Oxygen-dependentkau.diva-portal.org/smash/get/diva2:906974/FULLTEXT01.pdf · Det har varit en spännande och lärorik resa och jag har många att tacka för arbetet som ligger till

Oxygen-dependent regulation of key components in microbial chlorate respiration

Miriam Hellberg Lindqvist

Miriam

Hellberg Lindqvist | O

xygen-dependent regulation of key components in m

icrobial chlorate respiration | 2016:13


Contamination of perchlorate and chlorate in nature is primarily the result of various industrial processes. The microbial respiration of these oxyanions of chlorine plays a major role in reducing the society’s impact on the environment. The focus with this thesis is to investigate the oxygen-dependent regulation of key components involved in the chlorate respiration in the gram-negative bacterium Ideonella dechloratans. Chlorate metabolism is based on the action of the enzymes chlorate reductase and chlorite dismutase and results in the end products molecular oxygen and chloride ion. Up-regulation of chlorite dismutase activity in the absence of oxygen is demonstrated to occur at the transcriptional level, with the participation of the transcriptional fumarate and nitrate reduction regulator (FNR). Also, the chlorate reductase enzyme was shown to be regulated at the transcriptional level with the possible involvement of additional regulating mechanisms as well. Interestingly, the corresponding chlorate reductase operon was found to be part of a polycistronic mRNA which also comprises the gene for a cytochrome c and a putative transcriptional regulator protein.

DOCTORIAL THESIS | Karlstad University Studies | 2016:13 DOCTORIAL THESIS | Karlstad University Studies | 2016:13

ISSN 1403-8099

Faculty of Health, Science and TechnologyISBN 978-91-7063-692-9

Chemistry

DOCTORIAL THESIS | Karlstad University Studies | 2016:13



Print: Universitetstryckeriet, Karlstad 2016

Distribution:Karlstad University Faculty of Health, Science and TechnologyDepartment of Engineering and Chemical SciencesSE-651 88 Karlstad, Sweden+46 54 700 10 00

© The author

ISBN 978-91-7063-692-9

ISSN 1403-8099

urn:nbn:se:kau:diva-40698

Karlstad University Studies | 2016:13

DOCTORIAL THESIS



WWW.KAU.SE

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Abstract


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Tack

Jag är så oerhört tacksam för att jag har haft förmånen att arbeta vid Karlstads universitet med så många härliga och inspirerande människor. Det har varit en spännande och lärorik resa och jag har många att tacka för arbetet som ligger till grund för denna avhandling. Ett stort tack till min handledare Thomas Nilsson för att du alltid tagit dig tid att lyssna, kommentera och ge vägledning vad gäller både forskning och olika uppdrag som jag har haft under min doktorandtid. Ditt stöd och engagemang har varit väldigt värdefullt för mig. Jag vill även tacka min biträdande handledare Maria Rova som har bidragit med så mycket kunskap till detta projekt. Tack för alla goda råd och ditt stöd i mitt arbete på lab. Tack Anna för all din hjälp och support genom åren! Jag är så oerhört tacksam över att jag haft dig som doktorandkollega. Vad hade jag gjort utan dig? Jag vill också tacka alla övriga kollegor på min avdelning! Ett speciellt tack till Micke för att du alltid fixar allt och ger så kloka råd. Mina forna och nutida doktorandkollegor (och möjligtvis framtida doktorander) Hannah, Ola, Emelie, Christina, Anna-Sofia, Natasja, Erik, Alex, Sara och Maria, att ha fått dela doktorandtiden och dess olika vedermödor med er har varit en ynnest. Tack för all peppning när jag varit lite extra emotionell, alla trevliga fikastunder och djupa forskningsdiskussioner (och ibland inte så vetenskapliga samtal) under väntetider på lab. Slutligen, ett stort tack till min familj! Mamma och pappa för att ni alltid trott på mig. Er oerhörda kärlek betyder så mycket. Mina bröder Gustaf och Christian med familj, det betyder så mycket att veta att ni finns där i vått och torrt. Jag hoppas att jag inte alltid är en för bossig storasyster.

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Mina älskade små busfrön Siri och Milla. Tack för all den glädje och värme ni sprider omkring er. Ni är det finaste som finns! Per, min älskade livskamrat och bästa vän. Tack för att du finns vid min sida. Jag älskar dig!


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Table of contents

LIST OF APPENDED PAPERS ................................................................... 6

LIST OF ABBREVIATIONS ......................................................................... 8

1. INTRODUCTION ..................................................................................... 9

1.1. OXYANIONS OF CHLORINE ................................................................ 9 1.2. BIOLOGICAL WASTEWATER TREATMENT ........................................... 10 1.3. MICROBIAL ENERGY METABOLISM ................................................... 11

1.3.1. Aerobic and anaerobic bacteria .............................................. 11 1.4. CHLORATE- AND PERCHLORATE-RESPIRING BACTERIA ...................... 12

1.4.1. The biochemical pathway of (per)chlorate reduction .............. 12 1.4.2. Genes involved in (per)chlorate reduction .............................. 15

1.5. AIM OF PRESENT STUDY ................................................................. 18

2. THEORY ................................................................................................ 19

2.1. FROM DNA TO PROTEIN ..................................................................... 19 2.2. REGULATION OF GENE EXPRESSION ..................................................... 21 2.3. MOBILE GENETIC ELEMENTS ................................................................ 23

3. METHODS USED IN PRESENT STUDY ............................................... 24

3.1. POLYMERASE CHAIN REACTION ........................................................... 24 3.2. EXPRESSION ANALYSIS ....................................................................... 24

3.2.1. Reverse transcription polymerase chain reaction ..................... 24 3.2.2. Quantitative polymerase chain reaction ................................... 25

3.3. ENZYME ACTIVITY MEASUREMENTS ...................................................... 28 3.4. CREATION AND CLONING OF RECOMBINANT DNA .................................. 29

3.4.1. Reporter gene technology ........................................................ 30

4. RESULTS AND DISCUSSION .............................................................. 32

PAPER I ................................................................................................... 32 PAPER II .................................................................................................. 35 PAPER III ................................................................................................. 38 PAPER IV ................................................................................................. 44

5. CONCLUDING REMARKS .................................................................... 47

6. FUTURE PERSPECTIVE ...................................................................... 48

7. POPULÄRVETENSKAPLIG SAMMANFATTNING............................... 49

8. REFERENCES ...................................................................................... 54

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List of Appended Papers

I. Hellberg Lindqvist, M., Johansson, N., Nilsson, T., Rova, M. 2012. Expression of chlorite dismutase and chlorate reductase in the presence of oxygen and/or chlorate as the terminal electron acceptor in Ideonella dechloratans. Applied and Environmental Microbiology. 78:4380-4385.

II. Hellberg Lindqvist, M., Nilsson, T., Sundin, P., Rova, M.

2015. Chlorate reductase is co-transcribed with cytochrome c and other downstream genes in the gene cluster for chlorate respiration of Ideonella dechloratans. FEMS Microbiology Letters. 362:fnv019

III. Hellberg Lindqvist, M., Goetelen, T., Fors E., Nilsson T.,

Rova M. Anoxic induction of the chlorite dismutase gene of Ideonella dechloratans is dependent of the fumarate and nitrate reduction regulator, FNR. Submitted to Applied and Environmental Microbiology

IV. Hellberg Lindqvist, M., Nilsson, T., Rova M. An Fnr-type

transcriptional regulator is responsible for anoxic up-regulation of chlorite dismutase in Ideonella dechloratans. Manuscript

Reprints of Paper I and Paper III were made with permission from American Society for Microbiology. Reprint of Paper II was made with permission from Oxford University Press.

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My contribution to work in appended papers: Paper I: I performed the final experimental work published in this paper. Planning of experiments and preliminary results were performed by M. Rova and N. Johansson. I wrote parts of the manuscript. Paper II: I did all the experimental work in this paper. I wrote parts of the article. Paper III: I performed all the experimental work in this paper except for the mutagenesis and construction of plasmids pBBR1MCS-4-lacZ_cld(mut1), pBBR1MCS-4-lacZ_cld(mut2) and pBBR1MCS-2-lacZ_cld(82), which were created by T. Goetelen and E. Fors. I also wrote the first version of the manuscript. Paper IV: I performed all the planning and experimental work in this manuscript and part of the writing.

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List of abbreviations

ArcA Regulator protein, part of the two-component anoxic redox control system, ArcBA

ATP Adenosine triphosphate

cDNA Complementary deoxyribonucleic acid

Cld Chlorite dismutase

Clr Chlorate reductase

CRB Chlorate reducing bacteria

DNA Deoxyribonucleic acid

FNR Regulator protein for the fumarate and nitrate reduction

gDNA Genomic deoxyribonucleic acid

mRNA Messenger ribonucleic acid

PCR Polymerase chain reaction

Pcr Perchlorate reductase

qPCR Quantitative polymerase chain reaction

PRB Perchlorate reducing bacteria

qRT-PCR Quantitative real-time polymerase chain reaction

RNA Ribonucleic acid

RNAP RNA polymerase

RT-PCR Real-time polymerase chain reaction

TSS Transcription start site

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1. Introduction

The use of potentially harmful chemicals also includes the responsibility of appropriate management of these substances. Many human activities such as agriculture, transport and industry contribute to pollution of the biosphere. Whether the contaminants are placed in the water, air or soil, they can cause damage to ecosystems and can persist in nature for long time. It is very important to develop sustainable wastewater management systems to reduce the society’s impact on the environment.

1.1. Oxyanions of chlorine The present work deals with oxyanions of chlorine. Chlorine can combine with oxygen and form four different oxyanions; perchlorate (ClO4

−), chlorate (ClO3−), chlorite (ClO2

−) and hypochlorite (ClO−). The term (per)chlorate is used here for perchlorate and chlorate. Occurrence of these oxyanions in the environment is in general of anthropogenic origin such as by-products from industrial processes, disinfectants and herbicides. The main source of chlorate in the environment originates from the pulp- and paper industries when bleaching pulp with chlorine dioxide (Bergnor et al., 1987). Chlorate has historically been used as an herbicide and is also produced as a by-product in drinking water disinfection with hypochlorite. Chlorate is toxic to aquatic organisms, such as brown algae and marine microalgae (Rosemarin et al., 1994; van Wijk and Hutchinson, 1995), and can cause oxidative damage to red blood cells in mammals when continuously exposed (Condie, 1986; Couri et al., 1982). The oxyanion perchlorate has emerged as a contaminant in nature due to the use of perchlorate salts in products such as rocket fuel, missiles, airbags and fireworks. Perchlorate has been widely studied due to its inhibitory effect on iodine uptake in the thyroid gland in humans (Wolff, 1998).

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Both chlorate and perchlorate are chemically stable in water and occurrence in nature has become a problem due to their toxicity and long range aqueous migration capacity. Since the presence of oxyanions of chlorine in nature poses a threat to the environment and is a potential risk to human health (Coates et al., 1999; Renner, 2003; Urbansky, 2002; WHO, 2005), it is important with efficient wastewater treatment when using these compounds. Until recently, the only known natural occurrence of perchlorate in nature was as minor component in saltpeter deposits in Chile (Urbansky, 2002). However, it seems like both chlorate and perchlorate are produced during atmospheric processes (Kounaves et al., 2010; Rao et al., 2010), which can explain the ubiquitous presence of microorganisms metabolizing these chemicals on Earth.

1.2. Biological wastewater treatment For the removal of (per)chlorates different approaches have been tried, one of which is biological wastewater treatment (Malmqvist and Welander, 1992). This method has been shown to be efficient and simply involves confining naturally occurring microorganisms, mainly bacteria, which feed on chemicals in the wastewater. Although the process has been known for almost a century (Åslander, 1928) it is only in the recent decades that researchers have studied this process in more detail. Already in 1954 Bryan and Rohlich showed that microorganisms can reduce chlorate (Bryan and Rohlich, 1954), and today over 50 bacterial species capable of growth using chlorate or perchlorate have been identified (Achenbach et al., 2001; Bruce et al., 1999; Coates and Achenbach, 2004; Coates et al., 1999; Malmqvist et al., 1994; Rikken et al., 1996; Wallace et al., 1996; Waller et al., 2004; Wolterink et al., 2002; Wolterink et al., 2005). A major drawback with this technique is the conflict between effective removal of organic matter and effective removal of (per)chlorates in the wastewater. The efficient removal of organic matter requires oxygen. However, if the wastewater contains large amounts of oxygen, the microorganisms able to perform (per)chlorate reduction will use the oxygen instead of reducing chlorate (Yu and Welander, 1994).

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Different approaches are used in pulp mills today to generate oxygen-limited conditions for chlorate remediation.

1.3. Microbial energy metabolism Microorganisms need energy to support anabolic processes, such as the synthesis of biomolecules, and growth. Energy is obtained from catabolic processes where complex molecules are decomposed into smaller units. The energy provided from the catabolic processes can be stored as chemical energy in the form of adenosine triphosphate (ATP) and be used to drive energy-consuming anabolic reactions. To generate ATP, different approaches have evolved, including aerobic respiration, anaerobic respiration and fermentation. In the electron transport chain, electrons donated by an electron donor, are passed along a series of membrane-bound enzyme complexes to a terminal electron acceptor. The transfer of electrons is coupled to the translocation of protons across the inner membrane, producing a proton gradient which is used to drive the synthesis of ATP. Microorganisms can utilize a wide range of terminal electron acceptors for their respiration. In aerobic respiration the terminal electron acceptor is oxygen. In the absence of oxygen this pathway is not possible. The ability to use alternative electron acceptors, such as fumarate, nitrate and chlorate, allows electron transport when the access to oxygen is limited. The use of respiration using electron acceptors other than oxygen is called anaerobic respiration. These alternative electron acceptors have lower reduction potentials than oxygen and, hence, less energy is produced during anaerobic compared to aerobic respiration. Therefore oxygen is often the preferred electron acceptor in respiration.

1.3.1. Aerobic and anaerobic bacteria The need for, or tolerance or sensitivity to molecular oxygen (O2) varies widely among microorganisms. Bacteria that use oxygen as terminal electron acceptor for energy metabolism are called aerobes. If the requirement for oxygen is absolute, they are called obligate aerobes. The opposite, obligate anaerobes, are unable to use oxygen

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and cannot grow in the presence of oxygen. Facultative anaerobes, on the other hand, use oxygen as the primary electron acceptor when it is present but alternative electron acceptors when the supply of oxygen is low. The switch from aerobic to anaerobic respiration can require synthesis of alternative terminal reductases. There are also bacteria referred to as microaerobes, that require oxygen but only at low levels.

1.4. Chlorate- and perchlorate-respiring bacteria Early investigations of the chlorate reduction microbiology suggested that this action was performed by nitrate-respiring organisms and chlorate was assumed to be an alternative substrate for the nitrate reductase (de Groot and Stouthamer, 1969). This, however, could not explain the existence of chlorate reductase C in Proteus mirabilis, an enzyme only able to use chlorate as a substrate (Oltmann et al., 1976a). The nitrate and chlorate reduction pathways in different prokaryotes has been found to have many similarities (Oosterkamp et al., 2011) and membrane-bound respiratory nitrate reductases have been shown to utilize chlorate as an alternative electron acceptor (Bell et al., 1990). Today it is known that specialized microorganisms have evolved that are able to couple growth to (per)chlorate metabolism. These bacteria are phylogenetically diverse, with representatives in the α-, β-, γ- and ε-Proteobacteria (Bardiya and Bae, 2011; Coates et al., 1999; Nilsson et al., 2013). Common to all strains are that they are facultative anaerobes or microaerobes. The model organism chosen for this study is Ideonella dechloratans (Malmqvist et al., 1994) which is a gram-negative rod and belongs to the β-subclass of Proteobacteria. I. dechloratans is reported to be cytochrome c oxidase positive (Malmqvist et al., 1994) and can utilize both oxygen and chlorate as terminal electron acceptors.

1.4.1. The biochemical pathway of (per)chlorate reduction During the biodegradation of perchlorate and/or chlorate, chlorite and oxygen are sequentially produced by the oxidation of acetate or

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other electron donor illustrated in figure 1. Perchlorate reducing bacteria (PRB) can reduce both perchlorate and chlorate to chlorite (step 1 and 2) with the enzyme perchlorate reductase (Pcr) (Kengen et al., 1999), while chlorate respiring bacteria (CRB) can only reduce chlorate (step 2) and use the enzyme chlorate reductase (Clr) (Danielsson Thorell et al., 2003; Wolterink et al., 2003). Both PRB and CRB possess a chlorite dismutase (Cld) that can decompose the chlorite to chloride ions and molecular oxygen (step 3) (van Ginkel et al., 1996). For the reduction of chlorate six electrons are required, whereas the reduction of perchlorate requires eight electrons. The free energy from the redox reactions in the electron transport chain creates a proton gradient over the cell membrane which provides the driving force for ATP production by an ATP synthase. The molecular oxygen produced during this process is assumed to further be used as electron acceptor due to the facultative anaerobic nature of bacteria able to perform dissimilatory (per)chlorate reduction (Rikken et al., 1996).

Figure 1 The (per)chlorate is reduced via a multi-step pathway. Perchlorate reducing bacteria can reduce both perchlorate and chlorate (step 1 and 2) with a perchlorate reductase enzyme, while chlorate reducing bacteria, which use the enzyme chlorate reductase, only can reduce chlorate (step 2). In both perchlorate and chlorate reducing bacteria a chlorite dismutase can be found that enables the last step (step 3) in the (per)chlorate metabolism.

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The chlorate metabolism in the CRB I. dechloratans is catalyzed by the enzymes chlorate reductase, Clr (Danielsson Thorell et al., 2003), and chlorite dismutase, Cld (Stenklo et al., 2001) and takes place in the periplasm as illustrated in figure 2. The transport of donated electrons includes both a membrane-bound terminal cytochrome cbb3 oxidase and a periplasmic cytochrome c able to function as electron carrier (Smedja Bäcklund et al., 2009; Smedja Bäcklund and Nilsson, 2011).

Figure 2 The proposed electron transport in I. dechloratans. The reduction of one chlorate molecule requires six electrons passed from the quinone pool; two are used by the chlorate reductase and the remaining four is passed on to the membrane-bound terminal oxidase and is used for the reduction of O2 to H2O. X illustrates the suggested presence of additional cytochromes able to donate electrons to the terminal oxidase, but not to chlorate reductase. The accumulation of protons in the periplasmic space is used as driving force for the ATP synthase in the production of ATP.

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During anaerobic conditions the bacteria must be capable of using the oxygen produced in the chlorite dismutase reaction. Oxygen at higher concentrations, though, has been shown to inhibit (per)chlorate reduction (Chaudhuri et al., 2002; Song and Logan, 2004).

