The cyclic AMP-dependent catabolite repression system of Serratia marcescens mediates biofilm formation through regulation of type 1 fimbriae

Abstract

The mechanisms by which environmental carbon sources regulate biofilm formation are poorly understood. This study investigates the roles of glucose and the catabolite repression system in Serratia marcescens biofilm formation. The abilities of this opportunistic pathogen to proliferate in a wide range of environments, to cause disease, and to resist antimicrobials are linked to its ability to form biofilms. We observed that growth of S. marcescens in glucose-rich medium strongly stimulated biofilm formation, which contrasts with previous studies showing that biofilm formation is inhibited by glucose in Escherichia coli and other enteric bacteria. Glucose uptake is known to inversely mediate intracellular cyclic AMP (cAMP) synthesis through regulation of adenylate cyclase (cyaA) activity, which in turn controls fundamental processes such as motility, carbon utilization and storage, pathogenesis, and cell division in many bacteria. Here, we demonstrate that mutation of catabolite repression genes that regulate cAMP levels (crr and cyaA) or the ability to respond to cAMP (crp) confers a large increase in biofilm formation. Suppressor analysis revealed that phenotypes of a cAMP receptor protein (crp) mutant require the fimABCD operon, which is responsible for type 1 fimbria production. Consistently, fimA transcription and fimbria production were determined to be upregulated in a cyaA mutant background by using quantitative real-time reverse transcription-PCR and transmission electron microscopy analysis. The regulatory pathway by which environmental carbon sources influence cAMP concentrations to alter production of type 1 fimbrial adhesins establishes a novel mechanism by which bacteria control biofilm development.

FIG. 1.

Glucose stimulates S. marcescens biofilm formation. (A) Photographs of crystal violet-stained biofilms (black arrow) formed on glass test tubes. Biofilms were formed in at 30°C for 15 h under high-sheer conditions in LB supplemented with glucose or cAMP as indicated. (B) Biofilm levels from crystal violet-stained test tubes (results from three independent cultures per strain are shown, and error bars indicate 1 standard deviation). Growth in glucose-rich medium significantly increased crystal violet staining (P > 0.01). (C) Growth curve analysis shows culture turbidity as a function of time. Bacteria were grown in LB medium at 30°C and supplemented with 2% glucose (glu) or 10 mM cAMP as indicated. WT, wild type. This experiment was done with triplicate independent cultures. Error bars depict 1 standard deviation.

FIG. 2.

S. marcescens catabolite repression genes regulate biofilm formation and cAMP production. (A) Biofilms formed on test tubes that were grown at 30°C on a rotor for 14 to 15 h. Results are shown for mutation and complementation of cyaA, crr, and crp mutant strains. These tubes are representative images of reproducible biofilm phenotypes. WT, wild type. (B, C) Confocal microscopy of Syto-9-stained biofilms formed on a glass coverslip under constant-flow conditions at room temperature for 24 h. Panel B is a micrograph of the wild type, and panel C shows the cyaA2 mutant strain. Bar = 30 μm. This experiment was repeated with consistent results with two replicates per genotype per experiment. (D) Intracellular levels of cAMP, as determined by ELISA. Results are shown for a representative experiment done with triplicate independent samples per genotype. Error bars represent 1 standard deviation. *, P < 0.05. (E) Complementation of E. coli ΔcyaA mutant phenotypes for growth on M63 medium with glycerol as a sole carbon source by the S. marcescens cyaA gene on a multicopy plasmid.

FIG. 3.

Fimbriae are necessary for catabolite repression mutant biofilm phenotypes. (A) Mutation of fimC suppressed the hyperbiofilm phenotype caused by high levels of glucose or mutation of catabolite repression genes. For panels B and C, a kinetic assessment of yeast agglutination was performed. Positive agglutination results in lower absorbance readings. Here, absorbance is shown relative to time zero. WT, wild type. (B) The ability of S. marcescens to agglutinate yeast was dependent on fimbriae (fimC). Mutation of cyaA led to an increase in yeast agglutination that can be complemented by wild-type cyaA on a plasmid (pRMQS157) or by the addition of exogenous cAMP. (C) Kinetic agglutination of yeast by crr-1, mutants, and control strains. A crr fimC double mutant did not aggregate yeast (identical results were found with cya fimC and crp fimC double mutants [not shown]). All experiments were done with triplicate independent cultures on at least three different days, and the representative data from 1 day's experiment are shown. Error bars represent 1 standard deviation.

FIG. 4.

TEM analysis of fimbria production. TEM micrographs of negatively stained bacteria grown under shaking conditions for 15 h in LB medium at 30°C. The percentage value represents the percentage of cells that exhibit fimbriae (n ≥ 140 cells per strain) from multiple independent cultures. Black arrowheads indicate fimbriae. (A) Two wild-type (WT) cells with moderate levels of type 1 fimbriae are shown. (B) Most cells in the cyaA2 mutant culture exhibit high levels of fimbriae. (C) cyaA2 fimC4 double mutant cell devoid of fimbriae. (D) Bacteria from crp-1 mutant cultures are highly fimbriate. (E) TEM micrograph of the cyaA2 mutant grown in the presence of 10 mM cAMP. Note typical cyaA mutant morphology (black arrow) on one cell and lack of obvious fimbriae (white arrow) on another cell. The bar represents 100 nm, except in panel E, where the bar represents 500 nm. (F) Quantitation of TEM images from triplicate independent cya-2 cultures showing the percentage of cells with fimbriae grown in medium supplemented with (10 mM) or without cAMP. The asterisk represents a significant reduction in the percentage of cells with surface fimbriae (P < 0.02).

FIG. 5.

Transcription of fimA is negatively regulated by adenylate cyclase. Real-time qPCR was used to assess transcription of type 1 fimbria subunit coding gene fimA. RNA levels relative to those for the wild type (WT) are shown. The difference is significant (P < 0.03). The experiment shows an average of three independent cultures made on different days. Error bars indicate standard errors.