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In several Gram-negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB-like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti-sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three-hybrid system. We propose that both RssB and RssC are necessary for the ClpXP-dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.

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The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

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In bacteria, the 5'-end-dependent RNA degradation is triggered by the RNA pyrophosphohydrolase RppH converting tri/diphosphate to monophosphate transcripts. This study shows that in the soil bacterium Azotobacter vinelandii, inactivation of rppH gene negatively affected the production of bioplastic poly-ß-hydroxybutyrate (PHB) by reducing the expression at the translational level of PhbR, the specific transcriptional activator of the phbBAC biosynthetic operon. The effect of RppH on the translation of phbR seemed to be exerted through the translational repressor RsmA, as the inactivation of rsmA in the rppH mutant restored the phbR expression. Interestingly, in Escherichia coli inactivation of rppH also affected the expression of CsrA, the RsmA homolog. The level of the csrA transcript was higher and more stable in the E. coli rppH mutant than in the wild type strain. Additionally, and in contrast to the csrA mutants that are known to have a defective swimming phenotype, the E. coli rppH mutant showed a hyper-swimming phenotype that was suppressed by a csrA mutation, and the AvRppH restored to wild type level the swimming phenotype to the E. coli rppH mutant. We propose that in both A. vinelandii and E. coli, RppH activity plays a role in the expression of the translational regulator protein RsmA/CsrA.

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Late embryogenesis abundant (LEA) proteins constitute a large protein family that is closely associated with resistance to abiotic stresses in multiple organisms and protect cells against drought and other stresses. Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts. This bacterium possesses two genes, here named lea1 and lea2, coding for avLEA1 and avLEA2 proteins, both containing 20-mer motifs characteristic of eukaryotic plant LEA proteins. In this study, we found that disruption of the lea1 gene caused a loss of the cysts' viability after 3 months of desiccation, whereas at 6 months, wild-type or lea2 mutant strain cysts remained viable. Vegetative cells of the lea1 mutant were more sensitive to osmotic stress; cysts developed by this mutant were also more sensitive to high temperatures than cysts or vegetative cells of the wild type or of the lea2 mutant. Expression of lea1 was induced several fold during encystment. In addition, the protective effects of these proteins were assessed in Escherichia coli cells. We found that E. coli cells overexpressing avLEA1 were more tolerant to salt stress than control cells; finally, in vitro analysis showed that avLEA1 protein was able to prevent the freeze thaw-induced inactivation of lactate dehydrogenase. In conclusion, avLEA1 is essential for the survival of A. vinelandii in dry conditions and for protection against hyper-osmolarity, two major stress factors that bacteria must cope with for survival in the environment. This is the first report on the role of bacterial LEA proteins on the resistance of cysts to desiccation.

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In Azotobacter vinelandii, a cyst-forming bacterium, the alternative sigma factor RpoS is essential to the formation of cysts resistant to desiccation and to synthesis of the cyst-specific lipids, alkylresorcinols. In this study, we carried out a proteome analysis of vegetative cells and cysts of A. vinelandii strain AEIV and its rpoS mutant derivative AErpoS. This analysis allowed us to identify a small heat-shock protein, Hsp20, as one of the most abundant proteins of cysts regulated by RpoS. Inactivation of hsp20 did not affect the synthesis of alkylresorcinols or the formation of cysts with WT morphology; however, the cysts formed by the hsp20 mutant strain were unable to resist desiccation. We also demonstrated that expression of hsp20 from an RpoS-independent promoter in the AErpoS mutant strain is not enough to restore the phenotype of resistance to desiccation. These results indicate that Hsp20 is essential for the resistance to desiccation of A. vinelandii cysts, probably by preventing the aggregation of proteins caused by the lack of water. To our knowledge, this is the first report of a small heat-shock protein that is essential for desiccation resistance in bacteria.

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The transcription of genes involved in alginate polymerization and depolymerization, as well as the alginase activity (extracellular and intracellular) under oxygen-limited and non oxygen-limited conditions in cultures of A. vinelandii, was studied. Two levels of dissolved oxygen tension (DOT) (1% and 5%, oxygen-limited and non-oxygen-limited, respectively) strictly controlled by gas blending, were evaluated in a wild type strain. In cultures at low DOT (1%), in which a high molecular weight alginate (1200 kDa) was synthesized, the transcription levels of alg8 and alg44 (genes encoding alginate polymerase complex), and algX (encoding a protein involved in polymer transport through periplasmic space) were considerably higher as compared to cultures conducted at 5% DOT, under which an alginate with a low MW (42 kDa) was produced. In the case of genes encoding for intracellular and extracellular alginases, the levels of these transcripts were higher at 1% DOT. However, intracellular and extracellular alginase activity were lower (0.017 and 0.01 U/mg protein, respectively) in cultures at 1% DOT, as compared with the activities measured at 5% DOT (0.027 and 0.052 U/mg protein for intracellular and extracellular maximum activity, respectively). The low alginase activity measured in cultures at 1% DOT and the high level of transcription of genes constituting alginate polymerase complex might be mechanisms by which oxygen regulates the production of alginates with a high MW.

