Steven W. Hutcheson

Professor

Research Interests: Genetics of microbial pathogenesis; role and regulation of type III protein secretion systems; microbial genomics.

The primary research program of my laboratory is examining the role and regulation of type III protein secretion in the pathogenicity of Pseudomonas syringae. In addition, we are working to elucidate the extraordinary degradative abilities of Microbulbifer degradans, a marine bacterium isolated from apparently diseased salt marsh grass in the Chesapeake Bay.

Role and regulation of type III protein secretion in Pseudomonas syringae

The pathogenicity and host range of the gamma-proteobacterium Pseudomonas syringae is linked to a pathogenicity island known as the hrp gene cluster. At least 35 hrp/hrc genes have now been identified in this pathogenicity island, many of which are conserved in bacteria pathogenic to mammals. This gene cluster has recently been shown by my group and others to encode an apparently dedicated regulatory system, a type III protein secretion apparatus, and several secreted protein effectors that are responsible, in part, for the overall pathogenic phenotype of this bacterium.

We are presently characterizing a multi-component regulatory system that controls the environmental regulation of these genes including how the pathogen senses the host. The regulatory system involves an alternative sigma factor, two unusual enhancer-binding proteins and several negative-acting regulatory factors.  Current research is attempting to understand the role of regulated proteolysis in controlling the assembly and activity of the type III secretion system.. A second facet of this research program uses the hrp gene cluster as a means to elucidate the mechanism of type III protein secretion. Type III protein secretion is the most recently discovered mechanism by which bacteria secrete proteins and is best known for its role in the secretion of virulence factors by enteric bacteria pathogenic to mammals. This mechanism is distinct from classic signal sequence-dependent mechanisms or single step secretion systems and involves injection of protein effectors into the host cells. We are looking at chaperones and their role in secretion of effectors. A third facet of this research is attempting to understand the mechanisms by which the translocated effectors control the pathogenicity and host range of the bacterium. For example we have identified a translocated protein tyrosine phosphatase that interrupts signal transduction in the host to suppress defense responses.

Genome-wide analysis of the degradative abilities of Microbulbifer degradans 2-40.

Recently we have begun working on Microbulbifer degradans 2-40 in collaboration with Dr. Ron Weiner, Professor Emeritus whose former students initially characterized the unusual biochemical properties of this bacterium. M. degradans is capable of degrading at least 10 insoluble complex polysaccharides by assembling apparently dedicated enzyme complexes on its cell surface. Through conventional microbial genetics approaches and genome sequence analysis, we have determined that 2-40 expresses over 180 carbohydrases, several of which have very unusual structural properties. For example, the chitinolytic system has been shown to involve three secreted chitinases, two of which have lengthy polyserine domains (>40 residues). A survey of the 2-40 genome revealed that only secreted carbohydrases carry these polyserine domains that are rare in prokaryotes. These polyserine domains appear to be functioning as flexible linkers between anchoring, catalytic or substrate-binding domains. Unusual for carbohydrases, one of the chitinases has two catalytic domains; one of which functions as an endo-chitinase whereas the other has exo-chitinase activity. For the agarolytic system, two demonstrated and three candidate agarases have been identified in the 2-40 genome. Several unusual carbohydrate-binding domains were detected in these agarases that are currently undergoing crystallographic structure analysis. Agarases had previously been thought to be a group of non-orthologous enzymes but the analysis of the 2-40 agarases indicates that agarolytic microorganisms in general produce three families of agarases that are distinguishable by the resident glycosyl hydrolase domain. An apparent agarase-binding protein has been detected in the outer membrane that could be involved in surface localization. The 2-40 genome also appears to express a remarkable number of potentially surface-localized cellulases (20), xylanases (11) and pectate lyases (>7), many of which also have unusual domain structures. The poster presentation on these latter enzymes was awarded best graduate student presentation at the most recent Gordon Conference on Cellulases and Cellulosomes. Antibodies against several proteins of the agarolytic, chitinolytic and cellulolytic systems have just been raised to begin examining the localization of these proteins on the cell surface. Substrate-specific regulation of these systems is also under study. Both mass spectrometry-based proteomics and microarray analysis are being used in these latter studies.

Growth of enteric bacteria on seed sprouts.

After my sabbatical in the laboratory of J. Kaper (UMAB) where I studied the TTS system of an enteropathogenic E. coli strain, I was looking for a project that melded my expertise in plant-microbe interactions with a medically significant bacterium. Salmonella contamination of alfalfa sprouts and other seed sprouts has become a major public health problem. After touring a commercial alfalfa sprout facility to learn their production techniques and by applying methods developed by plant pathologists for studying seed-borne diseases, we showed that while Salmonella strains are generally able to grow on germinating alfalfa seeds under production conditions, this growth is limited to the period of maximal release of reducing sugars and other nutrients from the germinating seed and is independent of known pathogenicity determinants. Populations on germinating seeds were correlated with populations found in the irrigation wastewater. These observations provide the basis for monitoring procedures for Salmonella contamination of alfalfa sprouts during commercial production. The publication summarizing this research was identified as one of most significant papers appearing in any of the 11 journals published by the Amer. Soc. Microbiol. in January 2003 (ASM News 69: 139). We are currently looking at adhesion of Salmonella strains and other enteric bacteria to germinating seeds to understand better the colonization of germinating seeds.

