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Home , Crescentin, Cytoskeleton, MreB, ParM, Prokaryotic cytoskeleton

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List of Papers

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

I Bagchi, S., Tomenius, H., Belova, L. M., Ausmees, N. (2008) -like in and a cytoskele- tal function in Streptomyces. Molecular Microbiology, 70(4): 1037-1050 II Bagchi, S., Fuchino, K., Cantlay, S., Wu, D., Bergman, J., Ka- mali-Moghaddam, M., Flärdh, K., Ausmees, N. (2011) Co- operation between two coiled coil in polar growth in Streptomyces. Manuscript III Bagchi, S., Sandblad, L., Tomenius, H., Belova, L. M., Aus- mees, N. (2011) A preliminary model of in vitro assembly of a bacterial coiled coil cytoskeletal FilP. Manuscript IV Ausmees, N., Wahlstedt, H., Bagchi, S., Elliot, M. A., Buttner, M. J., Flärdh, K. (2007) SmeA, a small with multiple functions in Streptomyces sporulation including target- ing of a SpoIIIE/FtsK-like protein to division septa. Mo- lecular Microbiology, 65 (6): 1458-1473

Reprints were made with permission from the respective publishers.

Contents

Introduction...... 9 Prokaryotic ...... 10 homologues in ...... 10 homologues in prokaryotes...... 12 Prokaryotic intermediate filament-like cytoskeleton...... 13 The Streptomyces coelicolor...... 19 Germination and vegetative growth ...... 20 Formation of aerial hyphae...... 21 Sporulation ...... 22 and segregation during sporulation...... 23 The present study ...... 25 Aim of our study ...... 25 Coiled coil proteins are widely present among bacteria (Paper I) ...... 27 Spontaneous assembly of FilP filaments (Paper III)...... 30 FilP cytoskeleton in vivo (Papers I and II)...... 34 FilP deletion causes a morphological defect in S. coelicolor and slows down vegetative growth to some extent (paper I)...... 36 The interaction between FilP and DivIVA (Paper II) ...... 39 Characterization of a novel sporulation locus in S. coelicolor genome (Paper IV)...... 43 Swedish summary of the thesis...... 47 Acknowledgements...... 50 References...... 51

Abbreviations

Intermediate filament IF Deoxyribonucleic acid DNA PG Atomic Force Microscopy AFM Cyclic adenosine monophosphate cAMP ATP Enhanced green fluorescent protein EGFP

Introduction

The notion that bacteria are ‘just bags of ’ with no structural organi- zation, has been completely altered during the last 10-15 years. Within this relatively short period of time, bacteria have been demonstrated to be well- structured organisms with proteins that are homologues of, or similar to dif- ferent cytoskeletal elements present in (13, 14, 41, 54, 56, 57, 74, 91, 99, 101, 141). Thus, bacterial cytoskeleton is a fairly new concept. The interior of a bacterial cell appears to contain several different cytoskeletal elements, probably with a large number of associated components still to be discovered. Hence, the prokaryotic cell architecture is of immense interest for research nowadays. Eukaryotic cytoskeletons, which are readily visible in the electron micro- scope, have been intensively studied over a long period of time and their functions and working principles are well known by now. Historically, they were divided into three main groups according to the thickness of the corre- sponding filaments. are hollow tubes with a diameter of 25 nm, made up of - and -tubulin subunits. Actin filaments are approximately 7 nm thick, whereas intermediate filaments (IFs) have a diameter of around 8- 10 nm, and consist of various subunits, depending on the type of the filament and of the cell (3). Tubulin-derived microtubules and actin filaments are dynamic structures that undergo assembly, disassembly and redistribution within the cell in response to different cellular signals. They carry out dy- namic cellular tasks, such as chromosome segregation, cell division, cell movements, etc. Furthermore, both types have their own dedicated motor proteins, which move along cytoskeletal tracks and perform . Eukaryotic cytoskeletons include also stable filamentous struc- tures, such as intermediate filaments, which are thought to act as mechani- cally stable and resilient structural scaffold, maintain cell shape and integ- rity, confer elasticity, and protect against stress. The bacterial cytoskeleton can be described as filamentous structures that are based mainly on of a single class of proteins. Most bacterial cytoskeletal elements have also been shown to self-assemble in vitro into extended polymeric filamentous structures. Evidently, both eukaryotic and prokaryotic cytoskeletons share the common characteristics of their poly- meric and filamentous nature. Also, the performs similar functions as its eukaryotic counterpart, including cell division, chro- mosome segregation, cell shape determination, establishment of polarity, etc.

9 However, the differences between these two classes of organisms in terms of cytoskeletal elements are also quite clear. All characterized eukaryotic or- ganisms have actin- and tubulin-based cytoskeletons; metazoan organisms also have intermediate filaments. The presence of IFs in plants and fungi is still under debate. The vast majority of bacteria contain FtsZ, a tubulin homologue (13), but only a subset contains MreB, an actin homologue (74). Recent investigations have shown that intermediate filament-like proteins are present in bacteria quite widely. Thus, some bacteria are documented to contain counterparts to all eukaryotic cytoskeletal elements, with Caulobac- ter crescentus being the well-characterized example. Bacteria also possess cytoskeletal elements whose eukaryotic counterpart(s) are yet to be found, for example proteins either similar or homologous to MinD or ParA have not been found in any yet. Prokaryotic actin and tubulin proteins are by now well studied and shown to be conserved in bacteria and evolutionar- ily related to their eukaryotic counterparts, whereas the evolutionary rela- tionship between prokaryotic and eukaryotic intermediate filament-like pro- teins is still unclear. Moreover, it is apparent that different cytoskeletal classes in eukaryotes and prokaryotes use different mechanisms to perform analogous functions. For example, the tubulin filaments form the mitotic that is used to segregate the with the help of dedicated motor proteins. Intracellular vesicles and other cargo are also moved through cells on these tracks by motor proteins with defined direction of movements on microtubules. However, the bacterial tubulin FtsZ forms a contractile ring and directs cell division. Furthermore, no cytoskeleton- associated motor proteins have been characterized in bacteria so far.

Prokaryotic cytoskeleton All three eukaryotic cytoskeletal proteins, such as tubulin, actin and IF, have been found to have bacterial counterparts. They play crucial roles in sub cellular architecture in bacteria. The cytoskeletal elements operate, in gen- eral, as dynamic scaffoldings to regulate different important cellular func- tions (101) such as cell shape (MreB, DivIVA, ), chromosome segregation (ParA) and cell division (FtsZ).

Tubulin homologues in prokaryotes FtsZ is considered as the prokaryotic homologue of tubulin, irrespective of the fact that it shares only weak sequence similarity to its eukaryotic coun- terpart. This protein is present probably in all bacteria, and chloro- plasts (10, 96, 116, 144). FtsZ is found to polymerize in vitro in a tubulin- like, GTP-dependent manner (92, 130). Moreover, the crystal structure of FtsZ revealed a folding pattern very similar to that of tubulin (89). Both tu-

10 bulin and FtsZ are composed of two domains (114). All the contacts with the nucleotide are made by very similar N-terminal domains. The C-terminal parts of these two proteins differ significantly, where FtsZ lacks the charac- teristic long helices present in tubulin. Tubulin uses a so-called T7 loop (114) or a synergy loop (40) to insert into the nucleotide binding pocket of the next subunit of the protofilament and consequently activate hydrolysis. FtsZ probably uses a similar mechanism because it possesses a similar loop at the same position as T7 in tubulin (109, 134, 158). It was experimentally shown that FtsZ forms similar protofilaments as those of tubulin (90). Very interestingly, it has been demonstrated that similarly with tubulin filaments, GTP-bound FtsZ protofilaments are straight, but the GDP-bound forms pro- duce curved filamentous structures, like helices and rings (92). The confor- mational change is probably generated by phosphate release (104, 130). Thus, it seems unlikely that structurally very similar proteins with a con- served mechanism for dynamic filament formation arose independently dur- ing . It is possible that FtsZ and eukaryotic have a common evolutionary origin. In vivo FtsZ forms a ring structure at the cell division site, called the Z-ring (13). Several other cell division proteins, including the proteins involved in peptidoglycan synthesis, have been demonstrated to assemble at the impending division sites marked by the Z-ring (30, 43). This leads to constriction of the cells, formation of the septa, and consequently cell division and cell separation occurs (43, 93, 95). The Z-ring is actually proposed to be a helical structure with highly compressed pitch, whose as- sembly and disassembly involve several other interacting proteins. Other groups have reported that the Z-ring is not a continuous of FtsZ, but is discontinuous and consists of spatially separated arcs (88). It is not yet clear what causes the constriction of the Z-ring in vivo, but a membrane- anchored derivative of FtsZ was able to constrict lipid vesicles without any other factors (118). The location of the ring is specified by MinC–MinD proteins in certain bacteria (50). MinD binds to the membrane at the pole. The concentration of MinC-MinD complex is highest at the poles and lowest at the midcell position, where usually the Z-ring is formed. In some bacteria, there is another protein called MinE that displaces MinD from the membrane by increasing the ATP hydrolyzing rate (58, 124, 125). It is worth mention- ing here that this system is not present in all bacteria including, Caulobacter and Streptomyces. E. coli has the complete system with all three proteins MinCDE, whereas has only part of it with just MinCD and not the MinE. Another protein, called FtsA, is present in some bacteria and it inter- acts directly with FtsZ (59, 156). This protein probably is influencing at- tachment or modulation of the Z-ring. However, despite of being devoid of FtsA, many bacteria, including S. coelicolor, form Z-ring. (86, 139) present in several Bacillus species use a tubulin-based cytoskeletal element for segregation. pBtoxis encodes a TubZRC partitioning system for its segregation during host cell division. An adaptor

11 protein TubR binds a , tubC, and recruits the tubulin-like protein TubZ. TubZ forms a dynamic filament to push the plasmid towards the cell pole. TubZ has several properties typical to tubulin: it binds and hydrolyzes GTP, requires a relatively high critical concentration of TubZ-GTP subunits for assembly, and forms filaments containing predominantly TubZ-GDP. Recently the determination of the structure of the TubZ filament revealed a double helical superstructure, which is unusual for tubulin-like proteins (7). This rather resembles the structure of the actin-like filaments of ParM, an- other protein involved in plasmid segregation in bacteria (see below). It was suggested that there is a general mechanism how plasmid segregation can be executed by dynamic protein filaments, causing the similar shapes of these very different types of filaments via .

