The Global Arginine Regulator ArgR Controls Expression of argF in Pseudomonas syringae pv. phaseolicola but Is Not Required for the Synthesis of Phaseolotoxin or for the Regulated Expression of argK

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The Global Arginine Regulator ArgR Controls Expression of argF in Pseudomonas syringae pv. phaseolicola but Is Not Required for the Synthesis of Phaseolotoxin or for the Regulated Expression of argK
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    10.1128/JB.186.11.3653-3655.2004. 2004, 186(11):3653. DOI: J. Bacteriol. Alvarez-MoralesGarcidueñas-Piña, Alba E. Jofre-Garfias and Ariel José Luis Hernández-Flores, Karina López-López, Rogelio  argK  Expression of Phaseolotoxin or for the Regulatedbut Is Not Required for the Synthesis of pv. phaseolicola Pseudomonas syringae   in argF  Controls Expression of The Global Arginine Regulator ArgR http://jb.asm.org/content/186/11/3653Updated information and services can be found at: These include:  REFERENCES http://jb.asm.org/content/186/11/3653#ref-list-1This article cites 28 articles, 7 of which can be accessed free at: CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:  http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:  onM a y 1  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om  onM a y 1  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om   J OURNAL OF  B  ACTERIOLOGY , June 2004, p. 3653–3655 Vol. 186, No. 110021-9193/04/$08.00  0 DOI: 10.1128/JB.186.11.3653–3655.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved. The Global Arginine Regulator ArgR Controls Expression of   argF   in  Pseudomonas syringae  pv. phaseolicola but Is Not Required for theSynthesis of Phaseolotoxin or for the Regulated Expressionof   argK  † Jose´ Luis Herna´ndez-Flores, Karina Lo´pez-Lo´pez, Rogelio Garciduen˜as-Pin˜a, Alba E. Jofre-Garfias, and Ariel Alvarez-Morales*  Departamento de Ingenierı´a Gene´tica de Plantas, Centro de Investigacio´n y de Estudios Avanzados I.P.N.,Unidad Irapuato, Irapuato Gto., C.P. 36500, Me´xico Received 18 September 2003/Accepted 10 February 2004 In  Pseudomonas syringae  pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by  argF  , is negatively regulated by  argR , similar to what has been reported for  Pseudomonas aeruginosa . However,production of the phaseolotoxin-resistant OCTase encoded by  argK  , synthesis of phaseolotoxin, and infectivityfor bean pods occur independently of the ArgR protein.  Pseudomonas syringae  pv. phaseolicola causes halo blightdisease in common bean (  Phaseolus vulgaris  L.), which is char-acterized by water-soaked lesions surrounded by chlorotic ha-loes. Chlorosis is caused by a non-host-specific toxin known asphaseolotoxin [  N   -(  N   -sulfodiaminophosphinyl)-ornithyl-ala-nyl-homoarginine] (15, 16), which is produced ex planta whenbacteria are grown in minimal medium at temperatures be-tween 18 and 20°C and is not detected at 28°C (7, 14, 19, 26).The target of phaseolotoxin is the enzyme ornithine car-bamoyltransferase (OCTase) (EC 2.1.3.3) (4), which catalyzesthe conversion of ornithine and carbamoylphosphate to citrul-line in the arginine biosynthesis pathway.In  P. syringae  pv. phaseolicola the  argF   and  argK   genes codefor phaseolotoxin-sensitive OCTase and phaseolotoxin-resis-tant OCTase, respectively (5, 9, 10, 27).  argK   is expressed at18°C and ensures a supply of arginine for both cell growth andphaseolotoxin synthesis, since arginine is a substrate for anamidinotransferase encoded by  amtA  (8) that catalyzes thesynthesis of homoarginine and ornithine (12).  