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School of Veterinary and Life Sciences

Honours Projects

Dr Graham O'Hara

Position: Senior Lecturer
Phone: 9360 2583
Room: Chanc 1.009

Research

(In collaboration with Professor Michael Dilworth, Mr. John Howieson and Dr Ravi Tiwari)

Legumes are vital components of agricultural systems because of their ability to associate symbiotically with root nodule bacteria (RNB) that fix atmospheric nitrogen for them, thereby also providing soil nitrogen for other non-leguminous crops. They enhance the productivity and potential sustainability of farming systems. The Centre for Rhizobium Studies is researching a number of aspects of the root nodule bacteria (Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium) associated with legumes.

Current research projects are in the following general areas:

a) iron nutrition and siderophore production in Rhizobium leguminosarum bv viciae

b) molecular biology and physiology of acid tolerance genes in Sinorhizobium meliloti, Rhizobium leguminosarum and Rhizobium tropici

c) selection of improved rhizobial inoculants for crop and pasture legumes

d) properties of root nodule bacteria from indigenous Western Australian legumes

e) develooping superior inoculant technology

Iron nutrition and siderophore production in R. leguminosarum biovar viciae

Almost all organisms have an absolute requirement for iron. Some strains of root nodule bacteria grown in iron-deficient environments excrete iron-chelating compounds (siderophores) which complex iron from the environment. The iron-siderophore complexes are then taken up by specific transport systems. The ability to produce siderophores is widely scattered among genera and species of the root nodule bacteria, and its biological significance in the soil or in the legume is not well understood. Iron deficiency can limit nodulation, nitrogen fixation and yield of grain legume crops such as chickpea and lentils when they are grown in alkaline soils.

Projects

Iron Storage in root nodule bacteria

Iron storage may play a role in rhizobial persistence, particularly in calcareous or alkaline soils where iron is unavailable. It is not known if iron storage is common among the genera and species of root nodule bacteria. Only two root nodule bacteria, Rhizobium leguminosarum WSM710 and 8401pRL1JI have been shown to store iron. Under iron-deficient conditions, both R. leguminosarum WSM710 and 8401pRL1JI, excrete a trihydroxamate siderophore, vicibactin, and in replete conditions store iron. The amount of iron stored in R. leguminosarum WSM710 influences the repression of vicibactin synthesis, with low iron cells continuing to excrete the siderophore even when the external iron concentration is high. This project would screen a variety of genera/species of root nodule bacteria, including both siderophore and non-siderophore producers, for the ability to store iron. It would also investigate the external iron concentrations necessary for iron storage to occur, and the relationship of total iron content to the repression of siderophore biosynthesis.

Mechanism of synthesis of vicibactin by R. leguminosarum

Vicibactin is a trihydroxamate, made up of three residues each of D-ornithine acetylated at N2 and hydroxylated at N5 and 3-hydroxybutyrate, the bonds being alternating ester and peptide types. The sequence of steps leading to vicibactin synthesis from l -ornithine and 3-hydroxybutyrate theoretically require only 3 three genes - a hydroxylase, an acetylase and a non-ribosomal peptide synthetase. Collaborative work on the genetics between CRS and Prof. Johnston"s group at the University of E. Anglia in UK have now identified about 10 genes which actually affect vicibactin synthesis in one way or another. The hydroxylase and peptide synthetase have been unequivocally identified, but there are three potential acetylases in the cluster, mutants of which show a variety of phenotypes from no vicibactin production, through a non-acetylated vicibactin to the wild-type. Mutation in yet another gene which by homology is not an acetylase results in a non-acetylated vicibactin.

This project would be about using a variety of techniques, biochemical and others, to sort out which genes are responsible for which steps in biosynthesis. From the collaborative project, we have access to mutants with reporter gene insertions in each of the genes in the regulon.

Molecular biology and physiology of acid tolerance in Sinorhizobium meliloti, Rhizobium leguminosarum and Rhizobium tropici

The propagation of legumes is affected by harsh environmental and soil conditions that affect survival and growth of RNB, the microsymbionts. Low soil pH is one of the stresses that affect legume production in large areas of Australia and in many parts of the world. RNB display a range of sensitivity to low pH, Sinorhizobium meliloti being the most acid-sensitive. Lucerne, a perennial species with the potential to lower soil water tables and assist in controlling salinisation, requires this micro-symbiont for nodulation is therefore especially vulnerable in acid soils.

