Robert E. Andrews, Jr

Research Interests

In 1987, Joseph Naglich and I discovered that the conjugal transposon Tn916 was capable of conjugal transfer into Bacillus subtilis and Bacillus thuringiensis from the Enterococcus faecalis. In addition to self-transfer, Tn916 had the ability to mobilize many non-conjugative plasmids such as pUB110, pC194, and pE194 during matings between B. subtilis and B. thuringiensis. Since that time, the work in my laboratory has centered on the biology of Tn916. Two research questions are now the focus in my laboratory.

I) Environmental Genetic Exchange

Two properties of Tn916 cause concern if microbes containing the transposon are released into the environment. First, the organism is able to conjugally transfer between an extremely diverse group of microorganisms, including both Gram-negative and Gram-positive species. Second, in addition to our observations regarding the co-mobilization of plasmids, the laboratory of Gary Dunny (University of Minnesota) reported that Tn925 (which shares extensive homology with Tn916) has the ability to mobilize a broad range of unlinked chromosomal markers during matings between otherwise isogenic strains of B. subtilis. From an environmental standpoint, these observations are important because they may lead to further dispersal of antibiotic resistance genes in the environment. Moreover, if a genetically modified microbe contains a Tn916 element or acquires one after environmental introduction, the transposon may spread allochthonous genetic material to the normal flora of an environment.

Our hypothesis is that, when animal wastes are applied to the soil, broad-range conjugative elements such as Tn916 are able to mobilize themselves and other genetic information of both chromosomal and plasmid origin. The broad range conjugal elements provide not only for mobilization of their own antibiotic resistance, but, once in the soil, provide for fluidity of the gene pool, facilitating mobilization of antibiotic resistance genes of both soil and waste origin. Thus, the effect of manure application is to increase both the content of antibiotic resistance in the soil microbial population, and the diversity of the antibiotic resistant population. This mobilization plays an important role in the development of the antibiotic resistant microbial populations in both animals and humans.

To test this hypothesis, a soil system was chosen. We have demonstrated that there is reason for concern.

ÿ Tn916 transposition and transposon mediated gene transfer can be readily demonstrated in soil microcosms.

ÿ Fifty percent of fecal enterococci in swine wastes contain Tn916-like elements based on DNA homology studies (Southern Blots) and PCR directed against a 712 bp fragment of orf13 (described later).

ÿ When swine manure wastes are introduced into the soil both the Tn916 containing fecal enterococci and the genetic element itself persist in the soil for periods of several months.

ÿ Under some conditions, for example, warmer soils, the evidence suggests an en masse transfer of Tn916 to the soil microflora even when the enterococcal populations are in rapid decline.

Currently, we are adapting the PCR methodologies to detect the presence of Tn916-like elements in soil and surface water environments.

II) Mechanism of Tn916 conjugation

An interesting property of Tn916 (and related transposons, such as Tn925) is that their conjugative mechanisms are very non-specific. That is, they have the ability to mobilize themselves between many Gram positive and negative bacteria; to date, Tn916 movement has been reported for more than 50 species, including Escherichia coli. I have been intrigued by the notion of a non-specific conjugation mechanism. To better define this mechanism, we began a search of the Tn916 genome (18 kb) with the export signal probe TnphoA to identify genes that were targeted for protein export or for the cell membrane. Two genes, orf13 and orf15 appear to encode membrane target proteins, based on the TnphoA fusions. Further study of the TnphoA insertion mutants showed that these gene products were essential for conjugation but had essentially no effect on the intracellular transposition of Tn916. In addition, Tn5 mutagenesis helped identify a third gene, orf21, which is essential for both conjugation and intracellular transposition. Interestingly, the orf21 gene product shows extensive homology with the spoIIIE gene product of Bacillus subtilis. The SpoIIIE (the spoIIIE gene product) seems to function in the packing of the chromosome into the incipient prespore during sporulation, a process that appears much like a conjugation event.

Tn916 mobilizes both plasmid and chromosomal DNA during conjugation. Our initial approach to the problem was to propose that the plasmids mobilized by Tn916 contained a distinct mob region. Unfortunately, elimination of the mob region of pUB110 did not eliminate conjugative transfer. Next, we proposed that, as has been demonstrated for Tn916, an IncP site was the critical site on a mobilizable plasmid. Again elimination of this site had no effect. Our current hypothesis, which derives from the observation that orf21 gene product of Tn916 and spoIIIE of B. subtilis share extensive homology, is that the orf21 gene product recognizes a site on several genetic elements and induces gene transfer from these sites. Clewell and co workers identified an origin of transfer (oriT) on Tn916 and surmised that a 12 base pair sequence is the key binding site. Sites with similar homology exist on all plasmids that are mobilized by Tn916; unfortunately, these sites are located within the origin of replication and cannot be deleted. Homology searches of the B. subtilis chromosome show at least 28 sites with homology in 11 of the 12 base pairs.

Figure 1. Hypothetical model describing the roles of orf13, orf15, and orf21.

Figure 1 shows our current hypothesis in diagram form. orf13 and orf15 encode trans-membrane proteins (Orf13 and Orf15) that function in the conduction of DNA across the cell membrane. orf21 encodes a DNA binding protein (Orf21) that is probably a cytoplasmic component. After binding to either the covalently closed, circular intermediate of Tn916, the DNA-Orf21 complex binds a membrane structure for conjugation. The membrane structure, which includes Orf13 and Orf15, mediates conjugal transfer of the DNA. Alternatively, after binding of Orf21 to the Tn916 circular intermediate, the complex binds to an integration structure, which includes the integrase (Int-Tn) and other proteins, resulting in intracellular transposition. Conjugative transfer of other elements, such as plasmids and chromosome-borne loci occurs because a recognition site, the Orf21 binding site, exists in proximity to the genes of interest.