<p> Bacterial cell division is an essential process, which is initiated by forming the Z-ring as a cytoskeletal scaffold at the midcell site, followed by the recruitment of a series of divisome proteins. In <i>Escherichia coli</i> (Ec), at least 15 divisome proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsW, FtsN, FtsE, FtsX, ZapA, AmiC, EnvC) have been implicated in this process. The components of the cell division machinery proteins in <i>Neisseria gonorrhoeae</i> (Ng) differs from <i>E. coli. N. gonorrhoeae</i> possesses FtsA, but lacks FtsB. ZipA and FtsL in <i>N. gonorrhoeae</i> have low identity to ZipA and FtsL from <i>E. coli</i>. Our laboratory has studied the central division protein FtsZ in <i>N. gonorrhoeae</i>. Thus, my research investigated the role of <i>N. gonorrhoeae</i> FtsA in cell division and investigated the interactions between divisome proteins from <i>N. gonorrhoeae</i> to understand divisome assembly.</p> <p>This study determined the association of FtsA_Ng with FtsZ</sub>Ng and other divisome proteins in <i>N. gonorrhoeae</i> and identified the functional domains of FtsA<sun>Ng</sub> involved in these interactions using a bacterial two-hybrid (B2H) assay. FtsA_Ng interacted with FtsZ_Ng, FtsK_Ng, FtsW_Ng, FtsQ_Ng, and FtsN_Ng. Self-interactions of FtsA_Ng and FtsZ_Ng were also detected. FtsI_Ng, FtsE_Ng and FtsX_Ng did not interact with FtsA_Ng. The 2A₁, 2A₂ and 2B domains of FtsA_Ng were sufficient to interact with FtsZ_Ng independently. Domain 2A₁ interacted with FtsK_Ng and FtsN_Ng. Domain 2B of FtsA_Ng interacted with FtsK_Ng, FtsQ_Ng, and FtsN_Ng. Domain 2A₂ of FtsA_Ng interacted with FtsQ_Ng, FtsW_Ng, and FtsN_Ng. These data suggest that FtsA in <i>N. gonorrhoeae</i> plays a key role in interactions with FtsZ and other divisome proteins.</p> <p>The potential interactions between divisome proteins in <i>N. gonorrhoeae</i> were examined using B2H assays. The comparisons between the <i>N. gonorrhoeae</i> divisome protein interaction network and those of <i>E. coli</i> and <i>S. pneumoniae</i> indicates that the divisome protein interactome of <i>N. gonorrhoeae</i> is more similar to that of <i>S. pneumoniae</i> and differs from that of <i>E. coli</i>. The comparisons revealed that compared to the interactions in <i>E. coli</i> and <i>S. pneumoniae</i>, more interactions between divisome proteins upstream of FtsA_Ng (including FtsA_Ng) and downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i> while fewer interactions between divisome proteins downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i>. Possible reasons for this include the inability of ZipA_Ng to interact with other divisome proteins and the absence of FtsL and FtsB in <i>N. gonorrhoeae</i>, resulting in the lack of an FtsQ-FtsB-FtsL complex in <i>N. gonorrhoeae</i>. These results indicate a possibly different divisome assembly in <i>N. gonorrhoeae</i> from that proposed models for <i>E. coli</i>.</p> A model for FtsA_Ng structure was predicted based on structural homology modeling with the resolved crystal structure of <i>Thermotoga maritima</i> FtsA. Four domains on the molecule were identified, designated 1A, 1C, 2B and 2A (including 2A₁ and 2A₂). Domains 2A and 2B of FtsA were highly conserved based on multi-sequence alignments of FtsAs from 30 bacteria. FtsA_Ng located to the division site in <i>N. gonorrhoeae</i> cells and the ratio of FtsA to FtsZ ranged from 1:24 to 1: 33 in three <i>N. gonorrhoeae</i> strains, which gave a lower cellular concentration of FtsA compared to other organisms.</p> <p>I also determined that overexpression of FtsA_Ng in <i>E. coli</i> led to cell filamentous in rod-shaped <i>E. coli</i> and cell enlargement and aggregation in mutant, round <i>E. coli</i>. FtsA_Ng failed to complement an <i>ftsA</i>_Ec-deletion <i>E. coli</i> strain although the overexperssion of FtsA_Ng disrupted <i>E. coli</i> cell division. In addition, overexpression of FtsA_Ng only affected cell division in some cells and its localization in <i>E. coli</i> was independent of interaction with <i>E. coli</i> FtsA or FtsZ. These results indicate that FtsA_Ng exhibits a species-specific functionality and <i>E. coli</i> is not a suitable model for studying FtsA_Ng functionality.</p> <p>This is the first study to characterize FtsA from <i>N. gonorrhoeae</i> in cell division. I identified novel functional domains of FtsA_Ng involved in interactions with other divisome proteins. The <i>N. gonorrhoeae</i> divisome protein interaction network determined by B2H assays provides insight into divisome assembly in <i>N. gonorrhoeae</i></p>.

