Department of Microbiology, The Ohio State University, Columbus, OH 43210 (U.S.A.)
Received by A. Chakrabarty: 14 October 1991 Revised/Accepted: 4 November/11 November 1991 Received at publishers: 27 November 1991
SUMMARY
A two-component T7 expression system was developed for efficient expression of genes in the nonenteric bacterium, Pseudomonas aemginosa. The first component of the expression system is a bacteriophage-based transposable element that contains a lacUVS/laclq-regulated T7 RNA polymerase gene and a selectable antibiotic-resistance determinant. This ele- ment, designated miniD-180, was stably integrated into the P. aeruginosa PAOI chromosome. The second component of this system includes several improved broad-host-range expression vectors containing the T7 gene 10 promoter and mul- tiple cloning site (MCS). These vectors (pEB8, pEB 11, and pEB12) contain transcriptional terminators (T/4) upstream from the T7 promoter, and T7 terminators downstream from the MCS. Because the T7 promoter is somewhat leaky in these vectors, pEB 14 was constructed to decrease transcription of target genes by basal levels ofT7 RNA polymerase. This vector contains a core sequence of the lac operator located 19 bp downstream from the transcriptional start point of the T7 pro- moter, thereby providing a dually regulated system. The utility of this system was demonstrated by placing a promoterless chloramphenicol acetyltransferase (CAT) cassette under control of the T7 promoter and monitoring the isopropyl-/~-D- thiogalactopyranoside-dependent accumulation of CAT in cell-free extracts of P. aeruginosa. We observed up to nearly a 60-fold increase in CAT levels 4 h post-induction, at which time this polypeptide represented up to 20% of the total sol-
uble protein.
INTRODUCTION
Many Gram= bacteria, particularly Pseudomonads, possess an impressive range of biochemical activities that
allow them to derive their nutritional requirements from a large number of sources. In addition, several microorgaz~- isms within this group are also pathogenic and produce an arsenal of virulence factors (i.e., P. aeruginosa, P. cepa-
Correspondence to: Dr. A. Darzins, Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210 (U.S.A.) Tel. (614)292-0147; Fax (614)292-1538. * Present address: Department of Pathology, Case Western Reserve Uni- versity, Cleveland, OH (U.S.A.).
Abbreviations: Ap, ampicillin; attL, D3112 left terminus; attR, D3112 right terminus; bhr, broad host range; Ble, bleomycin; bp, base pair(s); BSA, bovine serum albumin; CAT, Cm acetyl transferase; cat, gene en- coding CAT; Cb, carbenicillin; Cm, chloramphenicol; A, deletion; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl-~-D-thiogalacto-
pyranoside; kb, kilobase(s) or 1000 bp; Kin, kanamycin; lacUVS, lac promoter carrying the UV5 mutation; LB, Luria-Bertani (medium); MCS, multiple cloning site(s); nt, nucleotide(s); oriT, origin of transfer; onV, origin of vegetative replication; P., Pseudomonas; PAGE, polyacrylamide- gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; PTT, T7 gene 10 promoter; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; R, resistance/resistant; rep, replicon; SDS, sodium dodecyl sulfate; s, sensitive/sensitivity; TI, terminator of the E. coil rrnB operon; 7"14, four tandem copies of TI; Tc, tetracycline; tetA, T¢-resistance-encoding gene of RP4; tetR, To-resistance regulator gene of RP4; To, T7 terminator; tsp, transcription start point(s); ' (prime), denotes a truncated gone at the indicated side; [ ], denotes plasmid-carrier state.
36
cia, P. maitophilia, P. pseudomalle~., P. mallei). Recombinant DNA technology has provided a powerful tool with which to investigate the genetic basis of these properties.
Central to the development of this technology has been the construction of genetic systems used to efficiently ex- press certain genes. In the past this was usually accom- plished in the heterologous prokaryotic host Escherichia cog. However, genes from bacterial hosts such as the op- portunistic pathogen P. aeruginosa as well as from other Pseudomonads can be poorly expressed in E. coii (Buckel and Zehelein, 1981; Clarke and Laverack, 1983; Minton et al., 1983). One possible reason for this difficulty is the significant variation of the Pseudomonas promoter structure from the E. colt consensus sequence (Rothmel et al., 1991). In addition, most of the genes that have been investigated are positively regulated and require specific activator mol- ecules that are presumably absent in E. coil (Hedstrom et al., 1986; Manch and Crawford, 1982; Pasloske et al., 1989). Lastly, some target proteins are produced in small quantities even in the presence of large quantities of spe- cific and stable mRNA. This poor translation is most likely due to the particularly high G+C content of Pseudomonas DNA (i.e., 67% for P. aeruginosa) which dictates a unique codon usage and results in an unfavorable distribution of rare codons (West and Iglewski, 1988). For th~se reasons, it is often des.;xable to perform expression experiments di- rectly in the homologous host under study.
A widely employed gene expression system is based on the ability of the E. coil bacteriophage T7 RNA polymerase to specifically recognize T7 promoters and efficiently tran- scribe downstream sequences (Studier and Moffatt, 1986; Tabor and Richardson, 1985). Relatively little is known concerning the use of T7 RNA polymerase systems in non- enteric bacterial hosts such as Pseudomonas. Davison et al. (1989) have described the T7 RNA polymerase-eontrolled expression of gaIK in Pseudomonas ATCCI9151 using a two-plasmid, heat-induction system. These investigators found a significant amount of GalK activity in uninduced cultures which demonstrated an incomplete repression of the T7 RNA polymerase. However, only a two-fold in- crease in GalK activity was achieved after heat induction. Here, we describe the development and use of a two com- ponent, Lad-regulated T7 expression system which allows the efficient expression of genes in a P. aeruginosa host. This system has been conveniently configured to contain the gene for T7 RNA polymerase in the chromosome under control of the inducible lacUV5 promoter and the target gene on a multicopy plasmid vector under control of the PT~ promoter.
RESULTS AND DISCUSSION
(a) Construction of a Pseudomonas aeruginosa strain where T7 RNA polymerase is prodded by induction of a chromosomal copy of the gene
The introduction of new genetic material into bacteria is an important step toward developing microorganisms with useful activities and usually involves the introduction of recombinant plasmids containing foreign genes that encode desirable gene products. However, plasmids grown in the absence of selective pressure may be unstable with respect to inheritance of the cloned gene. An alternative method to introduce genes on plasmids is the use of transposable el- ements as vehicles to introduce new or altered genetic ma- terial. The P. aeruginosa temperate bacteriophage D3112, like the E. coil phage Mu, uses the homology-independent process of transposition for its replication (Bukhari, 1976; Krylov et al., 1980; Rehmat and Shapiro, 1983). We have previously described the construction of transposable D3112 derivatives which are useful for genetic analysis of P. aeruginosa (Darzins and Casadaban, 1989a,b). These 'disarmed' mini-D3112 elements lack the genes essential for phage growth but retain the terminal sequences required in cis for transposition. Therefore, once established, the mini-D3112 insertions are stable and do not excise them- selves from the chromosome.
