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    Investigating the molecular basis of histidine catabolism in a human pathogenic bacterium Pseudomonas aeruginosa PAO1 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand
    (Massey University, 2021) Sreeja Jayan, Kiran
    Pseudomonas aeruginosa is an opportunistic and a nosocomial pathogen of significant medical concern, particularly for cystic fibrosis patients. The extensive metabolic flexibility coupled with an array of virulence factors make them ubiquitous and successful in causing persistent multi-drug resistant infections. Pathogens exploit nutrient-rich hosts, and thus nutrients can be considered as signals perceived by bacteria that allow host recognition and coordination of expression of metabolic and virulence genes for successful colonization. A deeper understanding of the metabolic pathways and host perception mechanisms are significant from a therapeutic perspective in the current era of antibiotic resistance. Histidine is an amino acid that can serve as a source of carbon and nitrogen to many bacteria. Histidine catabolism in Pseudomonas spp., is widely known to occur via a 5-step enzymatic pathway, and the genes for histidine utilization (hut) are negatively regulated by HutC protein. The enteric bacteria and some others utilize a 4-step enzymatic pathway for histidine catabolism, which differs from the 5-step pathway in the direct conversion of intermediate formiminoglutamate (FIGLU) to glutamate. However, P. aeruginosa contains an additional operon (dislocated from the hut locus) encoding for formimidoylglutamase enzyme and its regulator, which can break down FIGLU similar to 4-step pathway. Previous studies report the accumulation of histidine metabolites, urocanate and FIGLU, in the mammalian tissues and reduced virulence of P. aeruginosa defective in histidine catabolism towards animal models. But the implications of the presence of two pathways for histidine catabolism or mechanisms associated with virulence remain elusive. This prompted us to examine the hut pathways and mechanisms that link hut with virulence in P. aeruginosa PAO1. First, computational analysis identified a transporter gene (named figT) adjacent to formimidoylglutamase enzyme (FigA) and transcriptional regulator FigR. This led to a new hypothesis that the three genes (figRAT) are responsible for the uptake and utilization of FIGLU, and they are not involved in histidine utilization as previously thought. Genetic analyses utilizing site-directed mutagenesis and lacZ reporter fusions confirmed that figT encodes for a FIGLU-specific transporter whose expression is induced by FIGLU. The figT gene is co-transcribed with figRA, and its expression is activated by FigR. Furthermore, gene expression studies indicate that FIGLU is the physiological inducer of fig operon, while histidine and urocanate are indirect inducers (by virtue of conversion to FIGLU). Growth and fitness assays revealed that histidine is predominantly catabolised via the 5-step hut pathway (not via the FigRAT system). Together, our genetic and phenotypic data show that fig operon is involved in the direct utilization of FIGLU from the environment. Phylogenetic analysis showed that figRAT genes are highly conserved and present in all completely sequenced strains of P. aeruginosa, but we found no evidence for horizontal gene transfer events. Previous work in Zhang’s laboratory suggests that urocanate derived from host tissues could serve as a signalling molecule, eliciting P. aeruginosa infections via interaction with the HutC regulator. Here, we aimed to seek further genetic, biochemical, and phenotypic evidence to improve our understanding of the global regulatory roles for HutC beyond histidine catabolism and determine their potential contribution to the colonization of eukaryotic hosts. Utilizing in silico analysis, we predicted 172 novel HutC-target sites in the genome of P. aeruginosa PAO1 with a P value less than 10-4. Six selected candidates were subject to experimental verification for HutC binding by means of gel shift assays (EMSA) and/or DNAse I footprinting assays, and all were able to bind with purified HutChis6 proteins. Further, a hutC deletion mutant was constructed by site-directed mutagenesis and subjected to phenotypic characterization. Phenotypic analyses revealed that hutC is involved in biofilm formation, tobramycin-induced biofilm formation, cell motility, and pyoverdine production. Significantly, we found that mutation of hutC resulted in reduced killing of C. elegans by P. aeruginosa PAO1. Finally, we observed distinct binding patterns for HutC interaction with the hutF promoter DNAs in P. aeruginosa PAO1 and P. fluorescens SBW25 (a model plant-colonizing bacterium used for studies of histidine catabolism). Molecular investigations revealed that the differences were not caused by HutC proteins from either species, but HutC recognized a distinct site proximal to hutFSBW25. This site displayed sequence similarity with the NtrC-binding site and was called the Pntr site. Functional analysis of the significance of Pntr site identified that Pntr site is necessary for the wild-type level production of HutF in P. fluorescens SBW25 during growth on histidine. Overall, the results from this study enhance our understanding of hut catabolism in Pseudomonas and contribute to novel molecular mechanisms associated with the virulence of P. aeruginosa PAO1. The identification of fig operon for the utilization of FIGLU (accumulated in host tissues) and global regulatory role of HutC in gene expression have broader implications from a therapeutic perspective in treating P. aeruginosa PAO1 infections. The ability of HutC to recognise multiple distinct DNA-binding sites suggests complex modes of gene regulation mediated by HutC and promotes further studies to fully understand the functional significance of genes in the HutC regulon.
