Zoopharmacognosy and Your Animals Have you ever noticed your horse, dog or cat eating a certain plant in your garden or out in the fields? Well, if you have, you may be witnessing Zoopharmacognosy. This is a term coined by Dr. Eloy Rodriguez, a biochemist and professor at Cornell University. Zoopharmacognosy refers to the process by which animals self-medicate. In this process, animals select
Bardzo tanie apteki z dostawą w całej Polsce kupic viagra i ogromny wybór pigułek.
Rothlab.ucdavis.eduJOURNAL OF BACTERIOLOGY, Sept. 1997, p. 5827–5834 0021-9193/97/$04.00ϩ0Copyright 1997, American Society for Microbiology A Tn10 Derivative (T-POP) for Isolation of Insertions with Conditional (Tetracycline-Dependent) Phenotypes Department of Biology, University of Utah, Salt Lake City, Utah 84112 Received 29 January 1997/Accepted 14 July 1997 A new Tn10-based transposon has been constructed and used to isolate insertion mutations with tetracy-
cline-conditional phenotypes. Classes of mutants include conditional lethal mutations, conditional auxotrophs,
and conditional mutants of the eut (ethanolamine utilization) operon. The described mutations were made with
a new derivative of Tn10dTet that we have called Tn10d(T-POP). Others have noted that transposon Tn10dTet
directs weak tetracycline-inducible transcripts out of both ends of the element into adjacent sequences. We
have increased this level of outward transcription from Tn10dTet by selecting deletion mutations within the
element that presumably remove transcription-termination signals. Insertion of the Tn10d(T-POP) element
within an operon disrupts the target gene and makes expression of distal genes dependent on induction of
outward transcription by tetracycline. Insertion mutations made with Tn10d(T-POP) can cause tetracycline-
correctable conditional phenotypes based on expression of distal genes.
Transposable elements have been widely used in bacterial In this paper, we describe Tn10dTet derivatives (called T- genetics. The most commonly used elements are derived from POP) that provide higher regulated expression of adjacent transposon Tn10 or from bacteriophage Mu (for a review, see genes due to deletion mutations that allow more tetracycline- reference 19). Insertion of these elements within an operon has induced transcription to proceed out of the transposon. Isola- an extremely strong polar effect on the expression of genes tion of Tn10d(T-POP) insertions between any gene and its downstream of the insertion site. This absolute polarity limits promoter renders expression of that gene dependent on tetra- the usefulness of transposon insertion mutations in comple- cycline. These new Tn10d(T-POP) elements should prove use- mentation tests because multiple genes can be inactivated.
ful in genetic analysis since they avoid the problems of polarity Such polarity effects likewise prevent recovery of insertions in and allow isolation of single-gene null insertion mutations in operons that include essential downstream genes. These limi- tations would be removed by a transposon that provided forthe expression of distal genes.
MATERIALS AND METHODS
The Tn10dTet element is a derivative of transposon Tn10 made by introducing two deletions which remove the inside Bacterial strains. All strains are derivatives of Salmonella typhimurium LT2
(Table 1). The Tn10dTet element is a transposition-defective derivative of trans- ends of both IS10 elements and eliminate both transposase poson Tn10 from which the transposase gene and internal ends of IS10 have genes (31). The deletions leave intact the outside IS10 end been deleted (31). The MudA element is a transposition-defective derivative of sequences (transposase substrates) and the central region phage Mu that provides ampicillin resistance and carries a lacZYA operon for which encodes resistance to tetracycline. This central region creating transcriptional fusions (6, 18). The MudJ element is a transposition-defective derivative of phage Mu described by Castilho et al. (7) encoding includes two genes, tetA and tetR, which are transcribed from resistance to kanamycin. Plasmids encoding Tn10 transposase were kindly pro- divergent tetracycline-inducible promoters. The tetA gene en- codes a tetracycline efflux protein (for a review on Tn10-based Media. Complex medium was nutrient broth (NB; 0.8%; Difco Laboratories)
tetracycline resistance, see reference 16). The tetR gene en- supplemented with NaCl (0.5%). Minimal medium was Vogel and Bonner Emedium (12) with added glucose (0.2%) as a carbon and energy source. When codes a regulatory protein which prevents transcription from analyzing the metabolism of carbon sources other than glucose, minimal E the tetA and tetR promoters in the absence of tetracycline.
medium lacking citrate (NCE) (3) supplemented with the appropriate carbon Tetracycline interacts with the TetR protein to relieve repres- source (0.2%) was used. When required, nutritional supplements were added to sion and induce transcription of both the tetR gene and the tetA E and NCE media at previously recommended final concentrations (12). Thechromogenic ␤-galactosidase substrate 5-bromo-4-chloro-3-indolyl-␤-D-galacto- pyranoside (X-Gal) was added to solid minimal lactose medium (final concen- Studies of the rnc operon using the Tn10dTet element indi- tration, 20 g/ml) to help visualize Lacϩ colonies. Antibiotics were added at the cated that transcripts initiated at the tet promoters extend following concentrations: tetracycline, 20 g/ml (in complex medium) or 10 beyond the boundaries of this element and provide low-level g/ml (in minimal medium); kanamycin, 50 g/ml; ampicillin, 30 g/ml forsingle-copy resistance determinants (MudA elements) or 100 g/ml when main- regulated transcription of adjacent genes (30). This conclusion taining multicopy plasmids with resistance determinants. When selecting LacTD was based on the fact that an insertion of Tn10dTet within the mutants (tetracycline-dependent Lacϩ; see below), tetracycline was used at a rnc operon allows tetracycline-dependent expression of the final concentration of 2 g/ml. Solid media were prepared by the addition of agar distal, essential era gene. However, the level of outward tran- (1.5%; Difco) to NB or minimal medium.
