Seeing red; the development of pON.mCherry, a broad-host range constitutive expression plasmid for Gram-negative bacteria

Abstract

The development of plasmid-mediated gene expression control in bacteria revolutionized the field of bacteriology. Many of these expression control systems rely on the addition of small molecules, generally metabolites or non-metabolized analogs thereof, to the growth medium to induce expression of the genes of interest. The paradigmatic example of an expression control system is the lac system from Escherichia coli, which typically relies on the Ptac promoter and the Lac repressor, LacI. In many cases, however, constitutive gene expression is desired, and other experimental approaches require the coordinated control of multiple genes. While multiple systems have been developed for use in E. coli and its close relatives, the utility and/or functionality of these tools does not always translate to other species. For example, for the Gram-negative pathogen, Legionella pneumophila, a causative agent of Legionnaires' Disease, the aforementioned Ptac system represents the only well-established expression control system. In order to enhance the tools available to study bacterial gene expression in L. pneumophila, we developed a plasmid, pON.mCherry, which confers constitutive gene expression from a mutagenized LacI binding site. We demonstrate that pON.mCherry neither interferes with other plasmids harboring an intact LacI-Ptac expression system nor alters the growth of Legionella species during intracellular growth. Furthermore, the broad-host range plasmid backbone of pON.mCherry allows constitutive gene expression in a wide variety of Gram-negative bacterial species, making pON.mCherry a useful tool for the greater research community.

Fig 1. Construction of pON.mCherry.

(A) Promoter regions of pXDC50 (parental plasmid) and pON.mCherry. DNA fragments from a mutagenic PCR strategy targeting the lac repressor-binding site were cloned into pXDC50 as described in the Materials and Methods. Black circles identify nucleotide changes between the parental pXDC50 lacO1 operator and the mutated operator in pON.mCherry. (B) pON.mCherry plasmid map. Unique restriction sites are shown; R, EcoRI; N, NdeI; X, XbaI; Sa, SalI; Sp, SphI; H, HindIII. (C), (D) Screening pON.mCherry library in L. pneumophila demonstrates a variety of promoter strengths in the pON mutant operator library.

Fig 2. pON.mCherry yields constitutive mCherry expression in several Legionella species.

(A) & (B) Growth curves showing mCherry expression in (A) L. pneumophila and (B) L. longbeachae. Strains harboring the indicated plasmids were monitored for growth (left panels; log₂ scale) and mCherry expression (right panels; linear scale) in the presence and absence of 1 mM IPTG. (C) Photographs of mCherry expression in the indicated Legionella species.

Fig 3. pON.mCherry does not interfere with induction with other Ptac driven plasmids.

L. pneumophila KS79 harboring both pON.mCherry and pRKJ-GFP was cultured in a 96 well micro-titer plate and growth (A), mCherry production (B) and GFP production (C) were monitored over time in the presence and absence of 1 mM IPTG. As in Fig 2, increases in optical density are plotted on a log₂ scale.

Fig 4. Infection of amoebae with Legionella strains harboring pON.mCherry.

A. castellannii (A) and D. discoideum (B) were infected with the indicated strains of bacteria harboring pON.mCherry and incubated at 30°C or 25°C, respectively, for the indicated times. (C) Bacterial growth was monitored by colony forming units recovered from the infections of A. castelannii at the indicated days post infection for strains without a plasmid (solid lines) and also harboring pON.mCherry (dashed lines). (D) Representative micrographs of A. castellanii cells infected with L. pneumophila harboring pON.mCherry. Left panel, phase contrast image; Right panel, mCherry image. All infections were performed at a multiplicity of infection (MOI) of 1 and repeated in triplicate. Results from a representative experiment are shown.

Fig 5. pON.mCherry functions in several Gram-negative bacterial species.

Plasmids (pMMB207c, black bars; pXDC50, blue bars; pON.mCherry, red bars) were introduced into several Gram-negative bacteria, including (A) E. coli, (B) Acinetobacter baylyi ADP1 and (C) Salmonella typhimurium LT2. The resulting strains were grown for 16 hours in the presence of increasing concentrations of IPTG. mCherry expression is displayed as the mCherry fluorescence divided by optical density to normalize expression values to bacterial growth with error bars representing one standard deviation across three triplicate wells. The experiment was performed in triplicate and data from a representative experiment is shown. (D) Representative photographs showing colonies of the indicated strains harboring pON.mCherry after overnight growth on LB plates at 37°C.