<p> Bacterial cell division is an essential process, which is initiated by forming the Z-ring as a cytoskeletal scaffold at the midcell site, followed by the recruitment of a series of divisome proteins. In <i>Escherichia coli</i> (Ec), at least 15 divisome proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsW, FtsN, FtsE, FtsX, ZapA, AmiC, EnvC) have been implicated in this process. The components of the cell division machinery proteins in <i>Neisseria gonorrhoeae</i> (Ng) differs from <i>E. coli. N. gonorrhoeae</i> possesses FtsA, but lacks FtsB. ZipA and FtsL in <i>N. gonorrhoeae</i> have low identity to ZipA and FtsL from <i>E. coli</i>. Our laboratory has studied the central division protein FtsZ in <i>N. gonorrhoeae</i>. Thus, my research investigated the role of <i>N. gonorrhoeae</i> FtsA in cell division and investigated the interactions between divisome proteins from <i>N. gonorrhoeae</i> to understand divisome assembly.</p> <p>This study determined the association of FtsA_Ng with FtsZ</sub>Ng and other divisome proteins in <i>N. gonorrhoeae</i> and identified the functional domains of FtsA<sun>Ng</sub> involved in these interactions using a bacterial two-hybrid (B2H) assay. FtsA_Ng interacted with FtsZ_Ng, FtsK_Ng, FtsW_Ng, FtsQ_Ng, and FtsN_Ng. Self-interactions of FtsA_Ng and FtsZ_Ng were also detected. FtsI_Ng, FtsE_Ng and FtsX_Ng did not interact with FtsA_Ng. The 2A₁, 2A₂ and 2B domains of FtsA_Ng were sufficient to interact with FtsZ_Ng independently. Domain 2A₁ interacted with FtsK_Ng and FtsN_Ng. Domain 2B of FtsA_Ng interacted with FtsK_Ng, FtsQ_Ng, and FtsN_Ng. Domain 2A₂ of FtsA_Ng interacted with FtsQ_Ng, FtsW_Ng, and FtsN_Ng. These data suggest that FtsA in <i>N. gonorrhoeae</i> plays a key role in interactions with FtsZ and other divisome proteins.</p> <p>The potential interactions between divisome proteins in <i>N. gonorrhoeae</i> were examined using B2H assays. The comparisons between the <i>N. gonorrhoeae</i> divisome protein interaction network and those of <i>E. coli</i> and <i>S. pneumoniae</i> indicates that the divisome protein interactome of <i>N. gonorrhoeae</i> is more similar to that of <i>S. pneumoniae</i> and differs from that of <i>E. coli</i>. The comparisons revealed that compared to the interactions in <i>E. coli</i> and <i>S. pneumoniae</i>, more interactions between divisome proteins upstream of FtsA_Ng (including FtsA_Ng) and downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i> while fewer interactions between divisome proteins downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i>. Possible reasons for this include the inability of ZipA_Ng to interact with other divisome proteins and the absence of FtsL and FtsB in <i>N. gonorrhoeae</i>, resulting in the lack of an FtsQ-FtsB-FtsL complex in <i>N. gonorrhoeae</i>. These results indicate a possibly different divisome assembly in <i>N. gonorrhoeae</i> from that proposed models for <i>E. coli</i>.</p> A model for FtsA_Ng structure was predicted based on structural homology modeling with the resolved crystal structure of <i>Thermotoga maritima</i> FtsA. Four domains on the molecule were identified, designated 1A, 1C, 2B and 2A (including 2A₁ and 2A₂). Domains 2A and 2B of FtsA were highly conserved based on multi-sequence alignments of FtsAs from 30 bacteria. FtsA_Ng located to the division site in <i>N. gonorrhoeae</i> cells and the ratio of FtsA to FtsZ ranged from 1:24 to 1: 33 in three <i>N. gonorrhoeae</i> strains, which gave a lower cellular concentration of FtsA compared to other organisms.</p> <p>I also determined that overexpression of FtsA_Ng in <i>E. coli</i> led to cell filamentous in rod-shaped <i>E. coli</i> and cell enlargement and aggregation in mutant, round <i>E. coli</i>. FtsA_Ng failed to complement an <i>ftsA</i>_Ec-deletion <i>E. coli</i> strain although the overexperssion of FtsA_Ng disrupted <i>E. coli</i> cell division. In addition, overexpression of FtsA_Ng only affected cell division in some cells and its localization in <i>E. coli</i> was independent of interaction with <i>E. coli</i> FtsA or FtsZ. These results indicate that FtsA_Ng exhibits a species-specific functionality and <i>E. coli</i> is not a suitable model for studying FtsA_Ng functionality.</p> <p>This is the first study to characterize FtsA from <i>N. gonorrhoeae</i> in cell division. I identified novel functional domains of FtsA_Ng involved in interactions with other divisome proteins. The <i>N. gonorrhoeae</i> divisome protein interaction network determined by B2H assays provides insight into divisome assembly in <i>N. gonorrhoeae</i></p>.

