Genetic and Functional Heterogeneities Among Fluorescent Pseudomonas Isolated from Environmental Samples

J. Gen. Appl. Microbiol., 57, 101‒114 (2011) Full Paper

Genetic and functional heterogeneities among ﬂ uorescent Pseudomonas isolated from environmental samples

Inès Mehri,1,2 Yousra Turki,2 Mohamed Chair,1 Hanène Chérif,1 Abdennasser Hassen,2 Jean-Marie Meyer,3 and Maher Gtari1,*

1 Laboratoire Microorganismes et Biomolécules actives Faculté des Sciences de Tunis, Campus Universitaire, 2092, Tunis, Tunisia 2 Laboratoire Traitement et Recyclage des Eaux, Centre de Recherche et des Technologies des Eaux, Borj-Cédria, Tunisia 3 Laboratoire de Microbiologie et de Génétique, Université Louis-Pasteur, CNRS FRE 2326, Strasbourg, France

(Received July 29, 2010; Accepted January 13, 2011)

Fluorescent Pseudomonas from diverse environmental samples including wastes were identifi ed and screened for the solubilization of tricalcium phosphate, indole-3-acetic acid (IAA), production and inhibition of extracellular N-acylhomoserine lactone (AHLs) and characterized for their siderophores. Genotypic analysis by amplifi ed rDNA restriction analysis (ARDRA) and BOX-A1R- based repetitive extragenic palindromic-PCR (BOX-PCR) typing resulted respectively in 14 AR- DRA types and 24 different BOX-types with diverse incidence among the analyzed strains. Based on 16S rRNA sequence analysis the isolates were assigned to P. aeruginosa, P. otitidis, P. plecoglossicida, P. mosselii, P. monteilii, P. koreensis, P. taiwanenesis, P. frederiksbergensis and P. graminis. Of the 66 isolates, 56 (84.85%) isolates solubilized tri-calcium phosphate (TCP), 53 (80.30%) isolates produced plant growth hormone IAA, 62 (94%) produced bacteriocin and 34 (52%) isolates produced extracellular N-acylhomoserine lactone while 30 (45%) isolates were able to interfere with N-acylhomoserine lactone. Isolates were clustered into 17 siderotypes and 59Fe cross-incorporation experiments permitted assignment of all siderotypes but two into well- defi ned siderovars.

Key Words—AHLs; ARDA; BOX-PCR; IAA; PVD; Pseudomonas; TCP

Introduction sponsible for frequently lethal nosocomial infections (Ali et al., 1995; Fuchs et al., 2001; Römling et al., Due to their elevated metabolic versatility the 1994). Plant deleterious Pseudomonas, like Pseudomo- Pseudomonas are among the most ubiquitous bacte- nas syringae, produce toxins that affect the plant ria (Römling et al., 1994). Pseudomonas “sensu stric- growth (Fuchs et al., 2001). These bacteria are primar- to” group I (Kozo, 1995) is the largest of the groups, ily foliar pathogens producing diverse types of disease and includes both fl uorescent and non fl uorescent symptoms including necrosis, galls, and cankers bacteria. The type species of the group, Pseudomonas (Fuchs et al., 2001). Pseudomonas fl uorescens and aeruginosa, is an opportunistic human pathogen re- Pseudomonas putida are considered to be rhizobacte- ria that promote plant growth via enzymes and hormones such as phosphatase, indole-3-acetic acid * Address reprint requests to: Dr. Maher Gtari, Laboratoire Mi- croorganismes et Biomolécules actives Faculté des Sciences (IAA) and antifungal metabolites such as antibiotic and de Tunis, Campus Universitaire, 2092, Tunis, Tunisia. other toxic activity against various deleterious micoor- Tel: +216‒70‒860‒553 Fax: +216‒70‒860‒553 ganisms (microbial antagonism) (Latour et al., 2003; E-mail: [email protected] Loper and Henkels, 1999; Rangarajan et al., 2001). 102 MEHRI et al. Vol. 57

