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1. Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN 1.Bhawana Baloda- Introduction and Materials & Method 2.Sneha Talaviya- Materials&Methods 3.Vidhi Shah- Materials&Methods 4.Hardi Soni- Production & Isolation 5.Apurva S Kundaram- Mass Spectrometry 6.Avanti Raut- SDS Page analysis 7.Deeksha Sharma- Immunology analysis & Western Blotting 8.Shalini Kotnala -Protein inclusion as nutrient reserve& toxicity of protein inclusion. 9.Suchismita Sarkar -Discussion 10.Arushi Tiwari- Possible biological function Received 16 March 2001/Accepted 3 August 2001 Presented by: Submitted to:Dr.Ravindra Pal Singh

2. Family - Enterobacteriaceae Genus - photorhabdus Road shaped Bioluminescent bacteria key points Bacterium is Living in the symbiotic relation with entomopathogenic nematode (a tiny worm). Two types of morphologically distinect protein inclusion identified in the bacteria . The inclusions appeared during the bacterial exponential phase. These inclusions constitude about 40% of the total cellular protein. Hypothesis and implications - Initially, it was hypothesized that the inclusions might influence the symbiotic relationship and entomopathogenesis, but further research is needed to confirm their role. . INTRODUCTION 1 https://support.google.com/legal/answer/3463239?hl=en

3. 2 Bacteria and Culture conditions Materials and Methods Strains: Photorhabdus luminescens Hm (UC) and NC1 (UNC) Media: 2% proteose peptone no. 3 (PP3), 1.5% Bacto agar (Difco) Incubation Conditions: 30°C for 72 hours Storage: Room temperature, transferred monthly Secondary-Phase Variants (NC1): White secondary (nonpigmented), Yellow secondary (yellow pigmentation) Microscopy used in the experiment Phase-Contrast Microscopy Time-Lapse Microscopy Transmission Electron Microscopy Scanning Electron Microscopy Media Tested: Yeast extract (5%), Neopeptone (2%), Casitone (2%) Proteose peptone no. 3 (2%), Nutrient broth (2.5%) Peptone (10%), Trypticase (2%) Incubation: 30°C for 72 h. Analysis: Phase-contrast microscopy. Optimization of inclusion production

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5. 3 https://www.semanticscholar.org/paper/Mass-Spectrometry-as-a-Workhorse-for-Preclinical-on-Veeravalli-Madgula/7f94b8b3315018ef7b8f41d4f293589e9b01966d Figure 1 4

6. 5 Type 2 Inclusion protein from NC1 200 ug/ml protein adjusted and 10 ul analysed by SDS-PAGE Production of antisera Materials and Methods Type 1 Inclusion protein from NC1 Immunize white rabbits with inclusion protein Solubilized in NaOH Emulsification of protein in adjuvant 10 days after Complement inactivation in obtained serum Repeated injections upto significant production of antibodies Serum stored at -20°C Time course of inclusion protein production Serum Cells grown in 25ml PP3 broth 5 ml culture inoculated in 250 ml PP3 broth Cells grown on flask shaker at 30°C and 250 rpm Remove samples at various time and determine protein content Protein content Time Repeat for 2° phase cells grown for 96 hr

7. 6 Materials and methods Gel electrophoresis and Western blotting Stability of inclusion protein : Growth and Starvation Grow cells in PP3 broth Remove samples at various time and determine microscopic and viable count Starvation experiment Incubated flask Centrifuged flask Resuspended in sPBS Insect toxicity analysis Galleria mellonella larvae grown by H. C. Coppel method Protein sample preparation purified protein type 1 and type 2 solubilized with HCl or NaOH samples from 48 hr PP3 culture Protein samples administered either by feeding or injecting Bioassay of freshly prepared or frozen inclusion protein

