Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.06.15.496363

A library of promoter-gfp fusion reporters for studying systemic expression pattern of cyclic-di-GMP metabolism-related genes in Pseudomonas aeruginosa

Liu, D.·Wang, D.·Wei, Q.·Zhang, Y.·Ma, L. Z.
View original source ↗Export metadata ↓

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.

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Liu, D., Wang, D., Wei, Q., Zhang, Y., Ma, L. Z.. 2022-06-16. A library of promoter-gfp fusion reporters for studying systemic expression pattern of cyclic-di-GMP metabolism-related genes in Pseudomonas aeruginosa. https://doi.org/10.1101/2022.06.15.496363

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A population-scale landscape of the subgingival microbiome reveals divergent routes to periodontal dysbiosis

Periodontitis is an archetypical mucosal inflammatory disease in which microbiome dysbiosis at the tooth-epithelial interface interacts with host genetic and behavioral risk factors to drive immune-mediated tissue destruction. Although subgingival microbiome compositional shifts are thought to parallel disease severity, microbiome variation at the population-level and its relationship to periodontal clinical phenotypes and disease-modifying factors remain poorly defined. Here, we use unsupervised manifold learning to map the compositional landscape of the subgingival microbiome in 1,355 adults spanning periodontal health to severe periodontitis. We identified eight latent microbiome states organized along a branching continuum from eubiosis to dysbiosis. An intermediate microbial configuration marked ecological destabilization and bifurcation into two distinct periodontitis-associated dysbiotic trajectories, distinguished by links to gingival inflammation and smoking. Although the microbiome trajectories broadly tracked periodontal destruction, a minority of individuals showed discordant microbiome-clinical phenotypes, with some individuals with periodontitis retaining otherwise eubiotic microbiomes enriched for low-abundance pathobionts, while some cases of health or mild disease had highly dysbiotic communities, suggesting distinct host susceptibility. Together, these findings define a population-scale ecological landscape of the subgingival microbiome, reveal divergent trajectories to periodontal dysbiosis, and highlight heterogeneity in the relationship between microbial community structure and clinical disease expression.

microbiology↗
bioRxiv2026

Beta-lactam enhancement against methicillin-resistant Staphylococcus aureus by cell wall blockade is autolysis-dependent: a butyrolactone derivative as case in point

Methicillin-resistant Staphylococcus aureus (MRSA) is non-susceptible to beta-lactams. Blockade of cell wall biosynthesis is a potential target for beta-lactam enhancement but requires further investigation. A butyrolactone derivative enhanced beta-lactams against MRSA strains by reducing the availability of D-Ala-D-Ala. Unlike D-cycloserine, it did not inhibit D-Ala-D-Ala ligase (Ddl). Nor did it show an additive or synergistic effect when combined with cycloserine, indicating a unique mechanism for blocking cell wall precursor production that does not involve the traditional Lipid II pathway. Notably, beta-lactam potentiation by our chemical or D-cycloserine was highly dependent on the intrinsic autolytic ability of the tested MRSA strains. Strains that resisted lysis upon Triton X-100 exposure showed a minimal increase in beta-lactam susceptibility, whereas highly autolytic strains showed significant changes in their beta-lactam MICs. We have thus identified autolytic ability as the Achilles Heel in the strategy of targeting cell wall biosynthesis for beta-lactam potentiation.

microbiology↗
bioRxiv2026

Rapid and largely reversible shifts in the canine fecal metabolome during dietary change

Diet can rapidly change the fecal metabolome, but less is known about recovery after the original diet is restored. We used untargeted UPLC-MS metabolomics to analyze 72 fecal samples from nine Pumi dogs during an owner-managed switch from dry food to raw food and back to dry food. Diet phase accounted for a large proportion of variation in both ionization modes. More than 13,000 LC-MS features changed at the first sampling point after the switch to raw food, with a similarly large response after return to dry food. Among features significant in both comparisons, more than 99% changed in opposite directions. At the final sampling point, no positive-mode (ESI+) features and only 13 negative-mode (ESI-) features differed from the second dry-food baseline under the same threshold. BARF-associated patterns persisted in analyses excluding individual dogs and in pedigree-adjusted candidate models, although individual feature effects depended on normalization. Putative metabolites from several biochemical classes differed in their response and recovery. The fecal metabolome therefore changed rapidly and returned largely toward baseline, with differences among dogs.

microbiology↗