Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

Characterising novel anti-biofilm targets for the treatment of chronic Pseudomonas aeruginosa infection in the cystic fibrosis lung

The global rise in antibiotic resistance is a significant problem facing healthcare professionals. In particular within the cystic fibrosis (CF) lung, bacteria can establish chronic infection and resistance to a wide array of antibiotic therapies. One of the principle pathogens associated with chronic infection in the CF lung is Pseudomonas aeruginosa. P. aeruginosa can establish chronic infection in the CF lung partly through the use of the biofilm mode of growth. This biofilm mode of growth offers a considerable degree of protection from a wide variety of challenges such as the host immune system or antibiotic therapy. The threat posed by the emergence of chronic pathogens is prompting the development of next generation antimicrobials. The biofilm mode of growth is often central to the establishment of chronic infection and the development of antibiotic resistance. Thus, targeting biofilm formation has emerged as one of the principle strategies for the development of next generation antimicrobials. In this thesis two separate approaches were used to identify potential anti - biofilm targets. The first strategy focused on the identification of novel genes with a role in a biofilm formation. High throughput screening identified almost 300 genes which had a role in biofilm formation. A number of these genes were characterised at a phenotypic and a molecular level. The second strategy focused on the identification of compounds capable of inhibiting biofilm formation. A collection of marine sponge isolated bacteria were screened for the ability to inhibit the central pathway regulating biofilm formation, quorum sensing. A number of distinct isolates were identified that had quorum sensing inhibition activity from which, a Pseudomonas isolate was selected for further characterisation. A specific compound capable of inhibiting quorum sensing was identified using chemical analytical technologies in the supernatant of this marine isolate.

An analysis of selected secretion systems of Pseudomonas species

Direct secretion systems which deliver molecules from one cell to another have huge significance in shaping bacterial communities or in determining the outcome of bacterial associations with eukaryotic organisms. This work examines the roles of the Type III Secretion System (T3SS) and the Type VI Secretion System (T6SS) systems of Pseudomonas, a widespread genus including clinical pathogens and biocontrol strains. Bioinformatic analysis of T6SS phylogeny and associated gene content within Pseudomonas identified several T6SS phylogenetic groups, and linked T6SS components VgrG and Hcp encoded outside of T6SS gene loci with their cognate T6SS phylogenetic groups. Remarkably, such “orphan” vgrG and hcp genes were found to occur in diverse, horizontally transferred, operons often containing putative T6SS accessory components and effectors. The prevalence of a widespread superfamily of T6SS lipase effectors (Tle) was assessed in metagenomes from various environments. The abundance of the Tle superfamily and individual families varied between niches, suggesting there is niche specific selection and specialisation of Tle. Experimental work also discovered that P. fluorescens F113 uses the SPI-1 T3SS to avoid amoeboid grazing in mixed populations. This finding may represent a significant aspect of F113 rhizocompetence, and the rhizocompetence of other Rhizobacteria.

Interaction between Candida albicans and Pseudomonas aeruginosa

Fungal pathogen Candida albicans causes serious nosocomial infections in patients, in part, due to formation of drug-resistant biofilms. Protein kinases (PK) and transcription factors (TF) mediate signal transduction and transcription of proteins involved in biofilm development. To discover biofilm-related PKs, a collection of 63 C. albicans PK mutants was screened twice independently with microtiter plate-based biofilm assay (XTT). Thirty-eight (60%) mutants showed different degrees of biofilm impairment with the poor biofilm formers additionally possessing filamentation defects. Most of these genes were already known to encode proteins associated with Candida morphology and biofilms but VPS15, PKH3, PGA43, IME2 and CEX1, were firstly associated with both processes in this study. Previous studies of Holcombe et al. (2010) had shown that bacterial pathogen, Pseudomonas aeruginosa can impair C. albicans filamentation and biofilm development. To investigate their interaction, the good biofilm former PK mutants of C. albicans were assessed for their response to P. aeruginosa supernatants derived from two strains, wildtype PAO1 and homoserine lactone (HSL)-free mutant ΔQS, without finding any nonresponsive mutants. This suggested that none of the PKs in this study was implicated in Candida-Pseudomonas signaling. To screen promoter sequences for overrepresented TFs across C. albicans gene sets significantly up/downregulated in presence of bacterial supernatants from Holcombe et al. (2010) study, TFbsST database was created online. The TFbsST database integrates experimentally verified TFs of Candida to analyse promoter sequences for TF binding sites. In silico studies predicted that Efg1p was overrepresented in C. albicans and C. parapsilosis RBT family genes.