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    Investigation of the biosynthesis of exopolysaccharides within the biofilm matrix of Pseudomonas aeruginosa and Pseudomonas syringae pv. actinidiea : a thesis presented in partial fulfilment of the requirements for degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2017) Ghods, Shirin
    Polysaccharides are highly abundant natural biopolymers, which have biologically significant structural functions in living organisms. Various polysaccharides, with specific physicochemical properties, contribute to biofilm formation; defined as cell aggregations surrounded by extracellular polymeric substances. They are also important in the context of bacterial pathogenesis, while some have been harnessed for industrial and biomedical applications due to their unique chemical compositions and properties. In present study, we aimed at studying biofilm formation by Pseudomonas aeruginosa and P. syringae pv. actinidiae, respectively known as human and plant pathogens. In this context we focused on the production of exopolysaccharides, which predominantly constitute the biofilm matrix of these pathogenic bacteria. Here, we uncovered that the polysaccharide isolated from P. syringae pv. actinidiae biofilm mainly consists of rhamnose, fucose and glucose and it was cautiously introduced as a novel polysaccharide. In the context of disease control, and developing a management program, we provided some evidences for the effectiveness of chlorine dioxide and kasugamycin in the control of the bacteria living in both biofilm and planktonic modes. Furthermore, we investigated alginate biosynthesis as major polysaccharide contributing to mucoid biofilm formation by P. aeruginosa. We generated various mutants producing a variety of alginates with different chemical compositions. Also, this enabled us to analyse functional relationships of protein subunits involved in multiple steps of alginate biosynthesis including alginate polymerization, modification and secretion. We present evidence that while alginate unravelled that while alginate is polymerised and translocated across the membrane by a multiprotein complex, acetylation and epimerisation events positively and negatively correlated with the polymerization of the alginate or molecular mass, respectively. Analysis of the biofilms showed that biofilm architecture and cell-to-cell interactions were differently impacted by various compositions of the alginates. Also, this study provided insights into the c-di-GMP mediated activation of alginate polymerization upon binding to c-di-GMP as well as assigning functional roles to Alg8 and Alg44 including their subcellular localization and distribution. Here, we also used current knowledge of the alginate biosynthesis pathway to assess the production of alginate from biotechnologically accepted heterologous hosts including Escherichia coli and Bacillus megaterium strains. Primarily, we evaluated the production and functionality of the minimal protein requirements in nonpathogenic heterologous hosts, required for producing alginate precursor, and proceeding into polymerization and secretion steps. Overall, we concluded that polysaccharides play a major role in the formation of bacterial biofilms while chemical composition is a key determinant for biofilm architecture and development. This contribution to understanding the biosynthesis of bacterial polysaccharides and their properties could provide the necessary knowledge not only for developing novel therapeutics, but also for harnessing such biopolymers for various industrial applications and production via biotechnological procedures.
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    Understanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Wang, Yajie
    Alginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates.
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    Molecular mechanism of alginate polymerisation and modifications in Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements for degree of Doctor of Philosophy in Microbiology at Massey University, New Zealand
    (Massey University, 2015) Moradali, M. Fata
    Pseudomonas aeruginosa is an ubiquitous opportunistic human pathogen in immunocompromised patients. It is of particular relevance to cystic fibrosis (CF) patients where it frequently causes chronic bronchopulmonary infection and is the leading cause of morbidity and mortality. The decline in lung function is caused by the emergence of a mucoid variant showing excessive production of the exopolysaccharide, alginate. The alginate-containing biofilm matrix of this mucoid variant protects P. aeruginosa from the immune system and antibiotics. Here the alginate biosynthesis/modification/secretion multiprotein complex was investigated with regard to protein-protein interactions constituting the proposed multiprotein complex and the molecular mechanisms underlying alginate polymerisation and modifications. This study sheds light on the structure and function of various alginates from a material property and biological function perspectives. The binary interactions of AlgK-AlgE, AlgX-Alg44, AlgK-Alg44 and Alg8-Alg44 were identified proposing a new model for this multiprotein complex organisation. Proteinprotein interactions were found to be independent of c-di-GMP binding to PilZAlg44 domain. C-di-GMP-mediated activation of alginate polymerisation was found to be different from activation mechanism proposed for cellulose synthesis. This study showed that alginate polymerisation and modifications were linked. It was shown that the molecular mass of alginate was reduced by epimerisation, while it was increased by acetylation. It was determined that previously characterized proteins AlgG (epimerase) and AlgX (acetyltransferase) have mutual auxiliary and enhancing roles. Biofilm architecture analysis showed that acetyl groups lowered viscoelasticity of alginates and promoted cell aggregation, while nonacetylated polymannuronate alginate promoted stigmergy. Experimental evidence was provided that Alg44 boosted acetylation while the periplasmic domain of this protein was critical for protein stability and regulation of alginate modifications. Full-length Alg44 was purified and it was found to be a dimer in solution. Overall, this study sheds new light on the arrangement of the proposed alginate biosynthesis/modification/secretion multiprotein complex. Furthermore, the activation mechanism and the interplay between polymerisation and modification of alginate were elucidated and new functions and interactive role of alginate-polymerising and– modifying subunits were further understood.
