The virulence factor pyocyanin and the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP) play key roles in regulating biofilm formation and multi-drug efflux pump expression in Pseudomonas aeruginosa. However, the crosstalk between these two signaling pathways remains unclear. Here we show that BrlR (PA4878), previously identified as a c-di-GMP responsive transcriptional regulator, acts also as a receptor for pyocyanin. Crystal structures of free BrlR and c-di-GMP-bound BrlR reveal that the DNA-binding domain of BrlR contains two separate c-di-GMP binding sites, both of which are involved in promoting brlR expression. In addition, we identify a pyocyanin-binding site on the C-terminal multidrug-binding domain based on the structure of the BrlR-C domain in complex with a pyocyanin analog. Biochemical analysis indicates that pyocyanin enhances BrlR-DNA binding and brlR expression in a concentration-dependent manner.

Bacterial predation plays multiple essential roles in bacterial selection and mortality within microbial ecosystems. In addition to its ecological and evolutionary importance, many potential applications of bacterial predation have been proposed. The myxobacterium Myxococcus xanthus is a well-known predatory member of the soil microbial community. Its predation is commonly considered a collective behavior comparable to a wolf pack attack; however, individual M. xanthus cells are also able to competently lead to the lysis of a prey cell. Using a bacterial tracking technique, we are able to observe and analyze solitary predation by M. xanthus on Escherichia coli at the single-cell level and reveal the dynamics of both predator and prey during the process. The present study will not only provide a comprehensive understanding of M. xanthus solitary predation but also help to explain why M. xanthus often displays multicellular characteristic predatory behaviors in nature, while a single cell is capable of predation.

The broad-spectrum antimicrobial activity of Elsholtzia ciliate essential oil (ECO) has been previously reported, but its effectiveness against halitosis-causing bacteria such as Fusobacterium nucleatum and Porphyromonas gingivalis is not well understood. In this study, we investigated the bacteriostatic activity of ECO against planktonic cells and biofilms of F. nucleatum and P. gingivalis, as well as its ability to inhibit bacterial metabolism and production of volatile sulfur compounds (VSCs) at sub-lethal concentrations. Our findings revealed that ECO exhibited comparable activities to chlorhexidine against these oral bacteria. Treatment with ECO significantly reduced the production of VSCs, including hydrogen sulfide, dimethyl disulfide, and methanethiol, which are major contributors to bad breath. As the major chemical components of ECO, carvacrol, p-cymene, and phellandrene, were demonstrated in vitro inhibitory effects on F. nucleatum and P. gingivalis, and their combined use showed synergistic and additive effects, suggesting that the overall activity of ECO is derived from the cumulative or synergistic effect of multiple active components. ECO was found to have a destructive effect on the bacterial cell membrane by examining the cell morphology and permeability. Furthermore, the application of ECO induced significant changes in the bacterial composition of saliva-derived biofilm, resulting in the elimination of bacterial species that contribute to halitosis, including Fusobacterium, Porphyromonas, and Prevotella. These results provide experimental evidence for the potential clinical applications of ECOs in the prevention and treatment of halitosis.

C-di-GMP (3’,5’ -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity.