Lee, K.-S., W. W. Metcalf, and B. L. Wanner. 1992. Evidence for two phosphonate degradative pathways in Enterobacter aerogenes. J. Bacteriol. 174:2501-2510.
Vol. 174, No. 14
JOURNAL OF BACrERIOLOGY, July 1992, P. 4558-4575
0021-9193/92/144558-18$02.00/0 Copyright © 1992, American Society for Microbiology
TnphoA and TnphoA' Elements for Making and Switching Fusions for Study of Transcription, Translation, and Cell Surface Localization MARY R. WILMES-RIESENBERG AND BARRY L. WANNER* Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received 27 August 1991/Accepted 15 May 1992
We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene. Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene. We describe three kinds of elements for making each fusion type. Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance. The third kind is transposition defective (Tnp-) and encodes kanamycin resistance. In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions. Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances. Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype. Further, switching Tnp+ elements to Tnpelements reduces problems due to transposition that can arise in P1 crosses or cloning experiments. Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription. This feature is especially useful in the determination of operon structures. Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described. one element to a different type of fusion. TnphoA' elements are deleted of most of the phoA coding sequence, which in most cases is replaced with a lacZ gene. Several have the lacZ gene transcriptionally or translationally fused to phoA DNA near the fusion end of TnphoA such that they can form lacZ (op) or lacZ (pr) fusions by transposition. TnphoA and TnphoA' elements share identical DNA sequences on both outside ends, a condition that allows for efficient switching of one element for another by homologous recombination. Individual TnphoA, TnphoA'-lacZ (op), and TnphoA'-lacZ (pr) elements are available as transposition-proficient (Tnp+) elements encoding kanamycin or tetracycline resistance and as transposition-defective (Tnp-) elements encoding kanamycin resistance. Also, one TnphoA' element is available as a Tnp- element lacking a reporter gene and encoding chloramphenicol resistance, which was constructed as a tool to aid in switching fusions. Importantly, a fusion made with a Tnp+ TnphoA or TnphoA' element can be switched to a different fusion type simply by selecting transductants with a
Genetic fusions have proven valuable in innumerable studies on gene expression. In particular, lacZ operon (op) and lacZ protein (pr) fusions are useful in studying transcriptional and translational controls, while phoA gene fusions are useful in identifying genes that encode cell envelope proteins and in defining topological determinants of cytoplasmic membrane proteins (11, 16). The transposon TnphoA is an especially valuable tool in studies on cell surface localization, because it allows for the simple in vivo construction of phoA gene fusions by transposition (17). TnphoA fusions make a bacterial alkaline phosphatase (Bap) fusion protein that is active only when the fusion protein is localized to the cell surface. We faced two problems in using TnphoA to study the phosphate (PHO) regulon of Escherichia coli (40, 42). First, the wild-type phoA gene is our most useful reporter gene of the PHO regulon. Second, because the Pi sensor, PhoR, is thought to be a membrane protein, we had expected, but had not found, several Bap+ mutants with in-frame phoR:: TnphoA insertions (8). Therefore, we had to sequence a large collection of randomphoR::TnphoA mutations in order to identify ones that were in frame. The difficulty in finding fusions that produce an active protein is particularly a problem in the use of the TnphoA technique to determine the localization of a gene product whose localization (or sequence) is unknown. To circumvent both problems, we made elements based on TnphoA, called TnphoA' elements, which can form lacZ fusions and switched to TnphoA fusions. We made TnphoA' elements, as well as two new TnphoA elements, so that we could isolate mutants based on their Lac or Bap phenotype and then switch fusions made with *
different drug resistance. We describe the characteristics and use of TnphoA and TnphoA' elements in this paper. Detailed descriptions of the constructions of these elements and information on their molecular structures are in the Appendix.
