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Item Understanding the population genetic structure of bovine mastitis-causing Staphylococcus aureus in New Zealand to identify potential vaccine candidates using reverse vaccinology : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2021) Nesaraj, JabinStaphylococcus aureus is one of the major causes of bovine mastitis in New Zealand and worldwide, causing severe economic loss to the dairy industry. With significant advances in whole-genome sequencing (WGS) and associated bioinformatics, S. aureus from bovine mastitis has been studied worldwide, but the understanding of the organism's genomics is still incomplete. To my knowledge, there have been no published WGS studies of S. aureus in dairy cattle in New Zealand to date. As WGS has become more affordable, and the concomitant bioinformatic analysis offers high-resolution indiscriminating betweenS. aureus lineages, WGS analysis was applied to a sample of S. aureus isolates obtained from dairy cattle in New Zealand. The work is undertaken in this thesis utilised advanced WGS analyses to study the genomic epidemiology ofS.aureusfrom dairy cattle over a period of 15 years. The findings from the analysis enabled a subsequent reverse vaccinology analysis that identified a number of conserved peptides potentially useful for incorporation in subunit vaccines for cattle. The first study analysed the population genetic structure of the pathogen using 188 bovineS. aureus isolates were collected from dairy farms across New Zealand in 2002-03, 2013-14, and 2017-18. Ruminant-specific and non-specific clonal complexes(CCs) were identified. CC1 was the dominant CC, a unique feature not observed in dairy cattle in other countries. CC1 was predominant in cattle in the three periods of isolate collection, suggesting a stable and successful clonal lineage. Interestingly, CC1is also the predominant CC in humans in New Zealand, and is mainly associated with humans in other countries and is not commonly reported in cattle. The second study compared the genomes of the bovineS. aureus isolates from the first study with genomes of quasi-contemporaneous human, canine and feline(n=59), and small ruminant (n=30) isolates also collected in New Zealand. Comparative genomic analyses of the core and accessory genomes were used to assess the effect of the host species of origin on the phylogenetic clustering of the isolates, and to identify host-specific/host-adaptive genomic signatures. Comparative analysis of CC1isolates identified marked phylogenetic segregations of both the core and accessory genomes among cattle and humans, and the presence of previously described ruminant-adaptive genes in bovine isolates, but not in human isolates. To my knowledge, this is the first report of ruminant host adaptation within CC1. The third study compared the antimicrobial resistance results obtained using the Disk Diffusion test (DD) with a resistome analysis, to assess the potential usefulness of WGS analysis to predict the antimicrobial resistance phenotype of bovineS. aureus. The antimicrobial resistance genes identified were: theβ-lactamase geneblaZ, the erythromycin resistance gene ermC, the streptomycin resistance gene str, and the fusidic acid resistance gene fusC. WGS identification of theblaZgene had a sensitivity of 71% and a specificity of 100% in predicting resistance when the DD test was considered the reference standard. Similar analyses could not be performed with other antimicrobials due to the low frequency of other resistance genes in the sample. Finally, the information obtained from the first two studies was used to construct a reverse vaccinology bioinformatic pipeline to identify potential vaccine candidate (PVC) proteins for S. aureus mastitis. Eighteen PVC proteins were identified using a range of bioinformatics tools. Some of these proteins have previously been shown to be immunogenic through in vitro and animal studies, providing cross-validation to the pipeline, while others have not yet been tested. In summary, this thesis presents a detailed description of the population genetic structure of bovine mastitis-causing S. aureus in New Zealand over 15 years. The work provides new insights into the complex mechanisms of S. aureus host-adaptation to ruminants and identifies some obstacles for the successful application of genomic analysis for the prediction of the antimicrobial resistance phenotype of clinical S. aureus isolates. Potentially useful proteins to be included in subunit vaccines for cattle are also reportedItem