Park, Buem-Seek (2002) Responses of an Environmental Gram-negative Bacterium to Pollutant Stress. PhD thesis, Victoria University.

Abstract

Several strains of pseudomonads which are normally found in natural water and soil environments, including Pseudomonas fluorescens, Pseudomonas aeruginosa, and strains initially identified as Pseudomonas paucimobilis and Pseudomonas maltophilia, were used in this thesis and their stress responsive proteins were surveyed at the physiological level. Additionally, 16S rRNA gene comparison was employed to classify the pseudomonads and related species at the genetic level. In order to see to what extent the pseudomonads and related species responded to heat shock, cells were initially subjected to temperature upshift from 28 to 37 deegree centegrade then disrupted by sonication or cells lysed by boiling in sample buffer, and the extracted total proteins were resolved on 1-D or 2-D SDS-PAGE. Their growth characteristics in different media (varying from rich to minimal) were determined and the physiological impacts on growth were examined at various concentrations of selected chemicals (metal ions, phenolics), to establish toxic and sub-lethal levels for use in determining stress responses. Production of novel proteins or elevated levels of normal proteins following exposure to different concentrations of toxicants was examined in detail in strain VUN10,077 (formerly Ps. paucimobilis) by sampling throughout the growth cycle and using 35S-methionine incorporation into newly synthesized proteins. Indigenous strain VUN10,077 presented abnormal rRNA pattern in standard RNA gels and 16S-like rRNA was analysed using Northern blot associated with 16S rRNA gene analysis which confirmed the identity of the known pseudomonads. An bands of MW 2.5 kb (faint), 1.38 kb, 1.2 kb and 0.1 kb were detected in RNA gels. The 1.38 kb band corresponded to 16S rRNA gene analysis, as shown by Northern blot analysis. 16S rRNA gene sequencing homology showed that strain VUN10,077 was a Brevundimonas species. The pseudomonads and related species accumulated a GroEL-like protein under heat stress when cells were grown in LB broth and disrupted by sonication, and 2-D SDS-PAGE conditions were established using sonically disrupted cells of Stenotrophomonas maltophilia (formerly Ps. maltophilia). Cells lysed by boiling represented the total protein profile and included particulate or membrane-associated proteins, 70 kDa-like and 44-46 kDa proteins for strain VUN10,077. Selected pollutants impacted on the cell viability and sub-lethal levels of these were determined as follows; 2.0 gL-1 for CdCl2, 2.0 gL-1 for CuCl2.2H2O, 2.0 gL-1 for NiCl2.6H2O, 1.0 gL-1 for CoCl2.6H2O, 0.025 gL-1 for HgCl2, 0.25 gL-1 for SDS, 0.2 gL-1 for NaAsO2 and 0.5 gL-1 for phenol, from A600 readings and viable counts. Major HSPs were present at relatively lower levels or were barely increased compared to controls, whereas low molecular weight proteins were significantly changed. A 20 kDa protein was commonly found in cadmium, copper, cobalt, nickel, arsenite and phenol stresses. However, a DnaK-like protein strongly reacted against commercially available DnaK antibodies on Western blot analysis and a visibly increased signal was seen for heat, cadmium, zinc, cobalt, phenol stresses and control, where represents in that order, whereas changes in the GroEL-like protein were less specific. Several minimal or defined minimal media were developed and used in heat stress and different protein profiles were seen. Medium (2) contained M9 salts medium (supplemented with glucose) plus casamino acids and medium (5) contained M9 salts medium (supplemented with glucose), vitamin solution, trace elements solution and casamino acids. Media (2) and (5) were found to be suitable for studying the physiological changes in strain VUN10,077, but growth rates in medium (2) were much less than seen in medium (5). Strain VUN10,077 presented major HSPs (70, 58 and 18 kDa proteins) in medium (5), similar to seen in Brain Heart Infusion (BHI) media. Pulse-chase 35S-methionine labelling methods were established and known major HSPs were newly synthesised by strain VUN10,077 under heat and cadmium stresses, and these proteins gradually declined while 55 kDa, 50 kDa and 18 kDa proteins were detected under ongoing stress conditions. Presumptive groEL gene analysis was attempted and partial conserved