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The investigative study about role of hopanoides in stress tolerance and nutrient storage was carried out in Nostocpunctiforme which is a diazotrophic symbiotic cyanobacterium. Under starved condition N.punctiforme shows presence of photosynthetic vegetative filaments. Spores like akinetes are formed when N. punctiformeis exposed to other stresses like low light, phosphate starvation, unlike vegetative cell spores are capable of surviving under harsher condition. One of the well studied and described interaction of N. punctiformeis its symbiosis with hornwort Anthocerospunctatus. In this association the site of infection of N. punctiformeis mucilaginous cavities present at the growing edges of gametophytic tissues of plant, this infection develops visible symbiotic colonies. To determine role of hopanoide in this symbiosis association, the interaction was facilitated in between N. punctiforme shc mutant and Anthocerospunctatus; mutant was able to infect plant showing its resemblance towards WT strain for symbiotic properties. Thus hopanoide linked effects were not observed in symbiotic interaction. 

In case of N. punctiforme hopanoides are found in all cell type but more abundant in outer membrane of akinetes. In order to determine significance of hopanoides in temperature stress condition, along with wild type (WT) other mutants as shc, hpn and  shc complement were incubated at 13°C, 25°C and 40°C keeping all the other optimum conditions same. When compared with WT, at 25°C shc showed same doubling time as WT, hpnP showed slow growth than WT and shc compliment showed almost same doubling time as WT. In case of extreme temperatures i.e at 13°C shc showed faster doubling time than WT, shc compliment do not double its population that fast. At 40°C doubling time for shc was reduced whereas compliment shows almost same doubling time as WT. When observed growth rates of hpnP it did not showed significant difference in its growth rate from wild type at extreme temperatures. Lipid analysis of N. punctiforme(WT) and its shc mutant by LC-MS followed by PCA plot revels that at temperature extreme lipidome of shc clusters differentially from lipidome of shc at 25°C. Reason for such behavior of mutant strains with respect to WT can be stated as, at 40°C no 2-methyl bacteriohopanepentol was observed in WT, while bacteriohopanetetrol and 2-methylbacteriohopanetetrol were found abundantly than at 25°C.These data directs that function of hopanoide at extreme temperature is independent of the total amount of hopanoide available and possibly concerned with more or less specific type of hopanoide, such as 2-methylbacteriohopanetetrol.

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For nutrient storage and stress tolerance different assays were carried out including spot stress survival assay and desiccation assay, WT vegetative cells displayed reduced growth in spot dilution compared to optimum growth conditions, it  indicates that concentrations of stressors used was physiologically relevant.  However, normal growth of shc and hpnP mutant was observed like WT when subjected to stresses, suggesting hopanoides are not required for tolerance of these stresses in vegetative cells. Spores i.e. akinetes were induced  in WT and mutant strains of N. punctiforme and lysosomal survival experiment was  carried out which resulted in ­­~ 10 fold decrease in surviving population for akinetes of wild type cultures in contrast  shc akinetes exhibited  ~100 fold decrease in survival as compared to WT. In vegetative cell many inclusion bodies containing cyanophycin, a poly amino acid used for nitrogen and carbon storage were observed, whereas shc displayed less accumulation of these granules than WT. This shows that in akinetes of shc mutants due to lack of some specific hopanoides storage function is affected.

 

1.4.7 Hopanoid exposure

Hopanoids, a class of pentacyclic organic compounds with 27 to 35 carbon atoms in a naphthenic structure, are largely derived from the lipid fraction of once-living organisms (Peters et al., 2007) and are considered petroleum biomarker they are not present in gasoline and diesel fuel because they are in the higher boiling fraction of petroleum, although they are found in engine oil lubricant (Omar et al., 2006; Pakbin et al., 2009; Zakaria et al., 2001), and therefore are detected in auto exhaust aerosol emission, as well as in road dust (Bahry et al., 2009). A few studies suggested that hopane emission rates are not affected by alternative fuels such as biodiesel. However, recently, it was speculated that fuel type may affect the amount of lubricating released into exhaust through several mechanisms and that higher cetane number fuel may leads to reduced emission of hopanes. These discrepancies in emission rates may limit the use of hopanes as traffic-related tracers. A wide set of hopanoids were identified and quanitifed for the first time in particulated matter of less than 10 microns. In spite of the scarcity of studies, there is evidence that hopanes may pose risks to human health. Strong and statistically significant positive association were observed between hopanoid hydrocarbon and ischemis heart diseases, cardiac dysrhythmia and congestive heart failure. Hopanes were also associated with asthma disease (Sarnat et al., 2015). There is a significant association between exposure to hopane and shortness of breath in cohort of myocardial infraction survivals in Germany (McDonald et al., 2004). Hopanes were detected in the aerosol of road tunnels in Brazil. Hopanes may occur in the 17?, 21?(H) stereochemistry (??) as found in living organisms and fossil fuels, whereas the ?? or ?? configurations are observed in fossil fuels. The sterochemical configurations at the C-17 and C-21 positions of hopanes are frequently taken to evaluate the geological maturity of samples. Hopanes with 17?(H), 21?(H) configurations are considered immature, 17?(H),21?(H) are moderately mature and 17?(H),21?(H) are fully mature (Bi et al., 2008, Han et al., 2015). Different biomarker rations have been widely used to evaluate the organic matter thermal maturity and degree of preservation, whether for coal or for oil-related products. Homohopane index. The specification profiles of homohopane index obtained can provide inputs to chemical mass balance (CMB) receptor models. The database can also serve as a repository to categories emissions form flex fuel vehicle and to verify profiles derived from ambient measurements by multivariate receptor models. Further studies are needed to fully understand the mechnisms by which hopanes are exhausted from lubricating oils.

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