Natural variation in bacterial gene regulation : a thesis presented in partial fulfilment of the requirements for the degree of Ph.D in Microbiology & Genetics, Massey University College of Science, School of Natural and Computational Sciences(Massey University, 2021) Vlková, MarkétaIt has been over 160 years since Charles Darwin set out the theory of evolution by natural selection. This theory is broadly accepted these days. However, it is still not completely understood how natural selection shapes particular cell mechanisms and behaviours. There is a limited research about selection acting on gene regulation. To address the questions about how selection shapes gene regulation we used a collection of environmental E. coli isolates. We quantified the genetic variability of 605 promoters within this collection of highly diverged strains. We then selected ten promoters (aldA, yhjX, lacZ, aceB, mtr, cdd, dctA, ptsG, purA, and tpiA) which highly differ in their genetic variability to analyse their phenotypic variability. We used fluorescent reporter assays with flow cytometry to measure changes in gene expression with high-throughput and at single cell resolution. In order to discern natural selection acting on gene regulation we compared phenotypes from segregating promoter variants, which have been subject to natural selection and random promoter variants that have never been subject to natural selection. We generated the random variants using PCR random mutagenesis. Beside focusing on the changes in the overall expression (i.e., transcription and translation), we examine selection acting on transcription only. This we achieved be implementing self-cleaving ribozyme insulation. In this thesis we showed that natural selection towards high plasticity and low noise is common among regulated E. coli promoters. We also verify that the self-cleaving ribozyme RiboJ activity is highly effective and that this genetic tool can be used to detect changes in transcription alone. Utilizing the RiboJ we were then able to detect both directional and diversifying selection acting on lacZ promoter. This thesis thus broadens the knowledge about natural selection acting on gene regulation and provides a new insights into how promoters are shaped in nature by selection, including some most well-characterized bacterial promoters. This work also demonstrates a new application of RiboJ ribozyme that has not been published before.Item 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, KiranPseudomonas 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.Item Resistance of environmental bacteria to heavy metals and antibiotics in selected New Zealand soils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Wellington, New Zealand(Massey University, 2020) Heydari, AliThe usage of superphosphate fertilizers, animal remedies and other material containing heavy metals (HMs) in agriculture and horticulture is a problematic issue resulting in the accumulation of HMs in the soil. The presence of these HMs in the soil leads to the induction of resistance of environmental bacteria to these heavy metals and may co-select for resistance against a broad range of antibiotics (Abs). This co-selection may increase the health risk for both humans and livestock because of resistance to the wide range of Abs. As well as direct health effects, an increase in Ab resistance may impose a significant burden on the livestock industry and primary production, leading to potential for immense social and financial losses. The current project was aimed to investigate the resistance of soil-borne bacteria sampled from selected regions of New Zealand. Genetic diversity of these bacteria and molecular aspects of horizontal transfer of HM and Ab resistance genes to other bacterial hosts was also investigated. Soil samples with a different history of usage, including pastoral and arable with high levels of HMs (e.g. cadmium (Cd) and zinc (Zn)) were collected from the Waikato region (WR), as well as soil from an area of native bush (background) as control. Waikato Region (WR) is one of the regions in New Zealand with high levels of HMs in soil, due to the regular use of HM-containing superphosphate fertilizers and animal remedies. Belmont Regional Park (BRP) airstrip soil was used as a novel site to explore bacterial communities’ resistance to HMs, and any co-selected Ab resistance. A comprehensive investigation was performed to simulate the soil environment contaminated with various levels of HMs to interrogate induced resistance to HMs and Abs in soil bacterial