Phylogeny and identification of Pantoea species associated with plans

Phylogeny and identification of Pantoea species associated with plans, humans and the natural environment based on multilocus sequence analysis (MLSA).

By
Mabaso Anna Mangwase
201400348
A Research Proposal Submitted in fulfilment of the requirements for the degree of Bachelor of Technology in Biotechnology, to the Faculty of Science, University of Johannesburg
Supervisor: Dr Mahloro Hope Serepa-Dlamini
Date: 12 April 2018
DECLARATION
I Mabaso Anna Mangwase, hereby declare that this research proposal herewith submitted for the degree of Bachelor of Technology, has not been previously submitted by me to obtain a degree at any other University. The research described in this study was carried out in the Department of Biotechnology, Faculty of Science at the University of Johannesburg, South Africa.

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Signature…………………………………………………
Mabaso Anna Mangwase
Date of signature:
ABSTRACT
Enterobacteriaceae is a gram negative family that incorporate the genus Pantoea. Endophytes are microorganisms that occupy the epidermal part of plant tissues and they do not cause harm to the plant instead they a form a mutual relationship. The method that will be used to identify and categorise Pantoea species is called multilocus sequence analysis (MLSA) which is quick and accurate. The aim of this study will be to The aim of this study will be to identify Pantoea species from bacterial endophytes and to also determine their correct position on the phylogenetic tree using multilocus sequence analysis . The isolated genomic DNA will be amplified on PCR using primers and the PCR product will be taken to Inqaba Biotech for sequencing.

Keywords: MLSA, Pantoea, Endophytes, Sequencing, PCR, Genomic DNA.

ACKNOWLEDGEMENTS
I would like to acknowledge the department of Biotechnology; my supervisor; NRF which is a company that provides me with funding for this project and family members who will be supporting me throughout the project.

TABLE OF CONTENTS
Declaration…………………………………………………………………… 2
Abstract…………………………………………………………………………3
Acknowledgements…………………………………………………………………….4
1Introduction……………………………………………………………………6
1.1. Research problem…………………………………………………………6
1.2. Hypothesis………………………………………………………………….6
1.3. Aims and objectives………………………………………………………..6
1.3.1 Aim…………………………………………………………………………6
1.3.2 Objective……………………………………………………………………7
2. Literature review………………………………………………………………7
2.1 Isolation of endophytes……………………………………………………..7.

2.2. Identification of endophytes………………………………………………..7
2.3. Electrophoresis………………………………………………………………7
2.4. Polymerase Chain Reaction (PCR)………………………………………..8
2.5. Multilocus sequence analysis (MLSA)……………………………………..8
3. Materials and method…………………………………………………………..8
3.1. Materials and reagents………………………………………………………8.

3.2. Methodology…………………………………………………………………..8
3.2.1. Primer design……………………………………………………………….8
3.2.2. PCR and sequencing………………………………………………………8
4. Expected outcomes………………………………………………………………9
5. References………………………………………………………………………..9-10
INTRODUCTION
Pantoea is a genus of gram negative bacteria that is found in the family Enterobacteriaceae, there are several species found in the genus Pantoea and these species can be epiphytes or pathogens depending on how they are associated with the plant (Deletoile et.al, 2009). Referring to a study done by Deletoile et.al (2009), the genus Pantoea has different species identified such as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, Pantoea dispersa, Pantoea citrea, Pantoea punctate and Pantoea terrea.

Based on a study done by Nair and Padmavathy (2014), there are certain microorganisms such as fungi and bacteria which inhabit the healthy tissues of plants and stay there without causing any harm to the plant and the specific name for these microorganisms is endophyte. These endophytes are found everywhere; it can be on plants, on the environment or even on humans (Nair and Padmavathy, 2014). The intercellular spaces on plant tissues contain inorganic nutrients, carbohydrates and amino acids and become a good environment for endophytes to stay there (Suhandono et.al, 2016).

Endophytes can be obligate or facultative depending on their location on the host plant; obligate endophytes require plant metabolism in order for them to survive while facultative endophytes stay outside the plant tissue during a certain stage of their life cycle (Gouda et.al, 2016). The relationship between endophytes and plant tissue can either be mutualism where both organisms benefit, for instance, the endophyte produces various compounds that are needed for plant growth and also helping the plant to adapt to the environment or antagonism where one organism benefit and the other does not benefit (Nair and Padmavathy, 2014).

Research problem
Pantoea species are very identical and are closely related phenotypically, because of this characteristic, it becomes difficult to identify and classify them. 16S rRNA sequencing cannot clearly identify them, hence a new method called multilocus sequence analysis (MLSA) is used since it is quick and able to identify and classify Pantoea species correctly.

Hypothesis
Multilocus sequence analysis will classify and identify Pantoea species to species level.

Aims and objectives
Aim
The aim of this study will be to identify Pantoea species from bacterial endophytes and to also determine their correct position on the phylogenetic tree using multilocus sequence analysis.