MATERIALS AND METHODS Media and chemicals. Media and chemicals were usually the same as described previously (38, 44). Antibiotics were added to tryptone-yeast extract (TYE) or glucose M63 agar at 15 ,ug/ml for tetracycline, 50 p,g/ml for kanamycin, 12.5 or 25 ±g/ml for chloramphenicol, and 35 p,g/ml (each) for spectinomycin and streptomycin, except as noted in the text. 5-Bromo-4-chloro-3-indolyl-phosphate-p-toluidine (X-P) and
Corresponding author. 4558
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TnphoA AND TnphoA' ELEMENTS
4559
TABLE 1. Commonly used bacterial strains Straina
BW11397
Alac-169 AphoA8d creB510 supF58 supE44 hsdR514 galK2 galT22 trpR55 metBl tonA
BW13745 BW14697 BW14893
DE3(lac)X74 AphoA532e DE3(lac)X74 AphoA532 recA::cml-aadA DE3(lac)X74 AphoA532 AphnC?DEFGHIJKLMNOP(psiD)33-30F pBW30::TnphoA'-4 \pir/RP4::MuKan recA thr-1 leuB6 lacYl tonA21 supE44 pBW30::TnphoA'-1 Apir/RP4::MuKan recA thr-1 leuB6 lacYl tonA21 supE44 DE3(lac)X74 AphoA532 AphnC?DEFGHIJKLMNOP(psiD)33-30 A(srlC
BW16478
BW16948 BW16655
Pedigreeb
Genotype
Sourcec'
Pro' with P1 on BW7936 (21)
LE392 via BW9115 (Table Al) BD792 via BW13635 (21) BD792 via BW13745 BD792 via BW14331 (21)
Pro' with P1 on BW13247 (21) Cmr with P1 on BW10724 (44) Mel' with P1 on BW9766 (44)
SM10 (34)
Fig. A4
SM1o
Reference 22
BD792 via BW14893
Tcr with P1 on BW9356 (Table
Al)
recA)306::TnlO
BW16842
F'pOX38::TnlO-11/DE3(1ac)X74 AphoA532
BD792 via BW14893
Tcr exconjugant with BW7311
AphnC?DEFGHIJKLMNOP(psiD)33-30
a All strains are E. coli K-12. b Pedigree shows the parent strain from elsewhere and its immediate ancestor in this lab. Reference numbers are in parentheses. c See Materials and Methods. Reference numbers are in parentheses. dphoA8 is a deletion internal to the phoA gene which is also called DE10 or E15. e The AphoA532 deletion removes the PvuII fragment of the phoA gene which includes the promoter and proximal one-half of the structural gene. f The AphnC?DEFGHIJKLMNOP(psiD)33-30 deletion removes genes of the phosphonate transport system that also transports organophosphates such as X-P (21).
5-bromo-4-chloro-3-indolyl-j3-D-galactopyranoside (X-Gal; Bachem, Torrance, Calif.) are the blue dyes for detecting Bap and 3-galactosidase activities, respectively; both were used as described previously (38). [a-32PJdATP was from Amersham Corp. (Arlington Heights, Ill.). Synthetic oligonucleotides were made in the Laboratory for Macromolecular Structure (Purdue University). Bacteria. Strains used routinely are described in Table 1. Additional ones are listed in Table Al. S1247 (zbh-283:: TnlO) was from A. Campbell via B. Bachmann; SM10 (Xpir) and SY327 ()pir) were from J. Mekalanos; and SP265 [A(gal attX bio)76] was from R. Somerville. Other bacteria, including 71-18 (23), BW5104 (23), BW6050 (41), BW14879 (23), JC7623 (47), MC1061 (39), and MG1655Mc (23), were described previously. Plasmids. pGP703.6 was from J. Mekalanos; it is a derivative of pGP703.1 (24) with a BglII linker in the EcoRV site. pHP45fQ (28) and pIC20H (19) were from H. E. Umbarger, pSKS114 (33) was from M. Casadaban, and pUC118 (37) was from J. Messing. Plasmids described previously included F'pOX38::TnlO-ll (1), pBC6APstl (45), pBR322 (46), pBW20 (47), pBW120 (44), pEG5294 (23), pMW1 (46), pRS414, and pRS415 (1). pBW30 is described in Fig. 1; pMW2 is described in Fig. Al; pMW5, pMW6, pMW7, pMW8, pMW9, and pMW46 are described in Fig. A2; and pMWll (23), pMW12, pMW13, and M13mpl8'phoA are described in Fig. A6. pCS3 contains a promoterless lacZ gene cassette; it was made by cloning the 4.3-kbp Dral lacZY fragment of pRS415 (35) into the EcoRV site of pIC20H (32). pMW16 was made by cloning an EcoRI chromosomal fragment of a Xrex::TnphoA' lysogen, BW15185, into pUC118 by selecting kanamycin-resistant (Kanr) transformants (49); consequently, pMW16 carries the Xrex gene with a Tnp- TnphoA' insertion. pMW25 is described below. Phages. XcI857 b221 Pam3 rex::TnphoA (10) was from C. Manoil and is called X::TnphoA in the text. XG217 (imm434cI-2 Aint-9 h80) was from R. Weisberg. XpirR6K (12) was isolated from a RecA+ transductant of SY327 (Apir) following UV induction; it was used to make new Xpir lysogens that were verified by their immunity to XG217 and by their ability to replicatepir-dependent plasmids. XOam or
Pam phages with TnS-112, TnS-132, and TnSseql were described previously (22). XPam phages with TnphoA-132, TnphoAASacII, and TnphoA'-1 to TnphoA'-7 were made as described in the Appendix. All phages were plaque purified on an appropriate host before plate lysates were made by standard methods. Xam phages were grown on the suppressor-positive, Alac phoA+ host BW10168 (Table Al) or the suppressor-positive, Alac AphoA mutant BW11397 (Table 1). Genetic techniques. Conjugations, DNA transformations, and transductions with Plkc were done as described previously (44). SM10 has an RP4 derivative on the chromosome and was used as a donor to transfer RP4 oniT plasmids such as pBW30. ColEl plasmids were transformed into BW6050 (Alac recAl) or MC1061. Chloramphenicol-resistant (Cmr) or tetracycline-resistant (Tcr) transductants were selected after 20 min of adsorption. Kanr transductants were selected after 1 h of growth in Luria-Bertani broth to allow for phenotypic expression. All transductants were routinely purified at least once nonselectively before phenotypes were scored. Mutagenesis with TnphoA and TnphoA' elements. We isolated numerous mutants with TnphoA or TnphoA' insertions in the chromosome or high-copy plasmids (8, 22, 26, 49). To isolate mutants with chromosomal insertions, we selected drug-resistant (Drugr) transductants after infecting cells with Tnp+ XPam rex::TnphoA or XPam rex::TnphoA' phages at a multiplicity of infection of 0.2 to 2.0. To do this, we usually collected about 2 x 108 Luria-Bertani broth-grown, stationary-phase cells (0.1 ml) by centrifugation, resuspended the cells in 0.1 ml of 10 mM MgCl2-5 mM CaCl2, and then infected the cells with 5 ,ul of a phage lysate with a titer of about 1010 to 1011 PFU per ml. To allow for the subsequent switching of the insertions, we generally isolated such mutants from a RecA+, suppressor-negative host, such as BW13745 or BW14893 (Table 1). We measured transposition frequencies in recA AphoA Alac mutants by determining the number of Drugr transductants resulting from infection with a XPam rex::TnphoA or XPam rex::TnphoA' phage at a multiplicity of infection of 0.02 to 5.0. We isolated mutants of Xr E. coli and non-E. coli strains after mating them for 1 to