communities using microcosms with 6 weeks and 6 months incubation. The experiments carried out to investigate soil bacterial resistance to HMs and Abs were divided into two different categories, including physiological and molecular experiments. The physiological tests included plate culturing with a range of HMs and Abs concentrations and Pollution Induced Community Tolerance (PICT) analysis. Molecular investigations using Terminal Restricted Fragments Lengths Polymorphism (TRFLP) and Next Generation 16s rDNA were conducted to determine the probable changes in bacterial community structures induced by selection pressure of HMs presence in soil samples. Cd resistance genes were detected in individual bacterial isolates using specific oligomeric DNA primers via the polymerase chain reaction (PCR). Horizontal transfer of these genes to new bacterial recipients was investigated. Finally, Cd resistant bacterial isolates involved in Horizontal Gene Transfer (HGT) were identified using 16s rDNA Sanger Sequencing. Results clearly showed that there were significant differences between the levels of resistance to HMs and Abs in bacterial isolates from WR’s pastoral and arable soils compared to background soil (native bush). Differences between BRP soil samples with higher levels of HMs compared to those with lower HMs concentrations, and also microcosms’ with a range of HM levels showed there were significantly greater number of bacterial isolates resistant to HMs and Abs in soils with the higher initial levels of HMs. Pollution Induced Community Tolerance (PICT) analysis provided complementary results in concordance with the results of plate culturing experiments and showed the higher levels of bacterial resistance to HMs and Abs in soils with the higher initial levels of HMs. Terminal Restriction Fragment Length Polymorphism (TRFLP) and 16s rDNA Next Generation Sequencing experiments investigated HM-induced bacterial communities' structure changes and revealed significant differences among the bacterial community structures in the selected BRP and microcosms soil samples. The HGT experiments revealed the horizontal transfer of Cd resistance genes from donor isolates (from WR, BRP, and microcosms soils) to a characterised recipient bacterial strain in vitro, suggesting these genes were carried by mobile genetic elements. Overall, the result of the current project showed that there were higher levels of bacterial resistance to HM and also to Ab occurred while different levels of HMs were present in the soil. In addition, higher levels of HM and Ab resistance induction occurred in the presence of specific concentrations of HMs in microcosms’ soils. The bacterial community structures were changed in the presence of various levels of HM in soil. The investigation of bacterial community structures changes in microcosms containing background soil samples were greater compared to the microcosms containing pastoral soils; it is concluded in higher changes in bacterial communities in soils in presence of selection pressure of HMs. Cd resistance genes located on mobile genetic elements were able to be transferred horizontally form donor bacterial strains to recipients and the transconjugants showed resistance not only to Cd, Zn and/or Hg, but also to a range of Abs; it showed the possibility of spread of these HM resistance genes to the new bacteria in soil and conferring HM and subsequent Ab resistance in recipients.Item Regulation of histidine catabolism in Pseudomonas fluorescens SBW25 : a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand(Massey University, 2019) NarenThe pathway of histidine utilization (hut) has been a model for studying bacterial gene expression, particularly the coordination between cellular carbon and nitrogen metabolisms. Early studies in enteric bacteria led to the concept of catabolite repression, which explains the inhibitory effects of glucose on the utilization of alternative carbon sources such as histidine and lactose. Briefly, transcription of hut genes is activated by the catabolite-activating protein (CAP) charged with cAMP and the NtrBC/NAC cascade when histidine is used as a source of carbon and nitrogen, respectively. However, this well-defined paradigm does not hold for many non-enteric bacteria, including the closely related Pseudomonas. This work aims to define the molecular basis of hut gene expression in Pseudomonas, using the plant growth-promoting bacterium P. fluorescens SBW25 as a model. Previous work identified all hut genes involved in histidine uptake and subsequent