Actin homologues in prokaryotes Recent research has revealed several distinct actin-like proteins in prokaryo- tes. The actin-like proteins MreB and Mbl (MreB-like) (18, 19) form ex- tended, often helical and dynamic filamentous structures underneath the in most rod-shaped bacteria. The best characterized function of the MreB proteins is to affect cell shape of different bacteria (74). MreB-like proteins are responsible for the cylindrical growth of non-cocci bacteria, but they are not required to maintain all non-spherical cell types (91). MreB cytoskeleton interacts with transmembrane proteins MreC and MreD, and is believed to ultimately recruit the enzymatic machinery of peptidoglycan synthesis to specific sites (34), and thus ensure longitudinal elongation of the cell envelope. The crystal structure of MreB from Thermatoga maritima has been solved revealing similarity to eukaryotic F-actin, regarding both the monomer and protofilament structure (142). It appears to form polymers in mild conditions with strict requirement of ATP. Electron micrographs of polymerized MreB demonstrate both straight and curved filaments as well as small two-dimensional sheets. These structural and functional (cell shape determination) similarities between MreB-like proteins and actin suggest that they are true homologues, even though they have very low sequence identity. However, the actin sequences among eukaryotes are well- conserved. From the evolutionary perspective, this could indicate an early divergence phenomenon from a common ancestor of prokaryotes and eu- karyotes. It could also lead to the conclusion that may be these proteins are only needed to retain the nucleotide binding and hydrolysis domains for polymerization and interaction with other elements. Another actin-like protein called ParM is encoded in low-copy-number plasmids, such as R1 of E. coli (107). Low-copy plasmids encode (par) responsible for active partitioning of the plasmid DNA (6, 52, 115) prior to cell division (53). Orthologues of these par genes have also been found on bacterial chromosomes (106-108). The par locus of R1 contains

12 parM, parR and a centromere-like sequence parS. ParM forms filamentous actin-like structures both in vivo and in vitro. The assembly of these fila- ments is dependent on ATP and Mg2+, and their disassembly requires ATP hydrolysis. It has been demonstrated that ParM polymerizes into double helical protofilaments with a longitudinal repeat similar to filamentous actin (F-actin) and MreB filaments. However, the crystal structure of ParM re- veals major differences in the protofilament interface compared with F-actin, despite the similar arrangement of the subunits within the filaments. ParM also binds to the ParR protein. ParR recognizes the parS sequence and binds to it. ParM filament grows by insertion of new ParM-ATP subunits at the interface between the end of the ParM filament and the ParR/parC complex, associated with ATP hydrolysis. The unidirectional elongation of dynami- cally unstable ParM filamentous structures push plasmids apart during cell division in a way that is analogous to .

Prokaryotic intermediate filament-like cytoskeleton IFs are the most stable cytoskeletal structures in eukaryotic cells. They retain their normal arrangement in stress conditions where the cytoplasmic and nuclear constituents fall apart (73). Therefore, it was believed for a long period that the major function of IF was to protect cells from different me- chanical stresses by providing a fixed and stable infrastructure. Recent stud- ies suggest that IFs are highly dynamic structures and they are involved in several important physiological functions, such as (65), (117) and regulation (32, 35, 119). Their prokaryotic coun- terparts have been found recently in some bacteria. These IF like proteins seem to perform different functions in different bacteria. Crescentin gives the moon crescent-like shape to Caulobactor crescentus (4), whereas FilP provides rigidity and elasticity in the multicellular organism Streptomyces coelicolor (8).

Coiled coil domain architecture All IF proteins, found so far, share a common ‘tripartite’ architecture. It con- sists of a central ‘rod’ domain of alternating coiled coil segments and linkers, flanked by head and tail motifs (61). The coiled coil segments consist of heptad repeats in which the first and fourth amino acids are hydrophobic (120). This arrangement translates into an alpha-helical structure with a hy- drophobic seam. Two such structures bind together at the hydrophobic re- gion to form the coiled coil configuration (Figure 1) (51, 131, 136) . The elongated shape and the specific biochemical properties of an IF protein depend on the rod domain (121). The variations in the architecture and the sequences of the rod domains of eukaryotic and prokaryotic intermediate filament proteins are quite wide-ranged. Both coiled coil parts and non- coiled coil linker regions may have different lengths and sequences in differ-

13 ent IF proteins. Also, the head and the tail regions are also widely variable. This makes IF proteins unique among the other cytoskeletal proteins actin and tubulin, which are strictly conserved in all eukaryotes.

Figure 1. Schematic diagram of coiled coil protein showing alignment of seven amino acid repeats in each coil. a, d and a’, d’ represent two successive hydrophobic amino acids in each coil, respectively. Hence, the center of the coiled coil protein demonstrates the hydrophobic ‘seam’ that brings two coils together.

Crescentin The bacterial world is full of interesting shapes and sizes. During recent years cytoskeleton has emerged as an important determinant of cell shape also in bacteria. MreB proteins provide the cylindrical shape to many rod- shaped bacteria, such as E. coli and B. subtilis (74, 147). Supporting this view, it has been found that naturally round bacteria such as streptococci have no mreB homologues in their genomes. On the other hand, certain cy- lindrical bacteria such as rhizobia and corynebacteria are devoid of mreB homologues in their genomes (34). Thus, it appears that there are probably more factors involved in rod-shape determination than are known today. There are even more complex cell shapes found in the bacterial world than cylinders. What makes bacteria vibroid or helical? What is the biological

14 relevance of a complex cell shape? Some of these questions were addressed when the IF-like protein involved in cell architecture was found in Caulo- bacter crescentus. The protein was named ‘crescentin’ as it provided the moon crescent-like shape to the bacteria by forming a continuous filamen- tous structure along the inner curvature of the cells (4, 14, 15). It also spon- taneously formed filaments in vitro upon denaturation and subsequent rena- turation into physiological buffers without addition of nucleotides, divalent cations or other cofactors (4). This is a characteristic and distinguishing fea- ture of animal IF proteins. The assembly mechanisms of IF proteins into filaments and the roles of the various structural domains, such as the head, coiled coil segments, linkers, etc. have been extensively studied over many years, and a large body of data is available (33, 85, 145, 146, 157). The structural and functional analysis of the crescentin protein has demonstrated the importance of its distinctive IF-like domain organization and has re- vealed similar principles of assembly and similar properties of the filaments to those of eukaryotic IFs (16). The head domain of crescentin consists of two parts. The first amino terminal subdomain is required for its function, but not for the assembly of the filaments. The second part is needed for as- sembly. The rod domain is essential for the filament formation and the first linker (L1) is responsible for preventing formation of nonfunctional aggre- gates (16). The stutter and the tail domain are found to play important roles in stabilizing the filamentous structure against disassembly in the . De novo assembly of the crescentin filament in vivo has been found to be biphasic (23). Initially, bipolar longitudinal extension of the crescentin fila- ment takes place. When the structure ends reach the cell poles, crescentin subunits are inserted laterally to control the length of the filamentous struc- ture along the cell curvature in a cell size-dependent manner. Similarly to IFs the crescentin structure is relatively stable. Photobleaching experiments upon inhibition of new protein synthesis showed that there is no discernible ex- change of the subunits already incorporated into the filament (23). Similarly, another group concluded that the dynamics of the crescentin filamentous structure is very slow, especially the dissociation (44). Incorporation of new subunits lead to the half-time of fluorescence recovery of 26 ± 2 min, but it should be noted that these experiments were performed in a situation where new protein synthesis was permitted (44). New subunits can incorporate laterally and cause filament thickening (23). There is also growth from the ends, lengthening of the filament as the cell elongates. Elongated C. crescen- tus cells give rise to a continuous helical crescentin filament in vivo (4). This helical form of crescentin was observed even in the elongated straight cells (non-functional crescentin-GFP causes straight C. crescentus cells). This suggests that crescentin can assemble into an intrinsically helical polymer by itself and does not require the helical configuration of the bacteria. How does crescentin generate cell curvature? A main clue for was provided by the ob- servation that in its membrane-attached form the crescentin structure

15 stretches in a straight line from pole to pole along one side; but upon release it takes the form of a left-handed helix in the cytoplasm and shortens. This implies that the crescentin cable is elastic and attached to the cell membrane in a stretched-out state, and as such, applies strain on the underlying cell envelope. Another key experiment demonstrated that C. crescentus seems to synthesize less on the concave side compared to the convex one of the cells. Together these observations lead to a model. It is assumed that the peptide bonds of the PG sacculi are parallel to the cell membrane. They are elastic, and in a stretched configuration in normal conditions, probably due to the turgor pressure (81). This stretch facilitates hydrolysis of existing bonds and insertion of new cell wall precursors (81). In many rod-shaped bacteria new PG insertion occurs along the lateral wall, causing elongation of the cell cylinder. The strained crescentin structure exerts compressive force and reduces the degree of stretching of the underlying peptide bonds, causing lower rate of new PG synthesis at the site of its attachment to the cell envelope. The compressive force exerted by crescentin is propagated by the elastic PG fabric and a "stretch gradient" is formed, being lowest at the side of crescentin and highest exactly opposite it. Subsequently, a rate gradi- ent of new PG insertion is formed, resulting in a bent cell. Thus, crescentin's effect in generating cell curvature is mechanical, rather than biochemical. It has been suggested that proper assembly and organization of IFs in metazoan cells are actually dependent on the integrity of the tubulin and actin networks (22). Likewise, there is evidence that crescentin interacts with MreB. Cres- centin is anchored to the cell membrane at the inner curvature of Caulobac- ter crescentus and this cellular organization is MreB-dependent. This reflects a similar actin-dependent role of IF in metazoan cells (15, 17, 23). It is also likely that crescentin interacts with other factors in order to create the cellu- lar asymmetry. The next important quest is to study the existence and importance of IF- like cytoskeletons in a wide range of bacteria. We predicted that such pro- teins should be relatively common in prokaryotes (Paper I), and several very recent publications from other groups have supported this view. We have also experimentally characterized a novel IF-like cytoskeleton in Streptomy- ces coelicolor, a filamentous bacterium with complex cell shape and life cycle (see below and Papers I, II and III).

