Pathway analysis of the leptospiral polyhydroxybutyrate (PHB) synthesis

Abstract:

[Objective] Leptospira spp., including the pathogenic Leptospira interrogans and the saprophytic L. biflexa, can synthetise abundant bacterial storage in cell bodies, which may be one of the primary reasons why Leptospira spp. can survive in barren environments for a long time. In this study, the leptospiral polyhydroxybutyrate (PHB) storage was examined qualitatively and quantitatively. The major functional genes for PHB synthesis were complementarily annotated by the genome analysis. The integrity of the PHB synthesis pathway was verified by the molecular biology methods. This study laid foundation for further research on the relationship between the leptospiral storage synthesis and the stress tolerance. [Methods] The leptospiral PHB storage was determined qualitatively and quantitatively by the lipid-specific Nile Red dyeing and the concentrated sulfuric acid oxidation-ultraviolet spectrophotometer method. The storage synthesis genes were identified by the bioinformatics methods (BLAST and InterProscan/InterPro2Go) through the homolog analysis and the functional domain search. The expression of the predicted PHB synthesis genes were verified by gene cloning, sequencing and quantitative RT-PCR. [Results] By the Nile Red dyeing and the oxidation-colorimetric assay method, Leptospira spp. was proved to synthetise PHB, a common bacterial storage. The synthetic amounts of L. interrogans and L. biflexa were 42%?45% and 64%?68% of their dry weights, respectively. Though no integrated PHB synthesis pathways were defined in the leptospiral genomes, the homologs of the major functional genes for PHB synthesis (phbC) were identified in the genomes of L. interrogans and L. biflexa by the comprehensively bioinformatics analysis in this study. By cloning, sequencing and qRT-PCR, most of the PHB synthesis genes (phbA/B/C) were proved to be transcribed in Leptospira spp., which suggested that the leptospiral PHB synthesis pathways were primarily integrated, and some highly-expressed genes may be the main functional genes involved in the leptospiral PHB synthesis. [Conclusion] L. interrogans and L. biflexa all can synthetise PHB storage and have primarily integrated PHB synthesis pathways.