Research


Research Description
Background on leaf-associated bacteria Overview of the research in the Beattie laboratory Identification of the genetic and physiological traits that contribute to bacterial growth, survival and pathogenesis of leaves Evaluation of how the waxy cuticle of plants influences bacterial colonization of leaves Characterization of the microenvironments sensed by bacteria on leaves using targeted gene fusions. Effect of volatile organic pollutants on the chemical environment sensed by bacteria on leaves
Background on leaf-associated bacteria
Plant leaves are commonly colonized by large bacterial populations, as well as by other microorganisms including yeasts, mycelial fungi, and algae. Each leaf can be considered its own ecosystem - an ecosystem in which microorganisms battle among themselves for limited resources, forge strategies for surviving rapid changes in environmental conditions, and actively change their own microenvironment by instigating changes in their plant host. Among the bacterial species found on leaves, some can induce disease in the plant host induce frost injury by producing a protein that initiates the formation of ice at cold temperatures outcompete, kill, or exclude those microorganisms that cause disease or frost injury alter plant growth by producing plant growth hormones fix atmospheric nitrogen possibly promote plant growth and possibly degrade airborne pollutants that have collected on the leaf surface. In most cases, the potential for bacteria to perform these functions is dependent on the size of a population, and the size of the population, in turn, is dependent on bacterial survival and colonization. top		Phaseolus Vulgaris bean leaf surface Scanning electron micrographs of bacteria on bean leaves

Overview of the research in the Beattie laboratory
Our research is focused on the relations between the genetics and physiology of leaf-associated bacteria and their ecology. The bacterial genes and plant genes that influence bacterial leaf colonization are poorly understood. Similarly, although many physiological traits in bacteria have been proposed to enhance or diminish their colonization potential, only a few have been experimentally evaluated. We have proposed a working model of the events that occur during leaf colonization, and are continuing to examine this process, as well as the genetic and environmental factors that influence this process. We are currently pursuing several avenues of research:


	Environmental factors Characterization of the microenvironments sensed by bacteria on leaves using targeted gene fusions Effect of volatile organic pollutants on the chemical environment sensed by bacteria on leaves
	Plant factors Evaluation of how the waxy cuticle of plants influences bacterial colonization of leaves using plant mutants altered in wax production
	Bacterial factors Identification of the genetic and physiological traits that contribute to bacterial growth, survival and pathogenesis of leaves top

Identification of the genetic and physiological traits that contribute to bacterial growth, survival and pathogenesis of leaves

We are characterizing genes that contribute to the ability of the phytopathogen Pseudomonas syringae pv. syringae to survive, grow, and cause disease on leaves. We are currently focusing on two of 82 genes that were identified following a random mutagenesis and screen for altered epiphytic fitness. These two genes are restricted to a fairly narrow group of closely related pseudomonads. Sequence analysis indicates that both genes are unrelated to genes and proteins of known function. One of the genes appears to be the first gene in a constitutively expressed operon. This operon includes a number of genes that appear to have diverse functions, many of which may influence bacterial leaf colonization. Studies aimed at identifying the function of these genes and the identity and function of downstream genes in the operon are in progress.

We are also exploring the role of extracellular polysaccharides (EPS) in the ecology of leaf-associated bacteria. These polysaccharides are likely to have a dominant role because their presence defines the environment immediately surrounding the bacteria. As tools for these studies, we are using bacterial mutants that are deficient in the production of each of two types of EPS, EPS-specific antibodies, and fluorescence microscopy. In these studies, we are exploring such questions as when and where each type of EPS molecule is made, whether EPS production is induced by contact with a surface, how the loss of each type of EPS influences bacterial retention, growth, survival and entry into leaves, and whether the landscape on the leaf surface influences how each type of EPS contributes to bacterial colonization.

Selected publications:
Sabaratnam, S. and G. A. Beattie. 2003. Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic versus endophytic colonization of leaves. Applied and Environmental Microbiology 69:1220-1228.

Beattie, G. A. and S. E. Lindow. 1999. Bacterial colonization of leaves: a spectrum of strategies. Phytopathology 89:353-359.

Andersen, G. L., G. A. Beattie and S. E. Lindow. 1998. Molecular characterization and sequence of a methionine biosynthetic locus from Pseudomonas syringae.
Journal of Bacteriology 180:4497-4507.

Beattie, G. A. and S. E. Lindow. 1995. The secret life of foliar bacterial pathogens on leaves. Annual Review of Phytopathology 33:145-172

Beattie, G. A. and S. E. Lindow. 1994. Survival, growth and localization of epiphytic fitness mutants of Pseudomonas syringae on leaves. Applied and Environmental Microbiology 60:3790-3798.

Beattie, G. A. and S. E. Lindow. 1994. Comparison of the behavior of epiphytic fitness mutants of Pseudomonas syringae under controlled and field conditions. Applied and Environmental Microbiology 60:3799-3808.