Several Pseudomonas species of rRNA group I share pensions were serially diluted and 0.1 ml aliquots of the ability to produce and excrete, under iron limiting each dilution were spread onto King’s medium B (KB) conditions, soluble yellow green fl uorescence pig- agar in triplicate. After incubation at 28°C for 2 days, ments (Bultreys et al., 2003) named pyoverdines fl uorescent Pseudomonas colonies from replicate (PVDs) or pseudobactins, which act as siderophores plates were identifi ed under UV light (366 nm). Purifi ed for these bacteria (Meyer, 2000). These molecules are single colonies were further streaked onto KB agar thought to be associated with pathogenesis (Fuchs et plates to obtain pure cultures. Stock cultures were al., 2001). Pseudomonas species also employ com- made in Luria Bertani (LB) broth containing 50% (w/v) plex communication systems that link cell density and glycerol and stored at -80°C. gene expression to regulate a broad range of biologi- DNA extraction, ARDRA and 16S rRNA gene se- cal functions (Fuqua, et al., 2001; Miller and Bassler, quencing. A single bacterial colony was inoculated 2001). Such cell-to-cell communication is termed quo- into 5 ml LB and grown for 16 h at 30°C. Saturated rum sensing (QS). Of particular interest is the fi nding culture was harvested with centrifugation for 3 min at that QS regulates pathogenicity, or pathogenicity-re- 12,000 rpm. The cell pellet was resuspended and ly- lated functions, in bacteria of medical or environmen- sed in 200 µl of lysis buffer (40 mM Tris-acetate pH 7.8, tal importance (Bjarnsholt et al., 2010; De Kievit and 20 mM sodium-acetate, 1 mM EDTA, 1% SDS) by vigor- Iglewski, 2000; Venturi, 2006). ous pipetting. To remove most proteins and cell de- Consequently this prominent property makes the bris, 66 µl of 5 M NaCl solution was added and mixed Pseudomonas attractive candidates for use in biore- well, and then the viscous mixture was centrifuged at mediation and biocontrol activities (Palleroni, 1984). 12,000 rpm for 10 min. An equal volume of chloroform The overall goal of the present study is to analyze was added to the clear supernatant. Following centrif- the genetic and functional diversity of fl uorescent ugatinon at 12,000 rpm for 3 min, the extract superna- Pseudomonas isolates obtained from diverse environ- tant was precipitated with 100% EtOH, washed twice ments. Strains have been screened for the production with 70% EtOH, dried and dissolved in 50 µl 1× TE of a siderophore (pyoverdine), enzymes and/or phyto- buffer (Chen and Kuo, 1993). PCR amplifi cations were hormones such as phosphatase and indole-3-acetic performed using the following primers: forward primer acid. Furthermore, these microorganisms have been Ps-(5′-G GT CTGAGAGGATGATCAGT-3′) and reverse identifi ed as capable of inhibiting a wide range of bac- primer Ps-rev (5′-TTAGCTCCACCTCGCGGC-3′) for teria through the production of bacteriocin and/or quo- 16S rRNA gene (Widmer et al., 1998). rum-quenching enzymes. ARDRA profi les were determined using the following restriction enzymes: HaeIII, HinfI, AluI, RsaI, MspI and Materials and Methods HhaI (Promega, Madison, and WI, USA). The 16S rRNA gene PCR products were purifi ed from PCR reaction Bacterial strains. Pseudomonas strains described mixtures using the QIAquick Wizard PCR Purifi cation in the present study were collected from diverse envi- Kit (Promega), according to manufacturers instruc- ronments. Environmental samples were transported to tions. The sequences were determined by cycle se- the laboratory in sterile stomacher bags, stored at 4°C, quencing using the Taq Dye Deoxy Terminator Cycle and analyzed within 24 h. Strains whose designations Sequencing Kit (Applied Biosystems, HTDS, Tunisia), begin with PsWw, PsWs, PsWt and PsS were collected and underwent fragment separation in an ABI Prism respectively from waste water, sea water, thermal wa- 3130 DNA sequencer as previously described (Gtari et ter and soil. Strains designated PsC and PsTP were al., 2004). Similarity matrix of 16S rRNA gene sequenc- isolated from compost and a waste water treatment es with closest neighbors and identifi cation were plant. Clinical strains, kindly provided by Dr. Gouban- achieved using the EzTaxon server (http://www.eztaxon. tini, were isolated in the infectious disease service of org/) (Chun et al., 2007). The NCBI Accession Num- Rabta Hospital, Tunisia, and are designated PsCL. bers for the 16S rRNA gene sequences of the 66 iso- Isolation and growth conditions. Briefl y, soil sus- lates determined in this present study are HM627564‒ pensions were obtained by shaking 10 g or 10 ml sub- HM627629. samples in 90 ml of 0.1 M MgSO4･7H2O buffer for BOX-PCR. BOX-PCR was performed as described 10 min at 180 rpm on a rotary shaker. Resulting sus- by Gtari et al. (2004). Mixtures contained 1× PCR buf- 2011 Fluorescent Pseudomonas isolated from environmental samples 103 fer, 2 mM MgCl2, 0.1 mM dNTPs, 0.8 µM of BOX-A1R tion in minimal medium containing (per liter) KH2PO4, primer, 5% of dimethylsulfoxide, 1.3 U of Taq DNA 6.8 g; MgSO4･7H2O, 0.2 g; (NH4)2SO4, 2.0 g; citrate, polymerase and standardized 15 ng of genomic DNA 2.0 g; H3BO3, 0.006 g; ZnO, 0.006 g; FeCl3･6H2O, in a fi nal volume of 30 µl. Reactions were denatured at 0.0024 g; CaCO3, 0.02 g; and HCl, 0.13 ml, supplement- 94°C for 5 min, subjected to 35 cycles of 94°C for 1 min, ed with glucose (10 g) and L-tryptophan (100 µg ml-1), 45°C for 1 min and 72°C for 2 min and a fi nal extension using Salkowski’s reagent (Gordon and Weber, 1951). at 72°C for 10 min. PCR products were checked on The concentration of IAA in each culture medium was agarose gel electrophoresis. determined by comparison with a standard curve. Iso-Electric Focusing (IEF) analysis of PVDs and Phosphate solubilization. Cells were streaked onto PVD-mediated iron uptake. Iron-poor liquid growth Pikovskaya’s agar medium, which contains (per liter): medium was the Casamino Acid (CAA) medium, con- 0.5 g yeast extract, 10 g dextrose, 5 g Ca3(PO4)2, 0.5 g sisting of (per liter) 5 g of low-iron Bacto Casamino (NH4)2SO4, 0.2 g KCl, 0.1 g MgSO4･7H2O, 0.0001 g Acid (Difco), 1.54 g of K2HPO4･3H2O, and 0.25 g of MnSO4･H2O, 0.0001 g FeSO4･7H2O and 15 g agar. Af- MgSO4･7H2O, and was mainly used for PVD-IEF anal- ter 3 days of incubation at 28°C, strains that induced a ysis and PVD purifi cation through the Amberlite XAD-4 clear zone around the colonies were considered as (XAD) procedure as described previously (Meyer et positive (Katznelson and Bose, 1959). al., 2002). The cultures were incubated on a rotary Bacteriocin production. Culture supernatant was shaker (200 rpm) at 25°C.The model 111 mini-IEF cell prepared as follows: an overnight culture of each iso- from Bio-Rad was used. Casting of the gels (5% poly- late was centrifuged at 150 rpm. The resulting super- acrylamide containing 2% Bio-Lyte 3/10 ampholytes) natant was neutralized, sterilized by fi ltering and as- and electric focusing were performed according to sayed for the presence of an inhibitor in the broth the manufacturer’s recommendations. One-microliter following the Agar well diffusion assay technique samples of PVDs (aqueous XAD-purifi ed solutions (Barefoot and Klaenhammer, 1983) as follows. Nutri- [6.5 mg/ml]), or of culture supernatants (40-h CAA- ent agar was fi rst seeded with the indicator organism grown culture supernatant concentrated 20-fold by ly- (110 µl of overnight culture per 20 ml of agar) in sterile ophilisation) were used. PVD bands in the gel were Petri dishes, and after solidifi cation, dried for 15 min. visualized under UV light at 365 nm and photographed Wells of uniform diameter were bored in the agar. Ali- just after focusing. Their respective isoelectric pH val- quots of the cell-free supernatant were dispensed in ues (pI values) were determined with the Easy win 32 the wells, and the plates were incubated overnight at program as described by Fuchs et al. (2001). 30°C. Inhibition of growth was determined by an area For the PVD-mediated iron uptake experiment, as of inhibition surrounding each agar well. described previously by Meyer et al. (2002), iron- Quorum sensing and quorum quenching bioassays. starved cells are incubated in succinate medium un- AHL production and degradation ability of the isolates der non-proliferating conditions in the presence of a were detected by cross streaking against Chromobac- label mix containing 59Fe-PVD complex. Aliquots of the terium violaceum CV026 as the AHL biosensor (Swift bacterial suspension are withdrawn at different time et al., 1999). Supernatants from 7-day-old cultures of intervals and rapidly fi ltered on 0.45 µm porosity mem- the isolates were adjusted to pH 7 and 15 µl of each branes. The cells remaining on the fi lters are thorough- supernatant was directly used (for the AHL produc- ly washed and the radioactivity measuring the amount tion) or mixed with Pseudomonas aeruginosa PAO1 of label iron incorporated during the incubation time is supernatant (for the AHL degradation) and loaded into determined with a gamma radioactivity counter. Con- the hole of a bioassay plate overlaid with C. violaceum trol assays without bacteria were performed to verify CV026. the complete solubility of labeled iron through PVD complexation. Results Indole-3-Acetic Acid (IAA) production. Detection of indole-3-acetic acid (IAA) production was performed Identifi cation and genetic diversity of Pseudomonas according to the method described by Patten and isolates Glick (2002). The amount of auxin was measured Of a total of 438 cryopreserved strains maintained in spectrophotometrically at 535 nm after 72 h of incuba- the LTRE collection (Laboratoire Traitement et Recy- 104 MEHRI et al. Vol. 57 - + 9.03 TCP IAA Bacteriocin AHL - +( 3) + 0.11 +(11)