enzymatic breakdown, which are organized in three transcriptional units in the hut locus: hutF, hutCD and hutU-G. Like in enteric bacteria, histidine-induced expression of hut operons is mediated by the HutC repressor with urocanate, the first intermediate of the histidine degradation pathway, as the effector molecule. However, the precise interactions between HutC and its hut operator sites remain elusive; more importantly, recent progress suggests a new role of HutC in global gene regulation beyond histidine catabolism. Moreover, two two-component systems CbrAB and NtrBC are involved in hut activation, but it remains unknown whether they act in a direct or indirect manner. In this study, I first examined the molecular interactions between His6-tagged HutC protein and probe DNAs of the PhutU and PhutF promoters. Results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting indicate that HutC binds to a consensus sequence of TGTA-N2-TACA (named Phut site), and involves complex oligomerization in response to varying concentrations of urocanate. A novel weak HutC binding sequence (termed Pntr site) was identified in the PhutF promoter, which may help strengthen the repression of hutF. Significantly, this Pntr site shows no sequence similarity to the previously recognized Phut site, instead it is homologous to the NtrC-binding consensus sequence (GCACCA-N3-TGGTGC). Next, the Phut consensus sequence was used to predict HutC target genes in the genome of P. fluorescens SBW25. This led to the identification of 88 candidate promoters, eight of which were subject to experimental verification by EMSA and DNase I footprinting. Phenotypic analysis of the hutC deletion mutant showed that hutC is involved in cell motilities. The data is consistent with the predicted global regulatory role of HutC. Histidine utilization poses a significant challenge as it produces excess nitrogen over carbon. The rate of histidine utilization (hut) thus must be carefully regulated. Here we show, for the first time, that expression of hut genes is positively regulated by two global regulators CbrAB and NtrBC in a direct manner, while subjecting to histidine concentration-dependent negative control of the HutC repressor. hut expression is further regulated at the post-transcriptional level by the CbrAB-CrcYZ-Crc/Hfq cascade in response to the presence of succinate (the most preferred carbon source for Pseudomonas). When growing in nutrient-complex conditions such as a minimal salts medium supplemented with succinate and histidine wherein histidine is the sole nitrogen but less-preferred carbon source, CbrAB is involved in directly activating hut transcription but indirectly repressing hut translation. Under this condition, NtrBC plays the dominant role in transcriptional activation of hut genes, but it requires assistance from the HutC repressor. A combination of genetic and biochemical analyses show that HutC acts as a governor to monitor and control the histidine catabolic rate, preventing production of excess ammonium and consequent inactivation of the NtrBC system. HutC additionally recognizes the NtrC binding site responsible for ntrBC expression, which provides a negative feedback for NtrBC autoregulation. Together, data presented in this thesis extend our understanding of carbon catabolite repression to the cellular nitrogen catabolism of Pseudomonas: carbon/nitrogen metabolic balance is maintained by the interplay of CbrAB and NtrBC at the hut operator site, and it requires the local regulator HutC to prevent hut expression from exceeding a critical upper limit. The finding that the HutC regulator is capable of recognizing two distinct DNA binding motifs (Phut and Pntr) has broader implications in gene regulation. Further biochemical analysis is required to unravel the molecular basis of the observed dual site recognition.Item Temperature- and host-dependent transcriptional responses in the entomopathogenic bacterium, Yersinia entomophaga MH96 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany Campus, New Zealand(Massey University, 2020) Paulson, Amber RoseYersinia entomophaga MH96 is a virulent pathogenic bacterium that is infective towards a broad range of insects and is under development as a biopesticide. MH96 produces insecticidal toxin complex called Yen-TC that is secreted at temperatures of 25 °C and below and has been shown to be the primary virulence factor (VF) during per os challenge against the New Zealand grass grub, Costelytra giveni and other agricultural pests (Hurst et al., 2011a, 2019). New insights into