MOLECULAR IDENTIFICATION (16S rrna) OF BACTERIAL ISOLATES FROM GUNDARU RIVER BASIN AND SUBMISSION OF GENBANK AND CONSTRUCTION OF PHYLOGENETIC TREE

Transcription

1 CHAPTER IV MOLECULAR IDENTIFICATION (16S rrna) OF BACTERIAL ISOLATES FROM GUNDARU RIVER BASIN AND SUBMISSION OF GENBANK AND CONSTRUCTION OF PHYLOGENETIC TREE Introduction The conventional bacterial taxonomy a variety of characteristics of different strain or species are measured and these traits are then used to group the organisms. Characteristics of taxonomic value that are widely used include morphology, Gram reaction, nutritional classification (phototroph, chemoorganotroph and chemolithotroph), cell wall chemistry, presence of cell inclusion and storage products, capsule chemistry, pigments, nutritional requirements, ability to the various carbon, nitrogen and sulfur sources, fermentation products, gaseous needs, temperature and ph requirements (and tolerances), antibiotic sensitivity, pathogenecity, symbiotic relationship, immunological characteristics and habitat. It does not give reliable results. Ribosomal RNA s are ancient molecules, functionally constant, universally distributed and moderately well conserved across broad phylogenetic distances. The number of different possible sequences of large molecules such as ribosomal RNAs. There are three ribosomal RNA molecules, which in prokaryotes have sizes of 5S, 16S and 23S. The large bacterial rrnas, 16S and 23S rrna (approximately 1500 nucleotides and 2900 nucleotides respectively) contain several region of highly conserved sequence useful for obtaining proper sequence alignments. 91

2 Sufficient sequence variability in other region of the molecule to serve as excellent phylogenetic chromometers. 16S RNA is more experimentally manageable than 23S RNA it has been med extensively to develop the phylogeny of both prokaryotes and eukaryotes (using the 18S rrna counterpart of prokaryotic 16S rrna). The database of rrna sequences now numbers over 10,000 and can be accessed on the world wide web. Use of 16S rrna as a phylogenetic tool was pioneered in the early 1987s by Carl Woese at the university of Illinois, and the method is non widely used. Ribosomal RNA s are now relatively easy to analyze because they can be directly sequenced from crude cell extracts using reverse transcriptase and the dideoxy sequencing method. Although direct sequencing of ribosomal RNA is still used, newer methods are beginning to supplant this approach. Bacterial phylogenetic relationships The sequencing of proteins and nucleic acids provides a new and more powerful approach to measuring evolutionary relationships and a new way of looking at them in terms of the Evolutionary clock (Wilson et al., 1984). Genotyphic information i.e., sequence information, is superior in two main ways to phenotyphic information, the classical basis for relating and classifying organisms sequence information is (i) more readily, reliably and prelisely interpreted and (ii) innately more informative of evolutionary relationships then phenotyphic information. Molecular chronometers A molecule whose sequence changes randomly in time can be considered a chronometer the amount of sequence change it accumulates (formally a distance) is the product of a rate (at which mutations become fixed) x a time (over which the changes have occurred). The biologist cannot measure this change, 92

3 however by comparison of some original to some final state, since the original state, (ancestral pattern) is not accessible to him, Instead, the uses the fact that two (or more) visions of a given sequences that occurs in extent representatives of two (or more) lineages have ultimately come from the same common ancestral pattern, and so measures the sequence difference between the two (or more) extant versions which is roughly twice the amount of change that each lineages has undergone (assuming comparable rates of change in each) since they last shared a common ancestor. All sequences are not of equal value determining phylogenetic relationships. To be a use for chronometer, a molecule has to meet certain specification as to (i) clock like behavior (change in its sequences have to occur as possible) (ii) range (rates of change have to be commensurate with the spectrum of evolutionary distances being measured) and (iii) size (the molecule has to be large enough to provide an adequate amount of information and to be a smooth running). Phylogenetic range The world of bacterial evolution is vast in comparison to that of the eukaryotes with which we are familiar, in the meta 300. A billion years is a relatively short time in bacterial evolution, therefore the range of chronometers used to measure phylogenetic relationships among bacteria needs to be considerably greater than what is optimal for meta 300. Cytochrome c is an excellent chronometer for measuring match of eukaryote phylogeny (Fitch et al., 1967). Among the eubacleria its effective range is restricted to the subdivision level: it orders the α-purple bacteria but close not related these accurately to any other subdivision of the purple bacteria. The strength of 16S rrna analysis is its rapidity and simplicity, and it can therefore used to confidently recognize the level at which DNA pairing studies need to be performed. Similarities of greater than 97% of 16S rrna sequences will most probably equate to 70% DNA sequence homology. (Stackbrandt and Goebel, 1993). 93