groEL sequences were determined in the pseudomonads and related species, where similarity was 98-100%. In parallel, a water-based lux system was established to examine physiological impacts of selected pollutants at the cellular level. Lux genes cloned from a marine bacterium were introduced into the background of the indigenous strain VUN10,077, providing a potentially sensitive tool for toxicity screening for terrestrial and fresh water samples. To show the use of bioluminescence in the indigenous (naturally non-bioluminescent) organism, research was performed on stable marking and optimisation of light output including marking of the organisms with luxAB or the whole lux cassette, which necessitated establishing gene transfer systems using available transposition vectors, optimisation of conditions for light output and stability. This approach would alleviate the requirement for salt or osmotic stabilizers in the test system to maintain bioluminescence or viability, which currently is a requirement of the Microtox (TM) system which employs Photobacterium phosphoreum. Expression of bioluminescence genes in the indigenous strain employed the luminescence system of Vibrio species which is encoded by a gene cluster with a divergent transcriptional pattern. As strain VUN10,077 was a genetically uncharacterised natural isolate, this work was achieved by establishing the parameters necessary for introducing genes after determining antibiotic sensitivity to determine which marker genes could be used in these backgrounds. The genes for two polypeptides (LuxA, LuxB) which form luciferase were transferred into strain VUN10,077. Introduction of the luxAB bioluminescence genes into a range of bacteria has been facilitated by using plasmid, pUT::Tn5-luxAB (de Lorenzo et al., 1990), which carries a tetracycline resistance gene. The plasmid was introduced by bi- and tri-parental conjugation employing a helper plasmid pRK2013, if necessary, so that antibiotic resistance can occur in the transconjugants if transposition occurs. Alternatively, electroporation was used to introduce bioluminescence lux genes into strain VUN10,077 when cells were harvested at early-log and stationary phase. One of the engineered water-based luxAB-marked strain (VUN3,600) was used to detect the presence of toxic substances (determining selectivity and sensitivity). This bacterial luciferase system produces bioluminescence when supplied with an aliphatic aldehyde substrate, where the luxAB genes were expressed on the chromosome. The substrate delivery system and preparation of cells for bioluminescence toxicity test were achieved by diluting ndecyl aldehyde 10 (3)-fold in Milli-Q water and resuspending cells in tap-water after mild centrifugation, when cells were harvested at A600 1.0-1.2. When a variety of common pollutants were tested in the water-based monitoring system, cells of VUN3,600 responded sensitively, rapidly and differentially to Cd, Pb, Hg, Zn, Co ions and SDS, and with less sensitivity to Ni, and As; responses were also dose-related in a short-term assay. DMF, a solvent used to dissolve or extract several organic compounds in environmental testing, stimulated light output but in a controlled fashion. Selected substituted heterocyclic compounds tested and phenol, d- nitrophenol and strontium failed to cause dose-related inhibition, whereas di-, tri-, tetra- and penta-chlorophenol decreased light output in a long-term assay. DMF-dissolved PAH compounds were tested in the long-term assay and naphthalene, phenanthrene, fluorene, fluoranthene and benzo[a]pyrene presented dose-related responses while benz[a] anthracene and di-benzo[a]anthracence were not suitable for use in this type of assay. Although there is need to investigate extensive toxicity assays to gain broad acceptance, the water-based monitoring system showed repeatable and reliable toxicity assessment and has great possibility of usage in aquatic samples in either water-soluble or DMF-soluble forms. This study allowed explanation of the stress responses of strain VUN10,077 to be examined at a variety of levels. Bioluminescence was the most sensitive approach to determining the lowest concentrations of pollutants which affected cell physiology, as this was obvious at concentrations lower than required to cause changes in viability or induce stress responses.

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