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Summer Research Fellowship Programme of India's Science Academies

In- vivo protein-protein interaction studies in Xanthomonas oryzae pv. oryzicola (Xoc) using bacterial adenylate cyclase two hybrid system

Neha Bhandari

Banasthali Vidyapith, Rajasthan 304022

Dr. Subhadeep Chatterjee

Laboratory of Plant- Microbe Interactions, Centre for DNA Fingerprintng and Diagnostics (CDFD) Uppal, Hyderabad 500039

Abstract

Xanthomonas are a group of important pathogenic Gram negative bacteria which cause disease in more than 400 different economically important plants, resulting in severe loss to world-wide annual crop production. Xanthomonas oryzae pv. oryzicola (Xoc) is the cause of bacterial leaf streak (BLS) in rice. Our study is based on the Phosphate regulon and the DSF (Diffusible Signal Factor- that primarily mediates Quorum sensing) regulon and will focus on the protein-protein interactions between these two distinct regulatory networks and their possible role in maintenance of iron homeostasis. The in- vivo protein-protein interactions will be studied with the aid of Bacterial Adenylate Cyclase Two Hybrid (BACTH) approach. The phosphate regulon is efficiently regulated by the two component system PhoB and PhoR, where PhoR is the sensor Kinase and PhoB is its cognate response regulator; PhoU is the accessory protein involved in this regulation. RpfF is the DSF synthase and Fur is the ferric uptake regulator involved in regulation of ferric ion uptake by controlling various mechanisms including siderophore synthesis. This study will focus primarily on the in-vivo protein-protein interactions between PhoB, PhoR, PhoU, RpfF and Fur; and will provide an insight into the plausible crosstalk between the Pho regulon and Quorum Sensing with respect with the maintenance of iron homeostasis.

Keywords: phosphate regulon, DSF (diffusible signal factor), ferric uptake regulator

Abbreviations

Abbreviations
µg microgram
mg milligram  
g gram
µl micro litre
ml millilitre
nm nanometre
M molar concentration  
µM micro-molar concentration  
°C Degree centigrade
Tm Melting Temperature
Xoc Xanthomonas oryzae pv. oryzicola  
LB medium Luria-Bertani Medium
X-gal 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside
IPTG Isopropyl β-D-1-thiogalactopyranoside
Conc Concentration  
TCS   Two component system  
TAE Tris acetate EDTA buffer
PCI Phenol chloroform isoamyl alcohol
BACTH Bacterial Adenylate Cyclase Two Hybrid  

INTRODUCTION

Background

The phosphorus compounds serve as major building blocks of many biomolecules, and have important roles in signal transduction. Phosphate is involved in many biochemical reactions which involves the transfer of phosphoryl groups. All living cells sophisticatedly regulate the phosphate uptake, and survive even under phosphate-limiting condition, and thus phosphate metabolism is closely related to the diverse metabolism including energy and central carbon metabolism (Marzan, et al, 2011).

Adapting to changes in the environment is one of the hallmarks of life. For all life, phosphate is an essential nutrient. Bacteria have several mechanisms to scavenge phosphate that are only expressed when the level of available environmental phosphate is limited. Expression control of these genes is essential for optimal growth and has been implicated in the regulation of pathogenesis in several organisms (​Gardner, et al, 2015​).

In Xanthomonas a classic two-component signal transduction system, composed of the PhoR histidine kinase and the PhoB response regulator, is responsible for expression control of a group of genes called the Pho regulon. The Pho regulon is a huge regulatory network in bacteria. The phosphate (Pho) regulon is controlled by the two component-regulatory system PhoB and PhoR. s(2) Xanthomonas employs genes whose products sense environmental phosphate (Pi) and control the expression of the Pho regulon. These genes include phoB, phoR and phoU. Proper phosphate signaling is essential for robust growth of Xanthomonas oryzae pv. oryzicola and many other bacteria. Together, these genes are necessary and sufficient for Pi signal transduction (​Santos-Beneit, et al, 2015​).

