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					                                       Heterologous Production of Albicidin: a
                                       Promising Approach to Overproducing and
                                       Characterizing This Potent Inhibitor of DNA
                                       Gyrase
                                       Eric Vivien, Delphine Pitorre, Stéphane Cociancich, Isabelle
                                       Pieretti, Dean W. Gabriel, Philippe C. Rott and Monique
                                       Royer
                                       Antimicrob. Agents Chemother. 2007, 51(4):1549. DOI:
                                       10.1128/AAC.01450-06.
                                       Published Ahead of Print 12 January 2007.




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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2007, p. 1549–1552                                                                   Vol. 51, No. 4
0066-4804/07/$08.00 0 doi:10.1128/AAC.01450-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



      Heterologous Production of Albicidin: a Promising Approach to
        Overproducing and Characterizing This Potent Inhibitor of
                             DNA Gyrase
       Eric Vivien,1 Delphine Pitorre,1 Stephane Cociancich,1 Isabelle Pieretti,1 Dean W. Gabriel,2
                                          ´
                                Philippe C. Rott,1 and Monique Royer1*
                  ´                                                            ´
    Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement, UMR Agro.M-Cirad-Inra BGPI,
             Campus International de Baillarguet, TA 41/K, 34398 Montpellier Cedex 5, France,1 and Department of
                               Plant Pathology, University of Florida, Gainesville, Florida 326112
                  Received 20 November 2006/Returned for modification 18 December 2006/Accepted 27 December 2006

           The phytotoxin and polyketide antibiotic albicidin produced by Xanthomonas albilineans is a highly potent




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         DNA gyrase inhibitor. Low yields of albicidin production have slowed studies of its chemical structure.
         Heterologous expression of albicidin biosynthetic genes in X. axonopodis pv. vesicatoria resulted in a sixfold
         increase in albicidin production, offering promising strategies for engineering overproduction.


