Mutation of a Salmonella Serogroup-C1-Specific Gene Abrogates O7-Antigen Biosynthesis and Triggers NaCl-Dependent Motility Deficiency

Abstract

Several molecular detection marker genes specific for a number of individual Salmonella serogroups have been recently identified in our lab by comparative genomics for the genotyping of diverse serogroups. To further understand the correlation between serotype and genotype, the function of a Salmonella serogroup-C1-specific gene (SC_2092) was analyzed in this study. It was indicated from the topological prediction using the deduced amino acid sequence of SC_2092 that this putative protein was highly similar to the confirmed Wzx flippases. Furthermore, SDS-PAGE revealed that lipopolysaccharide (LPS) biosynthesis, specifically O-antigen synthesis, was incomplete in an SC_2092 in-frame deletion mutant, and no agglutination reaction with the O7 antibody was exhibited in this mutant. Therefore, it was revealed that this Salmonella serogroup-C1-specific gene SC_2092 encoded a putative flippase, which was required for O7-polysaccharide biosynthesis, and was designated here as wzxC1. Subsequently, the effects of the deletion of wzxC1 on bacterial motility and sodium chloride (NaCl) tolerance were evaluated. The wzxC1 mutant lacked swarming motility on solid surfaces and was impaired in swimming motility in soft agar. Moreover, microscopic examination and RT-qPCR exhibited that an increased auto-aggregation and a strong defect in flagella expression, respectively, were responsible for the reduced motility in this mutant. In addition, the wzxC1 mutant was more sensitive than the wild-type strain to NaCl, and auto-aggregation of mutant cells was observed immediately up on the addition of 1% NaCl to the medium. Interestingly, the motility deficiency of the mutant strain, as well as the cell agglomeration and the decrease in flagellar expression, were relieved in a NaCl-free medium. This is the first study to experimentally demonstrate a connection between a Salmonella serogroup specific gene identified by comparative genomics with the synthesis of a specific O-antigen biosynthesis. Also, our results show that the mutation of wzxC1 triggers a NaCl-dependent motility deficiency.

Two Novel Salmonella Bivalent Vaccines Confer Dual Protection against Two Salmonella Serovars in Mice

“…An early study suggested that the loss of surface O-antigen inhibits the swarming motility of Salmonella Typhimurium because it generally acts to increase the surface “wettability” required for bacterial colony expansion (Toguchi et al, 2000 ). Recent reports demonstrated that auto-aggregation and a defect in flagellar expression were responsible for the reduced swimming motility of a Salmonella Choleraesuis rough strain with a wzx gene deletion (Zhou et al, 2014 ), and the inactivation of genes involved in LPS synthesis decreases flagellar protein production in Salmonella Typhimurium (Liu et al, 2016 ). Thus, whether a similar mechanism for defective swimming motility occurs in the Salmonella Δ rfbN mutant and whether expression of the Salmonella Newport O-antigen has an adverse effect on flagellar expression still need to be addressed.…”

“…Salmonella Typhimurium mutant strains were constructed by allelic exchange using the suicide vector pRE112, which carries a chloramphenicol resistance gene and the sucrose-sensitivity gene sacB (Edwards et al, 1998 ), a gift from Dieter Schifferli (Addgene plasmid #43828), as previously described (Zhou et al, 2014 ). To delete the rfbN gene, the primer pairs D rfbN -1F/D rfbN -1R and D rfbN -2F/D rfbN -2R were used to amplify the regions ~400 bp upstream and downstream of the ATCC14028 genome, respectively.…”

The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology

“…O antigen is necessary for effective swimming and swarming motility in many bacteria [110][111][112][113][114][115][116][117][118][119][120], which has been demonstrated in genetic studies that investigated the effect on motility when genes involved in various steps of the O antigen synthesis and assembly pathway are deleted. Our group reported the phenotypes of several such mutants in P. aeruginosa.…”

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis

“…The auto-aggregation assay was performed based on the method previously described by Zhou et al [ 18 ]. Briefly, Salmonella cultures were statically grown in 5 ml LB medium at 37 °C for 16 h in test tubes.…”