Dataset Information

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

ABSTRACT: A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

SUBMITTER: Ochsner UA

PROVIDER: S-EPMC205310 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

Ochsner U A UA Koch A K AK Fiechter A A Reiser J J

Journal of bacteriology 19940401 7

A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosyn ...[more]

PMID: 8144472

Similar Datasets

Project description:Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death. Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions.Quantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments. We characterized hepP and its protein product HepP. hepP was cloned from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14?hepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14.Changes within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing.

Project description:As one of the most common pathogens of opportunistic and hospital-acquired infections, Pseudomonas aeruginosa is associated with resistance to diverse antibiotics, which represents a significant challenge to current treatment modalities. Phage therapy is considered a promising alternative to conventional antimicrobials. The characterization and isolation of new bacteriophages and the concurrent evaluation of their therapeutic potential are fundamental for phage therapy. In this study, we employed an enrichment method and a double-layer agar overlay to isolate bacteriophages that infect P. aeruginosa strains PAO1 and PA14. Three phages (named PA_LZ01, PA_LZ02, and PA_LZ03) were isolated and showed icosahedral heads and contractile tails. Following full-genome sequencing, we found that phage PA_LZ01 contained a genome of 65,367 bp in size and harbored 90 predicted open reading frames (ORFs), phage PA_LZ02 contained a genome of 57,243 bp in size and harbored 75 predicted ORFs, and phage PA_LZ03 contained a genome of 57,367 bp in size and carried 77 predicted ORFs. Further comparative analysis showed that phage PA_LZ01 belonged to the genus Pbunavirus genus, phage PA_LZ02 belonged to the genus Pamexvirus, and phage PA_LZ03 belonged to the family Mesyanzhinovviridae. Next, we demonstrated that these phages were rather stable at different temperatures and pHs. One-step growth curves showed that the burst size of PA_LZ01 was 15 PFU/infected cell, and that of PA_LZ02 was 50 PFU/infected cell, while the titer of PA_LZ03 was not elevated. Similarly, the biofilm clearance capacities of PA_LZ01 and PA_LZ02 were also higher than that of PA_LZ03. Therapeutically, PA_LZ01 and PA_LZ02 treatment led to decreased bacterial loads and inflammatory responses in a mouse model. In conclusion, we isolated three phages that can infect P. aeruginosa, which were stable in different environments and could reduce bacterial biofilms, suggesting their potential as promising candidates to treat P. aeruginosa infections. IMPORTANCE Phage therapy is a promising therapeutic option for treating bacterial infections that do not respond to common antimicrobial treatments. Biofilm-mediated infections are particularly difficult to treat with traditional antibiotics, and the emergence of antibiotic-resistant strains has further complicated the situation. Pseudomonas aeruginosa is a bacterial pathogen that causes chronic infections and is highly resistant to many antibiotics. The library of phages that target P. aeruginosa is expanding, and the isolation of new bacteriophages is constantly required. In this study, three bacteriophages that could infect P. aeruginosa were isolated, and their biological characteristics were investigated. In particular, the isolated phages are capable of reducing biofilms formed by P. aeruginosa. Further analysis indicates that treatment with PA_LZ01 and PA_LZ02 phages reduces bacterial loads and inflammatory responses in vivo. This study isolated and characterized bacteriophages that could infect P. aeruginosa, which offers a resource for phage therapy.

Dataset Information

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

Publications

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets