16S rDNA analysis for characterization of Pseudomonas sp. strain MF30 isolated from Rumex acetocella roots in northern Sweden (Received: 15.09 .2006; Accepted: 28.09.2006) Idress H. Attitalla*;Ali A. Bataw**; Jolanta J. Borowicz***and Sture Brishammar*** *Omar Al-Mukhtar University, Faculty of Science, Botany Department, Box 919, El-Beida, Libya **Omar Al-Mukhtar University, Faculty of Science, Zoology Department, Box 919, El-Beida, Libya ***Maselaboratoratorerna AB, Box 148, Uppsala, Sweden Corresponding author1. [email protected] ABSTRACT A bacterial strain obtained from the northern part of Sweden previously classified as Pseudomonas veronii based on biochemical and physioloical tests. In this study, phylogenetic tree was constructed using a nearly complete sequence within the 16S rDNA gene. The strain of Pseudomonas sp. subdivided into two rather distinctly related groups, neither of which is very close to the group within the Pseudomonas fluorescens cluster. Although the phylogenetic analysis is not conclusive, it is consistent with other observations, especially the capacities of the this strain as a biocontrol agent. Taken all together, the results suggest that the MF30 strain should be classifed as another Pseudomonas species, either Pseudomonas antarctica or P. meridina. Key words: Phylogenetic analysis, 16S rDNA gene, Pseudomonas species, P. veronii, P. antarctica, P. meridina. INTRODUCTION received attention even though they possess abilities to influence plant growth and acteria belonging to the fluorescent development through different mechanisms pseudomonads, known for the (Weller, 1988; O’Sullivan and O’Gara, 1992). B diversity of their metabolites They are now recognised as being antagonistic (Leisinger and Margaff, 1979), contain to several opportunistic soil-borne fungi species that are recognized as human and as (Weller, 1988; Keel et al., 1996) and to seed- animal pathogens (Nakazawa and Abe, 1996) borne fungi (Hökeberg et al., 1997). Also, and some are important phytopathogens some strains are responsible for significant (Jaunet et al., 1995). Most species are plant promoting effects (DeFreitas and however, saprophytic, and as a group, they are Germida, 1991; Kropp et al., 1996). ubiquitous in water (Verhille et al., 1997), soil Taxonomically, this flourecent Pseudo- (Lemanceau et al., 1995), plant spermosphere monas spp. belong to the rRNA group I of the (Fukui et al., 1994), phyllosphere (Rainey et gamma subclass of Proteobacteria (Palleroni, al., 1994) and rhizosphere (Lambert et al., 1993; Kersters et al., 1996). The species 1990). In recent years certain species, for within this group have been differentiated on example Pseudomonas chloroaphis, P. the basis of their DNA/Dna homologies fluorescens, P. veronii, P. putida, have (Johnson and Palleroni, 1989; Gardan et al., Arab J. Biotech., Vol. 10, No. (1) Jan. (2007):97-106. 98 Idress H. Attiatalla et al. 1992). This method of differentiating bacterial metabolities namely massetolides type A, a species has for several years been the most low molecular weight lipopeptide (Gerard et reliable, since traditional classification of al., 1997). This compound, as well as several bacteria based on phenotypic features does not derivatives (massetolides B-H), were recently always correlate with molecular taxonomy isolated from two Pseudomonas spp. isolates (Woese, 1987). Other methods based on of a marine algae and a marine tube worm cataloguing 16S rRNA have been shown to be origin (Gerard, 1997). To date, there only two reliable and effective (Woese et al., 1984; reports of in vitro anti-tuberculosis activity Woese, 1987). Indeed, using the 16-rDNA expressed by massetolides (El-Sayed et al., approach, Woese et al. (1990) were able to 2000). show definitively that life can be divided into In the present paper, we successfuly three major lines of descent presently named applied restriction fragment length Archea, Bacteria and Eukarya. polymorphism and sequencing of the 16S For bacteria belonging to the genus rDNA for clarifying the classification of the Pseudomonas, data collected on 16S rDNA strain MF30. This study indicates that it differs genes make taxonomical studies possible, from the P. veronii. However, since 16S rDNA including those in the rhizosphere. As a result data precluded the prospect of conclusive of such studies, many organisms classified as results from phylogenetic analysis, one can species of this genus have been reclassified rule out the possibility that the strain can (Kersters et al., 1996). However, classification represent a specific group or biotype of P. of the fluorescent pseudomonads has yet to be veronii. Using sequencing data from nearly clarified completely, in part due to the many complete sequence of the 16S rDNA gene new species being decribed (Elomari et al., together with their biological characters, we 1996). This paper a case in point. In earlier describe the strain MF30. We proposed that study, Attitalla et al. (2001) identified a this bacterium should be reclassified