Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

In nature, bacteria form biofilms by producing exopolymeric matrix that encases its entire community. While it is widely known that biofilm matrix can prevent bacterivore predation and contain virulence factors for killing predators, it is unclear if they can alter predator motility. Here, we report a novel "quagmire" phenotype, where Pseudomonas aeruginosa biofilms could retard the motility of bacterivorous nematode Caenorhabditis elegans via the production of a specific exopolysaccharide, Psl. Psl could reduce the roaming ability of C. elegans by impeding the slithering velocity of C. elegans. Furthermore, the presence of Psl in biofilms could entrap C. elegans within the matrix, with dire consequences to the nematode. After being trapped in biofilms, C. elegans could neither escape effectively from aversive stimuli (noxious blue light), nor leave easily to graze on susceptible biofilm areas. Hence, this reduced the ability of C. elegans to roam and predate on biofilms. Taken together, our work reveals a new function of motility interference by specific biofilm matrix components, and emphasizes its importance in predator-prey interactions.

Pseudomonas aeruginosa is a major pathogen that causes serious acute and chronic infections in humans. The type III secretion system (T3SS) is an important virulence factor that plays essential roles in acute infections. However, the regulatory mechanisms of T3SS are not fully understood. In this study, we found that the deletion of cysB reduced the T3SS gene expression and swarming motility but enhanced biofilm formation. In a mouse acute pneumonia model, mutation of cysB decreased the average bacterial load compared to that of the wild-type strain. Further experiments demonstrated that CysB contributed to the reduced T3SS gene expression and bacterial pathogenesis by directly regulating the sensor kinase RetS. We also performed crystallographic studies of PaCysB. The overall fold of PaCysB NTD domain is similar to other LysR superfamily proteins and structural superposition revealed one possible DNA-binding model for PaCysB. Structural comparison also revealed great flexibility of the PaCysB RD domain, which may play an important role in bending and transcriptional regulation of target DNA. Taken together, these results expand our current understanding of the complex regulatory networks of T3SS and RetS pathways. The crystal structure of CysB provides new insights for studying the function of its homologs in other bacterial species.These authors contributed equally to this work.