Enterobacterial common antigen integrity is a checkpoint for flagellar biogenesis in Serratia marcescens

Abstract

Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.

FIG. 1.

Phospholipase-negative mutant strains. (A) Egg yolk-LB agar plate showing the precipitation halo characteristic of lecithinase activity present in the wild-type (wt) strain and absent in the wec and ΔphlAB mutant strains. (B) Zymogram analysis of the spent culture medium from the wt and the wec mutant strains. Phospholipase activity is visualized as a white precipitate band of 26 kDa. The ΔphlAB strain was used as control. (C) Results of exoenzyme activity assays. Each activity assay was performed as described in Materials and Methods. −, negative result; +, positive result.

FIG. 2.

(A) Schematic representation of the wec cluster in S. marcescens. The sites of the mini-Tn5-Km transposon insertions are indicated with arrows. (B) Exopolysaccharide preparations of the wild-type and mutant strains were analyzed by immunodetection with anti-O14 antiserum to detect ECA (upper panel) or by silver staining to examine LPS (lower panel).

FIG. 3.

Motility assays. Swarming (LB-0.6% agar) and swimming (LB-0.25% agar) plates were incubated ON at 37°C or 30°C, as indicated in Materials and Methods, and photographed. The strains analyzed were S. marcescens RM66262 (wild-type) and wecD-, wzxE-, and wzyE-derived mini-Tn5-Km mutants. Results are representative of four identical individual assays.

FIG. 4.

TEM. Representative TEM observations of wild-type (wt) S. marcescens and mutants illustrating phenotype differences when cells were taken from (A) swarming plates (rim or center of the colony, as indicated) or (B) LB broth cultures, at the indicated growth temperatures. The strains analyzed were S. marcescens RM66262 (wild type) and wecD-, wzxE-, and wzyE-derived mini-Tn5-Km mutants. A detail of a hyperflagellated wild-type cell taken from the swarm center is shown. Cells were grown and stained for TEM as described in Materials and Methods. Bars = 2.0 μm (magnification is identical for all images, and the scale is included in the first image of each series).

FIG. 5.

Flagellin immunodetection. Whole-cell extracts from the wild-type (wt) and mutant strains isolated from the rim or the center of the swarming colony grown at 30°C (upper panel) or liquid LB cultures grown at 37°C or 30°C (lower panel) were analyzed by Western blotting developed with anti-flagellin polyclonal antibodies. Samples were standardized as described in Materials and Methods.

FIG. 6.

RT-PCR and qRT-PCR analysis of flagellar cascade genes and phlA expression. (A) Total RNA was extracted from the wild-type (wt) and the wecD, wzxE, and wzyE mutant strains. flhD, fliA, fliC, and phlA expression was analyzed by RT-PCR using specific primers (Table 1). 16S rRNA was used as an internal control. The PCR was carried out for 25 cycles for flhD, fliC, and 16S rRNA and 35 cycles for fliA and phlA. (B) Quantitative RT-PCR analysis of flhD and fliC expression. Average Ct values and n-fold changes in gene expression in each mutant strain compared with those of the wild-type strain were calculated from triplicate samples as described in Materials and Methods. Error bars indicate standard deviations.