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OBJECTIVE: This study aimed at investigating the significance of the skin-to-skin contact method with fathers, looking at their own experiences with their newborns. METHODS: The information was collected through in-depth interviews with 14 fathers who had used the skin-to-skin contact method with their newborns, after a cesarean delivery. The technique utilized for data analysis was the qualitative method of content analysis. RESULTS: Four principal themes emerged from the data: the preparation for the skin-to-skin method, the experiences of the fathers, the father's role, and effects of the method on the baby. CONCLUSIONS: The investigations performed highlighted the importance of the involvement of health professionals in the use of this method, which leads to a series of positive results for the organization related to satisfaction. We conclude that the skin-to-skin method is a simple technique, recommended for positive results for fathers and their babies.

OBJETIVO: Este estudo tem como objetivo investigar o significado do método "pele a pele" com pais, olhando para as suas próprias experiências com seus recém-nascidos. MÉTODOS: A informação foi coletada a través de "entrevistas em profundidade" em 14 pais que tinham usado o método de "pele a pele", com seus recém-nascidos após um parto por cesariana. A técnica utilizada no análise dos dados foi o método qualitativo de "análise de conteúdo". RESULTADOS: Quatro temas principais emergiram a partir dos dados: a preparação para o método "pele a pele", as experiências dos pais, o papel do pai e os efeitos do método no bebê. CONCLUSÕES: As investigações feitas destacam a importância do envolvimento dos profissionais da saúde na utilização deste método, o qual leva a uma série de resultados positivos para a organização relacionados com a satisfação. Concluímos que o método de "pele a pele" é uma técnica simples e recomendada com resultados positivos também para os pais e os seus bebês.

OBJETIVO: En este estudio se tuvo como objetivo investigar el significado del método "piel a piel" con padres, mirando hacia sus propias experiencias con sus recién nacidos. MÉTODOS: La información fue recolectada por medio de "entrevistas en profundidad" realizada a 14 padres que habían usado el método de "piel a piel", con sus recién nacidos después de un parto por cesárea. La técnica utilizada en el análisis de los datos fue el método cualitativo de "análisis de contenido". RESULTADOS: Emergieron cuatro temas principales a partir de los datos: la preparación para el método "piel a piel", las experiencias de los padres, el papel del padre los efectos del método en el bebé. CONCLUSIONES: Las investigaciones realizadas destacan la importancia del involucramiento de los profesionales de la salud en la utilización de este método, lo cual lleva a una serie de resultados positivos para la organización relacionados con la satisfacción. Concluimos que el método de "piel a piel" es una técnica simple y recomendada con resultados positivos también para los padres y sus bebés.

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We previously showed that in Azotobacter vinelandii, accumulation of polyhydroxybutyrate (PHB) occurs mainly during the stationary phase, and that a mutation in phbR, encoding a transcriptional regulator of the AraC family, reduces PHB accumulation. In this study, we characterized the roles of PhbR and RpoS, a central regulator during stationary phase in bacteria, in the regulation of expression of the PHB biosynthetic operon phbBAC and phbR. We showed that inactivation of rpoS reduced PHB accumulation, similar to the phbR mutation, and inactivation of both rpoS and phbR resulted in an inability to produce PHB. We carried out expression studies with the wild-type, and the rpoS, phbR and double rpoS-phbR mutant strains, using quantitative RT-PCR, as well as phbB : : gusA and phbR : : gusA gene fusions. These studies showed that both PhbR and RpoS act as activators of phbB and phbR, and revealed a role for PhbR as an autoactivator. We also demonstrated that PhbR binds specifically to two almost identical 18 bp sites, TGTCACCAA-N(4)-CACTA and TGTCACCAA-N(4)-CAGTA, present in the phbB promoter region. The activation of phbB and phbR transcription by RpoS reported here is in agreement with the observation that accumulation of PHB in A. vinelandii occurs mainly during the stationary phase.

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Azotobacter vinelandii is a soil bacterium that undergoes differentiation to form cysts that are resistant to desiccation. Upon induction of cyst formation, the bacterium synthesizes alkylresorcinols that are present in cysts but not in vegetative cells. Alternative sigma factors play important roles in differentiation. In A. vinelandii, AlgU (sigma E) is involved in controlling the loss of flagella upon induction of encystment. We investigated the involvement of the sigma factor RpoS in cyst formation in A. vinelandii. We analysed the transcriptional regulation of the rpoS gene by PsrA, the main regulator of rpoS in Pseudomonas species, which are closely related to A. vinelandii. Inactivation of rpoS resulted in the inability to form cysts resistant to desiccation and to produce cyst-specific alkylresorcinols, whereas inactivation of psrA reduced by 50 % both production of alkylresorcinols and formation of cysts resistant to desiccation. Electrophoretic mobility shift assays revealed specific binding of PsrA to the rpoS promoter region and that inactivation of psrA reduced rpoS transcription by 60 %. These results indicate that RpoS and PsrA are involved in regulation of encystment and alkylresorcinol synthesis in A. vinelandii.

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