4 Fox et al., 1992 pointed out that 16S rrna data are still a powerful tool for determining to which species a strain probably belongs once the relevant species are represented in the 16S rrna sequence database. Maidana et al., 1998 described an alternative molecular typing method that was applicable to almost all bacterial species, including those that had difficult to cultivate. They used a technique called multi - locus sequence typing in which DNA fragments of bacterial house keeping genes are amplified using PCR and then sequenced. Typing of strains were made by comparing the sequences of these genes between three and eight different loci. They proposed that laboratories in different countries and continents relate their local isolates from house keeping gene fragments to a central world wide database. The most important development in the application of 16S rrna sequences to bacterial systematics was the advent of PCR, which enabled the amplification of these sequences without necessarily culturing the microorganisms. (Wilson et al., 1990). Direct sequencing of 16S rrna requires microgram quantities of rrna per reaction, which cannot be obtained from bacteria that do not grow well in culture. Because some segments of rrna are conserved, whilst others are variable, it is possible to generate oligonucleotide primers that are complementary to segments specific for any level of the phylogenentic heirarchy. The use of specific 16S rrna probes has enabled researchers to amplify specific 16S rrna sequences from mixed cultures and therefore allows phylogenetic analysis, estimation of bacterial diversity and identification of isolates directly from clinical or environmental sites (Martinko et al., 1997 Wilson et al., 1990). 94

5 Further requirements of refinements of the PCR methodology has led to development of a capable of initiating enzymatic amplification of a phylogenetically and taxonomically wide range of bacteria and also primers capable of amplifying nearly full length 16S rrna sequences (Weisburg et al., 1991). The sequence of 16S and 18S rrna was obtained from a broad spectrum of organism (Woese et al., 1990) the bacteria are common organism that live in virtually all soil and aquatic environments. The most common used procedure to assess microbial diversity was originated in Pace s laboratory (Pace et al., 1986). The DNA is followed by polymerase chain reaction PCR (Mullis et al., 1987) has begun to applied to environmental detection of microorganism. Purified PCR products clone cells were sequenced using big dye terminator (Applied Biosystems) in Macrogen, Korea. Over the past few years, genotypic identification procedures had increasingly received attention as an alternative or complement to conventional phenotypic methods. Genotypic techniques involved the amplification of a phylogenetically informative target, such as the small subunit (16S) rrna gene. Broad range primers that recognized 16S ribosomal rdna sequences conserved among a wide variety of bacteria are used to amplify species specific variable regions of interest. In this study, we had evaluated the suitability of 16S rrna sequencing for the identification of mesophilic Gram negative rods under routine conditions 16S rrna sequences were compared with those available in the Gen Bank, EMBL and DDBJ databases. Although any gene might be used as Genetic marker, rrna genes offered distinct advantages. The extensive use of 16S rrnas for studies of microbial systematics and evolution had resulted in large computer data bases, such as the RNA Data Base project, which encompassed the phylogenetic diversity found within culture collections rrna genes are highly conserved and therefore could be used to examine distant phylogenetic relationships with accuracy. 95

6 Discussion Schmidt et al., 1991 studied 16S rdna sequence data of 10 Nitrosomonas species and Nitrococcus mobilis. 16S rrna sequences were determined for three Nitrococcus species isolates and for gamma subclass proteobacterium Nitrococcus halophilus. Richard et al., 1994 studied the divergence of 16S rdna sequences in marine sediment. They found twenty unique partial sequences among 33 cloned following PCR. Thirteen shared 82 to 91% similarity with sequences of delta subclass sulfate reducing bacteria. Three contained the target sequence for a sulfate reducing bacterium. Bourget et al., 1993 determined 16S rrna sequences of five Bifidobacterium strains representing four species and compared them with sequences available in Genbank database and used them to construct a distance tree and for a book strap analysis. They constructed phylogenetic tree based on 16S rrna sequence data. They found that two trees were similar, suggesting that the two types of molecules provided the same kind of phylogenetic informations and allowed them to confirm the validity of most of Bifidobacterium sp. Moore et al., 2002 reported that 16S rrna sequencing is 90% accurate to confirm Enterococcus genus. These results are in concurrence with reported findings of Jordan et al., (Jeane A Jordan, University of Pittsburgh, 2004). A molecular based approach for detecting a target like 16S rrna, which is present among all bacteria would be useful it provides invaluable information for physicians (Jordane et al., 2000). The 16S rrna gene (1500 bp) is large enough for information purposes. 16S rrna gene sequence can be used for multiplicity of purposes, there is 96

7 universal agreement of definitive and conclusive identification to the rank of species. (Mickael, 2005). 16S rrna gene analysis provides accurate identification at the species level and can clarify their clinical importance. Tang et al., compared a variety of identification systems including cellular fatty acid profiles, carbon source utilization and conventional biochemical identification with the 16S rrna gene sequence to elevate both unusual aerobic gram negative bacilli and coryneform organisms isolated from clinical specimens (Jensen, 1985, Jones et al., 1983). They found that 16S rrna gene sequence provide more rapid, unambiguous identification could translate improved clinical outcomes. In a large reference laboratory, Hall et al., 2003 found that 16S rrna gene sequence could identify 243 of 328 clinical isolates with phenotypic of < 1.1. Tortoli presented that the essential contribution made by 16S rrna gene sequencing of classically known species of slow and rapid growers into new groupings. Brouqui and Raulot using broad based PCR amplification of the 16S rrna gene, found the most common etiologic agents associated with culture negative endocarditis were Bactonella Quintana and Coxicella burnettii, both of which require special conditions to grow. The extensive use of 16S rrnas for studies of microbial systematics and evolution has resulted in large computer bases of data, such as the RNA Data Base project which encompass the phylogenetic diversity found with in culture collection. Various bacteria species could be identified by polymerase chain reaction analysis with two primers specific for highly conserved sequence region in the bacterial 16S rdna and by sequencing. The 16S rrna gene (15000 bp) is large enough for informatics purposes. It has been demonstrated that 16S rrna sequence data on an individual strain with a nearest neighbour exhibiting a similarity store of 97

8 <97% represents a new species, the meaning of similarity scores of 97% is not as clear. For bacteria that are difficult to grow or identify the identification rates were lower with 16S rrna sequencing than the values traditionally acceptable in the clinical laboratory (i.e., >90%). A 1995 study by Clayton et al., 1995 also revealed that at least 56% of 16S rrna gene sequence pairs (2 sequence deposited for the same species) in Genbank had >1% random sequencing errors and of these almost half had 75% random sequencing errors. Some researchers would never using a molecular identification over a conventional one 16S rrna gene sequencing is not infalliable and examples of such misidentification have been published. In 2000, Drancourt et al., made several recommendations concerning proposed criteria for 16S rrna gene sequencing on a reference method for bacterial identification. The isolate was identified and determined by means of DNA sequence analysis of a portion of the 16S rrna gene. The 16S rrna genes of isolates were partially amplified using primers 27F and 1492R and custom sequenced primer 341F by MWG Biotech (Ebersbey, Germany) PCR products were analysed on 2% TAE agarose gel. Comparison of 16S rrna have proven to be extremely useful for determining of phylogenetic relationships assay organisms from the level of domain to the level of moderately closely related species. The 16S rrna gene in universal in bacteria and so relationships can be measured among all bacteria. Usually PCR results can only be used to tell the presence or absence of the targeted species to functional group and it is generally accepted that an exact quantitative population composition from an environmental sample can never be confidently reported. Targeting a 16S rrna gene sequence for this purpose has many advantages; it is present as multiple copies per cell, and contains both highly conserved and variable regions. Laura et al., 2003 demonstrated 98

9 real time PCR methods for enterococcus and adenovirus quantification of both organisms in environmental waters. In the present study bacterial isolates are amplified by electrophoresis and shown in Fig. 25. In our study, phylogenetic and phonetic approaches to bacterial taxonomy were described and compared. From the isolated Salmonnella sp. 16S rrna sequencing were carried out. Although a variety of molecular techniques were used in estimating the phylogenetic relatedness of bacteria the comparison of 16S rrna is bacterial isolated were submitted to the Genbank NCBI US. Comparison of 16S rrnas have proven to be extremely useful for determining phylogenetic relationships among organisms from the level of domains to the level of closely related species 31. Phylogenetic tree generated using 16S rrna sequence of bacterial isolates from Gundaru river basin is shown in the Fig. 26. Alcaligens sp. encoded the Genbank accession number EF195165, EF195166, EF and EF Bacillus encoded the Genbank accession number of EF Achromobacter sp. encoded the Genbank accession number of EF Enterobacter aerogens and Enterobacter sp. encoded the Genbank accession number of EF and EF respectively. Salmonella typhi, Salmonella typhimurium, S.enterica, Salmonella enterica, Salmonella sp. encoded the Genbank accession number of EF195174, EF579648, EF579646, EF and EF respectively. Brevundimonas sp. encoded the Genbank accession number of EF Bacterial isolates from Gundaru river basin ad their Genbank accession numbers are reported in Table. 27. The results which were obtained in this study leads to the conclusion that the evolutionary relations exhibited by the isolates are such that these organism belong to the same eubacterial group. Bacterial diversity in environmental samples is usually determined by a characterization of isolated strains. After isolation of total bacterial DNA, variable 99

10 regions of 16S rrna gene are amplified by PCR and the resulting Diversity pattern are analysed and compared. (Bernard et al., 1995). The quickest way to survey the constituents of microbial ecosystems is through the use of PCR. (Saiki et al., 1988). To study the microbial diversity is thus important to solve new and emerging disease problems and to advance biotechnology. (Kapur et al., 2004). 16S rrna sequencing can be used for molecular characterization of microbial diversity in hyper saline ponds. Katherine, 2001 characterized the microbial diversity in hypersaline ponds by 16S rrna sequence San Salvador Island, Bahamas. 100