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Molecular Characterisation and Numerical Analysis of Novel Moderately Thermophile Anoxybacillus Sp

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Molecular Characterisation and Numerical Analysis of Novel Moderately Thermophile Anoxybacillus Sp Romanian Biotechnological Letters Vol. 23, No. 5, 2018 Copyright © 2018 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Molecular characterisation and numerical analysis of novel moderately thermophile Anoxybacillus sp. FMB1 DOI: 10.26327/RBL2018.150 Received for publication, September, 21, 2017 Accepted, April, 2, 2018 FATMA MATPAN BEKLER1*, SEÇİL YALAZ2, KEMAL GÜVEN3 1Dicle University, Science Faculty, Department of Biology, 21280 Diyarbakır, TURKEY 2 Dicle University, Science Faculty, Mathematics Department, 21280 Diyarbakır, TURKEY 3 Dicle University, Science Faculty, Department of Molecular Biology and Genetics, 21280 Diyarbakır, TURKEY *Address for correspondence to: [email protected] Abstract A moderately thermophilic bacteria was isolated from the water sample collected from Yozgat, in the Central Anatolia Region of Turkey. Morphological, physiological and biochemical characteristics of thermophilic isolate were determined in comparison to standard type strains, and they were all identified and classified into homogenous groups by using fundamental principles of numerical taxonomy. The UPGMA method was used for numerical taxonomy and the phenogram was drawn with MATLAB. Phylogenetic analyses were also performed by the neighbour-joining and UPGMA methods. According to the numerical definition result and 16S rRNA analysis, the strain FMB1 was most closely related to Anoxybacillus ayderensis AB04T. The results showed that MATLAB computer program using UPGMA method can be used as an alternative or additional method in bacterial classification, before 16S rRNA analysis performed. Keywords: Anoxybacillus, characterization, 16S rRNA, numerical analysis 1. Introduction The members of genus Anoxybacillus, which are known as rod-shaped, Gram positive, endospore-forming, anaerobes or facultative anaerobes and moderately thermophilic bacteria, are widely isolated from geothermal areas. Recently, many studies have been carried out on the members of this genus with increasing biotechnological and industrial interest for their thermostable products. Thus, the isolation of these bacteria and the determination of their phylogenetic relationship are of importance. Prokaryotes can be classified by using phenotypic characterization such as cell morphology, staining behaviour, ultrastructural characteristics, biochemical reactions, utilising carbon and nitrogen sources or by phylogenetic analysis. Phylogenetic analysis means inferring or estimating the evolutionary relationships (BRINKMAN & LEIPE [1]). The routine strategy for phylogeny is based on the correlation of a precise morphologic and phenotypic description of the isolate to be recognized. Generally, Bergey's Manual of Systematic Bacteriology or the Manual of Clinical Microbiology are used as a standard reference for morphologic and phenotypic identification by microbiologists. However, different computer programs and methods were designed for the evaluation and identification could differ among laboratories (CLARRIDGE [2]). Phylogenetic studies are carried out using molecular phylogeny in cases where morphological and phenotypic characterization is insufficient. Molecular phylogenies are based on the information about the evolution of the organisms. Deducing phylogenies from molecular information might be helped out through 13964 Romanian Biotechnological Letters, Vol. 23, No. 5, 2018 Molecular characterisation and numerical analysis of novel moderately thermophile Anoxybacillus sp. FMB1 the point analysis of similarities and differences in the studied sequences (ALBRECHT & BORGES [3]). The part of the DNA, which has been by far the most common housekeeping genetic marker for the phylogeny and taxonomy of bacteria, is the 16S rRNA gene (BOTTGER [4], JANDA & ABBOTT [5]). The 16S rRNA gene sequence, which is about 1,550 bp has been determined for a large number of strains because of some important reasons, including (i) to be common in almost all bacteria (ii) is a useful evolutionary chronometer, and (iii) the size of 16S rRNA gene is large enough to be used in bioinformatics (JANDA & ABBOTT [5]). Universal primers, which usually chosen as complementary to the conserved regions, are used for comparative taxonomy. GenBank, the largest databank of genetic sequence, has previously deposited sequences, helping out to compare the sequence of an unknown strain (CLARRIDGE [2]). In this study, a thermophilic bacterial strain designated as FMB1 was isolated from Yozgat, in the Central Anatolia Region of Turkey. The identification of the strain was carried out by numerical taxonomy utilising the phenotypic characteristics in comparison to 16S rRNA sequence analysis. 