International Journal of Health Medicine and Current Research Vol. 4, Issue 04, pp.1478-1483, December, 2019 DOI: 10.22301/IJHMCR.2528-3189.1478 INTERNATIONAL JOURNAL ORIGINAL ARTICLE OF HEALTH MEDICINE AND Article can be accessed online on: http://www.ijhmcr.com CURRENT RESEARCH SEQUENCE ANALYSIS OF 16SrRNA, rpoB, rpoC AND rpoD GENES FROM THE GENUS RHODOBACTER Edwin Thomas Apituley1* and Amos Killay1 1Jurusan Biologi Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Pattimura, Ambon. ARTICLE INFO ABSTRACT Article History: Molecular sequence data analysis become important tool to inferred Received 10th Sep, 2019 Received in revised form evolutionary relationship and systematic ordering among among organism. It 11th Oct, 2019 complementing traditional method such as comparison of morpholocical and Accepted 13th Nov, 2019 biochemichal characteristics. In this article, three genes sequnces encoding RNA Published online 31th Dec, 2019 polymerase subunits and 16S rRNA gene sequence are discussed for their evolutionary relationship among species of Rhodobacter, a genus of phototrophic α-proteobacteria, and between Rhodobacter species and several Key words: reference microorganism. Level of sequence identity among gene sequence Rhodobacter, phylogenetic analysis, 16S rRNA, rpoB, rpoC, rpoD. showed the lowest variation was in 16SrRNA gene sequences, and the the highest was in rpoD gene sequence. Phylogenetic tree constructions were done using Neighbour joining method and Kimura – 2 Parameter model to measure *Correspondence to Author: genetic distance. Bootstrap analysis was also applied with 1000 repeats. Edwin Thomas Apituley Members of Rhodobacter genus were divided into two major clusters based on Jurusan Biologi Fakultas phylognetic analysis of 16S rRNA gene sequence. There is ambiguity of Matematika dan Ilmu Pengetahuan Alam Universitas Pattimura, Ambon. Pararhodobacter sp. CCB-MM2 position between anlysis based on 16SrRNA gene sequnce and rpo gene sequences where analysis based on rpo genes sequences able to locate Pararhodobacter sp. CCB-MM2 on its own phylognetic E-mail: tree branch, separated from cluster of Rhodobacter species. [email protected] Copyright © 2019, Edwin Thomas Apituley. This is an open access article distributed under the creative commons attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Edwin Thomas Apituley1* and Amos Killay1, 2019 “Sequence Analysis Of 16SrRNA, rpoB, rpoC AND rpoD Genes From The Genus Rhodobacter”, International Journal Of Health Medicine And Current Research, 4, (04), 1478-1483. International Journal of Health Medicine and Current Research | 1478 INTRODUCTION Rhodobacter sp. LPB0142, Pararhodobacter sp. CCB- MM2, Rhodovulum sulfidophilum DSM 2351, DNA sequence analysis of evolutionary stable Rhodopseudomonas palustrisHaA2, Escherichia coli str. marker genes susch as the small-subunit (16S) rRNA K-12 substr. MG1655, Paracoccus denitrificans, gene is used in genotypic identification, clssification of Roseobacter denitrificans. Percentage of identity prokaryotes as alternative or complement to phenotypic between sequences was calculated as percentage number methods and phylogenetic study [1 – 3]. Because of its of identical nucleotides in a sequence relative to total important role in protein synthesis, 16SrRNA gene is number of nucleotides. Phylogenetic analysis was highly conserved among bacteria and other kingdoms conducted using MEGA 5 software [14] with Neighbor- [4]. Comparison of 16SrRNA gene sequence alows Joining Method [15] and Kimura 2-Parameter model to differentiation of bacteria at the genus level and built the tree [16]. Multiple sequence alignments were classification at species and subspecies level, however obtained using Clustal W [17]. Topology of the its presences as multiple copies in many bacteria became phylogenetic tree was evaluated by performing a its downside [5 – 6]. Researchers were looking for other bootstrap analysis using 1000 bootstrapped trials. alternative molecules, for example; GroEL [7], EF-Tu [8] and RNA polymerase subunit B [9]. Bacterial DNA RESULTS polymerase is an enzyme with function to catalyze transcription process. It consist of 2 subunit α, sub unit We have analysed fourteen species of β, subunit β’ and subunit ω, and also need σ factor for Rhodobacter and five reference organisms in this study. promotor recognition [10]. Subunit β and β’ are encoded The level of genes sequences identity among by rpoB and rpoC genes, while σ70 factor is encoded by Rhodobacter species were 93.4 – 99 % (16SrRNA), 81.7 rpoD gene. Rhodobacter genus has characteristics of – 99 % (rpoB), 81.8 – 99 % (rpoC) and 80.2 – 99.1 % gram negative bacteria with rod shaped morphology, (rpoD). The