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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Nilsson AI, Zorzet A, Kanth A, Dahlström S, Berg OG, An- dersson DI. (2006) Reducing the fitness cost of antibiotic resis- tance by amplification of initiator tRNA genes, Proc. Natl. Acad. Sci, USA 103(18):6976-81. II Zorzet A, Pavlov MY, Nilsson AI, Ehrenberg M, Andersson DI. (2010) Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance. Mol Microbiol. 75(5):1299- 1313. III Pavlov MY, Zorzet A, Andersson DI, Ehrenberg M. Initiation factor 2 mutants promoting fast joining of ribosomal subunits in the absence of initiator tRNA or GTP. Manuscript (2010). IV Zorzet A, Nilsson AI, Andersson DI. Compensatory evolution restores fitness to actinonin-resistant Staphylococcus aureus. Manuscript (2010).

Reprints were made with permission from the respective publishers.

Contents

Preface ...... 11 Antibiotic resistance ...... 13 Definition of resistance ...... 13 Resistance mechanisms ...... 14 Prevalence of resistance ...... 15 Selective pressure and its impact on antibiotic resistance ...... 16 Antibiotic levels in the environment ...... 16 Rational antibiotic use ...... 17 Factors contributing to distribution and rate of resistance ...... 19 Spread of antibiotic resistance ...... 19 Horizontal gene transfer ...... 19 Natural competence (transformation) ...... 19 Plasmid transfer ...... 20 Bacteriophage transfer ...... 20 Integrons and conjugative transposons ...... 20 Mutation supply rate ...... 21 Resistance mutations and fitness ...... 22 Compensatory mutations and compensatory evolution ...... 23 Gene amplification ...... 23 Recombination mechanisms ...... 24 The Cairns system ...... 24 Model organisms ...... 25 Salmonella typhimurium ...... 25 Staphylococcus aureus ...... 25 Antibiotic targets ...... 26 Antibiotics that target the ribosome ...... 27 Peptide deformylase inhibitors ...... 27 Translation in prokaryotes ...... 29 The structure of the ribosome ...... 30 Initiation ...... 32 Initiator tRNA and formylation ...... 33 Initiation factor 2 ...... 34 Elongation ...... 36 Termination and ribosome recycling ...... 37

Present investigations, results and discussion ...... 39 Paper I ...... 39 Actinonin resistance in S. typhimurium confers a slow-growth phenotype ...... 39 Fitness loss in actinonin-resistant strains can be ameliorated by amplification of initiator tRNA genes ...... 40 Paper II ...... 40 Fitness loss in actinonin-resistant strains can be ameliorated by point mutations in initiation factor 2 ...... 40 IF2 mutants can initiate translation in vitro with high efficiency using either formylated or unformylated Met-tRNAi ...... 42 IF2 mutants can initiate translation with deacylated tRNAi or elongator tRNA ...... 42 Evidence for mRNA-limited protein synthesis ...... 43 Paper III ...... 43 IF2 mutants can induce efficient ribosome subunit association in the absence of tRNA ...... 43 IF2 mutants can induce efficient ribosome subunit association with GDP ...... 44 Paper IV ...... 45 Fitness loss in actinonin-resistant Staphylococcus aureus mutants can be ameliorated ...... 45 Whole genome sequencing of compensated, actinonin-resistant S. aureus ...... 45 Concluding remarks and future aims ...... 47 Acknowledgements ...... 50 References ...... 52

Abbreviations

DNA Deoxyribonucleic acid RNA Ribonucleic acid tRNA Transfer RNA rRNA Ribosomal RNA mRNA Messenger RNA nt Nucleotide bp Base pair aa Amino acid Da Dalton Met Methionine Phe Phenylalanine IF Initiation factor EF Elongation factor RF Release factor PIC Preinitiation complex A-site Aminoacyl site P-site Peptidyl site E-site Exit site PTC Peptidyl transferase center SD Shine-Dalgarno GTP Guanosine triphosphate GDP Guanosine diphosphate ppGpp Guanosine 5'-diphosphate 3'- diphosphate SDS Sodium dodecyl sulphate IS Insertion sequence MIC Minimal inhibitory concentration PDF Peptide deformylase PDFI Peptide deformylase inhibitor MDR Multi-drug resistance PBP Penicilling-binding protein MRSA Methicillin-resistant S. aureus ESBL Extended-spectrum beta-lactamases

Preface

Antibiotic resistance is an increasing global problem. With the increased mobility of goods and people, events in far-off countries have a real and often direct impact on us. The fraction of resistant bacteria in some countries is alarmingly high and we will soon find ourselves in the same situation if present trends continue. The increased emergence of strains of bacteria with resistance toward several or even all available antibiotics could return us to the pre-antibiotic era, where currently treatable diseases such as pneumonia and meningitis could once again be fatal. In this thesis I have chosen to focus on the present situation regarding antibiotic resistance and what can be done to improve it. In recent years, more effort has been put into understanding the mechan- isms underlying the development of antibiotic resistance. What are the ge- netic reasons for resistance, and why do some antibiotics elicit resistance in the bacterial population very rapidly, whereas others do so very slowly? What determinants are important for fixing resistant bacteria in the popula- tion and what can we learn from these features? We have investigated the emergence of resistance to a new class of antibiotics targeting processes of bacterial translation and assessed these resistant mutants in terms of the clin- ical risk they might impose. We have evaluated the fitness of the resistant mutants as well as their propensity to acquire secondary compensatory muta- tions when evolved. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus au- reus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms.. In the course of this work we also ex- amined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.

11 12 Antibiotic resistance

Even before the first use of penicillin over 70 years ago, antibiotic resistance existed. The organisms that are the original producers of most of the antibio- tics that we use today (very few antibiotics are of purely synthetic origin) employ mechanisms that make them resistant to their own products. Addi- tionally, organisms that have shared the same ecological niche over hundreds of millions of years have evolved strategies to cope with their neighbors' toxic secretions. For example, it has been estimated that the pathways to synthesize streptomycin have existed for over 600 million years (Baltz 2005). In an investigation of ~500 spore-forming soil bacteria, all tested bacteria were resistant to at least one of 21 different antibiotics. Moreover, the average bacterium was resistant to 7-8 different antibiotics (D'Costa et al. 2006). Thus, for environmental bacteria living in ever-changing sur- roundings, multi-drug antibiotic resistance is the norm, rather than the ex- ception (Martinez 2008). Fortunately, this is not yet the case for most of the bacteria that are relevant for human infectious diseases. However, in recent years the development of multi-drug resistance (MDR) in pathogenic bacte- ria, commensals and opportunistic pathogens that infect immuno- compromised individuals shows that we might be heading in that direction.

Definition of resistance Some bacteria are intrinsically resistant to certain antibiotics. However, the clinical resistance problem is caused by bacteria belonging to drug- susceptible species that have become genetically altered, sometimes by mu- tation, and sometimes by acquisition of foreign genetic material. Resistant mutants are thus bacteria that have reduced susceptibility to an antibiotic compared to their susceptible wild type counterparts. For practical reasons, a relative scale was developed in Sweden that classifies bacteria as Sensitive (S), Indeterminant (I) or Resistant (R), referred to as the SIR-system (SRGA 1981). These breakpoints are defined for most pairs of antibiotics and spe- cies of bacteria and are useful from a clinical perspective as they take into consideration the dose of antibiotic that is possible for treatment. Bacteria that fall in the sensitive category can be treated with that particular antibiot- ic, whereas bacteria in the interdeterminant range should be treated with that antibiotic only if it is absolutely necessary. Needless to say, the antibiotic is

13 counter-indicated if the bacteria fall in the resistant category. The lowest level of antibiotic at which growth is inhibited is referred to as the minimal inhibitory concentration (MIC). It is important to remember that genetic resistance and resistance accord- ing to the SIR system are not the same thing. Bacteria may acquire mutations that lead to reduced susceptibility, i.e. resistance mutations, and still be clas- sified as clinically sensitive. Often, several mutations are collectively needed before a strain is classified as clinically resistant.

Resistance mechanisms As mentioned above, bacterial resistance can be divided into intrinsic and acquired resistance. Intrinsic resistance is inherent in the species without the need for any genetic alteration. The drug target might fundamentally differ in the resistant species, such as cephalosporin-resistant enterococci where their penicillin-binding proteins have low affinity for cephalosporins (Thornsberry et al. 1974; Georgopapadakou and Liu 1980; Williamson et al. 1985). On the other hand, a phenotypic characteristic of the organism might render it naturally resistant. Examples are vancomycin resistance in Gram- negative bacteria where the outer membrane is impermeable to the antibiotic or the presence of efflux pumps in Pseudomonas aeruginosa (Nikaido 1989; Strateva and Yordanov 2009). As for acquired resistance, bacteria have developed many different strate- gies. In Table 1 some of the most common resistance mechanisms are listed along with selected examples of relevant antibiotics that provoke that mode of resistance. In the case of beta-lactam antibiotics, for example penicillin, there are two common mechanisms of resistance. In the first case, enzymes called beta-lactamases hydrolyse the characteristic beta-lactam ring struc- ture, inactivating the antibacterial activity of the drug. Secondly, penicillin- binding proteins (PBPs) that normally bind penicillin are either altered to decrease binding or overproduced to sequester the drug (Georgopapadakou 1993; Drawz and Bonomo). The relative importance of a particular mechan- ism in the development of penicillin resistance frequently differs between bacterial species such as E. coli and S. pneumoniae (Georgopapadakou 2004).

