Construction of a Broad-Host-Range Tn
            <i>7</i>
            -Based Vector for Single-Copy P
            <sub>BAD</sub>
            -Controlled Gene Expression in Gram-Negative Bacteria

Pseudomonas aeruginosa thrives in multiple environments and is capable of causing life-threatening infections in immunocompromised patients. RsmA is a posttranscriptional regulator that controls virulence factor production and biofilm formation. In this study, we investigated the expression and activity of rsmA and the protein that it encodes, RsmA, in P. aeruginosa mucA mutant strains, which are common in chronic infections. We determined that AlgU regulates a previously unknown rsmA promoter in P. aeruginosa. Western blot analysis confirmed that AlgU controls rsmA expression in both a laboratory strain and a clinical isolate. RNase protection assays confirmed the presence of two rsmA transcripts and suggest that RpoS and AlgU regulate rsmA expression. Due to the increased amounts of RsmA in mucA mutant strains, a translational leader fusion of the RsmA target, tssA1, was constructed and tested in mucA, algU, retS, gacA, and rsmA mutant backgrounds to examine posttranscriptional activity. From these studies, we determined that RsmA is active in mucA22 mutants, suggesting a role for RsmA in mucA mutant strains. Taken together, we have demonstrated that AlgU controls rsmA transcription and is responsible for RsmA activity in mucA mutant strains. We propose that RsmA is active in P. aeruginosa mucA mutant strains and that RsmA also plays a role in chronic infections. IMPORTANCEP. aeruginosa causes severe infections in immunocompromised patients. The posttranscriptional regulator RsmA is known to control virulence and biofilm formation. We identify a new rsmA promoter and determine that AlgU is important in the control of rsmA expression. Mutant mucA strains that are considered mucoid were used to confirm increased rsmA expression from the AlgU promoter. We demonstrate, for the first time, that there is RsmA activity in mucoid P. aeruginosa strains. Our work suggests that RsmA may play a role during chronic infections as well as acute infections. Pseudomonas aeruginosa produces a myriad of virulence factors and can cause both acute and chronic infections (1, 2). To survive and persist, P. aeruginosa must coordinate gene expression in response to changing environmental conditions. Global regulatory networks respond to changing environmental conditions to allow the physiological changes necessary for survival to be made. The P. aeruginosa genome encodes many global regulatory systems, including posttranscriptional regulators, such as RsmA. RsmA is important in regulating several virulence factors involved in acute and chronic infections (3).Acute infections are characterized by motility and expression of the type III secretion system (4). RsmA positively regulates both motility and type III secretion system expression (4-6). A defining characteristic of chronic infections is biofilm formation, which includes the production of exopolysaccharides, such as alginate, and the type VI secretion system (T6SS) (7,8). RsmA is a negative regulator of the exopolysaccharide gene psl and the first hemolysin-coregulated se...

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“…For overexpression of algU, we used an arabinoseinducible promoter to control algU expression (43). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by Increasing rsmA Expression in a mucA22 Strain

Stacey

Pritchett

2016

J Bacteriol

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“…In order to characterize the functional roles of carO and carP in response to Ca 2ϩ , we obtained transposon mutants of each gene from the University of Washington two-allele library (69). Each mutation was complemented by cloning the respective gene into the single-copy Tn7 expression vector pTJ1 (46). To determine the effect of Ca 2ϩ on growth, each mutant strain and its respective complemented counterpart was cultured in BMM with or without 10 mM CaCl 2 .…”

Section: Resultsmentioning

confidence: 99%

The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

et al. 2016

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Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca 2؉ ) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca 2؉ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca 2؉ level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca . In addition, a mutation in carP had a pleotropic effect in a Ca 2؉ -dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca 2؉ and responding through its regulatory targets that modulate Ca 2؉ homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin. P seudomonas aeruginosa, a natural inhabitant of soil and water, is able to infect a variety of hosts, including plants and humans. In humans, it causes severe acute and chronic infections by colonizing respiratory and urinary tracts and burned or wounded epithelia, cornea, and muscles (1-3). The versatility of P. aeruginosa pathogenicity is associated with diverse metabolic capabilities, multiple mechanisms of resistance, a large repertoire of virulence factors, and adaptability, due in part to the tightly coordinated regulation of gene expression. A large portion of the P. aeruginosa PAO1 genome, approximately 9.4%, encodes transcriptional regulators (4, 5), including two-component regulators: 89 response regulators, 55 sensor kinases, and 14 sensorresponse regulator hybrids (2). The regulatory targets for most of these regulatory systems are unknown. IMPORTANCE During infectious disease,Calcium plays an important signaling role in both eukaryotic and prokaryotic cells. In prokaryotes, Ca 2ϩ is an essential nutrient, since it is a necessary cofactor for many enzymes. However, Ca 2ϩ can be toxic to cells at high concentrations; therefore, bacteria maintain a low-submicromolar intracellular concentration of Ca 2ϩ (6). P. aeruginosa may encounter environments where

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“…For Salmonella and E. coli, the single attTn7 site lies near the conserved glmS gene (16). Modifications have been made in an attempt to improve the flexibility of the Tn7 system, such as through the generation of new helper plasmids (17), helper plasmid and delivery plasmid hybrids (18), arabinoseinducible gene expression (19), and luciferase integration (20,21). Unfortunately, the Tn7 system (12,18) did not work in our hands for Salmonella.…”

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confidence: 99%

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Shivak

MacKenzie

Watson

et al. 2016

Appl Environ Microbiol

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Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin-and chloramphenicolresistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of 70 -dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10-to 30-fold boost in transposase gene expression and transposition efficiencies of 10 ؊8 to 10 ؊10 in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCEThis article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonella and E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae. This technology will allow for improved in vivo analysis of bacterial pathogens.T he ability to generate recombinant strains of Escherichia coli and Salmonella enterica has facilitated insight into their ecology and pathogenic mechanisms. In recent years, strain collections consisting of single-gene knockouts of the entire genomes of E. coli K-12 (1) and S. enterica serovar Typhimurium 14028 (2) have been assembled. These collections can be used for finely tuned analysis of gene function and host-pathogen interactions, as well as for strain fitness and competition experiments (3). There are also a wide array of reporter systems available to analyze gene expression in detail, for the ordering of hierarchical gene circuits (4), a deeper understanding of metabolism (5), or the development of biosensors (6). The use of these systems, coupled with next-generation sequencing approaches that have facilitated functio...

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Construction of a Broad-Host-Range Tn 7 -Based Vector for Single-Copy P _BAD -Controlled Gene Expression in Gram-Negative Bacteria

Cited by 25 publications

References 18 publications

Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by Increasing rsmA Expression in a mucA22 Strain

Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by Increasing rsmA Expression in a mucA22 Strain

The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

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Construction of a Broad-Host-Range Tn 7 -Based Vector for Single-Copy P BAD -Controlled Gene Expression in Gram-Negative Bacteria

Cited by 25 publications

References 18 publications

Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by Increasing rsmA Expression in a mucA22 Strain

Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by Increasing rsmA Expression in a mucA22 Strain

The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Contact Info

Product

Resources

About

Construction of a Broad-Host-Range Tn 7 -Based Vector for Single-Copy P _BAD -Controlled Gene Expression in Gram-Negative Bacteria