inhibition

- - + + 0.43 +(13) + + 0.43 +( 2) + + 0.86 +(29) ++ + 0.27 +(10) + 0.52 +( 9) + + 1.76 +(28) + + 0 +(11) + + 0 +(24) + + 0.31 +(25) ++ + 3.35 +( 5) + 0.47 +( 7) + + 0.50 +(12) AHL production a sp. (Pa6) + + 3.16 +(32) (Pa6) + + 1.41 +(23) (Pa6) + + 1.18 +(25) (Pa6) + + 0 +(19) (Pa6) + + 0.23 +(27) PVD-type isolates. species affi liation species affi P. aeruginosa P. (PAO1) P. aeruginosa P. Pseudomonas (Ag13) P. aeruginosa P. (PAO1) P. aeruginosa P. P. aeruginosa P. 27853) (ATCC P. aeruginosa P. 27853) (ATCC aeruginosa P. (PAO1) aeruginosa P. P. aeruginosa P. (PAO1) P. aeruginosa P. (PAO1) P. aeruginosa P. (PAO1) aeruginosa P. 27853) (ATCC P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. (PAO1) P. aeruginosa P. (PAO1) aeruginosa P. (PAO1) P. aeruginosa P. 27853) (ATCC I I I I I I I I I II II II II III III III III III XII Siderovar Pseudomonas 7 6 8 5 4 4 1 2 1 3 2 1 5 4 4 3 1 2 2 BOX BOX profi le profi A III A III A III A III AA III III AI A III AII AI AI A III AII AI A III AI AI AI E III profi le profi ARDRA LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG BCRC BCRC (AY953147) (AY953147)

P. otitidis P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. aeruginosa P. P. aeruginosa P. P. taiwanensis P. Closest 16S rDNA ) P. taiwanensis ) P. Genetic and functional diversity among sequence (%) with type strain (accession number) 100

(99.88) ( 1242T (Z76651) 1242T (Z76651) 17751T (EU103629) (99.88) 1242T (Z76651) 1242T (Z76651) (99.88) (99.88) (99.88) MCC10330T (100) 17751T (EU103629) 1242T (Z76651) 1242T (Z76651) 1242T (Z76651) 1242T (Z76651) (99.88) 1242T (Z76651) 1242T (Z76651) (99.88) 1242T (Z76651) (99.76) 1242T (Z76651) 1242T (Z76651) 1242T (Z76651) (99.88) 1242T (Z76651) 1242T (Z76651) /

Table 1. Table /

(DQ095903) (DQ095915) (AB088116) (AB088116) (AB088116) (AB088116) (AB088116) (AB088116) (AB088116) (AB088116) (AJ249451)/ (DQ464061)/ (AJ249451)/ (GQ217529) (99.88) (AB073312) (99.88) (J249451)/ (AJ249451) (AJ249451) (99.88) (AJ249451) (99.88) (AJ249451) (99.88) (AB126582) (99.88) (EF062513)/ (AJ249451)/ (AJ249451)/ (GU121439) (99.87) (AB073312) (99.76) (AJ876736)/ (AF064458)/ (AF064458) (EF523547) (DQ060242)/ (DQ060242)/ (accession number) Blast aeruginosa putida thermaerum