PhoB consists of an N-terminal phosphorylation domain that receives a phosphoryl group from PhoR and a C-terminal DNA binding domain. In low phosphate conditions, PhoR acts as a PhoB kinase. Upon phosphorylation, PhoB recruits RNA polymerase to promoters of the Pho regulon that contain a Pho box. In high phosphate conditions, PhoR acts as a phospho-PhoB phosphatase and removes the phosphate from PhoB to keep the expression level of Pho regulon genes very low. PhoR lacks a significant periplasmic domain that could detect phosphate abundance outside the cell and the PhoU protein play important roles in phosphate signaling to PhoR (​Gardner, et al, 2015​).

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    Model system of Xanthomonas showing interaction by a classic two-component signal transduction system, composed of the PhoR histidine kinase and the PhoB response regulator,  responsible for expression control of a group of genes of Pho regulon. Xanthomonas genes products sense environmental phosphate (Pi) and control the expression of the Pho regulon.

    Statement of the Problems

    Components of rpf (regulation of pathogenicity factor) cluster in Xanthomonas is involved in synthesis and perception of DSF. The gene rpfF encodes DSF synthase (RpfF), and is highly conserved across different Xanthomonas species. The rpfF gene is involved in production of a diffusible signalling factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes (​Ionescu, et al, 2013​ ​ ).

    In many bacteria, iron uptake and siderophore biosynthesis genes are tightly controlled by an active repressor Fur (Ferric uptake regulator) protein. Under the iron-replete condition, Fur dimer forms complex with co-repressor Fe2+ (holo-Fur) and binds to the conserved fur-box at upstream to the iron uptake and siderophore biosynthesis genes and consequently suppress the gene expression. In the E. coli, the ‘fur-box’ has been identified as the nucleotide sequence of 19bp palindromic inverted repeats (GATAATGATAATCATTATC). Under the iron-deplete condition, apo-Fur gets released from the promoter, which allows RNA-polymerase to binds at the promoter and consequently, gene expression starts .Role of Fur have been observed not only in the regulation of iron uptake and metabolism but also in the regulation of diverse cellular processes such as glycolysis, TCA cycle, respiration and oxidative stress (​Troxell, et al, 2013​ )

    It was observed that rpfF is involved in the regulation of Pho regulon genes especially under low iron condition in Xoc. Upon further study it was observed that the Pho regulon genes were also involved in iron hoemostasis by regulating the expression of siderophore. Thus the study of the interaction between the Pho regulon protein, RpfF and Fur was done in order to understand the intricacy of the crosstalk between the Pho regulon and DSF regulon via Fur with respect to iron and phosphate homeostasis by employing the BACTH (Bacterial Adenylate Cyclase Two Hybrid System( ​Rai, et al, 2015​ )

    The yeast two-hybrid method was initially developed by Fields & Song (1989). The bacterial two-hybrid variant was further developed to characterize interactions between two targeted proteins (Karimova et al., 1998). It has been extended to screen for previously unknown partners involved in antibiotic resistance (Domain et al., 2007) and bacterial viability ( ​Qin, et al, 2016​ )

    Bacterial two-hybrid system allows an easy in vivo screening and selection of functional interactions between two proteins. Two putative interacting proteins are genetically fused to two complementary fragments, T25 and T18, that constitute the catalytic domain of Bordetella pertussis adenylate cyclase. Association of the two-hybrid proteins results in functional complementation between T25 and T18 fragments and leads to cAMP synthesis. Cyclic AMP then triggers transcriptional activation of catabolic operons, such as lactose or maltose, that yield a characteristic phenotype. If two proteins interact colonies give the following phenotype on the indicator plates: blue on LB-X-Gal or red on MacConkey-maltose. Complementation can be detected in 1 - 4 days. If no interaction occurs colonies appear colorless (​​Qin, et al, 2016​​)

    METHODOLOGY

    Materials

    List of Media

    • Luria-Bertani Medium (LB medium)

    0.5% Yeast Extract

    1% Tryptone

    1% Sodium Chloride (NaCl)

    pH adjusted to 7.0 – 7.2 with 1N NaOH.

