Catabolite repression control of flagellum production by Serratia marcescens

Abstract

Serratia marcescens is an emerging opportunistic pathogen with a remarkably broad host range. The cAMP-regulated catabolite repression system of S. marcescens has recently been identified and demonstrated to regulate biofilm formation through the production of surface adhesions. Here we report that mutations in components of the catabolite repression system (cyaA and crp) eliminate flagellum production and swimming motility. Exogenous cAMP was able to restore flagellum production to adenylate cyclase mutants, as determined by transmission electron microscopy and PAGE analysis. A transposon-generated suppressor mutation of the crp motility defect mapped to upstream of the flhDC operon. This suppressor mutation resulted in an upregulation of flhD expression and flagellum production, indicating that flhDC expression is sufficient to restore flagellum production to crp mutants. Lastly, and contrary to a previous report, we found that flhD expression is controlled by the catabolite repression system using quantitative RT-PCR. Together, these data indicate that flagellum production is regulated by the cAMP-dependent catabolite repression system. Given the role of flagella in bacterial pathogenicity, the regulatory pathway described here may assist us in better understanding the putative role of motility in dissemination and virulence of this opportunistic pathogen.

Fig. 1

Adenylate cyclase is required for swimming motility and flagellum production by S. marcescens. A. Photograph of swimming zones through 0.3% agar by the cyaA (adenylate cyclase) mutant strain with either an empty vector or the wild-type cyaA gene under control of the P_lac promoter on a multicopy plasmid (pcyaA). B–C. TEM micrographs of wild-type (B) and cyaA mutant (C) cells. The black arrow denotes flagella and the gray arrows indicate fimbriae. The bar indicates either 500 nm (B) or 100 nm (C). D. PAGE analysis of surface protein fractions from stationary phase wild-type and cyaA cells with vector control or pcyaA. The flagellin protein was verified by mass spectroscopy.

Fig. 2

Flagellum production can be restored to a cyaA mutant by exogenous cAMP. A. The percent of cells with a flagellum observed by TEM microscopy in response to a cyaA mutation and increasing amounts of exogenous cAMP (n>200 cells per condition). No flagella were observed among cyaA mutant cells without exogenous cAMP (n>400). A statistical difference from the wild type was observed for each condition (p<0.02). B. PAGE analysis of surface protein fractions from stationary phase wild-type and cyaA cultures grown with increasing concentrations of exogenous cAMP.

Fig. 3

The scrp31 mutation suppresses the swimming and swarming deficiency phenotypes of a crp mutant strain. A. Swimming motility charted as a function of time. The crp scrp31 double mutant is able to swim, whereas the crp mutant strain is non-motile. B. Genetic map of the flhDC locus with the location of a predicted CRP binding site (black box with asterisk) and the location of a transposon insertion (scrp31), not drawn to scale. The transposon bears a P_tac promoter, which is directed toward the flhDC operon.

Fig. 4

The scrp31 mutation restores flagellum production to a crp mutant strain. A. TEM micrographs of a crp-1 mutant with no flagellum, but numerous fimbriae (gray arrows) and crp scrp31 double mutant cells covered with numerous flagella (black arrows). The size bar represents 500 and 100 nm respectively. B. PAGE analysis of surface protein fractions from wild-type culture, crp, crp scrp31 and scrp31 cultures showing flagellin production. C. Quantitation of flagellin levels using Image J analysis software. Surface fractions were taken from stationary phase cultures. Asterisks represent a statistically significant difference from the wild-type (p<0.01). D. Swarming motility on LB with 0.7% agar at 48 h. The crp mutant swarming defect is rescued by the scrp31 mutation. E. Plotting the percentage of positive swarming motility experiments at 48 h as a function of agar concentration shows that the scrp31 mutation confers a hyperswarming phenotype (n≥12 plates for WT and crp-1 scrp31 and n≥8 for the crp-1 mutant, performed on 4 separate occasions with similar results).

Fig. 5

Catabolite repression control of flhD expression. Quantitative RT-PCR analysis of flhD transcript levels relative to wild-type levels shows a significant decrease in transcript produced by the cyaA mutant, which is partially restored by the addition of exogenous cAMP to growth medium. The crp mutant was similarly reduced in the flhD transcript, and was rescued by the scrp31 mutation which caused a significant increase in flhD RNA. RNA was harvested from 3 or more independent cultures at an A₆₀₀ of 1.0. The asterisk indicates a significant difference versus the wild type (p<0.01).

Fig. 6

Model for coordinated catabolite repression control of attachment and motility processes. The catabolite repression system (CRS) is a signal transduction cascade that responds to environmental carbon. In response to less favorable carbon sources, adenylate cyclase (CyaA) is stimulated to generate cAMP. The activity of the global transcription factor CRP (cAMP receptor protein) is altered through binding with cAMP. The cAMP-CRP complex activates expression of flhDC, which in turn activates flagellum synthesis. At the same time, the cAMP-CRP complex directly or indirectly inhibits fimbriae production. Growth conditions with favorable carbon sources led to decreased adenylate cyclase activity and cAMP levels causing a decrease in production of flagella and derepression of attachment factor expression. The result of growth in an environment with a favorable carbon source(s) is decreased production of the flagellum and increased production of fimbriae steering a bacterium toward attachment and biofilm formation. Growth with less favorable carbon source(s) stimulates production of flagella and inhibits production of fimbriae to generate a motile bacterium that is more able to encounter hospitable environments.