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    Spatial and temporal localisation of exopolysaccharide gene expression in mucoid and non-mucoid Pseudomonas aeruginosa biofilms : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology, Massey University, Manawatu, New Zealand
    (Massey University, 2014) Holbrook, Stacey Ann Kashel
    The biofilm, or surface-associated microbial community, is the preferred method of growth for most bacteria. Pseudomonas aeruginosa is an ubiquitous, opportunistic pathogen capable of biofilm formation in a wide range of natural and clinical environments. In particular, biofilms formed by P. aeruginosa in the lungs of people with cystic fibrosis (CF) are responsible for a significant decline in the health and prognosis of these patients. Once established, P. aeruginosa biofilms begin to excrete an exopolysaccharide (EPS) called alginate which protects the bacterial microcolonies from antimicrobial molecules and confers a mucoid phenotype. Once this phenotypic switch has occurred, the biofilm becomes impossible to eradicate and ultimately leads to the death of the patient. Here, fluorescent signalling systems and confocal laser scanning microscopy (CLSM) have been used to spatially and temporally resolve the expression of three EPSs produced by P. aeruginosa; the pellicle-forming EPS (Pel), the EPS encoded by the polysaccharide synthesis locus (Psl) and alginate. In order to observe the effect (if any) of EPS production on spatial localisation of the cells within the biofilm, the biofilm-associated characteristics of three P. aeruginosa double-knockout mutants, each able to produce only one EPS has been observed. In analysing these biofilm structures, it was found that Pel has a role in facilitating an increased surface area of the biofilm, while Psl-producing mutants form a biofilm structure with a significantly increased biomass. By visualising fluorescent signals throughout a biofilm consisting of a mixture of the three mutants, the spatial localisation of EPS-producing bacterial populations has been observed. Here, Pel-producing mutants tended to aggregate at the attachment surface, suggesting a role in adhesion of the biofilm structure. Spatial and temporal localisation of EPS promoter activity was achieved by transforming the prototypic P. aeruginosa PAO1 strain with one of three plasmids encoding unstable gfp expression under the control of each EPS’s promoter sequence. Overall, this study has demonstrated the applications and limitations of fluorescence-based localisation of bacterial gene expression throughout P. aeruginosa biofilm development. Collectively, this information can help to guide future investigations into the expression and regulation of the genes associated with a biofilm phenotype, with the aim of identifying a target for effective therapy against this important pathogen.
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    The role of extracellular polymeric substances in Pseudomonas aeruginosa biofilm architecture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Ghafoor, Aamir
    Pseudomonas aeruginosa is an opportunistic pathogen. It causes chronic lung infections in the cystic fibrosis patients. These infections become highly resistant to antibacterial treatments. Bacteria develop this resistance because they become protected inside biofilms. Biofilms are microbial communities enmeshed in a partially self-produced and partially recruited, impregnable extracellular matrix. The matrix is composed of extracellular DNA, proteins, lipids and exopolysaccharides. The exopolysaccharides play an imperative role in architecture of the biofilm matrix. P. aeruginosa produces three distinct exopolysaccharides; Psl, Pel and alginate. In this study, non-mucoid strain PAO1 and mucoid (producing excessive alginate) strain PDO300 of P. aeruginosa were used to generate mutants deficient in one or more exopolysaccharides. Role of these three exopolysaccharides in biofilm formation was investigated. Results showed that the absence of alginate altered the architecture of biofilms in PDO300 as well as in PAO1, when compared to biofilms formed by the respective parent strains. Psl was found indispensable for mushroom-like shape of the biofilms in both strains. Pel was required for the compactness of the biofilms, but PAO1 formed mushroom-like structures even in the absence of Pel. However, Pel-deficient PDO300 did not form mature biofilm, suggesting differential role of Pel in the two strains. Psl-only as well as Pel-only, producing mutants were able to formed multilayer biofilm. Production of one type of exopolysaccharide appeared to influence production of the other types of exopolysaccharide. Psl-deficient mutants increased the production of Pel, while Pel-deficient mutants showed a ten-fold increase in the production of alginate. Furthermore, absence of negatively charged alginate in the biofilm was compensated by eDNA. Regulation of exopolysaccharide biosynthesis operons showed a high expression of psl operon in PAO1, whereas its expression in PDO300 was surprisingly low and confined to a few cells near the base. A high and uniform expression of the algD operon in PDO300 was observed at all times during biofilm development. A low expression of algD operon was also detected in PAO1. Expression of the pel operon was confined to the stalk of PDO300 and PAO1. The role of PelF, the only glycosyltransferase encoded by pel operon, in Pel biosynthesis was investigated and found to be a soluble glycosyltransferase which uses UDP-glucose towards Pel biosynthesis. Site directed mutagenesis revealed that conserved R-325 and K-330 were essential for the PelF activity