Closest 16S rDNA sequence P. P. monteilii P. plecoglossicida P. P. aeruginosa P. thermaerum P. P. aeruginosa P. P. thermaerum P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. aeruginosa P. P. thermaerum P. P. aeruginosa P. P. monteilii P. thermaerum P. P. guezennei P. otitidis P. putida P. plecoglossicida P. P. aeruginosa P. P. aeruginosa P. P. thermaerum P. P. aeruginosa P. P. aeruginosa P. P. thermaerum P. P. aeruginosa P. P. aeruginosa P. P. aeruginosa P. P. thermaerum P. aeruginosa P. treatment plant treatment plant Strains Origin PsS4 Soil PsWw168 Wastewater PsWw84 Wastewater PsC132 Compost PsWw127 Wastewater PsS150PsCLHMC1 Soil Clinical PsC12 Compost PsTp160 Wastewater PsS2 Soil PsC5 Compost PsWw174 Wastewater PsS3 Soil PsC99 Compost PsWt157 Thermal water PsTp179 Wastewater PsWt138 Thermal water PsCL10C Clinical PsWw175 Wastewater 2011 Fluorescent Pseudomonas isolated from environmental samples 105 - + 1.41 TCP IAA Bacteriocin AHL - +(11) + 0 +(13) +( 1) ++( 6) 0.91 + +( 8) 0.98 +(14) +(20) + 0.78 +(10) +( 9) + 0 +( 8)

+(24) + 0.82 +(13) +( 4) + 1.37 +(18) +(23) + 0 +(20) +(25) + 1.50 +(30) +( 4) + 1.11 +(12) +( 3) + 1.21 +(10) +(30) + 0.27 +(25) +(18) + 0 +(35) +(26) ++(23) 1.45 + +(17) 1.02 +(15) +(20) + 2.52 +(13) +(18) + 1.25 +(15) +(28) + 0.35 +(20) inhibition

------AHL production

a sp. sp. sp. (G176) (G168) (G168) (G176) (G176) (KT2440) (KT2440) (G168) (KT2440) (G176) (G168) (G168) (KT2440) (G176) (KT2440) (G176) PVD-type species affi liation species affi P. putida P. P. putida P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. P. putida P. Pseudomonas (Ag13) P. putida P. Pseudomonas (Ag13) Pseudomonas (Ag13) P. putida P. V V V V V XI XI XI XI XI XI XII XII XII VII VII VII VII VII Siderovar 4 5 5 7 8 9 1 1 6 2 3 13 13 10 11 12 13 13 13 BOX BOX profi le profi JIV JIV JIV EIV EIV EIV EIV EIV EIV EIV EIV EIV EIV EIV CIV CIV CIV CIV CIV Continued. profi le profi

ARDRA Table 1. Table BCRC BCRC BCRC BCRC BCRC BCRC BCRC BCRC BCRC BCRC BCRC BCRC BCRC BCRC CIP CIP CIP P. monteilii P. monteilii P. P. taiwanensis P. P. plecoglossicida P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. P. monteilii P. P. plecoglossicida P. P. taiwanensis P. P. taiwanensis P. P. taiwanensis P. P. taiwanensis P. Closest 16S rDNA ) P. taiwanensis ) P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. sequence (%) with type strain (accession number) 100 99.76 104883T (AF064458) 104883T (AF064458) (100) FPC951T (AB009457) (99.31) 17751T (EU103629) (99.57 17751T (EU103629) (99.73) FPC951T (AB009457) 17751T (EU103629) (99.76 17751T (EU103629) ( 17751T (EU103629) 17751T (EU103629) ( 17751T (EU103629) 17751T (EU103629) (99.76 17751T (EU103629) 104883T (AF064458) (100) 17751T (EU103629) 17751T (EU103629) (100 (100) 17751T (EU103629) 17751T (EU103629) (100 (100) 17751T (EU103629)

/ ) (DQ140383) (DQ095908) (DQ095898) (DQ095903) (DQ095882)/ (DQ140383) (DQ095903)/ (DQ095903) (DQ095903) (DQ09589) (DQ095899) (GU208206) (AF064458) (AF064458) (100) (GQ284481) (100) (AF064458)/ (EU430088) (99.76) (EU430088) (99.76) EF051575 (AE015451)/ (AF094741) (99.74 (EF051575) (99.74 (AE015451) (AF094737)/ (AB029257)/ (DQ229315) (GU186116)/ (D85998)/ (DQ060242)/ (AF094737)/ (DQ060242)/ (DQ060242)/ (DQ060242)/ (accession number) Blast monteilii

Closest 16S rDNA sequence P. putida P. P. plecoglossicida P. P. putida P. entomophila P. P. putida P. plecoglossicida P. P. plecoglossicida P. P. putida P. plecoglossicida P. P. monteilii P. P. monteilii P. P. P. putida P. plecoglossicida P. P. putida P. P. putida ( P. putida P. P. putida P. P. putida P. P. monteilii P. P. putida P. plecoglossicida P. monteilii P. P. putida P. plecoglossicida P. P. putida P. plecoglossicida P. P. monteilii P. plecoglossicida P. P. putida P. plecoglossicida P. putida P. plecoglossicida P. treatment plant treatment plant treatment plant treatment plant treatment plant treatment plant treatment plant Strains Origin PsTp169 Wastewater PsTp154 Wastewater PsS79 Soil PsS71 Soil PsS11 Soil PsTp139 Wastewater PsTp153 Wastewater PsTp172 Wastewater PsS46 Soil PsWs140 Sea water PsTp155 Wastewater PsS28 Soil PsC54 Compost PsS103PsS48 Soil Soil PsS15 Soil PsWw124 Wastewater PsTp156 Wastewater PsWs158 Sea water 106 MEHRI et al. Vol. 57 - - TCP IAA Bacteriocin AHL +(34) + 0+( 4) + +(30) 0 +(13) + 0+( 9) + +(19) 3.64 +(15) +(13) ++( 8) 2.67 + +( 3) 11.0 +(11) +(24) + 0 +(27) +(23) + 0 +(21) +(34) + 0 +(16) +( 3) ++(11) 0.82 + +(16) 4.83 +( 5) inhibition

