Mini-Tn7 Insertion in an Artificial attTn7 Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens

Abstract

Mechanistic studies of many processes in Agrobacterium tumefaciens have been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmid-based systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes in A. tumefaciens Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable.

Importance: Agrobacterium tumefaciens is a bacterial plant pathogen and natural genetic engineer. Thus, studies of essential processes, including cell cycle progression, DNA replication and segregation, cell growth, and division, may provide insights for limiting disease or improving biotechnology applications.

FIG 1

Construction of an a-attTn7 site in A. tumefaciens. (A) The native att site (underlined in red) is located at the 3′ end of the glmS gene (Atu1786) in A. tumefaciens. Insertion of Tn7 constructs into the native att site is not neutral, presumably due to the overlap of the glmS stop codon (red bar) with the start codon (blue bar) of the downstream gene encoding an AT (Atu1785). (B) The entire boxed sequence containing the att site in panel A was introduced into a cryptic tetracycline locus on the A. tumefaciens chromosome. Allelic exchange was used to replace the cryptic tetracycline locus (tetRA; Atu4205 to Atu4206) with the a-attTn7 site. The mini-Tn7 cassette contains an antibiotic resistance gene (Ab^R), the lacI repressor, and either a strong (P_tac) or weak (P_lac) promoter to drive a gene of interest (GOI). The left (Tn7L) and right (Tn7R) ends of Tn7 are indicated by the striped bars. (C) Replacement of the tetRA locus with a-attTn7 (dashed line) and subsequent insertion of a mini-Tn7 cassette (gray line) do not impact cell growth in ATGN compared to the wild-type parental strain (C58; solid black line).

FIG 2

Chromosomal and plasmid-based complementation of motility in Δrem. (A) Schematics of strains used, comparing single-copy chromosomal complementation (strains 1 to 4) and multiple-copy plasmid complementation (strains 5 to 8). (B) Strains 1 to 4 were assayed for swimming motility on ATGN soft-agar plates in the presence (black bars) or absence (white bars) of the inducer, IPTG. The data were normalized to the results obtained in the wild-type strain (strain 1). S.E., standard error of the mean. (C) Strains 5 to 8 were assayed for swimming motility on ATGN soft-agar plates containing kanamycin to maintain the plasmid in the presence (black bars) or absence (white bars) of the inducer, IPTG. In the absence of the inducer, we repeatedly observed an increase in the mucoidy at the center of the swim rings. In the Δrem strains, which are nonmotile, this increase in mucoidy leads to a slightly larger swim ring diameter. The data were normalized to the results obtained in the wild-type strain containing an empty vector (strain 5). Swim ring diameters were measured after 5 days of incubation at room temperature. The data shown are the means of the results of three independent experiments completed in triplicate.

FIG 3

Chromosomal and plasmid-based complementation of biofilm formation in ΔuppX. (A) Schematics of the strains used, comparing single-copy chromosomal complementation (strains 1 to 4) and multiple-copy plasmid complementation (strains 5 to 8). (B) Strains 1 to 4 were assayed for biofilm formation on vertical plastic coverslips immersed in ATGN medium in the presence (black bars) or absence (white bars) of the inducer, IPTG. The data were normalized to the results obtained in the wild-type strain (strain 1). (C) Strains 5 to 8 were assayed for biofilm formation in ATGN medium containing kanamycin to maintain the plasmid in the presence (black bars) or absence (white bars) of IPTG. The data were normalized to the results obtained in the wild-type strain containing an empty vector (strain 5). For all the strains, the coverslips were removed after 48 h of incubation at room temperature and rinsed to remove any loosely associated cells. Adherent biomass was determined as the absorbance of solubilized crystal violet (A₆₀₀), and the optical density of the planktonic culture (OD₆₀₀) was measured. Biofilm scores were calculated as the A₆₀₀/OD₆₀₀ ratio, and the data were normalized. The data shown are the means of three independent experiments completed in triplicate. Representative coverslips prior to crystal violet solubilization are shown for each strain.

FIG 4

GFP driven from P_tac or P_lac in the a-att site is rapidly depleted. (A) Schematics of strains used to compare expression of sfgfp from P_tac (top) and P_lac (bottom). (B) Western blots illustrating the depletion of GFP following the removal of the inducer (IPTG) as described in Materials and Methods. Expression of GFP driven from P_tac (top) is higher than expression of GFP driven from P_lac (bottom). (C) Depletion of GFP signal driven from P_tac (top) and P_lac (bottom) following the removal of the inducer (IPTG) was monitored in live cells using fluorescence microscopy.

FIG 5

Depletion of the essential cell cycle regulator CtrA results in the production of nonviable, rounded cells. (A) Schematic of the ctrA depletion strain. (B) Growth curves of the wild-type parent strain (dashed line) and the ctrA depletion strain in the presence (solid black line) and absence (gray line) of IPTG. (C) Cell viability was assessed as the ability to produce CFU on ATGN plates containing IPTG after the ctrA depletion strain was cultured in the presence (black bars) or absence (white bars) of IPTG for each indicated time point. The cell viability score was calculated as the number of CFU at the indicated time point divided by the number of CFU at the outset of the experiment. (D) Select images from 24 h of time lapse microscopy of individual ctrA depletion cells grown on ATGN agarose pads in the presence (top row) or absence (bottom two rows) of IPTG.