The molecular mechanics of bacterial cell division remain one of the most fundamental unsolved problems in bacterial cell biology. During bacterial cytokinesis, bacteria divide symmetrically to give rise to two identical daughter cells. This tightly regulated process is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. The divisome, which forms at the cell centre, is responsible for the invagination and fusion of the cell’s membrane and peptidoglycan layers. The Escherichia coli divisome is comprised of at least ten essential proteins whose individual functions are mostly unknown. These divisomal proteins are recruited in a semi-hierarchical order, with the early recruits being predominantly cytoplasmic and the later recruits being predominantly extracytoplasmic or multi-pass transmembrane proteins. DivIB and its ortholog FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria, respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. The α domain is located next to the transmembrane segment and is a polypeptide-transport-associated (POTRA) domain. The β domain comprises approximately half of the extracytoplasmic region, and has a unique three-dimensional fold. The most C-terminal domain, the γ domain, is relatively unstructured. This protein has been proposed to play a role in septal peptidoglycan cross-linking or lengthening. The primary aims of these studies were to further characterise the structure and function of the bacterial cell division protein DivIB as well as investigate the interactions this protein has with the other divisomal proteins. It was anticipated that the knowledge gained should aid in the development of antimicrobials that target this protein’s function or protein-protein interactions. A molecular dissection approach was used to determine which of DivIB’s domains are essential for its recruitment to incipient division sites and for its cell division functions. It was determined that DivIB has three molecular epitopes that mediate its localisation to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and this information was used to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localisation epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be individually nonessential. Yeast and bacterial two-hybrid studies have revealed that most of these proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate the interaction between pairs of interacting proteins. Recently, a heterologous septal targeting approach was introduced to study the interaction between Bacillus subtilis divisomal proteins in E. coli. This technique allows the interaction between pairs of divisomal proteins to be studied in vivo without the complications caused by other interacting proteins. This approach was used to perform a molecular dissection of the interaction between B. subtilis DivIB and the divisomal transpeptidase PBP 2B. Although both proteins have septal localisation determinants in their transmembrane domains, it was found that these regions do not mediate their interaction. Rather, it was shown that DivIB interacts with PBP 2B through its extracytoplasmic region. Dissection studies revealed that all three extracytoplasmic domains of DivIB are necessary for interaction with PBP 2B, suggesting that the two proteins make multiple interactions, each of which is not strong enough in isolation to mediate formation of a stable complex. Finally, it was shown that E. coli FtsQ localises to the division septum in B. subtilis but cannot complement a divIB null. Multi-angle laser light scattering (MALLS) analysis revealed that the extracytoplasmic domain of Geobacillus stearothermophilus DivIB is predominantly monomeric at high concentrations. This indicated that if DivIB does exist as a dimer in vivo, it dimerises through its cytoplasmic or transmembrane region. In vitro observations suggest the C-terminal residues of DivIB may play a role in peptidoglycan binding. Finally, attempts were made to determine the three-dimensional structure of the complete extracytoplasmic domain of DivIB. Although it proved impossible to determine the structure using NMR spectroscopy, crystals were obtained under many different crystallisation conditions. Despite diffracting to 3.5 Å, we were unable to solve the protein structure using X-ray crystallography. However, this work has laid the groundwork for future attempts at solving the structure of this protein using X-ray crystallography.

Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism. In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phase Bacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase. In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.

A divisão celular em B. subtilis inicia-se pela formação de um complexo multiprotéico, o divisomo, no sítio onde a bactéria irá se dividir. FtsZ é a primeira proteína a se localizar no futuro sitio de divisão, formando uma estrutura em anel (anel Z) que se estende por toda a circunferência da célula. O anel Z funciona como um arcabouço responsável por recrutar outras quinze proteínas de divisão que irão participar da montagem do divisomo. Nesta tese, utilizamos abordagens quantitativas e qualitativas de microscopia de fluorescência vital para estudarmos duas questões ainda não esclarecidas sobre o funcionamento do divisomo. A primeira delas é como o divisomo é montado. Para estudarmos a montagem do divisomo nós realizamos ensaios de co-localização entre o anel Z (FtsZ-mCherry) e as proteínas ZapA, EzrA, FtsW, FtsL, YpsB , DivIVA, e MinC fusionadas a GFP. Quanto maior a freqüência de co-localização entre FtsZ e outra proteína de divisão, mais inicial é a participação da proteína na formação do divisomo. Portanto, a medida da freqüência de co-localização entre o anel Z e as proteínas componentes do divisomo permite que se deduza uma cinética da montagem deste complexo. Estes ensaios demonstraram uma freqüência de co-localização de 97,33% para ZapA; 98,31% para EzrA; 83,90% para FtsW; 78,43% para FtsL; 50% para YpsB; 41,7% para DivIVA e 31,64% para MinC. Estes resultados sugerem que o divisomo seja formado em três etapas. ZapA e EzrA se associam ao divisomo imediatamente após a formação do anel Z, em seguida FtsW e FtsL são recrutados para o divisomo, e por último YpsB, DivIVA, MinC associam-se ao divisomo. A segunda questão que investigamos nesta tese foi o mecanismo da mudança de posição do divisomo que ocorre durante a esporulação em B. subtilis. Na fase de esporulação a célula divide-se assimetricamente, com a formação do septo próxima a um dos pólos. Durante o crescimento vegetativo a divisão não ocorre próxima aos pólos por causa da ação das proteínas MinC, MinD e DivIVA, importantes reguladores espaciais da divisão. MinCD e DivIVA são inibidores da formação do anel Z que