Overview

I would describe myself as a microbial geneticist interested in plant-microbe interactions. Skills students can learn in my laboratory include molecular and microbial genetics, gene cloning and characterization, genomics, proteomics, bioinformatics, and a variety of biochemical and immunological analyses. We function as a team so that students learn not only the techniques related to their own project, but they also learn, or at least hear about, the techniques and approaches used in the other projects. It is important to realize that the skills learned in theses projects are broadly applicable. Students studying the P. syringae hrp system also learn about closely related systems of mammalian pathogens, such as the Ysc system of Yersinia, as well as the basic processes of mammalian innate immunity. As I frequently speak at medical schools on this project, my students have the flexibility and skills to go on to study nearly any host-pathogen interaction. The Microbulbifer project incorporates many aspects of genomics, proteomics and microbial cell biology. Thus, students in any of these projects can expect to graduate with the skills necessary to be competitive in the current job market.

Teaching

I presently teach an undergraduate course on microbial genetics and administer the undergraduate Honors program for the Department. I also teach a graduate-level course on the "In and outs of grant proposal writing" alternate years. I work closely with students in the laboratory and try to work on the bench myself as much as time allows.

Recent Publications:

Elliott, S. J., V. Sperandio, J.A. Giron, J.L. Mellies, L. Wainwright, S.W. Hutcheson, T.K. McDaniel, and J.B. Kaper, 2000. The Lee-encoded regulator (Ler) controls the expression of both LEE and non-LEE encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infection Immunity 68: 6115-6127

Hutcheson, S.W., J. Bretz, T. Sussan, S. Jin, S. Heu, and K. Pak, 2001. The enhancer binding proteins HrpR and HrpS interact to regulate type III protein secretion in Pseudomonas syringae strains. J. Bacteriology 183: 5589-5598

Hutcheson, S.W., 2001. The molecular biology of hypersensitivity to bacterial pathogens. J. Plant Pathology 83: 151-172

Bretz, J., L. Losada, K. Lisboa, and S. Hutcheson, 2002. Lon protease regulates type III protein secretion in Pseudomonas syringae. Molecular Microbiology 45: 397-410

Howard, M., and S. Hutcheson, 2003. Growth dynamics of Salmonella enterica strains on alfalfa sprouts and in waste irrigation water. Applied Environmental Microbiology 69: 548-553 (ASM Journal Highlights; March 2003)

Charity, J., K. Pak, C.F. Delwiche and S. Hutcheson, 2003. Novel exchangeable effector loci associated with the hrp pathogenicity island of P. syringae: evidence for integron-like assembly from transposed gene cassettes. Molecular Plant Microbe Interactions 16: 495-507

Howard, M., N. Ekborg, L. Taylor, R. Weiner, and S. Hutcheson, 2003. Genome-wide analysis and initial characterization of the chitiniolytic system of Microbulbifer degradans strain 2-40. J. Bacteriology 185: 3352-3360

Bretz, J, N. Mock, J. Charity, S. Zeyad, CJ Baker and SW Hutcheson, 2003. A translocated protein tyrosine phosphatase of Pseudomonas syringae pv. tomato DC3000 modulates plant defense response to infection, Molecular Microbiology 49:389-400

Losada, L., K. Pak, T. Sussan, S. Zeyad, I. Rozenbaum, and S.W. Hutcheson, 2004. Identification of a novel virulence and avirulence determinant of Pseudomonas syringae Psy61 by a HrpL-dependent promoter trap assay.  Molecular Plant Microbe Interactions 17: 252-264

Howard, MB, RM Weiner, SW Hutcheson,2003. Identification and characterization of chitinases and other chitin modifying enzymes. J. Industrial Microbiology and Biotechnology 30:627-635

Howard, MB, NA Ekborg, LE Taylor II, RM Weiner, and SW Hutcheson, 2004. Chitinase B of Microbulbifer degradans 2-40 contains two glycosyl hydrolase family 18 domains with different chitinolytic activities. J. Bacteriology 186: 1297-1303

Howard, MB, N. Ekborn, L. Taylor, SW Hutcheson, and RM Weiner, 2004. Polyserine domains separate functional domains in 47 deduced carbohydrases of Microbulbifer degradans 2-40. Protein Science 13: 1422-1425

Bretz, J and SW Hutcheson, 2004. Role of type III effectors during pathogenesis in another kingdom, 2004. Infection and Immunity 72: 3697-3705

Losada, L. and S. Hutcheson, Lon-mediated degradation of effectors modulates type III protein secretion from Pseudomonas syringae strains, Molecular Microbiology (IN REVISION)

Ekborg, N., L. Taylor, M. Howard, R. Weiner, and S. Hutcheson, Genomic and proteomic analysis of the beta-agarases produced by the marine bacterium Microbulbifer degradans 2-40,  (SUBMITTED)

Taylor, L., M. Howard, N. Ekborg, S. Hutcheson, and R. Weiner, Genome-wide analysis of the cellulolytic system of Microbulbifer degradans 2-40.,(IN PREPARATION)

Weiner, R., N. Ekborg, M. Howard, L. Taylor, others and S. Hutcheson. Genome Wide Analysis of a Marine Bacterium Consequential in the Carbon Cycle of the Ocean. (INVITED SUBMISSION TO "SCIENCE").