Genetic techniques. Transductional crosses were mediated by the high-fre-
scription from Tn10dTet, as measured by lacZ operon fusion quency generalized transducing phage mutant P22 HT105/1 int-201 (27). Trans- ductants were single colony purified and made phage-free by streaking on non-selective green indicator plates (8). Cross-streaking to check phage sensitivitywas done with a P22 clear-plaque mutant, H5.
Selection of mutants that relieve termination of transcription from Tn10dTet.
* Corresponding author. Mailing address: Department of Biology, A Mud-lac insertion was placed downstream of a Tn10dTet insertion within University of Utah, Salt Lake City, UT 84112. Phone: (801) 581-3412.
either the eut or the his operon. The orientation of the Tn10dTet element was Fax: (801) 585-6207. E-mail: [email protected]
determined by PCR as described below. Selection for mutants that expressed the † Present address: Department of Biology, University of California, lac genes of the Mud-lac reporter was carried out by plating 0.1 ml (approxi- mately 108 cells) from independent, saturated NB cultures onto minimal NCE– ductants are due to transposition of the Tn10dTet element from the transducedparticle. To test the ability of the MudJ element to transpose, lysates from the Lacϩ mutant strains were used in crosses with recipient strain TT8353, whichexpresses the MuA and MuB genes from plasmid pLP103-6-3 (obtained from P.
Van de Putte). Kanamycin-resistant transductants with a Hisϩ phenotype indi- cated Mud transposition to a new chromosomal site.
Wild-type LT2/pLP103-6-3 (Ampr MuAϩ MuBϩ) Reconstruction experiments were done to determine if the LacTD phenotype resulted from modification of the Tn10dTet element or from unlinked mutations Wild-type LT2/pNK972 (Ampr tnpAϩ) that relieved transcription termination. By use of the mutant strains as donors, transductional crosses were done to recombine the Tn10dTet insertions into a recA1/pZT380 (Ampr lacI ptac-tnpA) clean MudJ background and to determine the linkage of the LacTD phenotype to Wild-type LT2/pNK2881 (Ampr tnpA*) the Tetr phenotype of the Tn10dTet element. Final evidence that the mutations eutA208::Tn10dTet (R) eutB8::MudA were confined to the Tn10dTet element was obtained by transposing the eutA208::Tn10dTet[del20] (R) eutB8::MudA Tn10dTet element out of its original insertion site and then transposing it back eutA208::Tn10dTet[del21] (R) eutB8::MudA into the original site in the his operon, a hot spot for Tn10 insertion (15). The eutA208::Tn10dTet[del22] (R) eutB8::MudA first transposition event was obtained by a transductional cross into the recArecipient strain TT14545 (as described above). The resultant strain (carrying an eutA208::Tn10dTet[del23] (R) eutB8::MudA insertion of the element at an unknown site) was then used to create a random hisG10175::Tn10dTet (A) hisC9955::MudJ pool of Tn10dTet derivative insertion mutants in a hisϩ background by a second hisG10175::Tn10dTet[del24] (A) hisC9955::MudJ cross into strain TT14545. This pool (containing at least 20,000 independent hisG10175::Tn10dTet[del25] (A) hisC9955::MudJ transposition events) was screened for HisϪ insertions as outlined below.
hisG10175::Tn10dTet[del26] (A) hisC9955::MudJ The Tetr transductants were pooled, and the free phage titer was reduced by hisG10175::Tn10dTet[del27] (A) hisC9955::MudJ washing the cells twice with E salts. A P22 lysate was prepared on this pool and hisG10175::Tn10dTet[del28] (A) hisC9955::MudJ used to transduce wild-type LT2. The resultant Tetr transductants were replica- hisG10176::Tn10dTet[del25] (R) hisC9955::MudJ eut-240 printed to identify HisϪ auxotrophs. Because the hisG gene includes a major hot hisG10175::Tn10dTet[del20,del25] (R) spot for Tn10 insertion (15), roughly half of the HisϪ insertions are at this single site within the hisG gene. This made it possible to return a mutant element (in hisC9955::MudJ eut-240(eut-cysA deletion) either orientation) to the same site at which it originated.
recA1 zzz-3831::Tn10Tet[del20,del25] Determining the orientation and position of Tn10dTet insertions. To deter-
proAB47/FЈ128 (pro lac) zzf-3832::Tn10Tet[del20] mine which tet promoter was directed towards the Mud-lac reporter fusion, proAB47/FЈ128 (pro lac) zzf-3833::Tn10Tet[del25] Tn10dTet insertions were oriented by the PCR. These reactions were run by use proAB47/FЈ128 (pro lac) zzf-3834::Tn10Tet[del20,del25] of an internal Tn10dTet primer specific for one side of the element and a second primer specific for a neighboring hisG or eutB sequence. This allowed determi- hisG10180::Tn10dTet[del20,del25] (A) hisC9955::MudJ nation of both the orientation of the insertions and their physical location within hisG10181::Tn10dTet[del20,del25] (R) hisC9955::MudJ the his or eut operons.
