GROWTH PHASE-DEPENDENT RPOS LEVELS IN ESCHERICHIA COLI LABORATORY AND ENVIRONMENTAL STRAINS

Transcription

1 GROWTH PHASE-DEPENDENT RPOS LEVELS IN ESCHERICHIA COLI LABORATORY AND ENVIRONMENTAL STRAINS

2 GROWTH PHASE-DEPENDENT RPOS LEVELS IN ESCHERICHIA COLI LABORATORY AND ENVIRONMENTAL STRAINS BY PARDIS HAGHIGHI, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Master of Science McMaster University Copyright by Pardis Haghighi, February 2016

3 MASTER OF SCIENCE (2016) McMaster University (Biology) Hamilton, Ontario TITLE: Growth phase-dependent RpoS levels in Escherichia coli laboratory and environmental strains AUTHOR: Pardis Haghighi, B.Sc. (Ferdowsi University of Mashhad, Mashhad, IRAN) SUPERVISOR: Dr. Herb E. Schellhorn NUMBER OF PAGES: x, 58 ii

4 ABSTRACT RNA polymerase and the sigma factors in Escherichia coli play role in maintaining the bacterial transcriptional profile in dynamic environmental conditions during growth cycle. Previously, transcriptom analysis between E. coli laboratory strain MG1655 and pathogenic strain EDL933 showed that a lot of genes are highly RpoS-dependent in EDL933 without RpoD to compete against RpoS. In the first project, the RpoD level was examined in these two strains. The same RpoD level between the two E. coli strains was shown suggesting the independent expression of RpoS regulon. In prototypical E. coli K12 MG1655 laboratory strain, RpoS levels, the stressresponse sigma factor, are not detectable during exponential phase while several genes are controlled by RpoS in this phase. Although, RpoS levels in E. coli pathogenic strain O157:H7 was higher during exponential phase, the RpoS expression pattern in other isolates of E. coli was not known. In second project, the E. coli environmental isolates were examined as we have access to a large number of isolates, collected from a variety of gut to aquatic sources. To examine if RpoS in environmental isolates has exponential phase expression similar to that of laboratory strains, immunoblots were performed on forty isolates at exponential phase (OD600 ~ 0.3). Twelve isolates showed higher levels of RpoS protein compared to laboratory strain during exponential phase. Also, the immunoblot analysis on some of the isolates at early stationary phase (OD600 ~ 1.5) and 24 h after subculture demonstrate the decrease in expression of RpoS protein at stationary phase to iii

5 overnight culture. This finding was different from the RpoS expression in E. coli K12 laboratory strain. In conclusion, the relatively lower levels of RpoS in the laboratory strains might occur due to selection for attenuation in RpoS levels in the laboratory strains after being cultivated for many years in laboratory conditions. iv

6 ACKNOWLEDGMENTS Pursuing graduate studies as an international student in a foreign country was a big challenge for me at the beginning and I could not handle it without the tremendous support from the amazing people in the Schellhorn lab. First and foremost, I would like to sincerely thank my supervisor, Dr. Herb Schellhorn, for his consistent support and constructive feedback on my work. I appreciate his guidance and patience during my time here, and I am very honored and thankful for being given the opportunity to learn and grow academically and as a person. I will always value our conversations and his advice on how to be a good grad student. I also thank my co-supervisor, Dr. Radhey Gupta for his guidance and helpful feedback on my project during my committee meetings. I would like to thank current and past members of Schellhorn lab (in no particular order): Mahi, Sakis, Shirley, Deepinder (especially for being good company while running the Western blots and correcting my English), Rachelle, Steve, Rachel, Paul, Cathy, Vivian, Ashish, Kristine and William. Thank you guys for making my academic life more fun and memorable. The Lab s mom will never forget any of those moments. Lastly, I have to say a big thank you to my family for their endless love and for supporting me even from miles away. And special thanks to my dearest friend and partner, Hooman, for being in my life, listening to all my worries during stressful times and his invaluable support. v

7 TABLE OF CONTENTS ABSTRACT... iii ACKNOWLEDGMENTS... v TABLE OF CONTENTS... vi LIST OF TABLES... ix LIST OF FIGURES... x Project 1: RpoD expression level in E. coli K12 laboratory strain and E. coli O157:H7 pathogen strain in exponential and stationary phases... 1 Chapter 1.1 Project Rationale and Overview Quantitative Immunoblot... 4 Chapter 1.2 Methods Sample collection SDS-PAGE Immunoblot... 6 Chapter 1.3 Results...7 Chapter 1.4 Discussion...7 vi

8 Project 2: Exponential expression levels of RpoS sigma factor in E. coli environmental strains Chapter 2.1 Introduction Escherichia coli RNA polymerase core enzyme Sigma factors in E. coli Sigma factors competition RpoS; a global regulator RpoS expression in exponential phase RpoS regulation and other transcriptional regulators Project rationale and objectives Chapter 2.2 Materials and Methods Bacterial strains Growth conditions and sample collection Immunoblot analysis Quantify the blots using ImageJ Chapter 2.3 Results...26 vii

9 2.3.1 Growth curve RpoS expression level in exponential phase Quantitation of RpoS protein expression using ImageJ Chapter 2.4 Discussion Possible factors involved in RpoS expression level in exponential phase Chapter 2.5 Conclusion and Future work...30 References Appendix Standard Operating Procedures...45 Thesis Defence Slides viii

10 LIST OF TABLES Table 2.1. Escherichia coli sigma factors Table 2.2. Summary of source types for the 40 environmental E. coli isolates used in this study Table 2.3. Environmental E. coli isolates used in this study ix

11 LIST OF FIGURES Figure 1.1. RpoD expression level in two strains of E. coli using Immunoblot performed by previous students Figure 1.2. Detecting the linear dynamic range using normalization experiment Figure 1.3. The linear range of detection using ImageJ 1.47 software Figure 1.4. RpoD expression level in E. coli K12 MG1655 and E. coli Ol57:H7 EDL933. (Replicate #1) Figure 1.5. RpoD expression level in E. coli K12 MG1655 and E. coli Ol57:H7 EDL933. (Replicate #2) Figure 1.6. RpoD expression level in E. coli K12 MG1655 and E. coli Ol57:H7 EDL933. (Replicate #3) Figure 2.1. Growth of E. coli environmental isolates in rich media Figure 2.2. Comparing RpoS expression level in 10 environmental isolates of E. coli as a factor of growth phase Figure 2.3. Quantitation of RpoS protein expression in exponential phase, stationary phase and overnight culture Figure 2.4. RpoS expression in 40 E. coli environmental isolates at exponential phase x

12 Project 1: RpoD expression level in E. coli K12 laboratory strain and E. coli O157:H7 pathogen strain in exponential and stationary phases 1

13 Chapter 1.1 Project Rationale and Overview Bacterial cells live in diverse environments such as mammalian gut, fresh water, marine water, and soil. They have a specific regulatory system that enables the cells to adapt to these diverse environments and survive (Ishihama, 2000). Bacterial sigma factors confer specificity upon RNA core polymerase to determine the genes that are transcribed under specific conditions. Among seven sigma factors known in E. coli, the protein expression level of two major sigma factors - the vegetative sigma factor RpoD ( 70 ) and the alternative sigma factor RpoS ( 38 ) - in pathogenic and laboratory strains of E. coli is of interest in this project. The pathogenic strain in this study is the E. coli O157:H7 strain EDL933, a Shiga toxin-producing E. coli (STEC) strain. Many serotypes of STECs are associated with gastrointestinal disease such as hemorrhagic colitis. However, O157:H7 is a dominant strain implicated in such diseases and associated outbreaks. The toxin produced by this strain is Shiga-like toxin and is structurally and functionally similar to Shiga toxin produced by Shigella (Dong & Schellhorn, 2010, Dong & Schellhorn, 2009b). The Shiga toxin, the type III secretion system and other virulence factors are encoded on the LEE pathogenicity island (McDaniel & Kaper, 1997, Elliott et al., 1998). Some of these virulence factors are controlled by RpoS, which are regulated by growth conditions and have differential expression in E. coli strain types (Dong & Schellhorn, 2009b). The laboratory strain E. coli K12 MG1655 is widely used to study transcriptional regulation at the cellular level. There are many phenotypic and genomic differences 2

