Differential control of Yersinia pestis biofilm formation in vitro and in the flea vector by two c-di-GMP diguanylate cyclases

Abstract

Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC) enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE), encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP) and two DGCs (HmsT and Y3730) control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

Figure 1. Pigmentation phenotype of Y. pestis strains on LB-Congo red agar.

See text and Table 3 for description of the strains.

Figure 2. Effect of Y. pestis GGDEF-, EAL-, and HD-GYP-domain genes on in vitro biofilm formation.

A, B. Relative amounts of adherent biofilm made by Y. pestis KIM6+ parent strain and isogenic derivatives deleted of (A) or overexpressing (B) one of the genes listed in Table 1. C. Quantitation of biofilm made by the hmsT hmsP y3730 triple mutant strain overexpressing one of the genes listed in Table 1, or the mutated y3730 GGAAF allele. The mean and standard deviation of two or more independent experiments are indicated.

Figure 3. Relative amount of in vitro biofilm made by the Y. pestis y3730, hmsT, and y3730 hmsT mutant strains after transformation with the empty plasmid vector, (white bars) or the plasmid containing wild-type y3730 (hatched bars), mutated y3730 (grey bars), or hmsT (black bars).

The mean and standard deviation of two or more independent experiments are indicated.

Figure 4. Expression of Y. pestis y3730 and hmsT in vitro and in the flea.

Relative amounts of y3730 and hmsT mRNA expressed in fleas (black bars), in vitro biofilm cultures (grey bars) and in vitro planktonic cultures (white bars) are shown. The mean and SD of three independent experiments is indicated. *P<0.05 by t-test.

Figure 5. y3730 and hmsT are required for Y. pestis autoaggregation.

The % sedimentation of bacterial growth in liquid cultures was determined by spectrophotometry after 1, 2, and 12 hours of stasis (white, grey, and black bars, respectively). The mean and SD of two independent experiments performed in duplicate are indicated.