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Studies of cryptic phytochromes in Rhodopsedomonas palustris Meng, Li 2008

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STUDIES OF CRYPTIC PHYTOCHROMES IN RHODOPSEUDOMONAS PALUSTRIS  by  MENG LI B.Sc (Hon.), Birmingham University, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Microbiology and immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2008  © Meng Li, 2008  ABSTRACT Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bphlike genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O2 does not inhibit the normal function of Bphs, although O2 was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double  ii  knockout of the regulatory genes ppsR1-2- led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression.  iii  TABLE OF CONTENTS  ABSTRACT ......................................................................................................................... ii TABLE OF CONTENTS ................................................................................................... iv LIST OF TABLES ............................................................................................................ viii LIST OF FIGURES ............................................................................................................ ix ABBREVIATIONS ............................................................................................................. xi ACKNOWLEDGEMENTS .............................................................................................. xii  CHAPTER 1 – THESIS INTRODUCTION ..................................................................... 1 1.1 Overview of phytochromes...... ....................................................................................... 1 1.2 The structure of phytochromes and chromophores ......................................................... 2 1.3 Bacteriophytochromes and photosynthesis...................................................................... 4 1.4 Bacteriophytochromes in Rhodopseudomonas palustris ................................................. 8  CHAPTER 2 – TWO PUTATIVE PHYTOCHROMES ENCODED BY RPA0122 AND RPA0990 ............................................................................................ 11 2.1. Introduction ..................................................................................................................... 11 2.2. Materials and Methods .................................................................................................... 13 2.2.1 Bacterial strains, growth conditions, and plasmids ............................................. 13 2.2.2 Construction and confirmation of RPA0122 and RPA0990 knockouts .............. 15 2.2.3 Phenotype of RPA0122 and RPA0990 knockouts .............................................. 18 2.2.3.1 Growth curve ............................................................................................. 18 2.2.3.2 Phototaxis .................................................................................................. 18 2.2.3.3 Absorption spectra of cells grown under standard photosynthetic conditions .................................................................................................. 19 iv  2.2.3.4 Absorption spectra of cells grown under irradiation with specific wavelengths ............................................................................................... 19 2.2.4 Construction of plasmids created for gene overexpression in E. coli.................. 20 2.2.5 Overexpression of RPA0122 and RPA0990 and purification from E. coli Rosetta DE3 ........................................................................................................ 21 2.2.5.1 Overexpression growth condition .............................................................. 21 2.2.5.2 Optimization of His-tag pulldown ............................................................. 22 2.2.6 Construction of plasmids for gene expression in R. palustris ............................. 23 2.2.7 Colony lift for screening for RPA0990 complemented strain ............................. 24 2.2.8 Evaluation of the stability of plasmid pJRD215 in the rpa0990 mutant strain ... 25 2.2.9 Purification of His-tagged RPA990 ..................................................................... 26 2.2.9.1 Expression growth condition .................................................................... 26 2.2.9.2 Purification of tagged RPA990................................................................. 26 2.2.10 Spectroscopic characterization of His-tagged RPA990..................................... 27 2.3 Results ........................................................................................................................... 27 2.3.1 Confirmation of rpa0122 and rpa0990 knockouts .............................................. 27 2.3.2 Overexpression and purification of His-RPA0122 and His-RPA0990 in E. coli .... ............................................................................................................................. 29 2.3.3 Construction of plasmids for gene expression in R. palustris ............................. 32 2.3.4 Expression and purification of His-RPA0990 in R. palustris.............................. 32 2.3.5 Absorption spectra data illustrated that His-RPA0990 is capable of switching between Pr and Pfr forms .................................................................................... 33 2.3.6 Phenotype of rpa0122 and rpa0990 mutants ...................................................... 37 2.3.6.1 Growth kinetics and yield .......................................................................... 37 2.3.6.2 Phototaxis .................................................................................................. 39 2.3.6.3 Absorption spectra of wild type and Bph mutants grown  v  photosynthetically ...................................................................................... 41 2.3.6.4 Absorption spectra of wild type and Bph mutants grown aerobically and illuminated with specific wavelengths ...................................................... 42 2.3.6.5 Construction of rpa0990 complemented strain ......................................... 44 2.4 Discussion ...................................................................................................................... 45 2.4.1 Purified His-RPA0990 has exhibited typical Bph spectroscopic properties ....... 45 2.4.2 Physiological function of RPA0990 requires future research ............................. 48 2.4.3 Expression and purification system used in this study ........................................ 50  CHAPTER 3 – EVALUATION OF POSSIBLE ANAEROBIC BILIN SYNTHESIS IN R. PALUSTRIS............................................................... 51 3.1 Introduction ................................................................................................................... 51 3.2 Materials and Methods .................................................................................................. 55 3.2.1 Strict anaerobic growth condition........................................................................ 55 3.2.2 Spectroscopy ........................................................................................................ 56 3.3 Results ........................................................................................................................... 56 3.3.1 Bph-dependent regulation of LH complexes persists in the absence of O2 ......... 56 3.4 Discussion ...................................................................................................................... 58  CHAPTER 4 – USE OF E.COLI LACZ GENE AS A REPORTER TO EVALUATE PROMOTER ACTIVITY IN R. PALUSTRIS ........................................ 65 4.1 Introduction ................................................................................................................... 65 4.2 Materials and Methods .................................................................................................. 67 4.2.1 Construction of lacZ transcriptional plasmids ..................................................... 67 4.2.2 β-galactosidase assays ......................................................................................... 69 4.3 Results….. ..................................................................................................................... 69  vi  4.3.1 Construction of lacZ transcriptional plasmids ..................................................... 69 4.3.2 β-galactosidase assays ......................................................................................... 70 4.4 Discussion ...................................................................................................................... 73 CHAPTER 5 – CONCLUSION........................................................................................ 77 REFERENCES .................................................................................................................. 79  vii  LIST OF TABLES Table 2.1:  Bacterial Strains and plasmids..................................................................... 14  Table 2.2:  Primers ......................................................................................................... 17  Table 2.3:  Comparison of short and long wavelength (λ) absorption peak, as well as ratio of 800 nm:870 nm absorption, in wild type and rpa0990 mutant strains grown under 700 nm illumination and in the dark ........................... 44  Table 3.1:  The 800:850 absorption ratios for R. palustris wild type, rpa3015-, rpa3016- and rpa1490- strains grown anaerobically at low light intensity .. 52  viii  LIST OF FIGURES Figure 1.1: The photocyle of phytochrome between Pr and Pfr forms ............................. 2 Figure 1.2:  Domain structure of typical phytochromes .................................................... 3  Figure 1.3:  Structural arrangement of a reaction center (RC) with its surrounding light harvesting complexes ..................................................................................... 6  Figure 1.4:  Mathematic deconvolution of LH absorption spectra of R. palustris ppsR2cells grown under high-aeration conditions ................................................... 7  Figure 1.5:  Genomic map of R. palustris showing bph0 through to bph5 ....................... 7  Figure 2.1:  Domain structure of putative bacteriophytochromes RPA0122 and RPA0990 of R. palustris ..................................................... 12  Figure 2.2:  Amino-acid sequence alignment of the region surrounding the chromophore-binding site for Bphs from R. palustris and D. radioduran .. 12  Figure 2.3: . Figure 2.4:  Map of rpa0990 showing the knockout scheme .......................................... 16  Figure 2.5:  Construction of plasmids created for gene expression in R. palustris ......... 24  Figure 2.6:  Confirmation of rpa0990 and rpa0122 knockouts showing the knockout scheme and the agarose gels with PCR products ......................................... 28  Figure 2.7:  Agarose gel showing the digestion products of recombinant plasmids pET28::rpa0122 and pET28::rpa0990 to confirm successful cloning ........ 29  Figure 2.8:  SDS PAGE showing the optimization of overexpression conditions in E. coli ................................................................................................................ 30  Figure 2.9:  SDS PAGE gels showing elutes of RPA0990 from Ni-NTA column under various conditions ........................................................................................ 31  Cloning scheme to create His-tagged Bphs overexpressed in E. coli .......... 21  Figure 2.10: SDS PAGE of partially purified His-RPA0990 ........................................... 33 Figure 2.11: Absorption spectra of tagged RPA0990 proteins expressed from R. palustris........................................................................................................ 35 Figure 2.12: Growth curves of R. palustris ..................................................................... 38 Figure 2.13: Doubling time of R. palustris wild type strain CGA009 and mutant strains in exponential phase as a function of illumination under anaerobic condition, and oxygen tension in the absence of illumination ............................................. 38 ix  Figure 2.14: Phototaxis experiments of wild type strain CGA009 and several different Bph mutants................................................................................... 40 Figure 2.15: Variability was observed within the same strain during phototaxis assay ... 40 Figure 2.16: Absorption spectra of intact cells grown under high light condition and low light condition ....................................................................................... 41 Figure 2.17: Absorption spectra of intact cells of R. palustris wild type strain CGA009 and rpa0990 mutant grown aerobically under dark, 750 nm and 700 nm light .......................................................................................... 43 Figure 3.1:  Absorption spectra of R. palustris wild type and mutant strains rpa3015- and rpa3016- grown at low-light intensity .................................... 52  Figure 3.2:  Cyclic heme is converted into linear biliverdin by heme oxygenase in the presence of O2 ........................................................................................ 54  Figure 3.3:  Absorption spectra of R. palustris strains grown under low light with different methods of manipulation of oxygen levels ................................... 57  Figure 3.4:  Sequences encoding heme oxygenase (HmuO) homologues in several bph operons .................................................................................................. 59  Figure 3.5:  Sequence alignment of heme oxygenase peptides ....................................... 62  Figure 3.6:  Transformation of ALA to biliverdin including two hypothetical pathways of making cofactor of Bphs in the absence of O2 ........................ 64  Figure 4.1:  Arrangement of the gene located around rpa1490 and rpa1491 in R. palustris CGA009 ........................................................................................ 66  Figure 4.2:  Promoterless lacZ gene was inserted into recombinant plasmid pJRD215::bph-puc downstream of the bi-directional promoter region ....... 68  Figure 4.3: Representations of the puc-bph::lacZ fusions ............................................... 70 Figure 4.4: β-Galactosidase specific activities ................................................................ 72  x  ABBREVIATIONS Bchl  Bacteriochlorophylls  Bph  Bacteriophytochrome  BV  Biliverdin  cfu  colony forming unit  Cph  cyanobacteria phytochrome  Fphs  Fungi phytochromes  FR  far-red  HO  heme oxygenase  IPTG  isopropyl-β-D-1-thiogalactopyranoside  kb  kilobase pair  kDa  kilodalton r  Km (Km )  kanamycin (kanamycin resistance)  LH  light harvesting  ORF  open reading frame  PCR  polymerase chain reaction  Pfr  far-red absorbing form  Phy  phytochrome  PM  a defined mineral medium (Kim et al. 1991)  Pr  red absorbing form  PU  photosynthetic unit  R  red  RC  reaction center  RT  room temperature  xi  ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor Prof. Dr. Thomas Beatty for providing me with this project as well as his guidance and support throughout the course of the thesis. A special thanks to Dr. Stephan Braatsch who guided me in the early stage of my M.Sc. project, for answering my countless questions and trying to make me a better scientist. Thank Kia Duthie for contributions to the early stage of this work. I would like to thank my committee members Dr. Rosemary Redfield and Dr. George Spigeman for all your valuable opinions and suggestions on the project. Thanks to Hai Xu from Davies Lab for suggestions in cloning techniques, to Christine Florizone and Katherine Yam from Eltis Lab for demonstrating the use of glove box, to Stacey Tom-Yew from Murphy Lab for teaching me colony blot and sharing the precious antibody solution, to Jie Liu from Eltis Lab and Helena Wang, Karen Lu from Davies Lab for being the big sisters not only in the lab but also in the spiritual life, to Du Xin and Nelson Chan from Bromme Lab for your all moral supports, to Joe for your contribution to the project. Last but not least, thanks to Paul Jaschke, Molly Leung, Jeanette Beatty, Kristopher Shelswell and Sarah Florizone of the Beatty Lab who are just great and made my time in the lab so enjoyable. Thanks to the Department of Microbiology and Immunology for giving me the opportunity to experience the field of microbiology and biochemistry research. Thanks to Dr. Michael Gold for coordinating and organizing MICB506 and MICB530, as well as helping me to improve my presentation skills. Thanks to Iris Liu, for not only carefully proof reading my thesis draft, but also supporting me by prayers through the hard times. Thanks to my family who have supported me to pursue my goals. It would not have been possible to reach it so far without their support.  xii  Chapter 1 – THESIS INTRODUCTION Light, the region of the electromagnetic spectrum that spans wavelengths detected by the human eye, affects many biological processes. Light-stimulated biological processes include photosynthesis (harvesting and use of light energy to drive conversion of CO2 to metabolic intermediates), as well as a variety of signal transduction pathways. This M.Sc. thesis investigates the function of genes predicted to encode signal transduction proteins, bacteriophytochromes.  1.1. Overview of phytochromes The phytochrome (Phy) superfamily is one of the three major classes of photoreceptors found in nature to mediate light-induced signal transduction, with the other two being cryptochromes and phototropins (Van der Horst et al. 2004). Phytochrome, first discovered in plant tissue (Butler et al. 1959), is known to be capable of responding to red/far-red light (625– 740 nm and 700-800 nm respectively) through photointerconversion between two states, the red absorbing form Pr and the far-red absorbing form Pfr, when bound to the chromophore (Fig 1.1). The cycle of photoconversion is comprised of a series of intermediate states. Phytochromes in plants respond to different light intensities by measuring the ratio of red/far-red light, stimulate light signaling pathways, and regulate many developmental processes such as germination, flowering and shade avoidance (Lamparter 2004). Recently the presence of phytochrome-like proteins has also been demonstrated in cyanobacteria (Cphs), fungi (Fphs) and both photosynthetic and non-photosynthetic bacteria (Bphs), and so phytochromes are not limited to photosynthetic organisms (Lamparter et al. 1997; Smith 2000).  1  Figure 1.1 The photocyle of phytochrome  between Pr and Pfr forms. Lumi-R and lumiF are the primary products upon illumination of Pr with red light or Pfr with far-red light. Several “meta” intermediates were also discovered under low temperatures (Furuya et al. 1994).  1.2 The structure of phytochromes and chromophores Modern technologies enable researchers to compare phytochromes at the predicted domain structural level. Plant Phys, Cphs, and most Bphs appear to share a domain structure consisting of an N-terminal photosensory module and a C-terminal regulatory region (Fig 1.2). The N-terminal module acts as a light-sensing region containing the chromophorebinding site. Several conserved domains were identified within this region, such as PAS, GAF and PHY. The PAS domain is named after three proteins that it occurs in: Per- period circadian protein, Arnt-Ah receptor nuclear translocator protein and Sim- single-minded protein. This domain is present in many proteins that regulate light and O2 signaling pathways through process where it is used as a signal sensor domain, as well as being involved in protein-protein interactions (Gomelsky et al. 1995; Aravind et al. 1997). The GAF motif, named after homologues detected in cGMP-specific and stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA, is also found to be involved in signaling pathways such as cGMP-binding. Deletion of GAF or PAS domains has resulted in unstable or misfolded proteins (Rockwell et al. 2006). A major breakthrough in phytochrome structure was the crystal structure of the PAS-GAF chromophore-binding domain (CBD) of DrBphP from Deinococcus radiodurans (Wagner et al. 2005). The cysteine residue within the GAF domain is highly conserved among various organisms and makes a linkage with the linear tetrapyrrole (a bilin, 2  often biliverdin (BV)) chromophore vinyl group via a thioether bond. The phytochrome (PHY) domain is unique to phytochromes. It has a low similarity to GAF, and is believed to regulate the photoconversion between Pr and Pfr forms. Deletion of the PHY domain resulted in failure of bilin binding by Phys Cphs and BphPs, which also exhibited reduced photoconversion efficiency (Karniol et al. 2005). The C-terminal region is known as a regulatory region, often comprised of a dimerization domain and a kinase (ATPase) domain (Hughes et al. 1999). The conserved histidine residue located on the dimerization domain is proposed to be where the autophosphorylation takes place. The structure of N-terminal PAS, GAF and PHY domains attached to C-terminal histidine-kinase domain typifies all classes of phytochromes.  Figure 1.2 Domain structure of typical phytochromes. Phytochromes from different organisms share the same N-terminal photosensory core consisting of PAS, GAF and Phy domains. The chromophore-binding site is located in the GAF domain and is highly conserved (Aravind et al. 1997). The PAS-GAF domain is thus known as chromophore binding domain (CBD). Different classes of phytochromes also share a common two-component histidine kinase (HK) domain in the C-terminal region. A dimerization domain is found within the HK domain which contains the histidine to be phosphorylated. Phys have additional two PAS domains that are important for light-mediated signaling (Chen et al. 2005) , whereas Fphs have a C-terminal regulator receiver (RR) domain.There are variations on these basic themes.  It was proposed that the photoconversion of holophytochome is caused by a Z-E isomerization about the C15-C16 double bond of the bilin, followed by proton transfers and  3  conformational changes of the protein matrix, as the apoprotein does not exhibit a typical phytochrome absorption spectrum (Furuya et al. 1994). Different subfamilies of phytochromes utilize various types of bilins: Plant Phys incorporate phytochromobilin (PΦB), Cph1s and Cph2s incorporate phycocyanobilin (PCB), while BphPs and Fphs use BV as chromophore (Rockwell et al. 2006). All subclasses of phytochromes have shown bilin lyase activity as they can all self-ligate to bilin in vitro in the absence of any cofactors. Again, this ligation property functions through GAF domain, whereas PAS and PHY domains are involved in the spectroscopic properties of the bound bilins (Wu et al. 2000). Phytochromes are thus photoconvertable photoreceptors that incorporate and utilize chromophore bilins.  1.3 Bacteriophytochromes and photosynthesis Bacteriophytochromes (Bphs) are believed to play various roles in bacteria such as in phototaxis (Yoshihara et al. 2004), and regulation of carotenoid, light-harvesting complex and photosystem synthesis (Giraud et al. 2002; Giraud et al. 2005). Bphs are homodimers (Evans et al. 2006) that share similar domain structure with plant phytochromes with an N-terminal chromophore binding domain and a C-terminal domain involved in autophosphorylation and dimerization. Biliverdin (BV), an open-chain tetrapyrrole, is the chromophore for proteobacteria phytochromes, compared to 3E-phytochromobilin (PΦB) and 3Z-phycocyanobilin (PCB) used by plant and cyanobacteria phytochromes respectively (Giraud et al. 2005). BV is known as a green pigment that is synthesized from heme by heme oxygenase (HO). Upon illumination with red or far-red light, the chromophore undergoes a cis-trans configuration change that is thought to lead to a conformational change in the holoprotein between the Pr form and the Pfr form (Van der Horst et al. 2004). The ratio of the Pr and Pfr forms is determined by light environment and a thermal process known as dark reversion (Rockwell et al. 2006). The phytochrome is slowly 4  converted to its dark stable form in the absence of light. Being more active in only one of these forms, Bphs act as autokinase and phosphodonor to downstream phosphotransfer signal transduction pathways for gene expression regulation in response to light changes. Two Bphs found in photosynthetic bacteria Bradyrhzobium and Rhodopseudomonas palustris are thought to regulate the synthesis of the photosynthetic unit (PU) (Giraud et al. 2005). This PU in most non-sulfur purple bacteria is comprised of at least two types light harvesting (LH) complexes and a photochemical reaction center (RC). The pigments chlorophyll a and carotenoid are assembled in the LH complexes that are responsible for absorption of light from the environment (Fig 1.3). LH complexes channel the energy from photons captured by the pigment molecules and transfer it to the RC, where electrons are therefore excited to higher energy levels to initiate a chain of electron transfer reactions. Electron transfer is coupled to protein translocation to create a chemiosmotic gradient. Type 1 light harvesting complexes (LH1) are relatively large-diameter rings in close contact with RC, whereas type 2 light harvesting complexes (LH2) are smaller rings arranged around the LH1 complexes (Fig 1.3). Different LH complexes are characterized by different absorption spectra. LH2 absorbs nearinfrared light at 800 and 850 nm whereas LH1 absorbs at about 870 nm (Fig 1.4). Under high light conditions, the ratio of LH2 complexes to RC/LH1 is low, whereas more LH2 complexes are made in response to low-light intensity illumination (de Ruijter et al. 2004). Under low-light condition, novel LH complexes are expressed to replace LH2: B800-820 (LH3) in Rhodopseudomonas acidophila strain 7050 and B800 (LH4) in R. palustris (Gardiner 1993; Hartigan et al. 2002). In R. palustris there are five gene pairs (pucBAa to pucBAe) encoding α and β peptides of a family of LH2 complexes and an LH4 complex (Tadros et al. 1989)(Fig 1.4). LH2 and LH4 have similar structure with repeated subunits (heterodimer of αβ peptides) forming a ring-shaped multimer. However they differ in the assembly patterns and the alignment of the 5  pigments, thus leading to different interactions between the pigment rings (Hartigan et al. 2002). It is thought that in changing light intensity conditions, Bphs help photobacteria obtain better light-trapping efficiency by regulating the synthesis of photosystem.  Figure 1.3 Structural arrangement of a reaction center (RC) with its surrounding light harvesting complexes. The light harvesting complex 1 (LH1) immediately surrounds the reaction center, while a second type of light harvesting complex (LH2) channels energy to the reaction center through LH1. Bchl, bacteriochlorophylls.  6  Figure 1.4 Mathematical deconvolution of LH absorption spectra of R. palustris ppsR2- cells grown under high-aeration conditions (Braatsch et al. 2006). The simulated absorption spectrum (dark solid line) is composed of three types of LH complexes: LH1 (blue line) absorbing at 880 nm, LH2 (red line) absorbing at 808 and 862 nm, LH4 (green line) absorbing at 808 nm. This spectrum is similar to the wild type grown under high light (low-aeration). Under low light conditions, the LH2 absorption decreases and the LH4 absorption increases.  Figure 1.5 Genomic map of R. palustris showing bph0 through to bph5 . The locations of LH2/4 operons are labeled as pucBA. Bph0 = rpa0122; bph1 = rpa0990.  7  1.4 Bacteriophytochromes in Rhodopseudomonas palustris Recent genomic studies of R. palustris strain CGA009 have revealed the unexpected presence of six Bph open reading frames (ORFs) (bph0 to bph5, Fig 1.5), compared to only one or two bphs in other bacterial genomes (Larimer et al. 2004). One of these Bphs (Bph3) is unusual: it has a signal sensor and transducer PAS domain at the C-terminus instead of the Nterminus. A homologue of Bph3 in the closely related species Bradyrhzobium is organized in a similar pattern. It is interesting to note that Bph3 lacks a kinase domain. However, it still controls the gene expression of photosynthetic gene clusters in R. palustris strain 2.1.6 (Giraud et al. 2002). Moreover, the Bradyrhzobium bph gene is downstream of, and presumably in the same operon as the PpsR2 transcriptional factor. This transcriptional factor represses the expression of Bchl, carotenoid and LH2 complexes genes in the absence of FR light (Braatsch et al. 2006). It has been suggested that FR light activates Bph3 to interact with PpsR via PAS domains and releases the repression of photosynthesis genes (Giraud et al. 2002). However in R. palustris strain CGA009, Bph3 (RPA1537) is one G residue frameshifted compared to the ones in strain CEA001 and Bradyrhzobium ORS278, thus was annotated as a pseudogene, and PpsR2 was proposed to be dysfunctional because of a base pair change compared to Bradyrhzobium (Giraud et al. 2004). In a recent study, the Bph3 in strain CGA009 was repaired, and it was demonstrated that PpsR2 is needed for 750 nm stimulation. Thus the model was proposed that in R. palustris strain CGA009, PpsR1 functions as a repressor for photosynthesis gene expression, whereas PpsR2 is not only a O2-responsive repressor but also a mediator of light signal transduction in concert with Bph3 (Braatsch et al. 2007). The five other Bphs in R. palustris share a similar domain arrangement with a Cterminal histidine kinase domain, except Bph1 (RPA0990), which has an additional CheYhomologous receiver domain predicted by homology modeling. Among these five ORFs, bph2 8  (rpa1490) is immediately downstream of the pucBAe genes, which encode one of the LH2 complexes, whereas a pair of genes bph4 (rpa3015) and bph5 (rpa3016) are located upstream of the pucBAd genes that encode the LH4 complex (Fig 1.5). Bph3 from R. palustris strain 2.1.6, Bph4 and Bph5 from CGA009 are capable of binding to chromophores to produce an active holoprotein that can be detected by absorption spectroscopy, yet they reveal different dark stable states: Pfr for Bph3 and Pr for Bph4 (Evans et al. 2005). Although Bph2 lacks the cysteine residue in the GAF domain that forms covalent linkage with chromophore biliverdin, mutating the bph2 gene results in changes to the LH absorption profile (Beatty lab, unpublished data, see Chapter 3). Thus Bph2 must also play a role in photosystem synthesis. In all, Bph2 through to Bph5 are all capable of regulating gene expression of photosynthesis system in response to illumination changes. The genes encoding the remaining two Bphs (RPA0122 and RPA0990) with functions currently unknown are located further away from the photosynthetic gene clusters (Fig 1.5). Thus it would be interesting to know whether these two putative Bph proteins also function as photoreceptors to regulate gene expression despite their unusual locations. Sequence alignments indicate that both of the Bph-like proteins encoded by these two ORFs contain the conserved cysteine residue that is responsible for making a covalent bond to the chromophore. My hypothesis is that in R. palustris strain CGA009 one or both of these Bph-like genes encode proteins that are capable of binding a chromophore and modulating some aspect of the cell’s physiology. Homology modeling has shown that RPA0990 contains a C-terminal region similar to a Che-Y receiver domain whereas RPA0122 contains only a histidine kinase domain. Another hypothesis is that these two Bph-like proteins work in concert via a two-component system pathway upon response to ambient light and regulate photosystem gene expression. An additional hypothesis is based on a previous study in Synechocystis sp. It was shown that the 9  TaxD1 protein contains domains that resemble both Bph and the methyl-accepting chemoreceptor proteins which mediate positive phototaxis for optimal light capture and efficient photosynthesis (Ng et al. 2003). Thus the presence of a CheY-receiver domain in RPA0990 may also indicate its potential function in phototaxis via phosphorelay pathways. R. palustris has developed complicated biochemical mechanisms including a network of phytochromes in order to adapt to different light environmental conditions. It has been a mystery: why are so many Bphs present in R. palustris? This thesis investigates a part of this story by studying the biochemical as well as the regulatory properties of the two cryptic Bphs RPA0122 and RPA0990.  