Molecular characterization of Tn10dTet derivatives. A PCR was performed
hisG10182::Tn10dTet[del20,del25] (R) hisC9955::MudJ with primers internal to the Tn10dTet element to investigate changes in the hisD10183::Tn10dTet[del20,del25] (A) hisC9955::MudJ regions of Tn10dTet distal to the tetR and tetA coding regions. The PCR products hisD10184::Tn10dTet[del20,del25] (R) hisC9955::MudJ obtained from these reactions were purified with the Wizard PCR purification kit hisD10185::Tn10dTet[del20,del25] (R) hisC9955::MudJ (Promega) and sequenced with the Mn2ϩ Reagent Kit for DNA Sequencing hisD10186::Tn10dTet[del20,del25] (R) hisC9955::MudJ (Sequenase version 2.0, kit no. 70130; U.S. Biochemicals).
hisC10187::Tn10dTet[del20,del25] (R) hisC9955::MudJ Placement of Tn10dTet derivatives on F episomes. To construct large pools of
recA1 zzz-3839::Tn10dTet[del20,del26] random insertion events, the Tn10dTet derivatives were moved onto FЈ plasmids proAB47/FЈ128 (pro lac) zzf-3840::Tn10dTet[del26] by transposition. The FЈ episome carries the pro and lac genes and was main-tained by selection for prototrophy in strains carrying chromosomal mutations in proAB47/FЈ128 (pro lac) zzf-3841::Tn10dTet[del20,del26] the proA (TR5656) or the proAB (TR1810) genes. Chromosomal Tn10dTet elements were transduced into a strain (TR1810) carrying the FЈ(pro lac) plas- hlpA1::Tn10dTet[del20,del25] mid. We then introduced into this strain plasmid pZT380, which carries an IPTG purB2372::Tn10d(T-POP) PurTD (isopropyl-␤-D-thiogalactopyranoside)-inducible Tn10 transposase. The addition pyrE2822::Tn10d(T-POP) PyrTD of IPTG (1.0 mM) to an exponentially growing culture of the resultant strain serA1477::Tn10d(T-POP) SerTD induces transposition of the chromosomal Tn10dTet insertion. Following the purB2371::Tn10d(T-POP) PurTD addition of IPTG, the culture was grown to saturation and the FЈ element was serA1478::Tn10d(T-POP) SerTD moved to a new strain (TR5656) by conjugation, with selection for Proϩ andcounterselection of the donor with streptomycin. Exconjugants (Proϩ Strr) were a Tn10dTet insertion orientation relative to downstream mud fusion: (R), tetR screened for tetracycline resistance to identify those that carry a Tn10dTet gene transcribed toward Mud-lac fusion; (A), tetA gene transcribed toward insertion on the transferred FЈ episome. Strains carrying this FЈ plasmid with the Mud-lac fusion. TD (superscript), tetracycline dependent.
Tn10dTet element were used as donors in subsequent transposition experiments;because the FЈ plasmid is of Escherichia coli origin, it does not recombine withthe Salmonella chromosome and the Tn10dTet element can be transductionallyinherited only by transposition.
lactose–X-Gal medium containing tetracycline (2 g/ml). Tetracycline was in- Determination of Tn10d(T-POP) insertion sites. Sequences adjacent to the
cluded to induce the tet promoters, allowing isolation of mutants in which Tn10d(T-POP) insertion in the hlpA gene were amplified by single-primer PCR transcription of the lac genes was dependent on tetracycline. Mutant Lacϩ done under low-stringency conditions (45 cycles of 1 min at 94°C, 1 min at 45°C, colonies arose on the plates after 2 days at 37°C, with additional mutants ap- and 45 min at 72°C; Idaho Technologies Thermocycler). Internal, outward- pearing upon additional incubation for up to 4 days. These Lacϩ colonies were directed primers specific to each side of the Tn10d(T-POP) element were used tested to determine whether their Lacϩ phenotype depended on the presence of individually for this initial PCR. PCR products having one endpoint within the Tn10d(T-POP) element were identified by comparison with the bands produced Genetic characterization of LacTD mutants. Transductional linkage between
with template DNA from an isogenic strain lacking the Tn10d(T-POP) element.
