Tightly regulated gene expression system in Salmonella enterica serovar Typhimurium

Abstract

A new Salmonella enterica serovar Typhimurium strain has been constructed to facilitate tightly regulated gene expression. Arabinose-inducible and glucose-repressible expression of a T7 RNA polymerase gene that has been integrated with an adjacent araC-P(BAD) control element into the bacterial chromosome allows dynamic control of T7 promoter-driven RNA transcription.

FIG. 1.

Schematic view of vector construction. Maps of the arabinose-inducible T7 RNA polymerase expression plasmids pSB/aadA/BADT7-1 and pAC/BADT7-1 and their construction intermediates are shown with relevant features. Products of the serial steps of construction are shown at the confluence of schematic arrows leading from that product's constituent parts: for example, pBAD/T7-1 results from the cloning of the NcoI-HindIII-delimited fragment containing T7pol into the pBAD/HisC plasmid. Plasmid names are in bold type, including those of vectors denoted by name alone (pBAD/HisC, pACYC184, and pSB890), which provided backbones for subsequent constructs. Cloned fragments used as inserts for construction steps are represented as ribbons that contain arrows denoting key functional components and their 5′-3′ orientations. The aadA gene in pUC/aadA is interrupted (by insertion into its PvuII site) during the construction of pUC/aadA/BADT7-1, and hence aadA is subsequently represented as two interrupted parts. Pertinent restriction sites are provided; restriction sites listed in parentheses [such as (SmaI) in pSB/aadA/BADT7-1] are destroyed by the cloning process. XhoI (Kl) is an XhoI site made blunt by the Klenow fragment.

FIG. 2.

Assessment of intermediate construction steps. (A) Expression of presumptive T7 RNA polymerase after induction with arabinose. Protein expression is shown for crude cell extracts of E. coli which were transformed either with pSB890 (lanes 1 to 3) or with pSBaadABADT7-1 (lanes 4 to 6) and then exposed during growth in liquid culture to the indicated arabinose concentrations. Lane 7 provides molecular mass markers: a 97.4-kDa band is left of the arrowhead. For lanes 5 and 6, the arrowhead denotes the expression of a protein of appropriate size (∼99 kDa) for T7 RNA polymerase. This expression occurs after induction with arabinose in E. coli transformed with pSBaadABADT7-1 (lanes 4, 5, and 6) but not in pSB890 transformants (lanes 1, 2, and 3), which lack the araC control element and the T7 RNA polymerase gene. (B) In vitro enzymatic activity of T7 RNA polymerase that was recovered from crude cell extracts of bacterial transformants. T7 RNA polymerase activity was assessed by detection of RNA (denoted by the arrowhead) with an ethidium bromide agarose gel, resulting from transcription in vitro of DNA behind a T7 promoter. Purified recombinant T7 RNA polymerase was used as a positive control (lane 8). Other specimens are crude cell extracts of E. coli (lanes 9, 10, and 11) or Salmonella serovar Typhimurium (lanes 12, 13, and 14) transformants. Both E. coli and Salmonella serovar Typhimurium contain plasmids encoding T7 RNA polymerase and the araC control element and either were subjected in liquid culture to treatment with glucose (for repression) or arabinose (for induction) or were not induced, as shown. E. coli cells were transformed with pSBaadABADT7-1 and were used as donors for subsequent conjugation. Salmonella serovar Typhimurium cells were transformed with pBADT7, an intermediate plasmid in the construction of pSBaadABADT7-1 (Fig. 1). (C) Detection of the T7 RNA polymerase gene in bacterial transformants and conjugation products by using PCR amplification. Arrowheads indicate PCR products corresponding with the presence of the T7 RNA polymerase gene. PCR amplification of the T7 RNA polymerase gene from plasmid or genomic DNA is shown for various Salmonella serovar Typhimurium constructs. Lane 15 is a negative control, the parent Salmonella serovar Typhimurium strain (SB300) prior to manipulation. Lanes 16 and 17 are Salmonella serovar Typhimurium transformants containing the T7 RNA polymerase gene in the plasmids pBADT7-1 and pACBADT7-1 (Fig. 1). Lane 18 is a large-scale plasmid preparation of pSBaadABADT7-1 from an E. coli transformant. Lane 19 is the PCR mixture without DNA. Lanes 20 and 21 are Salmonella serovar Typhimurium strains recovered after the selection and screening steps described in the text. The strains in lanes 20 (denoted SB300 A#1) and 21 (denoted SB300 A#14) are with and without a PCR-detectable T7 RNA polymerase gene, respectively. Both SB300A#1 and SB300A#14 were used for the in vivo experiment described in the legend for Fig. 3; SB300A#1 was used for the experiments reported in the legend for Fig. 4.

FIG. 3.

Basal and induced gene expression in E. coli BL21(DE3) and the new Salmonella serovar Typhimurium construct. Northern blots of constitutive and induced RNA transcription are shown for two systems: E. coli lac-based induction with IPTG (lanes 1 and 2) and the new Salmonella serovar Typhimurium system, ara-based induction with arabinose (lanes 3 to 6). The Salmonella serovar Typhimurium in lanes 3 and 4 (SB300A#1) has a PCR-detectable T7 RNA polymerase gene in genomic DNA, whereas the Salmonella serovar Typhimurium in lanes 5 and 6 (SB300A#14) lacks an integrated T7 RNA polymerase gene (Fig. 2C). Note the leaky transcription of T7 promoter-driven RNA even without induction in the E. coli lac system, in contrast to the off-on expression in the new Salmonella serovar Typhimurium ara system. Levels of constitutive RNA expression (assessed here by Northern blotting for the M1 RNA component of RNase P) are grossly similar in all samples, which were total RNA extracts from bacteria pelleted from liquid cultures.

FIG. 4.

Kinetics of gene expression control. Quantitation of on and off gene expression after induction with arabinose and repression with glucose. Northern blot signals of induced T7 promoter-driven RNA expression, normalized to constitutive RNA expression, are shown over time. Specimens are total RNA extracts of Salmonella serovar Typhimurium bacteria pelleted from liquid cultures at various times after the addition of the inducing agent arabinose (□), with arabinose induction followed by washing and by the addition of glucose (○), or without the addition of arabinose (▴).