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    The detection of plasmid transfer genes in Rhizobium species : a thesis presented in partial fulfilment of the requirement for the degree Master of Science in Microbiology at Massey University, New Zealand
    (Massey University, 1994) Fisher, Paul Jamie
    In order that Rhizobium tra genes responsible for Sym plasmid transfer might be found, DNA probes were constructed from Agrobacterium tra genes. Three probes were constructed from DNA containing;- 1) traR, a gene which regulates other tra genes on the Agrobacterium tumefaciens plasmid pTiC58, 2) an OriT site, at which nickases cleave the plasmid before conjugal transfer can take place, and 3) part of a gene required for construction of a mating bridge. All three probes were constructed by the ligation of tra areas from the A.tumefaciens strain C58 plasmid pTiC58 into broad host range plasmid vectors and subsequent electroporation into E.coli cells. The genomic DNA digests of several Rhizobium and Agrobacterium strains were blotted and probed with the three probes under various washing and hybridisation stringencies. A.tumef aciens strain LMG64 was the only Agrobacterium strain aside from strain C58 to have DNA homologous to any of the probes. Neither R.leguminosarum bv trifolii strain ICMP2163, nor R. leguminosarum bv trifolii strain ICMP2163::Tn5, nor R.leguminosarum bv trifolii strain PN165 had any DNA homologous to any of the probes. However, R.leguminosarum bv trifolii strain ATCC14480 showed homology to the tra I probe (containing traR), and R.loti strain ATCC33669, phylogenetically the most distant relative to A.tumefaciens strain C58 shared homology with the tra III probe (containing DNA responsible for mating bridge assembly). Therefore the distribution of tra genes from the Ti plasmid of A.tumefaciens strain C58 among the agrobacteria and rhizobia used in this study did not correlate to their phylogenetic relatedness to A.tumefaciens strain C58 or to one another.
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    Construction and utilisation of a bidirectional reporter vector in the analysis of two nod-boxes in of Rhizobium loti : a thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in Molecular Genetics at Massey University
    (Massey University, 1993) Parry, Simon Keith
    The nod-box is a 47bp cis-acting regulatory region which has been conserved amongst every species of Rhizobium studied to date. In species such as R. meliloti and R. leguminosarum, the nod-box has been shown to promote constitutive activity towards the regulatory nodD gene, and flavonoid-inducible expression towards the divergently-transcribed nodABCIJ operon. This bidirectional regulation of the so-called common nod genes was not observed in R. loti. A previous analysis of this species had shown that its nod-box promoted inducible activity towards the truncated 'nodD' gene, as well as the nodACIJ operon. It was the unusual arrangement of these R. loti nod genes that had initially aroused interest in this bacteria. To further investigate the role of the nod-box in the regulation of the R. loti common nod genes, a bidirectional reporter vector (pSPV4) was constructed. This novel vector allowed the promoter activity of a cloned nod-box-containing fragment to be concurrently measured in either direction using the same culture of cells. To achieve this construct, the gusA gene from pRAJ260 was blunt-end ligated into pUC21. An in-frame ribosome binding site (rbs) was cloned upstream of the gusA coding sequence to facilitate transcriptional fusions. The rbs and gusA gene were later excised as a functional unit and blunt-end ligated into pMP220 alongside the B-galactosidase reporter gene but in the opposite orientation. Hence, both reporter genes could be divergently transcribed from a common regulatory region cloned into the multiple cloning site that separated the genes. The fragments of DNA that were eventually cloned into the bidirectional vector were generated through the polymerase chain reaction. Each DNA insert contained the nod-box bracketed by differing lengths of flanking region. Once these PCR-generated fragments had been sequenced in pUC118 and subcloned into pSPV4, the resulting constructs were transformed into R. loti cells by electroporation. As the electroporation of these cells had not previously been reported, the conditions for this procedure were established and optimised. The results obtained from the bidirectional reporter assays disagreed with those observed in the earlier assays by Teo (1990). Neither the nodACIJ nod-box of NZP2037 nor the nodB nod-box of NZP2213, showed bidirectional inducible expression. In fact, both nod-boxes showed constitutive expression in the 'nodD' direction and inducible expression in the opposite direction. This indicates that the control of the nod genes in R. loti is fundamentally the same as that seen in other fast-growing Rhizobium species. Three regulatory elements affecting the levels of nod gene expression have tentatively been identified outside the nod-box sequence, though the results indicating their presence may simply be·due to spacing differences between the nod-box and the reporter gene.
