Physiological and genetic responses of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. LH128 to abiotic stresses

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental organic contaminants and have toxic, carcinogenic and mutagenic properties. Microorganisms are used in bioremediation processes to remove or recycle organic pollutants such as PAHs. However, they often fail to efficiently degrade these pollutants when introduced into or present in a contaminated environment. This is mainly due to different abiotic orbiotic stress situations in the environment. This study aimed at exploring and understanding the abiotic stress response features and gene functions that are involved in stress mitigation in the phenanthrene-degrading Sphingomonas sp. LH128. The first part of the study focuses on identifying the physiological and genome-wide transcription response of LH128 biofilm in relation to its PAH-degrading activity when exposed to either an acute (4 h) or chronic (3 days) solute stress invoked by 450 mM NaCl. Biofilm morphology, oxygen consumption and phenanthrene mineralization activities were not significantly affected after imposing acute or chronic solute stress. This is in agreement with the transcriptomic data since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to non-stressed condition. The strain maintained its catabolic activity and adapted to the solute stress byincreasing the degree of saturation of membrane fatty acids and increasing the expression of genes important for coping with solute stress, such as osmoregulatory genes. In the second part of the study, genes involved in survival and activity of the strain LH128 under solute and matric stress were identified by plasposon mutagenesis. About 5,000 mini-Tn5 plasposon mutants of strain LH128 were generated from which 11 and 14 mutants showed growth impairment when exposed to solute stress (450 mM NaCl) that is believed to simulate the osmotic stress in saline environments and matric stress (10% PEG6000, w/v) that is believedto simulate the dehydrating properties of low water-content habitats respectively. None of the disrupted genes in these mutants was shared between solute and matric stress affected mutants. One NaCl affected mutant (NA7E1) with a disruption in a gene encoding the outer membrane protein of Sphingomonas (OpsA) was affected at lower NaCl concentrations compared to other mutants. NA7E1 was also the only mutant that showed reduced growth and phenanthrene degradation in soil. It was suggested that opsA is a crucial gene for survival of strain LH128 in soil especially in case of reduced moisture content probably by mitigating effects of solute stress. The physiological response and phenanthrene catabolic activity of strain LH128 when inoculated in PAH-contaminated soil was then investigated in detail. Four hours after inoculation, the strain showsa rapid and large reduction (approximately 100-fold) in Colony Forming Units (CFU) after which the CFU number increases till a number similar to the inoculated cell density. However, microscopic counts of Gfp-containing and moving LH128 cells in soil at 4 h incubation corresponded with the initial culture cell density suggesting that the largest fraction of LH128 cells are alive but entered into a Viable But None-Culturable (VBNC)-like state. The VBNC-like cells were apparently not dormant butactive as suggested from the increase in the degree of saturation of membrane fatty acids and transcriptome analysis. Moreover, modeling and transcriptomics suggested that the VBNC-like cells were particularly active regarding PAH biodegradation. The latter showed highly increased expression of genes (up to 133-fold) involved in PAH degradation at 4 h incubation. In addition, genes involved in stress control showed increased expression suggesting that the largest fraction of cells experienced stress in soil. A similar approach was followed to identify the physiological and whole-genome transcription response of LH128 to long-term nutrient starvation. The results from microscopic counting of Gfp-containing cells and CFU suggested that the strain enters into VBNC-like state and survived starvation stress without drastic loss of viability. The strain adapted to starvation by reducing cell size and changingcell shape from rod to round morphology, and increasing the degree of saturation of membrane fatty acids. The strain also survived starvation stress by increasing the expression of gene functions involved in efflux systems, acquisition of stored foods such as by degradation of rRNA by ribonucleases and β-oxidation of fatty acids by acyl-CoA dehydrogenases and by decreasing the expression of many gene functions such asthose involved in ribosomal proteins biosynthesis, chromosomal replication, and cell division.In the last part of the thesis, a whole-cell bioreporter was successfully constructed in strain LH128 by means of a transcriptional fusion of the promoter region of phnA1f with gfp to detect PAH biodegradation activity and the bioavailable fraction of PAHs in soil. The newly developed reporter strain LH128PGFP detected the presence of PAHs in liquid culture and in PAH-contaminated soilboth on solid agar surface and in microcosms. Such a rapid and simple bioreporter-based diagnostic approach of detecting bioavailable PAHs can be helpful for policy makers in monitoring the effectiveness of bioremediation activities.In conclusion, Sphingomonas sp. LH128 shows several features of abiotic stress adaptation without compromising PAH degradation activity. Due to these attributes, LH128 and possibly other Sphingomonas strains can contribute to biodegradation of pollutantsin environments experiencing regular abiotic stresses and can be used for biosensor applications.status: publishe