1.4.2. Genes involved in (per)chlorate reduction The organization and transcriptional direction of the genes involved in (per)chlorate reduction differs between the bacterial strains possessing this trait. In PRB the operon pcrABCD can be found, encoding perchlorate reductase, and in CRB the operon clrABDC, encoding chlorate reductase, is found instead. Both PRB and CRB possess a gene encoding the chlorite dismutase (cld) which can be located either upstream or downstream the pcr or clr operon. The capability of (per)chlorate reduction has been shown to be subject of horizontal gene transfer over phylogenetic boundaries (Clark et al., 2013; Melnyk et al., 2011). This can explain that the key enzymes for (per)chlorate respiration have a wide dispersal over such a diverse group of prokaryotes. The genes involved in the reduction of (per)chlorate in PRB and reduction of chlorate in CRB are flanked by insertion sequences and have shown to be part of genomic islands. The concept of genomic islands and the relation to horizontal gene transfer will be discussed in more detail in section 2.2. The variability of gene content and orientation in the cluster together with the diversity of insertion sequences between organisms indicate that these mobile genetic element have evolved multiple times (Clark et al., 2013; Melnyk et al., 2011). However, except for the key enzymes Pcr/Clr and Cld not much is known of the functionality of other genes included in these clusters.

1.4.2.1. Perchlorate reductase and chlorate reductase

Several perchlorate and chlorate reductases have been isolated and studied from different bacterial strains (Danielsson Thorell et al., 2003; Giblin and Frankenberger Jr., 2001; Okeke and Frankenberger, 2003; Steinberg et al., 2005; Wolterink et al., 2003) and have been found to originate from different classes of enzymes regarding

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structure and biochemical aspects (Bardiya and Bae, 2011; Nilsson et al., 2013). The perchlorate reductases known today consist of two different subunits (pcrAB) compared to the identified chlorate reductases which are built up of three different subunits (clrABC). Phylogenetic analysis of PcrA (Bender et al., 2005) and ClrA (Danielsson Thorell et al., 2003) have shown that these proteins are members of the dimethylsulfoxide (DMSO) reductase family, which in turn belongs to the family of molybdopterin-containing enzymes (Kisker et al., 1999). DMSO reductases can degrade oxyanions and the reaction can be summarized as follows: XO + 2H+ + 2e− ⇄ X + H2O (McEwan et al., 2002). The transfer of one oxygen atom is coupled to the relocation of two electrons between substrates and other cofactors, such as iron-sulfur clusters and heme (Hille, 1999; Kisker et al., 1999). The pcrD and clrD genes included in each of the clusters likely encode chaperone-like proteins thought to be involved in the insertion of the molybdopterin cofactor (Bender et al., 2005; Danielsson Thorell et al., 2003) and the pcrC gene, found in PRB, is suggested to encode a cytochrome c involved in the electron transport (Bender et al., 2005). Chlorate reductase has previously been isolated from I. dechloratans (Danielsson Thorell et al., 2003). The purified protein was found to contain molybdopterin and iron. An optical spectrum of the protein indicated presence of heme b, which later could be confirmed as part of the cytochrome b subunit, encoded by clrC (Karlsson and Nilsson, 2005).

1.4.2.2. Chlorite dismutase

Chlorite dismutase is a heme b-containing protein that has the ability to catalyze the decomposition of chlorite into molecular oxygen and chloride ions (Schaffner et al., 2015; van Ginkel et al., 1996) and can be found in bacteria capable of (per)chlorate respiration (Mehboob et al., 2009; Stenklo et al., 2001; van Ginkel et al., 1996). The enzyme is vital for the organism due to the high toxicity of chlorite to bacterial

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cells (de Groot and Stouthamer, 1969; Oltmann et al., 1976b) and for the production of oxygen that can be utilized by a terminal oxidase. The reaction usually associated with the formation of a novel oxygen-oxygen bond is photosynthetic water oxidation and the production of molecular oxygen catalyzed by the Cld enzyme (Goblirsch et al., 2010; Streit and DuBois, 2008). The I. dechloratans Cld enzyme, located in the periplasm, has previously been isolated and purified (Stenklo et al., 2001) followed by the cloning and characterization of the cld gene (Danielsson Thorell et al., 2002).

1.4.2.3. Additional genes found in (per)chlorate respiring clusters

In the gene clusters for (per)chlorate reduction different families of c-type cytochromes have been identified (Bohlin et al., 2010; Melnyk et al., 2011). These are predicted to be involved in the electron transport of (per)chlorate reduction, but none of them have as yet a validated function in vivo. The putative cytochrome c gene (cyc) in the chlorate respiring cluster of I. dechloratans has previously been heterologusly expressed in E. coli, but it could not be verified if the reconstituted protein could donate electrons to purified Clr (Bohlin et al., 2010). Instead another chromosomally encoded cytochrome c, denoted cyt c-Id1, was shown to deliver electrons to chlorate reductase (Smedja Bäcklund and Nilsson, 2011), as illustrated in figure 2. Along with the cyc gene also a mobB gene and an arsR gene is found in the chlorate respiring cluster in I. dechloratans (Bohlin et al., 2010; Clark et al., 2013). The mobB gene is thought to encode a gene product that is involved in the molybdenum co-factor biosynthesis (Iobbi-Nivol et al., 1995) and arsR is a putative transcriptional regulator usually associated with sensing metals, but has been shown to be involved in other non-metal signals as well (Gao et al., 2012).

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1.5. Aim of present study The main focus of the research presented in this thesis was to investigate the oxygen dependent regulation of proteins involved in the anaerobic respiration of chlorate in I. dechloratans. It is important with deeper understanding regarding the regulation of these corresponding genes in response to different growth conditions for an efficient use of chlorate-reducing bacteria in bioremediation applications. The aims with the subprojects in this thesis have been to:

• Investigate the effect of oxygen on the expression of enzymes involved in chlorate respiration (paper I).

• Investigate the expression and regulation of additional genes (cyc and mobB) present in the chlorate respiring genomic island (paper II).

• Investigate potential regulatory sequences using a reporter system to obtain more detailed understanding of the mechanisms involved in the anaerobic induction of enzymes responsible for chlorate respiration (paper III and IV).

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2. Theory

The adaption to different growth conditions requires that the cell can regulate its content of metabolic enzymes to suit current needs. The specifications for all cellular proteins are encoded in the corresponding genes, which together constitute the genome.

2.1. From DNA to protein When a cell requires a specific protein, the DNA sequence encoding the corresponding gene is copied into a messenger RNA (mRNA) in a process called transcription. Following transcription, translation, i.e. the reading of the mRNA to make a protein, begins. Bacterial transcription is carried out by the multi-subunit RNA polymerase (RNAP). The subunits α2ββ'ω form the RNAP core enzyme which is capable of DNA-dependent RNA synthesis. However, the core enzyme cannot locate genes by itself and needs to interact with a sigma (σ) factor to form the RNAP holoenzyme (α2ββ'ωσ) capable of initiation of transcription. Once RNA synthesis has been initiated, however, the σ factor dissociates and the core enzyme proceeds with RNA transcription. The σ factor recognizes a sequence upstream of the gene known as promoter. Promoters are located close to the transcription start site (TSS) of genes. The TSS is the first nucleotide of the RNA transcript and is numbered +1, nucleotides upstream of the TSS are given negative numbers. Bacteria often have a set of alternative σ factors which can recognize different promoter elements. The association of appropriate alternative σ factor with the RNAP core enzyme enables the redirection of transcription initiation from different promoters in response to environmental changes. The majority of known σ factors today belong to the σ70-family. Four different DNA sequence elements have been found which has been shown to be important for promoter recognition by the σ70 factor. These are the -10 element, the -35 element, the upstream promoter (UP) element and the extended -10 element (Browning and Busby, 2004; Paget and Helmann, 2003). The consensus sequences, i.e. the calculated order

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of most frequent nucleotides, of these elements have been determined by the analysis of numerous prokaryotic promoters. The -10 and -35 element are located about 10 and 35 base pairs, respectively, upstream of the TSS (figure 3). The consensus sequence for the -10 element is TATAAT, where the last T and the first A is the most conserved bases and seems to be most important for promoter melting (Heyduk and Heyduk, 2014). The most conserved nucleotides in the -35 element seem to be the first three bases (TTG) of the consensus TTGACA. For the spacer between the -35 and -10 element there is no known consensus sequence, but a consensus length of usually 17 bp. The -10 extended motif (TGn) can be found in a number of prokaryotic promoters and is located direct upstream of the -10 element. Presence of this DNA motif can be particular important for promoters that contain a -35 element that not match the consensus sequence that well (Dombroski, 1997; Keilty and Rosenberg, 1987; Kumar et al., 1993) or has a longer spacer between the -10 and -35 element (Mitchell et al., 2003). In some promoters an UP element can be found which comprises an AT-rich region located around -40 to -60 bp relative the TSS.

Figure 3 A typical structure of a σ70 bacterial promoter with -10 and -35 regions.

Promoters can also contain an extended -10 element and an upstream promoter (UP) element. The consensus sequences for the -10 (TATAAT), extended -10 (TGn) and -35 (TTGACA) elements are shown. The transcription start site (TSS) are marked as +1.