The first component of our P. aeruginosa T7 expression system, a mini-D3112 element that contained an inducible T7 RNA polymerase gene and a selectable marker, was assembled initially on a bhr plasmid (Fig. 1) and subse- quently, with the aid of a D3112 helper phage, integrated into the bacterial chromosome. Briefly, the mini-D3112 element D 165 was removed from pADD165 (Darzins and Casadaban, 1989a) by EcoRI digestion and placed into the EcoRI site of the bhr vector pAD462. Vector pAD462 was constructed by cloning the 1.8-kb stabilizing fragment of pRO1600 (Olsen et al., 1982) and the RK2 oriT fragment from pTJS53 (Thomas et al., 1980) into a derivative of pUC19 in which the HindIII site was previously elimi- nated. The resulting plasmid containing mini-D165 was designated pADDI66. Next, a DNA 'cassette' containing both a regulated T7 RNA polymerase gene and the RP4 Tc R determinant was constructed. The first component of the cassette was constructed by replacing the promoter region of the iacl gene on pAR1219 (Davanloo et al., 1984) with the promoter region of the lacI q gene and then clon- ing the resulting 4.0-kb BamHI fragment into the BamHI site of pUC19. The resulting plasmid was designated pADD416. The second component of this cassette was constructed by cloning a 2.5-kb BglII-SmaI RP4 fragment that contained the entire Tc R determinant (Waters et'al., 1983) between the BamHI and Sinai sites of pUCI9. This plasmid was designated pADD353. The "regulatory'
37
Pstl
rep AP~ pROl 600
pAD462 5.3 kb
rep pMB1 oriT
EcoRI
pstl
r e a p ApR rep " ~ EcoRl/ pRO1600
I~pA4B| sacl SmmH~i oriT pADD166
10.2 kb
attL
Ble
Sinai Sa~cl ~.BamHI [-!ind1110848) Hindlll(3548) Sad
EcoRI,I)"~I'~L x,~ . ~ E c o R l ( 4 9 0 0 )
attL attR mini-D165
4.'9 kb
VPsII Sail
iac UV5
'~.-$ail • iSmai • / j ~:oRI ~ Sacl I ~--J- .... 2t.-vm ~.. ~v/es t l
Fig. 1. Construction of the bacteriophage D3112-based traasposable element DIS0 containing an inducible T7 RNA polymerase gene. Symbols EcoRl and HindIIl (next to arrows) indicate cleavage followed by ligation. The termini of the transposable phage D3112 are shown as square brackets. Plasmid pEBI harbors the mini-D3112 element DI80. The plasmids are not drawn to scale.
cassette was assembled by combining the EcoRI-Hindlll fragments of pADD353 and pADD416 (2.5 and 4.0 kb, respectively) with HindlII digested pUC 19. The resulting plasmid was designated pEB553 (Fig. 1). The last step in the construction of the transposable element involved re- moving the Km R fragment of pADD166 by HindlII diges- tion and replacing it with the HindIIl fragment containing the T7 RNA polymerase-laclq-Tc g cassette of pEB553. The resulting plasmid, designated pEB1, harbored the mini-D dement, D180, which would provide our expres-
sion system with a stable and inducible source of T7 RNA polymerase.
To generate mini-D 180 insertions into the P. aeruginosa chromosome pEB1 was introduced into the D3112 cts lysogen CDI0 by mobilization with pRK2013 (Figurski and Helinski, 1979)which provided Mob functions in trans. The bla gone was selected in P. aeruginosa to confer Cb g. A single Tc s Cb g transconjugant was used to prepare a D3112 lysate as previously described (Darzins and Cas- adaban, 1989a) and the resulting lysate was then used
38
to isolate Tc R transductants of P. aeruginosa PAO1 (Holloway, 1969). Any strain of P. aeruginosa that is sen- sitive to D3112 infection can be used to construct a T7 expression strain (Roncero et al., 1990). A single PAO1 Tc R Cb s transductant harboring mini-D 180 was designated ADD1976 and selected for further study.
