The Periplasmic Protein TolB as a Potential Drug Target in Pseudomonas aeruginosa

Sciuto, Alessandra Lo; Fernández-Piñar, Regina; Bertuccini, Lucia; Iosi, Francesca; Superti, Fabiana; Imperi, Francesco

doi:10.1371/journal.pone.0103784

Cited by 56 publications

(62 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This weak RBS would offset the effects of araC-ParaBAD leakiness by decreasing translational initiation and accumulation of the resulting protein gene product. Subsequently, miniCTX1 derivatives that preserve this weak RBS were constructed and used to study other essential envelope biosynthesis genes (16,44). As we demonstrated in our study, decreasing RBS strength can reduce the problem of promoter leakiness, but it also reduces the maximal induced amount of gene product and narrows the range within which the gene product can be modulated.…”

Section: Discussionmentioning

confidence: 68%

The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

Meisner

Goldberg

2016

Appl Environ Microbiol

View full text Add to dashboard Cite

The araC-ParaBAD inducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range in Escherichia coli, which has led to its widespread use in other bacteria. Although anecdotal evidence suggests that araCParaBAD is leaky in Pseudomonas aeruginosa, neither a thorough analysis of this inducible promoter system in P. aeruginosa nor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of the araC-ParaBAD system in P. aeruginosa. Using transcriptional fusions to a lacZ reporter gene, we determined that the noninduced expression from araC-ParaBAD is high and cannot be reduced by carbon catabolite repression as it can in E. coli. Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression in P. aeruginosa significantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of the araC-ParaBAD system, we found that the lacI q -Ptac and rhaSR-PrhaBAD inducible promoter systems had significantly lower noninduced expression and were inducible over a broader range than araC-ParaBAD. We demonstrated that noninduced expression from the araC-ParaBAD system supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis in P. aeruginosa, problems that were avoided with rhaSR-PrhaBAD. rhaSR-PrhaBAD is tightly controlled, allows gene expression over a wide range, and represents a significant improvement over araC-ParaBAD in P. aeruginosa. IMPORTANCEWe report the shortcomings of the commonly used Escherichia coli araC-ParaBAD inducible promoter system in Pseudomonas aeruginosa, successfully reengineered it to improve its functionality, and show that the E. coli rhaSR-PrhaBAD system is tightly controlled and allows inducible gene expression over a wide range in P. aeruginosa. Pseudomonas aeruginosa is a versatile Gram-negative bacterium that inhabits a variety of different environments. It is also an opportunistic human pathogen that causes acute infections in hospitalized patients as well as chronic infections in cystic fibrosis patients. Unfortunately, P. aeruginosa infections are becoming difficult to treat because of the increasing prevalence of multidrug (antibiotic) resistance (1). To improve the treatment of these infections, we need to understand which gene functions are essential for the growth of P. aeruginosa and develop new therapeutics to inhibit them. The study of essential genes is difficult because, by definition, inactivation of an essential gene is lethal to the cell. Analysis of essential genes generally involves the construction of conditional mutants, often accomplished by controlling the expression of a gene with an inducible promoter...

show abstract

Section: Discussionmentioning

confidence: 68%

The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

Meisner

Goldberg

2016

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Deletion of the region encompassing the lac promoter and the first 24 codons of the lacZ␣ gene was necessary to eliminate the transcriptional readthrough of RNA polymerase from the P lac promoter and the formation of a chimeric protein composed of LacZ␣ and the cloned insert. Subsequently, pVRL1b was digested at the NsiI (introduced with primer ΔlacP RV) and XhoI sites, to insert the arabinose-inducible araC-P BAD expression cassette amplified from miniCTX1-araC-P BAD tolB (14), using primers araC-P BAD FW and araC-P BAD RV. The resulting plasmid, named pVRL2 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

New Shuttle Vectors for Gene Cloning and Expression in Multidrug-Resistant Acinetobacter Species

Lucidi

Runci

Rampioni

et al. 2018

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Understanding bacterial pathogenesis requires adequate genetic tools to assess the role of individual virulence determinants by mutagenesis and complementation assays, as well as for homologous and heterologous expression of cloned genes. Our knowledge of pathogenesis has so far been limited by the scarcity of genetic tools to manipulate multidrug-resistant (MDR) epidemic strains, which are responsible for most infections. Here, we report on the construction of new multipurpose shuttle plasmids, namely, pVRL1 and pVRL2, which can efficiently replicate in spp. and in The pVRL1 plasmid has been constructed by combining (i) the cryptic plasmid pWH1277 from, which provides an origin of replication for spp.; (ii) a ColE1-like origin of replication; (iii) the gentamicin or zeocin resistance cassette for antibiotic selection; and (iv) a multilinker containing several unique restriction sites. Modification of pVRL1 led to the generation of the pVRL2 plasmid, which allows arabinose-inducible gene transcription with an undetectable basal expression level of cloned genes under uninduced conditions and a high dynamic range of responsiveness to the inducer. Both pVRL1 and pVRL2 can easily be selected in MDR, have a narrow host range and a high copy number, are stably maintained in spp., and appear to be compatible with indigenous plasmids carried by epidemic strains. Plasmid maintenance is guaranteed by the presence of a toxin-antitoxin system, providing more insights into the mechanism of plasmid stability in spp.

show abstract

“…Given the importance of the Tol-Pal complex in OM stability and bacterial cell division, it would be an attractive target for small molecule inhibition. This is especially so in some organisms, including the opportunistic human pathogen Pseudomonas aeruginosa, where the complex is essential for growth (Dennis et al, 1996;Lo Sciuto et al, 2014). The lack of understanding of the true function of the Tol-Pal complex, however, has impeded progress.…”

Section: Discussionmentioning

confidence: 99%

Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli

Shrivastava

Jiang

Chng

2017

Molecular Microbiology

View full text Add to dashboard Cite

Biogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well-studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans-envelope Tol-Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol-Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess PLs in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol-Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol-Pal complex may promote OM invagination during cell division.

show abstract

The Periplasmic Protein TolB as a Potential Drug Target in Pseudomonas aeruginosa

Cited by 56 publications

References 38 publications

The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

New Shuttle Vectors for Gene Cloning and Expression in Multidrug-Resistant Acinetobacter Species

Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli

Contact Info

Product

Resources

About