as a new bacterial strain (MF30) based on the Pseudomonas species, either P. antarctica or characteristics revealed by API NE 20 test P. meridina. (BioMerieux, Marcy l'Etoile, Marcy, France), the bacterial strain MF30 was assigned to the MATERIALS AND METHODS genus Pseudomonas, also partial sequences of Bacterial strain and cultivation 16S rDNA showed 99.6% similarity to Using the procedure described by Pseudomonas veronii and high similarity to Hökeberg et al., (1997), the bacterial strain other members of RNA group I of the MF30 was isolated from root of a wild plant Pseudomonas. Strain testing was done in (Rumex acetocella) in the mountain area of Deutsche Sammlung von Mikro-organismen northern Sweden: The strain MF30 from the aus Zelkulturen GmbH, Braunschweig, dicotyledonous plant. The strain was Germany (DSMZ), which also found that the maintained as freeze-dried or as deep-frozen (- profile of the cellular fattty acids was typical 70°C) stock culture. The bacterium was grown for that RNA-group, and although in 50% strength Tryptic Soy Broth (15g/k, physiological tests were unable to distinguish TSB, Difco) it shaking (120-rev min-1) at 20°C between the species in this group, yet they for 48 hours. were all closely related to biovars of Pseudomonas fluorescens. This strain has been shown to produce biologically active Arab J. Biotech., Vol. 10, No. (1) Jan. (2007):97-106. 16S rDNA of Pseudomonas strain MF30 in northern Sweden 99 Secretion of extracellular proteases DNA isolation and PCR amplification Proteolytic activity was estimated in 48 The bacterium was grown in 50% normal hours old cell free supernatant from bactrial strength TSB on a rotary shaker (120-rev min- culture grown in 50% normal strength TSB 1) at 20°C for 48 hours. The RapidPrepTM (15g/l) (Tryptic Soy Broth, Difco). Bacterial genomic DNA Isolation Kit for Cells and culture was incubated on rotary shaker (120- Tissues (Pharmacia Biotech) was used to rev min-1). Cells were removed by isolate DNA as advised by the manufacturer. centriifugation for 20 min at 16.000 g and DNA amplification was conducted on pure 2 supernatant was then filter sterilized. The to 3 µl DNA sample with about 150 ng of radial diffusion assay (Dow et al., 1990) in DNA per 1 µl of sample in a perkin Elmer milk agar plates was used. Fifty microliters of (Nowalk, CT) thermocycler. The fD1 and rD1 supernatant was applied to a well cut (6 mm in primers amplifying the 16S rDNA gene in diameter) in the middle of skim milk agar most of bacteria was used for DNA plates, which were then incubated at 37°C. amplification (Weisburg et al., 1991). PCR Clear zones (mm) around the well were reactions were conducted in a final volume of measured after 24 hours. The test was repeated 100 µl with 0.2 µM of each primer, 2.0 mM twice with two Petri-dishes per treatment. Mgcl2, 200µM dNTPs and 2.5 units of AmpliTag DNA polymearse mixed in the PCR Phenotypic characterization buffer (Perkin Elmer). DNA was amplified The strain was also characterized over 35 cycles of denaturation for 1 min at phenotypically with regard to production of 94°C annealing at 55°C for 1.5 min and fluorescent colonies on King’B medium, and extension at 72°C for 2 min. After the last for motalility in a swarming assay (Bondesson cycle, DNA was extended at 72°C for 10 min. and Haas, 2002; Thorsson, 2002). Amplification was confirmed by analyzing 5µl Fluorescence of PCR reaction mixture on 1% agarose gel Siderophore production was detected as (Promega). fluoresence under UV light (wave length = DNA sequencing 320 nm) when cells were spread on KB The PCR-product was purified using medium (King et al., 1954). Plates were QIAquik PCR Purification Kit (Qiagen), and incubated at 25°C for 2 days prior to visual sequenced using automatic ABI 310 DNA examination of fluorescence on UV a Sequencer, Big Dye Terminator Cycle transilminator. Sequencing Ready Reaction Kit, Perkin Elmer. Swarming The sequencing was performed in both To test swarming ability, 1 µl of bacterial direction using previously described primers suspension (a small loop of fresh bacterial (Lane et al., 1985; Lane, 1991). Sequencing cells suspended in 100 µl high purity water) of data was analzed by two different computer the strain was inoculated onto liquid medium alignment programs, DNAStar (DNASTAR, (LM plates containing 10g/l Bacto-trypton, Inc., USA) and Sequence Navigator (Perkin, Corp., USA). 6g/l Bacto-yeast extract, .5 g/l K2HPO4, 0.5 g/l NaCl, 0.4g/l MgSO4*7H2O). Determination of phylogenetic relationships The BLAST database (Altschul et al., 1997) of National Center for Biotechnology Arab J. Biotech., Vol. 10, No. (1) Jan. (2007):97-106. 100 Idress H. Attiatalla et al. Information was used to compare resolved an important bacterial strain. Attitalla et al., sequence of the MF30 strain with known 16S (2001) attempted to identify the MF30 strain rDNA sequences. Determination of on the basis of morphological and biochemical phylogenetic realtionships was analyzed by the characters; the strain was close to P.