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    The role of AlgK in alginate biosynthesis by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Wang, Yajie
    Alginate is a polysaccharide produced by brown seaweeds and two bacterial genera Azotobacter and Pseudomonas. While seaweed alginate finds numerous industrial and medical applications, alginate produced by Azotobacter and Pseudomonas spp., is important for cyst and biofim formation, respectively. A member of Pseudomonas, Pseudomonas aeruginosa, is the leading cause of death in Cystic Fibrosis (CF) patients. This pathogen over-produces alginate upon infection of the CF lung, protecting it from host immune responses and antibiotics while clogging up the patients’ airways leading to poor prognosis. Alginate biosynthesis occurs in four stages: (1) precursor synthesis in the cytoplasm (AlgA, D and C), (2) polymerisation at the inner membrane (Alg8 and Alg44), (3) periplasmic translocation and modification (AlgK, X, L, G, I, J and F), and (4) secretion (AlgE) across the outer membrane. The latter three stages are facilitated by a putative multi-protein complex spanning the entire envelope fraction. Currently, it is unknown how this complex is assembled and the roles certain components of the complex play in alginate biosynthesis are not clear. The periplasmic protein AlgK is a key component of this complex. This protein has multiple protein-protein interaction domains, suggesting that it could be critical for assembling functional alginate biosynthesis machinery. In the present study, an algK mutant was generated and used to determine the impact of AlgK’s absence on (i) alginate yield and size, and (ii) the stability of other components of the alginate biosynthesis machinery. This study demonstrates that AlgK is essential for polymerisation and is required for the stability of components involved in polymerisation (Alg44), translocation (AlgX), and secretion (AlgE). We also show that AlgK interacts with periplasmic AlgX but not with inner membrane Alg44 or outer membrane AlgE. Overall, this study sheds light on the role of AlgK in alginate production and the assembly of the alginate biosynthesis machinery.
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    Molecular mechanism of export of alginate in Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements for [the] degree of Doctor of Philosophy in Microbiology at Massey University, New Zealand
    (Massey University, 2013) Rehman, Zahid ur
    Pseudomonas aeruginosa is an opportunistic pathogen; infecting insects, plants and humans. It is of particular relevance to cystic fibrosis (CF) patients where it causes pulmonary infection and the leading cause of morbidity and mortality. The CF lung environment selects for a variant of P. aeruginosa characterised by the overproduction of an exopolysaccharide called alginate. It has been hypothesized that outer membrane protein AlgE forms a channel through which alginate is secreted into the extracellular environment. Furthermore, studies have suggested that proteins involved in the polymerisation, modification and export of alginate form a multiprotein complex that span the bacterial envelope. The aim of this thesis was to investigate the role of AlgE in polymerisation and secretion of alginate. For this purpose algE knockout mutant was created in PDO300. Results showed that AlgE does not have a role in alginate polymerisation however it has a role in secretion of alginate and stability of the alginate biosynthesis machinery. By performing FLAG epitope insertion mutagenesis the topology of AlgE was verified and site-directed mutagenesis further showed that the positive electrostatic field inside the AlgE lumen is required for efficient secretion of negatively charged alginate. By employing mutual stability analysis, evidence was provided for the existence of trans-envelope multiprotein complex required for alginate biosynthesis. Co-immuniprecipitaion assay suggest that AlgE interacts with periplasmic located AlgK and, most probably, this interaction is mediated by the peripasmic turn 4 of AlgE. Pull-down assays further showed that AlgK interacts with another periplasmic protein AlgX which in turn interacts with the inner membrane protein Alg44. Based on mutual stability analysis it was proposed that Alg44 interacts with Alg8 which might interacts with AlgG as well. Our results also support the existence of internal promoters for AlgE and AlgG.
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    Polymerisation and export of alginate in Pseudomanas aeruginosa : functional assignment and catalytic mechanism of Alg8/44 : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Microbiology
    (Massey University, 2007) Remminghorst, Uwe
    Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.