Resistance to antibiotics is already a significant and still increasing problem in our society also due to an unnecessary over-usage of these compounds. Bacteria utilize multiple strategies to obtain resistance against antibiotics, which causes problems due to the increasing number of resistant bacteria (Davies and Davies 2010). This issue will be discussed here with one specific example, the compound tetracycline (for review see (Griffin, Fricovsky et al. 2010)). Tetracycline was first introduced for medical usage in the late 1940s. Since Tet is a broad-spectrum antibiotic, it was heavily used against a wide range of Gram- positive and Gram-negative bacteria as well as chlamydiae and protozoan parasites for many years. Bacteria have developed several strategies to obtain resistance to this compound, including efflux pumps, ribosome protection proteins (RPPs), enzymatic inactivation of Tet and some uncharacterized mechanisms of resistance (for review see (Chopra and Roberts 2001)).
In 1948, the first member of the group of tetracyclines was isolated, namely chlortetracycline, that is naturally produced by Streptomyces aureofaciens (Duggar 1948). In general, the chemical structure of this class consists of a four-ring system (with the individual rings designated as A, B, C and D) and functional groups attached to it. Later, other tetracyclines were discovered, like the compound tetracycline (Tet) from several
Streptomyces species (1953), but also semi-synthetic approaches were accomplished with the production and marketing of doxycycline (Winkler and Weih 1967) and minocycline (Klastersky and Daneau 1972) which were essentially well tolerated. These compounds are known and can be referred to as first-generation (1948 to 1963) and second-generation tetracyclines (1965 to 1972). The next generation consists of the semi-synthetic group of
glycylcyclines (Chopra 2002), with tigecycline (Tgc) as one of the commercially used compounds (Tygacil®). In comparison to Tet, Tgc possesses a substitution at the C7 and C9 position of the chemical structure and displays an enhanced antimicrobial activity, including resistance against tet genes (Rasmussen, Gluzman et al. 1994; Bergeron, Ammirati et al.
1996).
As of 2001, twenty-nine different tetracycline resistance (tet) and three oxytetracycline resistance (otr) genes have been characterized, which encode either efflux proteins or RPPs. Efflux proteins have been extensively studied and all tet efflux genes encode for membrane- associated proteins which transport the drug via the cell membrane out of the cell. The pump functions with the help of a proton gradient, which means one H+ is exchanged against one molecule of tetracycline (Yamaguchi, Ono et al. 1990) resulting in a reduction of the intracellular drug concentration. In general, efflux proteins have a size of approximately 46 kDa and are divided into six groups according to their sequence identity. Tet efflux pumps show sequence and structure similarities to other known efflux proteins, for example AraB, an arabinose transporter described in Escherichia coli (Sheridan and Chopra 1991). Due to the encoding of many tet genes on plasmids, together with other favorable selection markers, the efflux genes are already widely distributed and this might increase even more during the next years.
Ribosome protection proteins (RPPs) are soluble, cytoplasmic proteins that confer resistance to tetracyclines by releasing the compound from the ribosome. In general, RPPs show homology to elongation factors EF-Tu and EF-G, including the GTP-binding domain (Sanchez-Pescador, Brown et al. 1988; Taylor and Chau 1996). Both proteins, Tet(O) and Tet(M), are well-characterized. It has been shown that EF-G and Tet(M) bind to the same region of the ribosome, however, Tet(M) has a higher affinity in comparison to EF-G (Dantley, Dannelly et al. 1998). Hence, there are functional differences between Tet(M) and EF-G, since the RPP cannot compensate for the function of EF-G in vivo and in vitro (Burdett 1996). GTP hydrolysis might be crucial for the release of the compound from the ribosome (Taylor and Chau 1996; Trieber, Burkhardt et al. 1998), but the exact mechanism as to how the compound is released from the ribosome is so far not well understood. Structural investigations using cryo-EM were performed on a complex consisting of E. coli 70S, Tet(O) and a non-hydrolyzable GTP analogue resulting in a structure at 16 Å resolution (Spahn, Blaha et al. 2001). It could be shown that the binding position of Tet(O) is close to the ribosomal A-site and adjacent to the Tet1 binding site similar to EF-G, however, the tip of
domain IV of EF-G reaches further into the A-site contacting intersubunit bridge B2a in the case of EF-G (Agrawal, Heagle et al. 1999) suggesting that Tet release from the ribosome is catalyzed by Tet(O) indirectly, probably due to conformational rearrangements in h34 (Spahn, Blaha et al. 2001).