    Media and solutions were either autoclaved at 121°C and 15 psi for 20 min or filtered through the membrane of 0.22 μM porosity.

    For LB agar 1.5% agar was added in LB medium.

    Buffers and solutions

    • Tris-EDTA (TE) buffer

    10 mMTris-HCl (pH 8.0)

    1 mM EDTA (pH 8.0)

    50X Tris

    • Acetic acid-EDTA (TAE) buffer

    242 g – Tris Base

    57.1 mL Glacial Acetic Acid

    100 mL 0.5 M EDTA

    Tris Base was mixed to approx. 600 ml miliQ water. Further added the EDTA and glacial acetic acid and brought the final volume to 1 L with miliQ H2O.

    • Working concentration of 1X TAE is

    40 mMTris base

    0.5 M EDTA

    • Phosphate-Buffered Saline (PBS)

    137 mMNaCl

    10 mM Na2HPO4

    2.7 mMKCl

    2 mM KH2PO4

    pH was adjusted to 7.3 and filtered through membrane of 0.45 μM porosity. PBS was prepared

    as a 10X stock solution.

    Buffers and solutions for plasmid and genomic DNA isolation and analysis

    • Resuspension buffer (P1)

    50 mMTris-HCl (pH 8.0)

    10 mM EDTA (pH 8.0)

    100 μg/ml RNase

    Volume was adjusted to 100 ml with miliQ water.

    • Lysis buffer (P2)

    200 mMNaOH

    1% SDS

    • Neutralization buffer (P3)

    3 M Potassium acetate (pH adjusted to 5.5 with acetic acid). 

    Antibiotics

    Antibiotics
    ANTIBIOTICS AND ANTIMETABOLITES Stock conc. (mg\ml) Conc. used on LB  
    Ampicillin 100 mg/ml 50 µg/ml
    Kanamycin 50 mg/ml 50 µg/ml
    IPTG 100mg/ml 50 µg/ml

    X-Gal is a widely used chromogenic substrate for β-galactosidase. It yields a dark blue precipitate at the site of enzymatic activity include detection of lacZ activity in cells. The stock conc. was 25mg/ml and conc. used on LB was 50µg/ml.

    Bacterial strains

    E.coli strains- BTH101 and XL1 Blue were grown aerobically in Luria–Bertani (LB) broth or LB agar at 30°C. To visualize bacterial two-hybrid interactions on solid medium, LB agar was supplemented with X-gal (50µg/ml), ampicillin (Amp; 50 µg /ml) and kanamycin (Kan; 50 µg /ml) as uninduced control plates and to check for induction additional IPTG (100 µM) was added.

    Reporter strain

    The non-reverting adenylate cyclase deficient (cya) E. coli reporter strains, BTH101 that was used as host organisms for detection of protein-protein interactions.

    Strain genotype- F-, cya-99, araD139, galE15, galK16, rpsL1 (Str r ), hsdR2, mcrA1, mcrB1

    XL1-blue strain was used for the cloning of the different genes in pKNT25 and pUT18 plasmids.

    Plasmids

    • pKNT25- The vector is a derivative of the low copy-number plasmid pSU40, expressing a kanamycin resistance selectable marker.
    • pUT18 is a derivative of the high copy number vector pUC19, expressing an ampicillin resistance selectable marker, and encodes the T18 fragment that is expressed under the transcriptional control of a lac promoter.

    Two-hybrid screens were carried out using the plasmid constructs pKT25pstN and pUT18trkA, which were then digested to remove the insert and gel purified. The different genes were amplified and cloned in the above mentioned plasmids and electroporated into XL1-blue. BTH101 cells were used for co-electroporation to study the protein-protein interaction. Plasmid constructs carrying phoR, phoB, phoU, rpfFand fur fusions to the T18 or T25 fragments were generated by cloning PCR fragments that incorporated HindIII, EcoR1, BamH1 restriction sites, into pUT18 and pKNT25 vectors individually.