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    The use of LAC fusions to analyse the regulation of NOD gene region of Rhizobium loti : a thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 1990) Gillman, Michelle Ann
    Two approaches where used in the analysis of common and host specific nod gene expression in Rhizobium loti strains NZP2213 and NZP2037. The first approach using the Tn3-HoHol transposon to generate lacZ transcriptional/translational fusions, produced 290 insertions within the 8.3kb EcoRI nod fragment of R.loti strain NZP2213. The position and orientation of all but one of these insertions was determined using restriction enzyme mapping and hybridisation. The sites of the insertion and orientation were generally found to be random. The lacZ fusions were transferred into R.loti strain NZP2213 where their B-­galactosidase activity was measured in the presence and absence of Lotus tenuis seed exudate. All insertions had a low level of B-galactosidase activity that was the same as the controls. This activity was independent of position or orientation. This lack of expression could be a result of the fusions being in regions that are not transcribed ie not downstream of either a nod inducible or other promoter, or that the appropriate conditions for constitutive or inducible activity were not achieved. The second approach to construct lacZ transcriptional fusions was less random and involved the cloning of three separate nod gene fragments: i) a 4.1kb Sall fragment that overlaps the nod region of the 8.3kb EcoRI fragment of R.loti strain NZP2213, ii) a 0.65kb EcoRl fragment isolated from the 4.1kb Sall fragment of R.loti strain NZP2213, and iii) a 1.4kb Sall fragment isolated from the 7.1kb EcoRI nod region of R.loti strain NZP2037. These three fragments (4.1kb, 0.65kb and 1.4kb) were isolated and cloned into pMP190, pMP220 and pMP190 respectively, in both orientations. Each lacZ fusion was transferred into the R.loti strains from which the fragment had originated, ie either NZP2213 or NZP2037. The B-galactosidase activity of these transconjugants was measured in the presence and absence of Lotus tenuis seed exudate. The 4.1kb Sall construct from R.loti NZP2213 was found to have consititutive activity in both orientations indicating that at least two consititutive promoters are located on this fragment. The activity of one orientation, corresponding to pPN38, was twice that of the reverse orientation corresponding to pPN37. V The smaller 0.65kb EcoRI fragment, that lies within the larger 4.1kb Sall fragment, contains a "nod box" and part of a nodD-like gene (Scott et al., In prep.). No significant 13-galactosidase activity was observed in either orientation with or without seed extract. These experiments showed that the "nod box" alone was insufficient for plant inducible expression. The 1.4kb Sall fragment from R.loti NZP2037, that was known to contain a nodA promoter (Emerson-Colins et al., pers. comm.) showed inducible expression for pPN39, corresponding to a fusion between nodA and lacZ. No significant activity was detected in the reverse orientation, pPN40, either with or without plant exudate.