the Tetr phenotype of the Tn10dTet element and the drug resistance of the The unique PCR products were excised and purified with the QIAquick gel downstream Mud element (Kanr for MudJ; Ampr for MudA) was tested. Link- extraction kit. The sequences were reamplified by high-stringency PCR with a ages substantially greater than those observed for the parent strain indicated that nested primer within Tn10d(T-POP) used in 100-fold excess with the original the Lacϩ mutant strain contained a deletion of the intervening region. For primer used for single-primer PCR. The final, somewhat-smaller PCR product mutant strains obtained from the hisG::Tn10dTet insertion, a loss of hisD gene was purified and sequenced as described above. This was done for both sides of expression (required for growth on minimal histidinol medium) also indicated Isolation of Tn10d(T-POP) insertions in the eut operon. New insertions of the
Any mutation (deletion or point mutation) that altered or removed the trans- Tn10d(T-POP) element in the eut operon were isolated by transducing the posase binding sites at the ends of a Tn10dTet element or MudJ element would Tn10d(T-POP) element (from strain TT18797) into a recipient strain expressing be expected to eliminate the ability to transpose. To test for Tn10dTet transpo- the Tn10 transposase from plasmid pNK2881 (19). The recipient also carried a sition, P22 lysates prepared on the Lacϩ mutant strains were used as donors in Mud-lac fusion element in the last gene of the eut operon (eutR); the Mud-lac a cross with a RecAϪ recipient strain (TT14545) that expresses the Tn10 trans- fusion is not expressed since eut operon transcription depends on the EutR posase from plasmid pNK972. The recipient recA mutation prevents inheritance protein (26). Insertions of the Tn10d(T-POP) element were isolated as Lacϩ of the donor Tn10dTet insertion by homologous recombination; all Tetr trans- transductants on minimal NCE-lactose-tetracycline medium and shown to be FIG. 1. Selection systems for modification of Tn10dTet. These abbreviated maps show the positions of Tn10dTet and MudJ insertions within the ethanolamine (eut) (A) and histidine (his) (B) operons of the parent strains. The eutA and eutB genes are the 11th and 12th genes in the ethanolamine operon. The hisG, -D, and -C genesare the first three genes in the histidine operon. The hisG::Tn10dTet insertion is at a hot spot for Tn10 insertion.
LacϪ on medium lacking tetracycline. These candidate insertions were then lactose-tetracycline medium. In the presence of tetracycline, transduced into eutRϩ backgrounds to test their Eut phenotype with and without the parental strain TT18784 containing the hisG::Tn10dTet insertion became HisDϩ, indicating that the low level of tet-racycline-induced transcription of hisD from the parental transposon is sufficient to produce histidine from histidinol.
Modification of left and right ends of Tn10dTet. Insertions
Mutants that expressed the lac genes to a much higher level of Tn10dTet were placed upstream of Mud-lac insertions, and were selected on minimal lactose medium with tetracycline.
the double mutant strains were used to select Lacϩ derivatives Revertants (Lacϩ) appeared at a frequency of 10Ϫ6 to 10Ϫ7/ with increased transcription extending from the ends of the cell plated. The majority of the revertants were shown to be Tn10dTet element into the Mud-lac element. Modification of Lacϩ with or without tetracycline. The LacTD strains were the left (tetR) end of Tn10dTet was selected by use of inser- tested for linkage between the Tn10 and MudJ elements and tions in the eut operon (Fig. 1A). The Tn10dTet insertion was for the ability of the Tn10 element to transpose. Eight inde- in the eutA gene and oriented so that tetR transcription pro- pendent LacTD mutants from the eutA::Tn10dTet insertion ceeds in the same direction as that of the eut operon. The and 16 from the hisG::Tn10dTet insertion were analyzed and MudA element was located in the eutB gene immediately 13 of them behaved as expected for having mutations within downstream. The Tn10dTet element blocks transcription of the Tn10 element. Classification of the LacTD mutants is de- the MudA lac operon from the main eut promoter; inducedtranscription from Tn10dTet is insufficient to provide a Lacϩphenotype. The Mud-lac fusion is not expressed unless a new TABLE 2. Mutant classes obtained in LacTD selection mutation provides for increased transcription.
For mutations affecting the right (tetA) end of Tn10dTet, a similar system was used (Fig. 1B) but in the his operon; the Tn10dTet element was located in the promoter-proximal hisG gene and was oriented such that the tetA gene is transcribed in the same direction as that of the his operon. The MudJ inser- tion was located in the downstream hisC gene. Transcription from the hisG::Tn10dTet element could be monitored by ex- Mutations internal to Tn10dTetd pression of the hisC::MudJ lac genes or by expression of the intervening hisD gene. Since the HisD enzyme converts his- tidinol to histidine, its expression allows a HisϪ strain to grow Assay of ␤-galactosidase activity allowed quantification Mutations internal to Tn10dTetd of the tetracycline-induced transcription emerging from the Tn10dTet element. For the original Tn10dTet element, the Total number of LacTD mutant classes from 10 independent saturated cul- tures of the parent strain TT18778. Mutations characterized in footnotes b to d addition of tetracycline to the growth medium results in a 4.5- are a subset of these LacTD mutants.
to 5-fold increased transcription of the Mud lac reporter ad- b Tn10dTet and MudJ elements were 100% linked (inseparable in transduc- jacent to the tetR or tetA ends of (see Table 3, rows 1 and 6, respectively); this was originally demonstrated by Takiff et al.