4560
J. BACTERIOL.
WILMES-RIESENBERG AND WANNER
ori pBW3OR6K rT7pBW30 CC
(3.6 kbp)
L8 e
i
oriT
bla
RP4
I :~Q~:
1 gc
LU~
FIG. 1. Structure of conditionally replicative plasmid. pBW30 has the R6K on region and therefore requires the it protein, the pir gene product, for DNA replication; it replicates only in strains with the pir gene on the chromosome or compatible plasmid. Also, because pBW30 has the RP4 onT region, pBW30 can be conjugatively transferred from strains in which the RP4 tra functions are provided in trans. pBW30 is a derivative of pGP703.6 in which we deleted a nonessential EcoRI fragment. pBW30 plasmids with TnphoA'-l and TnphoA'-4 insertions were made to use as suicide vehicles in transposition and recombinational switching (see text).
A. TnphoA'-IacZ (op) 'phoA GCG GGC GGC TTT TTT GG GAT CCG ACA ACC GAT GM Ala
Gly
Gly
Phe Phe Gly
Asp Pro
Thr
Thr
Asp Glu
AGC GGC GAC GCG CAG TTA ATCOCCA CAG CCG CCA GTT Ser
Gly
Asp
Ala
Gln
Leu
lie
Pro
Gin
Pro
Pro
Val
CCG CTG GCG GCA TmT TM CTTTCTTTATCACACAGGAAA Pro
Leu
Arg
Arg
Phe
lacZ
CAGCT ATG ACC ATG ATT ACG GAT TCA CTG GCC GTC GTT Met Thr
4 h with strains carrying TnphoA'-1 or TnphoA'-4 on the plasmid pBW30 (Fig. 1), such as BW16948 or BW16478 (Table 1), respectively. We selected Drugr exconjugants on agar on which the donor could not grow. We isolated ColEl plasmids with TnphoA or TnphoA' insertions in two ways. One way involved transposition from the chromosome onto a plasmid, which took advantage of the observation that the aph gene in TnS confers high-level kanamycin resistance when in multicopy (16). The second method involved mutagenesis of transformants with X:: TnphoA. Accordingly, we pooled Kanr transductants, isolated plasmid DNAs from several independent pools, and selected transformants of a AphoA or recA mutant with these DNAs on TYE X-P agar with kanamycin. The latter procedure was much more efficient. In both cases, we analyzed the resulting plasmids by restriction mapping and DNA
sequencing.
Recombinational switching of TnphoA, TnphoA', and TnS elements. We switched insertions by infecting Rec+ hosts carrying one TnphoA or TnphoA' element with XPam phages carrying an element with a different drug resistance marker and by selecting for the new drug resistance. We purified transductants nonselectively and then tested them for loss of the parental drug resistance and fusion phenotypes and for the presence of the incoming drug resistance and fusion phenotypes. We switched insertions on plasmids similarly, except that after selecting Drugr transductants, we isolated plasmid DNAs and transformed new hosts with these DNAs in order to identify ones that were switched. Molecular cloning of TnphoA and Tn,phoA' insertions. We used one of four vectors to clone TnphoA and TnphoA' fusion junction fragments. Each vector is described in Fig. A6. We cloned many insertions with the spectinomycin- and
streptomycin-resistant, mini-Mu plasmid pMWll or pMW12 by selecting Kanr transductants. We cloned some fusion junction fragments on the basis of complementation of a lacZct acceptor host by using the lacZa-negative plasmid pMW13. And we cloned some fusion junction fragments on the basis of restoration of a phoA+ gene to the phoAnegative phage M13mpl8'phoA by plating recombinants on X-P agar with an F' phoA host, such as BW16842 (Table 1). The choice of suitable restriction enzymes for such in vitro constructions was dependent on the actual TnphoA or TnphoA' element and was based on the presence of convenient restriction sites, as shown in Fig. A5 and A6. Recombinant DNA methods. Standard procedures were used (4). The dideoxy chain termination method was used to sequence double-stranded DNA templates by using a modi-
Met
lie
Thr
Asp Ser
Leu
Ala
Val
Val
B. TnphoA'-IacZ(pr) +
'phoA
lacZ
GCG GGC GGC TTT ITT GGG GAT CCC GTC GTT TTA CM CGT Ala
Gly
Gly
Phe Phe Gly
Asp Pro
Val
Val
Leu Gln
Arg
FIG. 2. Sequences of phoA-to-lacZ fusion junctions in TnphoAlacZ (op) and TnphoA-lacZ (pr) elements. The first base of both sequences is base 240 of the TnphoA element, which corresponds to base 550 of thephoA gene sequence (7). The vertical arrow marks the DraI-SmaI junction formed in creating TnphoA' elements. The first 254 bp of each element are identical; the first five amino acids shown are residues 68 to 73 of mature wild-type Bap (isozyme 1). (A) The last five phoA gene codons in TnphoA'-lacZ (op) elements, translated codons betweenphoA and lacZ DNA, and the first 11 codons of the lacZ gene. The asterisk marks a discrepancy between our sequence (shown) and the predicted one of pRS415 (35). (The predicted sequence has an additional guanine.) (B) Translated residues near the phoA-to-lacZ (pr) fusion junction in TnphoA'-lacZ (pr) elements, in which lacZ DNA begins at codon 10 of the lacZ gene. BamHI sites are underlined. The junctions were verified by DNA sequence analysis of the respective fusions on pMW7 and pMW8 (Fig. A2).
fied T7 DNA polymerase according to the manufacturer's protocols (Sequenase; U.S. Biochemicals Corp., Cleveland, Ohio). The primer 5'GGGTlTTlCCCAGTCACGACG3' was used to sequence the phoA-to-lacZ junctions on pMW7 and pMW8. The primer 5'AATATCGCCCTGAGCA3' was used to sequence the upstream junctions of TnphoA or TnphoA' insertions on plasmids. Enzyme assays. P-Galactosidase and Bap activities were measured in CHCl3- and sodium dodecyl sulfate-treated cells as described previously (38). Units are expressed as nanomoles of product made per minute at 28 or 37°C. Cell culture optical density (OD) was measured at 420 nm. RESULTS Description of TnphoA and TnphoA' elements. We made two new TnphoA and several TnphoA' elements for use in transposon mutagenesis and recombinational switching. Individual ones can form one of three types of fusions. One set
(TnphoA, TnphoA-132, and TnphoAASacII) can form phoA gene fusions; one set (TnphoA'-1, TnphoA'-2, and TnphoA'3) can form lacZ (op) fusions; and one set (TnphoA'4,
VOL. 174, 1992
>,~ ~
phoA
aph
phoA
tet
phoA
aph
TnphoA
7.7 kbp
Tnp+
De
TnphoA-132
7.7 kbp
Tnp+
&Mmismm
TnphoA ASadl 5.5kbp
Tnp-
TnphoA'-1
6.5kbp
Tnp+
TnphoA'-2
8.1 kbp
Tnp+
TnphoA'-3
5.9kbp
Tnp-
TnphoA'-4
7.8 kbp
Tnp+
TnphoA'-5
8.0 kbp
Tnp+
TnphoA'-6
5.1 kbp
Tnp-
TnphoA '-7
2.8 kbp
Tnp-
TnphoA'-9
5.2 kbp
Tnp-
='
smm
>-
TnphoA AND TnphoA' ELEMENTS
lacZ (op)
aph
lacZ (op)
tet
lacZ (op)
aph
lacZ (pr)
aph
lacZ (pr)
tet
lacZ (pr)
aph
4561
cat
lacZ (op)
aph
E] IS50L
LL
S IS50R 1 kbp | Ii~~~~~~~~~~. FIG. 3. Description of TnphoA and TnphoA' elements. The top line shows TnphoA (16). TnphoA-132, TnphoAASacII, TnphoA'-l, TnphoA'-2, TnphoA'-3, TnphoA'4, TnphoA'-S, TnphoA'-6, and TnphoA'-7were made as outlined in the legends to Fig. Al, A2, A3, and AS. TnphoA'-9 and TnphoA'-10 can result from recombinational switching. TnphoA'-9 is a recombinant of TnphoA'-2 with TnS-112; TnphoA'-10
TnphoA '-10
5.8 kbp
Tnp-
lacZ (pr)
aph 1
is a recombinant of TnphoA'-S with TnphoAASacII. TnphoA'-9 can form lacZ (op) fusions, but it is otherwise analogous to TnphoA'-6; TnphoA'-10 can form lacZ (pr) fusions, but it is otherwise analogous to TnphoA'-3. Restriction maps and detailed structures of each element are described in the legend to Fig. A5. The aph gene encodes aminoglycoside phosphotransferase and is also called the kan gene. The cat and tet genes encode chloramphenicol and tetracycline resistances, respectively.