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    Development of a DNA hybridisation method for the identification of Rhizobium and Bradyrhizobium: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in microbiology at Massey University, New Zealand
    (Massey University, 1990) Garman, Jean Heather
    The potential of a DNA hybridisation method, utilising a biotin-labelling system with a streptavidin/alkaline phosphatase detection system (ENZO Biochem), was investigated as an identification method for Rhizobium species and Bradyrhizobium sp. (Lotus) strains using nodule, colony and pure DNA. The method used for extracting DNA from colonies and crushed nodules and binding it to nitrocellulose did not purify the DNA sufficiently to stop non-specific binding occurring between the streptavidin-alkaline phosphatase conjugate and the sample. An alternative method of colony hybridisation that removed more of the cellular constituents was required. Only pure DNA could be used. The method was altered as follows: i) Tris/EDTA buffer was used to terminate the colour reaction in place of allowing the membrane to air dry; ii) 5% milk powder was used in place of 10% bovine serum albumin in the blocking buffer, complex detection buffer and washing buffer used in the detection of hybridised biotin-labelled DNA; iii) 5% dextran sulphate was included in the hybridisation buffer to decrease the minimum hybridisation time from 6hr to 3hr. Investigation of the effect of variable conditions on the intensity of colour produced showed that: i) the incubation of alkaline phosphatase with its substrate at room temperature resulted in fluctuation of the development time as the enzyme reaction rate is sensitive over this range of temperature (approximately 1s0 c to 30°C); ii) increasing the concentration of labelled DNA in the hybridisation buffer increased the intensity of colour produced, the minimum concentration that could be used without lowering the detection limit was 200 ng/ml; iii) continued incubation of alkaline phosphatase with its substrate after colour development in the negative control had begun gave an increased colour intensity in the sample but since this increase was not proportional to that of the negative control the net response (sample minus control) decreased. When genomic probes were hybridised with slot-blots containing homologous DNA the detection limit was between 63 and 125 ng of DNA. Both 32P-labelled and biotin­ labelled genomic Rhizobium leguminosarum biovar trifolii DNA probes were able to distinguish between Rhizobium leguminosarum and other Rhizobium species but not between the biovars of R.leguminosarum. To distinguish between closely related species or strains when using 32P-labelled or biotin-labelled probes a specific DNA sequence was required for use as the probe. Two distinct DNA homology groups have been described in Bradyrhizobium sp. (Lotus). From a gene library of Bradyrhizobium sp. (Lotus) strain cc814S (homology group I) 8 clones were isolated that contained sequences that distinguish a representative of homology group I (strain cc814S) from a representative of homology group II (strain NZP2076). This was achieved by hybridising total genomic DNA from strain cc814S with total genomic DNA from strain NZP2076 and removing the single stranded specific sequences with hydroxylapatite. The specific DNA was used to probe the gene library. Increased selection for group-specific sequences by substituting another homology group I strain (NZP2021) for strain cc814S and subcloning one of the clones isolated gave inconclusive results but indicated that a group specific sequence could be derived in this way.
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    Analysis of genomic rearrangement and plasmid conjugation of an inoculant strain of Rhizobium leguminosarum bv. trifolii : a thesis presented in partial fulfillment of the degree of Doctor of Philosophy
    (Massey University, 1989) O'Hara, Michael J
    Variation of plasmid profile is the hallmark of Rhizobium lequminosarum bv trifolii strains isolated from the nodules of pasture plants. Previous attempts to demonstrate that these variant types were derived from the original inoculant were inconclusive. Subsequent laboratory based simulations revealed that the broad host range plasmid RP4 was capable of generating stable alterations in the plasmid and total genomic DNA profile. This variation involved an apparent loss of pSym, with concurrent loss of the nod and nif genes, but these strains produced nodules on white clover plants. The strains recovered from the nodules, while not identical, were clearly derived from the pSym¯ strain. A plasmid closely corresponding in size to pSym was detected in the nodule re-isolates and repeated trials of this experiment involving antibiotically marked (apparently) sym¯ strains confirmed this observation. This left the conclusion that the DNA was still there but in a form which was difficult to detect by conventional DNA hybridization procedures, a result which is not totally without precedent (Downs and Roth, 1987). The second portion of this project involved an investigation of the transmissability of plasmids from the inoculant strain 2668. The strain was marked with Tn5 with the expectation that some of the movable DNA pieces would carry a Tn5 insert and could thus be selected, for. A transmissable symbiotic plasmid was detected, as had been previously observed in other rhizobia by Johnston et al (1978). The plasmid was shown to be transferable, in an altered form, to soil microorganisms of unidentified genera, to a sym¯strain of R. lequminosarum bv trifolii, to its original parent 2668 and to E. coli. In all strains, with the exception of E. coli the nodulation genes were functional, producing normal looking nodules (on the outside) and in many strains nitrogen was also fixed, though not generally as well as by the parent 2668. The significance of a self-transmissable broad host range symbiotic plasmid is discussed in the context of the microbial ecology Of rhizobia.