A set of cassettes and improved vectors for genetic and biochemical characterization of pseudomonas genes

Deretic, Vojo; Chandrasekharappa, Settara C.; Gill, John F.; Chatterjee, Deb; Chakrabarty, Ananda M.

doi:10.1016/0378-1119(87)90177-6

Cited by 79 publications

(31 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This fragment was cloned into pCR2.1 (Invitrogen) and then subcloned into pVDtac39 (11), creating the plasmid pVDtacPIL. The orientation of the insert relative to the tac promoter was determined by HindIII digest.…”

Section: Methodsmentioning

confidence: 99%

Identification of AlgR-Regulated Genes in Pseudomonas aeruginosa by Use of Microarray Analysis

et al. 2004

View full text Add to dashboard Cite

The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression. In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P. aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P. aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid. Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes. Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2). An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1. Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P. aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain. AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557. Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant. This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb 3 -type cytochrome PA1557.

show abstract

Section: Methodsmentioning

confidence: 99%

Identification of AlgR-Regulated Genes in Pseudomonas aeruginosa by Use of Microarray Analysis

et al. 2004

View full text Add to dashboard Cite

show abstract

“…When plasmids used for complementation assays were generated, the respective cnr genes were amplified from plasmid pMOL28 by PCR (which introduced suitable restriction sites), cloned into pGEM T-Easy (Promega), sequenced, and subcloned into the broad-host-range vector pVDZЈ2 (6). To measure the activity of the cnr promoters, cnrYp (bp 698 to 1284), cnrHp (bp 1516 to 1740), and cnrCp (bp 2250 to 2360) were fused upstream to a promoterless lacZ gene and into plasmid pVDZЈ2 in the orientation opposite to that of the lac promoter located on this vector plasmid (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…To study a possible interaction between cnr and the highly related nickelcobalt-cadmium resistance determinant ncc (34), the regulatory regions nccYXH and nccN were PCR cloned from A. xylosoxidans 31A into pVDZЈ2 (6). In the ⌬cnrYXH strain DN190(pMOL28-3), expression of nccYXH in trans did not mediate nickel-inducible expression of cnr (Table 2).…”

Section: Fig 2 Rt-pcr Of the Cnrc Regionmentioning

confidence: 99%

Regulation of the cnr Cobalt and Nickel Resistance Determinant from Ralstonia sp. Strain CH34

2000

View full text Add to dashboard Cite

Ralstonia sp. strain CH34 is resistant to nickel and cobalt cations. Resistance is mediated by the cnr determinant located on plasmid pMOL28. The cnr genes are organized in two clusters, cnrYXH and cnrCBA. As revealed by reverse transcriptase PCR and primer extension, transcription from these operons is initiated from promoters located upstream of the cnrY and cnrC genes. These two promoters exhibit conserved sequences at the ؊10 (CCGTATA) and ؊35 (CRAGGGGRAG) regions. The CnrH gene product, which is required for expression of both operons, is a sigma factor belonging to the sigma L family, whose activity seems to be governed by the membrane-bound CnrY and CnrX gene products in response to Ni 2؉. Half-maximal activation from the cnrCBA operon was determined by using appropriate lacZ gene fusions and was shown to occur at an Ni 2؉ concentration of about 50 M.Ralstonia sp. strain CH34 (formerly Alcaligenes eutrophus strain CH34 [3]) contains at least seven determinants encoding resistances to toxic heavy metals, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (180 kb [37]) and pMOL30 (238 kb [7,20]). The cnr determinant of plasmid pMOL28 mediates inducible resistance to Co 2ϩ and Ni 2ϩ in Ralstonia sp. strain CH34 (15). The cnr determinant is similar to ncc (nickel-cobalt-cadmium resistance) of Alcaligenes xylosoxidans 31A (34) and czc (cobaltzinc-cadmium resistance) on plasmid pMOL30 of Ralstonia sp. strain CH34 (28). All three resistances are based on cation efflux, which is best characterized for Czc (13,24,29). In analogy to Czc (9, 25, 28, 32), the products of the genes cnrA, cnrB, and cnrC are likely to form a membrane-bound protein complex catalyzing an energy-dependent efflux of Ni 2ϩ and Co 2ϩ , and the mechanism of action of the CnrCBA complex may be that of a proton/cation antiporter.Three regulators seem to control Cnr, an extracellular function (ECF) sigma factor (CnrH) and the products of two additional genes with unknown precise functions (CnrX and CnrY products, respectively) (15, 16). The genes cnrYXH are located upstream of cnrCBA and have the same direction of transcription. Transposon Tn5 insertion upstream of cnrH led to a constitutive expression of nickel resistance and to low zinc resistance as well (4, 15). However, this may be a polar effect and, additionally, a readthrough from a transposon promoter. As shown with Tn5-lacZ fusions (31), cnr is best induced by 128 M Ni 2ϩ . Other metals serve as less efficient inducers; however, this experiment was done with nickel-sensitive cnr-lacZ transposon-insertion mutants. In this study an improved cnrCBA-lacZ operon fusion was constructed, which mediates full nickel resistance and which was used to evaluate the physiology of cnr regulation.