Over the pas decade we have used three different approaches to characterise the molecular mechanism of low pH tolerance in root nodule bacteria.

(1) Low pH-sensitive mutants were generated from an acid-tolerant strain to identify the genes that are essential for growth at low pH.

(2) Low pH-regulated gene fusions were created, to identify the genes that are controlled by pH.

(3) 2 D gel electrophoresis, followed by N-terminal sequencing of selected proteins, has also been used to identify low pH-regulated proteins.

Construction of genetic tools for site directed mutagenesis

The genomes of Sinorhizobium meliloti and Mesorhizobium loti have already been completely sequenced. While this sequence information will describe the genes and the proteins encoded by them, it will not tell us when and how these genes will be expressed, nor what their regulatory elements are. New methods such as micro-array gene technology and proteome analysis will allow us to identify the mRNAs and proteins, respectively, produced by a genome under particular conditions. However, even these emerging technologies will be unable to identify the regulatory proteins and their targets.

In this post-genomic era research will therefore focus on understanding the function of gene products and identifying the control mechanisms of gene expression. To do this we will need methods for targeted inactivation of genes identified from the genomic DNA sequence. A comparison of protein patterns in the mutant and the wild type backgrounds can then pinpoint proteins that are regulated through that gene. To produce the required mutants will require faster methods of specific inactivation of a range of genes.

If you choose this project, you will be developing a new strategy that will speed up the process of targeted gene inactivation.

The Role of Alternative Sigma Factors in Resistance to Low pH and Other Stresses

Sigma factors provide specificity to RNA polymerase and dictate its DNA-binding properties. In response to environmental changes, or during the changes accompanying differentiation, bacteria use alternative sigma factors. Several alternative sigma factors like RpoS, RpoN, RpoH and ECFs (extra-cytoplasmic factors) are involved in the expression of specific sets of genes in response to a particular signal. Each of these sigma factors has the ability to recognize a specific promoter sequence that is not recognized by the normal housekeeping sigma factor, s70 , and thereby activate a specific set of genes for transcription.

Using the genome sequence for S. meliloti we have already identified 13 alternative sigma-factors in its genome. Two lines of evidence from our own work suggest an involvement of alternative sigma factors in response to acid. The first is that the promoter for the acid-inducible gene lpiA contains what appears to be a typical consensus sequence for s 54 . The second is that from proteomic assays proteins whose regulation involves alternative sigma factors (ClpP, DegP and GroES) are up-regulated in low pH conditions.

If you choose this project you will aim to:

(1) inactivate the genes for some of the alternative sigma factors in S. meliloti WSM419 and determine phenotypes for the mutants grown under low pH;

(2) use reporter gene fusions in the sigma factor genes to study their expression under different stress condition, particularly low pH.

Identification of the regulatory elements that control induction of IpiA by low pH

To understand the regulatory elements that control regulation signalled by low pH we have generated several pH regulated gusA fusions. WR101 (lpiA-gusA; low pH inducible) and several other minitransposon-induced mutants (RTL19, RT1721, WR1180, WR1181, WR1208 and WR1212) show several-fold induction of b-glucuronidase (GUS) activity by low pH. The regulation of these low pH regulated genes is as yet unknown. In the case of lpiA a small DNA region (~ 100 bp) has been identified that is required for up-regulation of the gene. Pinpointing the regulatory sequence in the lpiA promoter and the identification of the regulator that is required for up-regulation of lpiA will increase our understanding of the low pH regulatory mechanism.

To extend the host range of Rhizobium tropici to nodulate Medicago sativa

Root-nodule bacteria vary in their capacity to survive different physical (temperature and water potential) and chemical (acidity, alkalinity, salinity, toxic metals, etc) stresses. Medicagosativa (lucerne) is nodulated by S. meliloti, a low pH sensitive root nodule bacterium. The poor survival of S. meliloti in soils below pH 5.0 severely inhibits lucerne nodulation and has a dramatic effect on its productivity. In contrast to the acid-sensitivity of S. meliloti, Rhizobium tropici can survive in very acidic soils as low as pH 3.0.

In this project you will use the novel approach of increasing the host range of R. tropici, an acid tolerant organism, to overcome the problem of acid inhibition of lucerne nodulation.