14

Table 1: Resistance mechanisms and examples of antibiotics that select for that mode of resistance Mode of resistance Antibiotic examples Inactivation of the antibiotic by specific Beta-lactams, Rifampicin, Tetra- enzymes cycline Export of the antibiotic by efflux pumps Tetracycline, Fluoroquinolones Alteration of drug target Macrolides, Penicillin, Aminogly- cosides, Chloramphenicol, Van- comycin, Linezolid, Rifampicin, Tetracycline Overproduction of target Trimethoprim Bypass mechanisms Peptide deformylase inhibitors, Sulfonamides, Trimethoprim Ribosomal protection mechanisms Tetracycline

Prevalence of resistance In many countries, the fraction of resistant bacteria is increasing at an alarm- ing rate. For example, dysentery-causing Shigella flexneri that are resistant to at least one of the antibiotics, ampicillin, chloramphenicol, tetracycline or cotrimoxazole (which are first-line drugs used to treat dysentry) are now prevalent in Africa (Naik 2006). As a result, nalidixic acid (a second-line choice) has been used more often in recent years, but resistance rates to this drug have already risen from 0% to approximately 11% (Iwalokun et al. 2001). Studies of cholera, caused by Vibrio cholerae, in the USA show a rapid increase in resistance. The disease is not endemic to the USA but comes from third world countries and when isolates were studied, it was found that the frequency of bacteria resistant to at least one antibiotic in- creased from 3% to 93% in just two years (Mahon et al. 1996). There is also the additional problem of the increased emergence of strains of bacteria with resistances towards multiple antibiotics. A few of the more worrying exam- ples are methicillin-resistant Staphylococcus aureus (MRSA), extended- spectrum beta-lactamase (ESBL) producing Escherichia coli and vancomy- cin-resistant Enterococci spp (VRE). In Asia, multi-drug resistant Salmonel- la typhi are emerging at a high rate and mortality associated with typhoid fever has now risen to ~10%, approaching the ~13% mortality level of the pre-antibiotic era (Gupta 1994). Multidrug-resistant Mycobacterium tubercu- losis is found with frequencies of up to 30% in parts of Eastern Europe and Asia (Cox et al. 2004). Worldwide, an average of 3.6 % of all tuberculosis cases are multidrug-resistant and with several million new cases reported

15 each year it is easy to see that the outcome could be very problematic if re- sistance is not controlled (WHO 2010). In the case of opportunistic patho- gens, an example is multidrug-resistant Pseudomonas aeruginosa and Acine- tobacter baumannii that are becoming a major problem for tuberculosis pa- tients in the clinic (Falagas and Kopterides 2006). In some of the cases men- tioned above, we are quickly running out of treatment options (Giamarellou 2006). To improve the situation, we need more knowledge of how resistant bacteria emerge and spread and how or if development of resistance can somehow be avoided or delayed. For example, it is interesting to note that Streptococcus pyogenes has never developed resistance to penicillin, despite the use of this antibiotic to treat infections caused by this bacterium for dec- ades. On the other hand, penicillin-resistant isolates of Streptococcus pneu- moniae are found at an increasing and alarming rate (Linares et al. 2010). The latter pattern of resistance development is unfortunately the case for most combinations of bacteria and antibiotics: resistance develops faster than new classes of antibiotics are being developed.

Selective pressure and its impact on antibiotic resistance Selection pressure is the force that determines which individuals in a popula- tion that will survive or reproduce faster. Under a given set of conditions, those best adapted to the environment will survive and spread. Natural selec- tion of antibiotic resistant bacteria occurs because of the presence of antibio- tics in our environment (i.e. in patients, in animals and in the external envi- ronment). High concentrations ensure that only highly resistant bacteria will survive and spread but low concentrations of antibiotic, well below the mi- nimal inhibitory concentration may be important for selection of low-level resistant bacteria that could develop higher levels of resistance at a later stage. By limiting our use of antibiotics to when it is absolutely necessary, always using the correct antibiotic and not releasing antibiotics into the envi- ronment we could improve the situation. Strategic thinking as well as atti- tude changes and legislation also have a big impact on resistance develop- ment.

Antibiotic levels in the environment Antibiotics occur naturally in the environment but the use and over-use of antibiotics worldwide and over many years has increased the probability of bacteria encountering them. Human pathogens can acquire resistance genes already present in the environment that will allow them to survive and spread (Martinez 2008; Allen et al. 2010). Antibiotics are used unnecessarily, and sometimes in great excess, in an- imal husbandry. The same classes of antibiotics that are used to treat humans

16 are in many cases also used to treat animals (reviewed in (Sarmah et al. 2006). In addition, antibiotics such as amoxicillin and erythromycin are used prophylactically to ensure disease-free herds, or as growth promoters and to improve feeding efficiency (Aarestrup 2005). Sweden was the first country to ban antibiotics as growth promoters in 1986, and 20 years later the antibi- otic use in animals had decreased by 65% (Bengtsson and Wierup 2006). The Nordic countries followed suit and in the European Union, a ban was introduced on January 1st 2006, however the practice to use antibiotics as growth promoters continues in many countries. For medicinal purposes, human antibiotics are also used in animals such as the use of erythromycin in aquaculture (fish farming), because there are very few approved veterinary drugs for aquatic species (Esposito et al. 2007). Antibiotics that are used to treat humans are also used in horticulture. For example, streptomycin is sprayed on apple trees to treat fire blight caused by Erwinia amylovora. When blossoming trees have been sprayed, streptomycin has been found in apples picked three months later (Mayerhofer et al. 2009). Antibiotics can pass through the human body intact, and may end up in sewage systems (Sahlstrom et al. 2009; Yang et al. 2009) or in lakes and rivers, especially in those countries that have little or no waste-water treat- ment facilities (Minh et al. 2009). Horizontal gene transfer has been shown to occur in lake and sewage water and appears to be driven by the selection pressure of antibiotics in these environments (Shakibaie et al. 2009; Szczepanowski et al. 2009). Waste products from animals and humans are also used as fertilizers, thereby increasing the risk of spreading resistant bac- teria and resistance genes to nearby farms and the surrounding wildlife. A survey showed that the resistance pattern in nearby bird life closely matched the resistance profile found in the manure and sewage slurry that was used to fertilize the farms in the area (Blanco et al. 2009).

Rational antibiotic use In many cases, improper use of antibiotics is simply due to ignorance. For example, the continued use of “established” antibiotics even though they no longer have any therapeutic effect because a high fraction of bacteria are already resistant. Lack of knowledge can also lead to “preventive” use of antibiotics i.e. patients perceive an advantage in taking antibiotics in a prophylactic manner, which could drive resistance development. Information is the key, but since antibiotics are freely available without a prescription in many countries it is not sufficient to educate physicians, the general public must also be educated in the correct use of antibiotics. Also, there is no guarantee that what you purchase is actually what you obtain. Sampling of tetracycline capsules in Nigeria showed that they were of poor pharmaceuti- cal quality (Okeke and Lamikanra 1995) and sub-standard and counterfeit anti-malarial drugs are frequently found in many parts of Africa (Gaudiano

17 et al. 2007). Counterfeit medicines sold over the counter or over the internet are also a growing concern (Kelesidis et al. 2007). There is also the improper use of antibiotics against virus infections such as non-bacterial upper respiratory tract infections (Steinman et al. 2003). In the developed world, a key factor determining the level of antibiotic pre- scriptions is the expectations of the patient. Studies have shown that patients leaving their physician with a prescription are more satisfied (Pontes and Pontes 2003; Welschen et al. 2004). So, even though a patient might have a virus infection, most patients will still be more content receiving a (useless) antibiotic than receiving no treatment at all (Linder and Singer 2003). Again, information is paramount and studies have shown that receiving appropriate information on antibiotics and the risks of resistance can change patients’ opinions on the prescription of antibiotics (Pontes and Pontes 2005). Many patients chose their own healthcare providers, so keeping the “customer hap- py” is an important factor in medicine. In addition, selling drugs is one of the income sources for physicians in many countries such as China so there are sometimes also economical reasons behind prescriptions (Reynolds and McKee 2009). In the course of an antibiotic treatment, failure to adhere to the dose regi- men by quitting the course prematurely or not taking the prescribed number of pills can adversely affect the effective antibiotic concentration in the body. This in turn can lead to selection rather than extermination of resistant bacteria as the normal bacterial flora, sensitive to the antibiotic, is reduced while the resistant bacteria are allowed to persist. Studies have shown that patients are more likely to complete once-daily rather than twice-daily courses of antibiotics (Kardas 2007). Also, the number of patients that com- plete the entire duration of treatment increases if the course is short. It has been shown that between 10 and 40% of patients end the treatment prema- turely, when they start to feel well (Pechere et al. 2007). To help alleviate the selection pressure of a certain antibiotic, cycling dif- ferent antibiotics has met with some success (Pujol and Gudiol 2001). How- ever, this practice has also met with some opposition suggesting that cycling might even increase resistance rates so the simultaneous administration of several drugs has been proposed as an alternative strategy (Bergstrom et al. 2004). Regardless, both strategies require the development of new classes of antibiotics but unfortunately fewer and fewer research companies are willing to take on this endeavor. This is partly because research costs are high, while antibiotics are one of the cheapest drugs available and partly because resis- tance towards a new antibiotic may develop quickly, rendering the com- pound virtually useless (Projan 2003; Projan and Shlaes 2004; Overbye and Barrett 2005).

18 Factors contributing to distribution and rate of resistance There are several different factors contributing to the spread of resistant bac- teria and the rate of emergence of newly resistant strains. The dispersal of resistant bacteria can be due to spreading of a single strain due to lacking hygiene, or due to sharing of resistance genes between and within species of bacteria. The rate of emergence of resistant bacteria dependent on factors such as mutation supply rates, horizontal gene transfer rates and the fitness cost of resistance mutations.

Spread of antibiotic resistance Like their susceptible counterparts, most resistant bacteria spread naturally through infected water and lack of basic hygiene. Even in the western world, many epidemics of drug-resistant bacteria are clonal, i.e. many patients are infected with the same bacterial strain. A resistant strain can originate from a single patient and through inadequate hygienic procedures spread to hun- dreds of other patients.

Horizontal gene transfer There is also the possibility of dissemination of the resistance genes through horizontal gene transfer. The myriad of antibiotic resistance genes in the biosphere is sometimes referred to as the “resistome” and also includes cryp- tic genes and precursor genes that give low-level resistance (Wright 2007). With such a wealth of resistance genes available most bacteria, both com- mensal and pathogenic, can be viewed as reservoirs of antibiotic resistance genes. There are several different ways that bacteria can share their DNA, both within and between species. The different paths of DNA transfer from one bacterium to another are discussed below.