the pathobiology of MH96 during insect infection were gained from the in vivo transcriptome, including identification of a core secreted weaponry of co-expressed/co-secreted VFs, including Yen-TC and other exoenzymes; however, many other diverse types of VFs, including toxins, effectors, fimbriae, secretion systems, efflux pumps, iron acquisition, stress response and metabolic adaptation were also identified as highly expressed under in vivo conditions. A small DNA-binding protein, Yen6, was shown to be under thermoregulation at the transcriptional level and host-dependent-regulation at the post-transcriptional level and contributed to virulence during intrahemocoelic infection of Galleria mellonella at 37 °C. The in vivo transcriptome of Δyen6 and in vitro DNA-binding specificity analysis provided evidence that Yen6 is a novel LytTR-containing regulator that activates a ribose uptake/metabolism gene cluster, rbsD-xylG-rbsC-xylF-rbsK-ccpA, and represses a fructose uptake/metabolism gene cluster, IIA-fruK-IIB and a gene for RNA-binding protein yhbY during infection at 37 °C. Another small DNA-binding protein, Yen7, was also implicated as a potential temperature-dependent activator of Yen-TC component genes and over-expression of yen7 resulted in restored secretion by MH96 at 37 °C; however, deletion of yen7 did not abrogate Yen-TC production. Experimental investigations into potential regulatory linkages between Yen6 and yen7 were undertaken, and evidence to date does not support Yen6 as transcriptional repressor of yen7. A 17.5 Kb unstable element within the genome of MH96 with linkages to Yen-TC and toxin secretion, motility and cell shape was identified. Overall the findings presented in this thesis represent the most detailed investigation of MH96 pathogenesis to date, reinforcing MH96 as one of the most highly entomopathogenic bacteria known to humankind; yet suggesting MH96 has possibly maintained at least one core thermoregulatory mechanism more typical of an opportunistic pathogen.Item Pangenome-guided tools for investigating the role of epsilonproteobacteria in human gastroenteritis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Science at Massey University, Manawatu, New Zealand(Massey University, 2018) Cornelius, Angela JoyceGastroenteritis affects billions of people every year and current testing methods fail to identify the cause for approximately half of the samples submitted for microbiological testing. Epsilonproteobacteria contains Campylobacter jejuni, the most commonly reported cause of bacterial gastroenteritis in the world, and Helicobacter pylori, a gastric pathogen and class I carcinogen. This bacterial class also contains ≥20 additional species known, or suspected, of being human pathogens. To better understand the role some of these species play in human gastroenteritis, novel rapid, cost effective methods are needed. The growing number of whole genome sequences available for this class were exploited to first evaluate the classification of the genetically heterogeneous species C. concisus and then to identify taxon-specific CDS for a range of Epsilonproteobacterial taxa. Probes were designed to detect 28 of these CDS and incorporated into a single multiplex ligation-dependent probe amplification (MLPA) assay which was tested against DNA from 43 Epsilonproteobacterial species and then applied to DNA extracts from stool samples from a childhood gastroenteritis case control study undertaken in Belgium. The 22 C. concisus genomes consistently clustered into two genomospecies (GS) represented by ATCC 33237T (GS1) and CCUG 19995 (GS2). Taxon-specific genes were identified for 28 taxa, including the two C. concisus genomospecies, and concordant results were observed for the majority of MLPA probes and DNA extracts from pure cultures. The probes designed to detect C. lari subsp. concheus and H. pullorum failed to detect the target DNA; all of the urease positive thermophilic Campylobacter DNA extracts were also positive for the probe designed to detect C. subantarcticus, some probes lacked repeatability in the presence of elevated EDTA and the size differences between some probes needs to be optimised. C. jejuni was the most common Epsilonproteobacterial species isolated by culture and C. concisus was the most common species detected by MLPA. Both C. jejuni and C. concisus GS2 were detected in significantly higher numbers in cases than controls in a Belgian childhood case control study. This demonstrated the utility of the Epsilonproteobacteria MLPA assay and provides some evidence that