10  Chapter 2 - TWO PUTATIVE PHYTOCHROMES ENCODED BY RPA0122 AND RPA0990  2.1 Introduction Bacteriophytochromes (Bphs) are in a family of photoreceptors that sense light changes in order to regulate a set of biological systems in both photosynthetic and non-photosynthetic bacteria. They absorb red and far-red light and perform photoconversion between two forms (red absorbing Pr form and far-red absorbing Pfr form) by binding to linear tetrapyrrole chromophores. Most bacteria have only one or two Bphs, but the annotation of the genome of R. palustris has revealed six putative Bph open reading frames (ORFs) (Larimer et al. 2004). Four of these bph genes, rpa1537, rpa1490, rpa3015 and rpa3016, are close to photosynthetic gene clusters (Fig 1.5). The rpa1537 gene is located close to the pufBALM operon encoding reaction center (RC) and LH1 complexes. Its product has a dual role: it regulates both photosynthetic genes and Krebs cycle genes (Kojadinovic et al. 2008). The rpa1490 gene is located near pucBAe genes coding for LH2 complexes and the pucC gene that is involved in LH2 biogenesis. The product RPA1490 functions as an O2 sensor and regulates the expression of LH2 complexes (Vuillet et al. 2007). The rpa3015 and rpa3016 genes are adjacent and located close to pucBAd genes encoding the LH4 complex. These two Bphs phosphorylate a common response regulator RPA3017 upon autophosphorylation in their Pr forms, and were proposed to regulate the synthesis of LH4 complexes (Giraud et al. 2005). The remaining two Bphs (RPA0122 and RPA0990) have unknown functions and are located further away from the photosynthetic genes (Fig 1.5). However both of them are composed of typical Bph domain organization (Fig 2.1). Interestingly, RPA0990 contains a  11  response regulator domain homologous to CheY and might be involved in phototaxis. Sequence alignments indicate that both of the Bph-like proteins encoded by these two ORFs contain the conserved Cys residue that is responsible for making the covalent bond to the chromophore (Fig 2.2A). Also RPA0122 might use a Tyr residue instead of His to form an H-bond with a pyrrole nitrogen of bilin (Fig 2.2B). However, RPA0122 lacks the conserved His residue that is proposed as the phosphorylation site in the D. radiodurans Bph (DRA0050), thus it may have a reduced or no kinase activity (Fig 2.2C). My hypothesis is that one or both of these Bph-like genes encode proteins that are capable of binding to a chromophore and modulating some aspect of the cell’s physiology in R. palustris strain CGA009. My results will show that purified HisRPA0990 is capable of photoconversion and hence binds a bilin chromophore, and both of RPA0122 and RPA0990 may have a small effect on LH1 based on the absorption spectrum of rpa0122 and rpa0990 mutants.  Figure 2.1 Domain structure of putative bacteriophytochromes RPA0122 and RPA0990 of R. palustris. PAS: Per/Arnt/Sim repeats. GAF: chromophore binding domain. PHY: GAF related domain of eukaryotic phytochroms. HK: His-kinase domain. RR: response regulator domain.  Figure 2.2 Amino-acid sequence alignment of the region surrounding the chromophore-binding site for Bphs from R. palustris and D. radiodurans. The crystal structure of D. radiodurans Bph has revealed that: (A) Cys24 is the chromophore attachement site; (B) His260 forms H-bonds with pyrrole nitrogens of bilin; (C) His532 is a likely phosphorylation site (Wagner et al. 2005). Generated by ClustalW2 EMBL-EBI (http://www.ebi.ac.uk/Tools/clustalw2/index.html).  12  2.2 Materials and methods  2.2.1 Bacterial strains, growth conditions, and plasmids. The bacterial strains used are described in Table 2.1. The E. coli strains used for cloning and subcloning were DH5α (Table 2.1) and DH10B (Table 2.1). Strain S17-1 was used to conjugate plasmids into R. palustris. The E. coli strains were grown in LB medium (Sambrook J. 1989) supplemented when appropriate with kanamycin sulfate 50 µg/ml. The R. palustris strains were grown in PM medium (Berne et al. 2005) supplemented when appropriate with kanamycin sulfate, 100 µg/ml. The turbidity of cultures was monitored by measuring light scattering with a KlettSummerson photometer (filter #66, red); 100 Klett units represents approximately 109 colony forming units (cfu) per ml.  13  Table 2.1: Bacterial strains and plasmids.  Relevant characteristics Strains R.palustris CGA009 rpa0122 rpa0990  wild type CGA009 rpa0122::kixx, Kmr CGA009 rpa0990::kixx, Kmr CGA009 ppsR1/2::miniTn-5, Kmr  Source or reference (Larimer et al. 2004) This study This study (Braatsch et al. 2007)  ppsR1-2E.coli DH5α DH10B Rosetta(DE3)pLysS BL21(DE3)pLysS S17-1 Plasmids pET-28a(+) pJRD215 pHRP309  Expression vector with 6xHis at N-ter; Kmr (Studier et al. 1986) Broad-host-range cloning vector; Kmr (Davison et al. 1987) Broad-host-range lacZ transcriptional vector (Parales et al. 1993)  pJPUC  pJRD215 containing rpa1490-rpa1491 bidirectional promoter (BamH I to Xbal I) in puc orientation; Kmr  This study  pJBPH  pJRD215 containing rpa1490-rpa1491 bidirectional promoter (BamH I to Xbal I) in bph orientation; Kmr  This study  pJPUC::lacZ  pJPUC containing 3.2 kb lacZ fragment (Hind III to Xbal I); Kmr  This study  pJBPH::lacZ  pJBPH containing 3.2 kb lacZ fragment (Hind III to Xbal I); Kmr ); Kmr  This study  pJPUC::rpa0990  pJPUC containing 2.6 kb rpa0990 fragment (Hind III to Xbal I); Kmr  This study  pJBPH:rpa0122  pJBPH containing 2.4 kb rpa0122 fragment (Hind III to Xbal I); Kmr  This study  strain used for cloning strain used for cloning Cmr strain used for overexpression Cmr strain used for overexpression Tra+ strain used from plasmid mobilization  NEB NEB Novagen Novagen (Simon et al. 1983)  14  2.2.2 Construction and confirmation of rpa0122 and rpa0990 knockouts Mutant strains rpa0122 and rpa0990 were constructed by Kia Duthie as summarized below: R. palustris rpa0122 and rpa0990 mutant strains were created by double-crossover recombination of KIXX disrupted (Kmr) genes into the chromosomal rpa0122 and rpa0990 (Fig 2.3) loci. To create these mutant strains, plasmid pBlueScribe (pBS) was used to engineer the KIXX (Kmr) element from the plasmid pUC4KIXX (Barany 1985) flanked by DNA sequences containing 5’ and 3’ segments of the rpa0122 and rpa0990 genes. The bph-flanking sequences were amplified with primer pairs (122s/as and 990r/s, Table 2.2) and Pfx DNA polymerase (Invitrogen, UBC). The PCR products were digested with EcoR V (for rpa0990) or Hind III (for rpa0122), and Xbal I. The bph-flanking regions and the KIXX element were cloned into pBS such that unique EcoR V and Xbal I sites were located between the KIXX element and the upstream and downstream flanking region respectively. The EcoR V (Hind III for rpa0122) /Xbal I fragments containing the KIXX insertion were ligated into vector pJQ 200-SK. The suicide plasmid pJQ 200-SK was used to deliver interrupted rpa0122 and rpa0990 genes to the chromosome of CGA009 to yield strains rpa0122 and rpa0990. I used primer SB3/kixxA and SB4/kixxB (Table 2.2) for rpa0122 and SB5/kixxB and kixx2A/SB6A for rpa0990 disruption respectively to confirm the recombinations by PCR (see Result 2.3.1, Fig 2.6).  15  Figure 2.3 Map of rpa0990 showing the knockout scheme. The rpa0990 gene was PCR amplified with introducing an EcoR V or Xbal I site at each end and subcloned into vector pBS. KIXX cartridge replaced region between EcoR I sites.  16  Table 2.2: Primers.  Primer For knockouts 0122s 0122as 990r 990s For knockouts confirmation kixxA kixxB SB3 SB4 SB5 kixx2A SB6A For cloning 122ex-up 122ex-down 990ex-up 990ex-down bph_puc_E bph_puc_H bph_puc_F bph_puc_G For sequencing T7 T7-term pJRD215_seqF pJRD215_seqB FG_seqM1  Relevant characteristics  Source or reference  5'-GCAATCACCCTCGCGCTTCGT-3' 5'-GCCACGGGTGACCTTGAGCATC-3' 5'-TCGAACAGCAGATCTAGAGCGCCC-3' 5'-GCTGCCGACGTGATATCCCGG-3'  this study this study this study this study  5'-TGAAAGGTTGGGCTTCGGAATCG-3' 5'-GCCCTTGCGCCCTGAGTGC-3' 5'-CGATCTGTTTCCGACCGCCCC-3' 5'-CCTTCATCAGTCCAGCGGCCG-3' 5'-TCTCCGGCGGTGAGCAGCAG-3' 5'-GCCTGAGGTCACTGCGTGGATGG-3' 5'-GAGTTCGTGTATGCGATGTTCCCG-3'  this study this study this study this study this study this study this study  5'-CGCATATGGATGAGGCCGACAGC-3' 5'-ATTAAGCTTCTACGCATCGTGCCGTTCCTC-3' 5'-TACATATGCCGCGTAAGGTCGATCTCAC-3' 5'-ATTAAGCTTTCATTCGCGATCGTCGAGCATTG-'3 5'-GGCGGATCCCCTGAGCTCGGCAGAATTGT-3' 5'-GGCTCTAGAGATTCCGCGATCGTCAGACC-3' 5'-GGCTCTAGACCTGAGCTCGGCAGAATTGT-3' 5'-GGCGGATCCGATTCCGCGATCGTCAGACC-3'  this study this study this study this study this study this study this study this study  5'-TAA TAC GAC TCA CTA TAG GG-3' 5'-GCT AGT TAT TGC TCA GCG G-3' 5'-GGCACGTGCGTGGAGGCCATCAA-3' 5'-GGCTTCATACACGGTGCCTGACT-3' 5'-GGCGCTCAGGTCTAGATCCGATT-3'  (Studier et al. 1986) this study this study this study  17  2.2.3 Phenotype of RPA0122 and RPA0990 knockouts 2.2.3.1 Growth curve The R. palustris mutants and wild type strains were grown in the PM defined medium (Kim et al. 1991) with 10 mM succinate as the carbon source and 100 µg/ml kanamycin where indicated. Aerobic cultures of 10 ml were grown in test tubes (22 ml) at 30° C without illumination and rotated on roller drum (New Brunswick scientific) at 56 rpm. Semiaerobic cultures were grown at 30° C without illumination in Erlenmeyer flasks filled to 80% of nominal capacity and shaken at 150 rpm. The photosynthetic cultures were incubated in completely filled, sealed vessels or tubes at 30° C in an aquarium filled with water and illuminated with halogen lamps (Capsylite, Sylvania) at an intensity of ~10 µE m-2 s-1 for low-light and ~300 µE m-2 s-1 for high-light conditions. Light intensity was measured with a photometer equipped with a LI190SB quantum sensor (LI-COR Inc.). Growth was measured in a Klett photometer (100 Klett units equal ~109 cfu/ml). 2.2.3.2 Phototaxis Both aqueous and non-aqueous phototaxis experiments were performed for rpa0122 and rpa0990 mutants. Aqueous phototaxis experiments were done by Jean Huang in the Harwood lab at the University of Washington. Cultures were resuspended in PM medium lacking a carbon source (see 2.3.6.2, Fig 2.14). Light was presented on one side of the bottle, and over several days the movement of cells toward the side of the bottle closest to the light was observed by eye (Yoshihara et al. 2004). Non-aqueous (agar plate) phototaxis experiments were performed for both wild type CGA009 and bph mutant strains including rpa0122-, rpa0990-, rpa1490-, rpa3015- and rpa3016-.  18  Cells grown photosynthetically in PM medium were pelleted at both mid-log and late-log phases for comparison. PM or YPS agar plates containing Km if necessary were stabbed with a toothpick dipped into a cell pellet as described previously (Shelswell et al. 2005). Both aerobic and anaerobic (photosynthetic) growth conditions were tested. The photosynthetic cultures were incubated in a polycarbonate anaerobic jar at 30° C and illuminated with halogen lamps at high light or low light (see 2.2.3.1 for light intensity). The aerobic cultures were grown under similar condition but with the polycarbonate jar open to the air. Dark control plates were wrapped with aluminum foil. 2.2.3.3 Absorption spectra of cells grown under standard photosynthetic conditions Cultures of R. palustris cells at the mid-log phase of growth were centrifuged in microcentrifuge at 14 krpm for 2 min, and absorption spectroscopy of intact cells resuspended in 0.25 ml of PM medium plus 0.75 ml of 30% bovine serum albumin was performed, using a mixture (1:3) of PM medium and 30% BSA as the blank. Data were collected with a TIDAS II spectrophotometer (J& Analytische Mess- und Regeltechnik). Light-scattering at 650 nm was used to normalize the spectra, and the data from triplicate cultures were averaged as described previously (Braatsch et al. 2006). 2.2.3.4 Absorption spectra of cells grown under irradiation with specific wavelengths R. palustris CGA009 and RPA0990 mutant cultures (2 ml) were grown in PM medium at 30° C either in the dark or under continuous illumination at an intensity of 3~5 µE m-2 s-1 in stationary microtiter plates (Sarstedt) essentially as described (Braatsch et al. 2007). Far red light (750 ± 20 nm) was provided by light-emitting diodes (LED750-03AU; Roither Lasertechnik), whereas the red light was provided by a halogen lamp (NOVAFLEX Fiber Optic Illuminator, 19  WPI) filtered through a 700 ± 20 nm filter (Corion). Cultures with a starting OD650 = 0.05 underwent ~3 doublings during 72 hours of uninterrupted incubation before absorption spectroscopy of the intact cells was done as described in 2.2.3.3. 2.2.4 Construction of plasmids created for gene overexpression in E. coli The His6Bph fusion protein expression vectors (Fig 2.4) were constructed by inserting PCRamplified Bph ORFs from R. palustris chromosomal DNA into the vector pET-28a(+) (Table 2.2). The rpa0122 and rpa0990 sequences were amplified with primer pairs (122ex up/down and 990ex up/down), which introduced a Hind III or Nde I site at each end of the PCR product. Point mutations were unexpectedly generated during when Pfx DNA polymerase was used, possibly caused by the high GC content of gene fragment. This problem was overcome by using Pfx50 DNA polymerase (Invitrogen), which has a 50 x higher fidelity than Taq DNA polymerase. Recombinant plasmids were digested with Hind III and Nde I and the gel electrophoresis band pattern confirmed the correct pattern of cloning before sequencing. Sequencing was completed at University of British Columbia Nucleic Acid and Protein Service Unit (NAPS) using universal primers (T7 and T7-term, Table 2.2) from plasmid templates purified using QIAprep Miniprep kit (Qiagen). Verified expression constructs were heatshock transformed (Sambrook J. 1989) into E. coli Rosetta (DE3) (Table 2.1).  20  Figure 2.4 Cloning scheme to create His-tagged Bphs overexpressed in E. coli. PCR amplified fragments rpa0122 and rpa0990 from chromosomal DNA of R. palustris were cloned into pET28 vector by digestion with and ligation to Hind III and Nde I sites, with His6 tag at N-terminal.  2.2.5 Overexpression of RPA0122 and RPA0990 and purification from E. coli Rosetta DE3 2.2.5.1 Overexpression growth condition The expression strain E. coli Rosetta (DE3) carrying pET28::rpa0122/rpa0990 was grown at 37° C in LB medium (Km50) with shaking at 225 rpm until OD600 = ~0.5. Different expression temperatures and ITPG concentrations were tested and optimized to minimize the production of inclusion bodies. ITPG was added to a final concentration of 0.5 mM and induction proceeded at 20° C for 20 hours. Pelleted cells were frozen at -80° C.  21  2.2.5.2 Optimization of His-tag pull down Pelleted cells were resuspended in 50 mM Tris-HCl buffer (pH 8.0). Lysozyme (0.35 mg/ml), Triton X-100 (1% v/v), 0.01 mM MgCl2 and a few grains of DNAaseI were added. The samples were then incubated on ice for 2 hours, followed by disruption in a French Press. Large debris was removed by centrifugation in a JA-20 rotor (7,000 rpm, 10 min) and the supernatant was aspirated. The supernatant was centrifuged at 100 krpm in a TL-100 ultracentrifuge (Beckman) with a TLA 100.3 rotor for 15 min to pellet small debris. His-Bphs were purified using nickel-nitrilotriacetic agarose (Ni–NTA) beads (Qiagen, Valencia, CA). For batch purification, the 100 krpm supernatant (1 ml, ~3 mg of protein) was diluted 2-fold with dilution buffer [4 M NaCl, 20 mM imidazole, and 50 mM Tris-HCl (pH 8.0)]. For bead equilibration, Ni–NTA slurry (20 μL, in ethanol) and 200 μL binding buffer [2 M NaCl, 10 mM imidazole, 50 mM Tris-HCl (pH 8.0)] were added to the tube and incubated for 30 min at 4° C with gentle agitation. Following centrifugation at 5 krpm for 1 min in a microcentrifuge, the supernatant was discarded, and beads were washed three times with binding buffer as described in bead equilibration. The bound proteins were eluted with 3 x 20 μL of elution buffer [4 M NaCl, 300 mM imidazole, and 50 mM Tris-HCl (pH 8.0)]. A series of NaCl and imidazole concentrations in elution buffer were tested for optimal binding affinity. For purification with Ni–NTA affinity column chromatography, a Ni–NTA column was prepared using a 3 cc syringe (Becton Dickinson) with pyrex wool packed at the bottom, over which 2 ml of Ni-NTA was packed. The column was equilibrated with 5 ml binding buffer. The 100 krpm supernatant was applied to the Ni–NTA column and washed with 10 ml of wash buffer. Proteins were eluted with 3 ml of elution buffer. Samples were analyzed on 20% SDSpolyacrylamide gels (Novex) and visualized by staining with Coomassie blue (Bio-Rad).  