Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing

Abstract

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI(q)-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZalpha. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.

FIG. 1.

Structures of the pSRK family of broad-host-range expression vectors. The physical maps for each of the three vectors show the locations of the key genetic determinants, including replication (rep), antibiotic resistance (gentamicin [gnt, Gm], kanamycin [kan, Km], and tetracycline [tet, Tc]), and mobilization (mob). The expression cassette, composed of lacI^q, the lac promoter-O₁ complex (PlacZ), and the lacZα (α) coding sequence, is identical in the three vectors. The sequence of the multiple cloning site, located between the NdeI and NheI sites, is shown for each vector. While the sequence of the multiple cloning site is identical among the three vectors, the complements of unique restriction sites (underlined) differ for each.

FIG. 2.

Levels of expression of lacZα from the three pSRK vectors under repressed and induced conditions. Cultures of E. coli DH5α harboring one of the three pSRK vectors or a derivative of pSRKKm in which the lacZα coding region has been deleted (pSRKKm(α⁻) were grown in LB medium with (filled bars) or without (open bars) IPTG (1 mM final concentration) for 4 h. The cells were harvested and assayed for β-galactosidase activity, expressed as Miller units, as described in Materials and Methods. Error bars show standard deviations.

FIG. 3.

The lac promoter of the pSRK vector family is strongly controlled in diverse bacteria. Cultures of A. tumefaciens C58, B. abortus 2308, C. crescentus CB15, P. fluorescens 1855-344, R. leguminosarum 3841, and S. meliloti 1021, each harboring pSRKKm::uidA or pSRKKm(α⁻), were grown in the appropriate medium with (filled bars) or without (open bars) IPTG (1 mM final concentration) as described in Materials and Methods and Results. The cells were harvested and assayed for β-glucuronidase activity, expressed as modified Miller units, as described in Materials and Methods. Error bars show standard deviations.

FIG. 4.

Levels of expression from the lac promoter of the pSRK vector family can be controlled by inducer concentration. A culture of A. tumefaciens C58 harboring pSRKGm::uidA was grown in MG/L medium to early exponential phase and split into eight equal subcultures. IPTG at the indicated final concentrations was added to seven of the subcultures. The eight cultures were sampled after 4 h (open bars) and 8 h (filled bars) of additional incubation and assayed for β-glucuronidase activity, expressed as modified Miller units, as described in Materials and Methods.

FIG. 5.

traR, expressed from pSRKGm, is tightly controlled in Agrobacterium tumefaciens. A culture of A. tumefaciens NTL4 harboring pKPC12 (traR::lacZ) and pSRKGmtraR was grown in ABM minimal medium to early exponential phase, and the culture was split into two equal subcultures, to one of which IPTG (1 mM final concentration) was added. Incubation was continued, each subculture (▪, with IPTG; □, without IPTG) was sampled at the indicated times, and the cells were tested for donor competency in matings with A. tumefaciens C58C1RS as described in Materials and Methods. Growth of the culture, expressed as CFU per ml (○), was monitored by viable-cell counts of samples taken at each time point. Transfer frequencies are expressed as the number of transconjugants obtained per input donor. Error bars show standard deviations.

FIG. 6.

TraM inhibits basal levels of TraR and modulates the activity of induced levels of TraR in controlling Ti plasmid conjugative transfer. Cultures of A. tumefaciens NTL4 harboring one or more plasmids, as indicated, coding for traR (pSRKGmtraR), traM (pYZ1), and a TraR-dependent traA::lacZ reporter fusion (pZLB251) were grown in MG/L medium supplemented with IPTG (1 mM), N-(3-oxo-octanoyl)-l-HSL (AAI; 10 or 100 nM as noted), or both, as indicated, for 4 h; samples were harvested; and the cells were assayed for β-galactosidase activity. Enzyme activity is expressed as Miller units. Error bars show standard deviations.

FIG. 7.

Influence of the expression level of TraR on Ti plasmid transfer frequencies. A culture of A. tumefaciens NTL4 harboring pKPC12 (pTiC58traR::lacZ) and pSRKGmtraR was grown in ABM minimal medium to early exponential phase and divided into eight subcultures. One subculture was left unsupplemented (⋄), while IPTG was added to the remaining seven at final concentrations of 1 μM (▪), 10 μM (▵), 50 μM (♦), 100 μM (•), 250 μM (○), 500 μM (▴), and 1 mM (□). Incubation was continued, each culture was sampled at the indicated times, and the cells were tested for donor competency by mating with strain C58C1RS as described in Materials and Methods. Frequency of transfer is expressed as the number of transconjugants recovered per input donor. Error bars show standard deviations.