Mini-Tn 7 Insertion in an Artificial att Tn 7 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 plasmidbased 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. IMPORTANCEAgrobacterium 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. Members of the genus Agrobacterium are common soil-dwelling bacteria. Most are saprophytic and survive primarily by consuming decaying organic material; but some cause phytopathic diseases, including Agrobacterium rhizogenes (hairy root disease), Agrobacterium rubi (cane gall disease), Agrobacterium vitis (crown gall of grape) and Agrobacterium tumefaciens (crown gall disease). During infection, A. tumefaciens transfers a portion of its DNA (transfer DNA [T-DNA]) to plants, resulting in the formation of crown galls in susceptible flowering plants. Infected plants form tumors that limit transport of water and nutrients, leading to decreased vigor and crop yield (1, 2). Because of the ubiquity of A. tumefaciens and its ability to evade plant defenses, crown gall disease continues to be a problem, with documented economic impact on fruit and nut trees, particularly in nurseries (2). Thus, A. tumefaciens has become a model for the study of host-pathogen interactions (3-5) and processes related to pathogenicity, including type IV secretion (6), quorum sensing (7), and biofilm formation (8). Finally, mechanistic studies of A. tumefaciens-mediated DNA transfer into host cells have been harnessed for the genetic engineering of plants, fungi, and mammals (5, 9-11).While the processes related to phytopathogenicity have been subject to intense scrutiny, processes essential for A. tumefaciens survival in the environment, both in the rhizosphere and in association with hosts, are poorly understood. Recent studies have shown that A. tumefaciens cells exhibit a striking pattern of polar cell growth (12-14), leading to a vast array of experimental questions (15). Proper spatial and temporal regulation of the cell cycle must be required in order to coordinate key processes, such as chromosome replication and segregation, cell wall biogenesis, and cell division. Understanding the mechanisms underlying these essential processes will provide key insights into the basic biology of A. tumefaciens...

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division

“…All scale bars are set to 2 µm. promoter at a neutral site in the chromosome (Figueroa-Cuilan et al, 2016;Howell and Brown, 2016). Using western blot analysis, we have confirmed the depletion of FtsZ AT in the absence of IPTG (Supplemental Fig.…”

“…To characterize the function of each FtsZ homolog, we constructed deletions of ftsZ 1 and ftsZ 3 and a depletion strain of ftsZ AT . Since we were unable to construct a deletion of ftsZ AT , we used a depletion strategy in which ftsZ AT is present as a single copy under the control of an isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) inducible promoter at a neutral site in the chromosome (Figueroa‐Cuilan et al ., ; Howell and Brown, ). Using western blot analysis, we have confirmed the depletion of FtsZ AT in the absence of IPTG (Supplemental Fig.…”

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

“…5F; see also Movie S5). The branching phenotype observed when pph was induced in A. tumefaciens cells is reminiscent of cells exhibiting a block in cell division (57)(58)(59)(60)(61)(62)(63). This observation suggests that PPH may have a dual function, namely, blocking cell division and triggering cell lysis.…”

“…The C terminus of PPH may have a similar function leading to the inhibition of a cell division protein. Indeed, A. tumefaciens cells expressing PPH exhibited a branching phenotype similar to that in FtsZ-depleted cells (63) (Fig. 5F; Movie S5).…”

“…See Table 3 for a list of all primers used in plasmid construction and sequencing. All variants of pph were cloned into the vector pSRKKm-Plac-sfgfp, which allows for inducible expression of target genes under the control of the lac promoter using IPTG as the inducer (63). To construct pSRKKm-PPH, PCR using Phusion high-fidelity DNA polymerase (Thermo Scientific) was performed on the Atu_ph02 genomic DNA using primers PPH NdeI F and PPH BamHI R. PCR products were gel purified using the GeneJET gel extraction kit (Thermo Scientific).…”