C. concisus GS2 may have a role in childhood gastroenteritis.Item The analysis of plasmid rearrangements observed in the soil bacterium OR168 after the introduction of transposon Tn5 : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University(Massey University, 1994) Harte, David John GerardTransposon Tn5 mutagenesis has been used extensively in Escherichia coli and various other Gram-negative bacteria to produce both random and site directed mutants. The popularity of Tn5 as a mutagen stems from its apparent random insertion into the genome, leading to non-leaky polar mutations. It also confers on many bacteria resistance to aminoglycosides, providing a strong selectable marker. The site of insertion can be mapped by Southern DNA hybridisation against a specific Tn5 probe. Tn5-containing derivatives of the Rhizobia-like soil isolate, OR168, were produced using the broad host-range suicide plasmid vector pSUP1011. After the transfer of pSUP1011 to OR168 via heterogeneric bacterial conjugation, stable OR168::Tn5 exconjugants were selectively isolated at frequencies of approximately 10-4 per recipient. None of the 53 OR168::Tn5 exconjugants screened showed the parental plasmid profile. Visible alterations to the plasmid profile were common with respect to the native plasmid profile. These events generally showed large deletions from, or additions to, the native replicons of OR168. The alterations also included a low incidence of a decrease in plasmid number. Analysis of the exconjugant population shows that the insertion of Tn5 into the genome of OR168 may not be strictly random. It was shown that 66% of OR168::Tn5 exconjugants screened contain a plasmid-borne Tn5 element, with 90% of those involving Tn5 insertion in the same episome. There is evidence that events other than classical conservative transposition have occurred after the introduction of pSUP1011 into the OR168 genome. Screening of the isolated OR168::Tn5 population for pSUP1011 vector sequences revealed the presence of the pSUP1011-derived RP4-mob fragment in 33 of 35 OR168::Tn5 exconjugants containing a plasmid-borne Tn5 element. Analysis also revealed the acquisition of Tn5 alone, presumably by conservative transposition, occurred only twice in the 35 events involving a plasmid target. This suggests that another site within the RP4 fragment can act as a surrogate transposase recognition site. Alternatively, the insertion of the RP4-mob::Tn5 sequence into a plasmid target may involve a site specific recombination process peculiar to the OR168 isolate. No mechanism was elucidated for the formation of many of the alterations in plasmid mobility. Restriction fragment lengths in the immediate vicinity of the anomalous RP4-mob::Tn5 insertion arc identical in different plasmids. This may indicate sequence duplication among the OR 168 plasmids. Such duplication may precipitate, through homologous recombination processes, the plasmid instability observed.Item Effects of aerobic and anaerobic environments on bacterial mutation rates and mutation spectra assessed by whole genome analyses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2015) Shewaramani, SonalFor organisms that are exposed to different environments, the rates and types of spontaneous mutations that arise in each environment can vary, and potentially impact the direction of evolution as a whole. Oxidative stress is a major cause of mutation, but the effect of oxygen availability on the mutation rates and spectra of organisms grown in aerobic as compared to anaerobic environments is not well understood at the whole genome level. To investigate the mutation rates and spectra of a facultative anaerobic bacterium grown under strictly aerobic or anaerobic conditions, 24 mutation accumulation lineages, derived from Escherichia coli REL4536, were established and propagated through 180 and 144 single-colony population bottlenecks, respectively. Spontaneous mutation rates of 2.50 × 10-10 and 4.14 × 10-10 mutations per nucleotide per generation were obtained for aerobically and anaerobically grown cells, respectively. Mutations in the aerobic environment were significantly biased towards G T mutations and IS186 transposition, while C A, T G, A C mutations, gross chromosomal rearrangements (GCRs) and IS150 transposition were significantly more prevalent under anaerobic conditions. Transcriptional profiling, via RNAseq, of REL4536 grown under aerobic and anaerobic environments revealed that repair genes, especially those involved in the repair of GCRs, were generally up-regulated in the anaerobic