------+ + 0.62 +(20) + + 0.15 +(31) + + 0 +(23) + + 0 +(29) + + 0 +(26) AHL production a sp. sp. sp. sp. sp. sp. sp. bv.V bv.V bv.V bv.V bv.V bv.V bv.V / (Pfl 12) (Pfl G4R (PutC) (G176) PVD-type species affi liation species affi P. putida P. (=F317) P. fl uorescens fl P. (PL8) P. grimontii P. panacis P. (PL8) (PL8) P. fl uorescens fl P. uorescens fl P. P. putida P. Pseudomonas (HR6) Pseudomonas (Ag13) P. putida P. Pseudomonas (Ag13) Pseudomonas (Ag13) Pseudomonas (LBSA1) Pseudomonas (B10) (PL8) P. fl uorescens fl P. Pseudomonas (Ag13) X XI IV IV IV VI IV XII XII XII XII XV None XIII VIII XIV XVI XVII None + + 8.20 Siderovar 1 2 3 3 4 5 1 2 3 2 2 4 2 3 6 5 1 BOX BOX profi le profi IV LV FV K VII K VII K VII K VII EV EV BVI BVI BVI BVI B VII D VII NVI Continued. MV profi le profi

ARDRA Table 1. Table LMG LMG BCRC BCRC BCRC BCRC BCRC BCRC Ps Ps DSM CIP CIP CIP P. graminis P. P. taiwanensis P. P. otitidis P. P. otitidis P. P. aeruginosa P. aeruginosa P. P. plecoglossicida P. P. koreensis P. P. koreensis P. P. mosselii P. P. monteilii P. P. taiwanensis P. P. mosselii P. Closest 16S rDNA ) P. taiwanensis ) P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. ) P. taiwanensis ) P. sequence (%) with type strain (accession number) 100 100 (100) 105259T (AF072688) (99.28) 17751T (EU103629) MCC10330T (AY953147) (99.88) MCC10330T (AY953147) ( 17751T (EU103629) 104883T (AF064458) (100 1242T (Z76651) 1242T (Z76651) 11363T (Y11150) 17751T (EU103629) (100 (100) 17751T (EU103629) (99.73) FPC951T (AB009457) 17751T (EU103629) ( (100) 105259T (AF072688) 17751T (EU103629) 9-14T (AF468452) 9-14T (AF468452)

(EF645247) (DQ095915) (DQ095903) (DQ095903) (DQ095903)/ (DQ095908) (DQ095903) (CT573326)/ (CT573326)/ (GU208206)/ sp. (AB506051) (EU364810)(EU364810) (99.88) (99.88) (GU059580) (99.88) (GU059580) (99.87) (AJ876736) (AF064458) (100) (AF064458) (AM184223)/ (AF072688)/ (EF523547)(EF523547)/ (99.88) (AM184223)/ (DQ060242)/ (EU439423) (99.26) (EF153392) (GU186116)/ (AY686638)/ (AY686638)/ (AJ785569)/ (DQ060242)/ (AM184239)/ (DQ060242)/ (accession number) Blast Closest 16S rDNA sequence P. entomophila P. putida P. plecoglossicida P. P. mosselii P. putida P. entomophila P. Pseudomonas P. otitidis P. otitidis P. guezennei P. P. putida P. plecoglossicida P. P. aeruginosa P. aeruginosa P. putida P. plecoglossicida P. P. putida P. P. putida P. plecoglossicida P. P. monteilii P. P. monteilii P. P. putida P. plecoglossicida P. P. putida P. plecoglossicida P. P. putida P. plecoglossicida P. P. mosselii P. entomophila P. putida P. P. fl uorescens fl P. P. fl uorescens fl P. treatment plant Strains Origin PsWt146 Thermal water PsS83 Soil PsWw9PsWw118 Wastewater Wastewater PsS75 Soil PsWw121 Wastewater PsTp142 Thermal water PsS31 Soil PsC10 Compost PsWs147 Sea water PsS102 Soil PsS67 Soil PsWs173 Sea water PsTp171 Wastewater PsS18 Soil PsS89 Soil PsWw128 Wastewater 2011 Fluorescent Pseudomonas isolated from environmental samples 107 2.83 +(22) 4.49 +(18) 7.4 +(25) 1.53 +(12) 4.53 +(14) 1.30 +(23) 3.94 +(13) 2.32 +(22) 2.24 +(17) 1.18 +(28) ------TCP IAA Bacteriocin AHL inhibition

+ + + + + + + + + + + 0.03 +(23) + AHL production a ...... sp. sp sp sp. sp sp sp sp. sp. sp sp PVD-type species affi liation species affi Pseudomonas (PL9) Pseudomonas (PL9) Pseudomonas Pseudomonas (PL9) Pseudomonas (PL9) Pseudomonas (PL9) Pseudomonas (PL9) Pseudomonas (PL9) Pseudomonas (PL9) (PL9) Pseudomonas (PL9) Pseudomonas (PL9) IX IX IX IX IX IX IX IX IX IX IX Siderovar 6 6 6 6 6 6 6 7 8 6 6 BOX BOX profi le profi H VII H VII H VII H VII H VII H VII H VII H VII H VII H VII Continued. G VII profi le profi