Objective
In order to achieve the aim mentioned, the following objective will be achieved:
Genomic DNA must be properly extracted and identified using the alkali extraction method.

The extracted genomic DNA must be amplified using MLSA polymerase chain reaction (PCR).

The amplified genomic DNA will be sequenced using sequencing primers.

LITERATURE REVIEW
There are several benefits endophytes have on plants and these are as follows; they help the plant to grow and protect the plant from pathogens (Santoyo et.al, 2015). The endophytes have two mechanisms which they use to assist in plant growth and are: direct mechanism is when the endophytes transport important nutrients throughout the plant and indirect mechanism is when the plant is protected against pathogens by the endophyte (Santoyo et.al, 2015). Endophytes can be used in different industries such as agriculture, medicine, prevents pathogens from affecting plants; they produce secondary metabolites and bioactive compounds (Mousa and Raizada, 2013).

Isolation of endophytes
Different parts of plant such as meristem, roots, leaf and petiole are locations where endophytes can be isolated from (Nair and Padmavathy, 2014). The surface of plant tissue is firstly sterilized then the ground tissue extract is cultured on the media suitable for the microbe so that the endophyte can be isolated (Nair and Padmavathy, 2014).
Identification of endophytes
Morphological characteristics of bacteria, fungi and actinomycetes assist in the identification of endophytes, together with biochemical tests done on these microorganisms (Nair and Padmavathy, 2014). There is a certain stage where endophytes sporulate from the plant, once this occurs; it becomes easy to identify them using morphological and molecular biological techniques (Strobel and Daisy, 2003). Ribosomal DNA Internal Transcribed Spacer which is abbreviated as ITS is a sequence analyser that is widely used to identify these endophytes (Nair and Padmavathy, 2014).
Electrophoresis
Electrophoresis is a type of separating technique that uses insufficient equipment to separate molecules well; its principle is that, it works under the influence of an electrical field whereby charged molecules will move to the direction of electrode containing opposite charge (Westermeier, 2005).
Electrophoresis is done in the presence of a buffer which has a specific pH value and a constant ionic strength whereby the ionic strength should be as low as possible to achieve high speed of sample ions and their total current (Westermeier, 2005).Gel electrophoresis mostly agarose gel electrophoresis which forms a solid matrix is used to separate DNA based on size where small size will move faster than larger size (Westermeier, 2005).

Polymerase Chain Reaction (PCR)
DNA is amplified to form many copies of its sequences using a technique called PCR, the advantages of using PCR are that it is cheap, easy to use and quick (Joshi and Deshpande, 2010). Annealing where primers anneal to the DNA template, denaturation of DNA at high temperatures and extension where a complimentary strand is formed after primer has annealed are three steps involved in a PCR technique (Joshi and Deshpande, 2010).

Multilocus sequence analysis (MLSA)
In the beginning, 16S rRNA was used to detect bacterial phylogenies but with some species the phylogenic resolution was not clear hence the use of MLSA (Delamuta et.al, 2012). MLSA is a method used in studies of taxonomy and phylogeny; it is used to analyse several housekeeping genes and is preferred because the phylogenetic resolution obtained will be good and is effective (Delamuta et.al, 2012).
The primary DNA sequences from multiple conserved protein-coding loci is compared using MLSA so that the diversity and relationship of different isolates across related taxa can be compared with the use of an appropriate phylogenetic approach (Naser et.al, 2005).

MATERIALS AND METHODS
Materials and reagents
The following equipment’s and reagents will be used: bacterial culture, primers (both forward and reverse primers), DNA extraction kit and PCR, in addition, MLSA, pipettes, pipette tips and PCR buffer will be used (Drady et.al, 2008) . Furthermore, dNTP’s (deoxynucleotide triphosphates), sterile milliQ water, agarose gel, sequencing buffer, big dye sequencing reaction mix and montage seq 96 sequencing reaction clean-up kit will be used (Drady et.al, 2008).
Methodology
Strains investigated and DNA extraction
Bacterial strains that will be used will be kept in Bacterial Culture Collection, an alkali extraction method will be used to extract genomic DNA form each of the bacterial strains and the extracted genomic DNA will be stored at -20oC (Drady et.al, 2008).

Primer design
According to the sequence alignment of the bacterial strains, internal primers which will be used for sequencing and external primers which will be used for amplification will be designed (Drady et.al, 2008).
PCR and sequencing
The specific primers and conditions will be selected so that the complete 16S rRNA sequences will be determined for the bacterial strains selected of the Pantoea species (Drady et.al, 2008). The amplification primers will be used so that MLSA PCR will be done on the bacterial strains, each 50µl PCR will contain 5µl 10x PCR buffer, 5 µl of dNTP (200µM each), 0.5µl forward primer (50µM), 0.5µl forward primer (50µM), 1µl AmpliTaq DNA polymerase (1U/µl), 5µl template DNA and 33µl sterile milliQ water (Drady et.al, 2008).

