Analysis Of The Promoter Of SmrA Cluster In Sinorhizobium Fredii HN01

Sinorhizobium fredii is a fast-growing Chinese isolate with fewerextracelluar polysaccher that has been adopted as a model organism for thestudy of nitrogen fixation. In our previous work, a sulfur metabolism involvedgene smrA from S. fredii strain HN01 was cloned using Tn5gusA5 mutationstrategy. The smrA (ORFd) combined with other three genes ORFa, ORFb andORFc were found share one putative promoter and constitute an operon.Downstream of the smrA, ORFe with an other putative promoter was found hasthe same transcriptional directon. One of the aims of this work is to identify thestructure of the operon that smrA gene located in; The other is to investigate theeffect of sulphate on the regulation of this operon.In this study, we firstly amplified the two putative promoter fragment ofORFa and ORFe, named P1 (329 bp) and P2 (353bp), respectively. The twofragments were fused to the gus reporter gene encodingβ-glucuronidase andthen cloned to a suitable expression vector pRT102 generating two expressionplasmids. The two expression plasmids were then transferred into S. fredii HN01and GUS activities were tested using the two recombinant strains. The resultsindicated that ORFa, ORFb, ORFc and ORFd (smrA) formed an operon. For thepromoter P1, the highest GUS activity was achieved when HN01 harboring P1-gus fusion was cultured in MM medium containing glucose and NaNO₃while lower GUS activities were obtained when it was cultured in MM mediumwith glucose and NaNO₃ supplemented with sulfur source such as Na₂SO₄,Na₂SO₃, Na₂S₂O₃ and Met, respectively, suggesting that the promter activity ofP1 was restricted by these sulfur sources. After that, deletion analysis of P1promoter was carried out and four P1-gus deletions (P1 deletions were 215 bp,143 bp, 102 bp and 55 bp, respectively). Likewise, the corresponding expressionstrains were constructed by transforming the expression vector pRT102containing P1-gus deletions into S. fredii HN01, respectively. The promoteractivities of P1 deletions were tested in different sulfur sources with differentconcentrations. The results indicated that Met had stronger repression topromoter activity than Na₂SO₄, Na₂SO₃ and Na₂S₂O₃ when sulfur concentrationwas 100μM, and a 143bp fragment is necessary for a functional promoterregion.