The Tn10dTet element was unable to transpose when provided with trans- (30). However, the induced levels of ␤-galactosidase activity d LacTD mutation showed 100% linkage to the Tn10dTet element. The are insufficient to allow the parental strains to grow on minimal Tn10dTet element was able to transpose when provided with transposase.
FIG. 2. Position of deletion mutations within Tn10dTet. Deletions at the tetR end (left) were selected by use of the insertions in the ethanolamine operon; those at the tetA end (right) were selected by use of the insertions in the histidine operon. Numbers indicate the positions of each feature in the DNA base sequence of theparent transposon, Tn10dTet.
scribed in Materials and Methods, and the results are summa- (Fig. 2, del24 to del28). The published sequence (17) shows a rized in Table 2. Six eutA::Tn10dTet mutants and five stem-loop structure, characteristic of rho-independent termi- hisG::Tn10dTet mutants were sequenced; the chosen strains nators, just downstream of the tetA gene. Although this struc- were those showing the greatest induced expression of lacZ. ture seemed a likely candidate site for mutations that allow Based on the inducibility of the Lacϩ phenotype and the increased transcription out of the right end of Tn10dTet, only tetracycline resistance, the LacTD mutations were expected to two of the five deletions (del24 and del26) removed this puta- lie outside of the tetR and tetA coding regions. Oligonucleo- tides were designed to prime PCR amplification of the regions Quantification of transcription levels from Tn10dTet deriva-
between the tetR or tetA gene and the IS10 ends. All PCR tives. Transcription extending from the derivatives of Tn10dTet
products obtained from the mutant strains were smaller than was determined by ␤-galactosidase assays. Without tetracy- those from the respective parent strains, indicating that the cline, transcription from both tet promoters is essentially the LacTD phenotype resulted from deletions within the Tn10dTet element. Sequencing of the PCR products and comparison of the sequence to the parental sequence localized the endpoints.
parent strains (Table 3, column 5), which is consistent with the Five of the six tetR end deletions were characterized. Two of tight repression previously described for the tet promoters (5, the deletions appeared to be identical, leaving four different 10). The addition of tetracycline dramatically increased tran- deletions (Fig. 2, del20 to del23). As expected, none of the scription of downstream lacZ reporter genes to levels well over deletions disrupts the tetR coding sequence. Two deletions 300 ␤-galactosidase units (Table 3, column 6). For many of the (del20 and del23) extend into IS10 material, but neither re- strains containing deletions within the Tn10dTet elements, ex- moves the end sequences necessary for transposition. The col- pression was induced at least 30-fold, reaching a level 5- to lection of deletions at the tetA side was more heterogeneous 10-fold higher than the induced level of the parent strains TABLE 3. Expression of eut::MudA and his::MudJ lac fusions from Tn10 promoters a Tn10dTet insertion orientation relative to downstream Mud fusions: (R), tetR gene transcribed towards Mud-lac fusion; (A), tetA gene transcribed towards Mud-lac b Transcription of downstream lac fusion (in Miller units). For eut::Tn10dTet insertions, a eutB::MudA element was used, for his::Tn10dTet insertions, a hisC::MudJ c Inducing tetracycline was provided in growth medium at 2.0 g/ml.
FIG. 3. Construction of a transposon with two deletion mutations. The diagrammed cross was used to construct T-POP, the double deletion mutant Tn10dTet (del25, del20). The recipient carries mutation del25 but is oriented such that the end needed for expression of the MudJ lac genes has no deletion; this strain isphenotypically LacϪ. The donor strain has mutation del20 at the end nearest the MudA-lac operon. Therefore, this strain is phenotypically LacTD. In the cross, selectionis made for LacTD recombinants; this demands inheritance of the donor mutation del20 (events allowing this are indicated by roman numerals). The donor elementscannot be inherited at the recipient eut locus because it carries a large eut deletion mutation. All recombination events must occur within the two transposons sequencesshared by donor and recipient.
(Table 3, compare rows 1 and 6 to rows 2 to 5 and rows 7 to Tn10dTet element was moved out of the his operon and sub- sequently onto an FЈ plasmid by transposition as described in Construction of recombinant Tn10dTet derivatives with a
Materials and Methods. The element carrying both deletions deletion at both ends of the element. A transductional cross
(Tn10dTet[del20,del25]) has been named Tn10d(T-POP) or was done to form the desired double-mutant element by re- T-POP for the fact it confers (t)etracycline resistance and combination between two single-mutant Tn10dTet elements, utilizes the divergent (p)romoter-(o)perator-(p)romoter sys- one in the eutA gene and one in the hisG gene. This cross is tem for providing expression of adjacent genes. By similar diagrammed in Fig. 3. The donor strain (TT18779) carries a manipulations, a second double-mutant element that carries eutA::Tn10dTet[del20] insertion and a eutB::MudA(lac Ampr) the same tetR end deletion (del20) in combination with a dif- insertion; this strain is phenotypically Lacϩ in the presence of ferent tetA end deletion (del26) has been constructed; the tetracycline. The recipient strain (TT18792) contains an inser- second element has been designated T-POP2.
tion of the Tn10dTet[del25] element in the hisG gene with its Characterization of his mutants generated by the Tn10d(T-
unmodified (tetR) end oriented towards the hisC::MudJ ele- POP) insertion. Insertions of the new Tn10d(T-POP) element
ment; this strain is phenotypically LacϪ. The transduction was at new sites in the his operon were isolated by screening ap- done by selecting for Lacϩ recombinants on minimal lactose- proximately 35,000 independent insertion mutants. These HisϪ tetracycline medium supplemented with histidine and cystine.
insertions were mapped by a set of his deletions to identify The Lacϩ donor and the LacϪ recipient strains have different insertions in the hisGDC region.
sequences flanking their Tn10dTet and Mud elements (either Each of these new insertions of Tn10d(T-POP) was moved eut or his), and recombination in the eut region is prevented by into the hisC::MudJ background by transduction. The location the eut-cysA deletion in the recipient. Therefore, all Lacϩ re- and orientation of the new insertions were determined more combinants must arise by exchange between donor and recip- precisely by PCR. Based on the strong insertion site specificity ient Tn10dTet and Mud transposons. The desired recombinant of the Tn10 transposase (15), strains producing similarly sized type with deletions at each end is expected to have both theTn10dTet and Mud elements within the his operon, separatedby an intervening eut sequence.
Lacϩ transductants were isolated and tested for the LacTD phenotype. All recombinants were HolϪ (even with tetracy-cline) due to loss of the hisD gene, and all showed 100%linkage of the Tetr and Kanr phenotypes since nonhomology ofthe intervening eut sequence prevents recombination betweenthe two elements. The Tn10 elements of several Lacϩ trans-ductants were screened by PCR for possession of the deletionsat either side of the Tn10dTet. A recombinant (TT18793) FIG. 4. Positions of T-POP insertions in the histidine operon. Random in- sertions in the operon were isolated to test the behavior of T-POP at a variety of which showed the bands predicted for each of the two dele- sites. Positions were estimated by PCR. The behavior of these insertions is tions was saved for later use (data not shown). This composite TABLE 4. Transcription levels from his::Tn10d(T-POP) insertions a few his sites were tested and that the method used for thosesites is not very sensitive, since low levels of HisD enzyme are sufficient for growth. However, in many more tests of the eut operon, we have seen no evidence of antisense effects on up- stream gene expression (data not shown).
Isolation and characterization of conditional mutations gen-
erated by Tn10d(T-POP) insertion. To test the general useful-
ness of the Tn10d(T-POP) element, other chromosomal inser- tions were isolated from a pool of random Tn10d(T-POP) insertions. A lysate prepared on this pool of insertions was used to transduce LT2 on NB-tetracycline medium. Tetracy- cline-resistant transductants were replica-printed to minimal E-glucose medium lacking tetracycline to identify insertion a Tn10d(T-POP) insertions orientation relative to downstream Med fusion: mutants that caused auxotrophy or lethality in the absence of (A) tetA promoter directed towards MudJ insertion (R), tetR promoter directed tetracycline. From this initial screen, 21 Tn10d(T-POP) inser- tions were isolated. The classification of these mutants is given Transcription of downstream lac fusion (in Miller units); a hisC::MudJ ele- in Table 5. This same general distribution of mutant types has c Tetracycline was provided in growth medium at 2.0 g/ml.
been observed for a larger set of Tn10d(T-POP) insertionsisolated by a genetics laboratory course. We mapped the mu-tants whose growth phenotype was corrected by tetracycline; PCR products are likely to be insertions at the same site. A set the mapping method was that of Benson and Goldman (2).
of Tn10d(T-POP) insertions is diagrammed in Fig. 4. These One Tn10d(T-POP) insertion (TT19197) exhibited a tetra- strains were chosen based on their having either a distinct in- cycline-correctable, lethal phenotype and mapped to the 4- to sertion site or a distinct orientation. Tetracycline-induced tran- 7-min region of the Salmonella chromosome. To identify the scription levels from these Tn10d(T-POP) elements are pre- exact location of this conditional lethal Tn10d(T-POP) inser- sented in Table 4. Strains in which the lacZ gene is expressed tion, a low-stringency single-primer PCR (as described in Ma- from the tetR promoter showed variability in the levels of tran- terials and Methods) was used to amplify DNA adjacent to scription depending on the insertion site. Insertion of Tn10d the insertion site for sequencing. A FASTA analysis of the (T-POP) at the hisD10184 site exhibited high levels of induced sequences flanking the Tn10d(T-POP) insertion with se- transcription (Table 4, row 5), yet insertion at the hisG10182 quences from the E. coli genome database indicated that the site generated virtually no tetracycline-induced expression of Tn10d(T-POP) insertion was located in the Salmonella ho- ␤-galactosidase (Table 4, row 4). In contrast, transcription from molog of the E. coli hlpA gene. Alignments of the Salmonella the tetA promoter consistently showed a high level of induction sequence on either side of the Tn10d(T-POP) element and the for his operon insertions (Table 4, rows 1 and 2) as well as for E. coli hlpA sequence showed 84 and 85% nucleotide sequence insertions obtained in other operons (data not shown).
