealed 216genes that are speci?cally required for swarming (Inoue et al.,2007).These genes encode diverse functions including:carbon metabolism,DNA replication/repair,ion transport,protein chaperones,amino acid transport,signal transduction and synthesis of cell envelope components.

Motility in E.coli as well as other motile enteric bacteria is regulated via a hierarchical system of gene expression.Details of the regulation of ?agellar expression have been extensively reviewed (Aldridge and Hughes,2002;Fraser and Hughes,1999;Soutourina and Bertin,2003).The apex of this hier-archy is composed of the ?hDC operon (class 1)that is acti-vated by a number of regulatory proteins,some of which

*Corresponding author.Department of Microbiology and Immunology,3001Rollins Research Center,Atlanta,GA,USA.Tel.:t1(404)7285079;fax:t1(404)7287780.

E-mail address:prather@https://www.360docs.net/doc/732314967.html, (P.N.

Rather).Research in Microbiology 164(2013)31e

https://www.360docs.net/doc/732314967.html,/locate/resmic

0923-2508/$-see front matter Published by Elsevier Masson SAS on behalf of Institut Pasteur.https://www.360docs.net/doc/732314967.html,/10.1016/j.resmic.2012.09.002

respond to environmental stimuli(Aldridge and Hughes,2002; Soutourina and Bertin,2003).The?hDC operon encodes a heteromeric transcriptional activator,FlhD4C2,necessary for activation of a second class of genes designated class2(Wang et al.,2006).Class2genes include?agellar hook and basal body motor components as well as the?agella-speci?c sigma factor?iA(Sigma28).Sigma28is necessary for transcription of class3genes such as?iC(?agellin)which complete the ?agella as well as additional motor and chemotaxis proteins (Soutourina and Bertin,2003).

Both P.mirabilis and certain strains of E.coli possess the ability to colonize either host tissue or implanted devices such as catheters and produce a bio?lm community(Jones et al., 2005;Warren,1997).The association of cells within a bio-?lm provides a barrier to antibiotics as well as the host immune system(Jesaitis et al.,2003;Patel,2005).While some strains of?agella-de?cient or non-motile E.coli exhibit decreased or altered bio?lm formation(Pratt and Kolter,1998; Wood et al.,2006),non-motile(?iC mutant)enter-oaggregative E.coli forms a bio?lm indistinguishable from wild type on glass and plastic surfaces(Sheikh et al.,2001). Additionally,overproduction of curli can compensate for lack of?agella(Prigent-Combaret et al.,2001).

In a screen for mutants of P.mirabilis with enhanced swarming motility,a putative amino acid decarboxylase(DisA) was isolated(Stevenson and Rather,2006).When com-plementing the mutation,it was observed that disA in multicopy resulted in complete inhibition of swarming motility.Similarly, swimming motility in P.mirabilis was enhanced by a mutation in disA and abolished by overexpression.Analysis of?agellar gene transcript levels in swarmer cells of P.mirabilis demon-strated that class2(?iA),as well as class3(?aA,encoding ?agellin)gene expression was undetectable when disA was present in multicopy.However,class1(?hDC)expression was not signi?cantly altered.Since FlhD4C2is necessary for acti-vation of?iA,these results suggest that DisA affects?agellar regulation at a point after?hDC transcription and prior to?iA transcription.We propose that in P.mirabilis,DisA affects the post-translational regulation of protein subunits FlhD or FlhC through altering heteromeric(FlhD4C2)interaction,or FlhD4C2 DNA binding.

The putative DisA decarboxylase from P.mirabilis has homology to tyrosine decarboxylases from Carnobacterium divergens and Lactococcus lactis as well as a phenylalanine decarboxylase from Enterococcus faecium.In addition,the only members of the Enterobacteriaceae with a putative DisA homolog are the Providencia spp.Amino acid decarboxylases catalyze the enzymatic removal of the a-carboxyl group of an L-amino acid releasing CO2and forming an amine.Tyrosine decarboxylases catalyze the conversion of L-tyrosine to tyra-mine,while phenylalanine decarboxylase produces phenethyl-amine(Sandmeier et al.,1994).We propose that the decarboxylated product resulting from a DisA-mediated decar-boxylase reaction may regulate?agellar gene expression and motility.In fact,we have shown that in P.mirabilis,phenethyl-amine produces a similar pattern of?agellar gene regulation to that seen with disA in multicopy(Stevenson and Rather,2006).

