Partner-switching regulatory systems (PSRSs) are utilized by many different bacteria to regulate a wide array of cellular responses, from stress response to expression of virulence factors. The filamentous cyanobacterium Nostoc punctiforme can transiently differentiate motile filaments, called hormogonia, in response to various changes in the environment. Hormogonia utilize a Type IV pilus (T4P) complex in conjunction with a secreted polysaccharide for gliding motility along solid surfaces. This study identified three genes, designated hmpU, hmpW, and hmpV, encoding the protein components of a PSRS involved in regulation of hormogonium motility in N. punctiforme. Although mutant strains with in-frame deletions in hmpU, hmpW, and hmpV differentiated morphologically distinct hormogonium-like filaments, further phenotypic analysis demonstrated significant distinctions among the strains. The ∆hmpW strain contained a higher percentage of motile filaments that moved faster than the wild-type strain, while the ∆hmpU and ∆hmpV strains consisted of fewer motile filaments that moved at a slower rate compared to wild type. Immunoblotting and immunofluorescence of PilA, the major component of the pilus in the T4P system, showed that although all mutant strains appeared to express similar levels of PilA protein, the ∆hmpU and ∆hmpV strains displayed reduced extracellular PilA. Lectin blotting and staining with fluorescently-labeled UEA lectin demonstrated a decrease in extracellular hormogonium polysaccharide in the ∆hmpU and ∆hmpV strains, consistent with the current understanding that the polysaccharide is secreted via the T4P system. Epistasis analysis demonstrated that the ∆hmpW, ∆hmpV double-deletion mutant strain displayed reduced spreading in plate motility assays, similar to the ∆hmpV single mutant. Together, these results support a model in which the HmpU phosphatase and HmpW serine kinase control the phosphorylation state of the HmpV protein, modulating its activity on a downstream target to ultimately promote activation of the T4P motor complex and enhance hormogonium motility.

Cyanobacteria are prokaryotes capable of oxygenic photosynthesis, and for many species, nitrogen fixation, giving cyanobacteria an important role in global carbon and nitrogen cycles. Furthermore, multicellular filamentous cyanobacteria are developmentally complex, capable of differentiation into different cell types, including cells capable of nitrogen fixation and cells for motility, making them an ideal platform for studying development, as well as for practical use in biotechnology. Understanding how developmental programmes are activated require an understanding of the role of alternative sigma factors, which are required for transcriptional activation in bacteria. In order to investigate the gene regulatory network and to determine the role of alternative sigma factors in hormogonium development, real time PCR and Next Generation RNA-seq were used to measure expression levels of genes involved in hormogonium development and to further characterise the nature of the hormogonium developmental programme in the filamentous cyanobacterium Nostoc punctiforme. The results support a model where a hierarchal sigma factor cascade activates hormogonium development, in which expression of sigJ activates expression of the sigma factors sigC and sigF, as well as a wide range of other genes, including those involved in the type IV pilus (T4P), chemotaxis-like systems, and cell architecture. SigC and SigF have more limited roles: cell division genes are dependent on SigC and pilA expression was stringently SigF-dependent. Interestingly, SigC was also found to enhance expression of sigJ during hormogonium development, implying a potential positive feedback loop between sigJ and sigC.