Other coiled coil cytoskeletons in bacteria The first IF-like protein with IF-like coiled coil conformation was described in 1992 and it was shown that a bacterial protein, called TlpA in Salmonella typhimurium can actually organize itself into an ordered dynamic structure in the cells (64, 84). Later on several candidates of IF-like proteins were pre- dicted to be present in , a bacterium with helically curved cell shape. Recent work has shown that the helical shape of H. pylori is in- deed dependent on these coiled coil-rich proteins (Ccrp) which form fila-

16 mentous structures in vivo and in vitro (148). Four such Ccrp proteins have been identified so far in H. pylori (94, 148). These proteins with high se- quence variations are differentially needed for maintenance of cell morphol- ogy in different strains. For example, deletion of one ccrp called HP0059 caused total loss of helical cell shape in three out of the four strains tested. In the remaining strain (called 1061) the mutation also caused a similar pheno- type, but only in 85% of the cells. Deletion of another ccrp HP1143, on the other hand, caused a severe cell shape defect in the strain 1061, whereas it gave rise to relatively mild cell straightening in the other three strains. The HP1143 mutant of 1061 had lost the ability to maintain regular shape, the cells could be round, oval, bulgy rods, or otherwise irregularly shaped. Thus, HP1143 is critically needed for elongated rod and helical shape determina- tion in Helicobacter. Deletion of both genes caused a general exacerbation of the mutant phenotypes of the single mutants, indicating that each protein has a somewhat independent role in cell shape determination. All four Ccrp proteins have different oligomerization properties and they do not co-purify, suggesting their individuality in formation of polymers (94). Interestingly, MreB, although present, has no effect on cell shape in this basically rod- shaped bacterium (148). Another interesting coiled coil protein, named ‘rod-shaped morphology pro- tein’ (RsmP) is present in an actinobacterium Corynebacterium glutamicum and, as the name indicates, is needed for maintaining the rod-like cell shape of this bacterium (45). C. glutamicum belongs to the group of bacteria, which maintain their rod shape independently of MreB. Cell elongation is the result of new peptidoglycan insertion at cell poles, in strong contrast to the MreB-dependent insertion along the lateral sides. This polar growth is dependent on an essential coiled coil protein DivIVA (see below). RsmP was found as one of the proteins being induced by partial depletion of DivIVA. RsmP is also essential for viability, and its depletion caused a loss of the rod shape and adoption of the spherical shape by C. glutamicum cells. RsmP formed long filaments in vivo. Interestingly, the shape of the in vivo fila- ments was dependent on the phosphorylation status of RsmP, and phos- phorylation of this protein was necessary for directing growth at the cell poles. This resembles the role of phosphorylation in the assem- bly/disassembly of IFs. Also, similarly to IFs and crescentin, RsmP formed filaments spontaneously in the absence of any cofactor in vitro (8, 45). Sev- eral other coiled coil proteins are important for growth and of Actinomycetes, and will be more thoroughly described in the following sec- tion about Streptomyces, a main model organism of Actinomycetes. Tre- ponemes, members of spirochete family, have been shown to have a thick, helical cytoplasmic filament, consisting of the protein CfpA (68). These spectacular structures are probably historically the first bacterial cytoskeletal elements observed in the microscope. CfpA has no obvious homologues, but a distinct structural feature of CfpA is the segmented coiled coil architecture.

17 Although the coiled coil segments of CfpA are shorter than those in IF pro- teins and crescentin, and its general content of the coiled coils is lower, it has been proposed to be an IF-like protein (67). The function of this spectacular cytoskeleton is, however, poorly understood. A viable cfpA knockout mutant has been obtained in T. denticola (69). The mutation was pleiotrophic and caused several mild defects in cell division, , chromosome condensa- tion, etc. Future research will hopefully shed more light on the functions of this fascinating cytoskeleton.

18 The model organism Streptomyces coelicolor

Streptomyces species are naturally soil-dwelling, gram positive bacteria with G-C-rich genomes, belonging to the large group of Actinomycetes. They provide a large variety of and secondary metabolites with bio- logical significance. S. coelicolor has some striking and unique cellular fea- tures and a complex life cycle that make it a very useful model organism to explore different aspects of bacterial cell . For example, one intrigu- ing phenomenon is the polarity of the substrate hyphae, in which active growth occurs only at the tips of the hyphae. Obviously, the growing tips of these hyphae are the hot-spots where fascinating, and so far not well- understood processes and protein interactions take part. Streptomyces suc- cessively differentiates into several types of cells, starting from single spores, into vegetative hyphae, aerial hyphae, spore chains and ultimately individual exospores (Figure 2).

Figure 2. The developmental life cycle of Streptomyces coelicolor, demonstrating three major stages as follows: i) germination and vegetative growth, ii) formation of aerial mycelia and iii) sporulation.

19 Germination and vegetative growth Streptomyces coelicolor has three major stages in its life cycle, starting with germination from a single spore. Each spore gives rise to substrate mycelium with a multicellular meshwork of branching hyphae. Infrequent cross-walls (also known as septa) divide the vegetative hyphae into compartments with multiple copies of the genome, but cell separation does not occur. These compartments grow further by initiating new branches and the nascent branches then grow by tip extension. The frequency and spacing between the septa are variable. The mechanism behind the unequal division of vegetative hyphae by the cross-walls is unclear. Although the frequency and position of the branches are likely to be affected by the positioning of the cross-walls, it has been shown that both branching and tip extension happen independently of the cross-wall formation (99). The vegetative growth occurs only at the tips of the substrate mycelia by incorporation of new material into the cell wall (47, 123). The mechanism leading to polar incorporation of peptidoglycan is not completely known, but it involves a recently found essential protein, called DivIVA (46, 47). Di- vIVA is believed to recruit the protein complexes involved in peptidoglycan synthesis to cell poles. This polar growth is the opposite of the usual growth phenomenon observed in many rod-shaped bacteria (eg. E. coli, Bacillus subtilis) where the cells grow by the elongation of the lateral cell wall. The bacterial actin homologue MreB is believed to direct the insertion of pepti- doglycan into the lateral cell walls, while the poles remain inert during . The tip extension during the vegetative growth in S. coelicolor, in contrast, is independent of MreB, even though the mreB gene is present in its genome, but is instead required for maintenance of integrity of aerial hyphae and spores (97). Many non-sporulating rod-shaped bacteria with an apical growth pattern, such as corynebacteria and mycobacteria, are devoid of mreB genes (34, 62, 87, 111). Interestingly, depletion of DivIVA in Coryne- bacterium glutamicum leads to loss of polar cell wall assembly and forma- tion of spherical cells (87). Similar phenomenon has been observed in My- cobacterium smegmatis (111). In Streptomyces coelicolor, a new axis of cell polarity must be defined during initiation of branch formation. Enzymes involved in peptidoglycan assembly must be recruited to the initiation point of a new hyphal tip. It has been demonstrated recently that DivIVA is local- ized to the lateral wall as a focal point, from which a new branch emerges (60). This indicates the crucial role of DivIVA as a landmark protein that is involved in recruitment of cell wall biosynthetic machinery to new sites dur- ing hyphal branching. Formation of these new DivIVA foci in S. coelicolor is independent of the division sites or the existing poles. Obviously, as a ‘hot spot’ for growth, several important functions including peptidoglycan syn- thesis and assembly of other materials (such as teichoic acids, membrane lipids, cell surface proteins) for formation of the cell envelope must occur at

20 the hyphal tips (80). Little is known about these processes, and the localiza- tion of the relevant enzymes is unknown as well. One of the few proteins with characterized tip localization is the cellulose synthase-like protein, called CslA. It interacts with DivIVA and appears to be involved in the deposition of uncharacterized -linked glucan to the cell envelope (155). The cascade of events at the ‘busy’ hyphal tips is not complete yet, but DivIVA seems to be one of the crucial components involved in this process. Besides DivIVA, there are FilP and Scy, two other coiled coil proteins that have an important role in cell growth and morphology of S. coelicolor. FilP is the main subject of this thesis, and Scy is a large protein with many coiled coil segments (149). Deletion of Scy causes dramatic changes in cell morphology and integrity (our unpublished results). Bioinformatic analysis revealed a novel, distinct coiled coil repeat pattern in Scy, which is also pre- sent in FilP (8). It was proposed that these proteins might have their origin in ancient gene duplication. Thus, three different coiled coil proteins, FilP, DivIVA and Scy, with distinctly different functions collaborate in determi- nation of cell morphology and polarity.

Formation of aerial hyphae S. coelicolor reaches the stationary phase following the growth of substrate mycelia. The next phase of the developmental life cycle starts with produc- tion of aerial hyphae in the air, perpendicular to the vegetative hyphae (25). The growth of aerial hyphae is dependent on the release of nutrients from degraded vegetative parts (20, 102). The transition from primary to secon- dary as well as the production of antibiotics takes place at this stage of Streptomyces life cycle (21, 28, 29, 37). The entire complex cascade of cellular signaling involved in these processes including the birth of aerial hyphae from vegetative mycelia is still to be discovered. Extensive genetic analysis has revealed three groups of genes, namely ram, chp (31, 38, 82, 110, 154) and bld (63, 100, 112, 153) genes to be actively involved in the formation of aerial hyphae. A morphogenetic peptide, called SapB has been found to be important for aerial mycelium formation. It has been shown that many developmental (bald) genes are required for the of SapB. A collection of representative bald (bld) mutants, which are blocked in aerial mycelium formation, are all defective in the production of this peptide and regain the capacity to undergo morphological differentiation when SapB is supplied exogenously (153). SapB has also been described as a lantibiotic- like peptide (oligopeptide ) that is ribosomally synthesized (80). It is derived by posttranslational modification from the product of a gene (ramS) in the four-gene ram operon, which is under the control of the regula- tory gene ramR (82). ChpA-H, a class of eight hydrophobic secreted pro- teins, was shown to be expressed during the formation of aerial hyphae. De-

21 letion of chpABCDEH affected formation of aerial hyphae significantly. A purified protein mixture of ChpD-H was found to be highly surface active, and self-assembled into amyloid-like fibrils resembling those of a surface layer of aerial hyphae at the water–air interface. Possibly, the amyloid-like fibrils of ChpD–H lower the water surface tension to allow aerial growth and cover aerial structures, rendering them hydrophobic and perhaps ChpA–C help in binding ChpD–H to the cell wall (31, 38).

