A study on the diversity and production of microbial extracellular nucleases :potential anti-biofilm enzymes

PhD ThesisMicroorganisms have been viewed as planktonic, free living single cells but predominantly they exist as sessile multispecies communities in the natural environment forming ‘biofilms’. Biofilms are beneficial for organisms to survive in natural environment as well as for biotechnological applications such as microbial fuel cells and bioremediation. However, biofilms are associated with disease persistence and biofouling and are comprised of adhered microbes within a hydrated matrix rich in polysaccharides, proteins and extracellular DNA (eDNA). eDNA is an important structural component and its degradation by deoxyribonucleases may be a novel approach to eradicate biofilm related problems. The present work was undertaken in this context to discover and produce microbial nucleolytic enzymes for applications for the control of harmful biofilms. Eighty six out of 260 bacterial isolates which included thermophilic and psychrophilic strains, showed deoxyribonuclease activity. The diversity and function of extracellular nucleases was also investigated throughout the microbial world using bioinformatics tools. Sequence driven analysis suggested that major bacterial lineages contain diverse extracellular nucleases with biological function related to nitrogen, phosphate and carbohydrate metabolism, protection, survival and virulence. Production optimisation for one specific extracellular nuclease, NucB, from Bacillus licheniformis EI-34-6 was carried out. This enzyme was previously known to cleave eDNA causing biofilm dispersal, and may therefore be used commercially to remove biofilms. The understanding of B. licheniformis physiology was applied in order to enhance NucB production 10-fold. For further characterisation of the enzyme and to Abstract iv understand its biological mechanism in breaking down biofilms, NucB was expressed in the SURE expression host B. subtilis NZ8900. This allowed a 68-fold increase in protein yield. NucB protein has been purified to high degree purity with specific nuclease activity of 15000 U/mg of protein. Biophysical characterisation showed that the protein was thermally stable and could reversibly refold. Statistical optimisation of extracellular nucleases production in diverse bacteria grown at different temperatures was demonstrated as a promising methodology for enhancing key enzyme secretion. The effectiveness of biofilm disruption by NucB was successfully tested with different single species biofilms grown on polystyrene, glass, and stainless steel surfaces. Biofilm dispersal efficiency of other microbial nucleases ranged between 60 – 95 % of removal after 1 h. The results presented in this thesis demonstrate that bacteria were able to produce nucleases across broad temperature range. In context to biofilm dispersal, bioinformatic analysis speculates the ecological implication of secreted diverse microbial extracellular nuclease-like genes were to decide the fate of eDNA and play pivotal role in nutrient cycling of the eco-system. Bioprocess development confirmed process optimisation can reliably produce functional and well-folded recombinant NucB at levels suitable for applications where biofilm removal is needed. Production optimisation of extracellular nucleases from diverse bacteria expanded the availability of different nucleases with wide range of anti-biofilm properties. Evidence is also presented to show that extracellular nucleases can disperse preformed microbial biofilms on different substrata. Microbial extracellular nucleases therefore appear to be a rich and unexplored source of anti-biofilm enzymes

Secretion of DNases by Marine Bacteria: A Culture Based and Bioinformatics Approach

The vast majority of bacteria present in the natural environment are present in the form of aggregates and/or biofilms. Microbial aggregates are ubiquitous in the marine environment and are inhabited by diverse microbial communities which often express intense extracellular enzymatic activities. However, the secretion of an important group of enzymes, DNases, by bacteria from marine aggregates has not been studied, despite the importance of these aggregates in biogeochemical cycling of nutrients in the oceans. In this work, we therefore, employed both culture-based and bioinformatics approaches to understand the diversity of bacterial DNases in marine bacterioplankton. We found that 34% of 345 strains of attached and non-attached marine bacteria showed extracellular DNase activity. Most of these isolates belong to Proteobacteria (53%) and Firmicutes (34%). Secretion of DNases by bacteria isolated from marine gel particles (MGP) is reported here for the first time. Then, to further understand the wider diversity of the potential to produce DNases, sequences were compared using 2316 whole genome and 42 metagenome datasets. Thirty-nine different taxonomic groups corresponding to 10 bacterial phyla were found to encode genes responsible for DNase secretion. This study highlights the unexpected and widespread presence of DNase secretion in bacteria in general and in MGP more specifically. This has important implications for understanding the dynamics and fate of marine microbial aggregates in the oceans

Carbohydrate-degrading bacteria closely associated with Tetraselmis indica: influence on algal growth

In the present study, we examined the interactions between the algal species Tetraselmis indica and strains of bacteria with which it is closely associated. Three bacterial strains were isolated and sequence analysis of the 16S rDNA indicated that the organisms belong to the genera Pseudomonas, Acinetobacter and Ruegeria. Morphologies of the bacterial strains were studied using epifluorescence microscopy and scanning electron microscopy. Reassociation experiments were conducted with axenic cultures inoculated with the 3 bacterial strains in concentrations comparable to natural conditions, and the effect of each bacterial population on the growth of T. indica was determined. T. indica exhibited differential growth with the various bacterial cultures, and in particular Acinetobacter sp. was observed to promote growth of the algae. These experiments revealed that microbes associated with the alga differentially influence algal growth dynamics. Bacterial presence on the cast-off cell wall products of the alga suggested the likely utilisation of algal cell wall by bacteria. The bacterial strains were tested for carbohydrate metabolism using various sugars and screened for carbohydrase activity. Bacterial strains were found to produce carbohydrases for degradation of polysaccharides generally present in the cell wall of Tetraselmis (glucans, galactans, galactomannans and pectins), whereas no such utilisation was observed for other wall substrates (such as cellulose, arabinoxylan, rhamnogalacturonan). Pseudomonas sp. and Acinetobacter sp. showed carbohydrase activity with glucans, galactans, galactomannans and pectin, whereas Ruegeria sp. showed much less carbohydrase activity and only with pectin. The carbohydrate utilisation studies using artificial substrates suggested the potential utilisation of cast-off algal cell wall products