show abstract

“…The DNA sequence of the cloned insert was verified, and one of the positive clones, termed pCR17EA, was used for further manipulations. The 1.7-kb insert from pCR17EA was cloned as an EcoRI fragment into pVDtac39 (12) to generate pHY17EC. For the construction of pHYEG and pHYAG, the 1.7-kb insert was first cloned into a modified pUC12 vector (pUC12H) which had its HindIII site eliminated (by cutting pUC12 with HindIII, filling in the ends, and blunt end ligation), and the resulting plasmid was termed p12HEA.…”

Section: Methodsmentioning

confidence: 99%

Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa

1995

View full text Add to dashboard Cite

Mucoid colony morphology is the result of the overproduction of the exopolysaccharide alginate and is considered to be a major pathogenic determinant expressed by Pseudomonas aeruginosa during chronic respiratory infections in cystic fibrosis. Conversion to mucoidy can be caused by mutations in the second or third gene of the stress-responsive system algU mucA mucB. AlgU is 66% identical to the alternative sigma factor RpoE ( E ) from Escherichia coli and Salmonella typhimurium and directs transcription of several critical alginate biosynthetic and regulatory genes. AlgU is also required for the full resistance of P. aeruginosa to reactive oxygen intermediates and heat killing. In this work, we report that E. coli E can complement phenotypic defects of algU inactivation in P. aeruginosa: (i) the rpoE gene from E. coli complemented an algU null mutant of P. aeruginosa to mucoidy; (ii) the presence of the E. coli rpoE gene in P. aeruginosa induced alginate production in the standard genetic nonmucoid strain PAO1; (iii) the plasmid-borne E. coli rpoE gene induced transcription of algD, a critical algU-dependent alginate biosynthetic gene; and (iv) when present in algU::Tc r mutants, E. coli rpoE partially restored resistance to paraquat, a redox cycling compound that increases intracellular levels of superoxide radicals. A new gene, mclA, encoding a polypeptide with an apparent molecular mass of 27.7 kDa was identified immediately downstream of rpoE in E. coli. The predicted product of this gene is 28% identical (72% similar) to MucA, a negative regulator of AlgU activity in P. aeruginosa. The results reported in this study demonstrate that RpoE and AlgU are functionally interchangeable in P. aeruginosa and suggest that elements showing sequence similarity to those known to regulate AlgU activity in P. aeruginosa are also present in other bacteria.

show abstract

A set of cassettes and improved vectors for genetic and biochemical characterization of pseudomonas genes

Cited by 79 publications

References 29 publications

Identification of AlgR-Regulated Genes in Pseudomonas aeruginosa by Use of Microarray Analysis

Identification of AlgR-Regulated Genes in Pseudomonas aeruginosa by Use of Microarray Analysis

Regulation of the cnr Cobalt and Nickel Resistance Determinant from Ralstonia sp. Strain CH34

Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa

Contact Info

Product

Resources

About