ntrC Contributes to Nitrogen Utilization, Stress Tolerance, and Virulence in Acidovorax citrulli

Abstract

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Nitrogen, one of the most important limiting elements in the environment, is necessary for the growth and reproduction of bacteria. As a nitrogen-regulating gene, ntrC plays an important role in maintaining bacterial nitrogen utilization and biological nitrogen fixation. However, the role of ntrC has not been determined for A. citrulli. In this study, we constructed a ntrC deletion mutant and a corresponding complementary strain in the background of the A. citrulli wild-type strain, Aac5. Through phenotype assays and qRT-PCR analysis, we investigated the role of ntrC in A. citrulli in nitrogen utilization, stress tolerance, and virulence against watermelon seedlings. Our results showed that the A. citrulli Aac5 ntrC deletion mutant lost the ability to utilize nitrate. The ntrC mutant strain also exhibited significantly decreased virulence, in vitro growth, in vivo colonization ability, swimming motility, and twitching motility. In contrast, it displayed significantly enhanced biofilm formation and tolerance to stress induced by oxygen, high salt, and copper ions. The qRT-PCR results showed that the nitrate utilization gene nasS; the Type III secretion system-related genes hrpE, hrpX, and hrcJ; and the pili-related gene pilA were significantly downregulated in the ntrC deletion mutant. The nitrate utilization gene nasT, and the flagellum-related genes flhD, flhC, fliA, and fliC were significantly upregulated in the ntrC deletion mutant. The expression levels of ntrC gene in the MMX-q and XVM2 media were significantly higher than in the KB medium. These results suggest that the ntrC gene plays a pivotal role in the nitrogen utilization, stress tolerance, and virulence of A. citrulli.

Acidovorax citrulli type III effector AopU interferes with plant immune responses and interacts with a watermelon E3 ubiquitin ligase

“…Furthermore, to eliminate the effect of the vector, the empty vector pBBRNolac-4FLAG was transferred into the Aac5 and Δ aopU to generate strain Aac5-pBBR and Δ aopU -pBBR ( Fei et al, 2022 ). The screening and verification processes were conducted as described in previously ( Qiao et al, 2022 ; Liu et al, 2023 ).…”

Acidovorax citrulli Type IV Pili PilR Interacts with PilS and Regulates the Expression of the pilA Gene

“…Standardized bacterial suspensions were made by diluting suspensions to OD 600 = 0.3, using sterilized deionized water. A total of 2 µL of cell suspension was dropped on the center of a 0.3% semi-solid KB medium plate and cultured at 28 • C for 48 h, and the diameter of the halo was measured [28]. For each strain, 9 replicates were assayed.…”

A putative glucose 6-phosphate isomerase has pleiotropic functions on virulence and other mechanisms in Acidovorax citrulli

“…In addition, type II secretion system, type III secretion system, and quorum sensing have been identified as major virulence factors (Wang et al, 2016;Zivanovic and Walcott, 2017;Zhang et al, 2018;Kan et al, 2023). It is also reported that the ferric uptake regulator, YggS family pyridoxal phosphate-dependent enzymes, pyridoxal phosphate-dependent aminotransferases, glycerol-3-phosphate dehydrogenases, and 2, 3-bisphosphoglyceratedependent phosphoglycerate mutases, and the nitrogen regulator are associated with virulence in Ac (Liu et al, 2019;Kim et al, 2021;Lee et al, 2021;Lee et al, 2022b;Wang et al, 2022;Heo et al, 2023;Liu et al, 2023). However, these well-characterized factors/mechanisms involved are insufficient to comprehensively understand the virulence mechanisms of Ac.…”