(b) Construction and properties of bhr T7 expression vec- tors
Plasmid pADD617, a derivative of the lacZ protein fusion vector pRS414 (Simons et al., 1987; A.D., un- published data), was digested with SalI, blunted with Pollk, digested with BamHI and ligated to the 130-bp 8amHI-EcoRV To fragment from pET3a (Rosenberg et al., 1987). The BamHI site of this plasmid, designated pADD618, was subsequently removed by filling-in with Poilk. Plasmid pADD618ABamHI, was linearized with SmaI and ligated to the 182-bp Pvull T7 promoter/MCS fragment of pT7-1 (U.S. Biochemical Co. Cleveland, OH) to yield pADD619. The 3.9-kb StuI-PvuII fragment from pADD619 was combined with the 5.8-kb PstI (PolIk blunted)-PvuII bhr RSFI010 replicon fragment from the IncQ plasmid pKT240 (Bagdasarian et al., 1983). This fragment contains the RSFI010 sequences necessary for replication and mobilization (Scholz et al., 1989). This bhr mobilizable plasmid was designated pEB8 (Fig. 2).
• ~om ~r.~R_!
~p~
rep
pEB8 9,7 k b
lindlll
or iV
oriT
)R
T~
pEB11 9.2 kb
rep .. RSFI010
(c) T7 RNA polymerase dependent expression of cat in EschericMa coli
The promoterless cat gene from pCM7 (Close and Ro- driguez, 1982) was cloned into the HindIII site of pEB8 and this plasmid, designated pEBS-cat, was initially char- acterized in the E. cell T7 expression strain BL21(DE3) (Studier and Moifatt, 1986). Induction of BL21(DE3) [pEBS-cat] cultures with IPTG and SDS-PAGE analysis of whole cell lysates revealed the production of large quan- tities of at least two polypeptides, 25 and 28-29 kDa. The 25-kDa polypeptide was consistent with the size of mono- meric CAT while the large protein band at approx. 28 kDa was consistent with the size of the RepA/RepC replication proteins of RSF1010. Since the RepA and RepC coding regions are located approx. 5 kb downstream from the T7 promoter, this result suggested that the T7 RNA poly- merase was able to continue transcription around the plas- mid well past the T7 terminator. Transcription of these downstream sequences by T7 RNA polymerase was confirmed by the observation that IPTG induction also resulted in a severalfold increase in plasmid copy num- ber (data not shown), a result which is consistent with the findings of Hating et al. (1985). Taken together, these data strongly suggest that the one copy of the To in pEB8 is not sufficient to prevent transcription of downstream plasmid sequences.
EcoRI
TT, I~Ba m H I P Xt~l Hindlll
Pstl,, T ~ oriV ~ .L.~ApR T.e
I " pEBI2 oriV 10.2 kb oriT
rep ~SFI010
/ ~cat / ~ /.Hindlll I . . . . . . . . . . ~ B a m H I I . . . . pcol,t.car lacU ~ X b a l I '~v 8 . 0 k b P T ~ san
./UI R/ - - "17 / \Clal "l)stl Pvull
/Hindlll
~Xbal
\Clal Pstl
I ~,cy 7.2 kb ] | \pMB1 oriTJ,
Pvul l / '~ rep ~Sall pROl600 ~ 'Pst!
P,a/ Fig. 2. Physical m~ps of the T7 bhr expression vectors. The plasmids are not drawn to scale.
Ideally, expression systems should only direct the tran- scription of target genes, therefore, the contribution of ad- ditional copies of the To in preventing transcription ofthese downstream sequences was investigated. The HindllI- PvuII fragment of pEB8 containing the single To was re- placed with the I. l-kb HindIII-Eco RI fragment of pTG 105 which contains two tandem copies of the To (Giordano et al., 1989). This plasmid, designated pEBll (Fig. 2), was further modified by placing an additional two copies of the To from pTG105 into the HindIII and blunted SalI sites. This plasmid, which contained four tandem To termina- tors, was designated pEB12 (Fig. 2). Following transfor- mation of these plasmids into BL21(DE3), IPTG-induced T7 expression studies revealed a progressive decrease in the amount of the RepA and RepC proteins produced as the number of To copies increased. Four tandem copies of the To drastically reduced the production of the RepA/C proteins to the point where they could no longer be detected on Coomassie-stained PAGE gels. This decrease was also followed by a concomitant decrease in plasmid copy num- ber (data not shown).