22  2.2.6 Construction of plasmids for gene expression in R. palustris The bph1490-puc1491 (known as bph-puc hereafter) divergent promoter region was PCR-amplified from chromosomal DNA of R. palustris by primer pair bph_puc_E/H for puc promoter orientation and bph_puc_G/F for bph promoter orientation (Table 2.2). The promoter fragments were cloned into broad-host-range vector pJRD215 (Table 2.2) at BamH I and Xbal I sites (Fig 2.5). At first, it was difficult to generate a sufficient amount of low-copy plasmid pJRD215 for cloning. However the plasmids were eventually isolated by a large-scale preparation method: An E. coli DH5α carrying pJRD215 culture (250 ml) was grown to early stationary phase and harvested at 6 krpm in a JA-20 rotor. Cell pellets were resuspended in 10 ml P1 buffer (Qiagen QIAprep Miniprep kit), 10 ml P2 buffer (5 minutes incubation at RT), and 10 ml P3 buffer (30 minutes incubation on ice). The resuspension was centrifugated two times at 11 krpm in a JA-20 rotor for 30 minutes at 4° C. Isopropanol was added to the supernatant at a ratio of 2:3 and well mixed. The DNA pellet was collected by centrifugation at 10 krpm in a JA-20 rotor for 30 minutes at 4° C and dissolved in 0.5 ml elution buffer (Qiagen). The DNA solution was treated with phenol extraction, ethanol-precipitated, and washed with 70% ethanol to yield clean plasmid vector for cloning. Genes rpa0122 and rpa0990 from recombinant vector pET28::rpa0122/rpa0990 with His-tag coding sequence were cloned into pJRD215 at the Hind III and Xbal I sites, downstream of the bph-puc divergent promoter region (Fig 2.5). Sequencing was completed at the University of British Columbia Nucleic Acid and Protein Service Unit (NAPS) using universal primers (pJRD215_seqF, pJRD215_seqB and FG_seqM1, Table 2.2) from plasmid templates purified with the plasmid isolation method described above. The expression vectors were transformed into wild type R. palustris CGA009 by conjugation with E. coli S17-1. 23  Figure 2.5 Construction of plasmids created for gene expression in R. palustris. The PCR-amplified fragments containing the puc-bph divergent promoter region from chromosomal DNA of R. palustris were cloned into wide-host-range vector pJRD215 into BamH I and Xbal I sites. His-tagged RPA0990 and RPA0122 from recombinant vector pET28::rpa0122/rpa0990 were cloned into pJRD215 of Hind III and Xbal I sites, downstream of puc-bph divergent promoter region.  2.2.7 Colony lift for screening for RPA0990 complemented strain  The DNA probe against pJRD215 was digoxigenin-dUTP (DIG)-labeled using the DIG DNA labeling and detection kit (Roche), with Hind III-cut pJRD215 as template. The labeling efficiency was determined by comparing the direct detection between labeled probe and DIGlabeled control DNA. The labeled probe was shown to have the expected labeling efficiency.  24  The R. palustris RPA0990 knockout strain was conjugated with E. coli S-17 strain carrying pJPUC::rpa0990 (see Materials and methods 2.2.6 for a description of this plasmid). Approximately 300 colonies were screened on a conjugation plate (PM medium containing Km) by blotting onto a Biotrans nylon membrane. The membrane was then treated with denaturation solution (0.5 M NaOH, 1.5 M NaCl) for 15 minutes and neutralization solution (1.5 M NaCl, 1.0 M Tris-HCl, pH 7.4) for 15 minutes. The membrane was then washed with 2 x SSC for 10 min, and crosslinked by UV light at 150 mJ for 2 minutes to bind the DNA. The membrane was then soaked in proteinase K (50 mg/ml stock diluted 1:10 in 2 x SSC) at 37° C for 1 hour, and the debris were then removed by firmly pressing damp Whatman 3MM paper onto the membrane disc. The DIG-label probe was hybridized by incubation with membrane filters (blots) overnight at 50° C in 10 ml DIG Easy Hyb buffer. The blot was then washed twice for 5 minutes with 100 ml 2 x wash solution (2 x SSC, 0.1% SDS) at RT followed by two 15 minute washes at 68° C in 250 ml of 0.5 x wash solution (0.5 x SSC, 0.1% SDS). The blots were then incubated with the manufacturer’s antibody as described in Roche instruction manual for DIG High Prime DNA labeling and detection starter kit II. The membranes were then exposed to Versa Image for signal detection. Multiple exposures were taken over 48 hours. 2.2.8 Evaluation of the stability of plasmid pJRD215 in the rpa0990 mutant strain The rpa0990 mutant strain is resistant to Km (same marker as on the plasmid pJRD215) because of KIXX in the chromosomal rpa0990 gene as a result of the knockout construction. Therefore it was not possible to select for maintenance of Kmr. The plasmid pJPUC::lacZ was transformed into the rpa0990 mutant strain by conjugation with E. coli S17-1. Cultures were grown in liquid PM medium as described previously (Berne et al. 2005) containing the antibiotic  25  Kanamycin. After 3~4 doublings, cultures at late log phase were plated on PM kan30 Xgal40 agar, and colonies were screened for blue (lacZ+) phenotype. 2.2.9 Purification of His-tagged RPA0990 2.2.9.1 Expression growth condition The R. palustris cultures carrying the expression vectors were grown at 30° C without illumination in 2 L Erlenmeyer flasks filled to 80% of nominal capacity and shaken at 150 rpm. Typically, one preparation started with 15 flasks, about 24 L of culture. Cells were harvested at mid/late-stationary phase and stored at -80° C. 2.2.9.2 Purification of tagged RPA0990 Cells pelleted from a 24 L of culture in CEPA Z41 High Speed centrifuge (at the Single Cell Fermenter Suite at Life Sciences Center, UBC) were collected. Pelleted cells were resuspended in 30 ml of buffer as described in 2.2.5.2, incubated on ice for 2 hours, and disrupted with a 50 ml Dounce homogenizer followed by a French press. Crude lysate from French Press was centrifuged at 7 krpm in JA-20 rotor for 10 min, and the supernatant was centrifuged at 100 krpm for 30 min in TLA 100.3 rotor in an ultracentrifuge (Beckman). Supernatant was loaded on a Ni-NTA column (prepared as described in 2.2.5.2), followed by washed with 4 ml each of buffer 1 (20 mM NaH2PO4 pH = 8.0); buffer 2 (20 mM NaH2PO4, 300 mM NaCl, pH = 6.3); buffer 3 (20 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH = 8.0); buffer 4 (20 mM NaH2PO4, 20 mM imidazole, pH = 8.0). His-RPA0990 was eluted with 2 ml of 20 mM NaH2PO4 and 150 mM imidazole, pH = 8.0 followed by 2 ml of 20 mM NaH2PO4 and 300 mM imidazole, pH = 8.0. Ammonium  sulphate  precipitation  was  performed  using  stepwise  increasing  concentrations to determine conditions to use to increase the purity of RPA0990. Ammonium sulphate (0.1 g) was added to the 1 ml final elution (the second batch of elution with 150 mM 26  imidazole elution buffer) from Ni-NTA column and mixed well by vortex. The precipitated protein (10% ammonium sulphate) was recovered by centrifugation at 10 krpm in a microcentrifuge (Sigma) and resuspended in buffer A (50 mM Tris-HCl, pH = 8.0). The ammonium sulphate concentration was increased to 20% and 30% by adding portions of 0.1 g ammonium sulphate to the supernatant as above, and precipitated proteins were recovered. Samples were analyzed on SDS-polyacrylamide gels (see 2.2.5.2). 2.2.10 Spectroscopic characterization of His-tagged RPA0990 Measurements were made in a 700 µl volume cuvette using a Hitachi U3010-S spectrophotometer. Absorption spectra of RPA0990 were measured after irradiation with light at 700 nm (R) and 750 nm (FR) provided by filters placed in-line using custom filter holders, and filter optic cables to connect the light source to a cuvette containing the protein samples. The filters (Corion) have a half-height/half-maximal bandwidth of 20 nm. Other measurements were made after a prolonged incubation in dark.  2.3 Results 2.3.1 Confirmation of rpa0122 and rpa0990 knockouts I confirmed chromosomal knockouts recombinations by PCR with primer pairs SB5/kixxB and SB6A/kixx2A for rpa0990 (Fig 2.6A) and SB3/kixxA and SB4/kixxB for rpa0122 (Fig 2.6B) disruption respectively. The mutants were first screened against Kmr (the marker is located on the KIXX cartridge). The primers were designed in the way that they amplified in-between the KIXX catridge fragment and the neighboring ORFs. This shows that the chromosomal rpa0990 and rpa0122 genes were replaced by the KIXX-disrupted mutant. I conclude that the KIXX cartridge was inserted into chromosomal rpa0990 and rpa0122 genes by double-crossover recombinations (Fig 2.6). 27  Figure 2.6 Confirmation of rpa0990 (A) and rpa0122 (B) knockouts showing the knockout scheme and the agarose gels (1%) with PCR products.  28  2.3.2 Overexpression and purification of His-RPA0122 and His-RPA0990 in E. coli Both rpa0122 and rpa0990, which encode Bph-like proteins, were successfully amplified from R. palustris CGA009 chromosomal DNA and cloned into pET28a(+) expression vector with 6 x His tag at the N-terminus (see Materials and methods 2.2.4 and Fig 2.7A). DNA polymerase Pfx50 was used instead of Pfx, because Pfx generated point mutations during PCR.  Figure 2.7 Agarose gel (1%) showing the Hind III and Nde I digestion products of recombinant plasmids (pET28::rpa0122 and pET28::rpa0990) with comparing to empty vector (pET28) and PCR products (PCR122 and PCR990) to confirm successful cloning. The clones were then confirmed by DNA sequencing (data not shown).  His-tagged Bphs were initially expressed by IPTG induction at 37° C (Fig 2.8A). Improved conditions for overexpression were determined by loading whole cells and fractions of cell lysates on SDS PAGE gels (see Materials and methods 2.2.5.1). Growing in LB medium at 37° C or RT, induced with 1 mM or 0.5 mM IPTG, respectively, yielded high expression of the tagged proteins, but they appeared to be in inclusion bodies because most of the proteins were found in the pellet after centrifugation of cell lysates (Fig 2.8B and C). With a reduction of both temperature and IPTG concentration (16° C and 0.02 mM IPTG) a low expression level of  29  tagged protein was observed (Fig 2.8D). Thus an expression temperature must be chosen between RT (high expression with most of RPA0990 expressed in inclusion bodies) and 16° C (very low expression) in order to get maximum expression of soluble tagged protein. The expression conditions were eventually optimized to 20° C and 0.5 mM IPTG for 20 hours for maximum yield of soluble His-tagged protein (data not shown).  Figure 2.8 SDS PAGE showing the optimization of overexpression conditions in E. coli. (A) Tagged RPA0990 induction by 1 mM IPTG at 37° C in E. coli Rosetta DE3 strain, gel loaded with solubilized whole cells. (B) Most of tagged RPA0990 (expected size = 91.7 kDa) was expressed in inclusion body when induced at condition in A ([IPTG] = 1 mM, 37° C, t = 19 hr). W = whole cell; S = supernatant after centrifugation (7 krpm for 10 min in JA-20 rotor) of crude lysate; P = pellet after centrifugation of crude lysate; “-” = no IPTG control; “+” = 0.1 mM IPTG induction. (C) Most of tagged RPA0122 (expected size = 82.9 kDa) appeared to be expressed in inclusion bodies (Pellet lane in the figure) when the expression condition used [IPTG] = 0.5 mM, RT, t = 12 hr. (D) A significant decrease in expression of His-tagged RPA0990 was observed at condition of [IPTG] = 0.02 mM, 16° C, t = 11 hr.  A Ni-NTA column was used to specifically bind the hexahistidine tag of the overexpressed Bph proteins (see Materials and methods 2.2.5.2). The purification conditions were optimized to 2 M NaCl and 10 mM imidazole in binding buffer (Fig 2.9A and 2.9B). I observed proteolytic degradation after His-tag pulldown (Fig 2.9C, last lane) which was also 30  found in purification of other Bph-related proteins in another lab (T. Meyer, University of Arizona, personal communication). This indicated that certain specific E. coli proteases might be cleaving the R. palustris Bphs during purification. I tried using protease inhibitors (Complete Mini protease inhibitor cocktail tablets, EDTA-free, Roche) and expressing in E. coli BL21Star (DE) pLysS strain which lacks Lon and OmpT proteases and RNase E (Novagen), but neither of these approaches overcame the degradation problem. Also the absence of the cofactor might have caused the degradation. Therefore, I switched to expression of His-tagged RPA0990 in the native host, R. palustris.  Figure 2.9 SDS PAGE gels showing elutes of RPA0990 (expected size = 91.7 kDa) from Ni-NTA column under various conditions. The concentration of NaCl (A) or imidazole (B) in binding buffer was increased in batch experiment done in Eppendorf tubes. (C) Overexpression and purification of tagged RPA0990 with optimized expression (20° C and 0.5 mM IPTG, t = 20 hours) and purification (2 M NaCl, 10 mM imidazole in binding buffer) conditions. W = whole cell; S = supernatant after centrifugation (7 krpm for 10 min in JA-20 rotor) of crude lysate; P = pellet after centrifugation of crude lysate.  31  2.3.3 Construction of plasmids for gene expression in R. palustris There are no strong promoters characterized for the expression of cloned genes in R. palustris. However, it is known that LH2 proteins (encoded by puc operons) are among the most abundant proteins in the cell when fully induced (JT Beatty, personal communication). Furthermore, transcription from puc promoters can be induced simply by growing cultures under reduced aeration or under photosynthetic conditions (Braatsch et al. 2006). I chose the pucBe promoter because it is next to the divergently orientated rpa1490 (bph3) promoter, which could allow me to compare two promoters simply by inverting the PCR product in a plasmid. The bphpuc divergent promoter region was PCR-amplified from chromosomal DNA of R. palustris and cloned into the broad host-range vector pJRD215 in both orientations at BamH I and Xbal I sites, yielding plamids pJBPH and pJPUC (see Table 2.1). His-tagged rpa0122 and rpa0990 from recombinant vector pET28::rpa0122/rpa0990 were successfully cloned into plasmids pJPUC and pJBPH by ligating to Hind III and Xbal I sites, downstream of the bph-puc divergent promoter region (Materials and methods 2.2.6, Fig 2.5). The clones were confirmed by DNA sequencing (data not shown). The expression vectors were successfully constructed and introduced into wild type R. palustris by conjugation with E. coli S17-1.  2.3.4 Expression in and purification of His-RPA0990 from R. palustris The R. palustris cultures carrying the expression vectors were grown in PM medium containing Km under semi-aerobic condition without illumination. Cells were harvested at mid/late-stationary phase and a 27 g (wet weight) pellet was collected. Pelleted cells were resuspended as described in Materials and methods 2.2.8.2, and disrupted with a homogenizer followed by a French press. The crude lysate from French press was centrifuged, and the 32  supernatant was loaded on Ni-NTA column, followed by washing with a series of four washing buffers and eluting with two elution buffers sequentially (see Materials and methods 2.2.8.2). Ammonium sulphate precipitation was performed to increase the purity of RPA0990. A Ni-NTA elution using 2 ml buffer 5 (see Fig 2.10) was followed by 20% ammonium sulphate to precipitate His-RPA0990. The precipitate resumed in 1 ml was used to load on SDS PAGE 10 µl each (Fig 2.10). A protein of the approximated size of Bph RPA0990 was expressed in R. palustris with small yield compared to the E. coli expression system, yet most was in the soluble form and not degraded.  