Natural competence (transformation) Some bacteria have the ability to take up naked DNA from their surround- ings and incorporate it into their own genome. This ability is referred to as natural competence, and varies widely between species. Advantages asso- ciated with this mechanism are the ability to degrade the incoming DNA as a source of energy and the possibility of acquiring new genes that could give an advantage under selection (reviewed in (Solomon and Grossman 1996)). In the context of resistance it is interesting to note that the bacterium Strep- tococcus pneumoniae is naturally competent while its relative Streptococcus pyogenes is not and resistance towards penicillin has never been encountered in S. pyogenes, while it is relatively common in S. pneumoniae.

19 Plasmid transfer One of the most efficient ways of spreading resistance genes is through plasmid transfer. Plasmids are extra-chromosomal entities that do not con- tain core genes essential for growth and reproduction but typically harbor genes that are transiently useful such as resistance genes. Almost all classes of resistance genes are found on plasmids, notably those against commonly used antibiotics such as beta-lactams and fluoroquinolones. Plasmids can be transferred from one bacterium to another either by transformation (see above) or the process of conjugation. Conjugation occurs through direct cell- to-cell contact through a bridge-like connection. Most plasmids are circular and can vary in size from a few kbs to several hundred, and can therefore harbor a multitude of resistance genes. This means that multi-drug resistance can rapidly spread from one strain to another with the transfer of a single plasmid. For example, a hospital outbreak in Sweden of ESBL-producing Klebsiella spread from a single index patient to over 200 other patients (Ransjo et al. 2010). The bacteria harbored a ~220 kb plasmid with resis- tance genes for aminoglycosides, macrolides and trimethoprim- sulfamethoxazole in addition to penicillin and cephalosporin resistance genes (Lytsy et al. 2008). Also, the resistance cassettes in the plasmid have been shown to originate from E. coli (Sandegren L., personal communica- tion). This shows that a plasmid could potentially be maintained in the com- mensal flora and transferred to pathogens when selection pressure in the form of antibiotic treatment commences.

Bacteriophage transfer Viruses that are capable of infecting bacteria (bacteriophages) can occasio- nally inadvertently package some of their hosts’ DNA. Following a new infection this DNA can become incorporated into a new host genome. This horizontal gene transfer process is commonly known as transduction and through this process bacteria can acquire many different kinds of genes such as virulence factors and resistance genes. High-frequency transduction of resistance genes between isolates of Pseudomonas aeruginosa has been shown to occur and could be one factor contributing to the spread of resis- tance among these bacteria in clinical settings (Blahova et al. 1997; Blahova et al. 1999).

Integrons and conjugative transposons Integrons are a class of gene structures that can facilitate the transfer of genes and notably resistance cassettes (Stokes and Hall 1989). Integrons consist of an intI gene coding for an integrase, a recombination attI site and a strong promoter for the cassette genes. Using site-specific recombination, integrons can transfer from a chromosome to for example a plasmid (Partridge et al. 2009). Resistance genes can also be transferred by another

20 mobile genetic element called conjugative transposons (Clewell and Gawron-Burke 1986). Conjugative transposons have the ability to excise themselves from one DNA-molecule (for example a chromosome) and form a circular intermediate. This intermediate can promote its own conjugal transfer to another bacterial cell and re-integrate into the chromosome, thus transmitting its resistance genes (Salyers et al. 1995).

Mutation supply rate As described above, one way for bacteria to become resistant to antibiotics is to acquire new genes through horizontal gene transfer. Another possibility is de novo mutations in existing genes. Factors that influence mutation rates are discussed below. In nature, a fraction of bacteria can become mutators, i.e. gain a higher mutation rate that can in turn increase the probability of generating resis- tance mutations. The mutator phenotype is often due to defects in DNA re- pair and error correction systems such as the mismatch repair system (re- viewed in (Miller 1996)). However, if selection such as exposure to an anti- biotic occurs, the mutator phenotype can give a selective advantage. This is because a mutator cell is more likely to generate a beneficial (for the bacte- rium) resistance mutation required for survival. The probability of occur- rence of mutators increases with population size and the mutator population must become sufficiently large to have a chance of generating a beneficial mutation. From this follows that the larger the starting population, the larger the subpopulation of mutators having a high probability of obtaining a bene- ficial mutation (Le Chat et al. 2006). In the long run, an elevated mutation rate will lead to accumulation of many deleterious mutations, resulting in decreased fitness. The higher the advantage of obtaining a beneficial muta- tion is, the more likely the mutator phenotype is to hitchhike along. It has been shown that a stronger selective advantage of beneficial mutations leads to selection of stronger mutators (Tenaillon et al. 1999). This is because the mutator phenotype in itself leads to a cost due to the accumulation of delete- rious mutations and this cost must be balanced. When a population of E. coli was subjected to several rounds of exposure to antibiotics, the resulting sur- viving population consisted entirely of mutators (Mao et al. 1997). Support- ing this experiment is the discovery that in cystic fibrosis patients infected with Pseudomonas aeruginosa, 36% of the patients were colonized with mutator strains (Oliver et al. 2000). Furthermore, antibiotic treatment in mice selected for resistant strains with mutator phenotypes (Giraud et al. 2002). If the selection pressure disappears however, mutators are considered to be at a disadvantage (Funchain et al. 2000). Nevertheless, if a resistance mutation has occurred, there is always the possibility of reversal of the muta- tion or transfer and recombination of the beneficial gene into other, more fit non-mutator strains. The selective pressure acting on the mutation and re-

21 combination rates themselves is often referred to as second-order selection (reviewed in (Tenaillon et al. 2001)).

Resistance mutations and fitness Fitness in bacteria is the property that defines the survival, spread or viru- lence of bacteria in a certain environment. Thus, it follows that under a dif- ferent set of conditions, the exact same bacterium might have higher or low- er fitness. If the conditions change, mutations or other alterations such as up/down regulation of genes might increase or decrease fitness and thus the probability of survival. For simplicity, and to make measurements easier, fitness in bacteria is often defined as exponential growth rate. The validity of using exponential growth rate alone as a measure of fitness can be discussed since a prolonged lag phase could definitely decrease the ability to compete with other bacteria for example. Competition between a mutant and a wild- type strain, a method that encompasses the entire growth cycle, is an alterna- tive way of comparing relative fitness. In this work, fitness is assayed using one of these two methods. In the light of our knowledge of the vast reservoir of antibiotic resistance genes in the environment, and ongoing antibiotic selective pressures, one might ask why all bacteria are not already resistant? Bacterial fitness is a key parameter when discussing resistance, as resistant bacteria with low fitness are very likely to be out-competed by their susceptible counterparts when the selective pressure is removed. In the case of penicillin-sensitive S. pyogenes mentioned earlier, it has been suggested that penicillin-resistant mutants have never been isolated because resistance mutations are so costly that the resistant mutants never have the possibility to fix in the population. Experiments have revealed that the acquisition of resistance often confers a fitness burden on bacteria, typically displayed as a reduced growth rate and/or virulence. This cost can be explained by considering that key cellular functions such as components of translation have to be modified or that an already optimized expression pattern has to be altered for the bacteria to become resistant. If however, a resistance mutation has low or no cost the potential clinical problem caused by that strain, i.e. the probability of that particular strain of bacteria to propagate and take over even in the absence of antibiotics, is much greater. Mutations have indeed been found that confer little or no fitness loss (Björkman et al. 1998; Sander et al. 2002; Wichelhaus et al. 2002), and even in some cases increased fitness (Luo et al. 2005) as well as increased virulence (Yu et al. 2005). Also, relative fitness compared to a “wild-type” susceptible strain may vary under different growth conditions, where the resistant mutant actually has higher fitness under some growth conditions (Paulander et al. 2009). In addition, it has been shown that resistant bacteria can acquire secondary, compensatory mu- tations that partly or even fully restore fitness without loss of resistance

22 (Björkman et al. 2000; Levin et al. 2000; Nagaev et al. 2001; Maisnier-Patin et al. 2002; Maisnier-Patin and Andersson 2004; Besier et al. 2005).

Compensatory mutations and compensatory evolution Compensatory mutations are mutations that increase fitness of a strain of bacteria compared to a parental, less fit strain. In an effort to mimic compen- satory evolution in nature, we have used a simple system to evolve resistant bacterial strains to higher fitness. Slow-growing parental bacteria are serially passaged to fresh media using bottlenecks (~1000-fold dilution, 106 cells passaged). When fast-growers appear they are isolated and examined for compensatory mutations. The compensatory mutations found are typically mutations that increase growth rate compared to a slower-growing resistant strain, whilst preserving resistance. There are several different types of com- pensatory mutations where the most straightforward are intragenic mutations that occur within the gene that is altered to give the resistant phenotype. In the trivial case, the compensatory mutation is a revertant, but since a rever- tant loses its resistance, it is not further discussed here. One example of extragenic compensatory mutations is described for streptomycin resistant S. typhimurium. Resistance is conferred by a mutation in the rpsL gene (coding for ribosomal protein S12), and when evolved in LB, compensatory muta- tions in rpsD, rpsE, rpsL or rplS (coding for ribosomal proteins S4, S5, S12 and L19, respectively) arise. Apparently, an alteration in one ribosomal pro- tein which affects the proofreading step can be alleviated by second-site mutations in other ribosomal proteins, presumably restoring normal proo- freading activity (Maisnier-Patin et al. 2002). Another example is described in paper II (Zorzet et al. 2010), where mutations in initiation factor 2 com- pensate for fitness loss due to resistance to peptide deformylase inhibitors. Point mutations in the coding sequence are not the only mode of compensa- tion. Altered expression levels due to either promoter mutations or amplifi- cation of DNA have also been described (Paulander et al. 2010). In paper I (Nilsson et al. 2006), the genes encoding initiator tRNA were found to be amplified giving higher expression levels of tRNAi and thereby compensat- ing for the lack of formylated initiator tRNA. Taking all data on compensa- tory mutations into consideration, it is clear that when designing new antimi- crobial drugs one should not only look at the frequency of emergence of resistance, but also look at frequency and fitness-gain of compensatory mu- tations.