durante o crescimento vegetativo se localizam nos pólos das células.. Uma hipótese para explicar o uso dos sítios polares para a divisão durante a esporulação seria que as proteínas MinCD e DivIVA seriam removidas dos pólos celulares. Para testarmos esta hipótese, estudamos a localização das proteínas MinCD e DivIVA durante a esporulação. Nossos resultados demonstraram que MinCD e DivIVA se re-localizam e saem dos pólos celulares durante a esporulação. Porém esta dinâmica ocorre após a formação do anel Z assimétrico, sugerindo que o anel Z seja insensível a estes inibidores durante a esporulação. Por ensaios genéticos em B. subtilis demonstramos que a proteína SpoIIE, conhecida como provável proteína responsável por promover a formação do septo assimétrico, seja capaz de contrapor a ação de MinC no início da esporulação. Dessa maneira nós propomos um novo modelo de mudança da divisão simétrica para assimétrica durante a esporulação, diferentemente da simples saída do complexo MinCD dos pólos como é proposto na literatura. / Bacillus subtilis division begins through the formation of a multiprotein complex, the divisome, at the site of division. FtsZ is the earliest known protein to localize to the future division site where the protein forms a ring-like structure (Z-ring) that extends around the circumference of the cell. The Z-ring functions as a scaffold and recruits about fifteen other division proteins that compose the divisome. In this work, we used quantitative and qualitative methods of vital fluorescence microscopy to study two questions that have not been elucidated about the divisome dynamics. The first is how divisome is assembled. To address that problem, we made co-localization between Z-ring (FtsZ-mCherry) and proteins ZapA, EzrA, FtsW, FtsL, YpsB, DivIVA, and MinC fused to GFP. Higher is the match between GFP fusions to Z-ring, earlier is the assembly of division proteins to divisome. Therefore, the co-localization frequency between Z ring and divisome proteins will allow us to deduce the assemble kinetics of the divisome. This assays showed a co-localization frequency of 97,33% for ZapA; 98,31% for EzrA; 83,90 for FtsW; 78,43% for FtsL; 50% for YpsB; 41,7% for DivIVA and 31,64% for MinC. This data suggests that the divisome does not assemble in two but in three steps. ZapA and EzrA assemble into the divisome immediately after Z ring formation, secondly FtsW and FtsL were recruited to the divisome, and finally YpsB, DivIVA, MinC associated with the divisome. The second question that we investigated in this work is the mechanism responsible for change the divisome position that occurs during sporulation in B. subtilis. In sporulation the cell divides asymmetrically, with a septum formation near poles. During vegetative grown the divisiome does not occur near poles because of MinC, MinD and DivIVA action, relevant for spatial regulation of division. MinCD and DivIVA are inhibitors of Z ring formation that during vegetative growth are located at poles. A hypothesis to explain the use of polar sites for division during sporulation would be that MinCD and DivIVA would be removed from cellular poles. To test this hypothesis, we studied the location of MinCD and DivIVA proteins during sporulation. Our results demonstrated that MinCD and DivIVA re-localize and leave to cell poles during sporulation. However this process occurs after asymmetric Z ring formation, suggesting that Z ring would be unresponsive to this inhibitors during sporulation. Through genetics assays in B. subtilis we demonstrated that SpoIIE protein, known to probably play a role in asymmetric septum formation, would be able to contrapose MinC action during early sporulation. Therefore, we propose a novel model for change the symmetric to asymmetric division during sporulation, unlike the release of MinCD from pole proposed in the literature.

A divisão celular nas bactérias requer a formação do divisomo, um complexo protéico que tem como o primeira etapa a polimerização da proteína FtsZ, seguida pela associação de 15 outras proteínas conhecidas. Os mecanismos envolvidos na regulação espacial do divisomo são bem caracterizados, mas o controle temporal da divisão celular em relação a outros eventos do ciclo, como a replicação do cromossomo, segue controversa. Neste trabalho, aplicamos a metodologia de microscopia de série temporal para estudar duas questões fundamentais do processo de divisão: a montagem do complexo que executa a divisão e a possibilidade da oscilação periódica na expressão de um ou mais genes envolvidos em divisão possa participar do controle temporal da montagem do divisomo. Para investigar se há oscilação da expressão gênica, construímos inicialmente variantes instáveis GFP através da adição de sequências peptídicas C-terminais que encaminham para a degradação em B. subtilis e utilizamos estes repórteres para criar fusões transcricionais sob o controle de promotores de genes centrais do processo de divisão. Depois de otimizar as condições de microscopia de série temporal com fusões transcricionais usando a variante instável GFPAISV, observamos que a autofluorescência de B. subtilis interferia nas nossas quantificações. Como forma de contornar a autofluorescência, construímos então fusões transcricionais com duas variantes de YFP (proteína fluorescente amarela) e optamos por trabalhar com Ypet-AISV. A análise de filmes de células individuais, tanto com fusões a GFPAISV como a Ypet-AISV, indicou que apenas o promotor do operon ftsL-pbpB apresentava um padrão de oscilação significativamente diferente de um promotor artificial usado como controle negativo. Esta hipótese, no entanto, não foi confirmada por medidas estáticas de populações de células nas quais correlacionamos intensidade de fluorescência com posição no ciclo celular. Portanto, nossos dados não foram capazes de evidenciar flutuações na expressão dos genes ftsL-pbpB, minCD, ftsZ, ftsA e zapA ao longo do ciclo celular. Para estudar a cinética de montagem divisomo foram realizados experimentos de microscopia de série temporal de FtsZ-mCherry e Pbp2B-GFP, onde observamos que a associação de Pbp2B ao divisomo ocorre 3 minutos após a formação do anel de FtsZ em meio rico e 4 minutos em meio mínimo. Também realizamos experimentos de microscopia de série temporal com uma cepa contendo FtsZ-YFP e DivIVA-CFP, determinando que DivIVA é incorporado ao divisomo 16 minutos após a formação do anel de FtsZ em meio rico e 20 minutos em meio mínimo. Estes dados confirmam que a montagem do divisomo ocorre em três etapas, e não duas, como anteriormente proposto. / Cell division in bacteria requires the formation of the divisome, a protein complex that has as the first step polymerization of FtsZ, followed by the assembly of 15 other known proteins. The