ARDRA Table 1. Table P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. P. frederiksbergensis P. Closest 16S rDNA sequence (%) with type strain (accession number) (99.46) JAJ28T (AJ249382) (99.52) JAJ28T (AJ249382) JAJ28T (AJ249382) (99.52) JAJ28T (AJ249382) (99.40) JAJ28T (AJ249382) JAJ28T (AJ249382) (99.28) JAJ28T (AJ249382) (99.45) JAJ28T (AJ249382) (99.35) JAJ28T (AJ249382) (99.52) JAJ28T (AJ249382) (99.52) JAJ28T (AJ249382) (EU221415) (EU221415) (EU221415) (EU221415) (EU221415) (EU221415) (EU221415) (EU221415) (EU221392)/ (FJ422406) (99.52) (FJ422406) (99.52) (EU854430)/ (EU854430)/ (EU854430) (EU854430)/ (EU854430)/ (EU854430)/ (EU854430)/ (EU854430)/ (EU854430)/ (accession number) Blast ﬂ uorescens ﬂ

Closest 16S rDNA sequence P. fl uorescens fl P. thivervalensis P. P. fl uorescens fl P. thivervalensis P. P. fl uorescens fl P. P. thivervalensis P. P. P. fl uorescens fl P. thivervalensis P. P. fl uorescens fl P. P. fl uorescens fl P. thivervalensis P. P. fl uorescens fl P. thivervalensis P. P. fl uorescens fl P. thivervalensis P. P. fl uorescens fl P. thivervalensis P. P. fl uorescens fl P. thivervalensis P. ), the total number of strains inhibited.

Strains Origin , No activity; +, ( According to Meyer et al. (2002). a PsS93 Soil PsS90 Soil PsS60 Soil PsS29 Soil PsS91 Soil PsS39 Soil PsS23 Soil PsS26 Soil PsS73 Soil PsS25 Soil PsS49 Soil - 108 MEHRI et al. Vol. 57 clage des Eaux) showing fl uorescence under UV light, strains tested (data not shown). HinfI, HhaI and HaeIII 66 isolates were selected and used in the present were the enzymes that generated polymorphic band- study (Table 1). A unique fragment of approximately ing patterns and produced respectively four, six and 990 bp was amplifi ed for all the tested strains with the fi ve different restriction profi les among the tested fl uorescent Pseudomonas-specifi c 16S rDNA primers. strains (Fig. 1). Cluster analysis permitted the defi ni- Sequence analysis using Blast (NCBI database) as- tion of fourteen different haplotypes (A‒N) with differ- signed the isolates with different degrees of confi - ent incidence. Seven haplotypes were represented by dence. Isolates share the highest sequence identity 1 isolate each. Two haplotypes represented by 5 iso- with one (n=34), two (n=26), three (n=4) or four (n=3) lates each while fi ve haplotypes were represented by a closest identifi ed Pseudomonas species (Table 1). number of isolates varying from 3 to 18 (Table 1). To However using the EzTaxon server (http://www.eztaxon. gain insight into the genetic diversity and structure of org/) (Chun et al., 2007), a rigorous assignment was the isolates, BOX-A1R-based repetitive extragenic pal- obtained and the isolates were identifi ed as P. aerugi- indromic-PCR (BOX-PCR) was applied. Twenty-four nosa, P. otitidis, P. plecoglossicida, P. mosselii, P. mon- distinct profi les were obtained at a 60% similarity coef- teilii, P. koreensis, P. taiwanenesis, P. frederiksbergensis fi cient, having two to twelve reproducible bands and P. graminis. Amplifi ed Ribosomal DNA Restriction (Fig. 2). Clustal analysis of the banding patterns al- Analysis (ARDRA) was performed using six enzymes. lowed the delineation of seven groups (I‒VII). The restriction patterns of three of the enzymes (AluI, MspI and RsaI) produce only two pattern for all the

Fig. 1. Clustal analysis of ARDRA haplotypes showing the genotypic diversity of ﬂ uorescent pseudomonades isolates. The dendrogram was obtained from similarity coefﬁ cient (Dice) calculations and clustering was done using the unweighted pair-grouping method based on arithmetic averages (UPGMA) algorithm using MVSP software. The dendrogram resulted in 5 major clusters and 14 distinct ARDRA haplotypes. 2011 Fluorescent Pseudomonas isolated from environmental samples 109

Fig. 2. Cluster analyses of BOX-PCR fi ngerprints showing the genotypic diversity of fl uorescent pseudomonades isolates. The dendrogram was obtained from similarity coeffi cient (Dice) calculations and clustering was done using unweighted pair- grouping method based on arithmetic averages (UPGMA) algorithm using MVSP software. The dendrogram resulted in 5 major clusters and 25 distinct BOX profi les.