   Albicidin is produced by Xanthomonas albilineans which is                of the biosynthetic enzymes, (ii) to obtain large quantities of
a slow-growing bacterium and the causal agent of sugarcane                  albicidin, and (iii) to characterize albicidin through nuclear
leaf scald (13). Albicidin is involved in the pathogenicity of              magnetic resonance and mass spectrometry analyses. We
X. albilineans and inhibits the replication of chloroplastic                report here the construction of a two-plasmid expression
DNA (6, 7). Albicidin also inhibits DNA replication in Esch-                system harboring the complete albicidin biosynthetic gene
erichia coli at nanomolar concentrations (4), whereas mam-                  set; its transfer into a fast-growing heterologous host, X.
malian cells are unaffected at 8 g/ml (3). A recent study                   axonopodis pv. vesicatoria; and the subsequent production
showed that albicidin targets DNA gyrase with features of                   of albicidin.
inhibition that differ from those of other known antibiotics                  Two wide-host-range plasmids derived from IncW vector
(11). Albicidin is synthesized by a unique hybrid polyketide                pUFR043 (10) and IncP vector pLAFR3 (16) were used for
synthase-nonribosomal peptide synthetase (PKS-NRPS)                         the transfer of the complete albicidin biosynthetic machin-
pathway that does not resemble any other pathway de-                        ery. The previously described IncW plasmid pALB571 (12,
scribed to date. Three genomic regions (XALB1, XALB2,                       14) was used to transfer albI to albIX (the insert corresponds
and XALB3) involved in albicidin biosynthesis were cloned                   to nucleotides 19001 to 55839 of accession no. AJ586576).
and sequenced (12, 14, 15, 17). XALB1 contains the major-                   Plasmid pLAFRK7 was constructed by ligating together into
ity of albicidin biosynthetic genes in a cluster of 20 open                 pLAFR3 the three following genomic DNA fragments: (i)
reading frames (ORFs) (albI to albXX). ORFs albI, albIV,                    nucleotides 5896 to 19216 of accession no. AJ586576 (har-
and albIX encode three large PKSs and NRPSs, whereas the                    boring albX to albXX), (ii) nucleotides 1 to 2986 of accession
other ORFs are resistance, modifying, and regulatory genes.                 no. AJ586577 (harboring albXXI), and (iii) nucleotides 5510
XALB2 and XALB3 each contain a single biosynthetic gene,                    to 8119 of accession no. AM039979 (harboring albXXII),
albXXI and albXXII, whose products are, respectively, a                     respectively. Both plasmids were transferred by triparental
phosphopantetheinyl transferase and the heat shock protein                  mating into X. axonopodis pv. vesicatoria strain Xcv 91-118
HtpG. The antibiotic activity of albicidin against a wide                   (1), and bioassays for albicidin production were performed
range of gram-positive and gram-negative pathogenic bac-                    with exconjugants. Triparental matings were performed as
teria (Enterobacter aerogenes, E. coli, Haemophilus influen-                 described previously (12) except that exconjugants were se-
zae, Klebsiella pneumoniae, Shigella sonnei, and Staphylococ-               lected on SPA medium (2% sucrose, 0.5% peptone, 1.5%
cus aureus) is of interest for the development of new                       agar) supplemented with kanamycin at 50 g/ml (for the
antibacterial drugs (4, 5). Low yields of albicidin production              transfer of pALB571) and tetracycline at 12 g/ml (for the
in slow-growing X. albilineans have slowed studies of its                   transfer of pLAFRK7). Bioassays for albicidin production
chemical structure and potential therapeutic applications. A                were performed as described previously (12) except that (i)
heterologous system for albicidin overproduction is there-                  exconjugants were spotted onto SPA medium supplemented
fore highly desirable (i) to increase the levels of expression              with gentamicin at 3 g/ml, tetracycline at 12 g/ml, and
                                                                            morpholinepropanesulfonic acid (MOPS) buffer and (ii) the
                                                                            plates were overlaid with a suspension of E. coli DH5
  * Corresponding author. Mailing address: Centre de Cooperation
                                                             ´              harboring pUFR043 and pLAFR3 empty plasmids that confer
                                                     ´
Internationale en Recherche Agronomique pour le Developpement,              resistance to gentamicin and tetracycline, respectively. All excon-
UMR Agro.M-Cirad-Inra BGPI, Campus International de Baillarguet,
TA 41/K, 34398 Montpellier Cedex 5, France. Phone: (33) 4 99 62 48
                                                                            jugants harboring plasmids pALB571 and pLAFRK7 produced
44. Fax: (33) 4 99 62 48 08. E-mail: monique.royer@cirad.fr.                an antibiotic that inhibited the growth of E. coli DH5 . No anti-
    Published ahead of print on 12 January 2007.                            biotic production was observed with control exconjugants harbor-

                                                                     1549
1550      NOTES                                                                                                  ANTIMICROB. AGENTS CHEMOTHER.




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   FIG. 1. Analysis of antibiotics separated by TLC from supernatants of cultures of strains X. albilineans Xa23R1 and X. axonopodis pv.
vesicatoria Xves-alb. Samples prepared from bacterial supernatants were subjected to TLC, and TLC strips were laid on top agar layers containing
different E. coli indicator strains: DH5 (lane a); DH5 Albr (lane b), a spontaneous albicidin-resistant DH5 derivative described previously (12);
DH5 transformed with the pGEX-4T-3 empty vector (Pharmacia) (lane c); and DH5 transformed with pGEXAlbD (DH5 AlbD) and
expressing albD, an albicidin-detoxifying gene conferring albicidin resistance in E. coli (19) (lane d). All E. coli indicator strains were transformed
with pUFR043 and pLAFR3 empty vectors to confer resistance to gentamicin and tetracycline, respectively. The initial positions of the samples
on the chromatograms are indicated by “x”.



