Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli

Abstract

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae

“…This may be due to the different infection status of the bacterial cell at the time of sample processing or due to the inherent proprieties of the bacteria. Therefore, the results revealed the expression of FtsH protease modulator located in prophage of strains K3574 and K3320, which controls the lytic pathway ( 58 ), as well as the expression of the cupin protein located in plasmid in the strain K3574, a phosphomannose isomerase involved in lipopolysaccharide (LPS) synthesis, which is an important determinant of pathogenicity and phage susceptibility ( 59 ). In addition, proteins related to bacterial defense, resistance, and virulence and also to oxidative stress mechanisms ( 60 ) have been observed.…”

Molecular analysis of the interactions between phages and the bacterial host Klebsiella pneumoniae

“…This may be due to the different infection status of the bacterial cell at the time of sample processing or due to the inherent proprieties of the bacteria. Therefore, the results revealed the expression of FtsH protease modulator located in prophage of K3574 strain, which controls the lytic pathway (61), as well as overexpression of the cupin protein located in plasmid, a phosphomannose isomerase involved in LPS synthesis, which is an important determinant of pathogenicity and phage susceptibility (62). In addition, proteins related to bacterial defence, resistance and virulence and also to oxidative stress mechanisms (63) have been observed in the phage-infected strains.…”

FoCupin1, a Cupin_1 domain-containing protein, is necessary for the virulence of Fusarium oxysporum f. sp. cubense tropical race 4

“…Generally, proteins in the cupin superfamily contain one or two cupin structural domains ( Shutov and Kakhovskaia, 2011 ). Cupin proteins possess remarkable functional diversity, as they function as dioxygenase, lyase, epimerase, isomerases, and nonenzymatic storage proteins ( Uberto and Moomaw, 2013 ; Rahimi-Midani et al, 2021 ; Peng et al, 2022 ). The cupin superfamily is classified into at least 69 families in the Pfam database, including the Cupin_1 family to which FoCupin1 belongs ( Mistry et al, 2021 ).…”