Sporulation Growth of the aerial hyphae is followed by a significant change in the regu- lation of cell cycle, which transforms the filamentous, multinucleoid cells into chains of unigenomic spores and finally into stress-resistant exospores. During maturation of the spores, a thick spore wall encircles the genetic material to create an ovoid-shaped individual spore. Prior to the release of individual spores from the spore chain, the spores acquire a grey pigment that is related to polyketide antbiotics and is specified by genes of the whiE cluster (77). Streptomyces spores are present in the soil environment where they need to survive for a long time by preserving their DNA. These exo- spores lack the surface layers that are inherited from mother cells to the en- dospores of for example Bacillus species, and are much less resistant to ad- verse conditions compared to Bacillus endospores (39). Streptomyces exo- spores also lack Ca2+-dipicolinate or small acid-soluble spore proteins (SASPs) (12, 39) that are indispensible for Bacillus (133). Yet, the Strepto- myces spores are more resistant to heat, desiccation and physical stress com- pared to the vegetative cells. Several important events take place during the cellular differentiation of aerial hyphae into spores: simultaneous formation of multiple, regularly spaced Z-rings, condensation and segregation of the chromosomes, thicken- ing and maturation of the spore cell wall, cell division and separation of the individual spores. A complex regulatory network governs these dramatic changes in cell architecture. All aspects of this regulation are not elucidated yet, but a group of genes called whi genes, have been identified as regulators of sporulation. Mutations in these genes resulted in characteristic white colonies, indicating their contribution in formation of grey spores from the white aerial hyphae (24, 26, 63). These genes are parts of an intricate signal- ing cascade containing multiple crucial landmarks that have to be checked for successful sporulation to commence (24, 27). Sporulation requires RNA polymerase sigma factor WhiG (48, 78), which directly controls the expres- sion of two target genes whiH and whiI (1, 48, 128, 140). whiH encodes a member of GntR family of transcription factors, whereas WhiI is similar to response regulators (1, 128). whiG, whiH and whiI mutants are blocked early in sporulation and form aerial hyphae, which are defective in sporulation

22 septation, although some septa are formed, especially in the whiH mutant. A second, converging pathway, independent of whiG is represented by addi- tional two whi genes, named whiA and whiB (2, 26, 48). Upregulation of both whiA and whiB, independently of whiG, have been observed during sporulation. WhiA and whiB mutants exhibit characteristic long and tightly coiled aerial hyphae completely devoid of sporulation septa, indicating that, these genes are probably required to signal termination of growth in aerial hyphae and initiation of sporulation (24, 48). All mentioned whi mutants, except those of whiH, are also defective in chromosome condensation and the aerial hyphae of these strains contain uncondensed and un-segregated . This, together with the more frequent occurrence of sporulation septa in the whiH mutant indicate that sporulation is blocked somewhat later in this mutant. All mentioned whi genes are supposed to have a role in tran- scriptional regulation, but the search for direct and indirect targets of these sporulation regulators is still ongoing. Further research is needed to reveal the details of the signaling cascades and cellular events culminating in sporu- lation in Streptomyces.

Cell division and chromosome segregation during sporulation The process of sporulation involves developmentally induced upregulation of tubulin homologue FtsZ (49), which first polymerizes into helical fila- ments and subsequently forms a series of regularly-spaced FtsZ-rings along the hyphae. Interestingly, even though FtsZ is required for cell division in S. coelicolor, it is dispensable for its growth and viability. The vegetative my- celia of ftsZ deletion strain grow by spreading non-septated branches (99). The survival of ftsZ deletion mutants in S. coelicolor makes this organism unique among most other bacterial species. Some other division genes (often essential in most bacteria), namely ftsI, ftsK, ftsL, ftsW, divIC have been found to influence sporulation in Streptomyces, but mutants of these genes are not completely devoid of cell division (11, 12, 98, 105). Hence, these genes are not essential for growth and viability of Streptomyces, but they are definitely needed for efficient spore formation. Interestingly, some Z-ring associated proteins like FtsA that is commonly present in many bacteria is, however, absent in Streptomyces. Two genes called ssgA and ssgB have been found to be involved in the developmental control of cell division (76, 143). They belong to a group of paralogues found specifically in sporulating actin- omycetes. Both of these genes seem to execute important roles in sporula- tion, but the molecular mechanisms are not completely clear. However, a recent study has demonstrated direct recruitment of FtsZ by SsgB (152). SsgB seems to interact with FtsZ to promote polymerization of the latter. The localization of SsgB is mediated through SsgA, and premature expres- sion of the latter is enough to directly activate multiple Z-ring formation and hyper-division at early stages of the Streptomyces cell cycle (152).

23 During the synchronous multiple septation occurring along aerial hyphae a well-controlled, augmented system of DNA segregation and condensation is needed to secure inheritance of one copy of genome by each spore. S. coelicolor has 8.7 Mb linear genome with centrally located oriC region and long terminal inverted repeats (TIRs) (12). DNA segregation follows DNA replication that populates predivisional apical cells with several copies of the genome (127). Different studies have demonstrated contribution of several factors in partitioning, DNA transfer and genome segregation. ParA and ParB were found to play a part in nucleoid partitioning. ParB binds to parS sites at the oriC region and forms a nucleoprotein complex that facili- tates the partitioning of chromosome during division (70, 79). ParA assem- bles into a helical structure along the length of the aerial hyphae in order to assist an even organization for equally distributed ParB-oriC complexes (71). However, lack of ParA and ParB exhibited relatively mild phenotype in terms of spores containing aberrant quantity of DNA, suggesting that there are probably more contributors to this process. It is assumed that a mecha- nism for transport of DNA through the closing septa must exist in this bacte- rium as constriction of sporulation septa takes place prior to completion of nucleoid partitioning in Streptomyces (47, 103, 113, 132). Hence, S. coeli- color FtsK protein, which is homologous to the SpoIIIE/FtsK DNA translo- cases (42), is a strong candidate to be involved in sporulation-specific ge- nome segregation by pumping parts of the chromosome through the closing septum (5, 151). But, together with the involvement of FtsK, other mecha- nisms must contribute to chromosome partitioning, considering that most of the FtsK null mutant spores inherit intact chromosomes. Members of the SMC (structural maintenance of chromosomes) play a central role in chromosome dynamics from bacteria to eu- karyotes including humans. Aberrant DNA condensation and missegregation of chromosomes have been shown to be the primary effect of the deletion of smc gene in S. coelicolor. It has also been demonstrated that mutations in the genes encoding SMC-associated proteins ScpA and ScpB primarily affect nucleoid morphology in the prespore compartments, suggesting a minimal role in segregation (36). A transcriptomic survey of gene expression during sporulation recently revealed a developmentally regulated gene encoding HU-like protein (129). This study showed that there are two differentially regulated HU-like genes present in S. coelicolor genome. The gene hupA is preferentially expressed in the vegetative mycelia whereas hupS is developmentally controlled and highly upregulated in the sporogenic hyphae. HupS was shown to be in- volved in nucleoid condensation in the mature spores as well as it is required to produce sufficiently heat resistant spores. Thus, efficient chromosome segregation probably requires the joint action of FtsK, SMC, and ParAB, and perhaps other proteins. SMC proteins, ParAB and FtsZ appear to be highly expressed in response to the requirement of aerial hyphal maturation (83).

24 The present study

Recent advances in the field of bacterial cytoskeleton has unwrapped more questions than answers. This growing area of research is focused on explor- ing the existence of cytoskeletal elements among microbes, their origin and their functions in different bacteria. Obviously, the evolutionary aspect of these newly-found proteins is very intriguing. A prevalent view supports the idea that eukaryotic actin and tubulin cytoskeletons evolved from ancestral precursors directly related to those of the prokaryotes (41). This implies that cytoskeletal proteins evolved very early in the common ancestor of bacteria, archaea and eukarya. It has been suggested that they originally performed and cell shape determination: functions that they retained among bacteria and archaea, but lost across the eukaryotes in order to acquire new ones. The evolution of actin and tubulin in eukaryotes must involve the co- evolution with motor molecules and binding proteins, and once they have received necessary optimizations required for their specific functions, their sequence remained conserved throughout the species. This explains the fact that actin and tubulin are highly conserved among the eukaryotes, but they diverge distinctively from their prokaryotic counterparts. The evolutionary origins of the third major eukaryotic cytoskeletal element, intermediate fila- ments (IF), however, is much less understood. The IF sequences are not highly conserved among the eukaryotes, but they share a common domain architecture with coiled coil segments separated by non coiled coil linker.

Aim of our study In the present study we are addressing some fundamental questions. 1) How widespread is the presence of IFs in prokaryotes? 2) Study of IFs in Streptomyces coelicolor – an important model organ- ism. 3) What are the principles of spontaneous assembly of the coiled coil- rich proteins and the structure of the IF-like filaments? 4) What is the secret of sporulation in Streptomyces coelicolor? What are the roles of cytoskeletal elements in differentiation?

In an attempt to address these questions, we applied genetics and molecu- lar biology techniques using Streptomyces coelicolor as a model organism,

25 biochemical methods and in silico bioinformatic tools. The summarized ob- jective of this thesis is to understand the functions of cytoskeletal elements in prokaryotes and their similarities and differences compared to their eu- karyotic counterparts.