Gene amplification Bacterial adaptation to changing conditions and novel selection pressure can occur though several mechanisms. A rapid way of changing expression le- vels of cellular components is through amplification of selected gene regions

23 (reviewed in (Andersson and Hughes 2009)) As mentioned above, gene am- plification was found to be the mechanism of fitness compensation in several studies (Nilsson et al. 2006; Paulander et al. 2010). Bacterial genomes have high plasticity and amplification of regions of DNA occur at a high frequen- cy, ranging from >10-2 to 10-4 depending on genomic context (Anderson and Roth 1981). In the absence of selection however, segregation to one copy occurs rapidly (Pettersson et al. 2009). In the cases described above, the amplifications confer a selective advantage and are thus stabilized. Another consequence of gene amplification is the increase of the mutational target, which increases the probability of additional compensatory mutations occur- ring in one copy of the amplified gene (Roth and Andersson 2004b; Pettersson et al. 2009). If this happens, the selection on amplification is re- laxed and the amplified array can segregate down to one copy. These events can also have implications for the creation of new genes because selection pressure can keep a large section of DNA amplified. This allows more time for secondary mutational events to take place creating two slightly different gene copies in the so-called duplication-divergence model (Taylor and Raes 2004; Bergthorsson et al. 2007).

Recombination mechanisms Amplification occurs during recombination, although the specific mechan- isms are still debated. Recombination in bacteria can be divided into two classes, which can both give rise to duplications and higher order amplifica- tions: RecA-dependent and RecA-independent recombination. Recombina- tion facilitated by the strand transfer protein RecA is strongly dependent on homology between the interacting sequences, requiring a minimum of 25 bp of homology but occurring more frequently with larger repeats (Bianco et al. 1998; Morag et al. 1999; Lovett et al. 2002). RecA-independent recombina- tion is commonly referred to as illegitimate recombination and requires no sequence identity (Ehrlich et al. 1993).

The Cairns system The perhaps most studied system where amplifications have been found to compensate for a loss-of-function mutation is the lac system. This experi- mental system was originally designed to study adaptive mutation (Cairns and Foster 1991). A strain with a leaky lac frameshift mutation making the cells unable to grow on lactose was used. Plating these bacteria on minimal lactose media allows revertant, Lac+ colonies to accumulate. Over several days the number of Lac+ revertants increases making it seem like selection per se results in an increased rate of reversion. From these observations it was proposed that selective stress increases the general mutation rate thereby increasing the yield of Lac+ revertants (Rosenberg and Hastings 2004; Ponder et al. 2005; Slack et al. 2006). However, more recent findings sug- gest an alternative explanation for this phenomenon. In short, if a subpopula-

24 tion of the plated cells harbors a pre-formed duplication/amplification of the leaky lac-allele, this subpopulation of cells has an advantage in the selective environment as they are able to grow, albeit slowly (Roth and Andersson 2004a; Roth et al. 2006). During subsequent growth, further amplification of the lac-allele, or the occurrence of a lac+ mutation in any of the amplified copies, can generate the Lac+ phenotype. Studies showed that the Lac+ colo- nies contain cells with amplifications of the lac-allele and that these amplifi- cations had different origins. Some revertants originated from large-repeat, pre-formed duplications probably formed by recombination of IS3 elements (Kugelberg et al. 2006). Such duplication allowed modest growth on lactose as the size of the duplication is a potential metabolic burden for the cells, but provides a background of cells that can undergo one of two rare events: Ei- ther a lac+ reversion in any one of many clones or a deletion that shortens the repeat length. This subpopulation can then further increase in lac copy- number and subsequently obtain a lac+ reversion mutation. In conclusion, the existence of amplifications increases the probability of obtaining or re- establishing a gene function.

Model organisms

Salmonella typhimurium The model organism mostly used in this work is Salmonella enterica serovar Typhimurium (designated S. typhimurium henceforth). This Gram-negative, rod-shaped bacterium is a close relative of the popular model bacterium Escherichia coli (E. coli). S. typhimurium causes gastroenteritis in humans with symptoms such as diarrhea, fever and abdominal cramps. Salmonella infections are zoonotic and can spread between birds, mammals and reptiles. In the hosts’ body, Salmonella invades macrophages and replicates therein. The strains used in this thesis are all derived from S. typhimurium LT2, a strain that is avirulent due to its defective rpoS gene product (Swords et al. 1997). S. typhimurium LT2 is frequently used in molecular microbiology due to the excellent tools for genetic manipulation available for this organism.

Staphylococcus aureus Staphylococcus aureus (S. aureus) are Gram-positive cocci and are the lead- ing cause of Staphylococcal infections. S. aureus are carried asymptomati- cally by a substantial fraction of the population, usually in the nasal cavity (Kluytmans et al. 1997). However, these bacteria can cause a number of diseases, from minor skin infections to severe endocarditis and fatal sepsis. The emergence of methicillin-resistant S. aureus (MRSA), resistant to most

25 available antibiotics, has warranted renewed efforts in developing new, ef- fective antibiotics. The strains used in this thesis are derivatives of the fully sequenced S. aureus NCTC 8325-4 strain.

Antibiotic targets There are many different classes of antibiotics, each targeting a specific vital function in bacteria. A good target for an antibiotic typically involves an essential cellular function, such as replication, transcription or translation. Furthermore, the target should not exist in human cells or should be suffi- ciently different from its human counterpart to avoid toxic effects. That the target exists and is conserved in many bacterial species is also a distinct ad- vantage. Table 2 details the most commonly used antibiotic classes and their targets. Penicillin (a beta-lactam) was the first antibiotic discovered in 1928 by Alexander Fleming, but it was not commercially available until the 1940’s. The next drug, sulfapyridine of the class sulfonamides was discov- ered in 1932 and was also the first synthetic antibiotic compound. The two decades between 1942 and 1962 saw the advent of almost all antibiotic classes currently in use. However, after the discovery of fosfomycin in 1969, there was a 21-year long hiatus in finding new drugs until the year 2000 when linezolid was marketed.

Table 2: Historic and functional overview of classes of antibiotics. Adapted from (Walsh and Wright 2005). Year of Name Class Origin Target discovery 1928 Penicillin Beta-lactam Natural Cell wall synthesis 1932 Sulfapyridine Sulfonamide Synthetic Tetrahydrofolate synthesis 1944 Streptomycin Aminoglycoside Natural Protein synthesis 1947 Chloramphenicol Phenylpropanoid Natural Protein synthesis 1948 Chlortetracycline Tetracycline Natural Protein synthesis 1950 Erythromycin Macrolide Natural Protein synthesis 1955 Vancomycin Glycopeptide Natural Cell wall synthesis 1955 Virginiamycin Streptogramin Natural Protein synthesis 1955 Lincomycin Lincosamide Natural Protein synthesis 1959 Rifamycin Ansamycin Natural RNA synthesis 1962 Nalidixic acid Quinolone Synthetic DNA synthesis 1969 Fosfomycin Phosphonate Natural Cell wall synthesis 2000 Linezolid Oxazolidinone Synthetic Protein synthesis 2003 Daptomycin Lipopeptide Natural Cell membrane

In addition to the classes described above in Table 2, extensive enhance- ments have been made to existing drugs to create more efficient or less resis- tance-provoking drugs. An example is the development of several genera- tions of cephalosporins that have the same mode of action as penicillin, but

26 are less sensitive to beta-lactamases. As resistance has emerged towards the cephalosporins as well, the carbapenems have come into use. Carbapenems also have the same mode of action, but have an altered structure that makes them highly resistant to beta-lactamases.

Antibiotics that target the ribosome As can be seen in Table 3 above, many antibiotics target the process of pro- tein synthesis. The translation process is complex and involves many steps that are possible to inhibit or at least stall. Some examples include the ami- noglycosides that bind to the A or P sites on the 30S subunit and induce er- rors in translation. The tetracyclines also target the 30S subunit and block the binding of tRNA to the A-site. There are also several classes of antibiotics that target the 50S subunit. Macrolides binds in the exit tunnel where they hinder elongation of the nascent polypeptide. Lincosamides block peptide bond formation and linezolid decreases translational fidelity. Chlorampheni- col binds the 23S rRNA and blocks peptide bond formation by inhibiting peptidyl transfer (reviewed in (Poehlsgaard and Douthwaite 2005)). Because the translation machinery has several functional differences in prokaryotes compared to eukaryotes it makes a good target for further devel- opment of antibiotics. With the recent high-resolution structures of the ribo- some, both alone and in complex with different antibiotics, more rational design of antibiotics may be possible.

Peptide deformylase inhibitors The naturally occurring antibiotic actinonin has shown potential in treating infections of both Gram-positive and Gram-negative bacteria (Chen et al. 2000). Furthermore, it belongs to a new class of antibiotics that inhibits pep- tide deformylase (PDF). In recent years, there has been renewed interest in actinonin and other PDF inhibitors as agents against Mycobacterium tuber- culosis infections (Sharma et al. 2009). The PDF enzyme is present in bac- terial cells, but not in humans (except in the mitochondria). Initiation of translation in most eubacteria starts with a formylated methionyl-initiator tRNA (fMet-tRNAi) (Marcker and Sanger 1964; Newton et al. 1999). The enzyme responsible for formylation of the methionine is formyl-methionyl transferase (Fmt) (Guillon et al. 1992b). After the nascent polypeptide has been completed, the formyl group is removed by PDF (Adams 1968; Mazel et al. 1994). When PDF is inhibited by actinonin, formylated peptides accu- mulate in the cell, leading to subsequent inhibition of growth and ultimately, cell death. Because actinonin is too weak an inhibitor for human use, several modified peptide deformylase inhibitors have been developed, which are more potent (Chen et al. 2000). Two of these, intravenous compound BB- 83698 and oral compound LBM-415 (Figure 1) have entered phase I clinical trials (Guay 2007). Decreased susceptibility towards PDF inhibitors have

27 been described in E coli, S. typhimurium, S. aureus, S. pneumoniae, H. in- fluenzae and B. subtilis but all resistant mutants have had severely reduced growth rates (Guillon et al. 1992a; Margolis et al. 2000; Margolis et al. 2001; Nilsson et al. 2006; Dean et al. 2007; Duroc et al. 2009).