    Methods

    Polymerase chain reaction (PCR)

    PCR reactions were performed using gene specific primers to amplify the respective genes. The phoB, phoR, phoU, rpfC and fur were amplified with gene specific primers using high fidelity kappa taq polymerse

    Reaction mixture for PCR by kappa taq polymerse
    COMPONENTS VOLUME IN µl
    MQ water Adjusted according to the samples
    5X buffer 5
    MgCl2 1.5
    Dntp 0.5
    Forward primer 2
    Reverse primer 2
    Kappa Taq polymerase 0.1
    Template 3

    Reaction Conditions

    PCR Cycling conditions
      TEMPERATURE TIME
    Initial denaturation 98ºC 2 min
    Denaturation 98ºC 30 sec
    Annealing Tm-5 ºC 30 sec
    Extension 72ºC 50 sec
    Number of Cycles 31 cycles  
    Final Extension 72ºC 5 min

    Agarose gel electrophoresis

    The samples were mixed with appropriate volumes of the gel loading dye (6X- Bromo-phenol Blue) and subjected to electrophoresis with 1 kb ladder through 0.8-1% agarose gel in 1X TE buffer for 35 minutes. Ethidium bromide was used to visualize the DNA in the gel when viewed in a gel doc or UV eluminator.

    Gel extraction

    QIAquick Gel Extraction Kit was used to extract and purify DNA of 70 bp to 10 kb from standard or low-melt agarose gels in TAE or TBE buffer. The DNA fragment from the agarose gel with a clean, sharp scalpel was excised. For the complete solubilisation of the gel in 900 µl of QG buffer, Incubate at 50°C until the gel gets completely dissolved. To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. Isopropanol is added, mixed, transferred it into the column with collection tube.and centrifuged at 12,000 rpm for 1 minute. Discard the flow-through and add PE Buffer and centrifuge at 12,000 rpm for 2 minute. Discard the flow-through and additional spin is done. Place the QIAquick column into a clean 1.5 ml microcentrifuge tube. Elute DNA by adding 50 µl of Nucleus free water. Let the column stand for 10 minutes at room temperature. Centrifuge at 9,000 rpm for 3 mins to extract the DNA.Quantity of the plasmid DNA was determined by nanodrop reading.

    Restriction digestion

    Along with the respective restriction enzymes DNA samples and Vectors were digested in the presence of 10X fast digest buffer in a total reaction volume of 30-40 µl. The digestion mixture was placed at 37ºC for 2 hours for digestion. The digested vectors were subjected to gel electrophoresis along with DNA size markers (1kb ladder) to estimate the size of the digested fragments. After that gel was visualized in a gel doc or UV trans-eluminator and expected size band were removed from gel and preceded for gel extraction.

    GENE AND VECTORS RESTRICTION ENZYMES
    phoU HindIII and EcoRI
    phoR HindIII and EcoRI
    phoB HindIII and EcoRI
    fur HindIII and XhoI
    pKNT25ptsN HindIII and EcoRI
    pKNT25ptsN HindIII and BamHI
    pUT18CtrkA HindIII and EcoRI
    pUT18CtrkA HindIII and BamHI
    pET28A HindIII and XhoI

    PCI purification

     Purification of the PCR amplicons was performed by using QIAquick PCR Purification kit to avoid enzymes (polymerase, restriction enzyme), reaction components (dNTPs, primers, buffers, salts), proteins and various cell debriesfrom carrying over, contaminating, and negatively influencing the downstream experiments such as enzyme digestion, ligation, sequencing, and labelling experiments. The volume of the PCR product was made upto 500µl by sterile miliQ water. Equal amount of PCI (25:24:1) was added.  A well-mixed cell lysate–salt solution is then subjected to centrifugation at 12,000 rpm for 15 minutes at 4ºCto precipitate protein matters. The supernatant containing DNA is removed to a fresh new tube and it is combined with 85µl of sodium acetate and 350 µl of Iso propanol and it is incubated at -20ºC. For 20-30 minutes. The product was again centrifuged at 12,000 rpm for 30 minutes at 4ºC. 200 µl 70% Ethanol was added and spinned at 10,000 rpm for 15 minutes. Carefully decant the supernatant. Semi dry the pellet and dissolve it in 15 µl of Nucleus free water.