39
(d) T7 RNA polymerase-dependent expression of c a t in Pseudomonas aeruginosa
The extent of '£7 RNA polymerase-dependent cat gene expression by derivatives of pEB8, pEB11, and pEB12 harboring a promoterless cat cartridge is shown in Fig. 3. Lanes 2, 4, and 6 show that each of these constructs di- rected the production of significant quantities of the CAT protein in response to IPTG induction. Cultures contain- ing pEB8-cat and pEB1 l-cat produced comparable CAT levels, while pEB 12-cat cultures appeared to produce about twice as much CAT protein. Cell extracts of noninduced cultures appeared to produce low levels of the CAT pro- tein when compared with extracts from cells containing only the vector alone (data not shown). To provide quan- titative estimates of the level of CAT accumulation, the amount of CAT protein in cell extracts was determined by reaction with anti-CAT antibody in an ELISA assay (Table I). Consistent with the SDS-PAGE analysis, each uninduced culture contained a small amount of CAT. These levels most likely reflect the high basal activity of the un- regulated T7 promoter in pEB8, pEB 11, and pEB 12. Nev-
1 2 3 4 5 6 7. 8 9 10
Fig. 3. Induction of CAT protein synthesis in P. aeruginosa. Cells carrying plasmids were grown in LB media at 37°C to an A6oo of 0.5-0.6. Where in- dicated, the cultures were induced by the addition of IPTG (2 mM final cone.). After 4 h, the cells were harvested by centrifugation, resuspended in 10 mM Tris.HCi pH 8.0/1 mM EDTA/I mM PMSF and disrupted by passage through a French pressure cell. Cell debris was removed by centrifugation at 14 600 x g for 1 h. The protein content of each extract was determined by the method of Bradford (1976). Aliquots of each extract representing approx. 50 pg of protein were prepared for electrophoresis in 0.1 ~ SDS-12.5% polyacrylamide gels (Laemmli, 1970). The following pairs of lanes show cells without and with IPTG induction, respectively: 1 and 2, ADDI976[pEBS-cat]; 3 and 4, ADDI976[pEBI l-cat]; $ and 6, ADD1976[pEB12-cat]; 7 and 8, ADDI976[pEBI4-cat]. Lane 9 contains 2 pg purified CAT and lane I0 contains size standards of 66, 45, 36, 29, 24, 20.1, and 14.2 kDa (Sigma). The
Plasmids are shown in Fig. 2, See section a for strain and Fig. 3 legend for growth and induction conditions. b The amount of CAT in each cell lysate was determined by ELISA using anti-CAT antibody (5 Prime-3 Prime, Inc.). The amount of CAT present in each cell extract is expressed as ~g CAT/rag protein. The protein content of each extract was determined by the method of Bradford (1976) with BSA as a standard, These experiments were repeated twice with equivalent results, c Ratio of numbers in columns (2) and (3).
ertheless, induction with IPTG resulted in a 7-13-fold in- crease in the amount of CAT, a result which was also consistent with the SDS-PAGE analysis (Fig. 3). Further- more, the CAT protein from extracts of IPTG-induced cultures constituted a major portion (7-15~o) of the solu- ble protein fraction. Of the three vectors, pEB12 consis- tently directed the production of slightly higher mounts of CAT. This may be attributed to the presence of four tan- dem copies of Te downstream from the MCS which we have found leads to a more efficient termination of the T7 RNA polymerase.
