Kevin P. Bertrand

Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein

AcrAB of Escherichia coli, an archetype among bacterial multidrug efflux pumps, exports an extremely wide range of substrates including solvents, dyes, detergents and antimicrobial agents. Its expression is regulated by three XylS/AraC family regulators, MarA, SoxS and Rob. Although MarA and SoxS regulation works by the alteration of their own expression levels, it was not known how Rob, which is constitutively expressed, exerts its regulatory action. We show here that the induction of the AcrAB efflux pump by decanoate and the more lipophilic unconjugated bile salts is mediated by Rob, and that the low‐molecular‐weight inducers specifically bind to the C‐terminal, non‐DNA‐binding domain of Rob. Induction of Rob is not needed for induction of AcrAB, and we suggest that the inducers act by producing conformational alterations in pre‐existing Rob, as was suggested recently (Rosner, Dangi, Gronenborn and Martin, J Bacteriol 184: 1407–1416, 2002). Decanoate and unconjugated bile salts, which are present in the normal habitat of E. coli, were further shown to make the bacteria more resistant to lipophilic antibiotics, at least in part because of the induction of the AcrAB efflux pump. Thus, it is likely that E. coli is protecting itself by the Rob‐mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract.

Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322.

The Tn10 tetracycline resistance gene, tetA, encodes a tetracycline-inducible protein with an apparent Mr of 36 X 10(3). We have determined the nucleotide sequence of the Tn10 tetA gene. The extent of the tetA gene was determined by analysis of amino-terminal and carboxy-terminal deletion mutants. We conclude that a single Tn10 gene, the tetA gene, is sufficient to confer tetracycline resistance. The predicted Mr of the tetA protein is 43.2 X 10(3). The sequence homology between the Tn10 tetA gene and the pBR322 tetracycline resistance determinant (49% nucleotide homology, 44% amino acid homology) indicates that these phenotypically distinct tetracycline-resistance determinants must have evolved from a common ancestral sequence. The markedly hydrophobic character of the predicted amino acid sequences of the Tn10 tetA and pBR322 tet-coded proteins suggests that a substantial portion of these proteins may be embedded within the cytoplasmic membrane.

Development of recombinant ovalbumin-luteinizing hormone releasing hormone as a potential sterilization vaccine

The objective was to develop an immunogenic chimeric ovalbumin-LHRH (ova-LHRH) molecule using genetic engineering. Hybrid ova-LHRH genes with either four or seven LHRH inserts were constructed by cassette mutagenesis and oligonucleotide mismatch mutagenesis. Recombinant ova-LHRH proteins were over-expressed in E. coli strain BL21 (DE3) using a pET expression system, which expresses a target protein with a C-terminal His-Tag. The C-terminal His-Tag allows purification by metal chelation chromatography. The antigenicity and biological effects of these recombinant proteins were tested in mice. In experiment 1, 17 female 7 wk old BALB/c mice were randomly divided into three groups. Six mice were injected with 50 microg of the recombinant ovalbumin (ova) protein. Five mice were injected with 50 microg of the recombinant protein with four LHRH inserts (ova-LHRH-7). Six mice were injected with 50 microg of the recombinant protein with seven LHRH inserts (ova-LHRH-7). One primary immunization using Freunds complete adjuvant was followed by one booster using incomplete adjuvant. Mice were killed 2 wk after the booster, blood collected, and the reproductive tract removed and weighed. Only ova-LHRH-7 decreased (P < 0.01) uterine-ovarian weight (89+/-11 mg) vs control (138+/-6 mg) and ova-LHRH-4 (126+/-16 mg). The genetically engineered molecule with seven LHRH inserts induced LHRH antibody titers which were significantly correlated (r = -0.79) with biological response. In experiment 2, the recombinant ova-LHRH-7 was evaluated at two doses with the adjuvants Zmax and Immumax. Seventy female 6-8 wk old BALB/c mice were randomly divided into seven groups of 10 mice each. Anti-LHRH titers were detected in all of the ova-LHRH-7 immunized mice. Significant decreases were shown in uterine-ovarian weight of the mice by the immunization with 30 microg of ova-LHRH-7 and Zmax (P < 0.005) or 10 microg of ova-LHRH-7 with Immumax (P < 0.025). These data show that the recombinant ova-LHRH-7 protein could have potential as an effective sterilization vaccine.