26 Coiled coil proteins are widely present among bacteria (Paper I)

After the discovery of IF-like protein crescentin in , the obvious question to address was whether this is a unique feature of this particular bacterium, or is it a general property of bacteria, waiting to be characterized. The prokaryotic and eukaryotic and tubulins, despite of very limited overall sequence similarity, share conserved sequence motifs corresponding to the conserved functions of nucleotide binding and hydrolysis. IF proteins, on the other hand, have no specific sequence signatures, due to the lack of functional landmarks, such as signal sequence, DNA binding motif or enzy- matic activity etc. in the sequence. However, all eukaryotic IF proteins share a common domain architecture with a central rod domain consisting of coiled coil domains and non-coiled coil linkers (61). The rod domain is es- sential for the characteristic biochemical attribute of IF proteins to form in vitro filaments/polymers spontaneously in the absence of any external co- factors or energy (120, 131). The bacterial IF like protein crescentin also possesses an apparent rod domain with a different arrangement of coiled coil segments and linkers. It exhibits the specific biochemical property of spon- taneous polymerization without energy and cofactors, and determines cell shape of the bacterium Caulobacter crescentus (4). Due to the repetitive sequence of coiled coil proteins, search with IF proteins often pulls out sequences of proteins containing long coiled coil domains, such as SMC proteins, tail domains and others, but not necessarily prokary- otic IF homologues. Thus, we could not use, for example the Blast tool to identify candidates for IF-like proteins. One way to find potential IF proteins among prokaryotes, is to individually search for proteins with rod domains in different genomes. Hence, we began with in silico analysis of a set of bacterial genomes in order to identify coiled coil-rich proteins with a rod domain and with no putative function(s) designated to them. An algorithm called ‘COILS’ was applied on 26 bacterial genomes to predict the coiled coil content of all encoded pro- teins. Manual bioinformatic analysis was then carried out to identify and further analyze proteins with more than 80 amino acids in coiled coil con- formation, either as a block of continuous sequence or segmented by short non-coiled coil linkers. We excluded coiled coil proteins containing other

27 predicted functional domains (transmembrane segments, signal sequence, DNA-binding motifs) in order to sieve out the putative cytoplasmic IF pro- teins. Out of 26 genomes, 21 genomes encoded at least one candidate protein meeting our search criteria, and 16 genomes encoded several. As these ge- nomes were chosen to represent phylogenetically diverse groups of bacteria, it can be inferred that the presence of proteins consisting mainly of a coiled coil rod domain is common among bacteria. Further bioinformatics analysis unveiled a conserved rod-domain protein family with members from 14 ac- tinomycetes. The conserved sequence motifs are specifically positioned at the N-terminal confinements of two first coiled coil segments, regardless of the length or sequence of these segments or their intervening linkers in indi- vidual proteins. Crescentin does not share this conserved sequence trait. However, the general domain architecture of all these proteins resembles that of crescentin and the fact that it is not exactly the same, supports the notion of IF proteins being present in bacteria but hiding behind sequence dissimi- larity. After identifying a group of proteins with rod domains with no known func- tions, present in widely distributed bacterial species, the next step was obvi- ously to experimentally test the possibility of these proteins being IF-like proteins. First, we decided to test the characteristic biochemical property of spontaneous filament formation of these proteins, singled out in our in silico quest. We chose three proteins, called SCO5396 from Streptomyces coeli- color A3(2), Mb1709 from Mycobacterium bovis and JNB03975 from Jani- bacter sp. amongst the list of members of the actinobacterial rod domain family, considering their distinct domain architectures. These proteins were purified under denaturing conditions from E. coli using a recombinant tech- nology with an N-terminally added polyhistidine-tag. After renaturation in buffers with neutral pH and physiological salt concentrations, both scanning and transmission electron micrographs revealed the ability of all three pro- teins to form filaments spontaneously. These filaments from three different proteins were morphologically different. SCO5396 from S. coelicolor A3(2) formed two distinctly different types of filaments. We observed less frequent long, smooth and uniform rope-like structures, as well as more abundant striated and branched structures with varied diameter, the latter resembling strikingly filaments formed by nuclear under similar conditions (35). Mb 1709 from M. bovis formed laterally aggregating smooth filaments, whereas JNB03975 from Janibacter sp. polymerized into thin smooth fila- ments with no tendency of lateral aggregation. In vitro filament formation by three different proteins with distinctly different rod domain architecture from three different phylogenetically divergent actinobacterial species implies that assembly into regular structures is a common property of this conserved protein family. It also suggests that IF proteins are widely spread in bacterial genomes. The functions of these proteins in different bacteria might vary, but the analysed ones so far have demonstrated to be involved in some kind

28 of cytoskeletal function (see Introduction). In the following chapters, I will discuss one of these novel IF-like proteins; SCO5396 from S. coelicolor (the protein will be mentioned from now on as FilP for filament-forming protein) in more detail with some insight into its structure and function.

29 Spontaneous assembly of FilP filaments (Paper III)

The molecular building blocks of IFs are fibrous proteins that share a com- mon rod domain architecture consisting of coiled coil segments interrupted by non-coiled coil linkers. Interestingly, the primary sequences of all these proteins are divergent and the rod domain architectures are variable (Figure 4). How these divergent sequences are translated into formation of appar- ently similar polymers is an important quest. We have shown that bacterial coiled coil proteins polymerize into IF-like filaments. The pattern of the striated filaments formed by the S. colelicolor protein FilP strikingly resem- bled those of the eukaryotic IF nuclear A filaments (Figure 3). IF pro- teins lamins are the building blocks of . They maintain nu- clear shape, provide mechanical stability and are required for nuclear assem- bly, nuclear positioning, DNA replication, transcription regulation, chroma- tin organization and (35, 137). Mutations in human lamins are found to cause different heritable diseases as well as early aging (32, 119). However, the molecular mechanisms of this are not clear. Understanding the structural requirements for polymerization of FilP might not only help un- derstanding bacterial cytoskeleton, but also shed new light on the structure and function of animal IFs. In order to study the mechanism of polymerization of FilP, we first visual- ized FilP filaments by electron microscopy (both scanning and transmission) in different buffer conditions. The main result was that FilP formed either protofilaments with a beaded appearance or large striated filaments with dense bands. Both the beads and the dense bands were placed with regular intervals of approximately 64 nm (Figure 5). It has been shown that the 350 amino acids long rod domain of eukaryotic nuclear lamin is approximately 52 nm long (137). Similarly, since the central coiled-coil domain of FilP consists of 265 amino acids, the length of the rod domain should be ap- proximately 39 nm. The distance between the dark and light striations on FilP filaments is 64 nm on an average, as found by analyzing electron mi- crographs. Hence, the possibility of head-to-tail orientation in this assembly could be excluded and the head-to-head and tail-to-tail assembly was con- sidered as more likely.

30

Figure 3. Scanning electron micrograph of striated FilP filaments displaying uneven diameters

To study the orientation of the FilP subunits, His-FilP (a derivative of FilP with a hexahistidine tag in its N-terminus) together with gold nanoparticles coated with nickel ions (Ni-gold) were polymerized. These particles would specifically bind to the His-tags in the N-termini of FilP, and thus reveal the orientation of FilP subunits in the filaments. Filaments were then visualized with negative staining procedure. The gold particles, visible as electron-dark foci in the electron micrographs, did not stain the entire filament, but were found to be specifically clustered around the dark striations. Thus, we con- cluded that the dark striations were formed by overlapping head domains. Moreover, we studied dehydrated FilP filaments by using the tapping mode of the atomic force microscope (AFM). It revealed similar pattern of the filaments as seen in electron micrograph displaying striations. Height profile created for these filaments showed accumulation of significantly more mate- rial at the dark bands and additionally slight thickening corresponding to the position in the middle of the light striations, indicating that there is probably also an overlap between the tail regions. However, neither the head nor tail of FilP possesses globular domains, instead there are only short, approxi- mately 20 amino acids-long non-coil coiled stretches in both ends of FilP.

31 Thus, it was not obvious why the overlapping head domains cause a much denser band than overlapping tail domains. We propose that the first coiled coil domain folds back using a flexible linker and binds to the rod domain, thus creating a region with double density. The affinity of the first coiled coil segment towards the main body of the rod domain would also explain the formation of regular FilP networks. In the networks, the first coiled coil segment interacts with the main rod domain of another subunit, thus generat- ing a branch (Figure 6).

Figure 4. The rod domain architecture of crescentin, eukaryotic lamin A and FilP. The grey boxes represent coiled coil segments separated by the black straight lines representing non-coiled coil amino acid sequence.

Figure 5. Electron micrograph displaying the distance between two dark striations on FilP filament.

When we observed the filaments formed by His-FilP-mCherry (a derivative of FilP containing a His-tag in its N-terminus and a fusion to the red fluores- cent protein mCherry in its C-terminus), His-FilP-mCherry filaments seemed to retain the shape of His-FilP filaments, but were slightly shorter, thicker, and without striations in 50 mM Tris-buffer with and without 25 mM MgCl2. Interestingly, in a buffer containing 150 mM NaCl (a condition, which seemed to weaken the interaction between the tail domains of His-FilP), a weak striated pattern was discernible. This probably shows that, the globular mCherry domains increase the protein density at tail overlaps and causes an extra striation between those caused by head overlaps. We also looked at structures formed by a C-terminally truncated His-FilP (His-FilP-CT). This

32 derivative, lacking 30 C-terminal amino acids was not able to form fila- ments. However, the structures formed were not random aggregates, but appeared as a network composed of linear arrays of beads. This confirms that the C-terminal part is needed for filament formation. All data obtained so far are in agreement with our model of filament formation by FilP. Fur- ther experiments are needed to finally prove the model.

Figure 6. The hypothetical model exhibiting in vitro assembly of FilP filaments. The grey dots represent the N-terminal (head) domain.

