GENETIC CHARACTERIZATION OF QUORUM SENSING DEPENDENT BACTERIAL ISOLATES ASSOCIATED WITH LEGUMES

Abstract

The present study aimed at characterizing two Acyl Homoserine Lactone (AHLs) producing bacteria isolates nodulating (cultivated and wild) legumes. The isolate from cultivated Pisum sativum was named as Pcc1; while that isolated from the wild species Alysicarpus buleurifolius was called Pcc21. These isolates were selected from a set of twenty potential isolates obtained from the culture collection of Department of Plant Sciences, Quaid-i-Azam University. The initial screening of isolates was done for AHLs production using Chromobacterium violaceum (CV026) as indicator strain. These AHLs were further characterized for their diversity through TLC and LC/MS analyses. The TLC assay was done to study the size and range of the AHLs, revealing diverse nature of AHLs produced by the Pcc1 and Pcc21. The small bacteriocin assay revealed that Pcc1 produced AHLs which are a characteristic of CinI system, however, the Pcc21 was found negative. The phylogenetic analyses based on 16S rRNA sequences depicted the isolate Pcc1 as Rhizobium leguminosarum while that of Pcc21 as Sinorhizobium meliloti. Pcc1 being Rhizobium was further studied for the RhiR system which controls nodulation in host plants. Thus the RhiR gene was amplified and sequenced and using bioinformatics tools its protein sequences was generated. The sequence data thus generated and its comparison with the RhiR protein sequences in silico revealed useful information for the isolate (Pcc1) under study such as various domains homolgous to the LuxR transcriptional protein. To characterize it further mutation of RhiR locus was done using Pk19 mob vector, the induced mutation reduced the AHLs production and nodulation efficiency. The Pcc21 presented an interesting case. The 16S rRNA analysis revealed its identity as Sinorhizobium meliloti and it was isolated from root nodules of a wild legume species Alysicarpus bupleurifolius, this has been the first report on the novel symbiotic association between S. meliloti and Alysicarpus bupleurifolius. The study further characterized the AHL diversity produced in symbiosis. The TLC and LC-MS profiles revealed production of a diverse array of AHLs that ranged from short chain C4-HSL to long chain C12- HSL. It was observed that isolates thriving in younger nodules produced AHL abundance and points to the possible presence of more than one synthase system, as previously described in Rm1021 and AK63. The isolation of a strain producing wide ranging AHL molecules provides an opportunity to investigate the complex genetics of AHL production in S. melitoti. It was further revealed that the AHLs produced by Pcc21 were rather diverse. In an analysis the introduction of WspR plasmid in Pcc21 decreased the amount of exopolisaccharides produced in the system, however it did not affect the overall amount of AHLs produced.