ing either pALB571 or pLAFRK7 and one empty vector (and                       identical to the one observed with X. albilineans cultures or
therefore missing some albicidin biosynthetic genes). These data              with albicidin semipurified from X. albilineans. In addition,
demonstrated that transfer of the complete albicidin biosynthetic             several E. coli DH5 clones that spontaneously grew within
machinery into X. axonopodis pv. vesicatoria led to the heterolo-             the growth inhibition zone of Xves-alb were resistant to
gous production of an antibiotic that was named albicidinves. The             albicidin, confirming the cross-resistance between the two
exconjugant yielding the largest DH5 growth inhibition zone                   antibiotics. Albicidin and albicidinves appeared, therefore,
was designated Xves-alb and was used for further analyses.                    to display the same biological activities.
  Cross-resistance between albicidin and albicidinves was                        The growth of Xves-alb and the production of albicidinves
characterized by bioassays with agar cultures of Xves-alb                     were monitored in several rich liquid media supplemented with
using E. coli strains expressing a wide range of albicidin                    gentamicin at 3 g/ml and tetracycline at 12 g/ml: NYG
resistance determinants. Strain DH5 Albr (a spontaneous                       (0.3% yeast extract, 0.5% peptone, 2% glycerol), TY (0.3%
albicidin-resistant DH5        derivative [12]) and strain                    yeast extract, 0.5% tryptone, 5 mM CaCl2), and YM (0.3%
DH5 AlbD (expressing the albicidin-detoxifying gene albD                      yeast extract, 0.3% malt extract, 1% peptone). Maximum albi-
[19]) were completely resistant to albicidinves. Strains har-                 cidinves activity was obtained in early stationary phase from
boring albXIV (an albicidin efflux pump gene conferring                        shaken cultures grown at 28°C, and the best results were obtained
albicidin resistance in E. coli [8]), albXIX (an albicidin qnr                in NYG with an albicidinves titer of 970 g/liter. Albicidin was
gene conferring resistance in E. coli [11]), or sbmC (a gyrase                quantified with a bioassay, using the following formula devel-
inhibitor resistance gene [2]) were also resistant to albi-                   oped by Zhang et al. (20): free albicidin (ng/ml)           4.576
cidinves but to a lesser extent. This resistance pattern was                  e(0.315 inhibition zone diameter in mm). This titer was six times
VOL. 51, 2007                                                                                                                         NOTES          1551


higher than the titer of albicidin produced by X. albilineans in     replacement of TTG codons for the initiation of translation by
the optimized SP8 medium (0.5% sucrose, 0.23% peptone,               ATG codons in order to suppress any negative posttranscrip-
0.1% yeast extract, 3 mM K2HPO4, 1 mM MgSO4; pH 7). In               tional control due to the presence of these TTG codons; (iii)
order to minimize the presence of catabolites in the superna-        the overexpression of the albicidin efflux pump (and/or the Qnr
tant and to facilitate the subsequent purification of albicidinves,   albicidin resistance protein) in order to facilitate the excretion
we investigated growth of Xves-alb and the production of             of the antibiotic into the culture medium and potentially in-
albicidinves in minimal media without gentamicin and tetracy-        crease resistance; and (iv) the overexpression of the biosyn-
cline. We tested the XVM2 medium [20 mM NaCl, 10 mM                  thetic enzymes functioning in trans in order to improve the
(NH4)2SO4, 5 mM MgSO4, 1 mM CaCl2, 0.16 mM KH2PO4,                   efficacy of the PKS-NRPS system. This latter approach was
0.32 mM K2HPO4, 0.01 mM FeSO4, 0.03% Casamino Acids;                 successfully used to improve the production of leinamycin by
pH 6.7] (18) supplemented with a single carbon source (man-          Streptomyces atroolivaceus (9). The present study is a first sig-
nitol, glucose, fructose, sucrose, or glycerol). The best albi-      nificant step in the overproduction of albicidin that will be
cidinves titer (200 g/liter) was obtained in XVM2 supple-            necessary to purify large quantities of albicidin and to charac-
mented with 2% glycerol. A decrease of albicidin production in       terize this potent inhibitor of DNA gyrase that differs from
minimum media was also observed in X. albilineans (4).               other known antibiotics.
   The chromatographic behaviors of albicidin and albi-