CpxR activates MexAB-OprM efflux pump expression and enhances antibiotic resistance in both laboratory and clinical nalB-type isolates of Pseudomonas aeruginosa

Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. In this study, we demonstrate that CpxR, previously identified as a regulator of the cell envelope stress response in Escherichia coli, is directly involved in activation of expression of RND efflux pump MexAB-OprM in P. aeruginosa. A conserved CpxR binding site was identified upstream of the mexA promoter in all genome-sequenced P. aeruginosa strains. CpxR is required to enhance mexAB-oprM expression and drug resistance, in the absence of repressor MexR, in P. aeruginosa strains PA14. As defective mexR is a genetic trait associated with the clinical emergence of nalB-type multidrug resistance in P. aeruginosa during antibiotic treatment, we investigated the involvement of CpxR in regulating multidrug resistance among resistant isolates generated in the laboratory via antibiotic treatment and collected in clinical settings. CpxR is required to activate expression of mexAB-oprM and enhances drug resistance, in the absence or presence of MexR, in ofloxacin-cefsulodin-resistant isolates generated in the laboratory. Furthermore, CpxR was also important in the mexR-defective clinical isolates. The newly identified regulatory linkage between CpxR and the MexAB-OprM efflux pump highlights the presence of a complex regulatory network modulating multidrug resistance in P. aeruginosa.

Synergistic nisin-polymyxin combinations for the control of Pseudomonas biofilm formation

The emergence and dissemination of multi-drug resistant pathogens is a global concern. Moreover, even greater levels of resistance are conferred on bacteria when in the form of biofilms (i.e., complex, sessile communities of bacteria embedded in an organic polymer matrix). For decades, antimicrobial peptides have been hailed as a potential solution to the paucity of novel antibiotics, either as natural inhibitors that can be used alone or in formulations with synergistically acting antibiotics. Here, we evaluate the potential of the antimicrobial peptide nisin to increase the efficacy of the antibiotics polymyxin and colistin, with a particular focus on their application to prevent biofilm formation of Pseudomonas aeruginosa. The results reveal that the concentrations of polymyxins that are required to effectively inhibit biofilm formation can be dramatically reduced when combined with nisin, thereby enhancing efficacy, and ultimately, restoring sensitivity. Such combination therapy may yield added benefits by virtue of reducing polymyxin toxicity through the administration of significantly lower levels of polymyxin antibiotics.

A non-classical LysR-type transcriptional regulator PA2206 is required for an effective oxidative stress response in Pseudomonas aeruginosa

LysR-type transcriptional regulators (LTTRs) are emerging as key circuit components in regulating microbial stress responses and are implicated in modulating oxidative stress in the human opportunistic pathogen Pseudomonas aeruginosa. The oxidative stress response encapsulates several strategies to overcome the deleterious effects of reactive oxygen species. However, many of the regulatory components and associated molecular mechanisms underpinning this key adaptive response remain to be characterised. Comparative analysis of publically available transcriptomic datasets led to the identification of a novel LTTR, PA2206, whose expression was altered in response to a range of host signals in addition to oxidative stress. PA2206 was found to be required for tolerance to H2O2 in vitro and lethality in vivo in the Zebrafish embryo model of infection. Transcriptomic analysis in the presence of H2O2 showed that PA2206 altered the expression of 58 genes, including a large repertoire of oxidative stress and iron responsive genes, independent of the master regulator of oxidative stress, OxyR. Contrary to the classic mechanism of LysR regulation, PA2206 did not autoregulate its own expression and did not influence expression of adjacent or divergently transcribed genes. The PA2214-15 operon was identified as a direct target of PA2206 with truncated promoter fragments revealing binding to the 5'-ATTGCCTGGGGTTAT-3' LysR box adjacent to the predicted -35 region. PA2206 also interacted with the pvdS promoter suggesting a global dimension to the PA2206 regulon, and suggests PA2206 is an important regulatory component of P. aeruginosa adaptation during oxidative stress.

