Abigail A. Salyers
Professor of Microbiology
abigails@life.uiuc.edu
B.A. (Mathematics), George Washington University, 1963
Ph.D. (Physics), George Washington University, 1969
Assistant & Associate Professor (Physics), St. Mary's College, 1968-1972
Visiting Professor, Virginia Polytechnic Institute, 1973-1978
Antibiotic-resistance gene transfer; interaction of colonic bacteria with host; genetics of obligate anaerobes; conjugative transposons of Bacteroides
Resident microflora of the human colon
The human colon contains a complex population of microbes, most of which are obligate anaerobes. When I first entered this area, virtually nothing was known about the metabolic activities of these anaerobes and they were not amenable to genetic manipulation. I chose to work on Bacteroides, a genus of gram-negative obligate anaerobes that accounts for about 25% of colonic isolates, for several reasons. First, Bacteroides spp. are one of the major populations of colonic bacteria, and there was some evidence that Bacteroides spp. were largely responsible for the fermentation of dietary and host-derived polysaccharides that is one of the main activities of the colonic microflora. Second, Bacteroides spp. are opportunistic human pathogens, which are now causing serious problems because they are resistant to most antibiotics. Finally, Bacteroides is part of a phylogenetic group of bacteria that is distinct from both the gram-positive bacteria and the E. coli-Pseudomonas group of gram-negative bacteria. The Bacteroides phylogenetic group contains Prevotella and Porphyromonas, two genera that are suspected to have a role in periodontal disease. It also contains genera that are important members of the ruminal and intestinal microflora of livestock animals and genera that are found in soil ecosystems. Thanks largely to the efforts of my research group, Bacteroides spp. are now amenable to genetic manipulation, and there is a growing database on their metabolic activities. Until recently, Bacteroides spp. were the only members of this phylogenetic group that were genetically manipulable. Thus, Bacteroides has served as the "E. coli" for this group. The fact that Bacteroides spp. can be manipulated genetically has proved to be very important for studies of their physiology, because it enables us to determine which of the biochemically-detected activities are most important in the intact bacterium.
One of the questions we wanted to address was what activities were important for Bacteroides growing in the colonic environment. As our animal model for colonization studies, we have used the germfree mouse. Germfree mice are mice raised in completely aseptic conditions so that they have no resident microflora. We have done competition experiments between mutants and wild type to determine, for example, which polysaccharides are most important for survival of Bacteroides in the colon.
Mechanisms of polysaccharide utilization
Although my initial interest in Bacteroides polysaccharide utilization systems arose from an interest in what polysaccharides were important for bacteria growing in the colon, I have also become interested in the utilization systems themselves. It is generally assumed that bacteria degrade polysaccharides by secreting extracellular enzymes, which degrade the polysaccharide to subunits small enough to be transported into the cytoplasm. We have shown that Bacteroides spp. use a different strategy. Polysaccharides are first bound to the cell surface and are then translocated across the outer membrane into the periplasm. The degradative enzymes, most of which appear to be located in the periplasm, then break down the polysaccharides and the resulting products are transported across the cytoplasmic membrane into the cytoplasm. This type of strategy makes much more sense in a highly competitive ecosystem such as that found in the human colon than the extracellular enzyme strategy, because it helps to prevent products of polysaccharide breakdown from being lost to competing bacteria.
At present, we are focusing primarily in the binding and uptake steps of polysaccharide utilization. Not only is this probably the rate limiting step in polysaccharide utilization, but the polysaccharide binding complex may also mediate adherence of bacteria to plant particles or to the mucin layer. Although we have investigated utilization of a number of polysaccharides by Bacteroides species, the system we are now focusing on is the starch utilization system of Bacteroides thetaiotaomicron. We have obtained transposon-generated mutants that are deficient in starch binding and we have cloned and partially characterized a 20 kbp segment of the B. thetaiotaomicron chromosome which contains genes encoding binding proteins and regulatory proteins. Recently, we have located two other genetic loci that contain genes involved in starch utilization. We are now undertaking a detailed biochemical analysis of the proteins encoded in this DNA segment.
The significance of this work extends beyond the human colonic Bacteroides. Polysaccharide-degrading gram-negative bacteria related to Bacteroides are found in the rumen and gastrointestinal tracts of livestock. Polysaccharide fermentation by these bacteria contributes significantly to nutrition of the animal, especially in ruminants. Improving the efficiency of polysaccharide fermentation by these bacteria is of considerable economic importance. Also, polysaccharide fermentation plays a major role in the ecology of microbial communities found in landfills and in silts. Some of these polysaccharide-degrading bacteria may also employ a utilization strategy similar to that used by Bacteroides.