2.3.5 Absorption spectra data illustrate that RPA0990 is capable of switching between Pr and Pfr forms I discovered that purified RPA0990 with His tag has a Bph-like absorption spectrum (Fig 2.11A) and the capability of photoconversion upon red and far-red illumination (Fig 2.11B). The 33  absorption maxima of the Pr/Pfr forms are centered at 695 and 755 nm respectively (Fig 2.11C). Upon illumination with red (700 nm) light, a typical phytochrome transition from the Pr to the Pfr state was observed, with a decrease in the 695 nm peak and the appearance of a peak at 755 nm (Fig 2.11D (a) and E (a)). This transition can also be achieved by incubating RPA0990 at Pr form in the dark (D (c) and E (c)). Reversion from the Pfr state to the Pr state is obtained by farred light (750 nm) illumination (D (b)), with an increase in the 695 nm peak and decrease in the 755 nm peak (E (b)). After dark-adaptation, RPA0990 is in its Pfr form (Fig 2.11D (c and d)), without significant change in 695 nm peak but a small increase in 755 nm peak (E (d)). These results show that the rpa0990 gene encodes a functional Bph, at least in terms of light absorption and changes in optical properties in response to specific wavelength of light. Futhermore, the RPA0990 has a dark (‘ground’) state equivalent to the state after illumination with red (700 nm) light.  34  Figure 2.11 Absorption spectra of tagged RPA0990 proteins expressed from R. palustris. (A) Continuous line: Tagged RPA0990 expressed by puc promoter eluted from Ni-NTA column and exposed to standard laboratory light; dashed line: expression empty vector pJRD215 as control. (B) Tagged RPA0990 after 45 minutes illumination with 750 nm light (continuous line), 2-hour illumination with 700 nm light (dashed black line) and dark overnight incubation (dashed grey line). (C) Pr form minus Pfr form difference spectrum of RPA0990, obtained by subtracting Pfr from Pr.  35  (D) Time-lapse spectra demonstrating the transition between Pr and Pfr forms. (a) Pr form illuminated with red 700 nm light and reached full conversion within ~20 min. Spectra were measured at times 20 sec, 1, 7 min and 2 hrs. (b) Pfr form illuminated with far-red 750 nm light. Conversion was completed within ~20 min. Spectra were measured at times 15 sec, 1, 2 and 20 min. (c) Pr form incubated in dark. Spectra were measured at times 30 sec, 15, 60 and 120 mins. (d) Pfr form incubated in dark. Spectra were measured at times 1, 3, 45 min and overnight.  36  (E) Absorption spectra at 695 nm and 755 nm as a function of time. (a)-(d) labeling corresponds to the photoconversions in part D. (a) Pr form illuminated with red 700 nm light. (b) Pfr form illuminated with far-red 750 nm light. (c) Pr form incubated in dark. (d) Pfr form incubated in dark.  2.3.6 Phenotype of rpa0122 and rpa0990 mutants 2.3.6.1 Growth kinetics and yield Previous literature have reported that 770 nm illumination induced a decrease in the growth rate of R. palustris cultures grown at 8% oxygen tension (air has ~ 20% oxygen) under low light intensities (Kojadinovic et al. 2008). Kojadinovic et al. (2008) found that Bph1 (RPA1537) appeared to be the photoreceptor responsible for this growth rate limitation thus it is a repressor of the respiratory activity. However, unlike RPA1490, knockout of either RPA0122 or RPA0990 has not shown a clear effect on doubling time (Fig 2.12) or maximal culture density (Fig 2.13). The differences in Fig 2.12 and Fig 2.13 are attributed to experiment variations,  37  except for a possible small increase in culture density of the mutants grown anaerobically with high light intensities.  Figure 2.12 Growth curves of R. palustris. WT (dark diamond), rpa0122 (dark squares) and rpa0990 (grey triangles) were grown without illumination with different oxygen tension (aerobic and semiaerobic), or anaerobically with two light intensities (high light and low light). Cultures were inoculated at Klett = 10 (±2).  38  Figure 2.13 Doubling time of R. palustris wild type strain CGA009 and mutant strains in exponential phase as a function of illumination under anaerobic condition (high light and low light), and oxygen tension (aerobic and semi-aerobic) in the absence of illumination.  2.3.6.2 Phototaxis It has been proposed that a phytochrome-like photoreceptor in the cyanobacterium Synechocystis is essential for positive phototaxis (Yoshihara et al. 2004), and the purple photosynthetic Rhodobacter capsulatus is capable of photoresponsive flagellum-independent motility (Shelswell et al. 2005). Because of a putative CheY-receiver domain in RPA0990 (Fig 2.1, Chapter 2 Introduction), I hypothesized that RPA0990 has a function in phototaxis via phosphorelay pathways in a flagellum-independent way. However the results of experiments on single bph mutants in either non-aqueous (Fig 2.14A) or aqueous (Fig 2.14B) media did not support this hypothesis. In Fig 2.14A it can be seen that there was not a major difference between the wild type and the two mutants. The variability in the data suggests that the question could not be studied easily in R. palustris using this methodology (Fig 2.15). In Fig 2.14B it can be seen that both wild type and mutant cells were moving towards the light. Different cell densities were due to the differences in the starting densities. Methodology of studying phototaxis is still under investigation, and hopefully will be improved in the near future. Then, the function of RPA0990 composed of a CheY-receiver domain in phototaxis can be studied in a more precise and quantitative way. 39  Figure 2.14 Phototaxis experiments of wild type CGA009 and several different bph mutants. (A) Plates of motility assay stabbed in the center with wild type, rpa0122 and rpa0990 mutant strains. The direction of illumination was from the bottom of the figure (as the red arrow indicates). Cells moved towards the light at a similar angle for different strains. (B) Cultures were resuspended in medium under non-growing conditions. Light was presented on one side of the bottle (as the arrow indicates). Cells moved towards the edge of the bottle closest to the light. The bottles are labeled with the rpa# of the gene knocked out. (Fig B shows an experiment done by Jean Huang from the Harwood Lab in the Department of Microbiology, the University of Washington.  Figure 2.15 Variability was observed within the same strain during phototaxis assay. (A) Triplicate plates stabbed with rpa0990 mutant strain in the center. The direction of illumination was from the bottom of the figure (as the red arrow indicates). One would expect with the same light source and growth condition, the cells of same strain will behave similarly and move towards the same direction. But the triplicate plates showed different pattens of movement: either no movement or movement in different directions (more  40  data with movement towards different direction are not shown). (B) Dark control of rpa0990 mutant strain.  2.3.6.3 Absorption spectra of wild type and bph mutants grown photosynthetically R. palustris strains rpa0122 and rpa0990 were grown anaerobically while illuminated with white light and the absorption spectrum of intact cells was measured (see Materials and methods 2.2.3.3). There were no significant differences between the wild type and mutant strains in cultures grown with a high intensity of light (Fig 2.16A). Similar to the wild type strain, upregulation of the LH4 complex (~800 nm) was observed in both mutants under low light growth. However a small difference was reproducibly observed in the LH1 and LH2 long wavelength region of the absorption spectra (~850-870 nm) in the cultures grown under low light (Fig 2.16B).  Figure 2.16 Absorption spectra of intact cells grown under: (A) high light condition (~300 µE m-2 s-1); (B) low light condition (~10 µE m-2 s-1). Continuous line: R. palustris CGA009 wild type; dashed black line: RPA0122 knockout; dashed grey line: RPA0990 knockout.  41  2.3.6.4 Absorption spectra of wild type and bph mutants grown aerobically and illuminated with specific wavelengths The R. palustris wild type CGA009 and rpa0990 mutant strains were grown under aerobic conditions either in darkness, or illuminated with 700 or 750 nm light (see Materials and methods 2.2.3.4). These two wavelengths were chosen because purified His-tagged RPA0990 absorbs light at these wavelengths (Results 2.3.5 and Figure 2.11C). The absorption spectra of intact cells of the two strains grown under these three conditions are shown in Figure 2.17A, and the responses of the two strains appear similar. Direct comparisons between the two strains are shown in Figure 2.17B. There was little or no difference between the wild type and rpa0990 mutant strains after aerobic growth with 750 nm illumination (Fig 2.17B (b)). However, in the absence of illumination and with 700 nm illumination, the shape of the long wavelength peak that is from absorption by both LH1 and LH2 differed between the two strains (Fig 2.17 B (a) and (c)). There was higher variation within the same strain as well as greater difference between wild type and rpa0990 mutant strain compared to the similar short wavelength peak that is contributed by absorption of LH2 and LH4 (Table 2.3). This effect can also be seen in the ratio of 800:870 nm peak; the ratio is between 0.95~1 in wild type and 0.85~0.88 in the rpa0990 mutant strain when grown with 700 nm illumination, also slightly lower in the rpa0990 mutant strain (~0.77) compared to the wild type (~0.82) when grown in the dark (Table 2.3). These results correlate with the absorption spectra of intact cells grown anaerobically and illuminated with white light, as well as the status of the RPA0990 Bph. When the wild type and mutant strains were grown anaerobically with white light illumination, the difference between wild type and mutant strains was also found in the long wavelength peak (~850-875 nm) in cells grown under low light condition (Fig 2.16B). Also, in the absence of illumination and with 700 nm illumination, the RPA0990 Bph was found to be in the Pfr form, whereas with 750 nm light it 42  was in the Pr form (Fig 2.11B). These results are interpreted as showing that the Pfr form of RPA0990 Bph has a very subtle effect on the composition of photosynthetic components that absorb light in the ~850-875 nm region of the spectrum. The Pfr form of RPA0990 (in the dark or illuminated with 700 nm light) increases the long wavelength absorption of photosynthetic complexes, compared to the Pr form of RPA0990 (illuminated with 750 nm).  Figure 2.17 Absorption spectra of intact cells of R. palustris WT (Aa) and RPA0990 mutant (Ab) grown aerobically under dark (Ba), 750 nm (Bb) and 700 nm (Bc) light. Each spectrum is the average of three independent cultures, with variation at 862 nm ≤ 0.05A.  43  Table 2.3 Comparison of short (~803-805 nm) and long (~862-872 nm) wavelength (λ) absorption peak, as well as ratio of 800 nm:870 nm absorption, in wild type and rpa0990 mutant strains grown under 700 nm illumination and in the dark. Three cultures were grown, and the data from each are given in columns 1, 2 and 3. CGA009  rpa0990-  700 nm  dark  1  2  3  1  2  3  short λ peak  805  803  805  804  803  803  804  803  long λ peak  866  869  862  866  869  872  864  868  800/870 nm  0.872  0.888  0.85  0.870  0.984  0.94  1.011  0.978  short λ peak  804  805  805  805  805  805  805  805  long λ peak  866  864  866  865  863  862  864  863  800/870 nm  0.777  0.776  0.767  0.773  0.824  0.826  0.825  0.825  Mean  Mean  2.3.6.5 Construction of rpa0990 complemented strain The plasmid pJPUC::rpa0990 (which has the pucBe promoter driving transcription of rpa0990, Table 2.1) was conjugated into the rpa0990 mutant strain to express His-RPA0990. Since the rpa0990 mutant strain is already resistant to kanamycin and, the plasmid pJRD215 contains the same antibiotic resistance marker, it was not possible to select plasmid recipients by selecting for antibiotic resistance. Instead, potential conjugates were screened in a Southern blot using DIG-labeled probe against pJRD215 (see Materials and methods 2.2.7). To test for the stability of pRJD215-derived plasmids in a R. palustris strain that is resistant to kanamycin, The rpa0990 mutant strain containing the plasmid pJPUC::lacZ was plated on PM agar containing 100 µg/ml of kanamycin and 40 µg/ml of Xgal after a culture underwent 3~4 doublings in liquid medium in the absence of kanamycin. Less than half of the colonies were blue, suggesting that more than half of the cells lost the plasmid after 3-4 doublings. Because of this apparent instability of pJRD215-derived plasmids in R. palustris, and because the rpa0990 mutant was resistant to Km (the antibiotic used to select for pJRD215 and derivatives), it was not possible to study the effect of trans-complementation of the rpa0990 mutation. 44  2.4 Discussion 2.4.1 Purified His-RPA0990 exhibites Bph spectropic properties R. palustris is unique in having six bph ORFs compared to only one or two in other bacterial genomes. Four of the bph genes are close to photosynthetic gene clusters and were confirmed to regulate the synthesis of LH complexes in previous studies (Giraud et al. 2005; Vuillet et al. 2007; Kojadinovic et al. 2008). However, the two remaining bph genes (rpa0122 and rpa0990) are located further away from the photosynthetic gene clusters within the R. palustris genome. Questions arising include whether these two bph genes encode functional Bph proteins and, if so, which genes they regulate. In this thesis, I have discovered that purified RPA0990 with a His tag has a characteristic Bph absorption spectrum, and is capable of photoconversion upon red and far-red illumination. This means that the rpa0990 gene encodes a functional phytochrome. Based on sequence comparison to four other functional Bphs in the R. palustris CGA009 strain, it was hypothesized that both or one of RPA0122 and RPA0990 should also encode functional Bphs. Sequence comparison was performed between R. palustris Bphs, and a model Bph from Deinococcus radiodurans (DrBph) where the crystal structure has been determined to 2.5 Å resolution by Wagner et al (2005) (Fig 2.2). Cys 24 was shown to be the chromophore attachment site, and key amino acids form a solvent-shielded bilin-binding pocket, whereas His 260 forms hydrogen bonds with the pyrrole nitrogens of bilin, and has been demonstrated to be critical for biliverdin conjugation to DrBph (Bhoo et al. 2001; Wagner et al. 2005). Given the fact that RPA0122 and RPA0990 both contain the conserved Cys 24, I hypothesized that these two Bph-like proteins might incorporate the chromophore. I have failed in purifying RPA0122 due to the expression problem; however, the purified His-RPA0990 has an absorption spectrum that inter-converts between the red (Pr) and far-red (Pfr) forms. This proves that the RPA0990 45  protein incorporates the chromophore, because apophytochromes neither photoconvert nor exhibit a typical photochrome absorption spectrum (Furuya et al. 1994). The data not only suggest that RPA0990 binds to a cofactor such as linear tetrapyrrole, but also enable us to determine the photoconversion activity as well as the different absorption states of RPA0990. RPA0990 has a dark stable form resembling the Pfr form with a dominant absorption peak at 755 nm, whereas the Pr form absorbs mainly at 695 nm (Fig 2.11B). It was found that RPA1537 is active in the Pr form which is the opposite state to its dark stable form (Giraud et al. 2002). Evans et al. (2005) have supposed by analogy that RPA3015 is active in its non-dark stable state, which is Pfr form. Perhaps RPA0990, like RPA1537 is active in the Pr form, which is the non-dark stable state. In the future, phosphorylation experiments could be done to investigate the His-kinase activity of RPA0990. Cph1, with Pr being more active than Pfr, is well studied as a model phytochrome. In the proposed signaling mechanisms for Cph1, the photoconversion of Pr to Pfr causes a conformational change that facilitates autophosphorylation and phosphotransfer and initiates a cytoplamic output signal (Rockwell et al. 2006). I speculate that RPA0990 shows photoregulation similar to Cph1: RPA0990 is more phosphorylated in its Pr form, and the Pr form is the active form, whereas the Pfr form (the dark-stable form) is less phosphorylated and less active. It is interesting that the dark form of His-RPA0990 has a decreased peak at 700 nm compared to the Pfr form, although I suggested that the Pfr and dark forms are the same. This might be due to the incubation time: the Bph RPA0990 was incubated overnight in the dark in order to