 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°C
to 37°C 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[á]pyrene presented dose-related responses while benz[á] anthracene
and di-benzo[á,h]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. |
