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), produc- tion 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. pleco- glossicida, 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 hor- mones 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 G T 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).