Characterization of PVDs by IEF and Pyoverdine-medi- PVD-type species (I‒XVII), while two strains, PsWt146 ated iron-uptake capacities of the strain collection (XV) and PsWw118 (XVII), developed different and un- Electrophoresis of PVDs on ampholine-containing assigned PVD-IEF patterns (Table 1). In order to con- polyacrylamide gel (PVD-IEF) results in the separation fi rm the classifi cation reached by PVD-IEF, the 66 of the different molecular forms of PVD present in the strains were analyzed for their capacity to incorporate supernatant of an iron-starved fl uorescent Pseudomo- iron under the form of a PVD-iron complex. One strain nas culture in CAA medium. PVD isoforms appear on from each IEF siderotype, as defi ned in Table 1, was the electrophoresed IEF gel exposed to UV light as selected, and its pyoverdine was purifi ed and tested fl uorescent bands with various intensities, position for its capacity to mediate 59Fe iron uptake in each of (pHi) and number, depending on the respective con- the strains belonging to the corresponding siderovar centrations they reached in the culture supernatant (Fig. 4). All the strains within the same siderovar and during the bacterial growth (Meyer et al., 2002). As having an identical IEF pattern, are able to assimilate shown in Fig. 3, seventeen different PVD-IEF patterns effi ciently iron complexed by the corresponding py- were observed upon analyzing the culture superna- overdine. This had confi rmed the similarity in pyover- tants of the 66 isolates. Isolates developing an identi- dine structure produced by strains belonging to the cal PVD-IEF profi le were grouped together to form a same siderovar. The representative strains cross-re- so-called siderovar. The different patterns were com- acted with their own pyoverdine and with PVD-type pared to well-characterized PVD-type species avail- strains. All representative strains from each IEF sidero- able in the Laboratoire de Microbiologie et de Géné- type, their heterologous PVD and the species affi lia- tique, Université Louis-Pasteur, CNRS FRE 2326, tion are summarized in Table 1. Strasbourg, France. This had permitted the assignment of fi fteen PVD-IEF profi les to well recognized 110 MEHRI et al. Vol. 57

(A) (B)

(E)

Fig. 3. Isoelectrophoretic patterns of the pyoverdine isoforms produced by ﬂ uorescent Pseudomonas isolates. (A) Siderovar I: PsS.150 (lane 1), PsC.132 (lane 2), PsWt.138 (lane 3), PsWt.157 (lane 4), PsCL.10C (lane 5), PsWw.168 (lane 6), PsC.99 (lane 7), PsWw.175 (lane 8) and PAO1 (lane 9). (B) Siderovar II: PsWw.174 (lane 1), PsS.2 (lane 2), PsC.5 (lane 4), PsTp.179 (lane 5) and ATCC 27853 (lane 9). (C) Siderovar III: PsS.3 (lane 1), PsWw.127 (lane 3), PsC.12 (lane 4), PsCL.HMC1 (lane 6) and Pa6 (lane 8). (D) PSTp.172 (lane ‘9’), F317 (lane ‘8’), PL7 (lane 7), KT2440 (lane ‘6’), PsTp.139 (lane 5), G76 (lane 4), G168 (lane 3), PsWw.121 (lane 2), PL8 (lane 1). (E) PsS.28 (lane 1), PsS.26 (lane 2), PsWs.173 (lane 3), PsS.46 (lane 4), PsS.4 (lane 5), PsWs.147 (lane 6), PsS.31 (lane 7), PsS.18 (lane 8) and PsS. 75 (lane 9). Mix: internal standard for pI measurement (13).

Other potential plant growth promoting factors 11.00 µg ml-1 in 48 h. Of the 66 isolates tested, 56 Colorimetric assay for the detection of the plant strains (84.85%) produced phosphate solubilization growth hormone IAA was estimated for all the isolates, on Pikovskaya’s agar medium by inducing clear zones. 50 (75.75%) isolates are able to produce 0.03 to Sixty-two (94%) of the isolates produced bacteriocin 2011 Fluorescent Pseudomonas isolated from environmental samples 111

(a) inhibiting the growth of 2 to 35 strains (Table 1).

Quorum sensing and quorum quenching Violacein production in C. violaceum CV026 is induced by AHLs with a short chain (C4 to C8) of acyl or a 3-oxo-acyl side chain, while the addition of AHLs with a long side chain (C10 to C12) completely inhibits (b) the violacein synthesis induced by short-side-chain AHLs, such as C6-HSL (Cha et al., 1998; McClean et al., 1997). Using C. violaceum CV026 as an indicator for the production/inhibition of extracellular N-acylhomoserine lactone, 34 (52%) isolates were considered producers as shown by their ability to restore violacin while 30 (45%) isolates were considered as inhibitors (c) as shown by their ability to inhibit violacein production.