Tet can also be inactivated in an enzymatic way by the function of tet(X). Tet(X) is a 44 kDa cytoplasmic protein that encodes for an oxidoreductase and modifies the chemical structure of Tet in the presence of oxygen and NADPH. So far this gene was only found in anaerobic Bacteroides (Speer, Bedzyk et al. 1991; Yang, Moore et al. 2004). Additionally, one more gene is known to be involved in conferring Tet resistance, however, the mechanism is not described to date. tet(U) encodes for a small protein that shows some sequence similarity to known tet genes excluding the GTP binding domain (Ridenhour, Fletcher et al. 1996).
Due to bacterial strategies to obtain resistance against Tet, there was some time and effort invested to find and develop new tetracycline derivatives, such as the third- and fourth- generation glycylcyclines, that are immune to some of the above-mentioned resistance mechanisms (Chopra 2002). The most prominent and commercially used member of this group is tigecycline (Tgc, Tygacil®) that differs from the original Tet structure by having extensions at the C9 position (of ring D). This new compound shows higher affinity to the ribosome in comparison to the original compound (Bauer, Berens et al. 2004) and is immune against bacteria harboring tet resistance genes (Rasmussen, Gluzman et al. 1994; Bergeron, Ammirati et al. 1996). However, in recent studies it could be shown that TetX represents the first identified resistance mechanism against the broad-spectrum antibiotic Tgc (Volkers, Palm et al. 2011).
2 Objectives and Aims of the Thesis
The ribosome and the process of translation are central to the life of the cell and are therefore highly regulated. To date, a multitude of factors involved in ribosome function have been identified. This includes a variety of proteins that play a role during ribosome biogenesis, such as RNA modification enzymes and chaperones (see section 3: Dönhöfer, Sharma et al., 2009), but also protein factors that participate directly in the essential process of translation, e.g. the elongation factors EF-Tu and EF-G. Furthermore, the binding and interaction of factors to the ribosome can be modulated by the composition of the ribosome itself and therefore insights into translation regulation in different organisms, such as archaea or eukaryotic organelles (chloroplasts and mitochondria) can arise from differences in composition of their respective ribosomes. Finally, translation itself can be inhibited due to the action and binding of antibiotics to the ribosome. Interestingly, resistance to some antibiotics, such as the tetracyclines, can arise from the binding of specialized protein factors to the ribosome.
With this in mind, the thesis presented here aims to investigate these different regulatory events and is divided into four topics:
(I) Ribosome Biogenesis
The role of a group of factors involved in bacterial ribosome biogenesis has been so far only partially understood. This thesis concentrates on two different modification enzymes, namely the E. coli methyltransferase YhhF and RlmH as well as one chaperone-like protein,
E. coli RimM (see section 4). The aim for these projects was (i) to identify their exact binding position on either the 70S ribosome (in case of RlmH) or the small ribosomal subunit (in case of YhhF and RimM) by cryo-EM analysis and (ii) to gain more insights in the functional roles of these proteins. Moreover, ribosome assembly can be affected by the presence of several antibiotics. Here it was shown that premature ribosomal particles treated with specific compounds are able to maturate, but exhibit differences in the r-protein content in comparison to standard intermediate particles determined mainly by pulse labeling and mass spectrometry (see section 3: Siibak, Peil et al., 2011).
(II) Translation Factors
During the process of translation, the growth of the nascent polypeptide chain is facilitated through the movement of tRNAs on the ribosome in a cyclic manner accompanied by the connection of amino acids via peptide-bond formation. This process is catalyzed by EF-G in a GTP-dependent manner. One part of the thesis focuses on the translocation mechanism, in particular showing two structural sub-steps of translocation analyzed by cryo- EM (see section 3: Ratje, Loerke et al., 2010).
(III) Ribosome Composition
In a more global perspective, the composition of archaeal ribosomes was investigated with a particular focus on the r-protein content. This analysis elucidated the presence of novel r-proteins in general and identified possible binding positions of these proteins determined by cryo-EM (see section 3: Márquez, Fröhlich et al., 2011). Moreover, this work addresses the composition of ribosomes present in intracellular organelles, such as chloroplasts and mitochondria. Both organelles possess specific r-proteins that exhibit novel features or have a specific function on the ribosome. In chloroplasts, six plastid-specific ribosomal proteins (PSRPs) are known and one of these factors, called PSRP1, was studied here in detail from a biochemical and structural point of view (see section 3: Sharma, Dönhöfer et al., 2010). In mitochondria, a larger number of additional factors have been identified, but their functions and binding positions on the ribosome are only partially understood. Therefore, one part of this thesis also focuses on this topic (see section 5).
(IV) Antibiotic Resistance Proteins
One specific protein conferring resistance to the antibiotic tetracycline, namely Tet(M), was also investigated in this thesis (see section 6). The aim here was to confirm the binding position on the E. coli 70S ribosome in terms of ribosomal contacts as well as to obtain better insight into the exact mechanism of how the antibiotic is released from the ribosome due to the action of this protein factor.