Capillary electrophoresis with boron doped diamond electrode for amperometric detection of low molecular weight biological substrate

The research work in this thesis included the sensitive and selective separation of biological substance by capillary electrophoresis with a boron doped diamond electrode for amperometric detection. Chapter 1 introduced the capillary electrophoresis and electrochemical detection. It included the different modes of capillary electrophoresis, polyelectrolyte multilayers coating for open tubular capillary electrochromatography, different modes of electrochemical detection and carbon based electrodes. Chapter 2 showed the synthesized and electropolymerized N-acetyltyramine with a negatively charged sulfobutylether-β-cyclodextrin on a boron doped diamond (BDD) electrode followed by the electropolymerzation of pyrrole to form a stable and permselective film for selective dopamine detection. For comparison, a glassy carbon (GC) electrode with a combined electropolymerized permselective film of polytyramine and polypyrrole-1-propionic acid was used for selective detection of dopamine. The detection limit of dopamine was improved from 100 nM at a GC electrode to 5 nM at a BDD electrode. Chapter 3 showed field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles embedded in poly(diallyldimethylammonium) chloride, which has been investigated for the electrophoretic separation of indoxyl sulphate, homovanillic acid and vanillylmandelic acid. The detection limit of the three analytes obtained by using a boron doped diamond electrode was around 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfereing chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. Chapter 4 showed the selective detection of Pseudomonas Quinolone Signal, PQS for quorum sensing from its precursor HHQ, using a simply boron doped diamond electrode. Furthermore, by combining poly(diallyldimethylammonium) chloride modified fused silica capillary with a BDD electrode for amperometric detection, PQS was separated from HHQ and other analogues. The detection limit of PQS was as low as 65 nM. Different P. aeruginosa mutant strains were studied. Chapter 5 showed the separation of aminothiols by layer-by-layer coating of silica capillary with a boron doped diamond electrode. The capillary was layer-by-layer coated with the polycation poly(diallyldimethylammonium) chloride and negatively charged silica nanoparticles. All the aminothiols was separated and detected using a BDD electrode in an acidic electrolyte. It was a novel scheme for the separation and detection of glutathione reduced and oxidized forms, which is important for estimated overstressed level in the human system.

Investigation of polyhydroxyalkanoate production by an activated sludge microbial consortium treating artificial dairy wastewater

