Characterisation of pleiotropic regulators of gas vesicle biogenesis, antibiotic production, motility and virulence in Serratia sp. ATCC 39006
Serratia sp. ATCC 39006 (S39006) is a motile Gram-negative phytopathogenic bacterium known to produce two antibiotics – the β-lactam, 1-carbapen-2-em-3-carboxylic acid (a carbapenem) and the bioactive red pigment prodigiosin (a prodiginine). It is also the only known enterobacterium which naturally produces intracellular gas vesicles (GVs), enabling cells to float in static water columns. These hollow proteinaceous organelles are expressed from a 16.6 kb gene cluster comprised of 19 ORFs arranged in two contiguous operons separated by a 930-bp intergenic region. The left-hand gvpA1-gvpY operon mostly encodes GV structural components while the right-hand gvrA-gvrC operon is composed largely of regulatory genes. Other genetic regulators of GV biogenesis have been previously described which include the post-transcriptional regulator RsmA, the ribose operon repressor RbsR, the DeoR-family transcriptional regulator FloR, the sigma factor σ 54RpoN, and the transcription factor DksA. The regulation of GVs and secondary metabolites in S39006 can be coordinated but such pleiotropy is still poorly understood. PigP (an XRE family transcriptional regulator) and Rap (regulator of antibiotic production, a SlyA transcriptional regulator) are well-defined pleiotropic regulators of secondary metabolite production in S39006. However, their roles in the regulation of flotation have not been comprehensively investigated. In this study, quantitative proteomic analysis was used to compare the intracellular proteomes of pigP and rap mutants against the wild type. The abundances of proteins involved in antibiotic production, motility and GV biogenesis were consistent with the phenotypes of each mutant while known regulators also showed altered expression. Random transposon mutagenesis also identified two novel regulatory genes, draG (encoding an ADP-ribosylglycohydrolase) and xynR (encoding an IclR family transcriptional regulator), exhibiting diminished production of the carbapenem, prodigiosin, GVs and cellulase in S39006. Both mutants showed increased flagellar motility but attenuated virulence in planta and against C. elegans. Furthermore, by- pass mutagenesis of the draG mutant uncovered a putative inter-operonic sRNA located in the GV cluster (GV-sRNA) that repressed flotation of S39006. Finally, comparative quantitative proteomics was also undertaken on the draG, draG,GV-sRNA and xynR transposon mutants to investigate the wider impacts of each mutation and better understand their regulatory roles.