    Ligation

    The construction of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly digested vector. It is performed by the T4 DNA ligase enzyme. Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results in a single stranded overhang on the digested end of the DNA fragment. The overhangs, called "sticky ends", are what allow the vector and insert to bind to each other.

    Plasmids transformation in Escherichia coli cells through the electroporation

    Primary culture of XL1 Blue was streaked on LB plate. Primary culture was prepared by inoculating XL1 Blue in LB medium and incubated with shaking at 37°C overnight. Secondary culture was prepared by adding 1% of primary inoculum into LB medium and kept at 37°C with shaking until the OD600 reached 0.4-0.6. After centrifuging at 8,000 rpm at 4°C for 15 minutes the supernatant was discarded. The cells were washed with ice cold miliQ water and centrifuged at 8,000 rpm for 15 minutes at 4°C. This process was repeated thrice and water was removed gently without disturbing the pellet. After the third wash, the pellet was suspended with ice cold sterile miliQ water. 50µl of the competent cells were mixed and then subjected to electroporation, flowing which 1 ml of the fresh LB medium was added immediately after the shock and was mixed with gentle pipetting, transferred to 1.5 ml microcentrifuge tubes and then incubated at 30°C for 1 hr for the recovery. The microcentrifuge tubes were spinned at 6,000 rpm at room temperature for 5 minutes, supernatant was discarded leaving very small amount of media in microcentrifuge tubes and it was plated on LB plates containing appropriate antibiotics ampicillin (50 g/ml) or kanamycin (50 g/ml) and incubated at 30°C in the static incubator.

    Protocol for transformation

    The 100µl of DH5α or BL21 competent cells in one eppendorf was kept on ice. The required amount of plasmid DNA is added and incubated on ice for 10 minutes. This was then subjected to heat shock by placing them in a thermostat set at 42ºC for 90 seconds and transferred back to ice for 5 minutes. 1ml of LB was then added to the eppendorfs which were then placed shaker incubator for 37ºC for 60 minutes. Eppendorfs were then centrifuged at 6,000rpm at room temperature for 5 minutes, supernatant was discarded leaving very small amount of media in eppendorfs and the pellets were resuspended and plated on LB plates with respective antibiotic and incubated at 37 ºC overnight. The colonies obtained were further confirmed by colony PCR and nucleotide sequencing. 

    Colony PCR

    It is to determine the presence or absence of insert DNA in plasmid constructs. Individual transformants can either be lysed by PCR reaction in 20 µg miliQ water with heating temperature at 98 ºC for 15 mins. This initial heating step causes the release of the plasmid DNA from the cell, so it can serve as template for the amplification reaction. Primers designed to specifically target the insert DNA can be used to determine if the construct contains the DNA fragment of interest, to determine insert orientation. The presence or absence of a PCR amplicon and size of the product are determined by electrophoresis alongside a 1kb DNA size marker on an agarose gel.

    Plasmid DNA isolation

    Bacterial cells were grown in the presence of appropriate antibiotics required to maintain the plasmid DNA from 1-5ml overnight culture of E.coli in LB medium. Cells were harvested by centrifugation at 12,000 rpm for 5 minutes. Further, plasmids were extracted using QIprep Spin miniprep kit. Pellets were dissolved in 250 μl resuspension buffer (P1) and then added 250 μllysis buffer (P2) followed by incubation for 5 min at room temperature. Further, added 350 μl of neutralization buffer (P3), mixed by inverting gently 4-6 times and incubated in ice for 15 min. Then, spun down the tubes at 12,000 rpm for 10 minutes and supernatant was transferred to fresh column by decaning or pipetting. Centrifuge at 12,000 rpm for 10 min. Discard the flow-through and add 750μl PE Buffer and centrifuge at 12,000 rpm for 2 minute. Discard the flow-through and additional spin is done. Place the QIAquick column into a clean 1.5 ml microcentrifuge tube. Elute DNA by dissolving in 50 µl of Nucleus free water. Let the column stand for 10 minutes at room temperature. Centrifuge at 9,000 rpm for 3 minutes to extract the DNA Plasmid dissolved in nuclease free water. Quantity of the plasmid DNA was determined by nanodropreading and quality was checked by sequencing and thus the Plasmid was stored at -20°C.