In this work,we use E.coli and other members of the Enterobacteriaceae to demonstrate that inhibition of motility due to DisA overexpression can be extended to bacteria that do not contain the disA gene.As was observed in P.mirabilis, swimming motility was abolished in E.coli strains when disA was present in multicopy and the defect was due to decreased expression of class2and class3genes,but not the class1 (?hDC)genes.Moreover,we show that the decrease in ?agellar production when DisA is overexpressed can be rescued by?hDC in multicopy.Exposure of E.coli cells to exogenous phenethylamine,the putative product of the DisA decarboxylase,decreased motility and also reduced the expression of class2/3genes in the?agellar regulon.Both DisA overexpression and phenethylamine signi?cantly reduced bio?lm formation in E.coli.Finally,the expression of DisA or the presence of phenethylamine signi?cantly reduced motility in other enteric bacteria,such as S.marcescens, Enterobacter cloacae and Citrobacter koseri,but did not decrease motility of Acinetobacter baumannii,a non-enteric Gram-negative bacterium that is missing the?agellar gene regulatory cascade(Clemmer and Rather,2011).

2.Materials and methods

2.1.Bacterial strains and plasmids

All bacterial strains and plasmids are shown in Table1.All bacterial strains were electroporated by growing cells to mid-log phase in LB broth and harvesting25ml by centrifugation at4000?g.Cell pellets were washed twice with ice cold10% glycerol and resuspended in ice cold10%glycerol at1/100th the original volume for electroporations using0.2cm gap length cuvettes(Bio-Rad).

2.2.Motility assays

Swimming motility assays were done using LB broth containing0.2%Eiken agar for E.coli strains and0.3%for all other bacteria and incubated at37 C except for S.marcescens which was incubated at30 C.Depending on the plasmids present,the media was supplemented with ampicillin(100m g/ ml)or chloramphenicol(25m g/ml).Swarming assays were carried out by spotting equal density cultures onto LB plates with0.5%Eiken agar and0.5%glucose.Plates were incubated at37 C for16e24h.Phenethylamine(Acros)was prepared by adding to media followed by adjusting to pH7.0.For plates,the pH was adjusted to7and monitored using pH paper.Bacterial cultures were grown to mid-log phase and1ul was spotted onto either LB or LB containing phenethylamine plates.Plates were incubated for16h at37 C.

2.3.Construction of E.coli strains for b-galactosidase assays

Transcriptional fusion plasmids pVS175(?iC-lacZ), pVS177(?lA-lacZ),and pVD182(?hD-lacZ)(Sperandio et al.,2002)were electroporated into E.coli MC1061

32L.G.Stevenson et al./Research in Microbiology164(2013)31e37

containing vector control pACYC184or pAC.DisA and selected on chloramphenicol (25m g/ml)and ampicillin (150m g/ml).In addition,pVS175,pVS177and pVS182were electroporated into MG1655D lac for b -galactosidase assays in the presence of phenethylamine.For b -galactosidase assays,strains were grown overnight and diluted 1:1000in LB with antibiotics.Cells were collected at an OD 600of 0.5repre-senting mid-log phase cultures.b -galactosidase assays were carried out as previously described (Miller,1972).2.4.Genes in high copy that can restore motility to MG1655pAC.DisA

Genomic DNA from E.coli PB103was partially digested with Sau 3A and fragments were ligated into Bam HI digested vector pET21a.The plasmid library was electroporated into E.coli MG1655pAC.DisA.The resulting transformants were spread in a line on a 0.3%LB agar plate containing ampicillin (150m g/ml)and chloramphenicol (25m g/ml)and incubated at 30 C.Transformants with restored swimming motility migrated away from the line of growth and were puri?ed by streaking onto fresh agar.Plasmids which restored swimming upon retransformation to MG1655pAC.DisA were sequenced using a T7primer.2.5.Bio?lm assays

MG1655pSK or pSK.DisA was grown to an OD 600of 0.5in LB containing ampicillin (150m g/ml)and then diluted 1:100in fresh LB with ampicillin (150m g/ml).Similarly,MG1655was diluted in LB or LB containing 3mM and 6mM phenethylamine adjusted to pH 7.0with HCl.Dilutions were added to 6wells (200m l/well)of a polystyrene 96-well microtiter plate (Fisher Scienti?c)and plates were incubated at 30 C for the time indicated.Before removal of non-adherent cells,the OD 600was read from each well to measure bacterial growth.Plates were washed 3?in water to remove non-adherent cells before staining with 1%crystal

violet for 20min.Crystal violet was removed by washing in water until control wells were clear.Plates were dried prior to solubilizing the crystal violet with 33%acetic acid and absorbance was measured at 580nm.3.Results