identity. Furthermore, the hlpA gene is located at min 4.4 of Investigation of the effects of antisense transcripts from Tn10d
the E. coli genetic map (4). The lethal phenotype of the hlpA (T-POP) elements. With an element that produces transcripts
insertion is likely due to a polar effect on expression of essen- extending out of both ends, the possibility exists that the mes- tial downstream genes; this expression could be restored by the sage directed upstream (opposite that of the target operon) addition of tetracycline. The hlpA gene is part of an operon reduces expression of genes upstream of the insertion site. To that includes essential functions required for the synthesis of investigate this, we examined the HisD phenotype following lipid A, the hydrophobic anchor for lipopolysaccharides in the induction of transcription from a Tn10d(T-POP) element in- outer membrane (for reviews, see references 22 and 28).
serted in the downstream hisC gene (TT19186). Strain TT19186 Thus, the use of the T-POP element provides in vivo evidence showed no difference in growth on minimal E medium-glu- that the hlpA gene is dispensable but is located within an cose-histidinol plates than on identical plates with added tetra- operon that includes distal genes for essential proteins.
cycline (2.0 g/ml). This result and those obtained from Tn10d Identification of phenotypically silent genes in the eut oper-
(T-POP) insertions in the eut operon (see below) show that on. Genetic analysis of the ethanolamine utilization operon
antisense messages from the Tn10d(T-POP) element do not (eut) of Salmonella indicated six genes whose mutation caused cause major interference with the expression of genes up- an aerobic EutϪ phenotype (25). Subsequent sequence deter- stream of the insertion site. It should be kept in mind that only mination (14, 21, 29) has revealed 16 genes in the operon, TABLE 5. Characterization of Tn10d(T-POP) insertions 2 histidine auxotrophs, 2 tryptophan auxotrophs, 2 leucine auxotrophs, 2 proline auxotrophs, 1 methionine auxotroph, 1 arginine auxotroph, 1 with undetermined zusotrophy 2 serine auxotrophs (serA), 2 early purine auxotrophs (purB), 1 uracil auxotroph (pyrE) a Mutants classified by auxanography as described previously (13).
b Standard auxotroph, able to grow on NB medium but not on minimal medium with or without tetracycline; tetracycline-correctable auxotroph, able to grow on NB medium with or without tetracycline but grows on minimal E-glucose medium only if tetracycline is provided (specific gene classification based on auxotrophicrequirement and map position; includes strains TT19764 to TT19768); tetracycline-correctable lethal insertion, able to grow on either minimal or NB medium in thepresence of tetracycline [strain TT19197, hlpA1::Tn10d(T-POP)]; tetracycline-enhanced growth rate, growth on NB medium improved by but not requiring the presenceof tetracycline.
suggesting that many genes lack mutant phenotypes and there- general mechanisms of transcription termination. Currently, fore were missed by the genetic analysis. The Tn10d(T-POP) some termination is thought to occur at particular stem-loop element was used to test this hypothesis and generate single structures in RNA by mechanisms that are independent of dedicated protein factors; alternatively, termination can be Insertions of the Tn10d(T-POP) element in the eut operon caused by rho factor acting at sites that have proved difficult to were isolated and tested for correctability of their EutϪ phe- identify (11, 23, 24). Inspection of the sequences removed by notype by the addition of tetracycline. Two classes of mutants the Tn10 deletions reveals differences between the ends of the were obtained. Insertions in the previously identified eutD, -E, element. The region downstream of the tetA gene includes a -A, -B, -C, and -R genes were phenotypically EutϪ with or stem-loop structure characteristic of rho-independent termina- without tetracycline. Insertions of Tn10d(T-POP) in the eutS, tors. Our deletion mutations indicate that this structure is not -Q, -M, -J, -G, -K, and -L genes showed tetracycline-correctable a major contributor to transcription termination since only two aerobic Eut phenotypes as determined with MacConkey etha- of the five deletions (del24 and del26) remove it. Deletions indicator medium; previous mutant hunts had distal to this stem-loop may remove a region necessary for failed to identify these genes. The new Tn10d(T-POP) inser- rho-dependent transcription termination which is of major im- tions demonstrate that these genes were missed because they portance here. In support of this idea, we have isolated un- do not encode functions necessary for aerobic metabolism of linked suppressor mutations that allow tetA-promoted tran- scripts to extend out of the original Tn10dTet element. Of 20 A secondary use of Tn10d(T-POP) insertions is to select mutants isolated, 19 carried a rho mutation (data not shown).