    Coelectroporation of plasmid into BTH101 cells

    The gene of our interest along with the vector is coelectroporated into BTH101 cells and was plated on LB plates containing respective antibiotics ampicillin (50 μl/ml) and kanamycin (50 μl /ml) and kept at 30°C for 16 hours. The colonies were purified to get single colonies on LB plates containing respective antibiotics ampicillin (50 μl/ml) and kanamycin (50 μl/ml) with the help of streaking by toothpick. The plates were incubated under same conditions 30°C for 16 hours.

    X-gal and IPTG test for protein-protein interaction  

    The single purified colonies from LB plates containing respective antibiotics such as ampicillin (50μl/ml) and kanamycin (50 μl/ml) were inoculated into 5 ml of LB medium containing ampicillin (50 μl/ml) and kanamycin (50 μl/ml) along with BTH101 positive and negative controls grown overnight at 30°C. After incubating the primary culture overnight, the primary culture was serially diluted in 900 μl of phosphate buffer saline. Dilution spotting was then done with the help of template. 2 μl of spots were made on the LB containing ampicillin (50 μl/ml), kanamycin (50 μl/ml), IPTG(50µg/ml) and X-gal (50µg/ml) (Induced plates) and LB containing ampicillin (50 μl/ml), kanamycin (50 μl/ml), and X-gal (50µg/ml) (Uninduced plates). The plates were then incubated at 30°C for 4 days and scanned every day for the development of blue colour.

    RESULTS AND DISCUSSION

    Amplification of Genes of Interest

    The genes phoB, phoR, phoU, rpfF, rpfC and fur were amplified with gene specific primers from genomic DNA of Xoc. Agarose gel was run following PCR amplification.

    Doc14.jpg
      PCR Amplification

      Colony PCR

      To check the positive clone, colony PCR was performed by using gene specific primers sequence after PCR the gel run was carried out and it gives positive result.

      Doc17.jpg
      Colony PCR of PhoR, RpfC, PhoU
        Doc19.jpg
        Colony PCR of PhoB, fur, RpfF
          Colony PCR

          Observation of Protein-Protein Interaction

          A BATCH system was used to investigate interactions of proteins. For a qualitative analysis, cultures were spotted onto LB plates containing specific antibiotics ampicillin (50 g/ml), kanamycin (50 g/ml) X-Gal and IPTG. It was incubated at 30°C and observation was taken after every 24 hour till 96 hours. All samples shown were from the same plate.

          24 HOURS

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          CONCLUSION AND FUTURE PERSPECTIVES

          Conclusion

          To determine the protein-protein interaction Bacterial adenylate cyclase two hybrid (BATCH) technique was performed. Recombinant plasmids having the genes phoB, phoU, phoR, fur and rpfF in both pKNT25 and pUT18 vectors were cloned. The recombinant plasmids were co-electroporated into BTH101 background to study the interaction among the genes. The interaction was studied in terms of phenotypic characterization which was determined by the blue color formation on induction by IPTG on LBA plates containing ampicillin (50 μl/ml), kanamycin (50 μl/ml), IPTG (50µg/ml) and X-gal (50µg/ml). After incubating the plates at 30°C for 24 hours it was observed that the blue color was produced only in the positive control. After further incubating the plates at 30°C for 72 hours it was observed that the slight blue color was produced by the different interacting colonies, the blue colour was further enhanced on 96 hours of incubation. The color development was not seen in the BTH101 negative control. Thus it can be concluded that the genes phoB, phoU, phoR, fur and rpfF are interacting with each other, however the interactions are not so strong. The demonstration of this interaction shows possible crosstalk between the proteins of Pho regulon and the DSF regulon with respect with the maintenance of iron homeostasis.