Figure 1: Structure formulas for the natural peptide deformylase inhibitor actinonin and two synthetic derivatives.

28 Translation in prokaryotes

Translation is the process in which mRNA is decoded into amino acids to form a protein and can be mechanistically divided into three stages: initia- tion, elongation and termination. During initiation, the two subunits of the bacterial ribosome are assembled together with mRNA and initiator tRNA with the help of the three initiation factors IF1, IF2 and IF3. Initiation is rate- limiting and the efficiency of translation is often regulated at the level of initiation (Kozak 2005). During elongation, amino acids are added to the growing peptide chain and the process involves elongator tRNAs, the two elongation factors EF-Tu and EF-G, and the catalytic center of the ribosome. The termination phase begins when a stop codon is encountered on the mRNA and a release factor binds to it. The newly synthesized polypeptide chain is then released and the ribosome is recycled into its components. The steps of translation are described in more detail below and also in Figure 2.

29

Figure 2: Overview of the steps of translation: initiation, elongation and termination.

The structure of the ribosome The ribosome is the large and complex structure that is the center of protein synthesis. It is approximately 2.4 MDa in size in prokaryotes and consists of a large and a small subunit. The sedimentation coefficient is 30S for the small subunit and 50S for the large subunit and together the subunits form

30 the 70S complex. The ribosome is made up of two components, RNA and protein, where RNA accounts for approximately 65% of its mass. There are three ribosomal RNAs (rRNAs), the 16S rRNA in the small subunit and the 5S and 23S rRNAs in the large subunit. In addition, the 30S subunit contains around 20 ribosomal proteins and the 50S subunit around 35 proteins. The 16S rRNA binds complementary to specific regions on the mRNA and to the anticodon stem-loop of the initiator tRNA. The 23S rRNA catalyses the pep- tidyl transferase reaction leading to peptide bond formation (reviewed in (Bashan and Yonath 2008)). In the 1980’s, more and more evidence was gathered to suggest that the RNA was responsible for all catalytic functions of the ribosome, whereas the ribosomal proteins help stabilize the RNA structure. Experiments aimed at removing the proteins to investigate the peptidyl transfer ability of naked rRNA were performed. Peptidyl transfer was indeed achieved after treatment with proteinase K, SDS and phenol ex- traction, however 2-8% protein always remained (Noller et al. 1992). The theory was that these proteins were caged in by the RNA, thereby making them inaccessible to proteolytic cleavage. This lead to the proposal that the role of the proteins was to help maintain the structure of the rRNA, but it could not be completely ruled out that they played some catalytic role (Noller 1993). When crystal structures of the ribosome became available they showed that the active site for the formation of peptide bonds i.e. the peptidyl-transferase center (PTC), was composed solely of rRNA (Nissen et al. 2000). In structures of other species however, the proximity of protein L27 to the PTC implicated a role for this protein in peptidyl transfer (Selmer et al. 2006). However, subsequent studies showed that protein L27 is too far away to engage in peptidyl transfer (Trobro and Aqvist 2008; Simonovic and Steitz 2009; Voorhees et al. 2009). The three tRNA binding-sites on the ribosome are referred to as the aminoacyl site (A-site), the peptidyl site (P- site) and the exit site (E-site). The domains of the ribosome are named to indicate structural features (Fig. 3).

31

Figure 3: Schematic overview of the ribosomal 30S (black) and 50S (white) subunits. Images were made with PyMOL (DeLano 2002), from crystal structures of T. thermophilus (PDB codes 2J00 and 2J01 (Selmer et al. 2006).

Initiation Preceding the process of initiation, the 70S ribosome is split into its com- ponents by the joint action of the ribosome-recycling factor and elongation factor G. Initiation factor 3 (IF3) binds the 30S subunit to impede erroneous re-association with the 50S subunit (Karimi et al. 1999; Peske et al. 2005; Savelsbergh et al. 2009). To complete the 30S pre-initiation complex (PIC), mRNA and initiator tRNA binding is required (Simonetti et al. 2009). The mRNA contains a ribosome binding motif commonly known as the Shine- Dalgarno sequence (Shine and Dalgarno 1974; Steitz and Jakes 1975). The presence of an anti-Shine-Dalgarno (anti-SD) motif on the 16S rRNA com- plementary to the SD sequence secures the mRNA to the 30S subunit (Shine and Dalgarno 1974). The “strength” of the SD motif and thus the ribosome binding strength is determined both by the distance from the start codon and how well it complements the anti-SD sequence. In eubacteria, the sequence of the anti-SD core is commonly CCUCCU and hence a strong SD sequence would be AGGAGG. The distance between the SD sequence and the start codon is between 3 and 12 nucleotides, with the optimal spacing being 7-9 nts (Chen et al. 1994). IF3 proofreads the codon/anti-codon interaction, des- tabilizing any incorrect pairings (Gualerzi et al. 1977). The most commonly used start codon in E. coli is AUG (~90%) followed by GUG (~8%) and UUG (~1%) (Schneider et al. 1986). In addition, there is one occurrence of the codon AUU as a start codon in the gene infC coding for initiation factor 3 (IF3) (Gualerzi and Pon 1990). To ensure accurate positioning of the start

32 codon in the ribosomal P-site the presence of initiator tRNA is required (Hartz et al. 1989) and the initiation factors, especially IF3, play a role in the correct alignment of the start codon (La Teana et al. 1995). IF3 also prevents premature association of the 50S subunit, before initiator tRNA has bound to the 30S PIC (Subramanian and Davis 1970; Antoun et al. 2006b). The posi- tioning of the fMet-tRNAi itself occurs in three steps: codon-independent binding, codon-dependent binding, and fMet-tRNAi adjustment (Tomsic et al. 2000). The accuracy of selection of the correct tRNA is greatly aided by IF1, IF2 and IF3 (Antoun et al. 2006a; Antoun et al. 2006b) and IF2 is es- sential for fast binding of the initiator tRNA into the PIC (Wintermeyer and Gualerzi 1983; Antoun et al. 2006b). On the ribosome, the anticodon arm of initiator tRNA interacts with G1338 and A1339 of the 16S rRNA (Lancaster and Noller 2005). This interaction is monitored by initiation factor 3 (IF3) that can distinguish initiator from elongator tRNA and it has been found that initiator tRNA binding to the P-site requires the presence of the three con- secutive GC pairs in the anticodon stem (Seong and RajBhandary 1987; Hartz et al. 1989). Single mutations in the N-terminal domain of IF3 renders it defective in initiator tRNA selection as well as in start-codon discrimina- tion and inhibition of initiation on leaderless mRNA (Maar et al. 2008). When IF3 is no longer present in the 30S PIC, docking of the 50S subunit can proceed to form a translation-competent 70S ribosome.

Initiator tRNA and formylation In eubacteria, there are different species of Met-tRNA but there are essential differences between initiator Met-tRNA and elongator Met-tRNA to facili- tate differentiation between the two (Varshney et al. 1993). The first feature that sets the initiator tRNA apart are three consecutive GC base pairs in the anticodon stem. This attribute makes the anticodon loop less flexible and helps target the tRNA to the ribosomal P site and not the A site (Barraud et al. 2008). The second feature is a C1*A72 mismatch at the end of the accep- tor stem that is crucial for recognition of the initiator tRNA by the formyl- methionyl transferase (Fmt) responsible for formylation of the methionine (Guillon et al. 1992b). If this feature along with the three GC base pairs are transferred to an elongator tRNA, this tRNA will be able to participate in initiation of translation (Guillon et al. 1993; Varshney et al. 1993). The for- myl group and the CA mismatch are both features that aid selection of initia- tor tRNA by initiation factor 2 (Petersen et al. 1979; Hartz et al. 1989; Guillon et al. 1996). The final feature that is unique to the initiator Met- tRNA is the presence of a purine(11)-pyrimidine(24) base pair instead of the pyrimidine(11)-purine(24) found in elongator Met-tRNA (Laursen et al. 2005). The regions crucial for initiator tRNA discrimination from elongator tRNA are shown in Figure 4.

33 The major fraction of initiator tRNA in S. typhimurium is coded by the genes metW and metZ and in E. coli by metV, metW and metZ. A minor frac- tion in both species of bacteria is coded by metY, located in the nusA-infB operon (Ishii et al. 1984).

Figure 4: Side-by-side comparison of the initiator fMet-tRNA and elongator Met-tRNA of E. coli. Regions crucial for initiator tRNA identity are hig- hlighted. Adapted from (Varshney et al. 1993).