2. Materials and Methods 2.1 Chemicals All chemicals were purchased from Sigma (Sigma–Aldrich, St Louis, USA) and were of the highest quality available unless otherwise stated. All chemicals used were of analytical grade. 2.1 Location and sampling The water samples were obtained from Sorgun hot spring (39° 48' 14.0718" N, 35° 12' 31.0752" E), Yozgat, in the Central Anatolia Region of Turkey. The temperature and pH of hot spring water were 60 °C and 7.5, respectively. The water samples were inoculated into the modified liquid Thermus medium (TM) [Tryptone, 3 g/L; Yeast extract, 1 g/L; containing Nitsch’s trace element (0.5 ml/L) and Castenholz salts (100 ml/L)] and incubated at 50 °C for 24 hours in a shaker. After cultivation, samples were streaked on TM agar (TMA) and incubated at 50 °C for 2 days. Subsequently, different colonies developed in the media were selected and purified by subculturing. 2.3 Morphological, physiological and biochemical characterization Selected colonies were phenotypically characterized on the basis of shape, size, colour, surface, aspect, elevation and consistency, etc. The cell morphology was studied by the light microscopy of 24 hours old bacterial cultures growing on TMA plates. Gram staining to confirm the Gram reaction and spore position was investigated using light microscope according to the Dussault method (DUSSAULT [6]). The formation of spores was carried out by malachite green staining under light microscopy. Motility was determined by the hanging drop method. For the presumptive identification, data of phenotypic characteristics from the conventional methods such as catalase, indole, oxidase, citritase and urease activity, Methyl red test/Voges–Proskauer test, utilization of different carbon and nitrogen sources (glucose, galactose, lactose, fructose, maltose, arabinose, gelatine, starch, caseine and lipid) were used. The optimum growth temperature and pH were determined by incubating the isolate in TM liquid medium at different temperature (20 to 90 °C) and different pH range (4.0-11.0). The effect of NaCl on growth was examined in 100 mL TM liquid medium which was prepared in a phosphate buffer (50 mM) with final salt (NaCl) concentrations of 0.5%, 1.0%, 2.0%, 3.0% and 5.0% in a 300 mL Erlenmayer flask and 1mL of 18h old bacterial culture was inoculated in to this medium. Then, all flasks were incubated at 50 °C for 2 days in the shaker. The cell growth was measured hourly by spectrophotometer at 600 nm (OD600). All experiments were performed at least in duplicate. Romanian Biotechnological Letters, Vol. 23, No. 5, 2018 13965 FATMA MATPAN BEKLER, SEÇİL YALAZ, KEMAL GÜVEN 2.4 Numerical taxonomy and phenogram Numerical taxonomy was first developed and elaborated by Sokal and Sneath in 1963. In biological systematics, this system is useful to generate a taxonomy using numeric algorithms like cluster analysis rather than using subjective evaluation of their properties (SOKAL & SNEATH [7], SNEATH & SOKAL [8]). Phenetics is a field of numerical taxonomy. Classifications are formed based on the patterns of overall similarities. Clustering was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) which is based on the model sum of squares coefficient. UPGMA is a preferred as it is a useful tree-construction method for distance matrices (NEI & KUMAR [9]). The fundamental thought for UPGMA is to repeatedly combine a pair of operational taxonomic units (OTUs) with the smallest distance (or highest similarity) into a new OTU upto only one OTU is left and the distance between two OTUs is calculated by the arithmetic mean of all distances between their inter-pairs of sequences (or other kinds of data), where an inter-pair of sequences means that is in the one OTU and is in the other (TAKEZAKI & NEI[10]). The UPGMA algorithm constructs a rooted tree called dendrogram that reflects the structure present in a pairwise similarity or dissimilarity matrix. The UPGMA algorithm produces rooted dendrograms and requires a constant-rate assumption, distances from the root to every branch tip are equal. In biological systematics, dendogram is called as phenogram when the phenetics are used for classification. Numerical identification of the strain as Anoxybacillus species was performed with MATLAB computer program. The program uses UPGMA method to produce numerical taxonomy and phenogram and compares the test results of an unknown isolate with the known test result scales in these tests for a collection of related bacterial species, using established numerical methods. Nearly all of the characters existed in one or two mutually exclusive states and scored positive (+) or negative (-). Some characters could not be determined and scored as ND (not determined). Qualitative multistate characters, such as some of pigmentation and morphological tests, were
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