33 FilP cytoskeleton in vivo (Papers I and II)

To understand the function of the FilP cytoskeleton we needed to move from in vitro studies to in vivo studies. First, we attempted to study the in vivo assembly of FilP by live-cell fluorescence microscopy of strains containing FilP tagged with a fluorescent protein. Unfortunately, so far we have not been able to create a fully functional fluorescently tagged FilP. However, the partially functional fusions have given us useful information. In a filP-egfp strain (filP-egfp is expressed from the native promoter at an ectopic locus in the chromosome in a filP deletion mutant strain), we observed salient fluo- rescent filamentous signals in both young vegetative hyphae, as well as in aerial hyphae, indicating that FilP also forms filaments in vivo and suggest- ing a probable role of FilP in vegetative and aerial hyphal growth. However, this strain was not fully functional as the morphology of the cells resembled that of the filP deletion mutant strain. A merodiploid filP+/filP-egfp strain had an indistinguishable morphology from that of the wild-type. In this strain, we recorded strong fluorescent signals at growing tips of the majority of the vegetative hyphae, localized as filaments along one lateral side, or as more diffuse polar caps. This strong tip localization was dependent on active growth. Weaker filaments, together with bright fluorescent foci were also formed along the older parts of the mycelium. This clearly suggested that FilP cytoskeleton was important during tip elongation, but it was difficult to interpret these signals, partially due to their heterogeneity and due to high autofluorescence in S. coelicolor. Using immunoflurescence with affinity purified against FilP we finally revealed the true localization pat- tern of FilP, which was quite similar to that of the merodiploid filP+/filP- egfp strain (Figure 7). In actively growing young vegetative hyphae from exponentially growing liquid cultures FilP formed a network of filaments that was strongest close to the elongating tips, but present throughout the hyphae. The bright foci of the merodiploid strain proved to be an artifact, probably caused by aggregation of the GFP-tagged FilP. Parts of the network frequently appeared to coalesce into a thick filamentous cable localized asymmetrically along one side of the . Hyphae from stationary phase cultures had no polar gradient of FilP, but exhibited a network of FilP throughout the length of the hyphae, which frequently contained elements with helical morphology. Thus, FilP has a spectacular and unique localiza- tion pattern among other characterized bacterial cytoskeletons – a network of

34 cytoskeletal fibers stretching throughout the vegetative mycelium and form- ing polar, growth-dependent gradients in polarly growing hyphae.

Figure 7. Immunofluorescence of FilP, exhibiting its wild type localization in grow- ing vegetative hyphae (Figure kindly provided by Stuart Cantlay).

35 FilP deletion causes a morphological defect in S. coelicolor and slows down vegetative growth to some extent (paper I)

In the quest of finding the function of these incredible FilP structures in the cell, we deleted filP by replacing it with an apramycin resistance cassette. Testing growth of the wild type strain and the filP deletion mutant with equal inoculums in liquid and solid media, demonstrated a growth defect in the mutant. Growth curves indicated that the growth defect is pronounced at the beginning of growth (20-36 hours after inoculation). The biomass accumula- tion in the mutant strain started with a delay and proceeded slower than the wild type strain in liquid media. Since we did not record any obvious defect in branching frequency in the mutant, we infer that the total growth defect is a result of delayed germination and/or restricted linear growth. The mutant displayed distorted morphology of the vegetative hyphae, both when grown in an angle between inserted coverslips and the surface of the solid agar me- dia, as well as in the liquid media. This concludes that the morphological differences between the wild type and the mutant are not only due to possi- ble defects in the surface adhesion properties of the latter. Both the defect in morphology and the slower growth could be rectified by insertion of wild type filP in trans, confirming the defective phenotypes being result of ab- sence of filP. The normal phenotype of the vegetative hyphae in the wild type strain is spreading of the mycelium as straight hyphae over the surface, whereas in filP mutant we observed curved and curly hyphae. This led us to speculate about a role of FilP in strengthening the peptidoglycan (PG) exoskeleton. Possibly lack of FilP makes the hyphae less rigid and hence unable to grow in a straight wild type like fashion. Another possibility is that the FilP cy- toskeleton itself confers rigidity to the hyphae. To test our hypothesis, we used a special application of AFM to quantify hyphal rigidity of both wild type and the filP mutant strain. We recorded force curves (Figure 8) while the cantilever device approached, indented and retracted the hyphae of re- spective strains.

36

Figure 8. A. Schematic diagram of the recording of force curves by AFM. The X- axis represents the approach of the tip of the probe and the Y-axis shows the bending of the cantilever in relation with movement of the probe. The curves show the de- flection of the tip as it hits the surface. The ‘indentation’ demonstrates the difference between a hard and a soft surface as encountered by the tip of the probe. B. A typical force separation curve of wild type and filP mutant hyphae, indicating less compli- ance of the wild type compared to that of the mutant. The hysteresis effect (the loop between ‘load’ and ‘unload’ curves) was documented consistently in the mutant hyphae. This confirms more persistent deformation in the mutant hyphae compared to the wild type ones.

It can be concluded from the results that the wild type strain was much stiffer and more elastic than the filP mutant strain. Single typical force- separation curves of filP deletion mutant and wild type hyphae are shown in Figure 8 B. The force separation curves also demonstrate that the wild type hyphae undergo a fully reversible elastic deformation, while the mutant hy- phae suffer from a more permanent deformation, indicated by the hysteresis effect consistently present in all force curves. This hysteresis effect is mani- fested as a loop between the ‘load’ and ‘unload’ curves, caused by a delay in the elastic recovery of the mutant hyphae after every deformation created by the probe. This is probably because of softer cell surface and/or lower turgor pressure in the mutant hyphae. Hence, we conclude that indeed, FilP cy- toskeleton somehow provides rigidity and mechanical support to the cells. This was a pioneering study where the impact of a bacterial cytoskeleton was demonstrated by direct probing of live cells. We also observed that the filP mutant yielded more under the tip pressure and shear forces during im- aging, whereas the wild type hyphae remained undisturbed and demonstrated uniform height and width before and after the scanning. This is in accor- dance with our force curve measurements showing mutant hyphae being less rigid (softer) compared to that of the wild type. However, this pilot experi- ment could not definitely determine whether the increased compliance of filP mutant was due to softening of the cell envelope or lower turgor pres- sure in the cells. Presence of FilP at the growing tips and the unusual soft- ness at the tips of the mutant hyphae suggest a possible role of FilP in cell wall synthesis and/or maturation. However, analysis of thin sections of

37 growing hyphae by high resolution transmission electron microscopy did not reveal any differences in cell wall thickness and density between wild type and a filP mutant (our unpublished data). A cellulose synthase-like protein called CslA has recently been found in S. coelicolor that localizes at the tips and helps in accumulation of a -glycan polysaccharide (155), probably to reinforce the growing tips. Remarkably, avicel-binding protein (AbpS), an orthologue of FilP from S. reticuli, has been shown to bind avicel (a deriva- tive of cellulose), and oligomerize into larger structures. Thus, FilP most probably also can bind cellulose and perhaps via this activity affect cell wall composition. However, the subapical zone containing abundant FilP cy- toskeleton is much larger than the zone containing cellulose, which was shown to be restricted to the immediate tip area (150). FilP filamentous structure could also be imagined as a scaffold to provide mechanical support to the cells in a classical manner of an IF cytoskeleton in mammalian cells. While growing, bacteria must break the already existing cross-links of the cell wall by using lytic activity and incorporate new mate- rial to the cell wall. Not much is known about the mechanism of cell wall synthesis in Streptomyces, but the lytic activity must be most intense at the tips, where the primary synthesis of the peptidoglycan happens. So it is ob- vious to imagine the necessity of additional mechanical support at the grow- ing tips of S. coelicolor to stabilize this process and maintain cell integrity. It is also a possibility that FilP alters the composition and/or permeability of the cell membrane by some means yet to be found. By this alteration it might be affecting the turgor pressure inside the cells, and hence the rigidity of the hyphae. However, FilP is not a transmembrane protein and its putative effect on membrane permeability must in this case be indirect. Thus, our favoured hypothesis, which is most consistent with the existing preliminary data, is that FilP cytoskeleton acts as a mechanical scaffold and directly provides rigidity to the cells, similarly to IFs in mammalian cells. The function of FilP as a provider of rigidity in the growing hyphae seems biologically significant, as lack of stiffness can affect the fitness as well as survival of a naturally soil-dwelling organism like Streptomyces. Presence of FilP-like proteins in several actinomycetes and their ability to polymerize into filamentous structures in vitro indicate a conserved IF-like cytoskeletal function in this large group of bacteria.

38 The interaction between FilP and DivIVA (Paper II)

The next interesting questions to answer were: what is causing the spectacu- lar polar gradients of FilP cytoskeleton, and which proteins does FilP inter- act with? The polar growth in S. coelicolor is dependent on the essential, coiled coil protein DivIVA. It localizes at the growing tips of mycelia and also marks the new branching points in this organism. We showed that FilP also localizes to the regions of the growing tips of the mycelia. Obviously, the consistent presence of these proteins in close proximity led us to suspect a significant interaction between them. We studied in vitro interaction between these two proteins by co- immunoprecipitation. We could co-precipitate FilP with FLAG-tagged Di- vIVA and vice versa. We used total cell lysate of S. coelicolor for the co- immunoprecipitations and hence we could not exclude possible roles of other cellular components in this interaction. We also observed an interac- tion between FilP and DivIVA using the bacterial two hybrid (BTH) system. This method is based on reconstitution of a signal transduction pathway us- ing cyclic AMP (75). The proteins of interest are fused to the two subunits, T25 and T18 that together will constitute the catalytic domain of Bordetella pertussis adenylate cyclase. Interaction between fusion proteins triggers synthesis of cAMP, leading to transcriptional activation of catabolic operons, such as lactose or maltose and enables growth on minimal medium contain- ing these sugars or a blue colony phenotype on media containing X-Gal. It appeared that FilP interacted with itself and DivIVA whether cloned at the C- or N- terminus of the T18 subunit. However, DivIVA interacted with itself and FilP only when fused to the N-terminus of T18. This might indi- cate that the C-terminus of DivIVA is important for interaction with its part- ners and itself. However, these results showed that FilP and DivIVA were able to interact in absence of other Streptomyces proteins. The interaction between DivIVA and FilP and the previous localization stud- ies suggested that these two proteins might at least partially co-localize at the hyphal tips. We detected DivIVA-GFP (by GFP fluorescence) and FilP (by immunofluorescence) in the same cells, and to our great surprise we could not detect any co-localization between them. Instead, the FilP zone seemed to start immediately behind the apical zone occupied by DivIVA. In order to understand this puzzling result we asked if the interaction between DivIVA