argK   and  amtA have been shown to be located close to each other on thechromosome of   P. syringae  pv. phaseolicola (8), and their rel-ative proximity, as well as their G  C content, which differsfrom that of other genes in this bacterium, is consistent withthe hypothesis that the genes involved in phaseolotoxin syn-thesis were horizontally acquired (24, 25).There is evidence that  argK   is negatively regulated at 28°C,and it has been suggested that the repressor of   argK   binds tospecific sequences designated the thermoregulatory region(TRR) (22, 23). It has been shown previously that in fact  argK  is not directly regulated by temperature but most likely isregulated by a precursor of phaseolotoxin resembling car-bamoylphosphate (11). In  Pseudomonas aeruginosa argF   is re-pressed when growth occurs in media that provide high levelsof exogenous arginine, such as King’s medium B (KB), to-gether with the repressor protein ArgR, which also repressesthe  carAB  operon involved in carbamoylphosphate biosynthe-sis (20, 21). ArgR is a global regulator of the AraC/XylS family(3, 6, 20) and is also required for expression of genes involvedin arginine catabolism (  aru ) and uptake and transport (  aot- argR  operon) (18, 21). The following findings are interesting:(i) both  argF   and  argK   are negatively regulated; (ii) these twogenes code for enzymes that catalyze the same enzymatic re-action; (iii) the repressor molecule involved in  argK   regulationseems to be capable of interacting with carbamoylphosphate(11); (iv) an open reading frame with homology to a regulatorymolecule belonging to the AraC family has been reported to bein the phaseolotoxin gene cluster (28); and (v) in  Pseudomonas , ArgR is a global regulator that negatively regulates  argF   and isalso involved in regulation of carbamoylphosphate synthesis.Could the ArgR product of   P. syringae  pv. phaseolicola beinvolved in the regulation of   argK   or the synthesis of phaseo-lotoxin? By using  argR  from  P. aeruginosa  as a probe, thecorresponding gene from  P. syringae  pv. phaseolicola NPS3121 was identified and isolated from a genomic library. Analysis of this sequence with BLAST-N (1) and BLAST-X showed that itexhibited 54.5% similarity with  argR  from  P. aeruginosa  PAO1at the nucleotide level and that the level of similarity of thetranslation products is 81.5% (  argR  from  P. syringae  pv. phase-olicola, GenBank database accession number AAL35898). Construction of an  argR -defective mutant.  A 60-bp fragmentfrom the middle of the  argR  structural gene was replaced witha 1.8-kbp DraI/HpaI tetracycline resistance cassette frompUIRM504 (13). Double recombinants were selected as tetra-cycline-resistant clones and were confirmed by Southern blot-ting (data not shown). One mutant, UILH13R, was analyzedfurther, and its phenotype was identical to that reported for the  argR  mutant of   P. aeruginosa . Thus, UILH13R was not able touse arginine as a sole carbon source (20), and it constitutivelyexpressed  argF  ; in KB and M9 medium the OCTase specificactivities of the  P. syringae  pv. phaseolicola wild-type strain * Corresponding author. Mailing address: Departamento de Ing-enierı´a Gene´tica de Plantas, Centro de Investigacio´n y de Estudios Avanzados I.P.N., Unidad Irapuato, Irapuato Gto., Me´xico. Phone: 52462 62 39600. Fax: 52 462 62 45849. E-mail: aalvarez@ira.cinvestav.mx.† This work is dedicated to the memory of our friend and colleagueEsther de Ita Morales.3653   onM a y 1  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om    were 0.37  0.09 and 0.95  0.44 nmol of citrulline producedper  g of protein per min (mean  standard deviation;  n  3)at 37 ° C, respectively, whereas the speci fi c activities of theUILH13R  argR  mutant were 1.23  0.34 and 1.42  0.06 nmolof citrulline produced per   g of protein per min at 37 ° C,respectively. These values were determined by measuringOCTase speci fi c activity essentially as described by Ceriotti (2)by using 5-ml cultures of both  P. syringae  pv. phaseolicola andthe UILH13R mutant grown overnight at 28 ° C in KB or M9medium. Cells were disrupted with a VirTis sonicator (modelVirSonic 60). For the assay we used 3  l of crude extract. Thereaction mixtures were incubated for 20 min at 37 ° C, andOCTase activity was determined. The activity measured corre-sponded to the product of   argF  , the phaseolotoxin-sensitiveOCTase, and not to the phaseolotoxin-resistant OCTase en-coded by  argK  , since addition of a phaseolotoxin-containingsupernatant to the reaction mixture and preincubation for 25min before OCTase activity was determined eliminatedOCTase activity from the samples (data not shown). Binding of ArgR to the promoter region of   argF  .  