Massey Documents by Type
Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294
Browse
7 results
Search Results
Item Genome sequence of the entomopathogenic Serratia entomophila isolate 626 and characterisation of the species specific itaconate degradation pathway(BioMed Central Ltd, 2022-12) Vaughan AL; Altermann E; Glare TR; Hurst MRHBACKGROUND: Isolates of Serratia entomophila and S. proteamaculans (Yersiniaceae) cause disease specific to the endemic New Zealand pasture pest, Costelytra giveni (Coleoptera: Scarabaeidae). Previous genomic profiling has shown that S. entomophila isolates appear to have conserved genomes and, where present, conserved plasmids. In the absence of C. giveni larvae, S. entomophila prevalence reduces in the soil over time, suggesting that S. entomophila has formed a host-specific relationship with C. giveni. To help define potential genetic mechanisms driving retention of the chronic disease of S. entomophila, the genome of the isolate 626 was sequenced, enabling the identification of unique chromosomal properties, and defining the gain/loss of accessory virulence factors relevant to pathogenicity to C. giveni larvae. RESULTS: We report the complete sequence of S. entomophila isolate 626, a causal agent of amber disease in C. giveni larvae. The genome of S. entomophila 626 is 5,046,461 bp, with 59.1% G + C content and encoding 4,695 predicted CDS. Comparative analysis with five previously sequenced Serratia species, S. proteamaculans 336X, S. marcescens Db11, S. nematodiphila DH-S01, S. grimesii BXF1, and S. ficaria NBRC 102596, revealed a core of 1,165 genes shared. Further comparisons between S. entomophila 626 and S. proteamaculans 336X revealed fewer predicted phage-like regions and genomic islands in 626, suggesting less horizontally acquired genetic material. Genomic analyses revealed the presence of a four-gene itaconate operon, sharing a similar gene order as the Yersinia pestis ripABC complex. Assessment of a constructed 626::RipC mutant revealed that the operon confer a possible metabolic advantage to S. entomophila in the initial stages of C. giveni infection. CONCLUSIONS: Evidence is presented where, relative to S. proteamaculans 336X, S. entomophila 626 encodes fewer genomic islands and phages, alluding to limited horizontal gene transfer in S. entomophila. Bioassay assessments of a S. entomophila-mutant with a targeted mutation of the itaconate degradation region unique to this species, found the mutant to have a reduced capacity to replicate post challenge of the C. giveni larval host, implicating the itaconate operon in establishment within the host.Item Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background(John Wiley and Sons, Ltd, 2021-08-24) Vlková M; Morampalli BR; Silander OKThe expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between cis- or trans-genetic elements affect the behavior of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context-dependent cis-interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self-splicing ribozyme called RiboJ, which can be used to decouple upstream 5' UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans- and cis-genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis-effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis- nor trans-genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis- and trans-genetic changes.Item Dynamics of bacterial adaptation(Portland Press Limited on behalf of the Biochemical Society, 2021-04-12) Lai H-Y; Cooper TFDetermining pattern in the dynamics of population evolution is a long-standing focus of evolutionary biology. Complementing the study of natural populations, microbial laboratory evolution experiments have become an important tool for addressing these dynamics because they allow detailed and replicated analysis of evolution in response to controlled environmental and genetic conditions. Key findings include a tendency for smoothly declining rates of adaptation during selection in constant environments, at least in part a reflection of antagonism between accumulating beneficial mutations, and a large number of beneficial mutations available to replicate populations leading to significant, but relatively low genetic parallelism, even as phenotypic characteristics show high similarity. Together, there is a picture of adaptation as a process with a varied and largely unpredictable genetic basis leading to much more similar phenotypic outcomes. Increasing sophistication of sequencing and genetic tools will allow insight into mechanisms behind these and other patterns.Item Filamentous phage derived biological nanorods : development of a novel display system : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand(Massey University, 2018) Davey, Georgia RoseThe Ff filamentous bacteriophage are filament-like bacterial viruses approximately 900 nm in length. The F-pilus-specific filamentous phage are resistant to heat, pH-extremes, and detergents in combination with their structural properties and