The RNA polymerase is designed to transcribe RNA, but not all RNA molecules need to be synthesized in the equal amounts. For a fully function promoter it is not necessary with presence of all four upstream elements, but the combination of these sequences and

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similarity to the known consensus determines the rate at which the RNAP holoenzyme forms a stable initiation complex with the promoter. A variation in the consensus sequence of just one nucleotide can decrease the rate of binding dramatically. The result is that RNA polymerase will recognize some promoters well (referred to as a strong promoter) and others less well (referred to as a weak promoter). In bacteria, the mRNA can contain more than one cistron, i.e. a region of DNA that encodes a polypeptide. Such mRNAs are called polycistronic mRNAs (Snyder and Champness, 2003) and can be regulated in such fashion that each gene in the mRNA is translated into an individual polypeptide independently. The regions between the cistrons can be very short and the open reading frames may even overlap. If the polycistronic mRNA contains more than one ribosome binding site, it is possible with simultaneous translation of more than one region of the mRNA.

2.2. Regulation of gene expression Bacteria use their genes very efficiently and do not transcribe mRNA for proteins that are not needed at a certain moment. The regulation of gene expression can occur by a variety of mechanisms and can take place at any step in the production of a functional protein, e.g. at the transcriptional, translational or post-translational level. In addition of transcriptional control by different σ factors, bacteria can also regulate transcription by proteins which act either as up-regulators or down-regulators of genes (Barnard et al., 2004; Browning and Busby, 2004; Lee et al., 2012). Some activator proteins bind to specific sites on DNA and then recruits the RNA polymerase to the target promoter by protein-protein interactions (Ptashne and Gann, 1997). Alternative mechanisms instead involve activator proteins that change the conformation of the target promoter and induce binding of the RNA polymerase (Brown et al., 2003). Another possibility is by pre-recruitment which involves the formation of an activator-RNA polymerase complex before binding to the target DNA promoter (Griffith et al., 2002; Martin et al., 2002).

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The fumarate and nitrate reduction (FNR) protein of E. coli is a transcriptional regulator, encoded by the fnr gene and is required, directly or indirectly, for the expression of numerous genes involved in the anaerobic metabolism (Crack et al., 2004; Fleischhacker and Kiley, 2011). The active FNR protein acts as a homodimer and each subunit has a DNA-recognition helix in the C-terminal that interacts with the sequence TTGAT (Eiglmeier et al., 1989) and binds to the DNA consensus sequence TTGATNNNNATCAA, referred to as the FNR-box. Exposure to oxygen converts the protein-bound [4Fe-4S]2+ cluster to its [2Fe-2S]2+ form which decreases dimerization and causes loss of the specific DNA binding ability (Khoroshilova et al., 1997). FNR homologues have been found in a variety of taxonomically diverse bacteria, not all using the same signaling mechanism as the E. coli FNR protein (Spiro, 1994). Another transcriptional regulator is the ArcA-protein which is part of the two-component anoxic redox control system, ArcBA (Bekker et al., 2010). ArcB is a sensory kinase that phosphorylates and, hence, activates the cytoplasmic response regulator ArcA during oxygen-limited conditions. The active form of ArcA (ArcA~P) then binds to DNA and can either repress the genes of aerobic metabolism during anaerobic conditions (Unden and Bongaerts, 1997) or activate the expression of operons encoding enzymes important for the adaption to microaerobic or anaerobic conditions (Lynch and Lin, 1996; Nesbit et al., 2012).

The transcription of a gene into an mRNA does not automatically mean production of the corresponding protein. Degradation of mRNA occurs all the time by ribonucleases and the lifetime of individual RNAs in the bacteria have shown to be dependent on the RNA-ribosome association (Deana and Belasco, 2005). The production of a functional protein can also be regulated at the translational or post-translational level by for example translational blocking by antisense regulation, in which a short complementary RNA binds to the mRNA and prevent binding to the ribosome or create a target for mRNA cleavage by ribonucleases (Thomason and Storz, 2010).

23

2.3. Mobile genetic elements Mobile genetic elements are DNA segments that can move within or between genomes. Horizontal gene transfer refers to the movement of genes between different species of bacteria and has played a major role for bacterial evolution (Gogarten and Townsend, 2005; Ochman et al., 2000) and can be mediated by several mechanisms (Frost et al., 2005; Holmes and Jobling, 1996; Thomas and Nielsen, 2005). The acquisition of certain genes can enhance the survival chances for a bacterium, as for example with antibiotic resistance or virulence genes (Juhas, 2015; Rice, 2002).

A transposable element, or transposon, is a mobile element that encodes enzymes which enable it to translocate within the same or to another DNA molecule (Mahillon and Chandler, 1998) and can be found in both eukaryotes and prokaryotes. The simplest transposable elements are the so-called insertion sequences or IS elements (Mahillon and Chandler, 1998). If two IS elements with the ability to transpose are located close to each other, the intervening DNA sequence becomes mobile. These entities are termed composite transposons (Wagner, 2006) and contain one or more gene(s) in addition to those needed for transposition. The term genomic island is used for a region in the genome distinct from its surroundings, often with an origin in horizontal gene transfer (Bellanger et al., 2014). Examples of features used to recognize genomic islands are different GC-content and unusual codon usage compared to the chromosome of the host (Bellanger et al., 2014; Juhas et al., 2009). They are often relatively large (10-200 kb) (Hacker and Kaper, 2000) and can contain genes which enhance the fitness of the host such as novel metabolic pathways or resistance to antibiotics (Dobrindt et al., 2004). Some genomic islands may have been translocated by transposase or recombinase genes encoded by the island itself, but other mechanisms are known as well (Bellanger et al., 2014; Juhas et al., 2009).

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3. Methods used in present study

3.1. Polymerase chain reaction DNA with a known sequence can be conveniently and rapidly multiplied in vitro using the polymerase chain reaction (PCR). This method can be used both to detect and to amplify specific sequences for the construction of recombinant DNA molecules with starting from as little as a single copy. To be able to perform a PCR one needs template DNA, primers, heat-stable DNA polymerase (usually Taq polymerase) and free nucleotides. The amplification then is carried out with repeated cycles of heat denaturation of the DNA, primer hybridization and extension of the template DNA. The amount of the specific DNA sequence increases exponentially since amplified products from the previous cycle serves as template for the next cycle of amplification. Typically 20 to 30 cycles is enough to generate sufficient amounts of DNA to be detected in a subsequent agarose gel electrophoresis or used for recombinant cloning. The applications of the PCR are numerous and have led to a large number of variants of this method.

3.2. Expression analysis The expression of genes is carefully controlled by bacteria and can be increased or decreased in response to environmental changes. To provide insights into gene function it is important to understand where and when specific genes are used. Today several methods are known that can be used to quantitatively measure the mRNA level from a certain gene. Analysis of expression can also be determined by directly measure protein levels.

3.2.1. Reverse transcription polymerase chain reaction Reverse transcription polymerase chain reaction (RT-PCR) is a method performed with RNA as template which is reversed transcribed during the process into an equivalent amount of complementary DNA (cDNA) by the enzyme reverse transcriptase (RT). RNA is extremely susceptible to degradation and can be difficult

25

to analyze. The ability to synthetize cDNA from an RNA template opens the possibility to study RNA with the same methods used for the more thermostable DNA molecule. RT-PCR can be performed with sequence-specific primers, oligo(dT) primers or random hexamer primers, alternatively with combination of these. The use of sequence-specific primers offer the greatest specificity but a new cDNA synthesis must be performed for each gene to be studied. If the purpose is to synthesize large pools of cDNA, other primer options are more suitable. Oligo(dT) primers can be used for eukaryotic mRNA that have a 3´- poly(A) tail. For non-polyadenylated RNA, such as bacterial RNA, random hexamer primers are the ideal choice to produce a large pool of cDNA molecules. The mixture of random primers consists of oligonucleotides representing all possible hexamer sequences. These primers can bind to all types of RNA molecules and gives random coverage to all regions of the RNA. This gives rise to a cDNA pool with various lengths of cDNA. In present study both random hexamer primers and sequence-specific primers have been used to synthesize cDNA from bacterial total RNA preparations.

3.2.2. Quantitative polymerase chain reaction The PCR reaction can be used for the quantitation of the amount of template present in a sample. In this case, it is not sufficient to analyze the end product after temperature cycling. Instead, the reaction is followed and quantified in real time by the use of a fluorescent reporter that interacts with double stranded DNA molecules. It is possible to use either DNA or RNA as template. When using RNA as template the quantitative PCR (qPCR) must be preceded by a RT-PCR step. Combining these two methods yields quantitative reverse-transcription PCR (qRT-PCR) which can be performed in one or two steps (Bustin, 2000). To validate that the signal obtained from the target are from the specific amplicon to be observed, it is important to include certain

26

controls in the PCR. Such controls are the no-template control (NTC) and the no-reverse transcriptase control (no-RT) which is required for qRT-PCR reactions and is used to verify successful genomic DNA (gDNA) removal. Carryover of gDNA in RNA samples is a concern when determining expression levels. If gDNA is present in the samples, it can be co-amplified with the target transcript of interest and give rise to invalid data. Therefore, it is of utmost importance to include the no-RT control in qRT-PCR reactions. When the reaction is followed in real time, progress curves such as those seen in figure 3 are obtained. Initially, the amount of product and the fluorescence increase exponentially, and then level off as DNA polymerase activity and the supply of primers becomes limiting.

Figure 4 The amplification curve for target and reference DNA in quantitative

PCR. The fluorescence signal from each sample is plotted against cycle number. To obtain the relative expression level (∆CT), the target CT value is normalized to the reference CT value.