mechanisms that underlie spatial regulation of divisome assembly have been largely elucidated, but the temporal control that ties the timing of cell division to other cell cycle events, such as chromosomal replication, remains surrounded by controversy. In this work, we use time-lapse microscopy to address two issues in B. subtilis cell division: the timing of divisome assembly, and the possibility that a periodic oscillation in expression of one or more genes essential for divisome assembly may play a role in defining the timing of cell division. To study the possibility of oscilation in gene expression, we have first built unstable variants of GFP by adding to its C-terminus peptide sequences that target the protein for degradation and used those variants to build transcriptional fusions to access the promoter activity of core cell division genes. After optimizing time-lapse conditions with transcriptional fusions to cell divison genes with the unstable GFPAISV, we observed that B. subtilis autofluorescence was an issue to our quantifications. To improve our signal-to-noise ratio, we built transcriptional fusions with two variants of YFP (Yellow Fluorescent Protein), and decided to work with Ypet. In our single-cell analysis for GFPAISV and for Ypet-AISV, only the ftsL operon promoter presented an oscilating pattern different from our negative control. This was not confirmed, however, when we attempted to correlate fluorescence signal with cell cycle position in static single-cell measurements. Thus, we conclude that that there are no fluctuations in ftsL, pbpB, minCD, ftsZ, ftsA or zapA gene expression throughout the cell cycle. To study divisome assembly we performed time-lapse microscopy of FtsZ-mCherry and Pbp2B-GFP, and determined that the association of Pbp2B occurs 3 minutes after FtsZ polymerization in rich medium and 4 minutes in minimal medium. We also performed time-lapse microscopy with FtsZ-YFP and DivIVA-CFP, determining that DivIVA is incorporated to the divisome in 16 minutes after FtsZ polymerization in rich medium and 20 minutes in minimal medium. This data confirms the assembly of the divisome in three steps rather than two, as previously proposed.

O mecanismo de divisão mais comum entre procariotos é a divisão binária, na qual a célula- mãe reparte seu genoma e conteúdo citoplasmático de forma igual entre duas células filhas. Esse processo é mediado por um complexo protéico especializado, chamado divisoma, composto por cerca de 20 proteínas, que promovem a constrição da parede celular e membrana citoplasmática, formando o septo de divisão. O complexo é organizado em torno do anel Z, uma estrutura em anel composta pela proteína FtsZ, um homólogo de tubulina presente na maioria dos procariotos e em algumas organelas de eucariotos. Partindo de um levantamento detalhado da distribuição dos genes do divisoma em genomas completos de procariotos, aplicamos métodos de máxima verossimilhança para inferência de estados ancestrais e reconstruímos o conteúdo gênico do divisoma no ultimo ancestral comum das bactérias atuais. Estendendo essas análises com a aplicação de métodos filogenéticos, inferimos os eventos responsáveis pelas variações de composição deste complexo, observadas entre os diferentes grupos de bactérias. Nossos resultados mostram que o último ancestral comum de todas as bactérias já possuía a maior parte dos componentes conhecidos do divisoma, sugerindo a existência de uma parede de peptideoglicano e a presença de um aparato molecular tão ou mais complexo que o observado nas linhagens atuais, incluindo a presença de componentes considerados acessórios e de distribuição relativamente restrita, como as proteínas envolvidas na localização do anel Z (sistema Min) e alguns efetores positivos da polimerização de FtsZ. Observamos também que a evolução do complexo não foi muito afetada por eventos de transferência lateral, mas apresenta vários exemplos de perda de genes, em especial em linhagens com genoma reduzido, o que sugere a redundância de vários componentes já presentes no ancestral e a freqüente redução da complexidade, pelo menos dos componentes centrais do divisoma. Episódios de expansão de famílias de componentes do divisoma em linhagens específicas e os mecanismos evolutivos responsáveis pela incorporação de tais variações são discutidos. A caracterização da história evolutiva detalhada do divisoma, aqui apresentada, poderá servir como ponto de partida para novas análises evolutivas e como base para elaboração de experimentos funcionais. / The most common cell division mechanism among prokaryotes is binary fission, where a mother cell partitions its cytoplasm and genome equally among two daughter cells. This process is mediated by a specialized protein complex, known as the divisome, composed of around 20 proteíns, that promotes constriction of the cell wall and cytoplasmic membrane, thus forming the division septa. The complex is organized around the Z-ring, a ring-shaped struture composed by FtsZ, a tubulin homolog present in most prokaryotes and some eukaryotic organelles. After a detailed revision of the distribution of divisome genes among completely sequenced prokaryotic genomes, we applied maximum likelihood methods for the inference of ancestral states and reconstructed the gene content of the divisome in the last common ancestor of all extant bacteria. We then performed phylogeneticanalysis of all cell division genes and inferred the series of events responsible for the observed variations of the complex´s composition among bactérial lineages and their common ancestor. Our results show that the last common ancestor of all bacteria already possessed most of the known divisome components, thus suggesting the existence of a peptidoglycan cell wall and the presence of a molecular apparatus, perhaps more complex than those found in extant bacteria, including the presence of some accessory components with a somewhat restricted distribution, like the proteíns involved in the localization of the Z-ring (Min sistem) and some positive effectors os FtsZ polimerization. We also observed that the complex´s evolution was almost never the subject of horizontasl gene transfer events, but shows several examples of gene loss, specially in lineages displaying clear signs of genome reduction, thus suggesting the redundancy of several components in the ancestral divisome and a certain degree complexity reduction, at least for core components of the divisome. Lineage specific expansion of divisome component and the evolutionary mechanisms behind such processes are discussed. This characterization of the detailed evolutionary history of the divisome might serve as a starting point for new evolutionary analysis and as a basis for the design of functional experiments.