39 and FilP could be spatially limited to the region of contact between their repective zones. To demonstrate in situ an interaction between these two proteins, we used a novel system for bacteria, called the ‘proximity ligation assay’ or PLA. This is an immunoassay where PLA probes are used to detect probable interactions in the cells. PLA probes can be a pair of specific anti- bodies coupled to oligonucleotides (minus arm and plus arm) (135). If upon binding to their target proteins the PLA probes are brought into suffiently close proximity (less than 30 nm in case of the probes being primary anti- bodies), they will be able to host another pair of connector oligonucleotides and form a circle. This circle is then amplified by rolling circle amplification and visualized as a fluorescent signal under the microscope with desired filter (72). Hence, this method detects close proximity between proteins, and thus a possible interaction. We used anti-rabbit and anti-mouse as PLA probes, which bound to rabbit anti-FilP and mouse anti-Flag antibodies as primary probes (9). We frequently detected fluorescent foci close to, but not immediately at the hyphal tips, corresponding to the sub-apical positions of the convening edges of FilP and DivIVA zones. Signals were also ob- served at various locations along the hyphae, which possibly indicate the new branching points. As a control, we used a filP deletion strain, in which no signal was detected. Overexpression of DivIVA-FLAG caused more in- tense signals in a larger apical area, presumably occupied by FilP. Thus, there is a putative interaction also between soluble DivIVA molecules and the FilP cytoskeleton. Thus, the PLA assay confirmed that FilP and DivIVA exist in the cells in a proximity close enough (less than 60 nm, which is the minimum distance to produce a signal using secondary antibodies as PLA probes) to interact with each other. However involvement of other proteins cannot be excluded here. What is the significance of this spatially limited interaction between FilP and DivIVA? Could it be that DivIVA is directly involved in the apical localiza- tion of FilP and the formation of FilP apical gradients? Since DivIVA is needed for viability, we could not study how absence of DivIVA would af- fect localization of FilP. However, if we could change DivIVA localization pattern, and observe a corresponding change in FilP localization, we might be able to address this question. To this end we grossly overexpressed Di- vIVA-GFP, which caused a severe change in cell morphology. Instead of slim hyphae, the cells become very large, pear-shaped and swollen. Thus, the morphology of the cells and DivIVA apical zones was perturbed. Interest- ingly, FilP seemed to follow the abnormal localization of DivIVA in these cells. Most often a large zone of DivIVA, which extended along the enve- lope of the swollen cells, was immediately flanked by a FilP zone on the cytoplasmic side, tracing the shape of the DivIVA zone. Also, when DivIVA signal was observed elsewhere in the cells, it was in most cases accompanied by a FilP signal. However, like in the wildtype situation, there was no over- lap between DivIVA and FilP respective zones, which were always situated

40 just next to each other. Thus, it is likely that DivIVA is involved in directing FilP localization also in normal conditions. We also wondered whether perturbation of FilP localization would affect DivIVA in the cells. In a filP mutant DivIVA localizes to the tips of the growing hyphae quite normally. However, in a strain in which a FilP deriva- tive tagged with the red fluorescent protein mCherry (mCh-FilP) was over- expressed, we observed perturbations in DivIVA-GFP localization. Foci of mCh-FilP, which also contained wild type FilP, formed in the subapical parts of the hyphae, and trapped DivIVA-GFP. This is in agreement with the PLA experiment, showing interaction between the cytoplasmic DivIVA molecules and the FilP cytoskeleton. Trapping of DivIVA was seen only during a short time interval after induction of mCh-FilP, suggesting that this interaction was weak or transient, and that DivIVA was later released from the abnor- mal localization. Further indications that DivIVA is involved in formation of FilP apical gra- dients was obtained in an experiment where we observed changes in FilP localization and changes in total cellular concentrations of FilP and DivIVA during growth. It appeared that disappearance of FilP apical gradients coin- cided with arrival of the culture into stationary phase of growth and a sig- nificant decrease in total amounts of DivIVA. The amount of FilP stayed unchanged for several more hours. Thus, the disappearance of apical gradi- ents is a result of dynamic reorganization of the FilP cytoskeleton, not due to the decrease in FilP cellular concentration. It also supports the idea that Di- vIVA is involved in formation or maintenance of the FilP apical gradients. In conclusion, our study demonstrates cooperation between two coiled coil proteins forming distinct cytoskeletal structures in S. coelicolor cells, to en- sure integrity of the hyphae during polar growth. In the future, it would be interesting to understand in more detail the inter- play between DivIVA and FilP. What other proteins are involved? As the tip of the growing hyphae is the most active part of the mycelial network, sev- eral proteins must accumulate there and perform diverse functions individu- ally or together. Hence, there is obviously a possibility of FilP interacting with more partners. We have some more candidates that might be considered as the interacting partners of FilP. There are other coiled coil proteins with a characteristic rod domain found in this organism, which could potentially interact with FilP. One of these potential candidates, is a large coiled coil protein called Scy (149). The gene is located just upstream of filP. This gene is not conserved in bacteria. Deletion of scy causes somewhat similar, but more intense morphological defect in S. coelicolor as deletion of filP. An- other candidate protein encoded by SCO3114 is a much smaller coiled coil protein with a weak sequence similarity to FilP, but it lacks the characteristic conserved N-terminal sequence motifs. Interestingly, we detected an interac- tion between FilP and SCO3114 in the bacterial two-hybrid assay (our un- published data). Thus, future research is needed to reveal the protein interac-

41 tion network determining polar growth and cell morphology in this fascinat- ing bacterium.

42 Characterization of a novel sporulation locus in S. coelicolor genome (Paper IV)

Sporulation is a complex cell biological phenomenon. The aerial hyphae undergo a significant reprogramming of regulation and modification of morphogenesis as the multicellular filamentous cells differentiate into chains of unigenomic spores with thick cell walls and low metabolic activity to survive for a long time. All the components that are needed for successful completion of this complicated phenomenon of sporulation are yet to be found. Our aim was to identify new functions involved in sporulation. The study initiated with a search for new genes involved in spore develop- ment. We analyzed the gene expression profiles from a whole genome mi- croarray study of wild type and sporulation-deficient mutant strains during development. Together with some known genes influencing sporulation, we could identify some previously unknown genes with sporulation-specific expression patterns. Among them were two genes, one encoding a small putative membrane protein with no defined function, named SmeA, and the other encoding a member of the SpoIIIE/FtsK family, called SffA. The genes smeA and sffA form an operon, and they were upregulated at late develop- mental phase in the wild type strain, but not in any of the whi mutant we included. Thus they are dependent on products of whiG, whiA. whiI and whiH genes. SmeA (small membrane protein) contains a centrally located putative transmembrane domain as well as a putative N-terminal signal pep- tide. SmeA-like proteins seem to be widespread in Streptomyces and Frankia. Often SmeA and SffA can both be conserved, indicating a func- tional relationship. SffA contains an N-terminal transmembrane domain and a C-terminal ATPase domain, similar to that of SpoIIIE/FtsK-family pro- teins. What is the function of these proteins in sporulation? Deletion of both smeA and sffA caused several defects in spore maturation. First, a smeA-sffA mutant produced lightly pigmented spores compared to the dark grey ones made by wild type strain of S. coelicolor and the defect in phenotype was restored by in trans insertion of these genes in the double mutant strain. We also demonstrated that the pigmentation is probably a function primarily of SmeA, as the individual deletion of smeA produces lightly pigmented spores similar to that of the double mutant, but the sffA deletion strain give rise to dark gray spores similar to that of the wild type. Secondly, the smeA-sffA mutant strain was unable to condense the chromosomes to the wild type

43 level during sporulation. Thirdly, the presence of a small percentage of anu- cleated spore compartments in the mutant suggests also that lack of SmeA and SffA causes slight defect in chromosome segregation as well. Electron microscopy studies revealed even more defects caused by the lack of smeA- sffA. A study of thin sections of the spore chains by transmission electron microscopy revealed a defect in the layers of mature spores in absence of SmeA and SffA. The spores produced by the mutant strain had a much thin- ner and ‘paler’ envelope compared to the pronounced electron-dense outer layer of wild type spores. Additionally, scanning electron microscopy of the wild type displayed free dispersed spores in 8 day old cultures, whereas the mutant was found to produce almost exclusively only spore chains during that same time frame, indicating a defect in spore separation process. We also recorded uneven compartmentalization in the mutant spore chains which is almost never found in the wild type, thus SmeA and SffA are needed for proper positioning of the sporulation septa. The micrographs of smeA were indistinguishable from that of smeA-sffA, but sffA displayed apparently normal spore formation and maturation. However, it revealed an uneven septation defect similar to that of the double mutant strain. Consistent with the observed defect in spore envelope, the mutant spores exhibited more susceptibility to heat compared to the wild type ones, even though the mu- tant and the wild type spores were found to be equally resistant to detergent. To conclude, the products of smeA-sffA operon seem to be involved in suc- cessful maturation of spores including placement of division septa, chromo- some segregation and condensation, spore pigmentation and separation, maturation of spore envelope and development of heat resistance. As some of the phenotypes of our mutants resemble the defects inflicted by deletion of RNA polymerase sigma factor gene sigF, we speculated whether the transcription of smeA-sffA was sigF dependent. The gene sigF is known to affect the last stages of sporulation (122), is specifically induced in the sporulating hyphae (138) and is transcriptionally dependent on the early whi genes (78). We fused the promoter region, -binding site and the start codon of smeA to mCherry (a red fluorescence protein) gene and used the construct to monitor expression of SmeA in the cells. The results indi- cated that sigF is not required for sporulation-specific expression of smeA- sffA. Conversely, we have also shown that sigF expression is independent of SmeA-SffA and SigF itself. It has been speculated that septum formation can act as a trigger to initiate expression of the late sporulation genes (49). To check if expressions of SmeA and SffA are affected by the putative cell division check point, we wanted to record the timing of Z-ring formation in relation with smeA-sffA expressions. We introduced smeAp-mCherry reporter in a strain that ex- pressed both wild type FtsZ and FtsZ-EGFP. We detected both smeAp- mCherry and FtsZ-EGFP in most of the sporulating aerial hyphae. Interest- ingly, some unconstricted aerial hyphae displayed the Z-ring fluorescence,