TnphoA'-5, and TnphoA'-6) can form lacZ (pr) fusions. Each set includes, in order: one Tnp+ Kanr element; one Tnp+ Tcr element; and one Tnp- Kanr element. We also made one additional element called TnphoA'-7. TnphoA'-7 forms no fusion and is Tnp- and Cmr; it is used primarily in recombinational switching as described below. Each TnphoA'-lacZ (op) element has the same phoA-tolacZ fusion junction as does each TnphoA'-lacZ (pr) element. These phoA-to-lacZ junction sequences are shown in Fig. 2. Also, all elements have the same left and right ends, as illustrated in Fig. 3. All TnphoA and TnphoA' elements have 254 bp in common in the left end; all elements have 187 bp or more of ISSOR in common in the right end. Therefore, elements can be switched, one for another, by homologous recombination. Figure 3 also shows the structures of two additional elements called TnphoA'-9 and TnphoA'-10, which are formed by recombinational switching. Transposition of TnphoA and TnphoA' elements. We verified the transposition and fusion phenotypes of TnphoA and TnphoA' elements by using APam phages carrying these elements. Such phage can replicate only in amber suppressor hosts and thus act as a suicide delivery vehicle for the introduction of the elements into nonsuppressor hosts. Some
elements were expected to be Tnp+, while others were expected to be Tnp-. All elements showed the expected transposition phenotype (Table 2). APam phages carrying an element with an intact tnp gene (TnphoA, TnphoA-132, TnphoA '-1, TnphoA'-2, TnphoA'-4, and TnphoA'-5) gave 1.7 x 10-5 to 1.5 x 10-4 Drugr transductants per PFU, which are similar to values for TnS (6). XPam phages with Kanr Tnp- elements (TnphoAASacII, TnphoA'-3, or TnphoA'-6) gave no Kanr transductants. XPam phage carrying the Cmr Tnp- TnphoA'-7 gave a few Cmr colonies; however, they appeared to be spontaneous Cmr mutants. Tnp+ TnphoA and TnphoA'-lacZ elements produced Bap+ and Lac' transductants, respectively, thus verifying the function of their respective reporter genes. To show this, we selected Drugr transductants on X-P or X-Gal agar, as appropriate, and scored the number of blue and white colonies. In each case, we counted more than 1,100 transductants (Table 2). Both TnphoA and TnphoA-132 gave slightly more than 1% that were blue on X-P agar, in close agreement with other data on TnphoA (10). Tnp+ TnphoA'lacZ (op) elements gave about one-third that turned blue on X-Gal agar within 24 h; these transductants probably all had the lacZ gene transcriptionally fused to a nonessential gene
4562
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WILMES-RIESENBERG AND WANNER
TABLE 2. Transposition tests of TnphoA and TnphoA' elements Element
TnphoA TnphoA-132 TnphoAASacII TnphoA'-l TnphoA'-2 TnphoA'-3 TpphoA'4 TnphoA'-S
TnphoA'-6 TnphoA'-7
Transposition feuny frequency"
1.7 2.8
