In liquid media under different growth conditions Streptomycescoelicolor A3(2) does not produce any detectable hopanoids. But when S. coelicolor A3(2) was grown in solid media it produces hopanoids. Solid media allow differentiation of S. coelicolor A3(2) and process of sporulation (Poralla et al., 2000). In S. coelicolor A3(2), only during the transition period from a substrate to aerial hyphae hopanoids will be formed and protect spores from dehydration (Poralla et al., 2000). In contract to this, Streptomyces scabies 87-22 produces hopanoids both in solid and lipid media but the process of sporulation in S. scabies 87-22 is different and it produces spores both in solid and liquid media (Seipke and Loria, 2009). Bacillussubtilis produces the sporulene (XXXV) using enzyme squalene hopene cyclase. Sporulene (XXXV) in B.subtilis is produced only in sporulating stage which protect spores from oxidative stress (Kontnik et al., 2008).
1.4.5 Cell cycle
hpnN is the RND-family transporter homologous to the protein involved in eukaryotic steroidal trafficking. The knockout of the hpnN gene from Rps.palustris TIE-1 results in mislocalization of hopanoids in the cell (Doughty et al., 2011). There is an asymmetric distribution of hopanoids in daughter cell and mother cell (Doughty et al., 2011). At high temperature (38oC), ?hpnN mutant cells remains connected by their cell wall, forming a long filament. Hopanoid mislocalization rather than the absence of hopanoids in the outer membrane is responsible for the filamentation phenotypes. The asymmetric hopanoid distribution promotes cell division at elevated growth temperatures by participating in the formation of protein-lipid microdomains, resulting in the recruitment of cell division machinery to the proper subcellular region (Doughty et al., 2011).
1.4.6 Role of Hopanoids in stress tolerance and nutrient storage
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.
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.