Relative expression of selected swimming motility related genes.

<p>Total RNA was extracted from the wild-type and △wzx_C1 mutant grown in LB and NB medium. <i>flgA, motA, motB, cheA</i> and <i>cheB</i> expression was analyzed by quantitative RT-PCR. Average fold changes in gene expression in the mutant at each culture condition compared with those of the wild-type are shown. <i>recA</i> was used as an internal control. Errol bars indicate standard deviations. Stars indicate that the fold-changes in gene expression were significantly different (<i>p</i>-value, 0.05).</p

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

<div><p>Several molecular detection marker genes specific for a number of individual <i>Salmonella</i> 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 <i>Salmonella</i> serogroup-C1-specific gene (<i>SC_2092</i>) was analyzed in this study. It was indicated from the topological prediction using the deduced amino acid sequence of <i>SC_2092</i> 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 <i>SC_2092</i> in-frame deletion mutant, and no agglutination reaction with the O₇ antibody was exhibited in this mutant. Therefore, it was revealed that this <i>Salmonella</i> serogroup-C1-specific gene <i>SC_2092</i> encoded a putative flippase, which was required for O₇-polysaccharide biosynthesis, and was designated here as <i>wzx_C1</i>. Subsequently, the effects of the deletion of <i>wzx_C1</i> on bacterial motility and sodium chloride (NaCl) tolerance were evaluated. The <i>wzx_C1</i> 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 <i>wzx_C1</i> 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 <i>Salmonella</i> serogroup specific gene identified by comparative genomics with the synthesis of a specific O-antigen biosynthesis. Also, our results show that the mutation of <i>wzx_C1</i> triggers a NaCl-dependent motility deficiency.</p></div

Morphological features in the wild-type and <i>wzx_C1</i> deletion mutant strains revealed by TEM.

<p>TEM images of intact cells of wild-type <i>S.</i> Choleraesuis strain ATCC10708 (A and B) and the △wzx<b>_C1</b> deletion strain grown in medium with NaCl (C and D) or in NaCl-free medium (E and F). TEM thin sections of wild-type <i>S.</i> Choleraesuis strain ATCC10708 (G) and △wzx<b>_C1</b> deletion strain (H). The unknown intracellular crystals in the mutant cells were marked with the arrows.</p

Auto-aggregation of the wild-type and the △wzx_C1 mutant.

<p>Visual aggregation (A) on and percent aggregation (B) of wild-type (WT) and △wzx_C1 mutant (MT) cultures grown statically for 24 h at 37°C.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

The O₇-antigen synthesis was blocked in the △wzx deletion mutant.

<p>A) The presence of O-antigen was determined by SDS-PAGE separation of LPS preparations followed by silver staining. Lane 1, wild-type <i>S.</i> Choleraesuis strain ATCC10708; Lane 2, △wzx mutant strain; Lane 3, the △wzx complemented strain. The O-antigen latter is indicated. B) Agglutination was examined with somatic O₇ antiserum and pictures were taken within 5 min. Spot 1, wild-type <i>S.</i> Choleraesuis strain ATCC10708; Spot 2, the △wzx mutant strain; Spot 3, the △wzx complemented strain.</p

The effect of NaCl concentration on final culture density and cell morphology.

<p>The wild-type (WT), △wzx_C1 mutant (MT) and complemented mutant (MT-C) strains were grown in various concentrations of NaCl for 24 h. (A) The final OD₆₀₀ for cultures grown in different NaCl concentrations (0%–9%) was measured and (B) Morphologies at selected NaCl concentrations were recorded by light microscopy. CK: NB medium as a negative control. The data in (A) are from triplicate samples and images in (B) are representative of the three replicate samples. White bar  = 10 µm.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

MV tolerance in planktonic cells of different <i>L. monocytogenes</i> strains.

<p>The wild-type <i>L. monocytogenes</i> 4b G (G), <i>Δ</i>1771, <i>Δsod</i> and <i>Δ</i>1771<i>sod</i> were grown in TSB +1 mM MV broth respectively, measured the absorbance at OD₆₀₀ (A) and plated on TSB agar plates (B). The experiments were repeated three times and error bars indicate the standard errors of the mean.</p

Growth studies of the different <i>L. monocytogenes</i> strains in TSB at 37°C aerobically.

<p>(A) Growth curves of different strains were measured by spectrophotometry. Mean values are expressed as the measurements of OD₆₀₀±S.D. (n = 3). (B) Colony cultures of four strains in TSA plates for 20 h. Each strain was plated on TSA plates with triplicates.</p