ensure complete conversion from the Pr to the Pfr form, however the Pfr form was obtained from the Pr form after only 20 minutes of exposing to red-light illumination. One would expect to see a similar absorption spectrum for the Pfr form with a dominant peak at 750 nm but with a flatter shoulder at 700 nm if the Bph RPA0990 was illuminated with red-light for enough 46  time to convert the molecules. Alternatively, the bandwidth of the light filters used may have resulted in some overlapping of wavelengths, such that 755 nm light contained a small amount of 695 nm light, and vice versa. The conversion from Pfr/dark form to Pr form takes much less time: complete conversion from Pr to dark-stable form took overnight while 1 hour illumination of far-red light was sufficient to minimize the 755 nm peak. Dark reversion of phytochrome is not yet well characterized; however in the plants it was found that dark reversion can be reduced by interaction with other proteins or enhanced by missense mutations, suggesting that dark reversion plays an important role in Phy signalling (Elich et al. 1997; Sweere et al. 2001). Unlike RPA3015 and RPA3016 that are in their Pr forms after dark-adaption (Giraud et al. 2005), RPA0990 has Pfr form as the dark-stable form, as does RPA1537. RPA3015 and RPA3016 autophosphorylate in their dark-adapted Pr forms and transfer the phosphate to a common response regulator RPA3017. RPA1537 is not able to autophosphorylate because of the lack of a histidine kinase domain, but in its active Pr form, RPA1537 was proposed to release PpsR2 repression on the expression of photosynthesis genes by protein-protein interaction (Braatsch et al. 2007; Kojadinovic et al. 2008). Thus RPA0990 is expected to autophosphorylate and transfer its phosphate to a potential downstream response regulator in its active Pr form. On the other hand, because RPA0990 has a RR domain, it is possible that RPA0990 itself might be a response regulator. The other novel Bph, RPA0122, might autophosphorylate (because it has a histidine kinase domain) and transfer phosphate to RPA0990. Whether these two Bphs function in a two-component system could be investigated by in vitro phosphorylation and phosphotransfer studies once RPA0122 is purified. In conclusion, RPA0990 has an absorption spectrum resembling a Bph as well as being capable of photoconversion between Pr and Pfr forms. Being in the Pfr form after dark-adaption, RPA0990 is proposed to be phosphorylated and active under its Pr form. Future experiments 47  need to be conducted in order to investigate the downstream regulation activity of this Bph protein. 2.4.2 Physiological function of RPA0990 requires future research Whether the two Bph proteins RPA0122 and RPA0990 are involved in the regulation of photosynthesis is still a question. The genes encoding these two proteins are located far away from the photosynthesis gene clusters, in contrast to other bph genes that regulated photosynthesis genes. What makes it more complicated is that Bphs are also found in nonphotosynthetic bacteria, so that it is possible that these Bph-like proteins with unusual locations may be involved in physiological activities other than photosynthesis. My thesis research indicates that there may be a minor effect on LH1 synthesis resulting from both knockouts (Table 2.3; Fig 2.16B and Fig 2.17B). Different experiments have been conducted in order to study the phenotype of rpa0122 and rpa0990 mutants. No significant changes in growth kinetics and yield have been observed, indicating that these two Bph proteins may not be involved in growth rate control. Phototaxis assays were initiated on the hypothesis that the unique receiver domain (RR) of RPA0990 may be involved in phototaxis. A similar RR domain was found in Rcp1 that is the response regulator of cyanobacterial phytochrome Cph1 in Synechocystis PCC6803. It was suggested that a Cph1Rcp1 phosphorelay network is involved in red/far-red phototaxis (Choi et al. 1999). Thus, phototaxis experiments were designed to study the possible function of RPA0990 and RPA0122 in R. palustris phototaxis. However, in my study, the effect of knocking out these two bph genes did not affect phototaxis, also the methodology of phototaxis needs improvement to reduce the variability. It is also possible that the dimerization of RPA0990 is also regulated by phosphorylation through the RR domain, which might affect the interaction with downstream targets as proposed for the cyanobacterial Rcp1 by Im et al. (Im et al. 2002). 48  Absorption spectra, on the other hand, have suggested some interesting information. The absorption spectra of intact cells illuminated with both white light and light with specific wavelengths have showed a minor difference between wild type CGA009 and rpa0122/rpa0990 mutants. Under all light conditions, little or no differences were observed in the short wavelength peak region (800 nm) that is contributed by absorption of LH2 and LH4, indicating that knocking out these two bph genes may not effect the synthesis of LH2 or LH4 regardless of the light condition. However under illumination with white low light or low intensity 700 nm light, or incubating in the dark, the rpa0990 mutant had different absorption amplitude and shape of the long wavelength peak (~850-875 nm) compared to the CGA0090 wild type strain. This long wavelength peak is contributed to by both LH1 (absorbs at ~875 nm) and LH2 (absorbs at ~800 and 850 nm). As stated earlier, the synthesis of LH2 and LH4 (absorbs at ~800 nm) does not seem to be affected by rpa0122 or rpa0990 mutants because of the unaffected absorption peak at 800 nm, and so it is solely the LH1 complex that contributes to the difference between the wild type and mutant strains. Thus it is possible to propose that either or both of RPA0122 and RPA0990 regulate the synthesis of the LH1 complex under specific light wavelength (~750 nm) with low intensity (~10 µEm-2s-1) illuminations, or in darkness. However how these two Bph-like proteins function in regulating the expression of LH1 complexes remains to be determined, because up-regulation was observed with a white low-light intensity during phototropic growth (Fig 2.16), whereas down-regulation was found under illumination with 700 nm light or dark incubation during chemotropic growth. This regulation is probably part of a transcriptional and post-transcriptional network that controls the level of LH complexes to enable the cells to adapt to environmental conditions, as R. palustris is well known for its capacity to grow at extreme low-light intensities by adapting the LH apparatus (Tadros et al. 1993).  49  2.4.3 Expression and purification system used in this study Bphs RPA0122 and RPA0990 were first overexpressed in E. coli, but most of the proteins were degraded by an E. coli protease. The expression approach was later switched to R. palustris. Problems were first encountered with overexpression and purification of the Bphs in the E. coli expression system. Strategies were attempted to generate more soluble protein instead of inclusion bodies. To solve this issue, an expression protocol using low temprature (20° C) and low concentration of IPTG (0.5 mM) succeeded in generating ~20-25% of Bph present in supernatant. However, significant degradation of the peptides was observed with various E. coli strains for expression (Fig 2.9C), which suggested unknown E. coli proteases might function in cleaving the R. palustris Bphs during purification. The degradation was also observed in the purification of Bphs from other strains in other labs (T. Meyer, personal communication). Adding a protease inhibitor cocktail (Roche) did not prevent the degradation (data not shown). Thus an alternative approach was performed to express the Bphs in R. palustris, which presumably would result in non-degraded Bphs. A wide-host-range cosmid vector pJRD215 was used for cloning of bph gene fragments with the previously introduced His-tag in E .coli. Meanwhile a promoter region (471 bp) containing promoters for bph and puc operon with opposite directions was introduced for high level of the bph expression. RPA0990 was successfully expressed by the puc promoter under a semi-aerobic condition because the puc promoter is induced by reduced aeration (Braatsch et al. 2006). However the magnitude of Bph expression in R. palustris was not as high as in the E. coli overexpression system. In the future, a larger amount of His-RPA0990 could be generated by expressing the puc promoter under photosynthetic (anaerobic) condition, because puc expression is induced more than during growth in darkness with low aeration (see Chapter 4).  50  Chapter 3 - EVALUATION OF POSSIBLE ANAEROBIC BILIN SYNTHESIS IN R. PALUSTRIS  3.1 Introduction The annotation of the R. palustris genome sequence suggests that there are five “peripheral” LH complex operons (pucBA genes): three encode light-harvesting complexes type 2 (LH2), characterized by two absorption peaks at 800 and 850 nm; one encodes the LH4 complex that absorbs at only ~800 nm; and one contains a pseudo-gene that is considered as nonfunctional (Tadros et al. 1989; Tharia et al. 1999). It has been shown that in R. palustris certain bacteriophytochromes (Bphs) exert their function in light signal transduction pathways. The Bphs RPA3015 and RPA3016 function by covalent binding to the chromophore, presumably biliverdin, and regulating the expression of the genes for LH4 complex under anaerobic low light conditions or with red light illumination (Giraud et al. 2005). This is supported by the observation that the absence of either of these two Bphs results in an increase of LH2 complex levels, and a possible decrease in LH4, as seen in the absorption spectrum of intact cells grown photosynthetically (anaerobically) under low light intensities (Fig 3.1).  51  Figure 3.1 Absorption spectra of R. palustris wild type and bph mutant strains rpa1490, rpa3015 and rpa3016 grown at low-light intensity (unpublished data from Beatty lab). LH2 absorbs near-infrared light at ~800 and 850 nm whereas LH1 absorbs at about 875 nm. Under low light illumination, a novel LH4 (~800 nm) complex is expressed to partially replace LH2.  Table 3.1 The 800:850 absorption ratios for R. palustris wild type, rpa3015, rpa3016 and rpa1490 mutant strains grown anaerobically at low light intensity (corresponding to Figure 3.1).  The effects of bph gene knockouts on anaerobically grown cells are curious because Bphs require a tetrapyrrole such as biliverdin to sense light. Yet, the production of the biliverdin by the HO oxygenase requires O2 for cleavage of heme (Fig 3.2) (Furuya et al. 1994; Zhu et al. 2000; Kirkby et al. 2006). However, our experiments on bph knockout absorption spectra were done by inoculating tubes sealed with screw-caps. Thus the medium contained O2 at the time of inoculation, but it was assumed that the cellular respiration removed most of the O2 after a few minutes (JT Beatty, personal communication), whereas cells were not harvested until 24 hours after inoculation of cultures. If no O2 was accessible to these cultures, it is reasonable to assume 52  that no biliverdin was produced by HO through cleavage of heme. Therefore, after 3~4 doublings, the amount of Bphs would have dropped to 12.5~6.25% of the original amount in the pre-culture cells. Since the absorption spectra still show a significant decrease in LH synthesis (Fig 3.1), this indicates that Bphs are still functioning. One possible explanation is that a low amount of Bphs may still be sufficient for light signal transduction and regulation of LH synthesis in anaerobic cultures. Alternatively, it is conceivable that R. palustris has an enzyme which cleaves heme to produce a bilin anaerobically, perhaps by a novel hydrolysis mechanism in which H2O substitutes for O2 as a reactant. Finally, perhaps R. palustris can siphon off linear tetrapyrrole (hydroxymethylbilane; HBA) in the tetrapyrrole biosynthetic pathway leading to heme and Bchl (Beale 1995).  53  Figure 3.2 Cyclic heme is converted into linear biliverdin by heme oxygenase in the presence of O2 (Zhu et al. 2000).  In order to address the question of whether O2 is needed for the production of linear tetrapyrrole in R. palustris, I designed an experiment for growth of wild type cultures under rigorously anaerobic conditions. The assay for production of linear tetrapyrrole was the Bphdependent change in the LH absorption spectrum, as shown in Fig 3.1. The rationale was that if no bilin was produced in the absence of O2, the LH absorption spectrum under low-light condition would resemble that of bph mutant strains such as rpa3015 or rpa3016, with 800:850 nm absorption ratios of ~ 0.8-1.0, as opposed to the wild type strain with an 800:850 nm absorption ratio of 1.7 (Table 3.1).  54  3.2 Materials and methods 3.2.1 Strict anaerobic growth condition De-oxygenated photosynthetic cultures were grown in completely filled screw-cap tubes (17 ml), inoculated from photosynthetic pre-cultures under an inert N2 atmosphere using an Mbraun Labmaster glovebox (Stratham, NH, U.S.A.) maintained at 2 ppm O2 or less. The tubes in triplicate were stored for several days in an anaerobic glovebox until they were inoculated. In the glovebox, the tubes were inoculated and placed in a GasPak anaerobic jar with GasPakTM EZ Gas Generating Container Systems (BD) to eliminate residual O2, and BBLTM GasPakTM Disposable Anaerobic Indicator strip (BD) was used to indicate the level of O2 present. The GasPak anaerobic indicator strip consists of a foil envelope and a pad saturated with methylene blue solution. The methylene blue is blue in the presence of oxygen, but when the oxygen level is less than 1% the pad turns to colorless. The anaerobic jar containing sealed screw-cap tubes was sealed within the glovebox, and then removed and incubated under the photosynthetic condition as described in 2.2.3.1. Cultures grown under standard photosynthetic conditions, in filled screw-cap tubes filled with non-deoxygenated medium and incubated in an open container were used as controls.  55  3.2.2 Spectroscopy Intact R. palustris cells were collected by centrifugation at 14 krpm for 2 min in a microcentrifuge. Absorption spectroscopy of intact cells was performed with mixture of PM medium and 30% (wt%) BSA (1:3) as the blank. Data were collected in a Hitachi U3010-S spectrophotometer with scan speed of 600 nm min-1. Light scattering at 650 nm was used to normalize the spectra, and the data from triplicate cultures were averaged as described previously (Braatsch et al. 2006).  3.3 Results 3.3.1 Bph-dependent regulation of LH complexes persists in the absence of O2 Deoxygenated R. palustris wild type cultures (called DOLL, for DeOxygenated LowLight) were grown in deoxygenated media in screw-cap tubes that were additionally protected from O2 by incubation in an anaerobic jar, under low light condition (~10 µE m-2 s-1) (see Materials and methods 3.2.1). In contrast to the bph (rpa3015 and rpa3016) knockouts, no significant change in synthesis of LH complexes was detected through a series of four subcultures, with cells undergoing 4-5 doublings in each subculture (Fig 3.3A). Thus, the peak shapes and the amplitudes at the wavelengths absorbed by DOLL cultures resemble those absorbed by OLL (Oxygenated LowLight) cultures (compare Fig 3.3A and B), but differ greatly from the absorption spectra of bph knockout OLL cultures (rpa1490, rpa3015 and rpa3016; Fig 3.3C). The absence of a clear trend in the DOLL cultures, and the great difference from the rpa3015 and rpa3016 mutants, indicate that Bph-dependent regulation of LH complexes existed throughout all the subcultures. The minor differences in the DOLL and OLL cultures are attributed to experiment variation. Therefore, it appears that R. palustris is capable of producing functional Bphs and a linear tetrapyrrole in the absence of O2. 56  Figure 3.3 Absorption spectra of R. palustris strains grown under low light with different methods of manipulation of oxygen levels. (A) DOLL: cultures were inoculated with pre-deoxygenated media under anaerobic condition and grown in an anaerobic jar; DOLL1 is the first DOLL subculture, DOLL2 is the DOLL culture after second inoculation, and so on. (B) OLL: cultures inoculated with non-deoxygenated media under aerobic conditions and incubated under standard anaerobic (photosynthetic) conditions, in completely filled screw-cap tubes. (C) Absorption spectra of R. palustris wild type and mutant strains rpa3015 and rpa3016 grown as OLL cultures.  