in-frame deletions of the identified target region. This has The sequence removed by the Tn10dTet-internal deletions been applied to insertions in the eut operon to reinforce the does not fit well with the features of rho termination sites conclusion that some genes are nonessential. Tetracycline-cor- proposed by Alifano et al. (1); they provided evidence that rectable eut::Tn10d(T-POP) insertion mutants were plated, rho-dependent terminator regions encode mRNA rich in cy- without tetracycline, on medium that selects for a Eutϩ phe- tosine and poor in guanine. The two deletions removing the notype. Among the revertant clones were deletions which re- stem-loop show a slightly higher transcription read-through, moved the T-POP element and target gene sequences; the suggesting that the stem-loop structure may play a minor role remaining genes were sufficient for a Eutϩ phenotype. Se- in addition to a more important rho-mediated termination.
quencing of these revertants showed that the deletions did not The sequences between the tetR gene and the end of the disturb the reading frame or else had endpoints near the distal element agree with general features of rho-dependent termi- end of genes; these deletions must be essentially nonpolar to nators in that they show a significant excess of cytosine over provide the Eutϩ phenotype. Using this deletion procedure, we guanine. It is interesting to note that only deletions (no point have confirmed that the eutQ, eutT, eutM, eutN, eutJ, eutG, mutations) were found to reduce termination of messages.
eutH, and eutK genes are nonessential for aerobic utilization of This is reminiscent of the several mutations (all deletions) ethanolamine. The implications of eut gene insertions and de- found to eliminate a rho-dependent termination site within the letions will be detailed elsewhere (20).
Transcription from the tetA end of Tn10d(T-POP) provides strong, regulated expression of adjacent genes in a variety of DISCUSSION
contexts. This is surprising since many adjacent regions would We describe derivatives of transposon Tn10dTet that allow be expected to include rho termination sites. It is possible that regulated, high levels of transcription of chromosomal se- transcripts emerging from the tetA end of Tn10d(T-POP) may quences adjacent to the insertion site. The increased transcrip- be intrinsically resistant to rho termination; alternatively, the tion results from deletion mutations within the Tn10 element tetA promoter may be of sufficient strength that commonly that presumably remove transcription termination signals.
encountered rho sites do not reduce transcription significantly.
These elements (T-POP) should be useful in genetic analysis of Transcription from the tetR side of Tn10d(T-POP) shows lower bacteria and in the study of transcription termination.
expression and more variability in its ability to provide tetra- The use of Tn10d(T-POP) elements permits isolation of cycline-induced expression of downstream Mud-lac fusions insertion mutations with conditional (tetracycline-correctable) than that from the tetA end. Studies on the tet promoters have phenotypes due to transcriptional fusions between the regu- shown that mRNA transcripts from the tetR promoter have a lated tet promoters of the element and a functional chromo- shorter half-life than transcripts from the tetA promoter (21 somal gene nearby. In principle, these could be formed for any compared to 41 s) and that the tetR promoter initiates tran- gene by an insertion between that gene and its promoter.
scription 7 to 11 times less frequently than the tetA promoter However, since most genes are very close to their promoter, (10). Insertions at the same his target site but in opposite such fusions are more common for operons in which an exten- orientation allowed comparison of transcription levels from sive target region of dispensable genes is located between the the tetR and tetA promoters. These results indicate that the tetR gene providing the phenotype and its normal promoter. We promoter is 2.5 to 7.5 times less effective than the tetA pro- describe several auxotrophs, mutants defective for ethanol- moter (Table 4, rows 1, 2, 3, and 5) at expressing the nearby amine utilization, and a lethal mutant, whose growth pheno- lacZ gene. The difference between the earlier promoter studies types are corrected by tetracycline. Tn10d(T-POP)-generated and the ability of T-POP promoters to express nearby genes conditional mutations differ from traditional temperature-sen- probably reflects factors other than promoter strength; that is, sitive (i.e., missense) mutations in that nonpermissive condi- some termination signals may remain within the element or the tions prevent gene expression instead of altering the native two transcripts may have different sensitivities to degradation protein structure; such conditional expression avoids some po- or termination at sites within sequences adjacent to the inser- tential complications inherent in the use of temperature-sen- sitive mutations. The elements described here improve a gen- Both of the T-POP elements described here have been eral utility of Tn10dTet elements first pointed out earlier (30).
moved onto FЈ plasmids (TT18797 and TT19196) for use in Understanding how the described deletions increase tran- construction of pools of random Tn10d(T-POP) insertions in scription out of Tn10dTet may provide some insight into the Salmonella. These plasmids make it possible to introduce T- POP elements into E. coli by conjugation. Transposition of a manual for genetic engineering. Cold Spring Harbor Laboratory, Cold T-POP in E. coli could be selected by transducing the T-POP element from an FЈ plasmid-bearing strain into an FϪ RecAϪ 14. Faust, L. P., J. A. Conner, D. M. Roof, J. A. Hoch, and B. M. Babior. 1990.
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PREDICTING THE EFFECTIVENESS OF HYDROXYUREA IN INDIVIDUAL SICKLE CELL ANEMIA PATIENTS Homayoun Valafar, Faramarz Valafar, Alan Darvill and Peter Albersheim, Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, GA 30602 Abdullah Kutlar, Kristy F. Woods, and John Hardin, Department of Medicin