3.1.DisA inhibits motility in E.coli

We previously reported that a putative amino acid decar-boxylase (DisA)from P .mirabilis inhibited swimming and swarming motility when overexpressed from a plasmid vector (Stevenson and Rather,2006).Flagellar systems of P .mirabilis and E.coli have homologous protein components and are likely regulated by similar mechanisms (Claret and Hughes,2000;Furness et al.,1997).Also,detailed examinations of motility systems and ?agellar regulation in E.coli have been completed (Chilcott and Hughes,2000).Therefore,we chose E.coli as a model organism to test if the effect of DisA on motility was observed in other bacteria.E.coli strain MG1655was transformed with a high-copy plasmid,pBluescript.SK,containing the disA gene expressed from its native promoter (pSK.DisA)or with the vector only.As seen in Fig.1A,swimming motility was signi?cantly reduced on motility plates in cells containing pSK.DisA.The block in motility was not due to a decrease in growth rate,as growth was unaffected by the presence of pSK.DisA (data not shown).In addition,a similar loss of motility was observed when disA was present on the medium copy plasmid pACYC184(data not shown).Moreover,the presence of pSK.DisA blocked motility in other E.coli strains,indicating that this effect was not speci?c to MG1655(data not shown).

3.2.Isolation of multicopy suppressors that restore motility to DisA-overexpressing strains

To determine the pathway by which DisA inhibits motility,we initially attempted to isolate second-site suppressors that

Table 1

Bacterial strains and plasmids.Strain

Relevant motility genotype Source or reference Escherichia coli MG1655Motile Lab strain Escherichia coli MC1061Motile Lab strain Citrobacter koseri Motile Lab strain Serratia marcescens Motile Lab strain Enterobacter cloacae

Motile Lab strain Acinetobacter baumannii M2Motile

Lab

strain

Plasmid Relevant features

Source

pBluescript.SK High-copy,Ap R Stratagene pBC.SK High-copy,Cm R

Stratagene

pACYC184Medium copy,Cm R ,Tc R

Chang and Cohen,1978pAC.DisA pACYC184containing disA as a XbaI-SalI fragment This study

pSK.DisA pSKwith disA expressed from its own promoter Stevenson and Rather,2006pBC.DisA pBC.SK with disA expressed from its own promoter Stevenson and Rather,2006pQF1266pQF50containing a 1.3kb fragment with an This study

A.baumannii origin of replication,Amp R

pQF.DisA

pQF1266with disA as a XbaI-SalI fragment expressed

This study from its own promoter

L.G.Stevenson et al./Research in Microbiology 164(2013)31e 37

restored motility to E.coli overexpressing DisA.Despite repeated attempts,we were unable to obtain such suppressors.Therefore,we attempted to isolate multicopy suppressors of the motility defect in cells overexpressing DisA.Presumably,if DisA was blocking a key component of ?agellar gene expression,then overexpressing this protein should bypass any DisA-mediated inhibition.A partial Sau 3A E.coli genomic library generated in the vector pET21a was electroporated into MG1655pAC.DisA and transformants were screened for restoration of swimming motility.In this plasmid library,there is no expression from the T7promoter,but inserts with their own promoter can overexpress gene products due to their presence on a multicopy plasmid.

After screening approximately 6600transformants,two classes of plasmids were found to restore motility of MG1655pAC.DisA.Twelve of these isolates contained the same approximately 12kb insert which included the ?agellar master operon ?hDC as well as other motility and chemotaxis genes.A second class of plasmids that restored motility contained an approximately 4kb insertion with only the ?hDC operon.3.3.Class 2and 3?agellar gene expression in E.coli is blocked by DisA expression

To determine if DisA overexpression inhibited motility by targeting the ?agellar gene cascade,a panel of lacZ fusions to class 1(?hDC ),class 2(?iA )and class 3(?iC )genes was assayed.The expression of b -galactosidase from a plasmid-based ?hD-lacZ transcriptional fusion (pVS182)was exam-ined in the presence of the compatible plasmid pACYC184(vector only)or pAC.DisA (Table 2).In both cases,?hDC expression was similar (Table 2).The expression of a single-copy translational FlhDC-LacZ fusion was also similar in the presence or absence of DisA,indicating that DisA does not alter transcription or translation of ?hDC (data not shown).In contrast,the expression of multicopy transcriptional lacZ fusions to class 2(?iA )and class 3(?iC )genes were reduced 56and 55-fold,respectively,when disA was present in high copy (Table 2).