Beattie, G. A. and S. E. Lindow. 1994. Epiphytic fitness of phytopathogenic bacteria: physiological adaptations for growth and survival, pp. 1-27. In: J. L. Dangl (ed), Bacterial pathogenesis of plants and animals: molecular and cellular mechanisms. Springer-Verlag, NY

Lindow, S. E., G. Andersen and G. A. Beattie. 1993. Characteristics of insertional mutants of Pseudomonas syringae with reduced epiphytic fitness. Applied and Environmental Microbiology 59:1593-1601.

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Evaluation of how the waxy cuticle of plants influences bacterial colonization of leaves

We are investigating how specific leaf surface features influence epiphytic colonization. Since the major point of contact between epiphytic bacterial populations and the plant host is at the waxy layer known as the plant cuticle, the cuticle is likely to be a dominant plant trait influencing the leaf surface as a habitat for bacteria. For these studies, we are using 11 mutants of maize that differ in the composition of the cuticular waxes that they produce; thus, these mutants provide a range of chemically and topographically distinct landscapes for colonization by bacteria. We are exploring the ability of 3 distinct bacterial species to colonize these plant mutants, including a gram-negative phytopathogen, Pseudomonas syringae pv. syringae, a gram-positive phytopathogen, Clavibacter michiganensis subsp. nebraskensis, and a saprophyte, Pantoea agglomerans (previously known as Erwinia herbicola). Among the selected glossy mutants, we have identified both bacterial species-specific and non-species specific effects of the mutants on bacterial retention, growth and survival, and we are currently exploring the mechanisms underlying these effects.

Selected publications:
Marcell, L. M. and G. A. Beattie. 2002. The effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis. Molecular Plant-Microbe Interactions 15:1236-1244.

Beattie, G. A. and L. M. Marcell. 2002. Comparative dynamics of adherent and non-adherent bacterial populations on maize leaves. Phytopathology 92:1015-1023.

Beattie, G. A. and L. M. Marcell. 2002. Effect of alterations in cuticular wax biosynthesis on the physicochemical properties and topography of maize leaf surfaces. Plant Cell and Environment 25:1-16

Beattie, G. A. 2002. Leaf surface waxes and the process of leaf colonization by microorganisms, pp. 3-26. In: S. E. Lindow, E. I. Hecht-Poinar and V. J. Elliott (eds), Phyllosphere Microbiology, American Phytopathological Society Press, St. Paul, MN.

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Characterization of the microenvironments sensed by bacteria on leaves using targeted gene fusions.

Bacterial populations are highly variable among leaves, and this variability is likely due, in part, to heterogeneity in the growth and death rates of bacteria in distinct microsites within a leaf. This heterogeneity is likely a result of the nutritional and environmental conditions in those microsites. We are currently exploring the extent to which bacteria are exposed to limiting amounts of water in leaf microsites, since water availability has long been predicted to be a critical issue for bacteria on leaves. We have constructed bacteria that fluoresce green in response to water deprivation, due to the presence of a water stress-responsive reporter gene fusion. We are using these to investigate the exposure levels of both individual cells and populations of various bacterial species to water deprivation under various environmental conditions, and are relating these exposure levels to their effects on the physiology and growth of the colonization.

Selected publications:
Axtell, C. A. and G. A. Beattie. 2002. Construction and characterization of a proU-gfp transcriptional fusion that measures water availability in a microbial habitat. Applied and Environmental Microbiology 68:4604-4612.

Beattie, G. A. and C. A. Axtell. 2002. The use of a proU-gfp transcriptional fusion to quantify water stress on the leaf surface, pp. 235-240. In: S. A. Leong, C. Allen, and E. W. Triplett (eds), Biology of Plant–Microbe Interactions, vol. 3. International Society for Plant-Microbe Interactions, St. Paul, MN.

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Effect of volatile organic pollutants on the chemical environment sensed by bacteria on leaves

We are exploring the possibility that exposure of plants to volatile organic compounds (VOCs) changes the chemical environment that bacteria sense on leaves. Specifically, we are testing the hypotheses that 1) plants adsorb organic compounds that are present in the surrounding air, 2) plant cuticles are involved in this adsorption process, and 3) the adsorption of VOCs to plant leaves increases their availability to the resident bacteria. We have developed an experimental system that can measure small changes in the concentration of a target VOC in the air in closed systems with and without target plants, as well as in collaboration with others (link to abstract), have developed a VOC-responsive bacterial biosensor that is being used to investigate bacterial access to the adsorbed VOC.

Selected publications:
Casavant, C., D. Thompson, G. A. Beattie, G. J. Phillips, and L. J. Halverson. Use of a site-specific recombination-based biosensor for detecting bioavailable toluene and related compounds on roots Environmental Microbiology 5:238-249.

Casavant, N. C., G. A. Beattie, G. Phillips, and L. J. Halverson. 2002. Site-specific recombination-based genetic system for reporting transient or low-level gene expression. Applied and Environmental Microbiology 68:3588-3596.

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