A divisão celular em B. subtilis inicia-se pela formação de um complexo multiprotéico, o divisomo, no sítio onde a bactéria irá se dividir. FtsZ é a primeira proteína a se localizar no futuro sitio de divisão, formando uma estrutura em anel (anel Z) que se estende por toda a circunferência da célula. O anel Z funciona como um arcabouço responsável por recrutar outras quinze proteínas de divisão que irão participar da montagem do divisomo. Nesta tese, utilizamos abordagens quantitativas e qualitativas de microscopia de fluorescência vital para estudarmos duas questões ainda não esclarecidas sobre o funcionamento do divisomo. A primeira delas é como o divisomo é montado. Para estudarmos a montagem do divisomo nós realizamos ensaios de co-localização entre o anel Z (FtsZ-mCherry) e as proteínas ZapA, EzrA, FtsW, FtsL, YpsB , DivIVA, e MinC fusionadas a GFP. Quanto maior a freqüência de co-localização entre FtsZ e outra proteína de divisão, mais inicial é a participação da proteína na formação do divisomo. Portanto, a medida da freqüência de co-localização entre o anel Z e as proteínas componentes do divisomo permite que se deduza uma cinética da montagem deste complexo. Estes ensaios demonstraram uma freqüência de co-localização de 97,33% para ZapA; 98,31% para EzrA; 83,90% para FtsW; 78,43% para FtsL; 50% para YpsB; 41,7% para DivIVA e 31,64% para MinC. Estes resultados sugerem que o divisomo seja formado em três etapas. ZapA e EzrA se associam ao divisomo imediatamente após a formação do anel Z, em seguida FtsW e FtsL são recrutados para o divisomo, e por último YpsB, DivIVA, MinC associam-se ao divisomo. A segunda questão que investigamos nesta tese foi o mecanismo da mudança de posição do divisomo que ocorre durante a esporulação em B. subtilis. Na fase de esporulação a célula divide-se assimetricamente, com a formação do septo próxima a um dos pólos. Durante o crescimento vegetativo a divisão não ocorre próxima aos pólos por causa da ação das proteínas MinC, MinD e DivIVA, importantes reguladores espaciais da divisão. MinCD e DivIVA são inibidores da formação do anel Z que durante o crescimento vegetativo se localizam nos pólos das células.. Uma hipótese para explicar o uso dos sítios polares para a divisão durante a esporulação seria que as proteínas MinCD e DivIVA seriam removidas dos pólos celulares. Para testarmos esta hipótese, estudamos a localização das proteínas MinCD e DivIVA durante a esporulação. Nossos resultados demonstraram que MinCD e DivIVA se re-localizam e saem dos pólos celulares durante a esporulação. Porém esta dinâmica ocorre após a formação do anel Z assimétrico, sugerindo que o anel Z seja insensível a estes inibidores durante a esporulação. Por ensaios genéticos em B. subtilis demonstramos que a proteína SpoIIE, conhecida como provável proteína responsável por promover a formação do septo assimétrico, seja capaz de contrapor a ação de MinC no início da esporulação. Dessa maneira nós propomos um novo modelo de mudança da divisão simétrica para assimétrica durante a esporulação, diferentemente da simples saída do complexo MinCD dos pólos como é proposto na literatura. / Bacillus subtilis division begins through the formation of a multiprotein complex, the divisome, at the site of division. FtsZ is the earliest known protein to localize to the future division site where the protein forms a ring-like structure (Z-ring) that extends around the circumference of the cell. The Z-ring functions as a scaffold and recruits about fifteen other division proteins that compose the divisome. In this work, we used quantitative and qualitative methods of vital fluorescence microscopy to study two questions that have not been elucidated about the divisome dynamics. The first is how divisome is assembled. To address that problem, we made co-localization between Z-ring (FtsZ-mCherry) and proteins ZapA, EzrA, FtsW, FtsL, YpsB, DivIVA, and MinC fused to GFP. Higher is the match between GFP fusions to Z-ring, earlier is the assembly of division proteins to divisome. Therefore, the co-localization frequency between Z ring and divisome proteins will allow us to deduce the assemble kinetics of the divisome. This assays showed a co-localization frequency of 97,33% for ZapA; 98,31% for EzrA; 83,90 for FtsW; 78,43% for FtsL; 50% for YpsB; 41,7% for DivIVA and 31,64% for MinC. This data suggests that the divisome does not assemble in two but in three steps. ZapA and EzrA assemble into the divisome immediately after Z ring formation, secondly FtsW and FtsL were recruited to the divisome, and finally YpsB, DivIVA, MinC associated with the divisome. The second question that we investigated in this work is the mechanism responsible for change the divisome position that occurs during sporulation in B. subtilis. In sporulation the cell divides asymmetrically, with a septum formation near poles. During vegetative grown the divisiome does not occur near poles because of MinC, MinD and DivIVA action, relevant for spatial regulation of division. MinCD and DivIVA are inhibitors of Z ring formation that during vegetative growth are located at poles. A hypothesis to explain the use of polar sites for division during sporulation would be that MinCD and DivIVA would be removed from cellular poles. To test this hypothesis, we studied the location of MinCD and DivIVA proteins during sporulation. Our results demonstrated that MinCD and DivIVA re-localize and leave to cell poles during sporulation. However this process occurs after asymmetric Z ring formation, suggesting that Z ring would be unresponsive to this inhibitors during sporulation. Through genetics assays in B. subtilis we demonstrated that SpoIIE protein, known to probably play a role in asymmetric septum formation, would be able to contrapose MinC action during early sporulation. Therefore, we propose a novel model for change the symmetric to asymmetric division during sporulation, unlike the release of MinCD from pole proposed in the literature.