14 between E. coli O157:H7 and K12 strains. The O-island genes in E. coli O157:H7, which are one of the differences between this strain and E. coli K12, are possibly under control of RpoS rather than RpoD. About 10% of the genes regulated by RpoS in EDL933 are located on O-islands (Dong & Schellhorn, 2009b). Two different systems transcribe stationary phase genes in E. coli. One is RpoSdependent and transcription factor-independent, and the other one is RpoD-dependent and transcription factor-dependent (Ishihama, 2000). It is likely that RpoS evolved by duplication from RpoD, with which RpoS shares 59% identity in gene sequence. Furthermore, many RpoS-regulated genes can be transcribed by RpoD-associated holoenzyme in vitro, suggesting a strong functional similarity (Chiang & Schellhorn, 2010). The RpoS expression changes depends on strain background (King et al., 2004). RpoS expression level is higher in EDL933 than MG1655 strain in early stationary phase, while it is lower in overnight cultures (Dong & Schellhorn, 2009b). Since RpoS and RpoD share 30% similarity of DNA sequence and unlike other sigma factors that have specific promoter regions RpoD can transcribe most of RpoS-promoters in vitro (Hengge-Aronis, 2002), a question is how about RpoD level in these two strains? Is the level of RpoD lower or higher in EDL933 than MG1655 during exponential and stationary phases? Two previous students in our lab reported the lower expression of RpoD in EDL933 strain than in K12 MG1655 (Figure 1.1). I performed immunoblot experiments to test the expression. 3

15 1.1.1 Quantitative Immunoblot Western blot (protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. In Chemiluminescent detection method, the image is analysed by densitometry that quantifies the relative amount of protein staining by optical density. Chemiluminescent detection method determines the presence or absence, size, and modification or degradation states of target proteins and also quantitation of proteins. Possible detection problems stem from a low-dynamic range of detection and the difficulty in accurately determining the limit of detection (Taylor et al., 2013). For accurate protein quantitation, standard curves are needed because of the narrow quantifiable ranges. Elimination of the antibody is another important factor. The primary antibody influences the relation between the western blot signal and the protein amount on the membrane. This relation is non-linear and varies from one antibody to another. Using dilution series and the calibration curve is an asset in quantifying protein by western blot (Charette et al., 2010, Degasperi et al., 2014). Generating a standard curve based on a sample mixture can be used as an alternative to reliable standard proteins to determine the linear range of standard curves for Immunoblot quantitation (Taylor et al., 2013). Performing of dilution series should be done for every antibody in all experiments to increase the accuracy of protein quantitation. 4

16 Chapter 1.2 Methods Sample collection A single bacterial colony was inoculated into 5 ml of 1 LB in a sterile test tube and incubated overnight at 37 C on the rotating wheel. Cells were subcultured 1:10,000 into 50 ml of 1 LB (1:5,000 for rpos mutants to compensate for a longer rpos mutant lag phase). Samples were taken at the desired OD600. Chloramphenicol (final concentration of 150 µg/ml) was added immediately to stop protein synthesis. Samples were centrifuged at 11,000 g for 2 minutes. The supernatant was removed and pellets were washed with phosphate buffer. After second centrifugation the pellets were resuspended in 1 SDS loading buffer to a final cell concentration equivalent to OD600 of 2.0. The samples were boiled for 5 minutes and spun down at 10,000 g for 1 minute SDS-PAGE Ten µl of protein were loaded into wells for each gel. A protein ladder (Fermentas PageRuler Prestained Protein ladder) was included. Gels were run at 50 V for 0.5 h until protein has reached the separating gel, and then the voltage was increased to 100 V for h until protein has reached the bottom of separating gel. 5

17 1.2.3 Immunoblot Protein was transferred from gels to PVDF membrane at 20 V for 30 min. The membranes were blocked in 5% milk/tbst for 1 h at room temperature. Membranes were incubated in primary antibody O/N at 4 C (1:10,000 dilution). Then the membranes were washed with 1X TBST solution for 1 h (three times each 20 min). The membrane was incubated in secondary antibody (1:3,000) for 1 h at room temperature. The plates were gently shaken on the shaker at room temperature. The membrane was rewashed with 1X TBST three times each for 10 minutes. The membranes were soaked in ECL detection reagents (1:1 ratio of reagents 1 and 2) for 1 min and were wrapped in Saran wrap. They were developed on Amersham hyper film. 6

18 Chapter 1.3 Results The appropriate dilutions of samples are used to detect linear dynamic range for using densitometry analysis. The protein bands in the blot also indicated the decrease of expression as well as relative concentration (Figure 1.2 and 1.3). After developing the films in the dark room, the protein bands were visualized on the film. Figure 1.4A, 1.5A and 1.6A are the protein gels that stained with 0.1% Coomassie Blue solution and figures 1.4B, 1.5B and 1.6B are their relative blots respectively. Immunoblots shows high expression of RpoD in EDL933 which is not confirming the results reported by previous students (Figure 1.1). Chapter 1.4 Discussion The purpose of testing RpoD in these two theses was to have a control sigma factor for the experiments since RpoD is the housekeeping factor and is responsible for regulating essential genes. The results in the two previous theses have shown an unexpected low expression levels in pathogenic strains (and some environmental strains). One of the possible reasons explained is due to years selection of fast-grow E. coli K12 MG1655 strain in rich media under laboratory condition which leads to higher level of expression level of RpoD (Zheng, W. M.Sc. Thesis 2012). It is concluded that the expression of RpoD impress the environmental selection. While, as it is shown in the figures 1.4, 1.5 and 1.6 (three 7

19 replicates) there is no significant difference in RpoD expression level between these two strains. It is expected to see a constant and constitutive intracellular concentration of RpoD through exponential growth phase to stationary phase (Jishage & Ishihama, 1995). Differences in expression levels of genes regulated by RpoS between MG1655 and EDL933 might be due to different regulons in these strains or due to RpoS expression differences (Dong & Schellhorn, 2009b). The same explanation is reported for RpoS, which is the stress-induced sigma factor. There are slightly different results of RpoS expression in MG1655 and EDL933 strains between two reports. The slightly levels of RpoS expression is detectable from exponential phase OD (Zheng,W. M.Sc. Thesis 2012) which is different from the other report that RpoS expression begins near early stationary phase (Vijayakumaran, V. B.Sc. Thesis 2011). Further studies is needed to confirm these differences using quantitative immunolots. 8

20 A WT K12 rpos OD 600 Time of Incubation (h) ON 72 MG1655 EDL933 Lane B MG1655 WT K12 rpos OD 600 Time of Incubation (h) ON 72 EDL933 Lane Figure 1.1. RpoD expression level in two strains of E. coli using Immunoblot performed by previous students. (A) Wenjie Zheng M.Sc. Thesis (B) Vithooshan Vijayakumaran B.Sc. Thesis

21 A kda M Relative Concentration Lane B Relative Concentration RpoD Lane Figure 1.2. Detecting the linear dynamic range using normalization experiment. (A) SDS-PAGE protein gel electrophoresis of a 2-fold dilution series of the protein lysate of overnight culture of MG1655 strain. The final OD600 of the stock (undiluted sample) was 4.0. The samples were loaded in triplicates. M is the PageRuler Prestained Protein Ladder (Thermo Scientific #26616). (B) The immunoblot test. The RpoD anti-sera was used and the exposure time was one minute. 10

22 Relative Density y = x R² = Relative Concentration Figure 1.3. The linear range of detection using ImageJ 1.47 software. The plot shows the linear range of detection. The points indicated in the graph are the average relative density of three replicates for each dilution. The coefficient of determination (R 2 ) is shown in the graph. 11

23 A kda M MG1655 EDL OD B RpoD Lane MG1655 EDL OD 600 Lane Figure 1.4. RpoD expression level in E. coli K12 MG1655 and E. coli Ol57:H7 EDL933. (Replicate #1). (A) Coomassie blue staining of SDS-PAGE protein gel electrophoresis. (M is the PageRuler Prestained Protein Marker (Thermo Scientific #26616). (B) RpoD expression levels of E. coli Laboratory strain (MG1655) and pathogenic strain (EDL933). The exposure time was one minute. 12

24 A kda M MG1655 EDL OD B Lane MG1655 EDL OD 600 RpoD Lane Figure 1.5. RpoD expression level in E. coli K12 MG1655 and E. coli Ol57:H7 EDL933. (Replicate #2). (A) Coomassie blue staining of SDS-PAGE protein gel electrophoresis (M is the PageRuler Prestained Protein Marker (Thermo Scientific #26616). (B) RpoD expression levels of E. coli Laboratory strain (MG1655) and pathogenic strain (EDL933). The exposure time was one minute. 13

25 A E. coli K12 MG1655 E. coli O157:H7 EDL933 kda M K12 OD 600 (nm) WT rpos kda M K12 OD 600 (nm) WT rpos Lane Lane B OD O/N RpoD MG1655 RpoD EDL933 Lane Figure 1.6. RpoD expression level in E. coli K12 MG1655 and E. coli Ol57:H7 EDL933. (Replicate #3). (A) Coomassie blue staining of SDS-PAGE protein gel electrophoresis (M is the PageRuler Prestained Protein Marker (Thermo Scientific #26616). (B) RpoD expression levels of E. coli Laboratory strain (MG1655) and pathogenic strain (EDL933). The exposure time was one minute. 14

26 Project 2: Exponential expression levels of RpoS sigma factor in E. coli environmental strains 15

27 Chapter 2.1 Introduction In nature, bacteria are frequently exposed to changes in their environment. To adapt to the flux of environmental conditions, they possess specific genetic systems that mount appropriate responses (Ishihama et al., 2014). Within their genetic systems, mutations or expression of certain genes that help bacteria grow and survive under stress. Escherichia coli is a well characterized model organism whose gene expression in environmental strains differs from its commonly used laboratory strain. This difference arises due to their differing gene expression and cell composition that ultimately leads to contrasting bacterial phenotypes (Chiang et al., 2011). E. coli gene expression is not stable during bacterial growth. In fact, differential gene expression in E. coli laboratory strains has been elucidated (Dong et al., 2008). This reveals the possibility of differential gene expression in E. coli environmental strains during their growth cycle Escherichia coli RNA polymerase core enzyme RNA polymerase (RNAP) plays a key role in prokaryotic transcription. The RNA polymerase complex of Escherichia coli consists of a core enzyme with two α subunits, one β subunit, one β subunit and one σ subunit which enables the core enzyme to recognize the promoter region and to initiate transcription (Ishihama, 2000). 16

28 Two types of protein-protein interactions can change the distribution pattern of RNA polymerase. The first is the interaction of RNA polymerase holoenzyme with transcription factors that have a regulatory function, and the second is the interaction of core RNA polymerase with sigma subunits for promoter recognition (Ishihama et al., 2014). Knowing the intracellular concentration of the sigma subunits and transcription factors helps to understand the genome transcription regulation of E. coli under different stressful growth conditions in the environment (Ishihama et al., 2014) Sigma factors in E. coli Sigma factors (σ subunit) are transcription initiation factors (also known as promoter recognition factors) that provide specific binding of RNA polymerase to the promoters in bacteria to begin RNA synthesis. Sigma factors are the important global regulators in E. coli. Sigma factors regulate essential genes for survival and growth. Seven sigma factors are known for E. coli. The primary sigma factor, RpoD (σ 70 ), known as a housekeeping sigma factor, is responsible for the regulation of essential genes and the control of genes required for cell growth. The six remaining sigma factors (σ 54 (RpoN), σ 38 (RpoS), σ 32 (RpoH), σ 28 (RpoF), σ 24 (RpoE) and σ 19 (FecI)) are alternative sigma factors that control the expression of genes when the microorganism is under specific environmental stress. Sigma factors regulations occur in different environmental conditions, such as nitrogen limitation (σ 54 ), heat shock (σ 24 and σ 32 ) or iron limitation 17

29 (σ 19 ). In E. coli, σ 38 (RpoS) is the general stress response regulator in stationary phase (Hengge-Aronis, 1996). However, RpoS has different roles in other bacteria. For example, RpoS is important for the production of virulence factors in Pseudomonas aeruginosa (Landini et al., 2014). RpoS also plays an important role in exponential phase gene expression (Dong et al., 2008), bacterial biofilm formation and development (Sheldon et al., 2012) and bacterial virulence (Dong & Schellhorn, 2010, Dong & Schellhorn, 2009b) Sigma factors competition RNA polymerase core enzyme is the major regulatory system that rapidly adjusts promoter selectivity. Replacement of sigma subunits on the core enzyme is a mechanism for promoter selectivity and relies on many factors, such as types of stress conditions or the intracellular concentration of each sigma subunit. Thus, there is competition between sigma subunits for binding RNAP core enzyme after each round of RNA synthesis. The seven sigma subunits have different binding affinities for the core enzyme that is related to their intracellular concentration (Maeda et al., 2000, De Vos et al., 2011). Binding affinity is the tendency between ligand and protein core RNA polymerase and sigma factors. The overexpression of one sigma factor affects the function of other subunits by affecting the transcription of genes under their control (Jishage & Ishihama, 1999). The σ 70 subunit (RpoD) has the highest binding affinity for the core enzyme when compared with the other six sigma factors, while σ 38 (RpoS) has the weakest binding affinity (16- fold) (Maeda et al., 2000, Jishage & Ishihama, 1995). (Table 2.1) 18

30 2.1.4 RpoS; a global regulator RpoS protein is growth phase-specific and is expressed at different levels in each phase of growth. For example, in E. coli, K12 strain RpoS is highly expressed in stationary phase while it has very low to undetectable expression in exponential phase. RpoS protein expression is strain-specific. For example, in E. coli K12 strain RpoS expression level in early stationary phase (OD600 = 1.5) is lower than in E. coli O157:H7 pathogen strain (Dong & Schellhorn, 2009b). These two major specifications of RpoS are the basis of this project. Although much has been known regarding RpoS in stationary phase, there is less information on physiological functions of RpoS in exponential phase (Dong et al., 2008). The level of RpoS expression is very low in E. coli laboratory cultures, in which RpoS is more stable in stationary phase (Dong & Schellhorn, 2009a). Growth phase-dependent expression of RpoS in exponential phase is appreciably higher in the Shiga toxin-producing E. coli O157:H7 EDL933 strain compared to exponential expression of RpoS in the nonpathogenic K12 strain (Dong & Schellhorn, 2009b, Mand et al., 2013). In E. coli O157:H7 the level of RpoS in exponential phase is up to one-third of the levels found in stationary phase (Mand et al., 2013) RpoS expression in exponential phase RpoS positively regulates the expression of more than 400 genes in stationary phase (Patten et al., 2004, Lacour & Landini, 2004). In exponential phase, there are several genes 19

31 that are RpoS-dependent, such as osmy and aid (Schellhorn et al., 1998, Vijayakumar et al., 2004). By controlling the genes needed to adapt to exponential phase, RpoS plays role in growing cells. RpoS negatively regulates genes for metabolic pathways such as TCA cycle and glycolysis in exponential phase (Rahman et al., 2006). Many genes are RpoS-dependent only in exponential phase since when they enter into stationary phase they are no longer regulated by RpoS RpoS regulation and other transcriptional regulators There are specific mechanisms that modulate RpoS expression in exponential phase. Although RpoS expression level is low in exponential phase, RpoS regulates large sets of specific genes in exponential phase (Patten et al., 2004). This could be due to complex regulatory interactions between RpoS and other regulators such as H-NS (a nucleoid-associated DNA-binding protein which controls the expression of a large number of genes in E. coli and Salmonella), ppgpp, Hfq and Crl protein. The Crl protein is particularly interesting because it is one of the few RpoS associated regulatory factors that does not bind DNA, but instead it increases RpoS activity by stimulating the interaction between RpoS and the RNAP core enzyme in E. coli and Salmonella (Lelong et al., 2007, Bougdour et al., 2004). The Crl protein has an important role in enhancing the RpoS activity in exponential and early stationary phase when intracellular concentration of RpoS is low and RpoD is abundant in the cell (Typas et al., 20

32 2007, Dudin et al., 2013). Hence, RpoS even in low concentrations is capable of regulating many genes in exponential phase. Anti-sigma factors control gene expression by sequestering sigma factors that bind to the RNAP core enzyme (Trevino-Quintanilla et al., 2013). Controlling RpoS stability is one factor that changes the intracellular levels of RpoS in exponential and stationary phase. The RssB protein is a response regulator which binds to RpoS and leads to its degradation by ClpXP proteases (Landini et al., 2014). There are anti-adaptors that interact with RssB and inhibit its activity such as IraM, IraP and IraD which are three anti-adaptors in E. coli K12 to stabilize RpoS concentrations. Another anti-adaptor, IraL, is found in CFT073 strain of E. coli and Shigella. In these two strains, the level of RpoS is likely the same in exponential and stationary growth phases (Hryckowian et al., 2014). RpoD is the most abundant sigma factor in E. coli and has higher affinity for RNAP core enzyme than other sigma factors, but different factors in the transcription regulatory system in E. coli modulate the activity of this sigma factor. Rsd protein (Jishage & Ishihama, 1998) sequesters RpoD sigma factor and functions as an anti-sigma factor during exponential growth (Piper et al., 2009). The cross-linking and affinity binding experiments indicates that Rsd can affect RpoS binding to RNA core enzyme. That is how Rsd can influence the competition between RpoD and RpoS in binding to RNAP core enzyme (Hofmann et al., 2011). 21