          Future Perspectives

          Identifying the protein-protein interaction between Pho regulon and Quorum Sensing will help us understand the possible protein –protein ineractions involved in the maintenance of iron homeostasis and the plausible crosstalk between the two very different regulatory networks- Pho regulon and the DSF regulon, in Xoc.

          This study needs to be further confirmed by Pull Down assay to arrive at a definite conclusion.

          ACKNOWLEDGEMENTS

          I would like to thank Dr. Subhadeep Chatterjee for the continuous guidance and patience. Being my summer project I had lot of things to learn and the amount of knowledge and experience I have gained from him is matchless. I owe my sincere thanks to him.

          I thank Mrs. Prashantee Singh for her guidance in lab-talks and overall for making it a great learning experience.

          I would also like to thank Binod sir, Yasobanta sir, Biswajit sir, Raj kumar sir, Akanksha mam, Durga mam, Anindya sir and each member of the lab (LPMI) for all the support and discussions.

          I take this opportunity to sincerely acknowledge the Indian academy of sciences (IAS) and CDFD

          for selecting me as summer research fellow and giving this golden opportunity do this wonderful

          summer project and also for providing necessary infrastructure, resources and fellowship.

          I would also like to thank Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad

          for providing accommodation and other requirements hence making my stay comfortable.

          I thank all the Lab Assistants for providing me with strains and other chemicals during my work at LPMI.

          I thank Ms. Priyanka Sasmal, Ms. Athira Venugopal, Mr. Atharva Kaushik and all the other summer research fellows who made my stay in Hyderabad comfortable and fun.

          Also I indepted to my friends for their support, help and the valuable moments they gave me which made the days in CDFD very enjoyable.

          Finally, I thank my parents and my brother for their love, inspiration and encouragement without whom I would have never enjoyed so many oppurtunities.

          References

          • Marzan, Lolo and Shimizu, Kazuyuki (2011). Metabolic regulation of Escherichia coli and its phoB and phoR genes knockout mutants under phosphate and nitrogen limitations as well as at acidic condition. 10,

          • Gardner, Stewart G and Miller, Justin B and Dean, Tanner and Robinson, Tanner and Erickson, McCall and Ridge, Perry G and McCleary, William R (2015). Genetic analysis, structural modeling, and direct coupling analysis suggest a mechanism for phosphate signaling in Escherichia coli. 16,

          • Santos-Beneit, Fernando (2015). The Pho regulon: a huge regulatory network in bacteria. 6,

          • Gardner, Stewart G and Miller, Justin B and Dean, Tanner and Robinson, Tanner and Erickson, McCall and Ridge, Perry G and McCleary, William R (2015). Genetic analysis, structural modeling, and direct coupling analysis suggest a mechanism for phosphate signaling in Escherichia coli. 16,

          • Ionescu, M. and Baccari, C. and Da Silva, A. M. and Garcia, A. and Yokota, K. and Lindow, S. E. (2013). Diffusible Signal Factor (DSF) Synthase RpfF of Xylella fastidiosa Is a Multifunction Protein Also Required for Response to DSF. 195,

          • Troxell, Bryan and Hassan, Hosni M. (2013). Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria. 3,

          • Rai, Rikky and Javvadi, Sreegowrinadh and Chatterjee, Subhadeep (2015). Cell-cell signalling promotes ferric iron uptake inXanthomonas oryzaepv.oryzicolathat contribute to its virulence and growth inside rice. 96,

          • Qin, Ran and Sang, Yu and Ren, Jie and Zhang, Qiufen and Li, Shuxian and Cui, Zhongli and Yao, Yu-Feng (2016). The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium. 7,

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