Initiation factor 2 In bacterial translation, initiation factor 2 (IF2) is involved in several processes. One is recognizing the initiator tRNA (fMet-tRNAi) (aided by formylation of the methionine) and increasing the rate of its binding to the 30S subunit (Petersen et al. 1979; Hartz et al. 1989; Antoun et al. 2006a). There has been some debate on whether IF2 brings the tRNAi to the ribo- some similar to elongation factor EF-Tu with the elongator tRNAs or if IF2 first binds the 30S subunit and stimulates tRNAi binding. Recent results suggest that the latter model is the correct description of the process (Milon et al. 2010). The other role of IF2 is to stimulate subunit association of the 30S and 50S subunits (Antoun et al. 2003). IF2 is a GTPase that is present in all eubacteria and has homologues in archea and eukaryotes. The C-terminal protein sequence is very conserved in

34 most species whereas as the N-terminal sequence is highly variable or even absent. IF2 is commonly divided into four domains where domain I is the G- domain containing the GTP-binding site which is very conserved between species (Roll-Mecak et al. 2000). Domain II structurally resembles domain II of EF-Tu and EF-G. In E. coli, IF2 interacts with the 30S subunit via two different binding sites, one located in the N-domain and one in domain II (Caserta et al. 2006). Domains III and IV are connected by an outstretched flexible linker domain in crystal structures of the archaeal homologue of IF2, aIF5B (Roll-Mecak et al. 2000) and cryo-EM structures (Allen et al. 2005). In a solution structure, this linker domain is instead bent, bringing domain IV closer to domain II (Rasmussen et al. 2008). Domain IV is the tRNA- binding domain and the only domain in contact with the initiator tRNA (Roll-Mecak et al. 2000; Allen et al. 2005). Figure 5 outlines the domains and also shows the contact with the initiator tRNA. It was previously thought that all eubacteria required formylated Met- tRNAi for initiation of translation until it was discovered that Pseudomonas aeruginosa is able to initiate translation with both formylated and un- formylated Met-tRNAi (Steiner-Mosonyi et al. 2004). It was also demon- strated that IF2 is principally responsible in formylation-independent protein initiation. E. coli IF2 was not able to initiate translation using un-formylated Met-tRNAi in P. aeruginosa. However, when the E. coli IF2 was modified by reducing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNAi, it could function in formyla- tion-independent translation initiation. Also, increasing the positive charge of the cleft of the P. aeruginosa IF2 prevented the protein from participating in un-formylated translation initiation (Steiner-Mosonyi et al. 2004). IF2 is coded by the gene infB and is part of the nusA-infB operon, which is autoregulated in part by expression levels of NusA. Overexpression of IF2 however does not effect the regulation of the operon (Nakamura and Mizusawa 1985; Nakamura et al. 1985; Plumbridge et al. 1985b). In several bacterial species including S. typhimurium and E. coli, IF2 is present in three forms (Laursen et al. 2002). These isoforms are IF2- (97.3 kDa), IF2- (79.9 kDa) and IF2- (78.8 kDa) and are all translated from the same mRNA from three in-frame start codons (Plumbridge et al. 1985a; Nyengaard et al. 1991). All isoforms of IF2 are needed for optimal growth, but the proportion of each isoform changes in response to cold shock (Giuliodori et al. 2004). IF2 has also been implicated in functions outside of its role in translation initiation. It is known that IF2 is induced during cold shock (Jones et al. 1987) and that it can act as a chaperone (Caldas et al. 2000). Another exam- ple is the ability of IF2 to act as a metabolic sensor, as it has a similar bind- ing affinity to the alarmone ppGpp as its usual ligand, GTP. When levels of ppGpp are elevated in times of starvation a large fraction of IF2 is inacti- vated, slowing down the rate of translation until energy is replenished (Milon et al. 2006).

35

Figure 5: The domains of initiation factor 2 (The structure is based on the PDB le 1ZO1 and rendered with the program PyMOL (DeLano 2002).

Elongation The elongation process is divided in three phases: decoding, peptide bond formation and translocation of tRNA/mRNA (reviewed in (Agirrezabala and Frank 2009)). The decoding phase starts when an amino-acylated elongator tRNA in complex with elongation factor EF-Tu and GTP binds the ribosom- al A-site. To ensure fidelity of translation, discrimination between cognate

36 and non-cognate aa-tRNAs must be accomplished. Bases A1492, A1493, and G530 of the 16S rRNA ensures that at least two complementary Watson- Crick base pairs form between the codon of the mRNA and the anticodon of the tRNA. Cognate tRNA binding induces conformational changes in the 16S rRNA and domain movement of the 30S subunit ensuring that elonga- tion only proceeds when the correct tRNA is in place (Ogle et al. 2001). When a correct codon-anticodon interaction occurs, a signal is transmitted to the G domain of EF-Tu, likely through the tRNA and the ribosomal protein S12 (Li et al. 2008). GTP hydrolysis occurs and the release of the inorganic phosphate triggers a structural rearrangement of EF-Tu and its dissociation from the ribosome (Schuette et al. 2009; Villa et al. 2009). Once EF-Tu is released, the tRNA can be fully accommodated in the A site, bringing its 3’ end in contact with the peptidyl transfer center (PTC) on the 50S subunit. Peptide bond formation is swiftly catalyzed by the PTC between the amino group of the incoming amino acid and the terminal carboxyl group of the growing peptide chain attached to the tRNA in the P site (Pape et al. 1998; Simonovic and Steitz 2009). Immediately after, translocation of the tRNAs from the A and P sites to the P and E sites is catalyzed by elongation factor EF-G (Rodnina et al. 1997; Katunin et al. 2002). This motion can be seen as occurring in two parts and begins with the translocation of the mRNA and the two tRNAs relative to the large subunit and takes place before GTP hy- drolysis of EF-G. When the inorganic phosphate has been released, a second movement of mRNA and tRNAs relative to the small subunit occurs. This ratchet-like motion also leads to a one-codon movement of the mRNA so that the next codon can be presented in the A site. The deacylated tRNA now present in the E-site is later rejected when a new tRNA binds in the A-site, and a new cycle of elongation can begin (Wilson and Nierhaus 2006).

Termination and ribosome recycling Termination of translation occurs when a stop codon instead of a sense co- don is presented in the ribosomal A-site. There is no tRNA to recognize the stop codon; instead, the stop codons are recognized by so-called release fac- tors. In eubacteria, release factor 1 (RF1) recognizes UAG and UAA and RF2 recognizes UGA and UAA (Scolnick et al. 1968; Nakamura et al. 2000; Petry et al. 2008). In eukaryotes and archaea there is only a single release factor, which recognizes all stop codons (Nakamura et al. 1996). However, there are exceptions to the universality of stop codons as UGA is translated as tryptophan in mitochondria in some species and also in the bacterial spe- cies of Mycoplasma (Osawa et al. 1992). Stop codon identification is me- diated by short, tripeptide motifs in the prokaryotic class I RFs; P-(A/V)-T in RF1 and S-P-F in RF2 (Ito et al. 2000). Both class I RFs catalyze the release of the nascent polypeptide chain by hydrolyzing the ester bond that tethers it

37 to the P-site tRNA. This hydrolysis is mediated by a GGQ-motif present in all class I RFs and this motif is also universally conserved (Frolova et al. 1999). The GGQ-motif is located on a loop structure that is structurally or- dered when the factor is bound to the ribosome, and this loop reaches into the peptidyl transfer center (Petry et al. 2005). After hydrolysis is complete RF3, a class II prokaryotic release factor, also binds the ribosome while the tRNA is still present in the P-site. After RF3-induced ratcheting of the ribo- some, the tRNA is translocated to the E-site and the release of the class I RFs is stimulated (Freistroffer et al. 1997; Gao et al. 2007). To make the ribosome available for another round of translation, the entire complex must first be dissociated which requires the ribosome release factor (RRF). EF-G and IF3 also play important roles in subunit splitting and subsequent release of tRNA and mRNA (Karimi et al. 1999; Peske et al. 2005; Savelsbergh et al. 2009).

38 Present investigations, results and discussion

Paper I

Actinonin resistance in S. typhimurium confers a slow-growth phenotype The peptide deformylase inhibitor actinonin belongs to a new class of antibi- otics. Actinonin binds to and inhibits the essential enzyme peptide deformy- lase (PDF). Translation in eubacteria is initiated with Met-tRNAi that, unlike its eukaryotic counterpart, is formylated. Removal of the formyl group by PDF is required to produce active proteins, making PDF an attractive anti- bacterial drug target. To determine the value of deformylase inhibitors as a new antibiotic class, we studied the emergence of actinonin-resistant mutants in paper I. Salmonella typhimurium were plated on actinonin-containing LA plates and several resistant clones were isolated. The resistance mutations were identified and located in either of two genes, fmt or folD. The gene product of fmt is a formyl-methyltransferase (Fmt), an enzyme that attaches the formyl group to the Met-tRNAi. The folD gene product is a bifunctional enzyme required to produce 10-formyl-H4folate, which is the donor of the formyl group to Met-tRNAi. Inactivation of either fmt or folD leads to resis- tance to PDF inhibitors. To be clinically useful as an antibiotic, it is impor- tant that resistant mutants do not occur too frequently, and/or that resistance mutations have a high biological cost. We measured the exponential growth rate of the strains in LB medium and all strains with mutations in either fmt or folD had a severe slow-growth phenotype compared to the wild type. The loss of formylation capability clearly slows down the translation rate, pre- sumably by forcing protein synthesis to initiate with un-formylated Met- tRNAi, thus giving the mutants a severe growth disadvantage. For a subset of resistant mutants, growth and virulence were also measured by competition in a mouse model. Mice were injected with equal amounts of genetically tagged resistant and wild type bacteria and sacrificed after two days. The fraction of surviving bacteria of each type was determined, and all resistant strains except one had a growth disadvantage. In conclusion, actinonin resis- tant mutants generally exhibit severely reduced growth rates both in liquid culture and in a mouse infection model.

39 Fitness loss in actinonin-resistant strains can be ameliorated by amplification of initiator tRNA genes In paper I, we investigated the emergence of compensatory mutations that could alleviate the substantial fitness loss associated with actinonin resis- tance. Although resistant mutants have low fitness, if compensatory muta- tions that elevate fitness while maintaining resistance occur frequently, then resistant mutants could still pose a clinical threat. We evolved several of the slow-growing resistant mutants by serial pas- sage in rich medium. Each day, 106 cells were passaged into 1 ml of fresh medium and grown to full density. This procedure was continued until fast- growers appeared and when they constituted the majority of the population, one colony was randomly selected and frozen. Compared to the growth rate of the parental resistant strain the evolved mutants were substantially com- pensated. All compensated mutants except a revertant retained full resistance towards actinonin. We identified one group of compensatory mutations as 5- to 40-fold copy number amplifications of the metZ and metW genes (encod- ing initiator tRNAs) as assayed by Southern and Northern blots. These am- plifications confer increased gene dosage and expression of the tRNAi which by a mass-action effect increase the rate of initiation in the absence of tRNAi-formylation. The amplified arrays ranged from 2 kb to >90 kb in size and in some cases lacked sequence homology at their join points, implicating a RecA-independent mechanism for the initial duplication. These results may have broad implications for understanding how new genes can be created. The standard hypothesis that new genes arise by muta- tion of one copy of a duplicated gene has always had one serious caveat: that the segregation rate of duplications/amplifications is orders of magnitude greater than the duplication rate. The time window for acquiring beneficial mutations in one copy of a duplicate gene is thus very narrow and the prob- ability of such an event occurring is expected to be very small. However, if a positive selection pressure could retain an amplified array (as shown here), the probability that one of the gene copies could acquire a beneficial muta- tion would be greatly increased.

