Biofilms are complex and highly adapted communities of microorganisms found attached to surfaces. Among the best characterized infectious multi-cellular biofilms is the oral community known as dental plaque. Streptococcus mutans resides in the oral biofilm, and is one of the main causative agents of dental caries. Streptococci are known to monitor their population density using a peptide pheromone (CSP)/two component signalling system (ComDE) in a process classically known as quorum sensing (QS). Previous work in S. mutans has implicated the QS system in genetic competence, the stress response, bacteriocin production and biofilm formation. Our objective in this work was to thoroughly characterize the transcriptional and phenotypic response to CSP in S. mutans, and determine its role in biofilm formation. We have shown that the CSP pheromone is more than simply a QS signal, and is also an inducible ‘alarmone’ capable of communicating stress in the population. We have demonstrated that elevated concentrations of CSP such as those that occur during stress trigger autolysis in a fraction of the population. Importantly, we have shown that autolysis in S. mutans occurs via a novel mechanism of action: intracellular accumulation of a self-acting bacteriocin. We have also identified and characterized the autolysis immunity protein, which is differentially regulated from the bacteriocin to allow survival at low cell density. A second regulatory system was shown to govern expression of autolysis immunity in the absence of CSP signaling, and also contribute to the oxidative stress response in the biofilm. Finally, we present evidence that autolysis is involved in the release of DNA in the biofilm, which contributes to the architecture of the extracellular matrix and may provide a mechanism for the dissemination of fitness-enhancing genes under stress. Together, our data provides a mechanistic link between phenotypes previously ascribed to the CSP pheromone in S. mutans.

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.

Membrane biofilm reactors (MBfR) utilize membrane fibers for bubble-less transfer of gas by diffusion and provide a surface for biofilm development. Nitrogen removal was attempted using MBfR in various configurations - nitrification, denitrification and consecutive nitrification and denitrification. Effects of loading rate and dissolved oxygen on nitrification performance were primarily investigated in a stand-alone nitrifying MBfR. Specific nitrification rate increased linearly with specific loading rate, up to the load of 3.5 g N/m²d. Beyond that load, substrate diffusion limitation inhibited further increase of specific nitrification rate. 100% oxygen utilization was achievable under limited oxygen supply condition. Effects of mineral precipitation, dissolved oxygen and temperature on hydrogenotrophic denitrification were investigated in a stand-alone denitrifying MBfR. Mineral precipitation, caused by intended pH control, caused the deterioration of denitrification performance by inhibiting the diffusion of hydrogen and nitrate. Operating reactor in various dissolved oxygen conditions showed that the denitrification performance was not affected by dissolved oxygen in MBfR. Optimum temperature of the hydrogenotrophic denitrification system was around 28°C. Total nitrogen removal in a two-step MBfR system incorporating sequential nitrification and hydrogen-driven autotrophic denitrification was investigated in order to achieve nitrogen removal by autotrophic bacteria alone. Long-term stable operation, which proved difficult in previous studies due to excessive biofilm accumulation in autotrophic denitrification systems, was attempted by biofilm control. Nitrification performance was very stable throughout the experimental periods over 200 days. Performance of autotrophic denitrification was maintained stably throughout the experimental periods, however biofilm control by nitrogen sparging was required for process stability. Biofilm thickness was also stably maintained at an average of 270 µm by the gas sparging biofilm control. According to the cost analysis of denitrifying MBfR, hydrogenotrophic denitrification can be an economical tertiary treatment option compared to conventional denitrifying filter although its economic feasibility highly depends on the cost of hydrogen gas. Although this study was conducted in a lab-scale, the findings from this study can be a valuable stepping stone for larger scale application and open the door for system modifications in future.

The development of biofilms at solid-liquid interfaces has been investigated extensively, whereas the yield of planktonic cells from biofilms has received comparatively little attention. The detachment of single cells from biofilms has been attributed mainly to the erosive action of flowing liquid or the dispersal of cells from within biofilm microcolonies. The result has been an underestimation of the active role that biofilms can play in microbial proliferation through the production and release of planktonic cells to the environment. In this study, the cultivation of Pseudomonas sp. strain CT07 biofilms in conventional flowcells, glass tubes and a novel CO2 evolution measurement system was utilized to show that biofilm-derived planktonic cell yield was initiated within 6 hours of initial surface colonization and increased in conjunction with biofilm development. The magnitude of the yield was influenced by the metabolic activity of the biofilm, which was in turn dependent on environmental conditions, such as carbon availability. The physiologically active region of the biofilm was responsible for the yield of significant numbers of planktonic cells (~107 CFU.cm-2.h-1), whereas a less active biofilm zone was optimized for survival during unfavourable conditions and shown to be responsible for the subsequent re-establishment of biofilm structure, activity and cell yield. Despite the yield of numerically considerable numbers of planktonic cells (~1010 CFU), a carbon balance revealed that the carbon investment required to maintain this yield was insignificant (~1%) compared to the amount of carbon channelled into CO2 production (~54%). Together, these results indicate that biofilm-derived planktonic cell yield represents an efficient proliferation mechanism and support the view that the biofilm lifestyle affords microbes a dual survival-proliferation strategy, where the dominant strategy depends on the prevailing environmental conditions. An alternative model of biofilm development is presented to account for planktonic cell yield during all stages of biofilm development.

