The Self-protection Mechanism Of Albomycin Producing Strain Streptomyces Griseus ATCC 700974

Albomycins are peptide nucleoside antibiotics which have good antibacterial activity against a variety of gram-negative bacteria and some gram-positive bacteria.The good antibacterial activity of albomycins are due to its special chemical structure.Albomycins are composed of a iron carrier,a special thiofuranose and a highly modified cytosine.They can enter bacterial cells through the iron uptake systems,and then be hydrolyzed by proteases in the bacteria to release the active components SB-217452,which shows good antibacterial activity by inhibiting the activity of seryl-t RNA synthetase,thus it is called "Trojan horse" antibiotic.Normally,Streptomyces needs a set of self-defense system to deal with the suicides of antibiotics.The mechanism of action of albomycins has been studied clearly,but the self-protection mechanism of Streptomyces griseus ATCC 700974 is not clear.In this paper,we took Streptomyces griseus ATCC 700974 as the research object,carried out the following three aspects of research,and preliminarily revealed the self-protection mechanism of albomycin producing bacteria.First,abmK was identified as the resistance gene of albomycin.The biological activity of fermentation broth of abmk deletion mutant was analyzed,which indicated that the absence of abmKseriously affected the synthesis of albomycin;A mutant with abmK deletion was constructed,and the results showed that the deletion of abmK seriously affected the synthesis of Abomycin.In the sensitivity test of Streptomyces strains,we found that the strain Δabmand Streptomyces griseus IFO13350 were sensitive to albomycins,while the other Streptomyces strains were resistant to albomycins,which indicated that the albomycins had great harm to the producing bacteria.After abmK was introduced into the E.coli JM109 and Δabm,the sensitive strain showed tolerance to albomycins,the results showed that abmK could give the sensitive strain the ability to resist albomycins;The expression level of abmK was detected under the stress of albomycin at different concentrations.The results showed that abmK gene could increase its expression level in response to the changes of albomycins,so as to protect the producing bacteria from the damage caused by albomycins.Secondly,abmN,abmO,abmP and abmR may be involved in the transport of abromycin.By analyzing the conserved domains of AbmN,AbmO,AbmP and AbmR,it was found that AbmN and AbmR belong to ATP binding protein,while AbmO and AbmP belong to ABC transporter permease.Mutants with single transporter gene deletion and mutants with 4 genes deletion were constructed,respectively.The results showed that the intracellular and extracellular production of albomycins in all the mutants are decreased,which indicated that the destruction of the transport gene would affect the synthesis of albomycins,after the deletion of four transport genes at the same time,apomycin was still produced,which indicated that there were other transport genes involved in the export of apomycin in the strain;After the high expression of the transport gene,the antibacterial activity of the intracellular and extracellular products did not significantly improve;Introducing the transporter gene into the sensitive strains did not improve the tolerance of the strains,but made the strains slightly sensitive,the reasons need to be further explored and verified.Finally,we found a group of proteins involved in the internalization of albomycin.In the sensitivity test of Streptomyces to albomycins,the growth of Δabmmutant was seriously inhibited by albomycins,which indicated that there was an inward transport system of albomycins in Streptomyces griseus ATCC 700974,which transported it back to the cell and damaged the strain itself.In order to improve the tolerance and further analyze the self-protection mechanism of albomycins in Streptomyces griseus ATCC 700974,we carried out the following research work to find the inward transport system of albomycins in Streptomyces griseus ATCC 700974:(1)Through the competitive absorb experiments of three kinds of small molecule iron carrier compounds,it was determined that the ferrichrome transport system was the type of transport system for albomycins in Streptomyces griseus ATCC 700974.(2)Bioinformatics analysis was performed on the genome of Streptomyces griseus ATCC 700974 using the transport protein sequence of albomycin from Escherichia coli and Streptococcus pneumoniae,we found 10 groups of proteins that may be involved in the transport of albomycins.(3)CRISPR / cas9 technology was used to knock out the 10 groups of candidate importers respectively,and one group of proteins was found,which can improve the tolerance of the strain to a certain extent.To sum up,the self-resistance of microorganisms is a very complex process.albomycins as the antibiotic with unique mechanism of action and good antibacterial activity,the destruction of resistance gene and transport gene significantly affects the synthesis of albomycins,which indicates that the two self-protection mechanisms of resistance gene expression and timely antibiotic efflux are needed in producing bacteria to form multiple protection.Importantly,the discovery of inward transport system can further improve the self tolerance of strains to albomycins.