Construction of Mobilizable Mini-Tn
            <i>7</i>
            Vectors for Bioluminescent Detection of Gram-Negative Bacteria and Single-Copy Promoter
            <i>lux</i>
            Reporter Analysis

Damron, F. Heath; McKenney, Elizabeth S.; Barbier, Mariette; Liechti, George W.; Schweizer, Herbert P.; Goldberg, Joanna B.

doi:10.1128/aem.00640-13

Cited by 58 publications

(37 citation statements)

References 19 publications

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“…Overexpression/complementation studies. In order to overexpress algU, the coding sequence was amplified (Table 2) and cloned into the integrating vector pTJ1 for complementation or overexpression (36). Trimethoprim-resistant colonies were picked, cultured overnight, and diluted 1,000-fold on the following day.…”

Section: Methodsmentioning

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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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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Section: Methodsmentioning

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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“…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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“…The pHERD20T plasmid with alk genes and their respective promoters were electrotransformed into PAO1 and ATCC 33988 cells. The empty plasmid pHERD20T (57) was transformed into PAO1 wild-type cells and used as a control for the expression, growth, and Jet A degradation studies described below.…”

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confidence: 99%

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel

Gunasekera

Bowen

Zhou

et al. 2017

Appl Environ Microbiol

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Pseudomonas aeruginosa can utilize hydrocarbons, but different strains have various degrees of adaptation despite their highly conserved genome. P. aeruginosa ATCC 33988 is highly adapted to hydrocarbons, while P. aeruginosa strain PAO1, a human pathogen, is less adapted and degrades jet fuel at a lower rate than does ATCC 33988. We investigated fuel-specific transcriptomic differences between these strains in order to ascertain the underlying mechanisms utilized by the adapted strain to proliferate in fuel. During growth in fuel, the genes related to alkane degradation, heat shock response, membrane proteins, efflux pumps, and several novel genes were upregulated in ATCC 33988. Overexpression of alk genes in PAO1 provided some improvement in growth, but it was not as robust as that of ATCC 33988, suggesting the role of other genes in adaptation. Expression of the function unknown gene PA5359 from ATCC 33988 in PAO1 increased the growth in fuel. Bioinformatic analysis revealed that PA5359 is a predicted lipoprotein with a conserved Yx(FWY)xxD motif, which is shared among bacterial adhesins. Overexpression of the putative resistance-nodulation-division (RND) efflux pump PA3521 to PA3523 increased the growth of the ATCC 33988 strain, suggesting a possible role in fuel tolerance. Interestingly, the PAO1 strain cannot utilize n-C 8 and n-C 10 . The expression of green fluorescent protein (GFP) under the control of alkB promoters confirmed that alk gene promoter polymorphism affects the expression of alk genes. Promoter fusion assays further confirmed that the regulation of alk genes was different in the two strains. Protein sequence analysis showed low amino acid differences for many of the upregulated genes, further supporting transcriptional control as the main mechanism for enhanced adaptation.IMPORTANCE These results support that specific signal transduction, gene regulation, and coordination of multiple biological responses are required to improve the survival, growth, and metabolism of fuel in adapted strains. This study provides new insight into the mechanistic differences between strains and helpful information that may be applied in the improvement of bacterial strains for resistance to biotic and abiotic factors encountered during bioremediation and industrial biotechnological processes.

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Construction of Mobilizable Mini-Tn 7 Vectors for Bioluminescent Detection of Gram-Negative Bacteria and Single-Copy Promoter lux Reporter Analysis

Cited by 58 publications

References 19 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

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

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel

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