Identification of a Twin-Arginine Translocation System in Pseudomonas syringae pv. tomato DC3000 and Its Contribution to Pathogenicity and Fitness

Abstract

The bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) causes disease in Arabidopsis thaliana and tomato plants, and it elicits the hypersensitive response in nonhost plants such as Nicotiana tabacum and Nicotiana benthamiana. While these events chiefly depend upon the type III protein secretion system and the effector proteins that this system translocates into plant cells, additional factors have been shown to contribute to DC3000 virulence and still many others are likely to exist. Therefore, we explored the contribution of the twin-arginine translocation (Tat) system to the physiology of DC3000. We found that a tatC mutant strain of DC3000 displayed a number of phenotypes, including loss of motility on soft agar plates, deficiency in siderophore synthesis and iron acquisition, sensitivity to copper, loss of extracellular phospholipase activity, and attenuated virulence in host plant leaves. In the latter case, we provide evidence that decreased virulence of tatC mutants likely arises from a synergistic combination of (i) compromised fitness of bacteria in planta; (ii) decreased efficiency of type III translocation; and (iii) cytoplasmically retained virulence factors. Finally, we demonstrate a novel broad-host-range genetic reporter based on the green fluorescent protein for the identification of Tat-targeted secreted virulence factors that should be generally applicable to any gram-negative bacterium. Collectively, our evidence supports the notion that virulence of DC3000 is a multifactorial process and that the Tat system is an important virulence determinant of this phytopathogenic bacterium.The hallmark of the Pseudomonas syringae infection process is the injection of virulence effector proteins directly into host cells via the type III secretion mechanism (TTSS) (14). For efficient translocation of effector proteins, P. syringae must first establish productive contact with a target plant cell. This entails secretion of exopolysaccharides, cell wall-degrading enzymes, plant hormones, and several low-molecular-weight phytotoxins, such as coronatine and syringomycin (1, 30). Since many of these factors are secreted in a type III-independent manner and since deletion of many of these virulence factors often does not render P. syringae avirulent, it can be assumed that P. syringae virulence is multifactorial. Moreover, given the complexity of interactions that occur at the host-pathogen interface, there are likely many additional virulence determinants in P. syringae yet to be discovered.One possible contributor to P. syringae pathogenesis is the twin-arginine translocation (Tat) pathway that operates in the inner membrane of many gram-negative bacteria (4, 5, 56). Tat substrates are synthesized with cleavable N-terminal signal peptides that are characterized by a highly conserved twinarginine motif in the positively charged N-terminal region, a weakly hydrophobic core region, and a positively charged Sec pathway-avoidance signal in the C-terminal region (7,17). The most remarkable feat...

Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins

“…Moreover, virtually all phospholipase C orthologs in a wide variety of organisms have predicted Tat signal sequences (16), and there are bacterial Plc enzymes proven to be exported by the Tat pathway (9,36,46,60). In contrast to what was seen in M. smegmatis, when expressed in M. tuberculosis two species of PlcB-HA were evident by immunoblot analysis.…”

“…In several bacterial pathogens, the Tat pathway plays an important role in exporting virulence factors (9,10,17,30,36,42,46,60). Mycobacterium tuberculosis is the bacterial pathogen re-sponsible for tuberculosis, which kills 1.8 million people a year (64).…”

The Twin Arginine Translocation System Is Essential for Virulence ofYersinia pseudotuberculosis

“…Tat secretion has been shown to be important for the motility of the gram-negative pathogens P. aeruginosa, P. syringae pv. tomato DC3000, E. coli O157:H7, and Agrobacterium tumefaciens (11,21,43,50). In the latter two cases, the Tat-defective strains have been shown not to express flagellin, indicating that components of the flagellar secretion assembly and/or secretion system are Tat substrates.…”

“…However, the Tat system has also been shown to be involved in secretion of virulence determinants like phospholipase toxins in Pseudomonas aeruginosa (43,69), P. syringae pv. tomato DC3000 (11), and Legionella pneumophila (56), as well as Shiga toxin 1 in enterohemorrhagic E. coli (50).…”

Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis

“…Several studies indicate that a functional Tat system is required for many pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000, S. enterica, Campylobacter jejuni, and Ralstonia solanacearum (5,(48)(49)(50)(51), to efficiently re- spond to diverse forms of physicochemical stresses. Indeed, our transcriptomic analysis of the Y. pseudotuberculosis ⌬tatC mutant revealed that a number of prominent stress-responsive genes were induced at both temperatures, and this number was higher during stationary-phase growth.…”