57  3.4 Discussion Previous experiments in which the absorption spectrum of cells from standard anaerobic (photosynthetic) cultures of wild type and bph knockout cells were compared suggested that R. palustris has an O2-independent method to obtain bilin. This hypothesis was supported by the discovery that the absence of O2 does not inhibit the normal function of Bphs, which bind to the linear tetrapyrrole chromophore (presumbly biliverdin) that has been believed to be produced aerobically . Several R. palustris Bphs (RPA1490, RPA3015 and RPA3016) regulate the expression of photosynthetic complexes (Evans et al. 2005; Giraud et al. 2005). For example, RPA3015 and RPA3016 were proposed to operate in tandem to control the synthesis of the LH4 complex by measuring light intensity or wavelength upon binding to the chromophore biliverdin. RPA1490, however, lacks the key cys residue and hence is unable to bind the chromophore biliverdin, but appears to regulate LH2 synthesis as a sensor of O2 concentration (Giraud et al. 2005). Based on the current understanding, biliverdin is formed when heme is cleaved by heme oxygenase (HO) in the presence of O2 (Kirkby et al. 2006). The LH composition of wild type cells grown anaerobically under low light intensities differs from Bph mutant cells (Fig 3.1). This indicates that wild type cells contain Bph holoproteins (the Bphs bound with biliverdin) to modulate LH regulation under anaerobic conditions. One possible explanation invokes a ‘bilin pool’, either free or bound to Bphs, generated during pre-culturing in the presence of oxygen. These hypothetical bilin molecules would enable the function of Bphs for several doublings, even as the concentration of Bph holoenzymes decreases. The close location of the hmuO gene with the bphP gene in several species suggests that there is functional linkage between O2-dependent biliverdin formation and photoreceptor  58  function (Fig 3.4). For example, in R. palustris, the hmuO gene is immediately 3’ of an operon containing bph rpa1537 and repressor ppsR2 ORFs.  Figure 3.4 Sequences encoding heme oxygenase (HumO) homologues in several Bph operons. HmuO from R. palustris is not located in a Bph operon but is adjacent to bph3 (rpa1537).  My investigation of a possible O2-independent heme lyase was done by comparing the absorption spectrum of cells subcultured under standard low light conditions (i.e., transfer cells from a culture into a screw-cap tube containing medium off the laboratory shelf) with the absorption spectrum of cells subcultured by transfer into deoxygenated medium in an anaerobic glove box, followed by incubation of screw-cap tubes in an anaerobic jar (see Materials and methods 3.2.1). After cultures reached stationary phase (after 4~5 doublings), I used absorption spectroscopy to measure the amount of LH complexes synthesized and I also removed a sample for further subculture (in the anaerobic glove box). My study of whether Bphs are functional in the absence of O2 shows that the low-light (-O2) cultures, which were inoculated with deoxygenated medium in an anaerobic chamber and cultivated in screw-cap tubes placed in an anaerobic jar, had absorption spectra more similar to wild type cultures grown in nondeoxygenated media than to the bph mutants. This indicates that there is an additional pathway 59  of linear tetrapyrrole synthesis without utilizing O2 for heme cleavage (Fig 3.6). The mechanism for this putative O2-independent cleavage of heme is not known, but it may be catalyzed by a novel enzyme that uses H2O for hydrolysis of heme. It has been discovered that bacteriochlorophyll can be synthesized from the same substrate Mg-protoporphyrin monomethylester through either aerobic O2-dependent or anaerobic H2O-dependent pathway (Ouchane et al. 2004). Two separate Mg-protoporphyrin monomethyl ester cyclases, AcsF and BchE, are able to catalyse bacteriochlorophyll synthesis independently by using either O2 or H2O in purple photosynthetic bacteria. This finding suggests that R. palustris may also have an analogous pair of enzymes that function either aerobically or anaerobically to cleave heme to produce biliverdin. Two ORFs (rpa1539 and rpa3279) encoding HO homologues are present in R. palustris genome. I have examined the sequence alignment of these two putative HO proteins (Fig 3.5A) and found they only share 21.2% identity in peptide sequences. Interestingly, R. palustris shares some similarities with Pseudomonas aeruginosa, which is the first known organism to encode two functional HOs (PigA and BphO). Firstly, RPA3279 resembles PigA with identity of 39.5% (Fig 3.5B). PigA, with the crystal structure been solved, is known to produce unusual biliverdin isomers IXβ and IXδ (Wegele et al. 2004). Secondly, although only 26.5% in sequence identity, RPA1539 and BphO are similar in genetic organization: both of their coding sequences are adjacent to or in the same operon from a bph gene. Wegele et al. have discussed the functions of these two HOs, and presented that both of BphO and PigA function to oxidise heme to a BV but different isomers (Wegele et al. 2004). More surprisingly, RPA1539 has high similarity (29.6%) with a rat heme oxygenase-1 (HO-1) which oxidizes heme to biliverdin IXα (Fig 3.5C). It would be interesting to investigate the relationship and difference between the two hmuO homologs in R. palustris, whether they function to cleave heme in a  60  similar manner as P. aeruginosa HOs, and if yes, whether these heme degradation pathways are related to the presence of oxygen. In Vierstra’s previous work, DrBph expressed in E. coli was yellow, whereas when coexpressed with DrHmuO, a blue-green color was observed, which is the expected color of a Bph-bilin holoprotein (Bhoo et al. 2001). This indicates that Bphs are capable of spontaneously incorporating biliverdin produced by HmuO-cleavage of heme. It is not clear why the DrBph was yellow in the absence of HO expression. In the future, it would be interesting to construct an hmuO knockout of R. palustris and use this knockout to see whether the LH absorption spectra is similar to the wild type cells grown anaerobically with low light illumination. This knockout could also be used for His-tagged Bph purification from cells grown under aerobic and anaerobic conditions using the Bph-expression plasmid I made in this study (see Chapter 2). Whether a linear tetrapyrrole is produced can be determined simply in absorption spectra of Bph, because the Bph apoprotein (without cofactor bound) is not capable of absorbing red/far-red light or photoconversion. I speculate that a linear tetrapyrrole would be produced in the absence of hmuO, under anaerobic conditions. If so, this could further expand our understanding of the correlation between heme metabolism and photoreceptor function, and raise the possibility that a variety of anaerobes are capable of anaerobic cleavage of heme.  61  A). RPA1539 (220 aa) vs. RPA3279 (227 aa) RPA1539 MVVEAAKRGAESVVTALYVRTRQLHLEAEKSGILSEILHGTAGRDGYTLLLRNLHPAYRIEAGIERHRDA 70 :.: . . : : . :. : : :::... . ..:. :.:.: : .. ..: . RPA3279 MTVTDSAL-VLSRSKRLKAATQATH-ERLDSGIMAR--RPFSSRERYALFLLVQHRFHQDVDAFYRSAAL 66  RPA1539 NPILAPLAAHPLARTPAIESDLAALAGADWHER-LPV--------LPAAEAYAQRIAEVSEGDGSRLIAH 131 . .: : . : :: :: :. : . : :: .:.: .. .:: :. .. :. . RPA3279 GELLPALLER--RRLELIELDLRDLGVAPENSRATPVSIGEQPIDVPTALGWLY-VAEGSNLGAAFLLKQ 133  RPA1539 AYTRYLGDLNGGQIVRRLLEKTMQLSAGELAHYD-FSAIGDPATLKT--------DYREALERAGAAAPD 192 : . :.. :. :.:. :. :. :.. : : :.. : : .:. . . RPA3279 AGSLGLSETFGA---RHLVAAP----EGRGLHWKTFTTALDAAPLSAMEEERAIAGARAAYQRVHKLVNE 196  RPA1539 AAAVIEEGAVAFTCNIALSVAVQQHL---DA 220 .: :.: .. . . . . . .. RPA3279 LLVVAAESAGSLETDDGGPTRFRPDVKEGEG 227  B). Paeru PigA (198 aa) vs. RPA3279 (227 aa) Paeru PigA MDTLAPESTRQNLRSQRLNLLTNEPHQRLESLVKSKEPFASRDNFARFVAAQYLFQHDLEPLYRNEALAR 70 : . .:. ::.::. :. :.::.: . ...::.::. .: :. .:. :..:.. .::. ::.. RPA3279 MTVT--DSALVLSRSKRLKAATQATHERLDSGIMARRPFSSRERYALFLLVQHRFHQDVDAFYRSAALGE 68  Paeru PigA LFPGLASRARDDAARADLADLGHPVPEGDQ----SVREADLSLAEALGWLFVSEGSKLGAAFLFKKAAAL 136 :.:.: : : . . :: ::: .::... :. : ... :::::.:.:::.::::::.:.:..: RPA3279 LLPALLERRRLELIELDLRDLG-VAPENSRATPVSIGEQPIDVPTALGWLYVAEGSNLGAAFLLKQAGSL 137  Paeru PigA ELDENFGARHL-AEPEGGRAQGWKSFVAILDGIELNEEEERLAAKGASDAFNRFGDLLERTFA------- 198 :.:.:::::: : ::: :. ::.:.. ::. :. :: . : :: :..: :... .. RPA3279 GLSETFGARHLVAAPEG-RGLHWKTFTTALDAAPLSAMEEERAIAGARAAYQRVHKLVNELLVVAAESAG 206  Paeru PigA --------------------RPA3279  SLETDDGGPTRFRPDVKEGEG  227  C). HO-1 (267 aa) vs. RPA1539 (220 aa) 1DVE_A  MERPQLDSMSQDLSEALKEATKEVHIRAENSEFMRNFQKGQVSREGFKLVMASLYHIYTALEEEIERNKQ 70 : .... :: :...:..::.: .. .. .: ..:.:. :.. .:. : : .: :::... RPA1539 MVVEAAKRGAESVVTALYVRTRQLHLEAEKSGILSEILHGTAGRDGYTLLLRNLHPAYRAIEAGIERHRD 70  1DVE_A  NPVYAPLYFPEELHRRAALEQDMAFWYGPHWQEAIPYTPATQHYVKRLHEVGGTHPELLVAHAYTRYLGD 140 ::. ::: . : : :.:.:.: : :.: .: ::.. :..:. ::. :.:::::::::: RPA1539 NPILAPLA-AHPLARTPAIESDLAALAGADWHERLPVLPAAEAYAQRIAEVSEGDGSRLIAHAYTRYLGD 139  1DVE_A  LSGGQVLKKIAQKAMALPSSGEGLAFFTFPSIDNPTKFKQLYRARMNTLEMTPEVKHRVTEEAKTAFLLN 210 :.:::..... .:.: : :.:: :: . : .: .:. .: :: .. . : ::. .:: : RPA1539 LNGGQIVRRLLEKTMQL-SAGE-LAHYDFSAIGDPATLKTDYREALERAGAAAPDAAAVIEEGAVAFTCN 207  1DVE_A  IELFEELQALLTEEHKDQSPSQTEFLRQRPASLVQDTTSAETPRGKSQISTSSSQTP 267 : : .. ...: : RPA1539 IAL-----SVAVQQHLDA--------------------------------------- 220  Fig 3.5 Sequence alignment of heme oxygenase peptides using Genestream align tool (Pearson et al. 1997). (A) Sequence alignment of RPA1539 and RPA3279 reveals 21.2 % identity; (B) Alignment of Paeru PigA and RPA3279 reveals 35.9 % identity; (C) Alignment of rat heme oxygenase (HO-1) and RPA1539 reveals 29.6 % identity.  62  Alternatively, it is possible that the linear tetrapyrrole hydroxymethylbilane, the precursor of the heme precursor protoporphyrin IX (Fig 3.6), can be incorporated as the chromophore in R. palustris Bphs when cultures are grown in the absence of O2. Although hydroxymethylbilane is unstable in solution and it rapidly cyclises to uroporphyrinogen I (Beale 1995), I suspect Bphs might possibly make use of hydroxymethylbilane at very low level after it released from hydroxymethylbilane synthase. The nature of the Bph-bound linear tetrapyrrole present in R. palustris cells grown under anaerobic conditions could be determined, using a plasmid that I created (Materials and methods 2.2.6, Chapter 2). The plasmid pJPUC::rpa0990 expressing the 6-His tagged RPA0990 could be used in R. palustris cultures grown under rigorous anaerobic conditions, to purify RPA0990, and obtain the linear tetrapyrrole as described (Cornejo et al. 1992). Once the linear tetrapyrrole cofactor is obtained, we could collaborate with chemists to determine the mass of the linear tetrapyrrole and deduce the structure as BV, hydroxymethylbilane, or conceivably something else. If necessary we could use other methods such as NMR or X-ray crystallography to determine the structure of the bilin. In fact, the plasmid expression system I created could be used to express His-tagged versions of any of the Bphs, or, any protein in R. palustris.  63  Figure 3.6 Transformation of ALA to biliverdin including two hypothetical pathways of making cofactor of Bphs in the absence of O2. It is possible that the linear tetrapyrrole hydroxymethylbilane can be incorporated as a cofactor in Bphs, or alternatively, biliverdin can be made anaerobically by a novel anaerobic heme oxygenase.  64  Chapter 4 – USE OF THE E. COLI LACZ GENE AS A REPORTER TO EVALUATION PROMOTER ACTIVITY IN R. PALUSTRIS  4.1 Introduction Under anaerobic growth conditions, the purple non-sulfur bacteria synthesize a photosynthetic apparatus. The different types of photosynthetic apparatus are always composed of a reaction center (RC), and a light-harvesting complex type I (LH1), and many species contrain a peripheral light-harvesting complex type 2 (LH2) (Thornber et al. 1983). In the purple non-sulfur bacterium R. palustris, a novel peripheral light-harvesting complex type 4 (LH4) was also identified and characterized (Hartigan et al. 2002). This organism maintains light-trapping efficiency upon a decrease in light intensity by raising the proportion of LH2 with respect to LH1. Under very low light conditions R. palustris also replaces LH2 with LH4. The sequenced genome of R. palustris has revealed the organization of photosynthesis genes including those encoding LH complexes, and six predicted bph genes (Larimer et al. 2004). Specifically, divergent promoters of rpa1490 (bph3) and rpa1491 (pucBe) were identified (Fig 4.1). Because this divergent promoter region was used to express the Bph protein RPA0990 in another chapter of my thesis (see Chapter 2), I hope to thoroughly understand the expression activities of these two promoters in wild type and mutant strains grown under several standard conditions.  65  Figure 4.1 Arrangement of genes located around rpa1490 and rpa1491 in R. palustris CGA009. The dashed line is where the divergent promoter region is located, which drives transcription of bph3 and pucBe in opposite directions. The distance between the A of the ATG start codons of rpa1490 and rpa1491 is 346 bp.  In the R. palustris genome, there are five gene pairs (pucABa to pucABe) which encode the LH2 peptides (Tadros et al. 1989). Gene rpa1491 is annotated as pucBe that encodes the β chain E of the LH2 complex. The pucBe gene is located 5’ of pucAe and pucC (Fig 4.1). Among the five pucBA gene pairs, puce was shown to have the highest expression under both chemotrophic and phototrophic growth conditions (Tadros et al. 1993). Similar to the pucBA genes in Rhodobacter capsulatus, transcription from puc gene promoters in R. palustris is induced by reduced aeration (LeBlanc et al. 1993; Braatsch et al. 2006). Within a small region (346 bp between the A of the ATG start codons of rpa1490 and rpa1491), the promoters for both genes rpa1490 and rpa1491 are oriented in opposite directions. Gene rpa1490 is annotated as a bph homolog and its product Bph has been shown to regulate the synthesis of LH2 complexes (Vuillet et al. 2007). In most of the R. palustris wild type strains including CGA009, RPA1490 lacks the conserved Cys residue that binds the chromophore, however it is still capable of playing a role as histidine kinase without binding the chromophore biliverdin (Evans et al. 2005). In most R. palustris strains, the RPA1490 protein was proposed to function as an O2 sensor instead of sensing light, and modulate the affinity of a transcriptional 66  factor that binds to the pucBe promoter region by controlling its phosphorylation status (Vuillet et al. 2007). However, RPA1490 of R. palustris CGA009 strain, lacking the conserved Cys residue, was shown to affect LH complexes synthesis (Fig 3.1, Beatty lab, unpublished data). There are several regulatory systems that modulate transcription of photosynthesis genes in purple bacteria. Among them, PpsR plays an important role, because it directly or indirectly regulates most if not all of the photosynthesis genes by either activation or suppression (Elsen et al. 2005). Several PpsR knockout strains were previously made in Beatty lab including ppsR1-, ppsR2- and ppsR1-2- (Braatsch et al. 2007). In this thesis, I have studied the activity of rpa1490 and rpa1491 promoters in some of these ppsR mutants, to investigate how PpsR proteins regulate the expression of puc and bph genes. The activity of the divergent promoter region puc-bph was studied by fusion to the E. coli lacZ gene on a plasmid, and measurement of β-galactosidase activities. The results will advance our knowledge about the activity of these promoters as well as help in choosing the more efficient promoter in future experiments in which a plasmid-borne gene is to be expressed in R. palustris.  