Pkn44 And Pkn30, Two Genes Of The HstK Family Are Involved In Synthesis Of Heterocyst-Specific Glycolipid In The Anabaena Sp. Strain PCC 7120

Anabaena sp.strain PCC 7120 is a filamentous cyanobacterium capable of differentiating heterocysts,which account for 5 to 10%of the cells along each filament, upon limitation of a combined nitrogen source in the growth medium.Heterocysts provide an environment for nitrogenase to fix N₂ and support nitrogen source to vegetative cells.For nitrogenase that can be irreversibly inactivated by oxygen,three mechanisms are known to contribute to the formation of a microoxic environment to protect nitrogenase in heterocysts:first,formation of a thick cell envelope consisting of two distinct layers,the inner glycolipid layer and the outer polysaccharide layer,to limit oxygen penetration,secondly,inactivation of the oxygen-producing photosystemâ…¡,and thirdly,increased respiration rate.Heterocyst development proceeds in stages and constitutes a complex process of morphogenesis,many proteins related to signal transduction are involved in this process.The genome of Anabaena sp.strain PCC 7120 possesses a family of 13 genes which named the hstK family,all encoding proteins with a putative Ser/Thr kinase domain at their N termini and a His-kinase domain at their C termini.In this study,founction of two members of hstK gene family,pkn44(all1625) and pkn30(all3691),were identified.The double mutant D4.3 strain,in which pkn44 and pkn30 were both inactivated,is unable to sustain its growth in medium lacking a source of combined nitrogen and no obvious morphological detection were identified for heterocysts 24 h after nitrogen deprivation,however,in 48 h,the cells with a pattern similar to heterocysts could be observed,and weak alcian blue stained revealed the presence of heterocyst polysaccharides layer(Het⁺).These observations suggest that the heterocyst development in D4.3 was delayed and arrested at a relatively early stage.Nitrogenase activity of D4.3 after step-down of combined nitrogen was measured using an acetylene reduction assay, no acetylene reduction by D4.3 was detected under aerobic condition,and however,a low nitrogenase activity was detected under microaerobic condition(Fix⁺).Consistence with the nitrogenase activity,the expression of nifH in D4.3 could not be detected by Western blotting under aerobic condition,and only low expressional level detected under microaerobic condition.The nifH transcript was also determined by RT-PCR and real time RT-PCR,showing that nearly no transcripts were detectable in D4.3 under conditions provided except microaerobic condition.These results indicated that the ability for transcription and expression of nifH might not be lost in D4.3 and the high intracellular oxygenic level might be the main factor resulting in the defective procedures in D4.3,that maybe because of the incomplete heterocyst structure which might not prevent oxygen penetration.It was supported by which the ultrastructure of heterocysts in D4.3 by electron microscopy revealed that envelop of heterocysts was incomplete,the inner laminated glycolipid layer was absent and only a thin polysaccharide layer of the outer envelop was deposited.By the thin-layer chromatography,the mutant strain D4.3 had only one HGL corresponding to 1-(O-α-D-glucopyranosyl)-3,25-hexacosanediol,the other one corresponding to 1-(O-α-D-glucopyranosyl)-3-keto-25-hexacosanol was undetectable.The transcripts of genes involved in heterocyst glycolipid biosynthesis were abnormal compared with wild type through RT-PCR and real time RT-PCR.In Anabaena sp.strain PCC 7120,prpJ which encoding a PP2C-type protein phosphatases is another gene involved in only one HGL biosynthesis,one heterocyst-specific glycolipid,corresponding to 1-(O-α-D-glucopyranosyl)-3,25-hexacosanediol was completely missing in its mutant,while the other one corresponding to 1-(O-α-D-glucopyranosyl)-3-keto-25-hexacosanol could be detectable,prpJ2 also encodes a PP2C-type protein phosphatases and its deduced amino acid sequence is highly similar to that of PrpJ.The growth and heterocyst development were not defective in prpJ mutant,however,the heterocyst development was not be detected in the double mutant strain S19/S20 in which prpJ and prpJ2 were both inactivated.By real time RT-PCR,it showed that the the expression of prpJ2 was enhanced after step-down of combined nitrogen and this up-regulation was not detected in a ntcA mutant strain.EMSA were carried out with purified NtcA protein and a fragment from the prpJ2 upstream region containing the NtcA box,the retarded fragments were observed,indicating binding of NtcA.These results indicated that prpJ2 was directly under the control of NtcA.Through real time RT-PCR,the high expression level could not sustain in mutant stain S19/S20 after nitrogen deprivation compared with wild type.The defect in initiating heterocysts in S19/S20 might be due to the abnormal expression of hetR.hanA encodes historic-like protein,HU,in Anabaena sp.strain PCC 7120.In this study,ectopic expression of hanA defected heterocyst differentiation.The expression of hanA was enhanced at 24 hours after nitrogen step-down in wild type while the expression was not up-regulated in ntcA mutant.Combined with the result of EMSA that purified NtcA protein was able to bind upon a fragment from the hanA upstream region containing the NtcA box,hanA was directly under the control of NtcA.