8. 7 Culture Medium Inclusion proteins observed in phase contrast microscopy 2.5% Nutrient Broth Most cells (after 48 hrs.) 2% Peptone Most cells (after 48 hrs.) 2% PP3 more than 90% cells 2% Trypticase Soy Broth Approx. half of the total cells 2% Casitone 0 visible inclusions 5% Yeast Extract 0 visible inclusions 10% Peptone 0 visible inclusions (A) Production & Isolation of Inclusion proteins Result

9. 8 Observation of Inclusion proteins Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706 Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706 Figure 2 Figure 3

10. 10 PTM of Type 2 inclusion protein for ‘NC1' and ’Hm’ strain=maybe ‘Acetylation’ MALDI-TOF & SDS PAGE Single Protein Subunit Type 1 ‘NC1' inclusion ~>66 mass units ’Hm’ inclusion Type 1 inclusion | ‘cipB’ gene Type 2 inclusion | ‘cipA’ gene Solubility(1&2)- at pH 11 or 4 ~90% Insolubility(1&2)- at pH 5 and 10 http://www.chem.cmu.edu/cma/maldischem.html Figure 4: MALDI-TOF Diagram

11. 11 (C) Result of SDS-PAGE analysis and proteolytic degradation Figure 5: SDS PAGE Analysis Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706

12. 12 No degradation of inclusions from both strains was observed by indigenous cell proteases on incubation in cell lysate for one week Result of proteolytic degradation of Inclusion proteins from NC1 strain

13. 13 (D) Result by Immunological analysis and temporal regulation of inclusion protein accumulation Methods -Immunological analysis (polyclonal antisera) and western blot. Observation of inclusion bodies production in Photorhabdus luminescens with time Polyclonal antisera were raised against NC1 (type 1 and 2) for determination of inclusion protein production At 16 hours of growth the first type 1 and type 2 protein were detected At 24 hours both proteins were detected at high levels and 70% of cells contained small inclusions At 0 hours inclusion proteins are present because stationary phase cells are used as inoculum Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706 Figure 6

14. Type 1 antiserum Type 2 antiserum Cross reacts weakly with NC1 type 2 Cross reacts weakly with NC1 type 1 More weakly binds with 20 and 30 KDa of NC1 type1 More weakly binds with 20KDa in type 2 Strongly binds with 10KDa Strongly binds with 10 KDa Cross- reactivity of antisera and inclusion proteins Similar cross reactivity of antisera with Hm inclusion proteins Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706 14 14 Figure 7

15. Phase- contrast photomicrographs of cells of P.luminescens strain NC1 grown in PP3 broth and during starvation The first inclusions were visible at 16h after the inoculation of the culture. The inclusions remain present at all times after the shift to starvation conditions. (72 to 192 h) . (E) Insights on Protein Inclusions in Bacteria 1) Are the protein inclusions nutrient reserves? Growth and starvation of P.luminescens NC1. (Microscopic counts and viable-cell counts were determined at various times after inoculation. 15 Figure 9: Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706 Figure 8 : Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706

16. 2) Protein Inclusions in Dividing Cells 3) Toxicity of protein inclusions I ntact and solubilized P.luminescens Live G.mellonella larvae Larvae injected with viable P.luminescens G.mellonella larvae died within 24 hrs Photomicrographs of P.luminescens cells growing on PP3 agar Cells with large protein inclusions were capable of cell division. 16 Figure 10 Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706

17. 17 Two distinct intracellular protein inclusions were present. Nearly identical in molecular size , differ in amino acid content , susceptible to protease digestion and immuno cross reactivity High content of amino acids in the two protein classes (47% for type 1 and 42 % for type 2 ) DISCUSSION

18. 18 Mass spectrometry and SDS PAGE Analysis Type 1 inclusion is the cip B gene product and type 2 gene inclusion is the cip A gene product

19. Referance: Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens DAVID J. BOWEN† AND JERALD C. ENSIGN* Department of Bacteriology, The University of Wisconsin, Madison, Wisconsin 53706 THANK YOU