The threshold cycle (CT) is defined as the point at which the fluorescence of the reporter dye increases over an arbitrary placed threshold. It is a relative measure of the amount of target nucleic acid

27

in a sample provided that the threshold is chosen so that it is crossed during the exponential phase of amplification. Depending on the goal with the experiment there are different ways to determine the appropriate quantification method. We have studied the relative expression of genes at different growth conditions. The relative expression level (∆CT) is obtained when normalizing the CT value from the target to the CT value from a reference gene. Genes with constant expression levels, so called house-keeping genes, are often good choices as reference genes. In our studies we have used a gene for 16S ribosomal RNA as normalization gene. The variability of the reference gene value represents the cumulative errors that can occur during the different stages of the procedure including variations in extraction yield, variations in reverse-transcription yield and efficiency of amplification (Bustin et al., 2009). Hence, the normalization of the target gene to the reference gene enables direct comparisons of the same target in different samples. In our gene expression studies we have used comparative quantification to calculate the fold difference by the 2−∆∆CT method (Livak and Schmittgen, 2001) (Eq. 1 and 2). This method can be used when comparing a target gene of interest in one sample (growth condition A) with the same target gene in another sample (growth condition B). The results are presented as fold- up or down-regulation of the growth condition A compared to growth condition B. Each target gene is normalized to the reference gene. ∆∆CT = �CT target A − CT reference A � − �CT target B − CT reference B � (Eq. 1) Fold difference of relative expression levels = 2−∆∆C T (Eq. 2) Equation 2 can be used provided that the reaction efficiency for all target and reference genes is as similar as possible. The efficiency of a PCR reaction depends on several factors including choice of primers, master mix and sample quality and can affect the CT value. If the

28

reaction is 100 % efficient, the amount of target sequence is doubled in each cycle. In practice the efficiency may be lower and it is then important for quantitation that the efficiency for amplification of target and reference sequences are known. If there is a variation in efficiency between targets it is possible to use alternative quantitative models that correct for amplification differences (Pfaffl, 2001).

3.3. Enzyme activity measurements Enzyme activity measurements have been performed to compare the activity of the two enzymes Clr and Cld during different growth conditions. The Clr activity was determined colorimetrically by following the chlorate dependent oxidation of reduced methyl viologen (MV) under anaerobic conditions. In our measurements the assay mixture consisted of a bis-tris solution, MV and periplasmic extracts containing Clr enzyme. The MV is reduced by the addition of dithionite and when a stable absorbance is reached, the reaction is started by the addition of chlorate. In its reduced form, the otherwise colorless MV is blue with an absorption maximum at A600 nm. The oxidation of MV is monitored spectrophotometrically at 600 nm and the linear decrease in absorbance can be used to calculate the Clr activity. Values were normalized to the total protein content, and one unit of activity is defined as the amount of enzyme required to oxidize 1 µmol MV min-1 mg protein-1. The Cld activity was measured by following chlorite-dependent oxygen evolution using a Clark-type electrode. Periplasmic extracts (including Cld enzyme) and phosphate buffer with the addition of EDTA was added to the reaction chamber and the reaction was started by the addition of chlorite. The time course of oxygen evolution in this assay is non-linear, so it is important that the initial reaction rate is used to calculate the enzyme activity. Also in this assay the samples was normalized to the total protein content. The enzyme activity is expressed as mmol O2 produced min-1 mg protein-1.

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3.4. Creation and cloning of recombinant DNA The definition of recombinant DNA is an artificially created DNA molecule in which DNA sequences have been brought together forming a new molecule not normally found in nature. Recombinant DNA technology enables individual fragments of DNA to be inserted into a vector, such as bacterial plasmids or bacteriophages, and amplified in bacteria. The reproduction of the recombinant DNA inside the host cell contributing to the production of many copies of the specific sequence is known as cloning. This technique has numerous practical applications and enables the expression of genes, and hence produces proteins, not originally found in the organism. The basic procedure is to obtain many copies of the DNA to be inserted in the vector and this can be achieved with PCR. The DNA is then cut with two different restriction enzymes followed by ligation with a vector cut with the same set of restriction enzymes. By the use of two different restriction enzymes, it is possible to control the direction of the fragment when inserted into the vector. A thus constructed recombinant molecule can be introduced into bacterial cells in different ways. A common method is by transformation of chemically competent cells. Typically these cells are exposed to divalent cations (often calcium chloride) under cold conditions and thereafter heat shocked, this makes the bacteria’s cell membrane more permeable and increases the likelihood that the bacteria take up the new DNA molecule. Another method is electroporation, in which an electric field is applied to cells, creating holes in the bacterial membrane and enables the bacteria to take up the vector (Weaver and Chizmadzhev, 1996). Transformation is a highly inefficient process. Vectors must therefore contain selectable markers to allow the selection of transformed organisms. Common selective markers are genes that confer resistance to antibiotics. It is possible to co-transform a bacterium with more than one plasmid. However, if two plasmids use the same mechanism for replication, they belong to the same incompability group and cannot co-exist in the same bacterium (Bergquist, 1987). If the two plasmids

30

belong to different incompability groups, the regulation of plasmid replication, and hence copy numbers of the plasmids, are independent and both can be stably maintained in the population.

3.4.1. Reporter gene technology There are numerous different vectors available. Expression vectors are designed in such way that cloned fragment is inserted downstream of a promoter in the vector. The cloned insert can then, hopefully, often be transcribed and translated into a protein. In our studies we have instead used reporter vectors, and these are constructed for studying regulating elements upstream of genes such as promoters. The promoter of interest is cloned and ligated upstream of a reporter gene as illustrated in figure 5.

Figure 5 Reporter vectors are constructed for the insertion of regulating elements, such as promoters, upstream of a reporter gene. Followed digestion by restriction enzymes (1) and ligation with the promoter(2), the vector is transferred into a host bacterium (3).The produced reporter gene can then be measured at the mRNA- or protein-level (4 and 5) in the host.

31

Reporter gene technology can be used to study gene regulation, the structure of regulatory elements or signaling pathways (Naylor, 1999). The main considerations when selecting a reporter gene are (i) that the activity of the reporter gene should be easily distinguished from the endogenous proteins produced in the host cell; (ii) ensure that the reporter activity do not interfere with enzyme activities in the host cell; (iii) the chosen assay for detection of the gene product should be sensitive and reproducible. In this work reporter gene technology has been used to study clr and cld promoter functionality. This has been performed by the use of a vector containing a promoterless lacZ gene, encoding the enzyme β-galactosidase. By promoter-lacZ fusion constructs transcriptional expression encoded by promoter regulating elements can be studied. The amount of active β-galactosidase, transcribed from the upstream promoter, can be monitored a by using a colorimetric β-galactosidase assay (Miller, 1972). The measured enzyme activity is thereafter calculated with equation 3.

Units = 1000 × OD420−1.75 ×OD550𝑡𝑡×𝑣𝑣×OD600

(Eq. 3)

The OD600 denotes the cell density before the assay, OD420 the absorbance of the o-nitrophenol and 1.75 × OD550 the light scattering of cell debris. Included in the formula are also the time of the reaction specified in minutes (t) and milliliters of the culture used in the assay (v).

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4. Results and discussion

Paper I In this paper we wanted to investigate if the presence of oxygen effected the expression of the I. dechloratans enzymes Cld and Clr. Both the enzyme activity and the mRNA levels were investigated under three different growth conditions; aerobic without chlorate, aerobic with chlorate and anaerobic with chlorate. Whole cell extract preparations of the cultures were used for enzyme activity measurements. The Cld activity was measured as chlorite-dependent oxygen evolution and the Clr activity was measured as chlorate-dependent oxidation of reduced methyl viologen. The enzyme activities were normalized to total protein content and the comparisons between different growth regimes were performed on cell cultures at different OD600-values. The highest activity was found for cells in mid log phase (OD 0.4-0.6), and therefore subsequent measurements were performed with bacterial cultures harvested within this span.

Figure 6 The measured enzyme activities of Cld (A) and Clr (B), normalized to

total protein content. Black circles represent cells grown under anaerobic growth conditions and white circles aerobic growth conditions.

Enzyme activities for both Cld and Clr could be measured at basal levels at aerobic growth conditions with oxygen as sole electron acceptor (figure 6). An induction in enzyme activity could be observed after transfer to an anaerobic chlorate reducing environment, for Cld

33

about five times and for Cld 200 times. Absence of oxygen was required for enzyme activity induction since no significant difference could be observed when adding chlorate to the aerobic cultures. To study the genetic regulation of the cld and clrA genes, the corresponding mRNA’s were analyzed using qRT-PCR. RNA preparations were performed followed by cDNA synthesis with RT using random hexamer primers. All three growth conditions were then analyzed in the same qPCR with primers specific for 16S rRNA, cld and clrA. The mRNA levels of the two target genes, cld and clrA, were normalized to the reference gene 16S rRNA.

Figure 7 The relative expression ratio of the cld (white) and clrA (grey) genes for comparison of different growth conditions. Anaerobic compared to aerobic without chlorate and aerobic with chlorate compared with aerobic without chlorate. The error bars indicate standard errors of the mean (SEM; n=9).

The results show that both genes are transcribed at all three growth conditions, which is consistent with the results from the enzyme activity measurements. The expression of both the genes cld and clrA was shown to be induced under anaerobic conditions with basal expression levels at aerobic growth. The presence of chlorate during

34

aerobic conditions did not result in an induction of either the cld or the clrA gene (figure 7). Based on the results in this study both the genes cld and clrA seems to be regulated at the transcriptional level. At anaerobic growth an eight- to nine-fold increase in mRNA levels can be observed for both genes. For the cld gene this can account for the observed increase in corresponding enzyme activity of about five times. For the clrA gene, on the other hand, the difference in corresponding enzyme activity is much greater, suggesting that other regulating mechanisms are also involved.