33 2.1.7 Project rationale and objectives The expression level of RpoS changes dynamically. Although rpos is not essential, its exponential expression level might be the contributing factor to cell survivability or gene expression (Mand et al., 2013). RpoS as a possible predictor of growth and survival is reported in non-o157 and O157:H7 Shiga toxin-producing E. coli strains. There is a statistically significant association between levels of exponential phase rpos gene expression and survival in Shiga toxin-producing E. coli (Mand et al., 2013). It is still unknown if this finding can be extended to other strains of E. coli. The candidate strains in this project are the collection of 40 environmental isolates of E. coli derived from a larger collection (2,040 isolates) from diverse environmental sources provided by Dr. T. A. Edge (Table 2.2 and table 2.3). The environmental isolates can acquire mutations that have been selected and have variable phenotype with respect to RpoS-dependant functions (Chiang et al., 2011). Although RpoS levels are low in exponential phase, this level can still contribute to expression of a subset of genes. In some strains, RpoS levels are high enough in exponential phase to be a likely contributing factor to cell survival (Mand et al., 2013). The objective of this project was to understand if the RpoS expression level in exponential phase in environmental isolates of E. coli is higher than E. coli K12 MG1655 laboratory strain and if this is a factor for their associated phenotype. 22

34 Chapter 2.2 Materials and Methods Bacterial strains Laboratory Escherichia coli K-12 strain used in this study was MG1655. A total of 40 environmental E. coli isolates were collected from urban beaches (water and sand samples) and nearby fecal pollution sources (wastewater effluents and animal fecal droppings) in the cities of Hamilton and Toronto on Lake Ontario (Canada). Dr. Thomas A. Edge provided environmental E. coli isolates and source details Growth conditions and sample collection Isolates were streaked onto solid LB medium and incubated overnight at 37 C. Single colony isolates were used in all experiments. Overnight cultures were inoculated with single, independent colonies in triplicate, incubated aerobically overnight at 37 C at 200 rpm, and sub-cultured cells 1:10,000 to a starting OD600 of Cultures were harvested at OD600 = 0.3 after being maintained in exponential phase for nine generations, OD600 = 1.5 in stationary phase and 24 hours after subculture. Chloramphenicol (final concentration of 150 µg/ml) was added immediately to stop protein synthesis. Samples were pelleted at 14,000 g for 2 min. The supernatant was removed by pipette and pellets were washed in PBS buffer. After second centrifugation the pellets were resuspended in SDS loading buffer (125 mm Tris- Cl, ph 6.8; 2.5% β-mercaptoethanol; 8.7% glycerol; 23

35 1% SDS; 0.01% bromophenol blue) for a final cell concentration equivalent to an OD600 of Immunoblot analysis The resuspended pellets were boiled for 5 min. Ten µl of protein samples were resolved on 10% SDS polyacrylamide stacking and separating gel. Two gels were run at a time; one was stained for protein with 0.1% Coomassie blue dye to ensure equal protein loading. The other gel was used for immunoblot analysis. The resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Inc., Billerica, MA), using Bio-Rad semi-dry transfer cell. The membranes were blocked in 5% milk made in TBS-T (87 mm NaCl; 10 mm Tris-Cl, ph 8; 0.05% Tween 20) for 1 h at room temperature. Then it was incubated in 1:10,000 dilution of RpoS primary antibody (anti-σ s antibody; Neoclone, Inc., Madison, WI) overnight with gentle shaking at 4 C. After washing with TBS-T, the membrane was incubated in 1:3,000 dilution of the secondary anti-mouse antibody (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada) for 1 h at room temperature. After washing again with TBS-T, the membrane was soaked in 1:1 ECL detection reagent (Amersham GE Healthcare, Inc., Baie d Urfe, Quebec, Canada) for 1 min. The secondary antibody fluorophore activated in this reagent and signals were developed on Amersham Hyperfilm ECL. 24

36 2.2.4 Quantify the blots using ImageJ 1.47 ImageJ is a computational analysis tool used to identify alterations in expression of Immunoblot analysis. This software is a Java-based image processing program allowing for the comparison of exposure of fluorescent probes to a pre-determined value (control). ImageJ can calculate area and pixel value statistics of user-defined selections and intensity threshold objects. Blots were scanned in gray-scale TIFF format and the bands were analyzed by ImageJ software version The exposure was calculated to a value based on the threshold and transferred to Microsoft Excel for statistical analysis. The relative amounts of RpoS was quantified as a ratio of each RpoS protein band in exponential phase and stationary phase relative to the loading control (overnight culture of E. coli K12 strain). The relative value of expression levels for each sample was normalized to RpoB protein levels as an internal control which is expected to remain relatively constant. 25

37 Chapter 2.3 Results Growth curve Among a collection library of E. coli environmental strains we have in our lab, 40 isolates have been used for this study (Table 2.3). The isolates were cultured in LB media and growth was monitored spectrophotometrically. The optical density of the culture at 600 nm was recorded every hour until early exponential phase (OD600 ~ 0.3) and every 15 minutes until stationary phase (OD600 ~ 1.5) (Figure 2.1) RpoS expression level in exponential phase The protein extracts were run in electrophoresis gel. Immunoblot analysis was performed to detect RpoS protein bands for each sample. The expression of RpoS in E. coli K12 MG1655 strain was consistent with the previously published work (Dong et al., 2008). The RpoS expression level in exponential phase was not detectable while RpoS was highly expressed in early stationary phase (OD600 = 1.5) and 24 h culture. The environmental isolates showed different levels of RpoS expression in exponential phase. Among forty isolates, twelve strains (30%) showed high level of RpoS at exponential phase (Figure 2.4) including two environmental isolates, ECD08 from beach water and ABA08 from fresh fecal droppings of Canada goose (Branta canadensis) that had highest level of RpoS expression in exponential phase. RpoS expressed in exponential phase isolates of ECF09 and ECF01 from untreated combined sewer overflow sewage, four 26

38 isolates of ECA04, ECB06, ECA03 and ECC02 from beach water, ABF02 and ECD03 from beach sand and ABA01 from fresh fecal dropping of Canada goose (Branta canadensis). Slight expression of RpoS was detected in six isolates. Other environmental isolates did not show detectable expression of RpoS in exponential phase (Figure 2.4) Quantitation of RpoS protein expression using ImageJ 1.47 The protein bands were quantified with ImageJ 1.47 (Image Processing and Analysis in Java) and relative density values was measured. The quantification analysis was performed for one of the environmental isolates (ABA01). In this strain, the RpoS expression level in exponential phase was more than 3 fold of RpoS level in E. coli K12 strain MG1655 (Figure 2.3). 27

39 Chapter 2.4 Discussion RpoS is generally identified during starvation condition or when cells enter stationary phase (Dong et al., 2008) to slow down growth of E. coli and maximize survival rate. RpoS is regulating about 200 genes that are essential for survival. The change of RpoS expression levels will results in the change of RpoS regulated downstream essential genes expression levels. In E. coli K12 MG1655 strain the RpoS expression is not detectable in exponential phase (Dong et al., 2008), and the objective of this project was to determine if the RpoS expression level during exponential phase in environmental isolates of E. coli is higher than commonly used laboratory strain of E. coli K12 MG1655 to be a factor for their associated phenotype. In this study, all isolates were grown under the same laboratory conditions and all protein samples were harvested at the same growth points. Results showed higher RpoS expression during exponential phase in 30% of the environmental isolates compared to E. coli K12 laboratory strain (Figure 2.4). These results, confirms the strain-specific characteristics of RpoS and its dynamic expression level in each strain of E. coli. RpoS was expressed differentially during the growth of bacteria which confirms the growth phasespecific modulation of RpoS regulation in E. coli. The RpoS level can be appreciable in exponential phase (up to 1/3 of in stationary phase) in Shiga toxin strains of E. coli (Mand et al., 2013). The same results has been shown in figure 2.3 in ABA01 - one of the isolates that was collected from fresh fecal 28

40 dropping of Canada goose (Branta canadensis). The RpoS level of ABA01 strain in exponential phase is about 4 fold higher than this level in E. coli K12 strain Possible factors involved in RpoS expression level in exponential phase There is a lot of information on RpoS expression in stationary phase but less in exponential phase. By use of microarray studies, high levels of expression in most members of RpoS regulon in stationary phase is determined while many genes were regulated by RpoS only in exponential phase (Dong et al., 2008, Rahman et al., 2006). Although RpoS levels are low in exponential phase, this level can contribute to gene expression of a subset and regulate metabolic pathways. For example, as RpoS is negatively regulating genes that code for tricarboxylic acid (TCA) cycle (Patten et al., 2004), there is significantly higher activity of TCA cycle during exponential phase in rpos mutant than wild type (Rahman et al., 2006). The role of RpoS in exponential phase is dependent on many additional regulatory factors. RpoS regulators such as Crl protein (Typas et al., 2007), ppgpp (Dalebroux & Swanson, 2012) and Rsd protein (Piper et al., 2009) effects on RpoS binding to RNA polymerase. Also, anti-adaptors inhibit RssB activity to prevent degradation of RpoS by ClpXP proteases in stationary phase and exponential phase (IraL; an anti-adaptor in E. coli CFT073 pathogen strain and Shigella, play an important role in expression of RpoS during lag phase while this anti-adaptor is not active in E. coli K12 strain (Hryckowian et al., 2014)). Any of these factors can be a probable reason to explain the higher expression of RpoS in environmental strains than the laboratory strain during exponential phase. 29