A divisão celular nas bactérias requer a formação do divisomo, um complexo protéico que tem como o primeira etapa a polimerização da proteína FtsZ, seguida pela associação de 15 outras proteínas conhecidas. Os mecanismos envolvidos na regulação espacial do divisomo são bem caracterizados, mas o controle temporal da divisão celular em relação a outros eventos do ciclo, como a replicação do cromossomo, segue controversa. Neste trabalho, aplicamos a metodologia de microscopia de série temporal para estudar duas questões fundamentais do processo de divisão: a montagem do complexo que executa a divisão e a possibilidade da oscilação periódica na expressão de um ou mais genes envolvidos em divisão possa participar do controle temporal da montagem do divisomo. Para investigar se há oscilação da expressão gênica, construímos inicialmente variantes instáveis GFP através da adição de sequências peptídicas C-terminais que encaminham para a degradação em B. subtilis e utilizamos estes repórteres para criar fusões transcricionais sob o controle de promotores de genes centrais do processo de divisão. Depois de otimizar as condições de microscopia de série temporal com fusões transcricionais usando a variante instável GFPAISV, observamos que a autofluorescência de B. subtilis interferia nas nossas quantificações. Como forma de contornar a autofluorescência, construímos então fusões transcricionais com duas variantes de YFP (proteína fluorescente amarela) e optamos por trabalhar com Ypet-AISV. A análise de filmes de células individuais, tanto com fusões a GFPAISV como a Ypet-AISV, indicou que apenas o promotor do operon ftsL-pbpB apresentava um padrão de oscilação significativamente diferente de um promotor artificial usado como controle negativo. Esta hipótese, no entanto, não foi confirmada por medidas estáticas de populações de células nas quais correlacionamos intensidade de fluorescência com posição no ciclo celular. Portanto, nossos dados não foram capazes de evidenciar flutuações na expressão dos genes ftsL-pbpB, minCD, ftsZ, ftsA e zapA ao longo do ciclo celular. Para estudar a cinética de montagem divisomo foram realizados experimentos de microscopia de série temporal de FtsZ-mCherry e Pbp2B-GFP, onde observamos que a associação de Pbp2B ao divisomo ocorre 3 minutos após a formação do anel de FtsZ em meio rico e 4 minutos em meio mínimo. Também realizamos experimentos de microscopia de série temporal com uma cepa contendo FtsZ-YFP e DivIVA-CFP, determinando que DivIVA é incorporado ao divisomo 16 minutos após a formação do anel de FtsZ em meio rico e 20 minutos em meio mínimo. Estes dados confirmam que a montagem do divisomo ocorre em três etapas, e não duas, como anteriormente proposto. / Cell division in bacteria requires the formation of the divisome, a protein complex that has as the first step polymerization of FtsZ, followed by the assembly of 15 other known proteins. The mechanisms that underlie spatial regulation of divisome assembly have been largely elucidated, but the temporal control that ties the timing of cell division to other cell cycle events, such as chromosomal replication, remains surrounded by controversy. In this work, we use time-lapse microscopy to address two issues in B. subtilis cell division: the timing of divisome assembly, and the possibility that a periodic oscillation in expression of one or more genes essential for divisome assembly may play a role in defining the timing of cell division. To study the possibility of oscilation in gene expression, we have first built unstable variants of GFP by adding to its C-terminus peptide sequences that target the protein for degradation and used those variants to build transcriptional fusions to access the promoter activity of core cell division genes. After optimizing time-lapse conditions with transcriptional fusions to cell divison genes with the unstable GFPAISV, we observed that B. subtilis autofluorescence was an issue to our quantifications. To improve our signal-to-noise ratio, we built transcriptional fusions with two variants of YFP (Yellow Fluorescent Protein), and decided to work with Ypet. In our single-cell analysis for GFPAISV and for Ypet-AISV, only the ftsL operon promoter presented an oscilating pattern different from our negative control. This was not confirmed, however, when we attempted to correlate fluorescence signal with cell cycle position in static single-cell measurements. Thus, we conclude that that there are no fluctuations in ftsL, pbpB, minCD, ftsZ, ftsA or zapA gene expression throughout the cell cycle. To study divisome assembly we performed time-lapse microscopy of FtsZ-mCherry and Pbp2B-GFP, and determined that the association of Pbp2B occurs 3 minutes after FtsZ polymerization in rich medium and 4 minutes in minimal medium. We also performed time-lapse microscopy with FtsZ-YFP and DivIVA-CFP, determining that DivIVA is incorporated to the divisome in 16 minutes after FtsZ polymerization in rich medium and 20 minutes in minimal medium. This data confirms the assembly of the divisome in three steps rather than two, as previously proposed.