44 but no expression of SmeA-mCherry was recorded in them. However, we did not find any sporulating hyphae displaying smeAp-mCherry fluorescence without any FtsZ-EGFP signal. Hence, expression of smeA probably occurs after the Z-ring formation, and might be triggered by septation. To test this, we used a strain severely impaired in regular Z-ring formation as well as septa formation (55). This strain did not show any reduced fluorescence of smeAp-mCherry in the aerial hyphae indicating that that there is no quantita- tive dependence of smeA expression on either Z-ring or septa formation, but the possibility of a qualitative dependence cannot be ruled out, because the strain could still form a few sporulation septa. We did not manage to create a functional fluorescent fusion of SmeA, but SffA-GFP displayed signal at the middle of the sporulation septa, indicating a putative role as DNA translocase, due to its homology to FtsK/SpoIIIE proteins. To investigate the functional dependency between SmeA and SffA, we monitored SffA-EGFP signals in smeA background. SmeA appeared to be specifically required for the localization of SffA at the constricting sporu- lation septa. However, SffA could assemble at the vegetative-type septa in the basal part of the aerial hyphae in absence of SmeA. However, proper localization of SsfA cannot be the sole function of SmeA, because a sffA strain displays less severe sporulation defect compared to that of smeA and smeA-sffA. Our obvious next step was to check whether SffA and FtsK have overlapping or redundant functions. We monitored several DAPI- stained spore chains in wt, ftsK and smeA-sffA strains. We found that in wild type and in smeA-sffA strains the infrequently occurring anucleated spores were always flanked by a spore compartment containing two chromo- somes. This was not the case in a ftsK strain, in which anucleate spores were flanked by spores with normal amounts, or less than normal amounts of DNA. We concluded that FtsK is involved in removing chromosomal DNA that is trapped under closed septa to its adjacent compartment and hence forming proper unigenomic spores. But, if the septum placement or the chromosome positioning is defective, the trapped chromosome may be pumped in the wrong direction, producing an empty spore flanked by the one with double chromosome. As the smeA-sffA also displayed this phenome- non, it can be inferred that, this pumping of chromosome from closing septa is a function of FtsK and not SffA. In addition, we also showed that FtsK (tagged with mCherry) localizes at the middle of sporulation septa both in the wild type and smeA-sffA strain, indicating that unlike SffA, positioning of FtsK is independent of SmeA. Hence, SffA and FtsK are both putative DNA translocases, localizing at the sporulation septa, but their modes of action in localization and performing cellular functions are different. SffA seems to be functionally related to SmeA with a pleiotropic role in spore maturation. As deletion of smeA caused mislocalization of SffA, but the complex phenotype of smeA cannot be explained only by the mislocaliza-

45 tion, it is possible that, SmeA is needed for proper localization and mem- brane assembly of sporulation proteins in addition to SffA. In addition to similar localization of SffA and FtsK, several other properties are shared, at least partially, by these proteins. SffA has the typical domain architecture of the FtsK-HerA superfamily of pumping ATPases (66) with an N-terminal membrane spanning region and a C-terminal P-loop ATPase domain (126). However, some deviation in SffA amino acid sequence has been observed from that of the FtsK-HerA superfamily. The family of FtsK- SpoIIIE being large and present in most bacteria and archea, the functions of all the members of this family is not yet clear. Hence, the similarities and dissimilarities between SffA and FtsK make the former an interesting candi- date to study further.

46 Swedish summary of the thesis

Den mikroskopiska världen runtomkring oss är osynlig för blotta ögat, men den utgör en stor del av våra liv. Några ’goda’ mikrober bidrar till produk- tionen av drycker, mat och mediciner såsom öl, ost och penicillin, och några hjälper våra kroppar att smälta maten som till exempel probiotikan Laktoba- cillus och Bifidobacterium. Dessutom finns ett flertal ’dåliga’, sjukdoms- framkallande bakterier vilka påverkar vår hälsa och leverne. Det är intressant att utforska deras lilla värld, inte bara av nyfikenhet, utan också därför att de representerar en ’enklare’ livsform och det är relativt okomplicerat att veten- skapligt dissekera dem för att studera olika mekanismer och processer i syfte att lära sig mer om de ’högre’ stående livsformerna, som till exempel männi- skor. Bakteriecellens arkitektur är ett intressant ämne att studera. Olika vetenskap- liga rön under de senaste decennierna har skissat upp en realistisk bild av den strukturella komplexiteten i den prokaryota världen. Ett av de fascine- rande framstegen inom detta forskningsfält är upptäckten av cytoskelett- elementen i bakterier. Detta visar på att prokaryoter trots allt inte bara är en ’simpel’ livsform eller en ’påse enzymer’, utan att de har en bestämd cell- arkitektur, uppbyggd av cytoskelett och andra armeringsproteiner. Aktin, tubulin och intermediära filament är tre typer av cytoskelett som har hittats hos många olika arter, både i eukaryoter och prokaryoter. Det mest varieran- de bland dessa är de intermediära filamenten (IF). De bildar dynamiska fila- mentösa strukturer i cellen och utför olika funktioner i olika celltyper. Struk- turmässigt består IF proteiner av en ’stav’-domän med tvinnade spiral- segment så kallade ’coiled coil’ segment separerade av otvinnade länkar. Detta speciella arrangemang av aminosyror ger upphov till en av de karakte- ristiska biokemiska egenskaperna hos IF proteiner, nämligen att de spontant bildar filamentstrukturer in vitro utan krav på vare sig energi eller ko- faktorer. Vi har hittat, utgående från den typiska domänstrukturen och de karaterisk- tiska egenskaperna hos dessa proteiner, att förekomsten av IF-liknande pro- teiner är utbrett i bakterier. Vi har i detalj analyserat ett av de IF-liknande proteinerna i Streptomyces coelicolor och kallar detta protein FilP (filamen- tous protein). Det bildar långa strierade filament med ojämn diameter in vitro, utan krav på energi eller ko-faktorer. Vi misstänker att proteindimerer- na eller trimererna är ordnade ’huvud-mot-huvud’ och ’ända-mot-ända’ på ett sätt som ger upphov till ett ’pärlbands’- utseeende med omväxlande ljusa

47 och mörka band. Genom att använda oss av atomkraftmikroskopi (Atomic Force Microscopy) har vi visat att FilP antagligen ger rigiditet främst till de vegetativa hyferna hos S. coelicolor. I avsaknad av FilP proteinet blir hyfer- na mjukare och uppvisar en förvrängd cellmorfologi. Detta resultat visar att FilP har en biologisk funktion i Streptomyces celler som i hög grad liknar IF- cytoskelettets funktion i våra celler. Avsaknaden av FilP har även en negativ effekt på bakteriernas tillväxt. Den vegetativa tillväxten hos S. coelicolor sker endast genom extension av hyfspetsar, vilket också gör att cellväggen nära spetsarna är tunnare och mer sårbar än i andra delar av hyferna. Den polära tillväxten är helt beroende av ett protein, DivIVA, vilket lokaliserar till hyfspetsarna och styr inkorporeringen av nytt cellväggsmaterial. Vi fann att FilP bildar ett nätverk av tunna proteinfibrer genom hela längden av ve- getativa hyfer. Men under aktiv tillväxt är FilP-nätverket mycket kraftigare i den delen som är närmast den växande hyfspetsen, troligtvis för att ge extra stöd till de svaga delarna av cellen. Vi har visat med mikroskopi och bioke- miska metoder att dessa två proteiner möjligen interagerar med varandra. Vi kunde också påvisa denna interaktion i cellerna med hjälp av en ny metod kallad “proximity ligation assay” (PLA). Detta ledde till en hypotes att Di- vIVA styr lokaliseringen av FilP till hyfspetsarna under aktiv tillväxt. För att ytterligare bevisa detta följde vi lokaliseringen av FilP i cellerna som överut- tryckte DivIVA. Dessa celler blev mycket stora, päronformade och svullna, och den apikala zonen ändrade form och utseende. FliP verkade följa den onormala lokaliseringen av DivIVA nästan överallt i dessa celler, vilket styrker vår idé att DivIVA styr lokaliseringen av FliP även under normala förhållanden. I ett experiment kunde vi också påvisa det omvända, nämligen överproduktionen av ett FilP-derivat, taggad med det rödfluorescerande pro- teinet mCherry, påverkar lokaliseringen av DivIVA. Sammantaget visar dessa resultat att det sker ett samarbete mellan två coiled coil proteiner, som båda bildar olika cytoskelett-strukturer i S. coelicolor celler, och att detta samarbete sker i syfte att säkerställa integriteten av hyferna under polär till- växt.

Vår modellorganism S. coelicolor är en grampositiv, flercellig bakterie till- hörande den stora gruppen av actinomyceter. De producerar många olika antibiotika och sekundära metaboliter av biologisk betydelse. S. coelicolor genomgår olika faser av differentiering under dess komplexa livscykel. Den börjar med att gro sporer och fortsätter med tillväxt av vegetativa hyfer som bildar ett multicellulärt mycel. När näringen tar slut, bildas det lufthyfer som så småningom omvandlas till tåliga sporer med tjocka cellväggar och låg metabolisk aktivitet för att överleva under en lång tid. Sporulering är ett komplext cellbiologiskt fenomen och alla komponenter som behövs för en fullborda denna komplicerade process har ännu inte identifierats. Som en del av mina studier karakteriserade vi här två nya gener, smeA och ssfA, som är involverade i sporbildningen. Flera aspekter av sporuleringsprocessen var

48 påverkade i en mutant som saknade båda generna. Dels var cellväggen tun- nare än i vildtypen, dels blev även kromosomerna mindre kondenserade och ofullständigt segregerade. Detta resultat är av stor betydelse eftersom vi kunde avslöja nya gener och nya funktioner som sporbildningen, vilket är en viktig process.

49 Acknowledgements

This PhD thesis is not complete without expression of my deepest gratitude to numerous people who made this work possible. During my time as a PhD student at Uppsala University, I have met wonderful, most amazing and ex- tra-ordinary people. It is impossible for me to make the list of names of all the people who have influenced me one way or the other, because the list will be longer than my scientific contribution. However, I want to take this opportunity to thank all my friends and family, my teachers, supervisors, collaborators and co-workers for their constant encouragements.

My special thanks go to my supervisor Dr. Nora Ausmees, who gave me the chance to work on great projects and taught me all I know about microbiol- ogy and . Without her patience, I would not be able defend this thesis. Thank you, Nora.

There is one more person I need to mention specially and that is my father, Dr. Buddhadeb Bagchi. Thank you Baba for always listening to me and be my number one fan. I could not have done this without you.

50 References

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