The amplification conditions will be as follows: denaturation at 95?C for 5minutes which will be followed by 3 cycles of denaturation at 95?C for 1 minute; annealing will be done at 55?C for 2minutes and 15seconds and elongation will be done at 72?C for 1minute and 15seconds and will be followed by 30 cycles od denaturation at 95?C for 35 seconds, annealing at 55?C for 1minute and 15 seconds and elongation at 72?C for 1minute and 15seconds and a further 7 minutes of elongation a 72?C; An annealing temperature of 50?C will be used for several strains which would not amplify at 55?C (Drady et.al, 2008).The PCR products will be separated on 1% agarose gels at 75V for 45 minutes, the positive PCR products which will be having the expected size will be purified using NucleoFast 96 PCR plates (Machery-Nagel) (Drady et.al, 2008).
Sequencing reactions will be done using 3µl purified PCR product, 2µl 5x sequencing buffer, 0.2µl big dye sequencing reaction mix, 3µl primer (4µM) and 1.8µl sterile milliQ water, sequencing conditions will be done at 96?C for 5seconds for denaturation, 25 cycles of denaturation at 96?C for 10 seconds, annealing will be done at 55?C for 10 minutes and elongation will be done at 60?C for 4 minutes (Drady et.al, 2008). The Montag Seq 96 Sequencing Reaction Cleanup Kit (Millipore) will be used to purify the sequencing reactions. A Genesis Workstation 200 (Tecan) will be used to carry-out the PCR purification steps and set-up. The PCR product will then be taken to Inqaba Biotech for sequencing (Drady et.al, 2008).

EXPECTED OUTCOMES
At the end of the study, it will be expected to isolate genomic DNA from Pantoea species and be able to identify and categorize them to species level on the phylogenetic tree within the Enterobacteriaceae family using MLSA. The identification of these Pantoea species is important since they cause human and plant diseases, by doing this they will be able to be treated.
REFERENCES
Brady, C.; Cleanwerck, I.; Venter, S.; Vancanneyt, M.; Swings, J. and Coutinho, T. (2008). Phylogeny and Identification of Pantoea Species Associated with the Natural Environment Based on Multilocus Sequence Analysis (MLSA). 31. 448 and 452.

Delamuta, J.R.M.; Ribeiro, R.A.; Menna, P.; Bangel, E.V. and Hungria, M. (2012). Multilocus Sequence Analysis (MLSA) of Bradyrhizobium Strains: Revealing High Diversity of Tropical Diazotrophic Symbiotic Bacteria. Brazilian Journal of Microbilogy. 699.

Deletoile, A.; Decre, D.;Courant, S.; Passet, V.; Audo, J.; Grimont, P.; Arlet, G. and Brisse, S. (2009). Phylogeny and Identification of Pantoea Species and Typing of Pantoea agglomerans Strains by Multilocus Gene Sequencing. Journal of Clinical Microbiology. 47: 300.

Gouda, S.; Das, G.; Sen, S.K.; Shin, H. and Patra, J.K. (2016). Endophytes: A Treasure House of Bioactive Compounds of Medicinal Importance. Journal of Frontiers in Microbiology. 7: 2.

Joshi, M. and Deshpande, J.D. (2010). Polymerase Chain Reaction: Methods, Principles and Application. Journal of Biomedical Research. 5: 91.

Mousa, W.K. and Raizada, M.N. (2013). The Diversity of Anti-Microbial Secondary Metabolites Produced by Fungal Endophytes: An Interdisciplinary Perspective. 1-2.

Nair, D.N. and Padmavathy, S. (2014). Impact of Endophytic Microorganisms on Plants, Environment and Humans. The Scientific World Journal. 1-2.

Naser, S.M.; Thompson, F.L.; Hoste, B.; Gevers, D.; Dawyndt, P.; Vancanneyt, M. and Swings, J. (2005). Application of Multilocus Sequence Analysis (MLSA) for Rapid Identification of Enterococcus Species Based on rpoA and pheS Genes. Journal of Microbiology. 151: 2142.

Santoyo, G.; Moreno-Hagelsieb, G.; Orozco-Mosqueda, M. and Glick, B.R. (2015). Plant Growth –Promoting Bacterial Endophytes. Journal of Microbiological Research. 183: 93-95.

Strobel, G. and Daisy, B. (2003). Bioprospecting for Microbial Endophytes and their Natural Products. Journal of Microbiology and Molecular Biology.67: 495.

Suhandono, S.; Kusumawardhani, M.K. and Aditiawati, P. (2016). Isolation and Molecular Identification of Endophytic Bacteria from Rambutan Fruits (Nephelium lappaceum L) Cultivar Binjai. Journal of Biosciences. 23: 39.

Westermeier, D.A. (2005). One-Dimensional Gel Electrophoresis. Journal of Methods Enzymol. 463: 14 and 40.