Paper II

Fitness loss in actinonin-resistant strains can be ameliorated by point mutations in initiation factor 2 In paper I we identified resistance mutations resulting in actinonin resis- tance as mutations in the genes fmt and folD. These mutations led to a defi- ciency in formylation and required the bacteria to initiate translation using

40 unformylated initiator Met-tRNA. These resistant mutants had severely re- duced growth rates but their low fitness could be compensated by amplifica- tion of the initiator tRNA genes. We also found another class of compen- sated mutants that we identified in paper II as point mutations in initiation factor 2 (IF2). One function of IF2 is to enhance the binding of initiator tRNA to the 30S ribosomal subunit, which is one of the first steps in protein synthesis. Thus, it was not unexpected that compensation for lack of formy- lation of Met-tRNAi would yield mutations in IF2. Surprisingly no mutations were found in the tRNA-binding domain IV of IF2, which is the only part in contact with tRNAi. Instead, most mutations were clustered in domain III and the helical linker between domains III and IV (Fig. 6). To verify that these point mutations in IF2 were solely responsible for the observed com- pensatory effect, we cloned the mutated IF2s on a plasmid that was trans- formed into the formylation-deficient background and confirmed that com- pensation did occur.

Figure 6: Compensatory mutations found in initiation factor 2 (IF2) are marked in red. Mutations tested in the in vitro translation system are under- lined and Class A mutations are marked in blue.

41

IF2 mutants can initiate translation in vitro with high efficiency using either formylated or unformylated Met-tRNAi Using an in vitro translation system containing highly purified components, we investigated mechanisms responsible for the compensatory effects of these IF2 mutations. Two steps of translation initiation depend crucially on IF2: (i) binding of initiator tRNA to the mRNA-containing 30S subunit and (ii) subsequent docking of the 50S subunit to the 30S pre-initiation complex (PIC) to assemble an elongation-competent 70S initiation complex. These two steps of initiation were mimicked in in vitro experiments by rapidly mixing 50S subunits and initiator tRNAs with tRNA-lacking 30S PICs in a stopped-flow instrument and monitoring the formation of 70S initiation complexes by light scattering. We found that there were two types of mu- tants, class A and class B mutants that promoted translation initiation with different potency. Both types of mutants performed better than the wild type IF2 in initiation with unformylated tRNAi. The difference was most pro- nounced at the second step of initiation where the A-type mutants conferred a four-fold, and the B-class mutants a two-fold, increase in the rate of sub- unit joining compared to wild type IF2. Surprisingly, class A mutants were also slightly better than wild type IF2 at promoting subunit joining with for- mylated initiator tRNA. These results correlated well with the in vivo growth rates of the mutants, where the class A mutants gave the highest compensa- tory effect in a formylation-deficient strain.

IF2 mutants can initiate translation with deacylated tRNAi or elongator tRNA In a formylation-proficient strain, the class A mutants show the highest fit- ness cost and reduce growth rate by 5 to 25%. The higher cost was observed in poor medium and the lower cost in rich medium. The in vitro experiments showed that class A mutants were slightly better than the wild type IF2 at promoting subunit joining with formylated initiator tRNA, which excluded the possibility that their fitness cost was due to slow mainstream initiation. Further in vitro experiments showed that class A mutant IF2s promote an about five times faster formation of aberrant 70S initiation complexes than wild-type IF2, using either deacylated initiator tRNA or elongator Phe- tRNA. An aberrant initiation complex with deacylated tRNAi can be recy- cled in vivo by RRF and EF-G, albeit at the expense of consuming resources in the cell. In addition, initiation complexes formed with acylated elongator tRNA cannot be recycled back to subunits in vivo and will enter the transla- tion process leading to out-of-frame protein synthesis and accumulation of

42 erroneous proteins in the cell. Thus, we suggest that the reduced fitness con- ferred by the A-type mutations is due to their propensity to promote the for- mation of aberrant initiation complexes in vivo.

Evidence for mRNA-limited protein synthesis When wild-type IF2 was used to initiate translation in vitro it took 0.25 s with fMet-tRNAi and 2.8 s with Met-tRNAi. The corresponding in vivo gen- eration times were 25 and 101 minutes, respectively. Protein synthesis in the cell occurs at the rate of about 20 amino acids/second, and a protein of 500 amino acids (corresponding to 1500 nts) would therefore take around 25 seconds to translate. From this perspective, one must ask why an increase in initiation time of merely 2.55 s could cause a four-fold increase in generation time. We propose that the answer is mRNA-limited protein synthesis, i.e. that the ribosomes are in such an excess over mRNA that the total rate of protein synthesis is only proportional to the concentration of mRNA, and insensitive to the concentration of ribosomes. In this case, the rate-limiting step would be the loading of new ribosomes onto the mRNA. So, if the ini- tiation rate were slowed down even by a few seconds, it would hamper pro- tein production markedly due to a much lower density of ribosomes (a larger distance between ribosomes) in polysomes. In addition, a greater distance between ribosomes on mRNA could increase the probability of degradation by ribonucleases. This would reduce the concentration of mRNA in the cell, further amplifying the effect of slow initiation.

Paper III

IF2 mutants can induce efficient ribosome subunit association in the absence of tRNA In paper III we continued to investigate the IF2 mutants and their strong effects on subunit association using our in vitro translation system. In paper II we showed that the class A IF2 mutants could initiate translation with deacylated tRNAi and elongator tRNA with much lower reduction in rates than the wild type IF2. Here, we measured the ability of class A IF2 mutants to promote subunit association in the complete absence of tRNA. Most ex- periments in paper III were conducted in the absence of IF3 and hence the reductions in docking rates are not directly comparable between papers II and III. Surprisingly, we found that the complete removal of tRNA from the 30S subunit resulted in only a twofold reduction in the docking rates of the 50S subunit for class A IF2 mutants. In contrast, for class B mutants the rates of docking dropped ten-fold and for the wild type IF2, twenty-fold

43 upon tRNA removal from the 30S subunit. In addition, A–type IF2 mutants conferred substantially higher subunit association rates than B-type mutants and wild type IF2 even in the absence of initiation factor 1, tRNA and mRNA on the 30S subunit. We concluded from these experiments that A- type IF2 mutants can acquire their 50S docking conformation on the 30S subunit even in the complete absence of initiator tRNA. Moreover, these results indicate that the main role of initiator tRNA in the wild type case is to coerce wild type IF2 into its 50S docking conformation on the 30S subunit.

IF2 mutants can induce efficient ribosome subunit association with GDP IF2 is a GTPase as are many of the factors that participate in translation such as EF-Tu involved in elongator tRNA delivery, EF-G in translocation and RF3 in release factor recycling. Each of these factors undergoes conforma- tional transitions between an inactive form (GDP-form) and an active form (GTP-form). These GTPases, when in solution, typically remain in their inactive GDP-form even when GDP is replaced by GTP. In contrast, the same replacement on the ribosome triggers the switch to the active GTP- form. Replacement of GTP for GDP in a 30S pre-initiation complex (PIC) without IF3 but with fMet-tRNAi and with wild type IF2 results in a dra- matic 70-fold decrease in subunit association rate. In contrast, the same ex- periment using a class A mutant IF2 instead of wild type IF2 gives only a modest 30% decrease in subunit association rate. We also tested the effect of varying the concentration of GTP with wild type and mutant IF2. For wild type IF2, rates of subunit association reached half their maximum (GTP only) at around 40% GTP in the reaction mixture containing GTP and GDP. Remarkably, for class A mutants the rates reached half their maximum al- ready at 0% GTP, i.e. GDP only. In addition, rates were almost at maximum already in the presence of only 10-20% GTP. Similar results on the effect of the GTP fraction in the reaction mixture were also obtained in the presence of IF3. We suggest therefore that class A IF2 mutants are much less sensitive to the energy levels in the cell than wild type IF2. This may adversely affect the ability of the cells harbouring A-type mutations in IF2 to adjust their metabolism upon transition to growth under starvation-like conditions. Importantly, in the presence of only GDP and in the absence of fMet-tRNAi, subunit docking was very slow with both wild type IF2 and A-type IF2 mu- tants. We conclude therefore that fMet-tRNAi and GTP are both required to activate wild type IF2 on the 30S subunit for fast docking with the 50S sub- unit. In contrast, class A mutant IF2s can achieve their active conformation on the 30S subunit and change its state to a “50S docking-ready conforma- tion” in the absence of either tRNA or GTP. If both are absent however, the subunit association rate is very slow for both wild type and mutant IF2.

44

Paper IV

Fitness loss in actinonin-resistant Staphylococcus aureus mutants can be ameliorated In papers I and II, we studied actinonin resistance and fitness cost compen- sation in S. typhimurium because of the possibilities of performing advanced genetics in this species. As peptide deformylase inhibitors have their greatest effect against Gram-positive bacteria we also examined the more clinically relevant species S. aureus. Actinonin-resistant S. aureus mutants were iso- lated and all resistance mutations were mapped to the fmt gene whereas no mutations were found in folD. The mutants were slow-growing on rich me- dium agar plates. Several resistant mutants were subjected to compensatory evolution selection. The emergence of compensated mutants was much faster than in Salmonella, with fast-growers taking over the cultures within 40 generations and sometimes as fast as one cycle (10 generations). All 21 compensated strains that were isolated retained full resistance to actinonin. Attempts were made to identify these compensatory mutations by investigat- ing the genes that were altered in S. typhimurium. However, no mutations in fmt (Fmt) or infB (IF2) were found, and the copy number of the initiator tRNA and infB genes was equal to the wild type as assayed by Southern blots.

