87 Arab Univ. J. Agric. Sci., Ain Shams Univ., Cairo, 14(1), 87-103, 2006 NUMERICAL STUDY ON SOME ACTINOMYCETES ISOLATED FROM BURULLOS LAKE IN EGYPT [6] 1 2 Abu-Elela , Gihan M. and Nevin, B. Ghanem ABSTRACT Twenty nine actinomycetes isolates were isolated from Burullos Lake and char- acterized taxonomically for 62 phenotypic traits including morphological; biochemi- cal, nutritional, substrate utilization and anti-microbial activities. The results were analyzed by numerical techniques using the simple matching coefficient (SSM) and UPGMA clustering. At 54% similarity level, the majority of the isolates were grouped into six phena (A, B, C, D, E and F). Only two isolates were grouped sepa- rately and formed two single clusters at the same level of similarity. A representative isolate from each phenon was identified. The isolates were found to be Streptover- ticillum morookaense, Nocardia brasiliensis, Streptomyces alanosinicus, Streptomy- ces globosus and Streptomyces gancidicus Keywords: Burullos Lake, Actinobacteria, Numerical taxonomy, Fresh water habi- tats INTRODUCTION growth in aquatic habitats but can never- theless be recovered readily from fresh Class Actinobacteria (high G+C con- water, sea water and sediments samples tent, gram positive bacteria) has been (McCarthy and Williams, 1990). proposed by Stackebrandt et al (1997) Several studies demonstrated by culti- and includes members with unparalleled vation independent methods revealed that ability to produce diverse secondary me- the freshwater actinobacteria are present tabolites (Mellouli et al 2003). They rep- in a wide spectrum of ecologically differ- resent an ubiquitous group of microbes ent and globally distributed freshwater widely distributed in natural ecosystem habitats (Glöckner et al 2000; including soils (Xu et al 1998), marine Lindström and Leskinen, 2002; Hahn sediments (Ghanem et al 2000, Sabry et et al 2003). Zwart et al (2002) recently al 2004) and fresh water (Hahn et al identified 34 putative phylogenetic clus- 2003). Actinomycetes are not adapted for ters of bacteria which seem to contain 1- National institute of Oceanography and Fisheries Alexandria. Egypt 2- Botany Department, Faculty of Science, Alexandria University, Egypt (Received November 26, 2005) (Accepted January 2, 2006) 88 Gihan Abu-Elela and Nevin Ghanem typical freshwater inhabitants, 5 of the 34 MATERIAL AND METHODS clusters were affiliated with the class Ac- tinobacteria. Actinomycetes isolates Distribution of Actinobacteria in fresh water has been reported by Glöckner et Twenty nine actinomycetes isolates al (2000) who performed in situ hybridi- were selected from a total of 130 isolates zation. On the other hand, Pernthaler et previously isolated from sediments and al (2001) successfully enriched a phylo- water samples of Lake Burullous (Abu- type of one freshwater cluster of actino- Elela et al 2004). They were maintained bacteria in a continuous culture system. on a basal medium prepared with 1 liter Thus, despite the high number of cells of of Lake water of the following composi- actinobacteria observed in fresh water tion (g/l): soluble starch, 10.0; KNO3, samples no representatives of the fresh- 2.0; K2HPO4, 2.0; MgSO4 .7H2O, 0.05; water actinobacterial lineages have been CaCO2, 0.02; FeSO4, 0.01 and agar, 20.0 isolated so far (Zwart et al 2002). Zwart at 28 ºC (Küster and Williams, 1964). et al (2003) found that actinobacterial Culture stocks were maintained at -20 ºC cluster ACK-M1 is being well represent- in starch nitrate broth with 20% glycerol ed in most fresh water habitats. They de- (Kieser et al 2000). tected this cluster in all of the 81 lakes screened (from Belgium, The Nether- Characterization and numerical analy- lands, Denmark, Sweden and Norway), sis using reverse line blot hybridization. In the most recent published data (Van der Numerical analysis was not used for a Gucht et al 2005) the ACK-M1 (Actino- direct taxonomical purpose but also to bacteria) was the most representative in facilitate data handling and strains group- each of the studied lakes. These data con- ing. A total of 62 characters were coded firm those previously reported (Crump et as negative (0) or positive (1). The simple al 1999; Urbach, et al 2001). matching coefficient (SsM) (Sokal and Up to our Knowledge, few studies Michener, 1958) and the Jaccard coeffi- dealt with the distribution of class Ac- cient (Sj) (Sneath, 1957) were used and tinobacteria in aquatic habitats in Egypt clustering was achieved by unweighted (Al-Diwany and Cross 1978; Ghanem pair group average linkage (UPGMA), et al 2000). Lake Burullos is shallow Sneath and Sokal, (1973) and Sneath, brackish water that serves as fishery re- (1979). The computations were per- sources and reservoirs for drainage water. formed by using SYSTAT-PC program V It is contaminated