Diverse cyclic dinucleotide signals regulate Escherichia coli lifestyle transition

Abstract

Bacteria have the ability to change their lifestyle to adapt to various environmental conditions. Cyclic dinucleotides (cDNs) are ubiquitous second messengers that can regulate fundamental lifestyle switches, such as motility versus sessility and acute versus chronic virulence, in bacteria. Investigation of the diverse established cDNs, cyclic di-GMP, cyclic di-AMP, and the recently identified hybrid molecule cyclic GAMP, expanded our knowledge of the complexity of regulation of bacterial physiology by nucleotide-based second messengers and unravel common and distinct regulatory patterns.

In this thesis, we provided the molecular basis to assess the regulatory mechanisms of semiconstitutive rdar biofilm formation by Illumina Miseq or PacBio sequencing of the genomes of eight rdar biofilm forming E. coli strains (Paper I). By using phenotypic, genetic and biochemical approaches, we showed that animal commensal isolate E. coli ECOR31 expresses a semi-constitutive rdar biofilm morphotype on agar plates characterized by expression of the extracellular matrix components cellulose and curli fimbriae. This morphotype is conventionally dependent on the major biofilm regulator CsgD and positively regulated by cyclic di-GMP signaling (Paper II). As expected, flagella-dependent motility is negatively regulated by cyclic di-GMP signaling. Bioinformatic analysis suggested the presence of a dinucleotide cyclase DncV homolog, hypothesized to possess cyclic GAMP synthase activity, encoded by the E. coli ECOR31 genome. DncV synthesized 3’3’-cGAMP in vitro and in vivo and, via its catalytic activity, negatively regulated csgD expression at the transcriptional level with subsequent suppression of rdar biofilm formation and cell aggregation. DncV also suppressed swimming and swarming motility post-transcriptional of class 1 flagellar regulon genes. In liquid culture, expression of dncV restricted cell aggregation, but showed a complex temporal pattern of biofilm formation at the abiotic surface.

The patatin-like phospholipase CapV is a receptor for 3’3’-cGAMP. In this thesis, we showed that expression of CapVQ329R, a single amino acid variant of CapV, induced extensive cell filamentation in E. coli MG1655 independently of the 3’3’-cGAMP synthase DncV (Paper III). Moreover, overexpression of CapVQ329R repressed swimming motility by inhibiting flagella biosynthesis and reduced rdar biofilm formation and CsgD expression, possibly through modulation of cyclic di-GMP levels. The observed phenotypes of CapVQ329R are not restricted to E. coli MG1655, but common to other E. coli strains and S. typhimurium UMR1 suggesting that conserved pathway(s) are required for their expression. Based on our genome sequences, in the last study, we investigated the molecular basis of temperature-independent expression of the rdar biofilm morphotype and subsequently csgD expression in seven semi-constitutive rdar biofilm forming E. coli strains (Paper IV). Based on the observation that amino acid variations in cyclic di-GMP turnover proteins correlated with the expression of a semi-constitutive rdar biofilm, in particular, we demonstrated that distinct amino acid changes outside of conserved signature motifs that potentially lead to an alteration in the trigger activity of the hybrid cyclic di-GMP phosphodiesterase/diguanylate cyclase YciR contributed to regulate rdar biofilm formation and csgD expression in semiconstitutive rdar biofilm forming E. coli strains.

In conclusion, this thesis highlights that diverse cDN second messenger signals differentially regulate the bacterial sessile/motile lifestyle transition. Furthermore, the effect of CapVQ329R on bacterial phenotypes and physiology is an example of rapid evolution of protein functionality.