O mecanismo de divisão mais comum entre procariotos é a divisão binária, na qual a célula- mãe reparte seu genoma e conteúdo citoplasmático de forma igual entre duas células filhas. Esse processo é mediado por um complexo protéico especializado, chamado divisoma, composto por cerca de 20 proteínas, que promovem a constrição da parede celular e membrana citoplasmática, formando o septo de divisão. O complexo é organizado em torno do anel Z, uma estrutura em anel composta pela proteína FtsZ, um homólogo de tubulina presente na maioria dos procariotos e em algumas organelas de eucariotos. Partindo de um levantamento detalhado da distribuição dos genes do divisoma em genomas completos de procariotos, aplicamos métodos de máxima verossimilhança para inferência de estados ancestrais e reconstruímos o conteúdo gênico do divisoma no ultimo ancestral comum das bactérias atuais. Estendendo essas análises com a aplicação de métodos filogenéticos, inferimos os eventos responsáveis pelas variações de composição deste complexo, observadas entre os diferentes grupos de bactérias. Nossos resultados mostram que o último ancestral comum de todas as bactérias já possuía a maior parte dos componentes conhecidos do divisoma, sugerindo a existência de uma parede de peptideoglicano e a presença de um aparato molecular tão ou mais complexo que o observado nas linhagens atuais, incluindo a presença de componentes considerados acessórios e de distribuição relativamente restrita, como as proteínas envolvidas na localização do anel Z (sistema Min) e alguns efetores positivos da polimerização de FtsZ. Observamos também que a evolução do complexo não foi muito afetada por eventos de transferência lateral, mas apresenta vários exemplos de perda de genes, em especial em linhagens com genoma reduzido, o que sugere a redundância de vários componentes já presentes no ancestral e a freqüente redução da complexidade, pelo menos dos componentes centrais do divisoma. Episódios de expansão de famílias de componentes do divisoma em linhagens específicas e os mecanismos evolutivos responsáveis pela incorporação de tais variações são discutidos. A caracterização da história evolutiva detalhada do divisoma, aqui apresentada, poderá servir como ponto de partida para novas análises evolutivas e como base para elaboração de experimentos funcionais. / The most common cell division mechanism among prokaryotes is binary fission, where a mother cell partitions its cytoplasm and genome equally among two daughter cells. This process is mediated by a specialized protein complex, known as the divisome, composed of around 20 proteíns, that promotes constriction of the cell wall and cytoplasmic membrane, thus forming the division septa. The complex is organized around the Z-ring, a ring-shaped struture composed by FtsZ, a tubulin homolog present in most prokaryotes and some eukaryotic organelles. After a detailed revision of the distribution of divisome genes among completely sequenced prokaryotic genomes, we applied maximum likelihood methods for the inference of ancestral states and reconstructed the gene content of the divisome in the last common ancestor of all extant bacteria. We then performed phylogeneticanalysis of all cell division genes and inferred the series of events responsible for the observed variations of the complex´s composition among bactérial lineages and their common ancestor. Our results show that the last common ancestor of all bacteria already possessed most of the known divisome components, thus suggesting the existence of a peptidoglycan cell wall and the presence of a molecular apparatus, perhaps more complex than those found in extant bacteria, including the presence of some accessory components with a somewhat restricted distribution, like the proteíns involved in the localization of the Z-ring (Min sistem) and some positive effectors os FtsZ polimerization. We also observed that the complex´s evolution was almost never the subject of horizontasl gene transfer events, but shows several examples of gene loss, specially in lineages displaying clear signs of genome reduction, thus suggesting the redundancy of several components in the ancestral divisome and a certain degree complexity reduction, at least for core components of the divisome. Lineage specific expansion of divisome component and the evolutionary mechanisms behind such processes are discussed. This characterization of the detailed evolutionary history of the divisome might serve as a starting point for new evolutionary analysis and as a basis for the design of functional experiments.