41 Chapter 2.5 Conclusion and Future work E. coli environmental isolates are naturally growing in challenging environmental habitat while E. coli laboratory strains are cultivating in the laboratory condition for many years. The findings in this project contribute to the fact that the relatively lower levels of RpoS in the laboratory strains might occur due to selection for attenuation in RpoS levels in the laboratory strains. Also, the differences in RpoS expressions as a global regulator are probably because of mutagenesis during environmental selection, which helps environmental E. coli to survive and grow in the new environment (Chiang et al., 2011). There are other regulators involved in RpoS regulation in exponential phase. Crl protein increases RpoS binding to RNA core polymerase (Typas et al., 2007). Also, stimulates the expression of RssB and protects RpoS from being degraded by ClpXP protease. The fact that, Crl protein controls RpoS regulation even in low RpoS concentration (during exponential phase and early stationary phase) is a crucial point to study in modulating RpoS expression during exponential phase. In some of the environmental isolates RpoS level in stationary phase was much higher than in overnight culture (Figure 2.2). Screening this level in long term culture in the environmental isolates and the correlation between the survivability of these isolates could be a future direction for this project. There is a correlation between RpoS levels during exponential phase and the survival parameters in Shiga toxin-producing E. coli (Mand et al., 2013). This finding drives to the next step of this project which is determining if survival parameters in E. coli 30

42 environmental isolates are statistically associated with RpoS levels in exponential phase. The environmental isolates are more general and diverse criteria to be monitored compared to the limited groups of Shiga toxin-producing E. coli, so the results would vastly contribute to this field. 31

43 Table 2.1. Escherichia coli sigma factors. (Ishihama, 2000, Maeda et al., 2000) Sigma factor σ 70 (RpoD) Genes transcribed by each sigma factor Essential and growth-related genes Binding affinity for core polymerase a 1.0 Intracellular conc. (fmol/mg protein) b 160 σ 54 (RpoN) Nitrogen-regulated/stress response genes σ 28 (RpoF) Flagella-chemotaxis genes σ 32 (RpoH) Heat shock/stress response genes σ 19 (FecI) Ferric citrate transport /extracytoplasmic genes 6.65 < 1 σ 24 (RpoE) Heat shock/extracytoplasmic genes σ 38 (RpoS) Stationary phase/stress response genes 16.4 < 1 a The affinity is estimated from Kd value (dissociation constant) of the relative levels of core enzyme binding b In E. coli W

44 Table 2.2. Summary of source types for the 40 environmental E. coli isolates used in this study Source type Number of isolates Beach sand 15 Beach water 10 Untreated CSO sewage 6 Sewage plant final effluent 3 Canada goose (Branta canadensis) dropping 3 Combined sewage 1 Dog (Canis lupus familiaris) dropping 1 Gull (Larus delawarensis) dropping 1 33

45 Table 2.3. Environmental E. coli isolates used in this study Isolate Name Location Type Source ABB10 Eastwood Combined sewage T. Edge, CCTW ECF09 Eastwood Untreated CSO sewage T. Edge, CCTW ABD09 Eastwood Untreated CSO sewage T. Edge, CCTW ECF11 Bayfront Park beach Canada Goose (Branta canadensis) T. Edge, CCTW ABA01 Bayfront Park beach Canada Goose (Branta canadensis) T. Edge, CCTW ABA08 Bayfront Park beach Canada Goose (Branta canadensis) T. Edge, CCTW ABC08 Bayfront Park beach Gull (Larus delawarensis) T. Edge, CCTW ECA04 Hamilton Harbour Beach water T. Edge, CCTW ECA08 Hamilton Harbour Beach water T. Edge, CCTW ECB02 Hamilton Harbour Beach water T. Edge, CCTW ECB06 Hamilton Harbour Beach water T. Edge, CCTW ECD08 Hamilton Harbour Beach water T. Edge, CCTW ECA01 Bayfront Park beach Beach water T. Edge, CCTW ECA03 Bayfront Park beach Beach water T. Edge, CCTW ECD12 Bayfront Park beach Beach water T. Edge, CCTW ECC02 Bayfront Park beach Beach water T. Edge, CCTW ECC08 Bayfront Park beach Beach water T. Edge, CCTW ABB02 Bayfront Park beach Beach sand T. Edge, CCTW ABB03 Bayfront Park beach Beach sand T. Edge, CCTW ABC01 Bayfront Park beach Beach sand T. Edge, CCTW ABC03 Bayfront Park beach Beach sand T. Edge, CCTW ABC04 Bayfront Park beach Beach sand T. Edge, CCTW ABF02 Bayfront Park beach Beach sand T. Edge, CCTW ABH11 Bayfront Park beach Beach sand T. Edge, CCTW ECB07 Bayfront Park beach Beach sand T. Edge, CCTW ECD03 Bayfront Park beach Beach sand T. Edge, CCTW ECD05 Bayfront Park beach Beach sand T. Edge, CCTW ECE06 Bayfront Park beach Beach sand T. Edge, CCTW ABG06 Bayfront Park beach Untreated CSO sewage T. Edge, CCTW ECE10 Bayfront Park beach Untreated CSO sewage T. Edge, CCTW ECF01 Bayfront Park beach Untreated CSO sewage T. Edge, CCTW ECE12 Main and King Untreated CSO sewage T. Edge, CCTW ECG12 Hamilton SPCA Animal Shelter Dog (Canis lupus familiaris) T. Edge, CCTW ABE08 Hamilton bypass Sewage plant final effluent T. Edge, CCTW ABA03 Hamilton sewage treatment plant Sewage plant final effluent T. Edge, CCTW ECF07 Hamilton sewage treatment plant Sewage plant final effluent T. Edge, CCTW AZB10 Hamilton Beach sand T. Edge, CCTW AZB07 Hamilton Beach sand T. Edge, CCTW BNB03 Toronto Beach sand T. Edge, CCTW BNB04 Toronto Beach sand T. Edge, CCTW 34

46 Growth (OD 600 ) M.Sc. thesis P. Haghighi; McMaster University Biology 10 O 1 S 0.1 E MG1655 ABA01 ABA08 ECD08 ECF01 ABF02 ECF09 ECB Time (h) Figure 2.1. Growth of E. coli environmental isolates in rich media. Samplings indicated as exponential phase (E), stationary phase (S) and 24 h after subculture (O). 35

47 ABB10 Growth phases E S O AZB10 ABA03 ABC08 ABB02 ECG12 ECA01 ABA01 AZB07 BNB04 Lane Figure 2.2. Comparing RpoS expression level in 10 environmental isolates of E. coli as a factor of growth phase. In this experiment RpoS antibody was used to detect the level of RpoS protein. Samples were harvested at exponential phase (E) OD600 ~ 0.3, stationary phase (S) OD600 ~ 1.5 and overnight culture (O) in 10 E. coli environmental isolates. AZB07 and BNB04 are known as ΔrpoS. 36

48 RpoS RpoB Lane MG1655 ABA01 E E S S O O E E S S O O ΔrpoS Exponential phase Stationary phase Overnight culture RpoS relative density MG1655 ABA01 E. coli strains Figure 2.3. Quantitation of RpoS protein expression in exponential phase, stationary phase and overnight culture. RpoB antibody used as an internal control and ΔrpoS used as a negative control. 37

49 RpoS K12 K12 ΔrpoS ECD12 ECE10 ECF07 ECE12 ECD08 ECF09 ECF11 ECE06 ECF01 ECD05 RpoS K12 K12 ΔrpoS ABC04 ABC03 ABG06 ABF02 ABE08 ABC01 ABH11 ABA08 ABD09 BNB03 K12 K12 ΔrpoS ECC02 ABA03 ECB06 ECD03 ECA04 ECC08 ECA03 ECB07 ECA08 ECB02 RpoS RpoS Figure 2.4. RpoS expression in 40 E. coli environmental isolates at exponential phase. For each isolates, 10 µl of protein sample at OD600 of 0.3 was run on SDS-PAGE and RpoS protein bands were detected by immunoblotting using RpoS antibody. Overnight culture of E. coli K12 MG1655 used as positive control. K12 ΔrpoS used as a negative control. The exposure time for all the blots was one minute. 38