environment, consistent with findings that mutation rates, especially for GCRs, are higher in the anaerobic environment. GCRs have long been thought to play an important role in the evolutionary process, though their contributions to the process have not been specifically defined. SbcCD, an exonuclease, is involved in the repair of DNA secondary structures, and is thought to help prevent the occurrence of GCRs. Transcriptome analyses showed that in E. coli, sbcC was up-regulated during growth in an anaerobic environment, as compared to an aerobic environment. To investigate the impact of GCRs on adaptive evolution, an E. coli REL4536 strain with disrupted sbcC was constructed and evolved under anaerobic conditions for 1,000 generations in glucose-limited media in 14 parallel populations. Mutations that arose during evolution were determined by whole genome re-sequencing of selected clones, and evolved sbcC mutant strains displayed more GCRs and enhanced population-level fitness on average. Together, these results suggest that GCRs may play an important role in the rate of adaptation.Item Investigation of lactose utilization genes in Clostridium acetobutylicum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand(Massey University, 1988) Hancock, Kerrie RuthPreparatory to constructing a genomic library of Clostridium acetobutylicum, cell lysis and DNA preparation conditions leading to undegraded DNA were examined. Early to mid-exponential phase cells lysed more readily than cells at late-exponential or stationary phase. Lysis was facilitated by 0.3% w/v glycine in the growth medium. Achromopeptidase was a more effective lytic enzyme than either mutanolysin or lysozyme. All strains of C. acetobutylicum produced high levels of DNase activity, coinciding with the late-exponential and stationary growth phases. Chromosomal DNA isolated from all strains of C. acetobutylicum was rapidly degraded. This degradation was not prevented by the use of various protein inactivating agents. The adverse effect of oxygen and related radicals on the DNA of this strict anaerobe was considered to be responsible. Undegraded DNA was isolated by protecting the cells to be lysed from oxygen. A genomic library of C. acetobutylicum NCIB2951 in the cosmid vector pLAFR1, constituting 3,500 recombinant clones, was prepared. Clones from this library complemented various Escherichia coli auxotrophic mutations, showing that C. acetobutylicum genes are expressible in E. coli. Recombinant clones coding for the β-galactosidase of NCIB2951 were isolated from the genomic library using the chromogenic substrate X-gal. The lacY mutation of HB101 could not be complemented by these clones, suggesting that a classical lac operon system does not exist in C. acetobutylicum. The β-galactosidase (cbg) gene was further subcloned on a 5.2 kb EcoRI fragment, and was expressed when the fragment was cloned in either orientation. Cbg was thus expressed from its own promoter. The cbg gene is inducible by lactose in C. acetobutylicum. When cloned into E. coli, however, this gene was expressed constitutively, the level being unaffected by the presence of the inducer, IPTG or glucose. Six strains of C. acetobutylicum possessed a sequence highly homologous to the cloned β-galactosidase fragment. The β-gatactosidase gene region of NCIB2951 showed only low homology to the DNA from other Gram-positive bacteria (Streptococcus lactis ATCC7962, Streptococcus thermophilus DRI1424, Lactobacillus bulgaricus DRI20056, Lactobacillus helviticus DRI20064), and no detectable homology to DNA from Gram-negative bacteria (E. coli DC272 or Rhizobium loti PN2231). The β-galactosidase activity of the 5.2 kb fragment was inactivated by Tn5 insertion at either of two loci. Locus I (400 bp) was approximately 500 bp from locus II (approximately 3.2 kb). Maxi-cell analysis identified a 100 kDa protein as the β-galactosidase gene product. The 5.2 kb fragment was sequenced and analyzed. Three ORF's were identified. ORF1 (cbgA) coded for the structural β-galactosidase gene. Significant amino acid homology was detected with the amino acid sequences of the lacZ, ebgA (E. coli) and lacZ (Klebsiella pneumoniae). ORF2 (cbgR) coded for a small regulatory protein which shared homology with the amino acid sequence of the "0.3 kb gene" from Bacillus subtilis. ORF3 coded for a truncated protein which shared significant homology with the N-terminal amino acid regions of spo0A and spo0F (B. subtilis), two regulatory proteins of the two-component system. Hence, no lac operon exists in C. acetobutylicum.