amenability to DNA recombinant engineering has enabled their extensive use in modern biotechnology. However, the use of Ff-phage in vaccines and other such biological uses is controversial due to their ability to replicate in gut Escherichia coli, and the possibility of mobilisation and horizontal gene transfer of antibiotic resistance-encoding genes among the gut bacteria. As such, the novel system was established to create short, stable particles that cannot replicate, called NanoZap particles. However, this system has the disadvantage of often producing multiple-length particles, rather than the desired single-length particles; another disadvantage is that during packaging, one particle in a million packages the entire plasmid due to recombination that removes the terminator copy of the (+) ori, and given that these plasmids contain antibiotic resistance genes, this likely would spread antibiotic resistance throughout the surrounding environment. In this thesis several variations upon the original pNanoZap vector were created and tested to obtain monodisperse unit-length particles. The deletion of the complete multiple cloning site (MCS) that lied between the initiator and terminator of replication from the original pNanoZap vector achieved this aim. To eliminate rare antibiotic resistant particles that package the complete pNanoZap vector, the antibiotic resistance gene was removed from the pNanoZap 537 vector and replaced with an auxotrophic marker nadC; this vector was named pNanoZap 537N. It is yet to be seen if this new pNanoZap vector is capable of producing NanoZap particles. For high-sensitivity diagnostics it is desirable to construct high-avidity particles, containing large number of detector molecules. To achieve this a double-display (detector displayed on phage which in turn is displayed on the surface of florescent E. coli) was designed and tested. When the E. coli expressing red fluorescent protein TinselPurple were infected with the bacteriophage, the chromogenic protein was lost, thereby showing that a different method of colouring E. coli will need to be used in order to construct the double-display particles.Item The antibiotic sensitivity patterns and plasmid DNA content of gram-negative anaerobic bacteria isolated in Palmerston North, New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Science at Massey University(Massey University, 1987) Mooney, Christopher AllanOne hundred and seven Gram-negative bacteria, including 65 Bacteroides species, 28 fusobacteria and 14 veillonellae were isolated from 17 oral infections treated in two dental surgeries in Palmerston North. These bacteria, plus 37 isolates belonging to the B. fragilis group received from Palmerston North hospital, were surveyed for their antibiotic sensitivity levels, and their plasmid DNA content. The hospital isolates of the B. fragilis group were found to have sensitivity levels comparable with those of B. fragilis group isolates reported in the literature recently. The oral isolates were more sensitive to penicillin, cefoxitin, and tetracycline than isolates of the same species reported in the literature. Half the hospital isolates had plasmids, which were all between 8.5 and 2.7 kilobases (kb) in size except for one 60, and one 43 kb plasmid. Comparatively few of the oral anaerobes had plasmids. One Fusobacterium russii isolate had four plasmids, and five Bacteroides isolates had one plasmid each. These five Bacteroides isolates came from two specimens, R5 and R6. Restriction enzyme analysis of all plasmids revealed that the three 5.6 kb plasmids from sample R5 may be related to a group of 5.8 kb plasmids harboured by four of the hospital isolates. Two different species of Bacteroides isolated from sample R5 harboured the 5.6 kb plasmid, and two species of the B. fragilis group bacteria harboured the 5.8 kb plasmid. Plasmid DNA isolated from two tetracycline resistant hospital isolates was used to transform restriction negative E. coli to a low level of tetracycline resistance.Item Plasmids in Rhizobium phaseoli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1982) Ward, Lawrence James HenryFast growing strains of Rhizobium have been divided into four homology groups on the basis of DNA hybridization. Rhizobia from two of these homology groups can form effective nodules with beans (Phaseolus vulgaris). Large plasmids associated with nodulation have been demonstrated in Rhizobium sp. A study was undertaken to examine the plasmids in rhizobia from the two different homology groups capable of nodulating beans. The effectiveness of strains of Rhizobium phaseoli on bean plants were examined. Spontaneous antibiotic resistant mutants which retained the ability to nodulate beans were selected. Antibiotic resistance marked clones were incubated at elevated temperatures to produce an ineffective mutant stain of Rhizobium phaseoli NZP 5492. Methods of extracting large plasmids from Rhizobium phaseoli were developed. Plasmids were visualised by agarose gel electrophoresis