50 References Bougdour, A., C. Lelong & J. Geiselmann, (2004) Crl, a low temperature-induced protein in Escherichia coli that binds directly to the stationary phase sigma subunit of RNA polymerase. J Biol Chem 279: Charette, S.J., H. Lambert, P.J. Nadeau & J. Landry, (2010) Protein quantification by chemiluminescent Western blotting: Elimination of the antibody factor by dilution series and calibration curve. J Immunol Methods 353: Chiang, S.M., T. Dong, T.A. Edge & H.E. Schellhorn, (2011) Phenotypic diversity caused by differential RpoS activity among environmental Escherichia coli isolates. Appl Environ Microbiol 77: Chiang, S.M. & H.E. Schellhorn, (2010) Evolution of the RpoS regulon: origin of RpoS and the conservation of RpoS-dependent regulation in bacteria. J Mol Evol 70: Dalebroux, Z.D. & M.S. Swanson, (2012) ppgpp: magic beyond RNA polymerase. Nat Rev Microbiol 10: De Vos, D., F.J. Bruggeman, H.V. Westerhoff & B.M. Bakker, (2011) How Molecular Competition Influences Fluxes in Gene Expression Networks. PloS One 6. Degasperi, A., M.R. Birtwistle, N. Volinsky, J. Rauch, W. Kolch & B.N. Kholodenko, (2014) Evaluating Strategies to Normalise Biological Replicates of Western Blot Data. PloS One 9. 39

51 Dong, T., M.G. Kirchhof & H.E. Schellhorn, (2008) RpoS regulation of gene expression during exponential growth of Escherichia coli K12. Mol Genet Genomics 279: Dong, T. & H.E. Schellhorn, (2009a) Control of RpoS in global gene expression of Escherichia coli in minimal media. Mol Genet Genomics 281: Dong, T. & H.E. Schellhorn, (2009b) Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933. BMC Genomics 10: 349. Dong, T. & H.E. Schellhorn, (2010) Role of RpoS in virulence of pathogens. Infect Immun 78: Dudin, O., S. Lacour & J. Geiselmann, (2013) Expression dynamics of RpoS/Crldependent genes in Escherichia coli. Res Microbiol 164: Elliott, S.J., L.A. Wainwright, T.K. McDaniel, K.G. Jarvis, Y.K. Deng, L.C. Lai, B.P. McNamara, M.S. Donnenberg & J.B. Kaper, (1998) The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 28: 1-4. Gerhardt, P., (1994) Methods for General and Molecular Bacteriology. American Society for Microbiology. Hengge-Aronis, R., (1996) Back to log phase: sigma S as a global regulator in the osmotic control of gene expression in Escherichia coli. Mol Microbiol 21:

52 Hengge-Aronis, R., (2002) Signal transduction and regulatory mechanisms involved in control of the sigma(s) (RpoS) subunit of RNA polymerase. Microbiol Mol Biol Rev 66: , table of contents. Hofmann, N., R. Wurm & R. Wagner, (2011) The E. coli anti-sigma factor Rsd: studies on the specificity and regulation of its expression. PloS One 6: e Hryckowian, A.J., A. Battesti, J.J. Lemke, Z.C. Meyer & R.A. Welch, (2014) IraL is an RssB anti-adaptor that stabilizes RpoS during logarithmic phase growth in Escherichia coli and Shigella. MBio 5: e Ishihama, A., (2000) Functional modulation of Escherichia coli RNA polymerase. Annu Rev Microbiol 54: Ishihama, A., A. Kori, E. Koshio, K. Yamada, H. Maeda, T. Shimada, H. Makinoshima, A. Iwata & N. Fujita, (2014) Intracellular concentrations of 65 species of transcription factors with known regulatory functions in Escherichia coli. J Bacteriol 196: Jishage, M. & A. Ishihama, (1995) Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of sigma 70 and sigma 38. J Bacteriol 177: Jishage, M. & A. Ishihama, (1998) A stationary phase protein in Escherichia coli with binding activity to the major sigma subunit of RNA polymerase. P Natl Acad Sci USA 95:

53 Jishage, M. & A. Ishihama, (1999) Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D. J Bacteriol 181: King, T., A. Ishihama, A. Kori & T. Ferenci, (2004) A regulatory trade-off as a source of strain variation in the species Escherichia coli. J Bacteriol 186: Lacour, S. & P. Landini, (2004) SigmaS-dependent gene expression at the onset of stationary phase in Escherichia coli: function of sigmas-dependent genes and identification of their promoter sequences. J Bacteriol 186: Landini, P., T. Egli, J. Wolf & S. Lacour, (2014) sigmas, a major player in the response to environmental stresses in Escherichia coli: role, regulation and mechanisms of promoter recognition. Environ Microbiol Rep 6: Lelong, C., K. Aguiluz, S. Luche, L. Kuhn, J. Garin, T. Rabilloud & J. Geiselmann, (2007) The Crl-RpoS regulon of Escherichia coli. Mol Cell Proteomics 6: Maeda, H., N. Fujita & A. Ishihama, (2000) Competition among seven Escherichia coli sigma subunits: relative binding affinities to the core RNA polymerase. Nucleic Acids Res 28: Mand, T.D., D. Dopfer, B. Ingham, C. Ane & C.W. Kaspar, (2013) Growth and survival parameter estimates and relation to RpoS levels in serotype O157:H7 and non- O157 Shiga toxin-producing Escherichia coli. J Appl Microbiol 114:

54 McDaniel, T.K. & J.B. Kaper, (1997) A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol 23: Patten, C.L., M.G. Kirchhof, M.R. Schertzberg, R.A. Morton & H.E. Schellhorn, (2004) Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12. Mol Genet Genomics 272: Piper, S.E., J.E. Mitchell, D.J. Lee & S.J. Busby, (2009) A global view of Escherichia coli Rsd protein and its interactions. Mol Biosyst 5: Rahman, M., M.R. Hasan, T. Oba & K. Shimizu, (2006) Effect of rpos gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations. Biotechnol Bioeng 94: Schellhorn, H.E., J.P. Audia, L.I.C. Wei & L. Chang, (1998) Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli. J Bacteriol 180: Sheldon, J.R., M.S. Yim, J.H. Saliba, W.H. Chung, K.Y. Wong & K.T. Leung, (2012) Role of rpos in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival. Appl Environ Microb 78: Taylor, S.C., T. Berkelman, G. Yadav & M. Hammond, (2013) A Defined Methodology for Reliable Quantification of Western Blot Data. Mol Biotechnol 55:

55 Trevino-Quintanilla, L.G., J.A. Freyre-Gonzalez & I. Martinez-Flores, (2013) Anti- Sigma Factors in E. coli: Common Regulatory Mechanisms Controlling Sigma Factors Availability. Curr Genomics 14: Typas, A., C. Barembruch, A. Possling & R. Hengge, (2007) Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of sigmas activity and levels. EMBO J 26: Vijayakumar, S.R., M.G. Kirchhof, C.L. Patten & H.E. Schellhorn, (2004) RpoSregulated genes of Escherichia coli identified by random lacz fusion mutagenesis. J Bacteriol 186:

56 Appendix Standard Operating Procedures Reference: This method was modified by T. Dong and S. M. Chiang from (Gerhardt, 1994) Sample collection 1. Streak out the bacterial isolates onto LB plates and incubate overnight at 37 C. 2. Inoculate a single bacterial colony into 5 ml of 1 LB from overnight culture in a sterile tube and incubate aerobically overnight at 37 C, at 200 rpm. 3. Subculture cells 1:10,000 into 50 ml 1 LB in a sterile 250 ml Erlenmeyer flask to a starting OD600 of Collect the samples at OD600 = 0.3 for exponential phase and OD600 = 1.5 for stationary phase (take 6.6 ml and 1.5 ml of samples respectively to have final OD600 of 2.0 after adding SDS/loading buffer). For overnight culture, record the OD600 and calculate the sample amount needed. Add Chloramphenicol (final concentration of 150 µg/ml) to stop protein synthesis. 5. Centrifuge samples at 14,000 g for 2 min. Discard the supernatant using a pipette. 6. Resuspend the pellet in 500 µl PBS buffer and centrifuge at 11,000 g for 2 min. (Wash the pellet with PBS three times to remove the salt which cause wide bands in the polyacrylamide gel). 7. Resuspend the pellet in 1 ml of 1 SDS/loading buffer to have the final OD600 of 2.0 for the protein samples. 45