Discussion

In this study, comparison of the different methods Fig. 4. Heterologous PVD-mediated 59Fe incorporation by leads to the detection of similarities and differences in the PsWw.121 strain belonging to the fourth siderovar (a), PsTp.139 strain belonging to the fi fth siderovar (b) and PsS.75 relationships among the Pseudomonas isolates. There strain belonging to siderovar X (c). is general agreement between BOX-PCR, IEF and Ordinate values correspond to 59Fe-radioactivity incorporat- ARDRA classifi cation results. Out of 66 strains isolated, ed into the cells expressed in percentages, with 100% repre- the BOX-PCR, IEF and ARDRA technique discerned senting the incorporation obtained when using the homologous respectively 24, 17 and 14 groups. For the ARDRA typ- 59 pyoverdine as Fe-iron chelator (pyoverdine number 30 (a, b) ing method, the homogeneous nature of P. aeruginosa and number 26 (c) in abscissa). The other pyoverdines tested was clear (Dawson et al., 2002), whereas siderotyping on the abscissa (numbers 1 to 29 (a, b) as alkaline pyoverdines and pyoverdine-mediated iron uptake techniques clas- and numbers 1 to 25 (c) as acid pyoverdines) correspond to the structurally different pyoverdines synthesized by the following sifi ed the P. aeruginosa isolates into three different si- bacterial strains: (a, b) 1: G24, 2: G83, 3: G84, 4:G169, 5: derophore types (Meyer et al., 1997). However, BOX- Pseudomonas sp. CFML 96-312, 6: Pseudomonas sp. CFML PCR genomic fi ngerprints allowed the recognition of 96-318, 7: Pseudomonas sp. CFML 95-275, 8: Lille25, 9: 14 different profi les among the P. aeruginosa strains Pseudomonas sp. strain E8, 10: P. fl uorescens SB8.3, 11: A6, which refl ected a high degree of interspecies diversity 12: P. fl uorescens PL8, 13: P. kilonensis, 14: P. fl uorescens ATCC (Popavath et al., 2008). 13525, 15: P. fl uorescens 18.1, 16: P. aeruginosa PAO1, 17: P. Most techniques employed portrayed P. putida as putida CFML 90-136, 18: D46, 19: P. putida G168, 20: CFML 96- heterogeneous species (Meyer et al., 2007), produc- 192, 21: P. libanensis CFML 96-195, 22: Pseudomonas sp. PS6- ing diverse profi les and dispersed clustering. Hence, 10, 23: P. putida (Gwose), 24: Lille 40 (9AW), 25: P. fl uorescens ATCC 17400, 26: P. fl uorescens 1.3, 27: CFML 96-319, 28: the isolates identifi ed on the basis of 16S rDNA gene CHO59, 29: CIP 75.23. (c) 1: P. putida CFML 90-42, 2: syr, 3: sequencing as P. putida based on Blast utility were Pseudomonas sp. PL9, 4: P. putida C, 5: Pseudomonas sp. HR6, spread throughout the BOX-PCR and ARDRA dendro- 6: Pseudomonas sp. CFML 90-33, 7: P. putida CFML 90-40, 8: grams, confi rming the weak cohesion within this spe- G166, 9: G172, 10: P. putida G176, 11: G173, 12: P. putida ATCC cies. Moore et al. (1996) reported sequence differenc- 12633, 13: Pseudomonas sp. strain A214, 14: AP1, 15: AP15, es as high as 3% between different P. putida strains 16: G86 (G89), 17: G88, 18: Pseudomonas sp. Ag13, 19: Malar, and suggested that this taxon may encompass several 20: fl uo1 (CFT1), 21: Fb4, 22: Jn5, 23: Pseudomonas sp. Jn7, species. Moreover several investigations have depict- 24: Oc9, 25: Oc19. Pyoverdine numbers 1 to 29 (a, b) and numbers 1 to 25 (c) were XAD-purifi ed products, whereas the ho- ed the considerable diversity of P. putida, highlighting mologous pyoverdine was used without purifi cation, directly the need to clarify the taxonomy of this pseudomonal from the culture supernatant. group (Bossis et al., 2000) and to reorganize this group 112 MEHRI et al. Vol. 57 in future into several different species (Regenhardt et by AHL lactonase of P. fl uorescens strain, to reduce al., 2002). Meyer et al., 2007 analyzed the diversity of signifi cantly potato soft rot disease symptoms. Hence, 144 strains assigned to the P. putida species through the AHL-degrading bacteria such as PsTp171, PsTp172, siderotyping, showing a great diversity among these PsTp157, PsS102 etc. can disrupt and manipulate strains that could be subdivided into 35 siderovars and quorum-sensing signalling specially in agricultural proposing a novel Pseudomonas species. pathogenic bacteria, contributing to a reduction in The partial sequencing of fl uorescent Pseudomonas their saprophytic growth (Choo et al., 2006; Latour et demonstrates that this region, which is highly con- al., 2003). The concept of biological disease control, served, makes it diffi cult to separate this pseudomonal using non-pathogenic bacterial strains as a potential group, and there might be a high risk of incorrect iden- biopesticide, is considered an alternative to chemical tifi cation (Rangel-Castro et al., 2002). In most cases, control, because chemical pesticides result in the ac- different species show very similar or identical 16S cumulation of hazardous compounds toxic to soil biota rDNA sequences (Grimont, 2002; Gomila et al., 2007). (Costa et al., 2006; Gupta et al., 2001). In our study, identifi cation of some isolates has to be These bacteria being a pyoverdine producing rhizo- confi rmed due to the controversial information in data- sphere-competent bacterial strain, their PGPR traits bases and in some instance equal identity in 16S rDNA could not only control plants but also help to enhance sequence was shared with more than one species. As plant growth. an example, 16S rDNA sequence analysis indicated Consequently, fl uorescent Pseudomonas strains re- the presence of P. otitidis, a non-fl uorescent Pseudomo- ported in this study with biocontrol and biofertilizing nas, which is a highly unexpected result, since the se- properties could be effi cient bioinoculants in plant lection criteria included demonstration of fl uorescence rhizospheres (Latour et al., 2003). on King’s B medium (Chang et al., 2008; Clark et al., The results in this report showed multifunctional 2006). properties of some isolated strains, identifi ed by 16S In the present investigation, out of 438 fl uorescent rDNA sequencing as P. monteilii (PsS15), P. pleco- Pseudomonas strains maintained in the LTRE collec- glossida (PsTp156), and P. putida (PsTp172), and a tion, 66 strains have been screened for the production potential applicability in soil or the treatment of waste- of a siderophore (pyoverdine), enzymes and/or phyto- water. hormones such as phosphatase and indole-3-acetic These benefi cial microorganisms and some others acid. Furthermore, these microorganisms have been (P. mosselii (PsTp171) and P. monteilii (PsS102)) may identifi ed as capable of inhibiting a wide range of bac- be employed for bioremediation as inoculants into bio- teria through the production of bacteriocin and/or quo- reactors or mixed in wastewater, to suppress patho- rum-quenching enzymes. genic or phytopathogenic bacteria and reduce their Isolates such as PsS15, PsTP156, PsTp172 and opportunistic invasion (Antony and Philip, 2006). PsC54 revealed a broad spectrum antagonistic activity and produced plant-growth-promoting enzymes References and hormones. Moreover, the effi cient fl uorescent Pseudomonas isolated from a wastewater treatment Ali, N. J., Kessel, D., and Miller, R. F. (1995) Bronchopulmonary plant displayed higher activities then strains from other infection with Pseudomonas aeruginosa in patients infected with human immunodefi ciency virus. Genitourin. Med., 71, origins. 73‒ 77. 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