Petrochemical plastics/polymers are a common feature of day to day living as they occur in packaging, furniture, mobile phones, computers, construction equipment etc. However, these materials are produced from non-renewable materials and are resistant to microbial degradation in the environment. Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely considered as optimal replacement polymers, are the Polyhydroxyalkaonates (PHAs). Primary research in this area has focused on using recombinant pure cultures to optimise PHA yields, however, despite considerable success, the high costs of pure culture fermentation have thus far hindered the commercial viability of PHAs thus produced. In more recent years work has begun to focus on mixed cultures for the optimisation of PHA production, with waste incorporations offering optimal production cost reductions. The scale of dairy processing in Ireland, and the high organic load wastewaters generated, represent an excellent potential substrate for bioconversion to PHAs in a mixed culture system. The current study sought to investigate the potential for such bioconversion in a laboratory scale biological system and to establish key operational and microbial characteristics of same. Two sequencing batch reactors were set up and operated along the lines of an enhanced biological phosphate removal (EBPR) system, which has PHA accumulation as a key step within repeated rounds of anaerobic/aerobic cycling. Influents to the reactors varied only in the carbon sources provided. Reactor 1 received artificial wastewater with acetate alone, which is known to be readily converted to PHA in the anaerobic step of EBPR. Reactor 2 wastewater influent contained acetate and skim milk to imitate a dairy processing effluent. Chemical monitoring of nutrient remediation within the reactors as continuously applied and EBPR consistent performances observed. Qualitative analysis of the sludge was carried out using fluorescence microscopy with Nile Blue A lipophillic stain and PHA production was confirmed in both reactors. Quantitative analysis via HPLC detection of crotonic acid derivatives revealed the fluorescence to be short chain length Polyhydroxybutyrate, with biomass dry weight accumulations of 11% and 13% being observed in reactors 1 and 2, respectively. Gas Chromatography-Mass Spectrometry for medium chain length methyl ester derivatives revealed the presence of hydroxyoctanoic, -decanoic and -dodecanoic acids in reactor 1. Similar analyses in reactor 2 revealed monomers of 3-hydroxydodecenoic and 3-hydroxytetradecanoic acids. Investigation of the microbial ecology of both reactors as conducted in an attempt to identify key species potentially contributing to reactor performance. Culture dependent investigations indicated that quite different communities were present in both reactors. Reactor 1 isolates demonstrated the following species distributions Pseudomonas (82%), Delftia acidovorans (3%), Acinetobacter sp. (5%) Aminobacter sp., (3%) Bacillus sp. (3%), Thauera sp., (3%) and Cytophaga sp. (3%). Relative species distributions among reactor 2 profiled isolates were more evenly distributed between Pseudoxanthomonas (32%), Thauera sp (24%), Acinetobacter (24%), Citrobacter sp (8%), Lactococcus lactis (5%), Lysinibacillus (5%) and Elizabethkingia (2%). In both reactors Gammaproteobacteria dominated the cultured isolates. Culture independent 16S rRNA gene analyses revealed differing profiles for both reactors. Reactor 1 clone distribution was as follows; Zooglea resiniphila (83%), Zooglea oryzae (2%), Pedobacter composti (5%), Neissericeae sp. (2%) Rhodobacter sp. (2%), Runella defluvii (3%) and Streptococcus sp. (3%). RFLP based species distribution among the reactor 2 clones was as follows; Runella defluvii (50%), Zoogloea oryzae (20%), Flavobacterium sp. (9%), Simplicispira sp. (6%), Uncultured Sphingobacteria sp. (6%), Arcicella (6%) and Leadbetterella bysophila (3%). Betaproteobacteria dominated the 16S rRNA gene clones identified in both reactors. FISH analysis with Nile Blue dual staining resolved these divergent findings, identifying the Betaproteobacteria as dominant PHA accumulators within the reactor sludges, although species/strain specific allocations could not be made. GC analysis of the sludge had indicated the presence of both medium chain length as well short chain length PHAs accumulating in both reactors. In addition the cultured isolates from the reactors had been identified previously as mcl and scl PHA producers, respectively. Characterisations of the PHA monomer profiles of the individual isolates were therefore performed to screen for potential novel scl-mcl PHAs. Nitrogen limitation driven PHA accumulation in E2 minimal media revealed a greater propensity among isoates for mcl-pHA production. HPLC analysis indicated that PHB production was not a major feature of the reactor isolates and this was supported by the low presence of scl phaC1 genes among PCR screened isolates. A high percentage distribution of phaC2 mcl-PHA synthase genes was recorded, with the majority sharing high percentage homology with class II synthases from Pseudomonas sp. The common presence of a phaC2 homologue was not reflected in the production of a common polymer. Considerable variation was noted in both the monomer composition and ratios following GC analysis. While co-polymer production could not be demonstrated, potentially novel synthase substrate specificities were noted which could be exploited further in the future.

The role of bacterial interspecies communication in polymicrobial infection of the cystic fibrosis lung

There is an increasing appreciation of the polymicrobial nature of bacterial infections associated with Cystic Fibrosis (CF) and of the important role for interactions in influencing bacterial virulence and response to therapy. Patients with CF are co-infected with Pseudomonas aeruginosa, Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. Previous studies showed that DSF from S. maltophilia leads to altered biofilm formation and increased tolerance to antibiotics in P. aeruginosa and that these responses require the P. aeruginosa sensor kinase PA1396. The work in this thesis aims of further elucidate the influence and mechanism of DSF signalling on P. aeruginosa and examine the role that such interspecies signalling play in infection of the CF airway. Next generation sequencing technologies targeting the 16S ribosomal RNA gene were applied to DNA and RNA isolated from sputum taken from cohorts of CF and non-CF subjects to characterise the bacterial community. In parallel, metabolomics analysis of sputum provided insight into the environment of the CF airway. This analysis revealed a number of observations including; that differences in metabolites occur in sputum taken from clinically stable CF patients and those with exacerbation and DNA- and RNA-based methods suggested that a strong relationship existed between the abundance of specific strict anaerobes and fluctuations in the level of metabolites during exacerbation. DSF family signals were also detected in the sputum and a correlation with the presence of DSFproducing organisms was observed. To examine the signal transduction mechanisms used by P. aeruginosa, bioinformatics with site directed mutagenesis were employed to identify signalling partners for PA1396. A pathway suggesting a role for a number of proteins in the regulation of several factors following DSF recognition by PA1396 were observed.