Effects of bacterial colonization on the host
Recently, we have been collaborating with Jeff Gordon, a gastrointestinal cell biologist at Washington University, St. Louis, MO, to investigate the effects of bacterial colonization on the murine gastrointestinal tract. Using strains of colonic bacteria provided by us, Gordon's group has discovered the first specific molecular change in intestinal cells attributable to colonization by a specific bacterium: a fucosylated surface antigen that appears on enterocytes when germfree mice are colonized by Bacteroides thetaiotaomicron. A mutant of B. thetaiotaomicron that could not utilize fucose failed to elicit this response. We have been helping Gordon's group analyze the genetic locus in B. thetaiotaomicron that is responsible for this effect. We hope that this model system will help to elucidate ways in which bacteria influence activities of intestinal mucosal cells.
Resistance gene transfer elements of Bacteroides
In the process of developing a genetic system for Bacteroides, we became interested in some novel gene transfer elements called conjugative transposons, which are located in the chromosome. Our results suggest that these gene transfer elements are driving the spread of antibiotic resistance genes within the Bacteroides spp. These elements can also be transferred from Bacteroides spp. to E. coli. The Bacteroides conjugative transposons are at least 60 kbp in size and most carry a tetracycline resistance gene, tetQ. Some also carry other resistance genes. We have cloned and characterized a complex regulatory locus that controls the expression of transfer genes. Transfer functions are induced by low concentrations of the antibiotic, tetracycline. We have also sequenced an 18 kbp region that contains the structural genes that mediate transfer functions, and we are now biochemically characterizing the proteins encoded in this region. The process of broad host range transfer of DNA is not only significant clinically and environmentally, but also poses a fascinating problem in bacterial physiology: How does the transfer apparatus that mediates movement of DNA from donor to recipient form across the cell envelopes of the donor and recipient?
Another unique feature of the Bacteroides elements is that they can excise and circularize 10-12 kbp discrete unlinked DNA segments (designated NBUs, for nonreplicating Bacteroides elements). The excised and circularized NBUs are then mobilized to a recipient where they are integrated in the recipient's chromosome. We have cloned two different NBUs and are currently characterizing genes involved in their excision and mobilization. We have completed the DNA sequence of one of these NBUs and have nearly completed the sequence of the second one. We have identified the integrase gene, which proved to be remotely related to lambda integrase, and three genes that are involved in the excision step. Recently, we have shown that the NBUs can integrate in E. coli, a finding that should facilitate future investigations of the insertion mechanism. We are interested in learning more about the interactions between the conjugative transposons and the NBUs.
Genetic systems for other gram negative anaerobes
Our successful development of a genetic system for the colonic Bacteroides spp. has caused people working on other genera in the Bacteroides phylogenetic group to turn to us for vectors and genetic advice. Our vectors and transposon are now known to work in Porphyromonas and in cytophagas and are being used by a number of laboratories. We have also developed a system for introducing plasmids into the ruminal anaerobe, Prevotella ruminicola. Finally, several groups working on Flavobacterium species are trying our vectors on their strains.
Wang, Y., Wang, G.-R., Shoemaker, N.B., Whitehead, T. and Salyers, A.A. (2004) "Distribution of the ermG gene in bacterial isolates from porcine intestinal contents," Appl. Environ. Microbiol. Accepted pending revisions.
Sutanto, Y., DiChiara, J.M., Shoemaker, N.B., Gardner, J.F., and Salyers, A.A. (2004) "Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT," Plasmid. In press.
Wang, Y., Shoemaker, N.B., and Salyers, A.A. (2004) "Regulation of a Bacteroides operon that controls excision and transfer of the conjugative transposon, CTnDOT," J. Bacteriol. 186:2548-2557. [Abstract]
Gupta, A., Vlamakis, H., Shoemaker, N.B., and Salyers, A.A. (2003) "A new Bacteroides conjugative transposon that carries an ermB gene," Appl. Environ. Microbiol. 69:6455-6463. [Abstract]
Wang, Y., Wang, G.-R., Shelby, A., Shoemaker, N.B., and Salyers, A.A. (2003) "A newly discovered Bacteroides conjugative transposon, CTnGERM1, contains genes also found in Gram-positive bacteria," Appl. Environ. Microbiol. 69:4595-4603. [Abstract]
Whittle, G., Whitehead, T.R., Hamburger, N., Shoemaker, N.B., Cotta, M.A., and Salyers, A.A. (2003) "Identification of a new ribosomal protection type of tetracycline resistance gene, tet(36), from swine manure pits," Appl. Environ. Microbiol. 69:4151-4158. [Abstract]
View Publications by Abigail A. Salyers listed on the National Library of Medicine (PubMed)