Calcium nutrition of root nodule bacteria

Calcium is an essential nutrient for bacteria and this element may have a key role in the response of bacteria to environmental stresses such as acidity. We do know that high concentrations of calcium in the external environment can make root nodule bacteria more tolerant of acidity. Furthermore we know that rhizobia grown at an acidic pH contain 30-fold less cell associated calcium than cells grown at neutral pH. However we do not understand the physiological role of calcium in root nodule bacteria. This project aims to determine whether rhizobia can actively transport calcium and investigate the effect of acidity on the calcium uptake and location in rhizobia.

Selection of improved rhizobial inoculants for crop and pasture legumes

Nodulation in Biserrula pelecinus

Biserulla pelicinus is a monotypic pasture genus endemic to the Mediterranean basin, and this legume is nodulated by unique root nodule bacteria. Several cultivars of B. pelecinus have been released by CLIMA as alternate pastures for acid soils. However establishment and productivity of B. pelecinus on some soils is being compromised by delayed nodulation. At present the mechanism of nodulation in B. pelecinus is unknown, nor are the environmental factors affecting nodulation understood.

During legume nodulation the root nodule bacteria infect roots by one of three known mechanisms; root hair, crack or epidermal infection. This project would investigate the infection process leading to nodulation in B. pelecinus. Chemical components of the soil can inhibit legume nodulation and some of these factors may cause delayed nodulation of B. pelecinus. This study would determine the effect of environmental factors on nodulation. The results of this research will assist the development of suitable management practices for ensuring the successful establishment of this novel pasture legume.

Selection of suitable inoculant strains of rhizobia for perennial herbaceous legumes

Dryland salinity has arisen in southern Australia following the removal of a native perennial-based flora and its replacement with an annual flora that transpires less water. One indirect solution to salinity is to "perennialise" our agricultural systems through development of crops and pastures that maintain an economic return from the land whilst simultaneously functioning as mimics of the original vegetation with respect to moisture usage. Perennial plants may be woody or herbaceous. This project focusses on the latter and includes both forage legumes and grasses, as well as crops (which may be either).

Perennial forages are numerous in other agricultural systems. Lucerne, perennial clovers and grasses have wide international usage. The challenge is to identify germplasm well adapted to the target edaphic conditions, select appropriate microsymbionts for the legumes and to deliver this germplasm into the hands of those developing new farming systems based upon perennial species. An advantage in accessing germplasm is that we already have well developed research collaboration agreements with several International centres. Australian germplasm, too, should not be ignored. We have both indigenous grass and legume forage species that need to be evaluated in the new agricultural context. Finally, we need to identify salt tolerant material that can play a role in already salinized landscapes.

In this particular project we will focus upon developing suitable inoculant rhizobia for a range of pernnial clovers, lotus and related species.

Properties of root nodule bacteria from indigenous Western Australian legumes

WA native legumes and the specificity of their root nodule bacteria

While South-Western Australia has a very large range of indigenous legumes, very little is known about their root nodule bacteria. The data on some Acacia species indicates that both fast- and slow-growing root nodule bacteria are capable of nodulating them, and other data suggest that the slow-growing strains at least are promiscuous across the genus.

However, for most of the WA native legumes, little is known about what types of organism nodulate them, or about their capacity for cross-infection. This information would be very useful in the horticultural industry, where most horticultural species are sold non-nodulated, and the success of the plants depends markedly on their capacity to nodulate with root nodule bacteria in the environments where they are planted.

The project would involve selecting an appropriate set of genera/species (depending on seed availability, horticultural potential, etc.), nodulating them in soil from suitable locations, authenticating the isolates by nodulation testing, and then determining the cross-infection patterns for a limited range of species. Associate Professor Jen McComb would be a joint supervisor for the project.

Developing superior inoculant technology

Developing formulations that improve the survival of rhizobial inoculants applied to legume seed

For the rhizobial inoculation of legumes, the system of embedding cells in a sterile peat carrier was developed in the 1950s. This methodology remains as commercial best practice today, nevertheless it is limited to the application of peat-carrier onto seed which must then be sown immediately into moist soil. Even where this is achieved, the death rate of cells on seed can be as high as 90% per day primarily because of bacterial desiccation followed by rehydration. There is a fundamental requirement for improvement in technology that effectively delivers micro-organisms as inoculants. Whilst this has long been recognised in the Industry, until the recent research with vacuum-dried inoculants and polymer coatings that limit desiccation and oxygen transfer, there have been few promising technological breakthroughs.

This project aims to research means to more effectively deliver rhizobia as well as PGPR organisms to legumes by improved coating technology.

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