Gel retar-dation experiments were carried out by using a 275-bp DNA probe from the  argF   promoter region containing the  35 and  10 regions and the ArgR binding sequence TGTCGCN 8  AA (21). The reaction mixture [20 mM Tris (pH 8.0), 50 mM KCl,1 mM EDTA, 1 mM dithiothreitol, 10 ng of poly(dI-dC)  l  1 ,5% glycerol, 5   g of crude extract] was preincubated for 10min at room temperature, the radioactively labeled probe ([  - 32 P]dATP) was added, and the mixture was incubated for 15min at room temperature. Samples were loaded onto a 5%polyacrylamide gel in 0.5  Tris-borate-EDTA and electropho-resed for 60 min at 18 mA. A clear retardation signal of the  argF   promoter probe was observed when crude extract fromthe wild-type strain grown at 28 ° C in rich medium was added tothe retardation mixture, indicating that the ArgR repressor was bound to the operator of   argF   (Fig. 1). As expected, whencrude extract from the wild-type strain grown in minimal me-dium at the same temperature was used, only a weak signal wasobserved. When crude extract from UILH13R grown in richmedium was used, no retardation signal was detected, con fi rm-ing that the protein binding to  argF   was the product of   argR , which is not present in this strain. Speci fi c binding of ArgR to  argF   was demonstrated when the nonlabeled  argF   promoterprobe ef  fi ciently outcompeted labeled  argF   for binding and theretardation signal disappeared almost completely.To analyze the possibility that ArgR binds to  argK   DNA, weobtained an  argK   promoter probe containing both the coremotif G/CAAAG of the putative binding domain (TRR) in- volved in temperature-mediated regulation and identi fi ed in  argK   (23) and a sequence (TGTCG) similar to the core se-quence of the binding site of ArgR (TGTCGCN 8  AA) (21)located 80 bp upstream of the ATG initiation codon (17). Thenonlabeled  argK   promoter probe failed to compete the bindingof   argF   by ArgR, clearly indicating that the postulated proteinthat binds to the  argK   promoter is not the product of   argR .It could be argued that the inability of   argK   to compete forbinding of ArgR to  argF   could have been predicted because ithas been proposed that the repressor would bind to the TRR.However, only weak binding of the putative repressor proteinto the TRR sites in  argK   has been postulated (23). Further-more, the TRR domains in  argK   may not be involved in itsregulation since we have shown that this gene is not directlyregulated by temperature but most likely is regulated throughinduction mediated by a precursor of phaseolotoxin resem-bling carbamoylphosphate (11). Therefore, there seem to betwo proteins that bind to the TRR, one protein which bindstightly to strictly temperature-regulated promoters and a dif-ferent protein which acts as a repressor for  argK   with only weakbinding to the TRR and which is not temperature regulated. Synthesis of phaseolotoxin.  Forty-milliliter portions of M9medium were inoculated with the wild-type strain and mutantUILH13R to obtain an initial optical density at 600 nm of 0.1and incubated at 18 ° C. Parallel controls were grown at 28 ° C.Supernatants were collected after 60 h and used for phaseolo-toxin bioassays (26). Supernatants from the wild-type strainand UILH13R grown at 18 ° C produced similar inhibition pat-terns in the bioassay (Fig. 2), whereas cultures grown at 28 ° Cdid not produce phaseolotoxin, indicating that the absence of  ArgR did not have any qualitative or quantitative effect onphaseolotoxin synthesis. Role of the ArgR regulatory protein in pathogenicity.  Beanpod infection assays were carried out by using the wild-typestrain and the  argR  mutant. The results clearly showed that 1 2 3 4 5 6BA FIG. 1. Gel retardation assay performed with extracts from the  P. syringae  pv. phaseolicola wild-type strain and the  argR  mutantUILH13R. Lane 1, free  argF   promoter probe; lane 2,  argF   promoterprobe plus extract from the wild-type strain grown in rich medium at28 ° C; lane 3,  argF   promoter probe plus extract from the wild-typestrain grown in minimal medium at 28 ° C; lane 4,  argF   promoter probeplus extract from UILH13R grown in rich medium at 28 ° C; lane 5,competition with nonlabeled  argF   promoter probe; lane 6, competition with nonlabeled  argK  .  “ B ”  indicates the position of retardation signalsobserved in lanes 2 and 6 and very weak signals in lanes 3 and 5; noretardation signal was observed in lane 4.  “  A  ”  indicates the position of free probe. A B2121 FIG. 2. Phaseolotoxin bioassay. The assay was performed by usingsupernatants from  P. syringae  pv. phaseolicola (spot 1) and UILH13R(spot 2) grown in minimal medium at 18 ° C (A) and 28 ° C (B).3654 NOTES J. B  ACTERIOL  .   onM a y 1  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om   symptom development with the wild-type strain and symptomdevelopment with UILH13R were similar (Fig. 3).  P. syringae pv. tomato DC3000 was included in the assay to observe atypical hypersensitive response. The lack of ArgR in strainUILH13R did not lead to a hypersensitive response in theplants.The fact that ArgR does not act upon  argK  , the fact that thisprotein is not involved in the synthesis of phaseolotoxin, andthe fact that its absence does not interfere with pathogenicitysupport the idea that phaseolotoxin synthesis genes weregained by horizontal gene transfer during evolution (24, 25),and therefore there seems to be no metabolic link between theacquired set of genes for phaseolotoxin biosynthesis and theresident genes involved in arginine metabolism. We thank Beatriz Jime ´ nez-Moraila for sequencing and oligonucle-otide synthesis, Juan Campos Guille ´ n for technical assistance with theisolation of mutants, Gustavo Acevedo for help with the retardationassays, and Gustavo Herna ´ ndez-Guzma ´ n for critically reading themanuscript.We acknowledge support from a grant from the National Councilfor Science and Technology (CONACYT-Me ´  xico) to A. Alvarez-Mo-rales and from a fellowship to K. Lo ´ pez-Lo ´ pez. REFERENCES 1.  Altschul, S. F., T. L., Maddan, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman.  1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res.  25: 3389 – 3402.2.  Ceriotti, G.  1974. Ornithine carbamoyltransferase, p. 691 – 698.  In  H. V.Bergmeyer (ed.), Methods of enzymatic analysis, 2nd ed., vol. 2. VerlagChemie International, Deer fi eld Beach, Fla.3.  Egan, S. M.  2002. Growing repertoire of AraC/XylS activators. J. Bacteriol. 184: 5529 – 5532.4.  Ferguson, A. R., and J. S. Johnston.  1980. Phaseolotoxin: chlorosis, ornithineaccumulation and inhibition of ornithine carbamoyltransferase in differentplants. Physiol. Plant Pathol.  16: 269 – 275.5.  Ferguson, A. R., J. S. Johnston, and R. E. Mitchell.  1980. Resistance of   Pseudomonas syringae  pv  phaseolicola  to its own toxin, phaseolotoxin. FEMSMicrobiol. 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Evol. 49: 627 – 644.26.  Staskawicz, B. J., and N. J. Panopoulos.  1979. A rapid and sensitive assay forphaseolotoxin. Phytopathology  69: 663 – 666.27.  Staskawicz, B. J., N. J. Panopoulos, and N. J. Hoogenraad.  1980. Phaseolo-toxin insensitive ornithine carbamoyltransferase of   Pseudomonas syringae  pv.  phaseolicola : basis for immunity to phaseolotoxin. J. Bacteriol.  142: 720 – 723.28.  Zhang, Y. X., and S. S. Patil.  1997. The  phtE  locus in the phaseolotoxin genecluster has ORFs with homology to genes encoding amino acid transferases,the AraC family of transcriptional factors, and fatty acid desaturases. Mol.Plant-Microbe Interact.  10: 947 – 960. 1 2 3 4 FIG. 3. Pod inoculation assay. Fully developed green pods fromsusceptible bean plants (  P. vulgaris  cv. Flor de Mayo) were inoculatedby puncturing them with toothpicks soaked in fresh cultures of theUILH13R mutant (spot 1),  P. syringae  pv. phaseolicola (spot 2),  P. syringae  pv. tomato DC3000 (spot 3), and sterile distilled water (spot4). A typical hypersensitive response was observed in spot 3, and nodifference in lesion formation was observed between wild-type bacteria(spot 2) and the  argR  null mutant (spot 1). Inoculated pods wereincubated inside a sealed plastic container with a wet paper towel in agrowth room at 28 ° C. At this temperature, phaseolotoxin production was not expected to occur, but lesion development was clear. Thelesions were examined 4 days postinoculation.V OL  . 186, 2004 NOTES 3655   onM a y 1  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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