4.2 Materials and methods 4.2.1 Construction of lacZ transcriptional plasmids The lacZ transcriptional fusion plasmids (Fig 4.2) were constructed by inserting the lacZ gene into pJBPH or pJPUC plasmids (previously made in Chapter 2). The divergent promoter region bph-puc was previously cloned into plasmid pJRD215 (Table 2.2) in both orientations between BamH I and Xbal I sites (see Materials and methods 2.2.6, Fig 2.5). The lacZ gene was cut out from pHRP309 (Table 2.2) by Hind III and Xbal I sites and cloned into pJBPH and pJPUC plasmids downstream of the divergent promoter region. Sequencing was completed at the 67  University of British Columbia Nucleic Acid and Protein Service Unit (NAPS) using primers (pJRD215_seqF and pJRD215_seqB, Table 2.2) from plasmid templates purified with the plasmid isolation method described in 2.2.6. PCR-verified expression constructs were transformed into E. coli S17-1 and conjugated into R. palustris wild type and ppsR1-2- strains (Table 2.1) by conjugation (Braatsch et al. 2007). Positive exconjugants were identified by visual inspection for blue colonies on PM Km30 Xgal40 plates (a genetic screen), because the recipient ppsR1-2- strain has the same antibiotic resistant marker (Kanr) as the pJRD215 plasmid derivatives and so selecting for Kmr could not be used.  Figure 4.2 Promoterless lacZ gene (Xbal I-Hind III) from plasmid pHRP309 was inserted into recombinant plasmid pJBPH downstream of the divergent promoter region.  68  4.2.2 β-galactosidase assays β-galactosidase assays (Miller 1992) were performed to study the activity of the puc/bph promoter in plasmids where transcription of the lacZ coding region was dependent on the promoter in question. Plasmids pJPUC::lacZ and pJBPH::lacZ were transformed into wild type CGA009 and ppsR1-2- mutant strains by conjugation with E. coli S17-1. Cells grown in the absence of illumination under aerobic and semiaerobic conditions, or anaerobically under highlight or low-light conditions (2.2.3.1), were harvested at mid-log phase. The cells were disrupted in a FastPrep® Cell Disrupter (Abiogene Inc.) at speed 6 for 3 x 30 seconds. The supernatant after bead-beating was collected after centrifugation at 14 krpm for 5 min in a microcentrifuge and used for β-galactosidase assays. β-galactosidase activities were determined by a colorimetric assay with ortho-nitrophenyl-β-D-galactopyranoside (ONPG). Activities were reported as Miller units, where 1 unit of β-galactosidase is the amount of enzyme that produces 1 µmol of onitrophenol min-1 at 420 nm at 37° C or RT and pH 7 using ε = 4.5 X 103 M-1 cm-1, and with the method of Lowry et al. (1951) used for protein estimation with bovine serum albumin as the standard.  4.3 Results 4.3.1 Construction of lacZ transcriptional plasmids The lacZ gene was cut out from broad host-range vector pHRP309 (Table 2.2) using Hind III and Xbal I and ligated with Hind III/Xbal I-cut pJPUC and pJBPH vectors, downstream of the divergent puc-bph promoter region (Fig 4.3). The pJPUC::lacZ and pJBPH::lacZ transcriptional plasmids (Table 2.1) were introduced into R. palustris CGA009 wild type and ppsR1-2- mutant strains by conjugation with E. coli S17-1. 69  Figure 4.3 Representation of the puc-bph::lacZ fusions used to investigate puc and bph gene expression.  4.3.2 β-galactosidase assays The specific activities of β-galactosidase were measured in cell extracts of R. palustris wild type CGA009 and ppsR1-2- double knockout mutant strains (Table 2.1) containing puc::lacZ and bph::lacZ fusions, grown aerobically or semi-aerobically without illumination, or anaerobically with two light intensities (high light and low light), to mid-log phase. Comparison of β-galactosidase specific activities between wild type strain CGA009 grown under different oxygen levels shows that expression of both puc::lacZ and bph::lacZ were higher when induced under anaerobic photosynthetic conditions (Fig 4.4A). However the difference was much greater in puc::lacZ expression: it was 17-fold higher in cultures that were grown under high light compared to the aerobic condition, whereas bph::lacZ had the highest expression under low light condition that was only 2-fold greater than the lowest expression in semi-aerobic induction. The overall expression of bph::lacZ was relatively low under all conditions used (<100 Miller units). Fusions puc::lacZ and bph::lacZ were also expressed in the R. palustris ppsR1-2- double knockout mutant strain (Braatsch et al. 2007). Expression of the puc promoter was lower in the ppsR1-2- knockout grown under all four conditions: the β-galactosidase activity was 12% and 70  42%, respectively, in the ppsR1-2- strain grown under aerobic and semi-aerobic conditions compared to the wild type strain; whereas the ppsR1-2- strain had only 2% activity under anaerobic photosynthetic conditions with both high and low light intensity (Fig 4.4B). In contrast, expression of the bph promoter was upregulated in ppsR1-2- double knockout strain under most conditions, but no significant change in β-galactosidase activity was observed in ppsR1-2- knockout under low light condition (Fig 4.4C). Comparison of β-galactosidase specific activities between wild type strain and ppsR1-2- mutant strain shows that expression of the bph::lacZ fusions is 2-fold and 2.5-fold higher in the mutant strain than the wild type strain under aerobic and semi-aerobic conditions, respectively (Fig 4.4C). The upregulation of bph expression by ppsR1-2- is 1.6-fold when induced under high light condition.  71  Figure 4.4 β-galactosidase specific activities. Cultures were grown aerobically or semi-aerobically without illumination, or anaerobically with two light intensities (high light and low light). Cells were harvested at mid-log phase. (A) Comparison of rpa1491 (puc::lacZ) (white bar) and rpa1490 (bph::lacZ) (black bar) promoter activity in wild type CGA009. (B) and (C) Strain differences in β-galactosidase activities of (B) puc::lacZ and (C) bph::lacZ plasmidborne gene fusions. The β-galactosidase activities in the wild type strain CGA009 (white bars) and the ppsR1-2- double knockout strain (grey bars) are compared.  72  4.4 Discussion A 346-bp divergent promoter region between the RPA1490 and RPA1491 coding regions was used to express the Bph protein RPA0990 in Chapter 2 of my thesis. There were no plasmids containing strong promoters for expression of cloned genes in R. palustris when I began my work. However, it was known that the LH2 proteins encoded by puc are among the most abundant proteins in the cell when fully induced (J.T Beatty, personal communication). This chapter has focused on the expression activity of two promoters pucBe (rpa1491) and bph3 (rpa1490). My results show that the pucBe promoter has much higher activity than the bph3 promoter, and reaches the maximum expression under anaerobic (photosynthetic) high-light induction. I also studied the effects of a ppsR1 and ppsR2 gene double knockout on the level of expression of a lacZ gene driven by the bph and puc promoters. I found that double knockout of ppsR1 and ppsR2 led to an increase in bph::lacZ expression, and a decrease in puc::lacZ expression. How exactly the PpsR proteins regulate pucBe (rpa1491) and bph3 (rpa1490) expression however remains unclear and needs further research. Transcription from LH2 promoters (five promoters, driving expression of five pucBA gene pairs) can be induced simply by growing cultures under reduced aeration (Braatsch et al. 2006). In R. capsulatus and now in R. palustris, the puc promoter has been used to express plasmid-borne cloned genes. In previous work by Tadros et al. (1993), the pucBe promoter from R. palustris strain 1e5 was proven to have the strongest expression among all five puc promoters (pucBAa to pucBAe), based on lacZ fusions. This chapter of the thesis on the expression of a pucBe-bph3 (rpa1491-rpa1490) divergent promoter region has compared β-galactosidase specific activity from puc::lacZ and bph::lacZ fusions. It was shown in this study that pucBe from R. palustris wild type strain CGA009 indeed is a strong promoter, especially when induced under anaerobic photosynthetic 73  conditions. It was previously reported that the promoter pucBe of R. palustris strain 1e5 fused to lacZ yielded high expression (18085 U) when grown anaerobically under high light intensities and lower expression (830 U) under low light intensities (Tadros et al. 1993). This 22-fold difference in pucBe promoter activity under high light vs. low light compared to the 1.2-fold difference in my study may rise from several differences: 1) strains, 2) start and end point of promoter region used, and 3) light intensities: the difference between high light and low light intensities in Tadros et al.’s work (1993) was 1000 fold while it is only 30 fold in my study. This indicates that higher light intensity induces higher expression of pucBe promoter, which is in agreement with a previous proposal that the number and composition of photosynthetic units is highly variable and depends on the light intensity and oxygen partial pressure (Tadros et al. 1993). In all, the findings suggest that instead of being induced by reduced aeration in darkness, the puc promoter can be induced to even higher levels under anaerobic, illuminated conditions. On the other hand, bph3 (rpa1490) was shown to be a relative weak promoter. Also the differences in bph3 promoter activity over different growth conditions were not greatly different (did not exceed a factor of 2) compared to puc promoter (30 fold when comparing extreme values). This indicates that the expression of Bph3 (RPA1490) is not highly regulated under the aeration and light conditions used in my work; perhaps RPA1490 is more-or-less constitutively transcribed and functions to trigger downstream phosphorylation pathways by a kinase activity regulated by light in addition to the previously described redox condition (Vuillet et al. 2007). Although it lacks the conserved Cys residue that binds the bilin chromophore biliverdin (Fig 2.2), RPA1490 affects the light-intensity regulation of expression of photosynthesis genes as indicated by absorption spectra of rpa1490 mutant cells (Fig 3.1). I speculate that RPA1490 might function as a phosphotransfer protein that connects a Bph kinase and a response regulator.  74  PpsR proteins have been shown to be transcriptional regulators with a dual role in photosynthetic bacteria. Most PpsR proteins are repressors when cells are grown under high aeration, but one appears to be a low aeration inducer of some genes (Steunou et al. 2004). Most species have a single PpsR protein, but it was found that there are two PpsR proteins in R. palustris, both of which function as oxygen-responsive transcriptional repressors (Braatsch et al. 2007). However I found that double knockout of PpsR1 and PpsR2 led to an increase in bph::lacZ expression and a decrease in puc::lacZ expression. One possibility is that Bph3 (RPA1490), as one of the proteins involved in photosynthesis that regulate LH2 synthesis according to the light or redox conditions (Kojadinovic et al. 2008), is repressed by PpsR proteins. Alternatively, one would expect Bph3 activity to change, but not necessarily Bph3 levels. Yet as observed in the results of my β-galactosidase assays, the repression was small: only 2.6-fold under semi-aerobic condition and 2-fold under aerobic condition. Interestingly, Kojadinovic’s recent work (2008) has reported 104 genes potentially regulated by PpsR2 in R. palustris based on the presence of palindromic sequence [T,C]G[A,T]N12[T,A]C[G,T,A] in putative promoter regions, among which Bph3 is the only Bph out of six Bph-like proteins identified to be regulated by PpsR2 . If this prediction is true, it would be interesting to know why PpsR2 functions to regulate only Bph3 but not other Bphs. It is interesting to speculate whether this is due to the unique redox-sensing ability of Bph3, because PpsR2 is predicted to be an activator of respiration activity (Kojadinovic et al. 2008). It was surprising that the double knockout of ppsR1 and ppsR2 led to a decrease in puc expression. PpsR proteins have been shown to regulate expression of LH2 antennae in many photosynthetic bacteria, such as an activator of pucBA expression in R. gelatinosus (Steunou et al. 2004), a repressor of photosystem synthesis (Braatsch et al. 2006), and an activator of LH2 in R. palustris (Vuillet et al. 2007). Vuillet et al. have shown that PpsR2 from R. palustris CEA001 75  strain binds to pucBAb/e promoters and represses the synthesis of the entire photosynthetic unit, and they also proposed PpsR1 functions as an activator for LH2 synthesis (Vuillet et al. 2007). However Braatsch et al. (2006) reported that the inactivation of either of these PpsR proteins in CGA009 leads to derepression of most photosystem genes. The β-galactosidase data in my study support the proposal that the absence of both of the two PpsR proteins results in activation of pucBe expression (Fig 4.4B). The model of PpsR protein functions in R. palustris needs further investigation, as the control of photosystem synthesis is complicated and differs between species. My work on the activity of a puc promoter under different environmental conditions enables us to make better use of this puc promoter for expressing cloned genes.  76  Chapter 5 -THESIS CONCLUSIONS This work was initiated by the discovery of the presence of two particular ORFs encoding Bph-like proteins in the R. palustris genome sequence. Unlike the other four bph genes, rpa0122 and rpa0990 are located further away from the photosynthetic gene clusters (Fig 1.5). In Chapter 2, I expressed and purified His-tagged RPA0990 in R. palustris and found that it is capable of binding chromophores, and switching between Pr and Pfr forms. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990 were determined to be 695 nm and 755 nm, respectively (Fig 2.11B). The Pr form is predicted to be the active form and capable of phosphorylation. Investigation of the phenotype by comparing the absorption spectrum of the rpa0122 and rpa0990 mutants to that of the wild type strain revealed that both of these Bphs may have a small effect on the amount of LH1 per cell (Fig 2.16B and Fig 2.17B). Chapter 3 described an experiment of growing wild type cultures under rigorously anaerobic conditions, because it appeared that the absence of O2 does not inhibit the normal function of Bphs that utilize a linear tetrapyrrole such as biliverdin. The results showed that there is no significant change in the absorption spectrum of R. palustris wild type strain CGA009 after more than 10 doublings, when growing under a low-light, rigorously anaerobic condition (Fig 3.3). Thus I conclude that a linear tetrapyrrole such as biliverdin is made under this rigorously anaerobic condition, and suggest two possible pathways of making biliverdin anaerobically: 1) linear tetrapyrrole can be made through heme cleavage (perhaps hydrolysis) by a novel anaerobic enzyme; 2) or directly from the heme precursor hydroxymethylbilane (Beale 1995) without ring cleavage. Future experiments include expressing His-tagged RPA0990 from R. palustris cultures grown under both rigorous anaerobic and aerobic conditions. Upon purifying His-tagged RPA0990 holoprotein, the bound cofactor can be released and the structure deduced from the mass in mass spectrometry. This may answer the question of whether a different linear 77  tetrapyrrole is made in response to the presence or absence of O2 level. Furthermore, two hmuOhomologous ORFs (rpa1539 and rpa3279) both encoding a putative HO, can be knocked out to investigate the potential pathways of making a linear tetrapyrrole under aerobic vs. anaerobic conditions. In Chapter 4, I evaluated the activity of a divergent promoter region by using the E. coli lacZ gene as a reporter. The results showed that the pucBe promoter is a strong promoter and has much higher (30-fold) activity than the bph3 promoter (Fig 4.4A). I also found that high light intensity induces high expression of pucBe promoter (Fig 4.4A). It is interesting to notice that the double knockout of the ppsR1 and ppsR2 regulatory genes led to an increase in bph3::lacZ expression, and a decrease in pucBe::lacZ expression. These results indicate that PpsR proteins might function to suppress the expression of Bph3, and also suggest that at least one of the two PpsR proteins functions as an activator for pucBe expression.  78  REFERENCES Aravind, L. and C. P. Ponting (1997). 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