level of genes sequences identity between able to form vesicular intrasitoplasmic membrane as Rhodobacter species and and reference organisms were well as ability to grow anaerobically and 77.8 – 94,8 % (16SrRNA), 64.5 – 87.5 % (rpoB), 63.8 – photoheterotrophically using sulfide as electron donor 86 % (rpoC), 62.5 – 86.4 % (rpoD). The range between [11]. Some members of the genus have ability to fix the highest and lowest level of sequence identity for nitrogen and have been used as model system to study each gene was calculated and the data is presented in photosynthesis and membrane development [12]. figure 1. Rhodobacter genus is a genus of phototrophic purple Figure 1 show that the range of level identity nonsulfur α-proteobacteria,a class in the phylum of among 16SrRNA gene sequence of Rhodobacter species Proteobacteria. Close phylogenetic relationship between is the smallest and on the other side, range of level phototrophic and non-phototropic members of identity among rpoD gene sequence is the bigest Proteobacteria made communities analysis of compare to other gene sequncesanalysed. The same Rhodobacterbase solely on 16SrRNA gene become result is also shown for comparison of the level identity complicated [13]. range of genes sequences between Rhodobacter species The aim of this study was to investigate benefit to reference organisms. of sequence analysis of 16SrRNA, rpoB, rpoC and rpoD genes for infering phylogeny and identification 0,3 tools of Rhodobacter genus. 0,25 METHODS 0,2 0,15 Nucleotide sequences of 16SrRNA genes, rpoB 0,1 genes, rpoC genes and rpoD genes were retrieved from 0,05 National Center for Biotechnology Information (NCBI) % Identity Data Bank, United States. The source of these genes 0 sequences were Rhodobacter megalophilus DSM 18937, 16S rRNA rpoB rpoC rpoD Rhodobacter capsulatus SB 1003, Rhodobacter sphaeroides 2.4.1, Rhodobacter johrii JA 192T, Rhodobacter ovatus JA 234, Rhodobacter azotoformans Figure 1. Range between highest and lowest percentage KA 25, Rhodobacter blasticus 28/5, Rhodobacter identity for each gene tested, measure among Rhodobacter veldkampii DSM 11550, Rhodobacter aestuarii DSM species (filled bars), between Rhodobacterspecies and 19945, Rhodobacter vinaykumarii DSM 18714, reference organisms (open bars). Rhodobacter maris JA 276, Rhodobacter viridis JA 737, International Journal of Health Medicine and Current Research | 1479 Phylogenetic based on 16SrRNA sequence topology of the tree has high similarity to topology of showed that all species members ofRhodobacter genus tree based on 16 S rRNA gene sequence,except for are separated into two major clusters (Figure 2). First Paracoccusdenitrificanis movedin second cluster of cluster of Rhodobacter genus consist of R. Rhodobacter genus and Pararhodobacter sp. CCB – megalophilus, R. sphaeroides, R. johrii, R. ovatus, R. MM2 is placed on its own tree branch separated from azotoformans, R. blasticus and the second cluster consist clusters of Rhodobacter (Figure 3).. Pararhodobacter of R.veldkampii, R. aestuarii, R. vinaykumarii, R. maris, sp. CCB – MM2 is also placed at its own branch in R. capsulatus, R. viridis and Rhodobacter sp. LPB0142. phylogenetic tree based on rpoC gene sequence. Pararhodobacter sp. CCB – MM2 is placed in the first Paracoccus denitrificans together with R. veldkampii cluster of Rhodobacter genus eventhough it is not the and R. vinaykumarii are shifted to first cluster of member of this genus. Roseobacter denitrifican and Rhodobacter (Figure 4). Each of Paracoccus denitrificans are clustered together. Pararhodobactersp.CCB – MM2 and Paracoccus Rhodovulum sulfidophilum and Rhodopseudomonas denitrificans are placed at their own branch on the palustris, two other phototrophic Proteobacteria are phylogenetic tree based on rpoD gene sequence however placed on their own tree branch. Gene sequence of Rhodovulum sulfidophilum and Rhodobacter Escherichia coli was used as an outlier to root the vinaykumarii are placed together in one cluster (Figure tree.rpoB sequence based phylogenetic tree showed that 5). Figure 2. Phylogenetic tree based on analysis of 16SrRNA gene sequence. Genetic distance is indicated by scale. Figure 3. Phylogenetic tree based on analysis of rpoB gene sequence. Genetic distance is indicated by scale. International Journal of Health Medicine and Current Research | 1480 Figure 4. Phylogenetic tree based on analysis of rpoC gene sequence. Genetic distance is indicated by scale. Figure 5. Phylogenetic tree based on analysis of rpoD gene sequence. Genetic distance is indicated by scale. DISCUSSION genes sequence. Pararhodobactersp.CCB-MM2 was previously known as Rhodobacter sp. CCB-MM2 before The result of phylogenetic analysis