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Paper II In addition to the enzymes chlorate reductase and chlorite dismutase, chlorate respiration requires a carrier for the transport of electrons from the membrane-bound components of the respiratory chain to the periplasmic chlorate reductase. A c cytochrome, cyt c-Id1 encoded by the I. dechloratans chromosome, has been shown to be able to donate electrons to chlorate reductase and constitutes a major part of the periplasmic c cytochromes (Smedja Bäcklund and Nilsson, 2011). A candidate cytochrome c gene (cyc) is present in the gene cluster for chlorate respiration, but the corresponding protein has hitherto not been detected in the periplasm. This raises the question of the expression of the cyc gene. The gene has previously been cloned and heterologously expressed in E. coli, but the refolded gene product could not be shown to serve as an immediate electron transfer to Clr (Bohlin et al., 2010). The work presented in this chapter was initiated to investigate if the cyc gene is transcriptionally active in I. dechloratans, and if so, whether there is any difference in expression under different growth regimes. We were able to verify the presence of cyc mRNA in total RNA preparations from I. dechloratans by qRT-PCR. It was found that the mRNA levels increased about nine-fold when transferring cells from aerobic to anaerobic conditions. Moreover, the calculated ∆CT -values, that is the amount of cyc mRNA normalized to the reference gene, were very similar to those found for clrA under aerobic and anoxic conditions in paper I, indicating the same absolute copy numbers for cyc and clrA under both conditions. This suggested the possibility that both sequences are present in the same mRNA molecule, which is possible since the gene for cyc follows directly downstream the gene for clrC (figure 8). To investigate a co-transcription scenario, PCR reactions were set up with total cDNA as template. The primer pairs were constructed to span two open reading frames (ORF) with the forward primer specific for the 3´end in one ORF and the downstream primer specific for the 5´end in the nearest downstream ORF (figure 8). Precautions were taken to ensure elimination of gDNA before cDNA synthesis. A PCR

36

product could only be formed if the template polynucleotide, in this case the mRNA, contains both genes in the same fragment. PCR products with expected sizes for all reactions could be verified on an agarose gel. The PCR products were then ligated into the T-tailed vector pGEM-T Easy and transformed into E. coli XL-1 Blue cells followed by sequencing of the insert. The correct sequence targeted by the primers with cDNA as template could be confirmed. This shows that the genes clrABDC, cyc, mobB, arsR are co-transcribed in a polycistronic mRNA.

Figure 8 Gene arrangement in the chlorate metabolism cluster of I. dechloratans. clrA, clrB, clrC encode the α-, β- and γ-subunits of chlorate reductase respectively, clrD is assumed to encode a specific chaperone, cyc encodes a c-type cytochrome, mobB is assumed to encode a protein involved in molybdenum cofactor biosynthesis, arsR is a putative member of the ArsR transcriptional regulator family and h denotes a hypothetical protein. ISIde1 is an insertion sequence positioned between the clrABDC and cld genes and ISAav1 is one of the two insertion sequences flanking the composite transposon (Clark et al., 2013). Positions of primer pairs used in the PCR reactions are indicated with arrow heads. This figure is reproduced and reprinted from paper Hellberg Lindqvist, M., Nilsson, T., Sundin, P., Rova, M. 2015. Chlorate reductase is co-transcribed with cytochrome c and other downstream genes in the gene cluster for chlorate respiration of Ideonella dechloratans. FEMS Microbiology Letters. 362:fnv019, with permission from Oxford University Press.

An additional cDNA synthesis was performed to confirm this. This time a specific primer was used instead of random hexamer primers for cDNA synthesis. The reverse primer in the hypothetic protein was the only primer added to this reaction with total RNA as template.

37

PCR reactions were set up with cDNA as template, primer pairs used for the different reactions were: clrA-clrB, clrD-clrC and arsR-hypothetic protein. The products could be verified on an agarose gel as single bands, whereas no products could be detected in the no-RT controls from the cDNA synthesis containing all components without addition of RT. This verifies complete elimination of gDNA before cDNA synthesis. In paper II the expression of the cyc gene could be verified at the mRNA level. The transcript is quite long (over 5.1 kbp) and includes genes that are quite dissimilar regarding characteristics and function. If all genes included in the mRNA are translated into functional proteins still needs to be further investigated. It is not certain that the bacteria needs equal amounts of all corresponding proteins and the most logical scenario would then be an additional regulation at the translational level. Despite several attempts, the cyc gene product has not been found in cell extracts and it is possible that a role as electron donor to Clr has been taken over by the chromosomally encoded cyt cId-1 in I. dechloratans.

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Paper III In order to investigate the presence of regulating elements in the cld and clr promoters the sequences upstream of these genes were cloned into a broad host range reporter plasmid upstream of a lacZ gene and transformed into E. coli. The pBBR1MCS vector series (Kovach et al., 1995) was known to be possible to electroporate into I. dechloratans from earlier work and was chosen with the intention of future expression studies in I. dechloratans, after initial characterization in E. coli. The sequence upstream of the clr gene was ligated correctly to the broad-host range plasmid, as verified by sequencing, but did not function as a promoter in the reporter system. The sequence upstream of cld, on the other hand, was found to function as a promoter in the reporter system, with expression sensitive to the oxygen level and the presence of a functional FNR protein in the host. These results are presented in this paper. In a search for recognition sequences for known bacterial transcription factors (using the Virtual footprint server, http://www.prodoric.de/vfp), a putative binding site for FNR and a possible ArcA binding region have been noted (figure 9). To assess the role of FNR and/or ArcA as possible anaerobic transcriptional regulators, four reporter constructs were made; one with the wild type cld promoter (pBBR1MCS-4-lacZ_cld(200)), one with a shortened version of the cld promoter (pBBR1MCS-2-lacZ_cld(82)), one with a four-nucleotide substitution in the putative FNR-box (pBBR1MCS-4-lacZ_cld(mut1)), and one construct with a four-nucleotide substitution in the possible ArcA-binding region (pBBR1MCS-4-lacZ_cld(mut2)) (figure 9). All four promoter-lacZ fusion plasmids were transformed into E. coli XL-1 Blue cells. The functionality of the cloned regions as promoters was analyzed in the reporter system with a β-galactosidase assay as described in section 3.3.1. and the results are presented in figure 10.

39

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40

The β-galactosidase activity from cells containing the plasmid pBBR1MCS-4-lacZ_cld(200) grown under aerobic conditions was clearly detectable, and at a level about eight times above the background level obtained with the reporter plasmid without insert (pBBR1MCS-4-lacZ). After anaerobic growth, significantly higher activity was obtained. At anaerobic conditions an about four-fold induction of the reporter enzyme could be observed compared to the activity from aerobically grown cells and this is in the same order of magnitude as the induction of Cld activity measured in I. dechloratans (paper I). This result show that the regulatory sequences that account for the anaerobic induction of cld from I. dechloratans is present in the cloned fragment and are functional in the used E. coli reporter system.

Figure 10 Results from the β-galactosidase assays with E. coli XL-1 Blue cells or

RM101 harboring plasmids pBBR1MCS-4-lacZ (200, mut1 or mut2) or pBBR1MCS-2-lacZ (82). Neg K1 and Neg K2 show reported values obtained with plasmids pBBR1MCS-4-lacZ and pBBR1MCS-2-lacZ, respectively, without insert which serves as negative controls. The white bars represent values from aerobically grown cells and the grey bars anaerobically grown cells. The error bars indicate standard error of the mean (SEM; n=6-12).

41

The site-directed mutagenesis of four nucleotides in the putative FNR-box in plasmid pBBR1MCS-4-lacZ_cld(mut1) was found to knock out the transcription the downstream lacZ gene. No induction of β-galactosidase activity could be observed in the absence of oxygen and both the aerobic and anaerobic values were in the same order of magnitude, reduced to about two times the background level. This indicates that this region is critical for the transcription of the downstream gene, irrespective of cultivation conditions. The I. dechloratans genome was found to contain a gene that might encode a FNR homologue. It appeared therefore possible that the anaerobic induction of the cld gene is dependent of an FNR-type transcription factor. To further investigate the role of FNR as transcriptional regulator of the cld gene the plasmid pBBR1MCS-4-lacZ_cld(200) was transferred to the fnr-negative E. coli strain RM101 (Bekker et al., 2010). Under aerobic conditions the observed β-galactosidase activity was about nine times higher compared to the background level. This is comparable to the basal aerobic activity observed in E. coli XL-1 Blue with the same plasmid. No induction could be seen in RM101 cells grown under anaerobic fermentative conditions compared to the aerobically grown cultures (figure 10). This result demonstrates that the E. coli FNR protein is involved in the anaerobic induction from the cld promoter in the reporter constructs. FNR-dependent class II promoters usually have a single FNR binding site centered at-41.5 bp relative the TSS (Browning and Busby, 2004). Based on the position of the FNR binding site the most likely -35 and -10 promoter elements found is AACACAN14TGCGAAGAT and includes a putative -10 extended motif, as shown in figure 9. The plasmids pBBR1MCS-4-lacZ_cld(mut2) and pBBR1MCS-2-lacZ_cld(82) was shown to contain functional promoters. The relative β-galactosidase enzyme activity from both plasmids was about 40 % at aerobic conditions and >60 % at anaerobic conditions compared to measured values for the plasmid pBBR1MCS-4-lacZ_cld(200) containing the full-length wild type cld promoter. It thus appears that the mutated nucleotides in plasmid pBBR1MCS-4-lacZ_cld(mut2)

