A biofilm is a complex aggregate of microorganisms that embed din extracellular
polymeric substances (EPS) and can develop on almost all the surface. Biofilm studies
using model species in laboratory have been extensively performed. However, biofilm
communities in natural environments are much complex and the molecular mechanisms
governing these biofilm development remain largely unexplored. Herein we applied
artificial surface-based biofilm as an ideal model to study biofilm development in
marine environments. The integration of ecogenomic techniques, including
metagenome, metatranscriptome, metaproteome and single genome binning, facilitates
understanding of mechanisms governing biofilm development in intertidal zone and
deep-sea cold seep system.
In the first part of the th...[
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A biofilm is a complex aggregate of microorganisms that embed din extracellular
polymeric substances (EPS) and can develop on almost all the surface. Biofilm studies
using model species in laboratory have been extensively performed. However, biofilm
communities in natural environments are much complex and the molecular mechanisms
governing these biofilm development remain largely unexplored. Herein we applied
artificial surface-based biofilm as an ideal model to study biofilm development in
marine environments. The integration of ecogenomic techniques, including
metagenome, metatranscriptome, metaproteome and single genome binning, facilitates
understanding of mechanisms governing biofilm development in intertidal zone and
deep-sea cold seep system.
In the first part of the thesis, biofilms were developed on artificial surfaces deployed in
intertidal and subtidal zones near Hong Kong Water. Using comparative metagenomics,
mechanisms governing biofilm development in the intertidal zone were illuminated.
The genes responsible for resistance to metal ion and oxidative stresses were enriched
in both 6-day and 12-day intertidal biofilms, compared to the subtidal biofilms. We
proposed that a complex signaling network was used for stress tolerance and a model
illustrating the relationships between these functions and environmental metal ion
concentrations and oxidative stresses. This was the first successful use of metagenomic
analysis on artificial surface-based biofilm model.
The second part of the thesis work focuses on biofild development under extreme
environment. In the Thuwal cold seeps II, which comprises a brine pool and the
adjacent normal bottom water (NBW). Biofilms were developed on the surface of
different materials, iucluding aluminium (Al), polyether ether ketone (PEEK),
polyvinyl chloride (PVC), polytetrafluoroethene (PTFE), stainless steel (SS) and
titanium (Ti). The comparative metagenomic results demonstrated that biofilm
developed on different surfaces used similar mechanisms. We presented different
mechanisms of biofilm development from previous biofilm models. The role of
polysaccharides in biofilm development was more complex and fundamental than
previously reported (as a component of the matrix). The replacement of oxygen by
nitrate as an electron acceptor was an important process in brine biofilm development.
Remarkably, exploiting and organizing niche-specific functional features through inter-species
interaction and generation of special micro-environments would be important
mechanisms of biofilm-dependent adaptation.
We also used the artificial surface-based biofilm in the cold seep system as a model to
test ecological sheories, such as species sorting, that is, the filtering by local
environmental conditions is important in assembly of bacterial communities. We
designed an experiment to investigate the effects of environmental switching between
the brine pool and the NBW on biofilm assembly, which could reflect environmental
filtering effects during bacterial immigration to new environments. Analyses of 16S
rRNA genes of 71 biofilm samples suggested that the microbial composition of biofilms
established in new environments was determined by both the source community and
the incubation conditions. The results shed new light on microbial community assembly
in special habitats and bridges the gaps in species sorting theory.
As a direct extension of the cold seep biofilm work, we performed a large scale genome
binning work to study the environment-microbe interaction using cold seep biofilms as
a model. Genome analyses of 66 biofilm members spanning seven phyla exemplified
by Proteobacteria, Marinimicrobia and Cloacimonetes reveal genes coding for
fermentative metabolism of recalcitrant carbohydrates and energy generation.
Polysaccharide metabolism is further evidenced by metatranscriptomic reads mapping
and in vitro characterization of enzymes catalyzing deconstruction of both α- and β-D-glucosides.
The results suggested that polysaccharides affect both genomic content and
post-genomic activity; simultaneously expression of diverse fermentative respiratory-related
elements, together with enzymatic flexibility, underwrite microbial
polysaccharide utilization.
To complement the naural biofilm study, we also performed study on the model single
species, Pseudomonas aeruginosa biofilm. We applied iTRAQ-based quantitative
proteomics to quantitify matrix-associated proteins isolated from different phases of
Pseudomonas aeruginosa ATCC27853 biofilms. The increased abundance of stress
resistance and nutrient metabolism-related proteins during biofilm development was
consistent with the hypothesis that biofilm matrix forms micro-environments in which
cells are optimally organized to resist stress and use available nutrients. Further analysis
revealed complex interactions among these modulated proteins, and the mutation of
selected proteins attenuated biofilm development. Interestingly, there was a good
correlation between the abundance changes of matrix-associated proteins and their
expression. Collectively, this work presents the first dynamic picture of matrix-associated
proteins during biofilm development, and provides evidences that the
matrix-associated proteins may form an integral and well regulated system that
contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of
biofilm.
In summary, the main contribution of thesis work lies in establishment of a biofilm
model to address ecologigical questions from multiply aspects. It integrates omics,
molecular and biochemical methods to study biofilms. It contributes to understanding
of classical ecological theories, microbial roles in biogeochemical cycles, microbial
adaptation straties to extrem envrionments, as well as controlling strategy of biofilm
development in the biofouling and bioinfection frameworks.
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