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School of Veterinary and Life Sciences

Honours Projects

Dr Wayne Reeve

Position: Senior Lecturer
Phone: 9360 2631
Room: BioSc 3.031

Research

Research in my laboratory is focused on microbial physiology and genetics with a particular emphasis on molecular microbiology. The research areas of particular interest include:
1. Molecular communication.
2. Genes required for stress tolerance.
3. Stress response, adaptation and mechanisms related to virulence of agrobacteria.
4. Microbial ecology, saprophytic competence and competition.
5. Transcription regulation by RNA polymerase sigma factors and other regulators.
6. Structural and functional genomics.

Students are encouraged to choose from one of these areas but other projects will also be considered. Please feel free to discuss your ideas with me.

APPLICATION OF RESEARCH.
A particular emphasis of the research is microbiology pertaining to microsymbionts of agricultural importance. The root nodule bacteria (RNB) establish a symbiotic relationship with legumes enabling atmospheric nitrogen to be fixed into a form that can be utilized by the plant. This process enhances the productivity and potential sustainability of farming systems. The research into RNB is conducted in the Centre for Rhizobium Studies located in the Biological Sciences Building on the third level.


SPECIFIC RESEARCH PROJECTS.

1. Molecular communication.

Prokaryotes respond to signal molecules through signal transduction mechanisms or quorum sensing. The latter being a cell density dependent phenomenon. Two component signal transduction systems have been identified in S. medicae that are required for stress response and tolerance. Quorum sensing plays a role in the symbiotic process and in the production of exopolysaccharide synthesis in S. medicae.

The main research interest in this area is to identify how the signals are perceived and to explore the range of genes regulated through these circuits.

2. Genes required for stress tolerance.

The propagation of legumes is affected by harsh environmental and soil conditions that affect survival and growth of the microsymbionts. Low soil pH is one stress that affects legume production in large areas of agricultural land. Root nodule bacteria display different sensitivity to low pH. For example, the genus Sinorhizobium is one of the most acid-sensitive whereas Rhizobium tropici is one of the most acid-tolerant. Lucerne, a perennial species with the potential to lower soil water tables and assist in controlling salinisation, requires the Sinorhizobium microsymbiont for effective nodulation and hence productivity is especially vulnerable in acid soils.
Four different approaches to characterise the molecular mechanisms of low pH tolerance in root nodule bacteria are in use

(i) Construction of low pH-sensitive mutants to identify genes essential for growth at low pH. For more information see Reeve et al., 2002; Mol Microbiol 43: 981-991.

(ii) Creation of pH-regulated fusions to identify genes controlled by pH. For more information see Tiwari et al., 2004; JMMB 7: 133-139.

(iii) Proteomic analysis to identify low pH-regulated proteins. For more information see Reeve et al., 2004; JMMB 7: 140-147.

(iv) Transfer symbiotic genes from Sinorhizobium into a more acid-tolerant background. For more information see Combio conference poster.

3. Stress response, adaptation and mechanisms related to virulence of agrobacteria.

Several low pH-regulated gene fusions have been identified in R. leguminosarum, R. tropici and S. medicae. The lpiA gene has been identified in Agrobacterium tumefaciens, R. tropici and S. medicae and is the most acid-induced gene described to date in S. medicae. How these low pH-regulated genes are expressed is as yet unknown. In the case of lpiA a small DNA region (~ 100 bp) upstream of the start codon is required for up-regulation of the gene. Pinpointing the regulatory sequences of acid-responsive promoters and identification of regulators required will increase our understanding of stress response in these important bacteria.

4. Microbial ecology, saprophytic competence and competition.

The microsymbiont Rhizobium leguminosarum biovar trifolii nodulates clover and fixes nitrogen. Agronomists have targeted perennial clovers for their potential role in Australian agriculture. However, the rhizobia for annual clovers are generally ineffective with perennial clovers. This presents a challenge to introduction of perennial clovers into the an Australian setting where agriculture is still very reliant on nitrogen fixation from T. subterraneum. Studies must be designed to increase our understanding of these symbiotic interactions so that we can develop strategies to improve the management of legume-rhizobia interactions to extend (rather than restrict) the use of legumes in new environments (Yates et al., 2005; AJEA 45: 189-198).
The genus Sinorhizobium has the ability to effectively nodulate the legume host Medicago. However, each species has a different host range. For example, S. meliloti can establish an effective symbiotic relationship with M. sativa but not M. murex, M. arabica and M. polymorpha. In contrast, S. medicae can form an effective relationship with all of these hosts. This project will identify genes required for microbe-plant interaction. The research findings will have practical applications to prevent introduction of incompatible strains and for screening strains for enhanced nitrogen fixation characteristics with a particular host.

5. Transcription regulation by RNA polymerase sigma factors and other regulators.

Sigma factors provide specificity to RNA polymerase and dictate its DNA-binding properties. Bacteria use alternative sigma factors to transcribe genes in particular conditions (ie in response to stress challenge or during differentiation). 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, sigma-70 , enabling transcription of gene sets.
Fourteen sigma-factors have been identified in the genome of S. meliloti Rm1021; thirteen of these are alternative sigma factors. Two lines of evidence suggest an involvement of alternative sigma factors in response to acid. The first is that the promoter for the acid-inducible gene lpiA contains a typical consensus sequence for sigma-54 (ComBio conference poster). The second is that proteomics has revealed proteins (ClpP, DegP and GroES) up-regulated in low pH conditions and the regulation of transcription of the genes encoding these proteins is subject to control by alternative sigma factors in other organisms.
It is the aim of this project to:
(i) Inactivate the genes for some of the alternative sigma factors in S. meliloti WSM419 and determine phenotypes for the mutants challenged to acid and other stresses,
(ii) create reporter gene fusions to sigma factor genes to study their expression under different stress conditions, and
(iii) determine the role of the sigma factors in Sinorhizobium.
For preliminary work in this area please see poster 1 and poster 2 presented at the Australian ComBio conference in 2004.

6. Structural and functional genomics.

In collaboration with the Joint Genome Institute (USA), the complete genome sequence of Sinorhizobium medicae will be established by 2006/7. This sequence information will essential for a complete description of genes and proteins found in S. medicae and will be used for comparative and functional genomics. New methods such as micro-array gene technology and proteome analysis are being employed to identify the mRNAs and proteins, respectively, produced by the genome under particular conditions. Methods for targeted inactivation of genes can be employed to construct specific mutants. A comparison of protein patterns in mutant and wild type backgrounds can be used to pinpoint proteins that are regulated by a particular regulator. A large scale functional genomics strategy is currently being developed in conjunction with Prof. Turlough Finan at McMaster University in Canada to monitor the expression of genes and create knockout mutants. This is an exciting time to become involved in genomics to identify the role of genes in S. medicae.

 

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