42

and the region missing in plasmid pBBR1MCS-2-lacZ_cld(82) affect the transcription pattern in the same way. The region upstream of the putative FNR-box seems to be needed for maximal expression at both aerobic and anaerobic conditions, but has greater impact on the aerobic expression. To be able to draw any conclusions regarding possible involvement of an ArcA protein in the reporter-system used, additional work is needed. However, no gene encoding an arcA homologue has been found in the I. dechloratans genome, and it is therefore questionable if the cld gene is regulated by an active ArcA~P protein in vivo in the host bacterium. Previous work revealed only one possible TSS in the cld promoter from I. dechloratans (Danielsson Thorell et al., 2002). The findings in this study, however, raise the question of the presence of alternative TSS, one for the basal aerobic expression and one for FNR-dependent anaerobic transcription (figure 9). Similar examples with overlapping promoters are known for other bacteria. As example the E. coli focApfl operon can be mentioned, which is under the control of two alternative TSS, separated by 10 nucleotides (Kaiser and Sawers, 1997). The transcription under aerobic growth is regulated by a factor independent promoter. In contrast, the anaerobic transcription is dependent on the presence of a functional FNR protein and transcribes from the alternative TSS. An analogous scenario for cld expression would be that aerobic transcription occurs from the promoter upstream of the previously determined TSS (figure 11). This promoter would then account for the basal aerobic expression of the cld gene. The anaerobic expression, on the other hand, takes place from the promoter downstream from the FNR-box and is dependent on the presence of a functional FNR protein.

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Figure 11 Illustration of a possible scenario with two alternative overlapping promoters in the region upstream of the cld gene. a) The anaerobic promoter consists of TSS (1), -10 (1) and -35(1) and the transcription from this promoter is dependent on the binding of a functional FNR protein. b) The aerobic promoter consists of TSS (2), -10 (2) and -35 (2) and account for the basal Cld activity observed at aerobic growth.

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Paper IV In this paper the candidate FNR homologue in the I. dechloratans genome is further investigated. The expression of the fnr gene in I. dechloratans was confirmed by the detection of the corresponding mRNA using qRT-PCR. These findings thereby support the conclusion in paper III suggesting an FNR-dependent induction from the cld promoter in the absence of oxygen. The expression levels of the I. dechloratans fnr gene was shown to be equal independent of cultivation conditions (aerobic vs. anaerobic). Previously studies of the expression from the cld promoter in plasmid pBBR1MCS-4-lacZ_cld(200) in the fnr-negative E. coli strain RM101 showed that only basal expression of the downstream gene could be observed at both aerobic and anaerobic growth (paper III). Therefore we wanted to investigate if the I. dechloratans FNR homologue could complement the fnr mutation in RM101. The I. dechloratans fnr gene, including promoter region, was amplified by PCR and cloned into the plasmid pBR322, creating pBR322_Id(fnr). RM101 was then co-transformed with pBR322_Id(fnr) or plasmid pBR322 together with plasmid pBBR1MCS-2-lacZ_cld(prom) or pBBR1MCS-2-lacZ. This resulted in RM101 clones with four different plasmid combinations presented in table 1.

Table 1 Designations of RM101 cells containing four different plasmid combinations.

Clone Plasmids

RM101(𝑓𝑓𝑓𝑓𝑓𝑓+𝑐𝑐𝑐𝑐𝑐𝑐+) pBR322_Id(fnr) pBBR1MCS-2-lacZ_cld(prom)

RM101(𝑓𝑓𝑓𝑓𝑓𝑓+𝑐𝑐𝑐𝑐𝑐𝑐−) pBR322_Id(fnr) pBBR1MCS-2-lacZ

RM101(𝑓𝑓𝑓𝑓𝑓𝑓−𝑐𝑐𝑐𝑐𝑐𝑐+) pBR322 pBBR1MCS-2- lacZ_cld(prom)

RM101(𝑓𝑓𝑓𝑓𝑓𝑓−𝑐𝑐𝑐𝑐𝑐𝑐−) pBR322 pBBR1MCS-2-lacZ

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The four clones RM101(𝑓𝑓𝑓𝑓𝑓𝑓+𝑐𝑐𝑐𝑐𝑐𝑐+), RM101(𝑓𝑓𝑓𝑓𝑓𝑓+𝑐𝑐𝑐𝑐𝑐𝑐−), RM101(𝑓𝑓𝑓𝑓𝑓𝑓−𝑐𝑐𝑐𝑐𝑐𝑐+) and RM101(𝑓𝑓𝑓𝑓𝑓𝑓−𝑐𝑐𝑐𝑐𝑐𝑐−), were grown in a defined medium under aerobic or batch fermentative conditions followed by a β-galactosidase assay. The clones RM101(𝑓𝑓𝑓𝑓𝑓𝑓−𝑐𝑐𝑐𝑐𝑐𝑐−) and RM101(𝑓𝑓𝑓𝑓𝑓𝑓+𝑐𝑐𝑐𝑐𝑐𝑐−) lacking the cld promoter served as negative controls. The measured activity from clone RM101(𝑓𝑓𝑓𝑓𝑓𝑓−𝑐𝑐𝑐𝑐𝑐𝑐+) was about eight times higher than the negative controls, independent of culture conditions. This result shows that the cld promoter is compatible with the transcription machinery in RM101. The basal transcription from the cld promoter is in the same order of magnitude independent of growth conditions and presence of a functional FNR protein. These data confirms previous results presented in paper III, but with another plasmid.

Figure 12 The result from the β-galactosidase assay from RM101cells with four

different plasmid combinations. The white bars represent aerobically grown cells and the grey bars anaerobically grown cells. The error bars indicate standard error of the mean (SEM; n=9).

The presence of I. dechloratans fnr gene in clone RM101(𝑓𝑓𝑓𝑓𝑓𝑓+𝑐𝑐𝑐𝑐𝑐𝑐+) resulted in an induction of β-galactosidase of about five times during anaerobic fermentative conditions compared to aerobic conditions.

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This shows that the I. dechloratans fnr gene is able to complement the ∆fnr mutation in RM101 and that the gene product can induce expression from the cld promoter to the same extent as seen with the E. coli XL-1 FNR protein in paper III. These findings thereby support the result in paper III indicating that the anaerobic transcriptional regulation of the cld gene is mediated by a functional FNR protein in vivo. In the E. coli FNR an N-terminal cluster of four cysteine residues can be found. Three of these together with one internal cysteine residue have been found to serve as ligands for the iron-sulfur cluster (Green et al., 2001). Corresponding regions in the I. dechloratans putative FNR homologue was also found to contain four cysteine residues; three in the N-terminal and one internal. This argues in favor for that the I. dechloratans gene encodes a true FNR homologue. Hitherto no attempts have been made to try to find this protein in the host bacterium and this remains to be done to confirm that the detected fnr mRNA actually is translated into a functional protein in vivo. The finding that a gene encoded outside the genomic island for chlorate respiration serves an important function is an interesting parallel to the role of cyt c-Id1 discussed in paper II, and may reflect integration of a function acquired by horizontal gene transfer into the regulatory machinery of the host.

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5. Concluding remarks

The aim with this study was to clarify how the genes involved in the chlorate respiration in I. dechloratans are regulated in response to different growth conditions. We have demonstrated that both the genes cld and clrA are transcribed both at aerobic and anaerobic conditions with an upregulation of about eight to nine times in the absence of oxygen. This can correspond for the upregulation seen for Cld enzyme activity of about five times at anaerobic growth compared to cells grown under aerobic conditions. For Clr enzyme activity, on the other hand, which is upregulated about 200 times in the same comparison it is possible that other regulating mechanisms are involved as well. The presence of chlorate itself during oxygen rich conditions did neither affect the transcription nor the enzyme activity of either of the two proteins. Interesting, the work presented in paper II demonstrates that clrABDC, cyc, arsR and mobB are co-trancribed in a polycistronic mRNA. It still needs to be clarified, though, if the genes downstream of the clr operon are translated. Due to difficulties in the transformation process of the bacterium I. dechloratans, we decided to work with an E. coli reporter system in paper III and IV. Unfortunately, we were not able to clone a functional clr promoter, but the anaerobic induction from the cld promoter, on the other hand, could be demonstrated to be dependent on the presence of a functional FNR protein in the host. Presence of fnr mRNA was demonstrated in I. dechloratans and the gene product was able to complement the ∆fnr mutation in E. coli strain RM101. In conclusion, these findings thereby support that the reported anaerobic induction of the Cld enzyme is mediated by a functional FNR homologue in vivo.

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6. Future perspective

To obtain a deeper understanding of the mechanisms involved in the anaerobic induction of enzymes responsible for chlorate respiration, also functional regions in the clr promoter have to be studied in more detail. It would be very interesting if one could be able to create functional clr promoter:lacZ fusion construct to be analyzed into a reporter system as for the cld gene. When using a reporter system there is always a risk that regulatory sequences present in the promoter region are not recognized by the transcription machinery in the reporter. Therefore, a complementary approach would be to study both cld and clr promoter:lacZ fusion construct into the host bacterium I. dechloratans. The pBBR1MCS-series of vectors were chosen for this study as they can be electroporated into the bacterium, although with very low efficiency. Several attempts have been made to electroporate I. dechloratans with the cld promoter:lacZ construct, unfortunately with as yet limited success. To pursue this line of work, improvement of transformation, as well as the establishment of conditions for reporter enzyme assay in I. dechloratans is needed. The cyc and arsR genes downstream of the clr operon were shown to be transcribed in paper II. The arsR might encode a transcriptional regulator and the cyc gene may encode a cytochrome c involved in the electron transport to Clr. Further investigations are needed to find out if these genes are translated.