and purified by cesium chloride - ethidium bromide density gradient ultracentrifugation. We demonstrated the presence of plasmids of molecular weight range 66 Md to 316 Md in Rhizobium phaseoli strains. Some strains contained a single plasmid while others contained multiple plasmids. Comparisons were made between whole plasmids and restriction endonuclease digests of the plasmids from the two groups. The fragment pattern obtained from Eco RI digests showed differences in fragment numbers and size between plasmids from DNA homology group 1 and DNA homology group 2. Further studies using gel blotting and hybridization techniques are required to ascertain the degree of homology between the plasmids, both within groups and between groups. Rhizobium phaseoli NZP 5479 and NZP 5547 a non-nodulating mutant of strain NZP 5479 were examined. Both strains had plasmids of estimated molecular weights 186 Md and 288 Md. There was no detectable difference in the size of the plasmids in the non-nodulating mutant compared to the effective parent strain. Rhizobium phaseoli NZP 5492 B5/8 (effective) and NZP 5492 B5/1 an ineffective mutant obtained from strain NZP 5492 had plasmids of similar molecular weight. Differences were observed in the Eco RI fragment pattern and possible rearrangements to the DNA to account for these differences are presented.Item Lactococcal plasmid replicon ; vector construction and genetic organization : 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, 1990) Xu, FengfengThe 5.5 kb high-copy number cryptic plasmid pDI25 from Lactococcus lactis subsp. lactis 5136 was isolated and used as the basis to construct a series of vectors. The vector pFX1 (5.5 kb) was first made by ligating the 4.5 kb HpaII-MboI fragment of the lactococcal plasmid to the 1 kb chloramphenicol transacetylase gene from the staphylococcal plasmid pC194. Plasmid pFX1 was further modified by deleting a non-essential 1.9 kb ClaI region to construct pFX2 (3.6 kb). Deletion analysis showed an essential region for plasmid replication was located within a 1.2 kb CfoI-ThaI-CfoI fragment. The vector pFX3 was constructed by incorporating the α fragment of the Escherichia coli lacZ structural gene, a multiple cloning region and the T7 and T3 promoters from pUBS into pFX2. Recombinant plasmids constructed in E. coli using X-gal selection could be subsequently electroporated into lactococci. pFX3 could also be used directly for transcription studies or DNA sequencing of cloned inserts. A set of lactococcal translational gene-fusion vectors was constructed by incorporating the E. coli lacZ gene fusion system (pNM480,481,482) into pFX2. These constructions, pFX4, pFX5 and pFX6, permit the fusion, of cloned genes to lacZ in all three reading frames. Gene expression can be readily and quantitatively monitored by measuring β-galactosidase activity. All the pFX vectors were efficiently transformed into lactococci and E. coli by electroporation (104-106 cfu/μg DNA in each host) and maintained stably in both organisms (> 95% cells carrying the Cm marker after 100 generations growth without drug selection). A cell-wall bound proteinase from Lactococcus lactis subsp. cremoris H2 was isolated and characterized as a PI type proteinase since it preferentially degraded β-casein. A 6.5 kb HindIII fragment of plasmid pDI21 (63 kb) was initially cloned and expressed this enzymatic activity in E. coli using vector λNM1149. The restriction map of this pDI21 prt gene fragment had minor differences from those of other published lactococcal prt fragments. Using pFX1, the pDI21 prt gene fragment was recloned and directly electroporated into lactococci where it was efficiently expressed. The effectiveness of pFX3 was demonstrated by initially cloning a pDI1 4.4 kb EcoRI tagatose 1,6-bisphosphate aldolase gene fragment into E. coli from where it was electroporated into lactococci. Using the translational fusion vectors pFX4, pFX5 and pFX6, the 6.5 kb HindIII prt gene fragment of pDI21 was identified as having two promoters with opposite orientations. The pDI21 2.0 kb EcoRI galactose-6-phosphate isomerase gene fragment was shown to carry a promoter and the direction of gene transcription was determined. The complete DNA sequence of the lactococcal portion of pFX2 (2508 bp) was determined and the genetic organization analyzed. A lactococcal plasmid plus origin and two replication protein coding regions (repA and repB) were located. RepA had an αhelix-turn-αhelix motif, a geometry typical of DNA-binding proteins. RepB showed high homology to the plasmid replication initiation proteins from other Gram-positive bacteria and Mycoplasma. The transcribed inverted repeat sequence between repA and repB could form an attenuator to regulate pFX2 replication. Upstream of the plus origin site, and in a region nonessential for replication, a 215 bp sequence identical to the staphylococcal plasmid pE194 and carrying the RSA site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with the pE194 group of plasmids.