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?

We are currently focusing on the regulation of excision and transfer a widespread type of Bacteroides CTn, CTnDOT.  Both excision and transfer are stimulated by tetracycline.  We have identified and characterized a complex cascade of regulatory proteins, including the three proteins that start the regulatory process. RteA, RteB and RteC.  Recently, we have found that the genes encoding proteins involved in excision of CTnDOT (orf2c, orf2d and exc) have a second function – regulating the expression of transfer genes.  The excision proteins are necessary and sufficient for increased expression of the transfer genes.  Moreover, we have discovered a small regulatory RNA, RteR, that inhibits production of the transfer proteins.  This is the first small RNA to be identified in the Bacteroides phylogenetic group.  We are currently investigating the mechanism by which this small RNA controls transfer.

Figure. Excision and conjugative transfer of a Bacteroides conjugative transposon, CTnDOT. The first step is excision of the CTn in the donor cell to form a transient circular intermediate. The genes involved in this process are the integrase gene (int) and the excision genes (orf2c, orf2d, exc). A single stranded copy of the circular intermediate is then transferred by conjugation to the donor and both copies are made double stranded before the CTn integrates back into the chromosome.

Representative Publications

Song, B. and A. A. Salyers. 2009. An unexpected effect of tetracycline concentration: Growth phase-associated excision of the Bacteroides mobilizable transposon NBU1. J. Bacteriol. 191:1078-1082.

Malanowska, K., J. Cioni, B. Swalla, A. Salyers, and J. F. Gardner. 2009. Mutational analysis and homology-based modeling of the IntDOT core-binding domain. J. Bacteriol. 191:2330-9.

Jeters, R., G-R Wang, K. Moon, N. B. Shoemaker and A. A. Salyers. 2010. Tetracycline-associated transcriptional regulation of transfer genes of the Bacteroides conjugative transposon CTnDOT. Microbial Drug Resistance 15:309-315.

Park, J., and A. A. Salyers. 2011. Characterization of the Bacteroides CTnDOT Regulatory Protein RteC. J. Bacteriol. 193: 91-97.

Wang, G-R, N. B. Shoemaker, and A. A. Salyers. In press. Interaction between two components of CTn12256, a chimeric Bacteroides conjugative transposon. Plasmid.

Complete Publications List