Polyamines et differenciation induite par l'hexamethylene bis acetamide (HMBA) de cellules erythroleucemiques de souris : role cle de la desacetylation de l'HMBA dans cette differenciation

SIGLECNRS TD Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

FixJ: a Major Regulator of the Oxygen Limitation Response and Late Symbiotic Functions of Sinorhizobium meliloti

Sinorhizobium meliloti exists either in a free-living state in the soil or in symbiosis within legume nodules, where the bacteria differentiate into nitrogen-fixing bacteroids. Expression of genes involved in nitrogen fixation and associated respiration is governed by two intermediate regulators, NifA and FixK, respectively, which are controlled by a two-component regulatory system FixLJ in response to low-oxygen conditions. In order to identify the FixLJ regulon, gene expression profiles were determined in microaerobic free-living cells as well as during the symbiotic life of the bacterium for the wild type and a fixJ null-mutant strain. We identified 122 genes activated by FixJ in either state, including 87 novel targets. FixJ controls 74% of the genes induced in microaerobiosis (2% oxygen) and the majority of genes expressed in mature bacteroids. Ninety-seven percent of FixJ-activated genes are located on the symbiotic plasmid pSymA. Transcriptome profiles of a nifA and a fixK mutant showed that NifA activates a limited number of genes, all specific to the symbiotic state, whereas FixK controls more than 90 genes, involved in free-living and/or symbiotic life. This study also revealed that FixJ has no other direct targets besides those already known. FixJ is involved in the regulation of functions such as denitrification or amino acid/polyamine metabolism and transport. Mutations in selected novel FixJ targets did not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa roots. From these results, we propose an updated model of the FixJ regulon

Flow effects on the viability and lysis of suspended mammalian cells

A mouse myeloma cell line growing in suspension was subjected intermittently to flow through a sudden contraction and turbulent flow in a capillary tube. The probability of lysis per pass through the capillary tube increased with average wall shear stress level and with residence time per pass in the tube. Lysis was first observed at a threshold average wall shear stress level of 1800 dyn/cm^2. Although the flow caused lysis, it had no effect on cell viability

Relationship between ornithine decarboxylase activity and hexamethylene bisacetamide-induced differentiation of murine erythroleukemia cells.

International audienceA transitory increase in ornithine decarboxylase (ODC) activity is shown not to be a prerequisite for the differentiation induced by hexamethylene bisacetamide (HMBA) in murine erythroleukemic (MEL) cells. On the contrary, conditions are described, where inhibition of the ODC activity with alpha-difluoromethyl ornithine (DFMO) stimulated the induced differentiation. Polyamine analysis demonstrated that a reduction in intracellular putrescine and spermidine occurred in MEL cells before commitment to erythrodifferentiation. The presence of DFMO increased the rapidity and the amplitude of these changes. No effect of dexamethasone on these changes in ODC activity or intracellular polyamines was observed

Which role for nitric oxide in symbiotic N2-fixing nodules: toxic by-product or useful signaling/metabolic intermediate?

The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible with nitrogenase activity. The present review analyses the different roles of NO in functioning nodules, and discusses the role of plant and bacterial (flavo)hemoglobins in the control of NO level in nodules

Tyrosine Nitration of Flagellins: a Response of Sinorhizobium meliloti to Nitrosative Stress

International audienceRhizobia are bacteria which can either live as free organisms in the soil or interact with plants of the legume family with, as a result, the formation of root organs called nodules in which differentiated endosymbiotic bacteria fix atmospheric nitrogen to the plant's benefit. In both lifestyles, rhizobia are exposed to nitric oxide (NO) which can be perceived as a signaling or toxic molecule. NO can act at the transcriptional level but can also modify proteins by S-nitrosylation of cysteine or nitration of tyrosine residues. However, only a few molecular targets of NO have been described in bacteria and none of them have been characterized in rhizobia. Here, we examined tyrosine nitration of Sinorhizobium meliloti proteins induced by NO. We found three tyrosine-nitrated proteins in S. meliloti grown under free-living conditions, in response to an NO donor. Two nitroproteins were identified by mass spectrometry and correspond to flagellins A and B. We showed that one of the nitratable tyrosines is essential to flagellin function in motility.IMPORTANCE Rhizobia are found as free-living bacteria in the soil or in interaction with plants and are exposed to nitric oxide (NO) in both environments. NO is known to have many effects on animals, plants, and bacteria where only a few molecular targets of NO have been described so far. We identified flagellin A and B by mass spectrometry as tyrosine-nitrated proteins in Sinorhizobium meliloti in vivo. We also showed that one of the nitratable tyrosines is essential to flagellin function in motility. The results enhanced our understanding of NO effects on rhizobia. Identification of bacterial flagellin nitration opens a new possible role of NO in plant-microbe interactions

The Nitrate Assimilatory Pathway in Sinorhizobium meliloti: Contribution to NO Production

The interaction between rhizobia and their legume host plants culminates in the formation of specialized root organs called nodules in which differentiated endosymbiotic bacteria (bacteroids) fix atmospheric nitrogen to the benefit of the plant. Interestingly, nitric oxide (NO) has been detected at various steps of the rhizobiumlegume symbiosis where it has been shown to play multifaceted roles. It is recognized that both bacterial and plant partners of the Sinorhizobium meliloti-Medicago truncatula symbiosis are involved in NO synthesis in nodules. S. meliloti can also produce NO from nitrate when living as free cells in the soil. S. meliloti does not possess any NO synthase gene in its genome. Instead, the denitrification pathway is often described as the main driver of NO production with nitrate as substrate. This pathway includes the periplasmic nitrate reductase (Nap) which reduces nitrate into nitrite, and the nitrite reductase (Nir) which reduces nitrite into NO. However, additional genes encoding putative nitrate and nitrite reductases (called narB and nirB, respectively) have been identified in the S. meliloti genome. Here we examined the conditions where these genes are expressed, investigated their involvement in nitrate assimilation and NO synthesis in culture and their potential role in planta. We found that narB and nirB are expressed under aerobic conditions in absence of ammonium in the medium and most likely belong to the nitrate assimilatory pathway. Even though these genes are clearly expressed in the fixation zone of legume root nodule, they do not play a crucial role in symbiosis. Our results support the hypothesis that in S. meliloti, denitrification remains the main enzymatic way to produce NO while the assimilatory pathway involving NarB and NirB participates indirectly to NO synthesis by cooperating with the denitrification pathway