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.

It is well established that bacteria cause pulpal and periradicular disease (Kakehashi et al. 1965). Of the bacteria recovered from failing root canals, Enterococcus faecalis is one of the most prevalent species (Molander et al. 1998; Sundqvist et al. 1998). Many laboratory studies have investigated the effectiveness of root canal irrigants and medicaments against E. faecalis. Most used planktonic cultures, which are not representative of the in vivo growth conditions of an infected root canal system, where bacteria grow as a biofilm adhering to the dentinal wall (Nair 1987). Organisation of bacteria within biofilms confers a range of phenotypic properties that are not evident in their planktonic counterparts, including a markedly reduced susceptibility to antimicrobial killing (Wilson 1996). Objectives: The aims of this study were: 1) To compare the efficacy of commonly used endodontic medicaments against E. faecalis cultured as a biofilm. The medicaments tested were Ledermix paste, calcium hydroxide, Odontopaste, 0.2% chlorhexidine gel and 50:50 combinations of Ledermix/calcium hydroxide and Odontopaste/calcium hydroxide. 2) To compare the antimicrobial effect achieved through exposure to endodontic medicaments with that achieved by exposure to a constant concentration of sodium hypochlorite for varying times. Methods: A biofilm was established using a continuous flow cell. E. faecalis inoculum was introduced into the flow cell and allowed to establish on human dentine slices over 4 weeks. Each test medicament was introduced into the flow cell for a period of 24 or 48 hours, while sodium hypochlorite was evaluated after 1, 10, 30 and 60 minutes. Biofilms were harvested by sonication in sterile PBS. Cellular protein levels were measured to quantitate the amount of biofilm harvested. Cellular viability was determined using serial plating. The number of colony forming units was then adjusted for cellular protein levels to allow treatment protocols to be compared. Qualitative SEM analyses of the biofilm was performed following exposure to each test agent. Results: Sodium hypochlorite was the only agent that achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis biofilm, while calcium hydroxide and 50:50 combinations of calcium hydroxide with either Ledermix or Odontopaste were able to reduce viability by > 99%. Conclusion: When used in isolation, antibiotic containing medicaments had no appreciable effect on the viability of Enterococcus faecalis. Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2009

It is well established that bacteria cause pulpal and periradicular disease (Kakehashi et al. 1965). Of the bacteria recovered from failing root canals, Enterococcus faecalis is one of the most prevalent species (Molander et al. 1998; Sundqvist et al. 1998). Many laboratory studies have investigated the effectiveness of root canal irrigants and medicaments against E. faecalis. Most used planktonic cultures, which are not representative of the in vivo growth conditions of an infected root canal system, where bacteria grow as a biofilm adhering to the dentinal wall (Nair 1987). Organisation of bacteria within biofilms confers a range of phenotypic properties that are not evident in their planktonic counterparts, including a markedly reduced susceptibility to antimicrobial killing (Wilson 1996). Objectives: The aims of this study were: 1) To compare the efficacy of commonly used endodontic medicaments against E. faecalis cultured as a biofilm. The medicaments tested were Ledermix paste, calcium hydroxide, Odontopaste, 0.2% chlorhexidine gel and 50:50 combinations of Ledermix/calcium hydroxide and Odontopaste/calcium hydroxide. 2) To compare the antimicrobial effect achieved through exposure to endodontic medicaments with that achieved by exposure to a constant concentration of sodium hypochlorite for varying times. Methods: A biofilm was established using a continuous flow cell. E. faecalis inoculum was introduced into the flow cell and allowed to establish on human dentine slices over 4 weeks. Each test medicament was introduced into the flow cell for a period of 24 or 48 hours, while sodium hypochlorite was evaluated after 1, 10, 30 and 60 minutes. Biofilms were harvested by sonication in sterile PBS. Cellular protein levels were measured to quantitate the amount of biofilm harvested. Cellular viability was determined using serial plating. The number of colony forming units was then adjusted for cellular protein levels to allow treatment protocols to be compared. Qualitative SEM analyses of the biofilm was performed following exposure to each test agent. Results: Sodium hypochlorite was the only agent that achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis biofilm, while calcium hydroxide and 50:50 combinations of calcium hydroxide with either Ledermix or Odontopaste were able to reduce viability by > 99%. Conclusion: When used in isolation, antibiotic containing medicaments had no appreciable effect on the viability of Enterococcus faecalis. Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2009

Zugl.: Braunschweig, Techn. Univ., Diss., 2005