Epistatic effects of promoter and repressor functions of the Tn10 tetracycline‐resistance operon on the fitness of Escherichia coli

We have been studying the effects of expression of plasmid‐borne, Tn10‐encoded, tetracycline resistance on the fitness of Escherichia coli K12. We previously demonstrated large reductions in fitness resulting from induced or constitutive expression of the resistance protein; however, any residual expression by the repressed operon was so slight that possession of an inducible resistance function imposed essentially no burden in the absence of antibiotic. Here, we demonstrate two distinct disadvantages for inducible genotypes relative to isogenic constitutive constructs. During the transition from antibiotic‐free to antibiotic‐containing media, the inducible genotype experiences a longer lag phase prior to growth. In the sustained presence of antibiotic, full induction of the resistance function in the inducible genotype is prevented by the continued action of its repressor. However, these disadvantages may be reduced by increasing the strength of the promoter for the resistance gene in the inducible genotype. Simultaneous consideration of the mode of gene regulation (i.e. constitutive or inducible) and the strength of the resistance‐gene promoter (i.e. maximum level of expression) indicates an adaptive landscape with very strong epistasis and, perhaps, multiple fitness peaks.

Mutations in the Tn10 tet represser that interfere with induction. Location of the tetracycline-binding domain

Tetracycline induces transcription of the Tn10 tetracycline resistance gene (tetA) by binding to the tet repressor, thereby reducing the repressors affinity for two operator sites that overlap the tet promoters. We characterized mutations in the tet repressor (tetRs mutations) that interfere with induction of tetA expression. The mutations were isolated on multicopy Tn10 tet plasmids by selecting for resistance to the inducer 5a,6-anhydrotetracycline. Under these conditions, maximal induction of tetA expression inhibits the growth of Escherichia coli K-12. DNA sequence analysis of 25 spontaneous tetRs mutations identified amino acid changes at 13 different positions clustered near the middle of the 207 amino acid residue sequence of tet repressor. This region (residues 64 to 107) is distinct from the bihelical DNA-binding motif of tet repressor (residues 26 to 47). The capacity of tetRs repressors to bind tet operator DNA and to respond to inducer was examined in vivo in tetA-lacZ fusion strains. In three cases, the capacity of tetRs repressors to bind tetracycline was examined in vitro using cell extracts enriched in repressor. Mutations 64Y (His64----Tyr) and 82H (Asn82----His) reduce the repressors affinity for tetracycline more than 1000-fold and more than 100-fold, respectively, suggesting that His64 and Asn82 may be part of the inducer-binding site or directly involved in maintaining its conformation. Mutation 103I (Thr103----Ile) reduces the repressors affinity for tetracycline less than tenfold, yet it interferes with induction to a greater extent than either 64Y or 82H, suggesting that 103I may also reduce the repressors capacity to undergo a conformational change required for induction. The properties of tetRs mutants suggest that the region of amino acid residues 64 to 107 is involved in inducer binding and in signalling between the inducer-binding and operator-binding domains of the repressor.

Immunization of Male Mice with Luteinizing Hormone-Releasing Hormone Fusion Proteins Reduces Testicular and Accessory Sex Gland Function