An even higher level of control over the T7 RNA poly- merase was achieved with the construction of a dually reg- ulated system. Central to the development of this dually regulated system was the construction of a bhr derivative of pTG105. This pBR322-based plasmid harbors a copy of the promoterless cat gene downstream from the T7 pro- moter. More importantly, however, this plasmid also con- tains the core of the lacO sequence 19 bp downstream from the T7 promoter. Plasmid pTGI05 has previously been shown to provide extremely tight levels of CAT repression, yet allowed high levels of expression following induction in E. coli (Giordano et al., 1989). Plasmid pTG105 was linearized with AvaI, blunted with Pollk and ligated to a 2.7-kb blunt-ended fragment from pAD461 which contains the stabilizing fragment of pRO1600 and the RK2 oriT se- quence. Plasmid pAD461 is identical to pAD462 except for the presence of a HindllI site adjaeer~t to the Pstl site distal to the oriT fragment. The resulting plasmid, desig- nated pEB 14-cat (Fig. 2), was also found to direct the syn- thesis of large quantifies of CAT upon IPTG induction (Fig. 3, lane 8). In addition~ quantitative CAT assays of
ADD1976[pEB 14-cat] cell extracts revealed that this pro- teha constituted approx. 20% of the total soluble cellular protein at 4 h after induction (Table I). More important, however, was the finding that IPTG induction produced nearly a 60-fold increase ha the mount of CAT. Therefore, the use of a regulated T7 promoter in conjunction with an inducible T7 RNA polymerase provided a greater control of target gene expression. Deletion of the HindIII cat cas- sette from pEBl4-cat resulted in the expression vector pEB 14 (Fig. 2).
Even higher levels of repression, especially important for the cloning and expression of potentially toxic gene prod- ucts in P. ae~uginosa may be achieved by the addition of a copy of the lad and/or T7 lysozyme gene (gene 3.5) di- rectly onto the vectors we have described in this report. An additional copy of the lacl gene would increase the mount of the Lac represser inside the cell and serve to further decrease the low-level expression of the T7 RNA poly- merase from the chromosome. In the case of pEB 14 this would also serve to block more efficiently the entry of T7 RNA polymerase to the template at the T7 promoter. The presence of the T7 lysozyme protein has been found to modulate the activity of basal levels of the T7 RNA poly- merase in E. cell (Moffat and Studier, 1987).
The potential use of the T7 expression system for spe- cific [35S ]methionine labeling of proteins in P. aeruginosa distinguishes it from other promoter/expression systems such as the tac promoter (Bagdasarian et ai., 1983) and the XylS-activated p,, promoters of the TeL plasmid (Mer- mod et ai., 1986). We tested the ability of ADD1976 [pEB 14-cat] cultures to synthesize CAT in pulse-chase ex- periments. In the presence of IPTG and rifampicin (200/zg/ml), cell extracts of pEB 14-cat containing cultures were found to contain large quantities of a single 25-kDa polypeptide (CAT) following SDS-PAGE and fluorogra- phy. Only minute quantities of CAT were synthesized in the absence of IPTG (data not shown). We have recently used this system successfully to specifically express the P. aeruginosapiIT gene which is involved in pilus retraction and twitching motility (Whitchurch et al., 1991; P. Truax and A.D., unpublished data).
(e) Conclusion (1) We have constructed a two-component T7 expres-
sion system for use in P. aeruginosa that should be appli- cable to most strains of P. aeruginosa.
(2) This system was conveniently configured to contain the gene for T7 RNA polymerase in the chromosome under control of the inducible lacUV5 promoter and the target gene on a multicopy plasmid vector under control of the T7 o10 promoter.
(3) The utility of this system was demonstrated by plac- hag a promoterless cat cassette under control of the PT7
promoter. We observed up to nearly a 60-fold increase in CAT levels 4 h post-induction at which time this polypep- tide represented up to 20% of the total soluble protein.
ACKNOWLEDGEMENTS
This research was supported, in part, by a grant to A.D. from the American Lung Association. E.B. was the recip- ient of an Ohio State University Honors Scholarship. We wish to thank W.T. McAllister for kindly providing us with pTGI05. We would also like to thank D.R. Galloway, C. Sauer and L.C. Freck for helpful discussions and critical reading of the manuscript.
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