3.4.Phenethylamine inhibits motility and expression of class 2and class 3genes in E.coli

The DisA decarboxylase shares strongest homology with tyrosine and phenylalanine decarboxylases.In P .mirabilis ,phenethylamine,but not tyramine,speci?cally altered class 2/3gene expression (Stevenson and Rather,2006).Therefore,the effect of phenethylamine on motility was examined with E.coli MG1655,which exhibited a strong inhibition of swim-ming motility at 12mM phenethylamine (Fig.1B).Since DisA overexpression speci?cally altered class 2(?iA )and class 3(?iC )gene expression,the effect of phenethylamine on the expression of these genes was also examined.In the presence of 12mM phenethylamine,the expression of ?iA-lacZ was reduced (3-fold)and a ?iC-lacZ fusion was reduced (2.2-fold).As a control,phenylalanine at 12mM had no signi?cant effect on ?iA or ?iC expression (data not shown).3.5.DisA overexpression and phenethylamine decrease bio?lm production in E.coli MG1655

To determine if DisA overexpression affected bio?lm formation,a time course was carried out with bio?lm measurements taken at 14,and 24h post inoculation.Bio?lm was inhibited by DisA at each time point,with a 30%decrease at 14h and a 60%decrease at 24h (Fig.2).Given that both DisA and phenethylamine inhibited ?agellar gene expression

Table 2

Effects of DisA overexpression on ?agellar gene expression.Fusion Plasmid

b -galactosidase a Fold-reduction pVS182(?hD-lacZ )pACYC18422498?962pVS182(?hD-lacZ )pAC.DisA 21863?38911pVS177(?iA-lacZ )pACYC18430214?183pVS177(?iA-lacZ )pAC.DisA 537?11556pVS175(?iC-lacZ )pACYC18416052?521pVS175

(?iC-lacZ )

pAC.DisA

293

?19

Determined in Miller units from cells harvested at an O.D 600?0.5.Values represent quadruplicate samples from two independent

experiments.

Fig.1.Effect of DisA overexpression and phenethylamine on swimming motility in E.coli .In panel A,E.coli MG1655containing pSK or pSK.DisA were grown to an OD 600of 0.6and 1m l was spotted onto 0.2%motility agar for swimming assays.Plates were grown at 37 for 16h.In panel B,E.coli MG1655cultures were grown to an OD 600of 0.6and 1m l was spotted onto 0.2%motility agar containing either LB (A)or phenethylamine at a concentration of 12mM (B).Plates were incubated at 37 for 16h.

34L.G.Stevenson et al./Research in Microbiology 164(2013)31e 37

in a similar pattern,bio?lm formation was examined in the presence of phenethylamine.At 24h,in 3mM and 6mM phenethylamine,bio?lm formation was decreased 53and 52%,respectively (Fig.3).These concentrations did not signi?cantly alter growth rate (data not shown).3.6.DisA overexpression can inhibit motility in other members of the Enterobacteriaceae

To determine if the inhibition of FlhD 4C 2activity by DisA could be extended to other members of the Enterobacteriaceae,strains of S.marcescens , E.cloaceae and C.koseri were transformed with high copy plasmids containing the disA gene.In all cases,the presence of disA in multicopy resulted in a signi?cant inhibitory effect on swimming motility (Fig.4).Each of these bacteria contains a ?agellar regulatory system

similar to that of E.coli with FlhD and FlhC proteins that are highly similar.In each of these bacteria,motility was also inhibited at least 70%by 12mM phenethylamine (data not shown).