Abstract Genes for ovalbumin-luteinizing hormone-releasing hormone 7 (LHRH-7) and thioredoxin-LHRH-7 fusion proteins (containing seven LHRH inserts) were constructed by cassette and mismatch mutagenesis and expressed in Escherichia coli. In experiment 1, 10 μg of either ovalbumin-LHRH-7 or thioredoxin-LHRH-7 were suspended in Z-max adjuvant and injected three times at 4-wk intervals into postpubertal male BALB/c mice. In experiment 2, the fusion proteins were suspended in Immumax adjuvant and administered in equimolar quantities (0.4 nmol per injection) to postpubertal male BALB/c mice. In addition to injection of these two proteins alone, the proteins were also administered in different sequences or together in a mixture. Both LHRH fusion proteins induced significant antibody titers, which resulted in a significant decrease in vesicular gland and anterior prostate weight (measure of biological response) in both experiments. Vesicular gland and anterior prostate weight and LHRH antibody titers were significantly correlated in experiments 1 (r = −0.64) and 2 (r = −0.53). Percentage of animals responding to treatment varied from 40–60% in experiment 1 and from 11–89% in experiment 2, with the highest responses in treatments that used a combination of both fusion proteins. The variation in responders and nonresponders was evaluated by estimating antibody KD from displacement curves. Part, but not all, of the high antibody nonresponders can be explained by antibody affinity.

Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.

Nucleotide sequence of class D tetracycline resistance genes from Salmonella ordonez.

SummaryPlasmid pIP173, isolated from Salmonella ordonez strain BM2000, confers resistance to tetracycline and a number of other antibiotics. We determined the nucleotide sequence of the pIP173 tetR repressor and tetA resistance genes. The pIP173 tetR gene is essentially identical to the class D tetR gene from plasmid RA1. The pIP173 tet genes are flanked by directly repeated copies of the insertion sequence IS26. Interestingly, the 3′ end of the tetR gene, encoding the C-terminal 16 amino acids of the TetR protein, extends into the flanking IS26 sequence. The relationships between the class A, B, C, and D TetA sequences parallel the relationships between the corresponding TetR sequences; class D is more closely related to class B than to either class A or C. Overall, the four TetA sequences show 38% identity and 57% similarity.

Trp repressor protein controls its own structural gene

Abstract To study the regulation of expression of the trpR gene of Escherichia coli , fused transcriptional units consisting of the promoter-operator region of trpR and a functional but promoterless lacZ gene were constructed by techniques of cleavage and ligation in vitro . In fusions of this type, the β-galactosidase levels of trpR + haploid strains and strains deleted for the trpR gene were essentially identical. Significant reduction of β-galactosidase production resulted when strains harboring trpR-lacZ fusions were transformed with plasmids that cause overproduction of Trp repressor. This result confirms earlier suggestions (Singleton et al. , 1980) that the trpR gene is regulated autogenously.

Immunization Against Luteinizing Hormone-Releasing Hormone Fusion Proteins Does Not Decrease Prostate Cancer in the Transgenic Adenocarcinoma Mouse Prostate Model

This study was undertaken to test the effect of immunization against luteinizing hormone-releasing hormone (LHRH) fusion proteins on the development and progression of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Two LHRH fusion proteins, ovalbumin with seven LHRH peptides (OV-LHRH-7), and thioredoxin with seven LHRH peptides (TH-LHRH-7) were used in a cocktail vaccine. Two groups of male TRAMP mice were immunized with the cocktail. Primary immunizations were at either 4 or 8 weeks of age. LHRH immunized mice (n = 19) were compared with castrated (n = 19) and intact mice (n = 18) for testosterone concentration, tumor weight, and lifespan. Immunization against LHRH in the TRAMP mice resulted in significant production of antibodies to LHRH compared with surgically castrated and intact control mice. Testicular weight was significantly reduced in the LHRH immunized groups compared with intact control mice. Serum testosterone was reduced (P < 0.05) in the immunized mice compared with intact control mice and was not different from that of castrated mice (P > 0.05). Tumor weight was variable and inconsistent throughout all treatment groups. Lifespan was not increased by immunization against LHRH or castration. Intact control mice (lived the longest (227 ± 11 days), whereas immunized mice lived 206 ± 11 days and castrated mice lived 213 ± 13 days. Tumors from immunized TRAMP mice appeared more aggressive than tumors of castrated and intact mice, as demonstrated by 35% expression of gross lung tumors in the immunized mice whereas none were observed in the castrated or intact TRAMP mice. Prostate cancer is initially dependent upon androgens for growth and development, but cells have the ability to escape androgen dependence and progress to an androgen independent state, which was evident in this study. The TRAMP mouse model immunized against LHRH may have utility in future studies and treatments of the androgen independent prostate cancer.