with anthropogenic 7 (Wilkinson et al 1992) on an IBM materials (Abu-Elela et al 2004). There- computer. fore, this study is an attempt to evaluate The selected isolates were phenotypi- the distribution of members of class Ac- cally characterized as described by tinobacteria in Lake Burullous using phe- Pridham and Gottlieb (1948); Shirling notypic, physiological and biochemical and Gottlieb, (1966) and Tresner et al data, depending on numerical taxonomic (1968). All tests were carried out at 28ºC procedures. for up to 3 weeks. Arab Univ. J. Agric. Sci., 14(1), 2006 Numerical study on Actinomycetes from burullos lake 89 For morphological characterization, Chemotaxonomic study was per- the following media were used: Krass- formed by the detection of dia- linikov SRL agar, Glycerol- nitrate agar, minopimelic acid (DAP) isomers and Czapek- Dox agar after slight modifica- diagnostic sugars according to Becker et tion, Glycerol- asparagine agar and Inor- al (1964) and Lechevalier et al (1977) in ganic salts- starch agar (Shirling and AL-Azhar University Fermentation Bio- Gottlieb, 1966). technology and Applied Microbiology Additional phenotypic characteriza- Center. Identification was carried out in tion was performed using the standard comparison to reference strains. procedures, catalase and urease produc- tion were detected, melanin production Scanning electron microscopy according to Shirling and Gottlieb, Electron micrographs were made with (1966), nitrate reduction (Williams et al Jeol model ISM 5300 scanning electron 1983), sulphide precipitation (Cowan, microscope (SEM) operating at 15 KeV 1974) and indole production (Molin and (Molitoris et al 1996). Trenstrom, 1986). Lecithinase was per- formed on egg-yolk medium according to RESULTS AND DISCUSSION the method of Nitsch and Kutzner, (1969), Lipase (Elwan et al 1977), prote- In a previous survey on the distribu- ase (Ammar et al 1991), pectinase, am- tion of Actinobacteria in Lake Burullos, ylase (Ammar et al 1998) production 130 isolates were obtained (Abu-Elela et was investigated. Also Degradation ac- al 2004). Based on colony morphology, tivities of some substance such as starch, 29 isolates were selected which represent cellulose, chitin, gelatin and tyrosine different morphotypes. In this study, we were performed according to Nonomura aimed to cluster those isolates for the and Ohara (1969). purpose of identification and classifica- The antagonistic activity of the tested tion. isolates against Escherichia coli HP101, Actinomycetes isolates were studied Staphylococcus aureus ATTC 29523 and for 62 characters listed in Table (1). Bacillus subtilis was determined as de- Analysis of the 62 characters using the scribed by Goodfellow et al (1990). simple matching coefficient (SSM) and Zones of inhibition were scored as posi- UPGMA clustering yielded the den- tive results after 24 h at 28C°. Resistance dogram in Fig. (1). Data showed that the against phenol (0.002, 0.01 w/v), crystal majority of the isolates were grouped at violet (0.0001, 0.001 w/v), and sodium 54% similarity level, into six phena (A, azide (0.01w/v) was tested. Antibiotic B, C, D, E and F). Only two isolates were resistance was examined as the ability to grouped separately and formed two single grow on medium supplemented with clusters at this level. antibiotics one at a time using gentamy- cin (998 μg.ml-1), erythromycin (804 Phenon A: The four isolates grouped in μg.ml-1), tetracycline (990 μg.ml-1) and phenon A possess several features that cefixine (997 μg.ml-1) according to Wil- are common to members of genus Strep- liams et al (1983). toverticillium. It is thus suggested that the Arab Univ. J. Agric. Sci., 14(1), 2006 90 Gihan Abu-Elela and Nevin Ghanem Table 1. Comparison of the frequencies of positive and negative characters for the six clusters of actinomycetes obtained by numerical taxonomy analysis Phenon A B C D E F Character No. of 4 3 6 5 3 6 isolates Growth on Krasslinikov SRL agar 100 100 100 0 33 83 Glycerol-asparagine agar 100 100 100 100 100 100 Glycerol –nitrate agar 100 100 100 100 100 100 Czapek-Dox agar 100 100 100 100 100 100 Inorganic salts –starch agar 75 100 100 100 100 100 Substrate mycelium Yellow brown 50 67 33 80 0 33 Reddish brown 25 0 50 0 67 50 Blue 25 0 0 0 0 0 Violet 0 33 17 20 33 17 Aerial mycelium Gray yellowish pink 0 33 0 0 67 0 Pink gray 0 67 17 0 0 0 Gray 0 0 67 80 0 50 Whitish gray 50 0 0 0 0 0 Blue 25 0 0 0 0 0 Violet 0 0 17 20 0 17 Yellow 25 0 0 0 33 33 Diffusible pigment Unpigmented 0 100 100 80 67 50 Reddish brown 0 0 0 20 33 0 Yellowish brown 100 0 0 0 0 50 Growth at ºC 20 50 100 100 100 100 100 30 100 100 100 100 100 100 40 100 100 100 100 100 100 50 25 0 0 0 0 17 Growth at pH 5 50 100 50 20 0 50 6 75 100 100 100 100 100 7 100 100 100 100 100 100 8 75 100 100 100 100 100 9 75 67 100 100 100 100 Arab Univ.