Hcp of the Type VI Secretion System (T6SS) in Acidovorax citrulli Group II Strain Aac5 Has a Dual Role as a Core Structural Protein and an Effector Protein in Colonization, Growth Ability, Competition, Biofilm Formation, and Ferric Iron Absorption

Abstract

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3 medium+. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.

Global transcriptional analysis ofGeobacter sulfurreducens gsu1771mutant biofilm grown on two different support structures

“…In the Δ gsu1771 biofilm grown on the electrode, a group of genes encoding proteins homologous to the type VI secretion system (T6SS) were upregulated, including gsu3167 ( vasJ ), gsu0433 ( vasG ), gsu0428 ( tssJ ), gsu3166 ( ImpK ), gsu3166 ( impL ), and gsu3174 ( hcp ). In bacteria, T6SS has been linked to virulence, antibacterial activity, metal ion uptake, transport, and biofilm formation (Mougous et al, 2006; Chen et al, 2015; Gallique et al, 2017; Fei et al, 2022). In P. aeruginosa, P. fluorescens , and Acidovorax citrulli , T6SS has been related to the formation of mature biofilms (Gallique et al, 2017; Chen et al, 2020, Fei et al, 2022).…”

“…In bacteria, T6SS has been linked to virulence, antibacterial activity, metal ion uptake, transport, and biofilm formation (Mougous et al, 2006; Chen et al, 2015; Gallique et al, 2017; Fei et al, 2022). In P. aeruginosa, P. fluorescens , and Acidovorax citrulli , T6SS has been related to the formation of mature biofilms (Gallique et al, 2017; Chen et al, 2020, Fei et al, 2022). In P. aeruginosa and A. citrulli , mutations in the hcp gene had negative effects on the development of mature biofilms and exopolysaccharide production (Chen et al, 2020; Fei et al, 2022).…”

Acidovorax citrulli Effector AopV Suppresses Plant Immunity and Interacts with Aromatic Dehydratase ADT6 in Watermelon

“…However, little is known about the molecular interaction between A. citrulli and host plants. Acidovorax citrulli secretes T3Es as a major pathogenicity strategy [ 28 ], however, only a few effectors have been studied in A. citrulli thus far. In 2020, Jimenez-Guerrero successfully identified some A. citrulli T3E proteins by machine learning and by comparing the transcriptomic differences between the A. citrulli wild-type strain M6 and the hrpX mutant strain [ 32 ].…”

“…Despite this, the molecular interactions between A. citrulli and host plants are not well-defined, and effective BFB management methods, including commercial sources of resistance, are lacking. At present, the research on A. citrulli mainly focuses on type II, type III, and type VI secretion systems, among which the research on the T3SS has made progress [ 25 , 26 , 27 , 28 ]. The T3SS is a key pathogenicity factor of A. citrulli [ 26 ].…”

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

“…The disease was first reported in Georgia, USA, in 1965 and has been reported in many countries [ 1 ]. Acidovorax citrulli uses a variety of virulence factors, such as the Type III secretion system (T3SS), the Type VI secretion system (T6SS), Type IV pili (T4P), polar flagella, quorum sensing, and biofilms [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ] to induce disease. Acidovorax citrulli strains can be divided into two major groups (I and II) [ 11 , 12 ].…”