Made available in DSpace on 2015-03-26T13:51:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1357885 bytes, checksum: 94cbad5284c486e24d0ad442975d1eda (MD5) Previous issue date: 2010-06-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / In Nostoc isolates, the vegetative cells multiplication and differentiation of some of them in heterocyst is the life cycle phase in which biomass production occurs. In other phase, many environmental changes can trigger hormogonium differentiation, a transient and a non-growth state. The use of cyanobacteria strains in biotechnological processes have been studied for many years, however, the production of biomass is influenced, and can be limited, by the fact that the colonies growth intensify the selfshading. Consequently, changes in light intensity and quality received by cells can occur. The aims of this work were: 1) to characterize biomass and pigments production by Nostoc isolates in response to changes in light intensity; 2) to analyze the effect of pre-cultivation and exposure in different light intensities in the same parameters and in cellular differentiation processes; 3) and to relate, for isolate Nostoc CCLFM XXI, growth phases to predominant cellular differentiation processes. The greater biomass production was achieved at 20, 45 and 75 μmoles m-2 s-1, respectively in Nostoc CCLFM I, VIII and XXI. In Nostoc CCLFM I, only phycoerithrin content changed with light intensity, been maximum at 15 μmoles m-2 s-1, decreasing with increasing light intensities. Pigment contents, in Nostoc CCLFM VIII did not vary with light intensities. In Nostoc CCLFM XXI phycocyanin and alophycocyanin contents varied with light intensity, reaching a maximum at 45 μmoles m-2 s-1, been constant up to 105 μmoles m-2 s-1. Biomass pre-cultivated at 15 μmoles m-2 s-1, when exposed to lower light intensities led to an intense akinetes differentiation in Nostoc CCLFM VIII and XXI, fact that did not occur in Nostoc CCLFM I. When biomass pre-cultivated at 75 μmoles m-2 s-1 were exposed to lower light intensities, filaments with smaller cells than vegetative ones were observed, indicating probably, the occurrence of hormogonium differentiation in Nostoc CCLFM I and VIII. When cultivated at 15 μmoles m-2 s-1, Isolate XXI showed intense akinetes differentiation, and when cultivated at 60 μmoles m-2 s-1 it showed distinct cellular differentiation patterns in phases in which biomass production was not observed. Hormogonia were observed only in the early non-growth phase, while akinetes were observed in middle and late non-growth phases. Therefore, there is relation between light intensity and patterns of cellular differentiation. In lowest light intensities the akinetes differentiation predominates over other differentiation process. However, in higher intensity hormogonia and akinetes were observed, with hormogonia associated with the first non-growth phase and akinetes with middle and late nongrowth phase, indicating a sequence in which cellular differentiation occur, probably related with light energy available to photosynthesis. / Em isolados do gênero Nostoc, a multiplicação das células vegetativas e a diferenciação de algumas células em heterócitos em intervalos regulares é a etapa do ciclo de vida em que ocorre a produção de biomassa. Em outra etapa do ciclo de vida, vários fatores do ambiente podem induzir a diferenciação de hormogônios, filamentos nos quais não ocorre produção de biomassa. A aplicação biotecnológica de cianobactérias pode ser limitada pela intensificação do auto-sombreamento durante o crescimento destas em foto-biorreatores. Em conseqüência, pode ocorrer diminuição na intensidade e alteração da qualidade espectral da luz que atinge as células. Os objetivos deste trabalho foram: 1) caracterizar a produção de biomassa e de pigmentos por isolados do gênero Nostoc cultivados em diferentes intensidades luminosas; 2) analisar o efeito do pré-cultivo e subseqüente exposição a diferentes intensidades luminosas sobre os mesmos parâmetros e sobre os processos de diferenciação celular e 3) caracterizar durante o cultivo de Nostoc CCLFM XXI em duas intensidades luminosas, a relação das fases de crescimento com os processos de diferenciação celular predominantes. As maiores produções de biomassa foram obtidas a 20, 45 e 75 μmoles m-2 s-1, respectivamente em Nostoc CCLFM I, VIII e XXI. Em Nostoc CCLFM I, apenas a concentração de ficoeritrina variou com a intensidade luminosa, apresentando-se máxima a 15 μmoles m-2 s-1, e diminuindo com aumentos na intensidade luminosa. As concentrações de pigmentos em Nostoc CCLFM VIII não variaram com a intensidade luminosa. As concentrações de ficocianina e aloficocianina, em Nostoc CCLFM XXI, variaram com a intensidade luminosa, atingindo um máximo à 45 μmoles m-2 s-1, e mantendo-se constantes nas maiores intensidades. O pré-cultivo a 15 μmoles m-2 s-1 e exposição às baixas intensidades luminosas levou, em Nostoc CCLFM VIII e XXI a uma intensa diferenciação de acinetos, o que não ocorreu para Nostoc CCLFM I. Quando o pré-cultivo foi realizado a 75 μmoles m-2 s-1, observou-se, em Nostoc CCLFM I e VIII, filamentos com células menores que as células vegetativas, indicativo de diferenciação de hormogônios. Nostoc CCLFM XXI quando cultivado a 15 μmoles m-2 s-1 apresentou intenso padrão de diferenciação de acinetos, ao passo que o cultivo a 60 μmoles m-2 s-1 apresentou distintos padrões de diferenciação celular nas faixas de parada de produção de biomassa. Nas fases iniciais de cultivo houve predominância de hormogônios, e de acinetos nas fases intermediária e final da curva. Desta forma, há uma relação entre intensidade luminosa e diferenciação celular, sendo que as mais baixas levam à diferenciação de acineto. No entanto, em maiores intensidades, observase tanto diferenciação de hormogônios quanto de acinetos, sendo os primeiros observados na primeira parada de produção de biomassa e os acinetos nas faixas mais tardias de parada, indicando uma sequência na ocorrência destes processos relacionada à disponibilidade de energia luminosa adequada à fotossíntese.