A library of promoter-gfpfusion reporters for studying systemic expression pattern of cyclic-di-GMP metabolism-related genes inPseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism, which is notorious for its resistance or tolerance to antibiotics due to the formation of biofilms. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains 41 genes that encode enzymes to participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools to investigate the systemic expression pattern of those genes. Here, we constructed a promoter-gfp transcriptional fusion reporters’ library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c-di-GMP metabolism-related (CMR) genes from P. aeruginosa PAO1 strain, thus each promoter-Gfp fusion reporter can be used to detect the promotor’ activity as well as the transcription of corresponding gene. The promoters’ activity was tested in P. aeruginosa and Escherichia coli respectively. Among the 41 genes, the promoter of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and sub-inhibitory concentrations of antibiotics on transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed different growth conditions did impact different genes’ transcription, while the promoter’ activity of a few genes kept at the same level under several different growth conditions. In summary, we provided a promoter-gfp fusion reporters’ library for systemic monitoring or study of the regulation of CMR genes in P. aeruginosa and the functional promoters can also be used as a bio-brick for synthetic biology studies.ImportanceThe opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans and it is one of main pathogens in nosocomial infections. Biofilm formation is one of most important causes for P. aeruginosa to persist in hosts and evade immune and antibiotic attacks. c-di-GMP is an important second messenger to control biofilm formation. In P. aeruginosa, there are 41 genes that are predicted to participate in the making and breaking this dinucleotide. A major missing information in this field is the systemic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter-gfp transcriptional fusion reporters’ library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.