57 8. Boil the samples in boiling water for 5 min. 9. Before adding into the gel, centrifuge the samples at 10,000 g for 1 min. SDS-PAGE Prepare the buffer and reagents before starting the experiment. 1.5 M Tris-Cl, ph 8.8 or 1 M Tris-Cl, ph 6.8 (Store at 4 C): 91 g Tris base for 1.5 M Tris-Cl, 60.6 g for 1 M Tris-Cl, 300 ml ddh2o, adjust the ph with 1N HCl, and add ddh2o to 500 ml. 10 Running Buffer with SDS (Store at room temperature): 30 g Tris base, 144 g Glycine, 10 g SDS, add ddh2o to 1 L. 10 TBT-T (Store at room temperature): 43.5 g NaCl, 50 ml 1 M Tris-Cl ph 8, 2.5 ml Tween 20, add ddh2o to 500 ml. 10 Transfer Buffer (Store at room temperature. Before use, dilute into 1 transfer buffer and place on ice or at 4 C): 6.05 g Tris base, 28.8 g Glycine, 400 ml Methanol and add ddh2o to 2 L. 2 SDS/loading Buffer (Store at room temperature. For long term storage keep at 4 C): 12.5 ml 1 M Tris-Cl ph 6.8, 8.7 ml Glycerol, 2.5 ml β-mercaptoethanol, 10 ml 10% SDS,1 ml 1% Bromophenol blue, 15.3 ml ddh2o. 0.1% Coomassie Blue solution (Store at room temperature. You can re-use it 2-3 times): 0.5 g Coomassie Brilliant Blue, 200 ml Methanol, 50 ml Acetic acid, 250 ml ddh2o. Destain solution: 450 ml Methanol, 100 ml Acetic acid, 450 ml ddh2o. 46

58 1. To set the apparatus, first rinse and dry the spacers and short plates and wipe with 70% ethanol. 2. Fix the plates in a casting frame, and assemble in a casting stand. Important Note: make sure the gaskets are completely dry. Wet gaskets result in leaking the gel. 3. Prepare the separating gel by adding the reagents as detailed below (From S. M. Chiang Ph.D. thesis 2012). Make sure to add in order and swirl gently after each addition. Reagents Volume (ml) for making 2 gels ddh2o M Tris-Cl. ph % SDS % Acrylamide 0.8% Bis % APS 0.1 TEMED (N,N,N`,N` Tetramethylethylenediamine 4. Right after mixing the reagents, pipette it between gel glasses up to ~ 5.5 cm from bottom. Add water on top to avoid forming the bubbles inside the separating gel. 5. Let gels solidify for about min. (You will be able to see the solidified gel line). Pour out water and use a Kim-wipe to drain the glasses for adding the stacking gel. 6. Prepare the stacking gel by adding the reagents as detailed below (From S. M. Chiang Ph.D. thesis 2012). Make sure to add in order and swirl gently after each addition. 47

59 Reagents Volume (ml) for making 2 gels ddh2o M Tris-Cl. ph % SDS % Acrylamide 0.8% Bis % APS 0.05 TEMED (N,N,N`,N` Tetramethylethylenediamine 7. Right after mixing the reagents, pipette the mixture between gel glasses up to the top. Insert comb. Note: the gels start to solidify quickly, so add the comb as soon as pouring the gel. Let the gel solidify for about 30 min. 8. Remove comb, and assemble the gels into the apparatus. Fill the unit with 1 running buffer with SDS. (For running two gels, fill only half of the unit) 9. Load 10 µl of protein sample into wells. Load the appropriate protein ladder (In this study Fermentas PageRuler Prestained Protein ladder Thermo Scientific #26616 was used). 10. Plug in the apparatus and set the voltage at 50 V for 0.5 h for running protein through stacking gel. After, increase the voltage to 100 V for 1.5 h (until protein reach the bottom of the separating gel). Warning: over running the gel results in running protein samples out of the gel. 11. After resolving the gels in electrophoresis apparatus, remove them from casting glasses. Gently, cut the stacking gel. Use one gel for protein staining and the second gel for immunoblotting. 48

60 12. Soak the protein gel in 0.1% Coomassie Brilliant Blue solution and shake it gently and evenly for 1 h. Destain the gel with destain solution for 30 min. replace the destain solution every 10 min. 13. Add 10% acetic acid and store the gel. Take photos. Immunoblotting 1. Transfer the protein from second gel after SDS-PAGE to a polyvinylidene difluoride (PVDF) membrane by semi-dery transfer method: a. Cut 7 9 cm PVDF membrane and soak it in methanol for 2-5 min. b. Cut six 7 9 cm filter papers. Soak the filter papers, the protein gel, and PVDF membrane (after soaking in methanol) in ice-cold 1 transfer buffer. c. Assemble sandwich by (bottom up): Base (anode), three filter papers, PVDF membrane, separating layer of gel, three filter papers, Lid (cathode). Eliminate air bubbles between layers by using a roller. d. Run transfer cell at 20 V for 30 min. 2. After transferring protein from the gel into the membrane, take out the membrane from the sandwich and incubate in 50 ml of 5% milk (2.5 g in 1 TBS-T) shaking for 1 h at room temperature. (Or overnight at 4 C to avoid high background). 3. Discard the 5% milk, and cut the edges of membrane to fit within small Petri dishes. 4. Incubate the membrane in 10 ml of 1:10,000 dilution of primary antibody overnight at 4 C with gentle shaking. 5. Wash membrane with 1 TBS-T on a shaker at room temperature for 30 min. Change the buffer each 10 min. 49

61 6. Add 10 ml of 1:3,000 dilution of secondary antibody at room temperature with gentle shaking. 7. Wash membrane with 1 TBS-T on a shaker at room temperature for 30 min. Change the buffer each 10 min. 8. For chemiluminescent detection, use 1:1 ECL Western Blotting Detection Reagent (Amersham GE Healthcare, Inc.). Mix 5 mil of reagent 1 and 5 ml of reagent 2 in a Petri dish. 9. Take the membrane out of TBS-T buffer and drain off the excess buffer. Soak the membrane in Amersham ECL Detection reagent for 1 min with gentle shaking at room temperature. 10. Seal membrane in Saran wrap. Eliminate the leak by covering the sealed membrane in paper towels. 11. Put the membrane in X-ray film cassettes and take it into the dark room. Expose Amersham Hyperfilm ECL to the membrane for appropriate amount of time (1 s to 1 min) and develop the film. 50

62 Quantify the blots using ImageJ 1.47 Reference: ImageJ is a java-based program by Wayne Rasband from National Institute of Health (USA) and is available for free download at: This software relatively quantifies the protein bands on Immunoblot films (not the absolute values). The relative amounts of protein can be quantified as a ratio of each protein band relative to the loading control. 51

63 52

64 Outline Background Growth phase-dependent RpoS levels in Escherichia coli laboratory and environmental strains Pardis Haghighi M.Sc. Defence February 16, 2016 Hypothesis Objective Methodology Results Conclusion Future directions Project (1) description Bacterial growth curve Transcription regulation in E. coli Stationary phase Expression pattern of E. coli genome Controlled by Number of cells (log) RNA polymerase distribution pattern Core RNAP Sigma subunits RNAP holoenzyme Transcription Factors Lag phase Time Measure the intracellular concentration of the sigma subunits and transcription factors under exponential and stationary phase Ishihama, A., et al. (2014). Journal of Bacteriology 196(15): E. coli sigma factors RpoS (σ 38 ) RpoS in stationary phase Sigma factor Genes transcribed by each sigma factor Reference σ 70 (RpoD) Essential and growth-related genes (Paget & Helmann, 2003) σ 54 (RpoN) Nitrogen-regulated/stress response genes (Zhao et al., 2010) σ 28 (RpoF) Flagella-chemotaxis genes (Zhao et al., 2007) σ 32 (RpoH) Heat shock/stress response genes (Zhao et al., 2005) σ 19 (FecI) Ferric citrate transport /extracytoplasmic genes (Maeda et al., 2000) σ 24 (RpoE) Heat shock/extracytoplasmic genes (Dartigalongue et al., 2001) σ 38 (RpoS) Stationary phase/stress response genes (Hengge-Aronis et al., 1991) o Well-studied o High intracellular levels o Controls many sets of genes RpoS in exponential phase o Less studied Growth phase-dependant Strain-specific o Undetectable intracellular levels in laboratory strains o Rapidly degraded by proteases 53

65 Why is studying RpoS level in exponential phase important? RpoS regulates sets of exponential phase-specific genes even in low concentration in E. coli laboratory strains [1] o Genes for metabolic pathways [2] Is RpoS expressed differentially among environmental E. coli isolates? Detectable RpoS levels in E. coli pathogenic strains during exponential phase [3,4] Project hypothesis [1] Dong, T., et al. (2008). Molecular Genetics and Genomics 279(3): [2] Rahman, M., et al. (2006). Biotechnology and Bioengineering 94(3): [3] Dong, T. and H. E. Schellhorn (2009). BMC Genomics 10: 349. [4] Mand, T. D., et al. (2013). Journal of Applied Microbiology 114(1): Hypothesis Objective RpoS expression levels during exponential phase in environmental isolates of E. coli are higher than laboratory strain To investigate the RpoS expression level of E. coli environmental isolates in exponential phase (OD 600 = 0.3) Origins of environmental isolates used in this study Source No of isolates Beach sand 15 Beach water 10 Untreated CSO sewage 6 Sewage plant final effluent 3 Canada Goose (Branta canadensis) dropping 3 Combined sewage 1 Dog (Canis lupus familiaris) dropping 1 Gull (Larus delawarensis) dropping 1 Isolate Name Location Source Type ABB10 Eastwood Combined sewage ECF09 Eastwood Untreated CSO sewage ABD09 Eastwood Untreated CSO sewage ECF11 Bayfront Park beach Canada Goose (Branta canadensis) ABA01 Bayfront Park beach Canada Goose (Branta canadensis) ABA08 Bayfront Park beach Canada Goose (Branta canadensis) ABC08 Bayfront Park beach Gull (Larus delawarensis) ECA04 Hamilton Harbour Beach water ECA08 Hamilton Harbour Beach water ECB02 Hamilton Harbour Beach water ECB06 Hamilton Harbour Beach water ECD08 Hamilton Harbour Beach water ECA01 Bayfront Park beach Beach water ECA03 Bayfront Park beach Beach water ECD12 Bayfront Park beach Beach water ECC02 Bayfront Park beach Beach water ECC08 Bayfront Park beach Beach water ABB02 Bayfront Park beach Beach sand ABB03 Bayfront Park beach Beach sand ABC01 Bayfront Park beach Beach sand ABC03 Bayfront Park beach Beach sand ABC04 Bayfront Park beach Beach sand ABF02 Bayfront Park beach Beach sand ABH11 Bayfront Park beach Beach sand ECB07 Bayfront Park beach Beach sand ECD03 Bayfront Park beach Beach sand ECD05 Bayfront Park beach Beach sand ECE06 Bayfront Park beach Beach sand ABG06 Bayfront Park beach Untreated CSO sewage ECE10 Bayfront Park beach Untreated CSO sewage ECF01 Bayfront Park beach Untreated CSO sewage ECE12 Main and King Untreated CSO sewage ECG12 Hamilton SPCA Animal Shelter Dog (Canis lupus familiaris) ABE08 Hamilton bypass Sewage plant final effluent ABA03 Hamilton sewage treatment plant Sewage plant final effluent ECF07 Hamilton sewage treatment plant Sewage plant final effluent AZB10 Hamilton Beach sand Strains used in this study The isolates provided by Dr. T. A. Edge (Canada Centre for Inland Waters) 54

66 Subcultured from O/N culture Methodology Growth of E. coli environmental isolates in rich media Collected samples at desired OD600 Added Cm to stop protein synthesis 10 Washed with PBS Growth (OD600) Resuspended in SDS/loading buffer Boiled the samples Ran SDS-PAGE gel O 1 S 0.1 ABA08 ECD08 ABA01 ABC08 10 AZB07 ECA01 AZB10 ABB10 ECG12 ABB03 K12 ΔrpoS K12 ΔrpoS ABB02 K12 K ECD05 7 ECF01 ECF11 9 ECF09 8 ECE06 7 ECD08 6 ECE12 5 ECF07 ECE10 ECD ABC01 ABH ECB02 ECA03 8 ECA08 ECC08 7 ECB07 ECA04 6 ECD03 5 ECB06 4 ABA03 ECC02 3 ABE08 5 E ABA08 ABG06 ABC03 ABC Samples collected at exponential phase (OD600= 0.3) Positive control: K12 (Overnight culture) Negative control: K12 ΔrpoS (Overnight culture) RpoS 1 E 5 RpoS expression in 40 E. coli environmental isolates at exponential phase Conclusions and future directions ABA01 S S 9 10 O 11 O ΔrpoS E: Exponential phase (OD600 = 0.3) S: Stationary phase (OD600 = 1.5) O: Overnight culture 30% of environmental isolates showed more than 2 fold expression of RpoS compared with laboratory strains during exponential phase Exponential phase Stationary phase 1.4 RpoS as a strain-specific sigma factor RpoS as a Growth phase-dependent sigma factor Overnight culture 1.2 RpoS relative density 4 O 4 BNB ABD09 2 MG1655 S S O 3 8 ABF02 1 RpoS quantification 2 2 K12 ΔrpoS E: Exponential phase (OD600 = 0.3) S: Stationary phase (OD600 = 1.5) O: Overnight culture AZB K12 ABA01 RpoB Lane 4 6 Time (h) RpoS ECA01 E 2 K12 ΔrpoS ECG12 E 1 K12 ABB02 RpoS 3 2 RpoS ABC08 Lane 2 0 RpoS ABA03 BNB04 1 ECF09 ECB06 BNB Growth phases E S O AZB10 E. coli Environmental strains ECF01 ABF02 Quantified the bands using ImageJ ABB10 E. coli K12 Laboratory strain MG1655 ABA01 E Immunoblot Growth phase-dependent RpoS levels in 10 environmental isolates of E. coli E: Exponential phase (OD600 = 0.3) S: Stationary phase (OD600 = 1.5) O: Overnight culture MG1655 ABA01 E. coli strains 55

67 Cont. Project 1: Selection for attenuation in RpoS levels in the laboratory strains Additional modulating factors relevant to RpoS action o RpoS regulation depends on other transcriptional regulators, such as Rsd, 6S RNA, ppgpp and Crl o Anti-adaptors Further studies on survival parameters in environmental isolates Statistically associated with RpoS level in exponential phase Is the expression level of RpoD sigma factor lower in E. coli K12 laboratory strain (MG1655) and pathogenic strain O157:H7 (EDL933)? (Confirming previous students experiments) RpoS levels in environmental isolates during stationary and post-stationary phase Immunoblot analysis of RpoD expression in E. coli O157:H7 strain EDL933 and E. coli K12 MG1655 performed by previous students RpoD level in E. coli pathogenic and laboratory strains A. Wenjie Zheng M.Sc. Thesis B. Vithooshan Vijayakumaran B.Sc. Thesis RpoD level in E. coli pathogenic and laboratory strains A MG1655 EDL933 M OD 600 kda RpoD level in E. coli pathogenic and laboratory strains A kda M MG1655 EDL OD B Lane MG1655 EDL OD 600 B Lane MG1655 EDL OD 600 RpoD RpoD Lane Lane

68 Acknowledgments Dr. Schellhorn Dr. Gupta Thanks Dr. Kolasa All members in Schellhorn lab The audience Supplementary information E. coli sigma factors Binding affinity for Intracellular conc. core polymerasea (fmol/mg protein)b Sigma factor Genes transcribed by each sigma factor σ70 (RpoD) Essential and growth-related genes σ28 (RpoF) Flagella-chemotaxis genes σ54 (RpoN) Nitrogen-regulated/stress response genes σ32 (RpoH) σ19 (FecI) Ferric citrate transport /extracytoplasmic genes σ24 (RpoE) Heat shock/extracytoplasmic genes σ38 (RpoS) < < Stationary phase/stress response genes athe Heat shock/stress response genes 1.4 affinity is estimated from Kd value (dissociation constant) of the relative levels of core enzyme binding bin E. coli W3110 at exponential phase [1] Ishihama, A., (2000). Annual review of microbiology 54: [2] Maeda, H., N. Fujita & A. Ishihama, (2000). Nucleic Acids Res 28: Is RpoS expressed differentially among E. coli pathogenic strains? Linear dynamic range using normalization experiment A RpoS is detectable in pathogenic strains of E. coli during exponential growth although expression levels are lower than stationary-phase cells. [1] 1 kda M In E. coli O157:H7 EDL933 the level of RpoS in exponential phase is up to one-third of the levels found in stationary phase. [2] Relative Concentration Lane 1 B Dong, T. and H. E. Schellhorn (2009). "Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933." BMC Genomics 10: 349. Mand, T. D., et al. (2013). "Growth and survival parameter estimates and relation to RpoS levels in serotype O157:H7 and non-o157 Shiga toxin-producing Escherichia coli." J Appl Microbiol 114(1): [1] 1 RpoD [2] 57 2 Lane Relative Concentration

69 The linear range of detection using ImageJ 1.47 software IraL; Inhibits RssB activity in logarithmic phase The level of RpoS is comparable in exponential (LOG) and stationary (STAT) growth phases in: Relative Density y = x R² = osome E. coli isolates including uropathogenic E. coli strain CFT073 oshigella strains Relative Concentration Hryckowian AJ, et al. IraL is an RssB anti-adaptor that stabilizes RpoS during logarithmic phase growth in Escherichia coli and Shigella. mbio 5(3):e (2014) 31 58