Tetracycline-Associated Regulation of the Transfer Genes of the Bacteroides Conjugative Transposon CTnDOT

125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.Many human colonic Bacteroides spp. harbor a conjugative transposon (CTn) called CTnDOT. CTnDOT carries two antibiotic resistance genes tetQ and ermF. A distinctive feature of CTnDOT is that both its excision and transfer are stimulated by tetracycline. The regulation of excision has been described previously. I provide here the first characterization of the organization and regulation of the CTnDOT transfer (tra) genes. RT-PCR analysis of the region containing the tra genes showed that these genes are transcriptionally coupled in an operon and that expression of this operon is regulated at the transcriptional level. The transcription initiation site of the operon was located 290 bp upstream of the start codon of the first tra gene, traA.A previous study had shown that the excision proteins not only to participate with the integrase to catalyze the excision reaction but are also required to regulate transfer. Expression of the excision gene operon is controlled by a regulatory protein RteC. To determine whether the excision proteins affect tra gene expression directly or act indirectly through RteC, the excision gene operon was placed under control of a heterologous promoter, thereby removing RteC from the regulatory pathway. This construct, in the absence of rteC, was able to support enhanced expression of the tra operon. Thus, the excision proteins act directly to up-regulate tra operon expression and increase the transfer frequency of CTnDOT.Although the entire orf2c operon is responsible for an increase in transcription of tra genes, transfer of CTnDOT was inhibited by a DNA segment that contained the 3' end of one of the excision genes (exc). This segment contained a small open reading frame, rteR. Previous work in our lab found that RteR is acting as a small RNA rather than as a protein. Primer extension analysis showed that rteR was transcribed in the same direction as exc. Unlike exc, rteR was expressed constitutively, not regulated by tetracycline. My work shows that RteR did not affect stability of the tra mRNA, nor did it have a detectable effect on the expression of a traA:: uidA translational fusion. If the effect of RteR was measured instead by RT-qPCR, however, it mediated a 5-fold decrease in transcription of tra genes. In this respect, the effect of RteR differs from that of most other small RNAs, which act at the post-transcriptional level. Taken together, my results show that the regulation of CTnDOT transfer is complex and involves both activator proteins, which also participate in the excision process, and an inhibitory small RNA that is encoded within the excision operon. This regulatory cascade, which links excision to regulation of transfer gene expression may function to ensure that transfer does not occur before excision is complete.CTnDOT has transferred extensively among bacteria found in humans. The same is true of another CTn, Tn916. Both appear to be stably maintained in these human-associated bacteria, but this stable maintenance could be due at least in part to the periodic exposure to antibiotics experienced by such bacteria. I was able to gain access to samples taken from the vaginal tracts of non-human primates (baboons and mangabeys), and this provided an opportunity to ask whether antibiotic resistance genes normally associated with CTns like CTnDOT and Tn916 are similarly widespread in animals that are rarely if ever exposed to antibiotics. I found that tetM, a gene carried by Tn916, and tetQ , a gene carried by CTnDOT were detectable by PCR in many of these samples. Also, using PCR analysis, I was able to show that at least some of these genes were associated with the corresponding CTn. This finding supports the contention that CTns such as CTnDOT are stably maintained in the absence of antibiotic selection. Also, my finding that genes associated with these CTns are surprisingly widespread in the primate vaginal microbiota raises the question of whether there is another condition besides tetracycline in stimulating transfer and maintenance of the CTns.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Antibiotic Resistance Genes in the Vaginal Microbiota of Primates Not Normally Exposed to Antibiotics

Previous studies of resistance gene ecology have focused primarily on populations such as hospital patients and farm animals that are regularly exposed to antibiotics. Also, these studies have tended to focus on numerically minor populations such as enterics or enterococci. We report here a cultivation-independent approach that allowed us to assess the presence of antibiotic resistance genes in the numerically predominant populations of the vaginal microbiota of two populations of primates that are seldom or never exposed to antibiotics: baboons and mangabeys. Most of these animals were part of a captive colony in Texas that is used for scientific studies of female physiology and physical anthropology topics. Samples from some wild baboons were also tested. Vaginal swab samples, obtained in connection with a study designed to define the normal microbiota of the female vaginal canal, were tested for the presence of two types of antibiotic resistance genes: tetracycline resistance (tet) genes and erythromycin resistance (erm) genes. These genes are frequently found in human isolates of the two types of bacteria that were a substantial part of the normal microbiota of primates (Firmicutes and Bacteroidetes). Since cultivation was not feasible, polymerase chain reaction and DNA sequencing were used to detect and characterize these resistance genes. The tet(M) and tet(W) genes were found most commonly, and the tet(Q) gene was found in over a third of the samples from baboons. The ermB and ermF genes were found only in a minority of the samples. The ermG gene was not found in any of the specimens tested. Polymerase chain reaction analysis showed that at least some tet(M) and tet(Q) genes were genetically linked to DNA from known conjugative transposons (CTns), Tn916 and CTnDOT. Our results raise questions about the extent to which extensive exposure to antibiotics is the only pressure necessary to maintain resistance genes in natural settings

The Uranium from Seawater Program at the Pacific Northwest National Laboratory: Overview of Marine Testing, Adsorbent Characterization, Adsorbent Durability, Adsorbent Toxicity, and Deployment Studies

The Pacific Northwest National Laboratory (PNNL) is evaluating the performance of adsorption materials to extract uranium from natural seawater. Testing consists of measurements of the adsorption of uranium and other elements from seawater as a function of time using flow-through columns and a recirculating flume to determine adsorbent capacity and adsorption kinetics. The amidoxime-based polymer adsorbent AF1, produced by Oak Ridge National Laboratory (ORNL), had a 56-day adsorption capacity of 3.9 ± 0.2 g U/kg adsorbent material, a saturation capacity of 5.4 ± 0.2 g U/kg adsorbent material, and a half-saturation time of 23 ± 2 days. The ORNL AF1 adsorbent has a very high affinity for uranium, as evidenced by a 56-day distribution coefficient between adsorbent and solution of log K_D,56day = 6.08. Calcium and magnesium account for a majority of the cations adsorbed by the ORNL amidoxime-based adsorbents (61% by mass and 74% by molar percent), uranium is the fourth most abundant element adsorbed by mass and seventh most abundant by molar percentage. Marine testing at Woods Hole Oceanographic Institution with the ORNL AF1 adsorbent produced adsorption capacities 15% and 55% higher than those observed at PNNL for column and flume testing, respectively. Variations in competing ions may be the explanation for the regional differences. Hydrodynamic modeling predicts that a farm of adsorbent materials will likely have minimal effect on ocean currents and removal of uranium and other elements from seawater when farm densities are <1800 braids/km². A decrease in uranium adsorption capacity of up to 30% was observed after 42 days of exposure because of biofouling when the ORNL braided adsorbent AI8 was exposed to raw seawater in a flume in the presence of light. No toxicity was observed with flow-through column effluents of any absorbent materials tested to date. Toxicity could be induced with some non-amidoxime based absorbents only when the ratio of solid absorbent to test media was increased to part per thousand levels. Thermodynamic modeling of the seawater−amidoxime adsorbent was performed using the geochemical modeling program PHREEQC. Modeling of the binding of Ca, Mg, Fe, Ni, Cu, U, and V reveal that when binding sites are limited (1 × 10^–8 binding sites/kg seawater), vanadium heavily outcompetes other ions for the amidoxime sites. In contrast, when binding sites are abundant, Mg and Ca dominate the total percentage of metals bound to the sorbent