Whole genome sequencing of compensated, actinonin-resistant S. aureus Due to the difficulty of genetic manipulation in S. aureus, whole genome sequencing was performed using 454 sequencing technology. A parental, slow growing resistant strain and three compensated, fast growing mutants were selected for sequencing. Two of the compensated strains were evolved from the sequenced parental strain and one came from a different parental strain. Initial results showed alterations in three different genes for at least one of the compensated mutants compared to the parental strain. The first was SAOUHSC_01699, coding for shikimate 5-dehydrogenase, which is involved in the synthesis of aromatic amino acids. A second mutation was found in SAOUHSC_00945, coding for a magnesium transporter that func- tions in the maintenance of cellular Mg2+ concentration. Lastly an alteration was found in SAOUHSC_02264, coding for accessory gene regulator pro- tein C (AgrC) which is involved in quorum sensing and virulence. None of these mutations could be linked in an obvious way with compensation of a

45 growth defect associated with initiation of translation with unformylated tRNAi. When sequencing these three genes in the remaining compensated strains, only mutations in agrC were found. Of the 16 mutants successfully sequenced, 11 contained mutations in the agrC gene. The mutations found were 1 bp insertions and deletions as well as point mutations in early posi- tions of the gene. To rule out that the mutations found were conferring me- dium-adaptation effects, we evolved a wild-type susceptible strain in the same manner as the actinonin-resistant strains. The wild type failed to evolve an increased growth rate, indicating that the suppressor mutations are spe- cific. However, agrC has been known to mutate at a high rate in clinical isolates and in the laboratory. Thus, in future experiments we will sequence the evolved wild type susceptible strains to investigate if the agrC mutations are the result of random high-frequency mutational events or if they are in fact mutations involved in adaptation to loss of formylation.

46 Concluding remarks and future aims

Bacterial resistance to antibiotics continues to increase and very few new classes of antibiotics are being developed. In this thesis I have investigated the mechanisms of resistance and adaptation to the inhibitor actinonin, a peptide deformylase inhibitor. In paper I, we studied the emergence of mu- tants resistant to actinonin in Salmonella typhimurium. We localized the site of the resistance mutations to the genes fmt and folD. Both of these genes are required for formylation of initiator fMet-tRNAi and their disruption forces translation to initiate with unformylated Met-tRNA. This causes a severe growth rate defect also seen in several other species of bacteria. We then performed compensatory evolution on several resistant, slow-growing mu- tants and selected fast-growers that were still resistant to actinonin. One class of compensatory mutations consisted of copy number amplification of the genes metZ and metW, coding for initiator tRNA. In paper II we contin- ued to identify compensatory mutations and found several point mutations in initiator factor 2 (IF2) that could also compensate for loss of formylation albeit at a lower level. Selected mutants were tested in a cell-free in vitro translation system to study their effect on initiation. Ribosomal subunit asso- ciation rates were studied and a subset of the mutants (class A) were found to promote subunit association efficiently with both formylated and unfor- mylated initiator tRNA. A weaker effect was seen in class B IF2 mutants but these mutants still promoted faster subunit association with unformylated tRNAi than wild type IF2. These rates correlated well with growth rates in vivo, where class A mutants had a stronger compensatory effect than class B mutants. In a formylation proficient background, class A IF2 were alone in conferring a fitness cost which ranged from 5 to 25% when measured in rich and poor media. In an in vitro translation system, we found that class A IF2 mutants could promote subunit association in the presence of deacylated tRNAi or elongator Phe-tRNA. We propose that the fitness cost seen in vivo is due to the propensity of the class A mutants to form aberrant initiation complexes. In paper III we expanded our study of the A-type IF2 mutants in vitro and found that they could in fact promote subunit association with- out any tRNA present, in the absence of IF3. Subunit association rates with- out the presence of tRNA were tenfold higher with class A IF2 mutants than with wild type IF2. In addition, A–type IF2 mutants conferred substantially higher subunit association rates than wild type IF2 even in the absence of initiation factor 1, tRNA and mRNA on the 30S subunit. We also studied the

47 effect of exchange of GTP for GDP in the same in vitro system. Remarkably, subunit association rates were almost 70-fold higher with class A IF2 mu- tants than with wild type IF2. If tRNA was also removed however, subunit association rates dropped to close to wild type levels. We propose that class A mutant IF2s can achieve their active conformation on the 30S subunit and change its state to a “50S docking-ready conformation” in the absence of either tRNA or GTP. In paper IV, we studied the emergence of actinonin- resistant mutants in S. aureus. In accordance with earlier studies, we identi- fied resistance mutations in fmt. We then performed compensatory evolution as in Salmonella (paper I). Compensated mutants arose much faster than in Salmonella, within 40 generations and sometimes in less than 10 genera- tions. The mutants were fully resistant to actinonin and clearly compensated on plates, but exponential growth rates in liquid media were not higher than the parental resistant mutants. The known compensational targets for formy- lation deficiency from papers I and II were not altered, so the entire ge- nomes of one slow-growing strain and three compensated mutants were se- quenced. Three genes were found to be different between the slow-growing strain and the compensated mutants; SAOUHSC_01699 coding for shiki- mate 5-dehydrogenase, SAOUHSC_00945 coding for a magnesium trans- porter and SAOUHSC_02264, coding for accessory gene regulator protein C (AgrC). None of these mutations are obvious candidates for compensating lack of formylation. When sequencing these three loci in the remainder of the compensated strains, mutations in agrC were found in 11 of 16 strains. It remains to be seen if these mutations are due to general adaption for slow growth or specific compensation for loss of formylation. Finally, below are some thoughts on what could be done to follow-up these studies and expand on them: The IF2 mutants we have identified were only compensated up to ~65% of the growth rate of the wild type, so it would be interesting to continue to evolve them to investigate if further compensation is achievable. Alterna- tively, a second round of mutagenesis of infB could be done to try and find double mutants of IF2 that are more strongly compensated and then evaluate these mutants in the in vitro translation system. As mentioned above, the IF2 mutants have the ability to activate the 30S subunit for 50S subunit docking, but we do not know how this is achieved. Further studies on the exact mechanisms of this process are needed. There is an evident cost in vivo that is not due to a lowered ability of mu- tated IF2 to initiate translation in a normal, formyl-proficient background. We have proposed that this cost is due to aberrant initiation complex forma- tion as mutated IF2 can induce subunit association with deacylated initiator tRNA and elongator tRNA, but in vivo evidence for this hypothesis is lack- ing. We have only begun to investigate compensations of formylation defi- ciency in S. aureus, and more work is needed to investigate the role of the

48 mutations found in agrC. We have passaged wild type bacteria without not- ing any improvements in colony size but we will sequence the agrC gene locus to see if this gene is mutated. Another idea is to perform compensatory evolution on other slow-growing S. aureus mutants and look for mutations in agrC. These experiments will yield important clues to whether agrC muta- tions are due to environmental adaption, to general compensation for slow growth or to specific compensation for loss of formylation.

49 Acknowledgements

First and foremost, I would like to thank my supervisor Dan Andersson for giving me this opportunity to explore the world of research. Your enthu- siasm is mildly contagious and your sense of humor really is the stuff of legends!

I would also like to thank my co-supervisor Diarmaid Hughes for always lending an ear and helping out with proofreading this thesis.

All the past and present members of the lab, we truly had some amazing times! Especially Sanna – my companion in crime and a true friend. Life here in the lab would have been so much duller without you and I sincerely hope we will stay friends. Linus, you are the glue that kept the lab together at times, and I love your (our) sense of humor. Maria, (almost) always le- velheaded, and someone you can really count on to take time to listen. Miss Chris – thanks for letting me have half your lab bench, cheering me up and never saying no to a (light) beer. Peter – always the mysterious guy, keep on sneaking! Thanks for always taking the time for my technical questions on various things. Song, you will soon be the senior PhD student, and you will do great! Jocke – Hopefully, you will be a big innebandy star one day, but for now you have to be content with being a star of translation. Thank you for the help with the ribosome pics! Marlen, Hava, Erik and Lisa– you have started out great and I am sure you will have as much fun as we have had. Hervé and Amira, hope you will enjoy your post docs here in Sweden!

In addition I would like to thank Sanna, Linus, Jocke, Chris and Maria for your constructive criticism and proofreading of this thesis.

A big thank you to all my collaborators in other labs, especially Michael Pavlov, Måns Ehrenberg, Jenny Göransson and Mats Nilsson. Thanks also to all my co-authors, especially Annika who showed me the ropes when I first started out in the lab.

Past and present corridor members: The virus girls Heidi, Ellenor, Monika and Anna -always time to chat for a while or to share your lab skillz. Lena – your help in the lab has been invaluable and I wish you all the best as you

50 start the next phase of your life. Nizar, Amani and Göte - always a smile to greet me, means a lot on bad days.

The guys and gals in IPHAB throughout the years –pubs, lussefika, crayfish parties – you name it, we did it!

The administrative and technical staff – Tony, Nalle, Ted, Olav, Kerstin, Marianne, Barbro, Erika, Rehné, Malin. Thank you for all your assistance throughout the years!

My friends – what would I ever have done without you? Viktor, you have a magical ability of always being able to cheer me up. Don’t ever lose your positive outlook on life! And thank you for being my go-to guy in all things graphical and doing a great job with the cover of this thesis. Katta, you have stood by me for more than 15 years now, and I hope and know it will be longer still. Thank you for always lending an ear when I needed it and for keeping me company all those times at Maj’s café. Moa, I miss our lengthy discussions about nothing and everything over giant cups of tea. When your time come you will be brilliant. Yes you will! Ville, you are a great guy and one of the most thoughtful people I know, thank you for all the cat-sitting! Your quirky sense of humor is (almost) always good for a laugh or two. The party crowd: Mike, Per, Claes and Carro. No one knows how to party as you guys, hope to see it at my dissertation party! Anders, for lovely distrac- tions while writing this thesis – I sorely needed it!

Sist men inte minst, tack till min familj: mamma Agneta, papi Euro och systeryster Sandra. Jag har ofta sagt, men kanske inte till er, att ni är den bästa familj man kunnat önska sig! Jag har alltid känt ert stöd och vetat att ni är på min sida, vad som än händer.

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