Cyclic dinucleotides (cDNs) act as intracellular second messengers, modulating bacterial physiology to regulate the fundamental life style transition between motility and sessility commonly known as biofilm formation. Cyclic GMP-AMP (cGAMP), synthesized by the dinucleotide cyclase DncV, is a newly discovered cDN second messenger involved in virulence and chemotaxis in Vibrio cholerae O1 biovar El Tor. Here we report a novel role for horizontally transferred DncV in cGAMP production and regulation of biofilm formation and motility in the animal commensal strain Escherichia coli ECOR31. ECOR31 expresses a semiconstitutive temperature-independent rdar (red, dry, and rough) morphotype on Congo red agar plates characterized by the extracellular matrix components cellulose and curli fimbriae which requires activation by the major biofilm regulator CsgD and cyclic di-GMP signaling. In contrast, C-terminal His-tagged DncV negatively regulates the rdar biofilm morphotype and cell aggregation via downregulation of csgD mRNA steady-state level. Furthermore, DncV sequentially promotes and inhibits adhesion to the abiotic surface after 24 h and 48 h of growth, respectively. DncV also suppresses swimming and swarming motility posttranscriptional of the class 1 flagellum regulon gene flhD. Purified DncV produced different cDNs, cyclic di-GMP, cyclic di-AMP, an unknown product(s), and the dominant species 3′3′-cGAMP. In vivo, only the 3′3′-cGAMP concentration was elevated upon short-term overexpression of dncV, making this work a first report on cGAMP production in E. coli. Regulation of rdar biofilm formation and motility upon overexpression of untagged DncV in combination with three adjacent cotransferred gene products suggests a novel temperature-dependent cGAMP signaling module in E. coli ECOR31. IMPORTANCE The ability of bacteria to sense and respond to environmental signals is critical for survival. Bacteria use cyclic dinucleotides as second messengers to regulate a number of physiological processes, such as the fundamental life style transition between motility and sessility (biofilm formation). cGAMP, which is synthesized by a dinucleotide cyclase called DncV, is a newly discovered second messenger involved in virulence and chemotaxis in the Vibrio cholerae biovar El Tor causing the current 7th cholera pandemic. However, to what extent cGAMP exists and participates in physiological processes in other bacteria is still unknown. In this study, we found an elevated cGAMP level to possibly regulate biofilm formation and motility in the animal commensal E. coli strain ECOR31. Thus, we detected a novel role for cGAMP signaling in regulation of physiological processes other than those previously reported in proteobacterial species.

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K‐12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K‐12, the 4.88 Mbp Fec10 genome is characterized by distinct single‐nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two‐component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpG_GI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand‐alone disaggregation activity, but observed distinct biochemical characteristics of ClpG_GI‐Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpG_GI‐SG17M. Furthermore, we noted a unique contribution of ClpG_GI‐Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food‐borne heat‐resistant strains in the human gut.

Agar plate‐based biofilm of enterobacteria like Escherichia coli is characterized by expression of the extracellular matrix components amyloid curli and cellulose exopolysaccharide, which can be visually enhanced upon addition of the dye Congo Red, resulting in a red, dry, and rough (rdar) colony morphology. Expression of the rdar morphotype depends on the transcriptional regulator CsgD and occurs predominantly at ambient temperature in model strains. In contrast, commensal and pathogenic isolates frequently express the csgD‐dependent rdar morphotype semi‐constitutively, also at human host body temperature. To unravel the molecular basis of temperature‐independent rdar morphotype expression, biofilm components and c‐di‐GMP turnover proteins of seven commensal and uropathogenic E. coli isolates were analyzed. A diversity within the c‐di‐GMP signaling network was uncovered which suggests alteration of activity of the trigger phosphodiesterase YciR to contribute to (up)regulation of csgD expression and consequently semi‐constitutive rdar morphotype development.

Biofilm formation is a fundamental part of life cycles of bacteria which affects various aspects of bacterial-host interactions including the development of drug resistance and chronic infections. In clinical settings, biofilm-related infections are becoming increasingly difficult to treat due to tolerance to antibiotics. Bacterial biofilm formation is regulated by different external and internal factors, among which quorum sensing (QS) signals and nucleotide-based second messengers play important roles. In recent years, different kinds of anti-biofilm agents have been discovered, among which are the Chinese herbal medicines (CHMs). CHMs or traditional Chinese medicines have long been utilized to combat various diseases around the world and many of them have the ability to inhibit, impair or decrease bacterial biofilm formation either through regulation of bacterial QS system or nucleotide-based second messengers. In this review, we describe the research progresses of different chemical classes of CHMs on the regulation of bacterial biofilm formation. Though the molecular mechanisms on the regulation of bacterial biofilm formation by CHMs have not been fully understood and there are still a lot of work that need to be performed, these studies contribute to the development of effective biofilm inhibitors and will provide a novel treatment strategy to control biofilm-related infections.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapV_Q329R, but not CapV, causes pronouncedsulA-independent pyridoxine-inhibited cell filamentation in theEscherichia coliK-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapV_Q329Rphenotypes. Furthermore, CapV_Q329Rproduction substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenicE. colistrains andSalmonella typhimuriumresponded with cell filamentation and modulation in colony morphotype formation to CapV_Q329Rexpression. In conclusion, this work identifies the CapV variant CapV_Q329Ras a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.