To investigate if the inhibitory effect of DisA over-expression on motility could be observed in bacteria that do not contain the FlhD and FlhC proteins,the disA gene was transformed into A.baumannii ,a motile Gram-negative bacterium.In contrast to the previous bacteria,the over-expression of DisA in A.baumannii did not reduce motility (Fig.4A).4.Discussion

In this study,it has been demonstrated that a putative phenylalanine decarboxylase (DisA)isolated from P.mirabilis could alter ?agellar-mediated motility of E.coli and other members of the Enterobacteriaceae that contained the FlhD and FlhC proteins.The overexpression of DisA also decreased bio?lm formation,which can involve ?agellar-mediated motility.Through the use of ?agellar gene fusions in E.coli ,it was shown that expression of class 2and class 3,but not class 1,gene expression was decreased in the presence of DisA.This corresponds to previous work in P.mirabilis where a similar pattern of gene regulation was observed by northern blot analysis of genes in the ?agellar regulon (Stevenson and Rather,2006).These data suggest that a conserved mecha-nism for the action of DisA is present in both organisms.In an attempt to identify multicopy suppressors of the DisA-mediated inhibition of motility,an E.coli library was screened for the restoration of swimming motility to MG1655pAC.DisA.These plasmids all contained the ?hDC operon.The use of lacZ fusions demonstrated that disA in multicopy did not inhibit ?hDC transcription or translation.In addition,the stability of the FlhD or FlhC proteins is not altered by DisA expression (Stevenson and Rather,2006).Therefore,it

Fig.2.Effect of DisA overexpression on bio?lm formation.Bio?lm formation was measured by crystal violet staining of E.coli MG1655containing vector (pSK)or disA in multicopy (pSK.DisA).Cultures of equal density were diluted 1:100in LB containing ampicillin and aliquoted into 96-well polystyrene plates.Density was measured at 14and 24h after incubation at 30 prior to washing and staining with crystal violet for bio?lm formation.The average of 6wells per strain is shown.The growth rates of cells containing pSK and pSK.DisA were similar.P -values for the bio?lm assays were less than 0.05between MG1655/pSK and MG1655/pSK.DisA for all

conditions.

Fig.3.Effect of phenethylamine on bio?lm formation.E.coli MG1655was diluted 1:500in LB only or LB containing various concentrations of phene-thylamine (PEA)and aliquoted into 8wells per sample of a 96-well poly-styrene microtiter plate.Bio?lm formation was measured after 24h by crystal violet staining of adherent cells.P -values were less than 0.05between LB and the two concentrations of phenethylamine tested.The growth rate of cells in either concentration of phenethylamine was not signi?cantly different than in LB only.

L.G.Stevenson et al./Research in Microbiology 164(2013)31e 37

suggested that FlhDC is regulated by DisA at the post-translational level by altering the formation of heteromul-timers or affecting the ability of FlhD 4C 2to bind DNA.

Based on our previous studies in P .mirabilis (Stevenson and Rather,2006),we propose that phenethylamine or a closely related molecule formed by the DisA decarboxyl-ation reaction is directly mediating the inhibition of FlhDC activity at the post-translational level.Although exogenous phenethylamine did not inhibit class 2?agellar gene expres-sion to the same extent as DisA overexpression,this may be due to the inability to achieve the same intercellular concen-tration of decarboxylated product.For example,DisA over-expression may generate higher intracellular amounts of this product than can be transported when it is supplied exoge-nously,possibly due to inef?cient uptake into the cell.It is also possible that phenethylamine is not the actual product of the reaction catalyzed by DisA,but suf?ciently similar in structure to have partial activity.

The ?nding that ?agellar gene regulation and motility of E.coli expressing DisA is similar to that in P .mirabilis indicates that both organisms have a conserved target for the DisA product.E.coli does not encode an obvious DisA ortholog;however,a functionally similar enzyme may be present in E.coli with limited similarity to DisA.In addition,amino acid decarboxylation products such as phenethylamine are found in the human digestive tract and may in?uence bacterial attach-ment to intestinal mucosa by their effects on ?agellar-mediated motility (Lyte,2004;Fischer et al.,2010).In support of this,E.coli possesses genes which convert phene-thylamine to phenylacetic acid,suggesting the possibility that

cells may encounter phenethylamine in the environment (Hanlon et al.,1997).This study reveals a potentially impor-tant application of phenethylamine for the inhibition of motility in the Enterobacteriaceae.For example,the devel-opment of urinary tract catheters impregnated with phene-thylamine could signi?cantly reduce both motility and bio?lm formation on catheter surfaces and reduce the incidence of catheter-associated urinary tract infections.Acknowledgments

We are grateful to V .Spernandio for providing the ?agellar gene transcriptional fusion plasmids.This work was supported by a Merit Review award to P.N.R.from the Department of Veterans Affairs.P.N.R.is also a recipient of a Research Career award from the Department of Veterans Affairs References

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