The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

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.

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.