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cidinves were then compared by thin-layer chromatography               We thank Sandrine Duplan for technical support. We thank Felipe
(TLC). Albicidinves was analyzed from cultures of Xves-alb           Moreno for supplying the SbmC-producing pMR100 strain and Lian
grown to early stationary phase (48 h) in 5 liters of XVM2           Hui Zhang for supplying the AlbD-producing E. coli strain.
                                                                       E.V. was supported by a fellowship from the Direction Scientifique
medium supplemented with 2% glycerol and without any                 du CIRAD.
antibiotic, and albicidin was analyzed from cultures of X.
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an antibiotic activity at a 1,000-fold dilution were pooled           2. Baquero, M. R., M. Bouzon, J. Varea, and F. Moreno. 1995. SbmC, a stationary-
                                                                         phase induced SOS Escherichia coli gene, whose product protects cells from the
and vacuum dried to a final volume of 0.5 ml. Then, 2- l                  DNA replication inhibitor microcin B17. Mol. Microbiol. 18:301–311.
samples were separated by TLC chromatography with silica              3. Birch, R. G. 1983. Phytotoxin production by Xanthomonas albilineans, the
gel 60F254 TLC aluminum sheets (20 by 20 cm; Merck,                      sugarcane leaf scald pathogen. Ph.D. thesis. University of Hawaii, Honolulu.
                                                                      4. Birch, R. G., and S. S. Patil. 1985. Preliminary characterization of an anti-
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Subsequently, strips of the TLC plate were placed for 2 h at             Escherichia coli. J. Gen. Microbiol. 131:1069–1075.
                                                                      5. Birch, R. G., and S. S. Patil. June 1985. Antibiotic and process for the
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taining an E. coli indicator strain. Inhibition zones, observed       6. Birch, R. G., and S. S. Patil. 1987. Correlation between albicidin production
after overnight incubation at 37°C, documented the posi-                 and chlorosis induction by Xanthomonas albilineans, the sugarcane leaf scald
                                                                         pathogen. Physiol. Mol. Plant Pathol. 30:199–206.
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albicidin were tested (Fig. 1). All indicator strains harbored           replication. Physiol. Mol. Plant Pathol. 30:207–214.
                                                                      8. Bostock, J. M., G. Huang, S. M. Hashimi, L. Zhang, and R. G. Birch. 2006. A
pUFR043 and pLAFR3 empty plasmids that confer resis-                     DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albi-
tance to gentamicin and tetracycline, respectively (antibiot-            cidin antibiotic resistance in Escherichia coli. J. Appl. Microbiol. 101:151–160.
                                                                      9. Cheng, Y. Q., G. L. Tang, and B. Shen. 2003. Type I polyketide synthase
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with the two DH5 derivative sensitive indicator strains for          10. De Feyter, R., and D. W. Gabriel. 1991. Use of cloned DNA methylase genes
                                                                         to increase the frequency of transfer of foreign genes into Xanthomonas
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absence and a significant intensity reduction of inhibition               The phytotoxin albicidin is a novel inhibitor of DNA gyrase. Antimicrob.
                                                                         Agents Chemother. 51:181–187.
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                                                                         Xanthomonas albilineans. J. Bacteriol. 178:4590–4596.
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   In conclusion, we describe here the heterologous produc-              J. C. Comstock, B. J. Croft, and A. S. Saumtally (ed.), A guide to sugarcane
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                                                                     14. Royer, M., L. Costet, E. Vivien, M. Bes, A. Cousin, A. Damais, I. Pieretti, A.
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those observed with X. albilineans. In addition to growing               2004. Albicidin pathotoxin produced by Xanthomonas albilineans is encoded
faster than X. albilineans, this heterologous production sys-            by three large PKS and NRPS genes present in a gene cluster containing also
                                                                         several putative modifying, regulatory, and resistance genes. Mol. Plant-
tem has the substantial advantage of being more easily mod-              Microbe Interact. 17:414–427.
ifiable by genetic engineering because albicidin biosynthetic         15. Royer, M., D. W. Gabriel, R. Frutos, and P. Rott. November 2006.
                                                                         Complete biosynthetic gene set for synthesis of polyketide antibiotics,
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din in this heterologous system: (i) the replacement of na-          16. Staskawicz, B., D. Dahlbeck, N. Keen, and C. Napoli. 1987. Molecular
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1552       NOTES                                                                                                         ANTIMICROB. AGENTS CHEMOTHER.

    P. C. Rott, and M. Royer. 2005. Xanthomonas albilineans HtpG is required       19. Zhang, L., and R. G. Birch. 1997. The gene for albicidin detoxification
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