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7. Populärvetenskaplig sammanfattning

Miljögifter i naturen kan finnas kvar länge och påverka oss människor i generationer. Med användingen av potentiellt skadliga kemikalier så ingår också ett ansvar som innefattar lämplig hantering av dessa. Blekning av pappersmassa med klordioxid inom pappers- och massaindustrin ger upphov till biprodukten klorat, en kemikalie som kan ha negativ inverkan på den omgivande miljön om den släpps ut i närliggande vattendrag. Kloraten som bildas kan renas bort från avloppsvattnet genom biorening, det vill säga rening med bakteriers hjälp. Nedbrytningen av klorat sker i två steg och resulterar i slutprodukterna syre och kloridjoner med hjälp av de två enzymerna kloratreduktas (Clr) och kloritdismutas (Cld). Syftet med denna avhandling har varit att studera om, och i så fall hur, dessa två enzymer påverkas av tillgången på syre i bakterien Ideonella dechloratans. Biorening

Själva bioreningstekniken har varit känd sedan början av 1900-talet och används idag världen över för att rena avloppsvatten från industrier och hushåll. Cirka 30 000 av de ämnen som återfinns i vår omgivande miljö har sitt ursprung i kemikalier som vi använder varje dag. Konceptet med biorening är relativt enkel och man använder sig av naturligt förekommande bakterier som bryter ner komplexa molekyler till mindre komplexa föreningar. Då det inte är möjligt för en enskild organism att bryta ner alla ämnen krävs en uppsättning med många olika sorters bakterier för att kunna ta hand om alla kemikalier som förekommer i avloppsvattnet. Hur effektiv nedbrytningen av vissa ämnen är bestäms även delvis av yttre faktorer såsom tillgång på syre, temperatur och pH. Det klorat som förekommer i naturen har generellt hamnat där på grund av olika utsläpp från industriella processer. Kemikalien har också historiskt använts som ogräsmedel och produceras som en biprodukt vid disinfektion av dricksvatten med hypoklorit. Klorat har

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visat sig vara giftigt för alger i Östersjön och kan orsaka hemolytisk anemi (vilket innebär att de röda blodkropparna går sönder) hos däggdjur om det intas via dricksvatten i höga doser under lång tid. Det är idag känt att kloratrespirerande bakterier kan användas för biologisk nedbrytning av klorat, I. dechloratans som vi studerar är en av dem. Som tidigare nämnt så sker nedbrytningen av klorat i I. dechloratans med hjälp av de två enzymerna Clr och Cld. Clr katalyserar nedbrytningen av klorat till klorit och Cld katalyserar nedbrytningen av klorit till kloridjoner och syre (figur 1). Det molekylära syre (O2) som bildas används av bakterien själv i en reaktion med vatten som sluprodukt. Hela denna kedja av reaktioner sker i bakterien för att i slutänden kunna producera kemisk energi i form av ATP (adenosintrifosfat). Denna energi kan sedan användas av bakterien för att exempelvis tillverka proteiner och att dela sig.

Figur 1 Nebrytningen av klorat till klorit katalyseras av enzymet kloratreduktas (Clr). Enzymet kloritdismutas (Cld) katalyserar i sin tur nedbrytningen av klorit till kloridjoner och syre. Syret som produceras används vidare i en ytterligare reaktion och ger upphov till slutprodukten vatten.

Om det finns syre närvarande i det omgivande avloppsvattnet kan detta ställa till med problem för nedbrytningen av klorat. Då syre finns att tillgå är det mer förmånligt för bakterien att använda detta än att bryta ner klorat och bakterien stänger av förmågan att bryta ner klorat temporärt. Denna förmåga kan dock återupptas igen när allt syre är konsumerat men fördröjningen kan vara lång. På grund av att den biorening som sker av andra ämnen i avloppsvattnet från ett pappersbruk ofta kräver syre, kan det här uppstå en konflikt mellan att effektivt bryta ner andra ämnen och nedbrytningen av klorat. Det är viktigt att öka förståelsen för hur och varför biorening av klorat

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fungerar för att på så vis kunna utveckla denna reningsmetod till att bli så effektivt som möjligt. Genreglering

Även om en bakterie väljer att stänga av produktionen av ett visst protein, till exempel tillverkningen av enzymerna Clr och Cld, så finns ändå generna som kodar för detta protein kvar. Genom att styra vilka gener som är aktiva och vilka som inte är det kan bakterien enbart producera de proteiner som behövs just för tillfället. Produktionen av proteiner är en mycket kostsam process och därför tillverkar bakterier enbart de proteiner som de har användning för just för tillfället.

Figur 2 Tillverkningen av proteiner i cellen kan delas in i två steg; i det första

steget (a), transkriptionen, så utgör genen på DNA-molekylen en mall för hur mRNA-molekylen ska se ut, i det andra steget (b), translationen, så översätts mRNA-molekylen till ett protein.

Koden för hur varje enskilt protein (inklusive enzymer) i en cell ska se ut finns lagrat i cellens DNA. En DNA-molekyl innehåller gener vilka utgör en mall för tillverkningen av mRNA-molekyler (efter engelskans messenger RNA) i en process som kallas transkription. I nästa steg,

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translationen, så används mRNA-molekylen som en ritning för tillverkningen av själva proteinet (figur 2). Alla celler, inklusive bakterier, kan själva styra vilka proteiner som bildas vid en given tid genom att reglera transkriptionen av en gen eller translationen av ett mRNA. Regleringen kan även ske av det färdiga proteinet. Om regleringen av en gen sker på transkriptionsnivå så påverkar det hur många mRNA-molekyler som bildas. En nedreglering innebär att färre mRNA-molekyler bildas (och därmed färre proteinmolekyler) medan en uppreglering är det motsatta och medför att fler mRNA-molekyler bildas (och även fler proteinmolekyler). Vad har denna avhandling bidragit till?

Arbetet i denna avhandling har handlat om att försöka förstå hur de gener som är involverade i nedbrytningen av klorat i I. dechloratans regleras i närvaro respektive frånvaro av syre och även tillgången på klorat. Vi har därför låtit bakterier få växa under tre olika förhållanden; i medium rikt på syre men inget klorat, i medium med både syre och klorat, i medium utan syre men med klorat. Efter några timmars tillväxt har bakteriecellerna löst upp (lyserats) och enzymaktiviteten av Clr och Cld har mätts i cellextrakten med lite olika metoder. När bakterierna får växa i medium som är rikt på syre (aerobt) så kan man bara uppmäta mycket låga aktiviteter för enzymerna Clr och Cld i cellextrakten. Det innebär att bakterien inte kan bryta ner klorat i någon större utsträckning när syre finns tillgängligt i odlingsmediet. Den aktivitet som har uppmätts i cellextrakt från bakterier som istället har fått växa utan syre (anaerobt) men med klorat är betydligt högre för båda enzymerna. Närvaron av klorat i sig hade ingen effekt så slutsaten blev att det är frånavon av syre som leder till en ökning av enzymaktiviteten för både Clr och Cld. Varken Clr eller Cld fyller någon funktion när syre finns att tillgå och det är onödigt kostsamt för bakterien att tillverka en stor mängd aktivt enzym som inte ska användas. Det var inte känt innan detta

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arbete om regleringen av Clr och Cld i bakterien I. dechloratans skedde på transkriptions- eller translationsnivå. Förutom att mäta mängden aktivt enzym vid de tre olika odlingsförhållandena valde vi därför att även mäta mängden mRNA som bildades av de båda enzymerna Clr och Cld. Intressant nog så visade dessa mätningar att ett större antal mRNA-molekyler producerades av både kloratreduktas och kloritdismutas vid anaeroba förhållanden vilket innebär att dessa båda enzymer regleras på transkriptionsnivån. Vi kan dock inte utesluta att även andra reglermekanismer kan vara involverade i regleringen av Clr. Aktiveringen av Cld har visat sig vara beroende av ett protein, kallat FNR. Denna typ av protein finns hos vissa bakterier och är endast aktivt i frånvaro av syre. Under anaeroba förhållanden hjälper FNR-proteiner till att slå på gener som behövs och stänga av gener som inte behövs när syre inte finns att tillgå. Hos bakterien I. dechloratans så binder troligtvis ett FNR-liknande protein till en sekvens på DNA-molekylen precis innan genen för kloritdismutas under anaeroba förhållanden. Detta leder till att produktionen av kloritdismutas mRNA-molekyler uppregleras i frånvaro av syre och det i sin tur innebär att många fler Cld enzymer bildas. Det är därför vi kan uppmäta en högre enzymaktivitet av Cld i cellextrakt anaerobt än aerobt. Förutom att vi kan uppmäta ett större antal mRNA-molekyler av kloratreduktas i frånvaro av syre, så råder det fortfarande en viss osäkerhet kring hur induktionen av Clr sker under anaeroba förhållanden. Detta spännande arbete hoppas jag att framtida doktorander i biokemi får möjlighet att undersöka djupare.

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