[23] Genetic systems in Pseudomonas

Rothmel, Randi K.; Chakrabarty, Ananda M.; Berry, Alan; Darzins, Al

doi:10.1016/0076-6879(91)04025-j

Cited by 46 publications

(31 citation statements)

References 73 publications

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“…2b). No k35 consensus sequence could be identified ; however, this is a common finding for P. aeruginosa regulated promoters (Rothmel et al, 1991). Analogous primer-extension experiments performed with a primer complementary to phoP failed to detect any transcriptional start sites immediately upstream of the phoP gene (data not shown).…”

Section: A Single Mg 2m -Regulated Promoter Situated Upstream Of Oprhmentioning

confidence: 85%

Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides

2000

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Resistance to the polycationic antibiotic polymyxin B and expression of the outer-membrane protein OprH in the opportunistic pathogen Pseudomonas aeruginosa both involve the PhoP-PhoQ two-component regulatory system. The genes for this system form an operon with oprH, oprH-phoP-phoQ, that responds to Mg 2M starvation and PhoP levels. In this study, the Mg 2M -regulated promoter for this operon was mapped upstream of oprH by primer-extension experiments. An oprH ::xylE-Gm R mutant H855 was constructed and measurement of the catechol 2,3-dioxygenase activity expressed from this transcriptional fusion provided evidence for a second, weak promoter for phoP-phoQ. Wild-type P. aeruginosa PAO1 strain H103 was found to exhibit Mg 2M -regulated resistance to the α-helical antimicrobial cationic peptide CP28 in addition to its previously characterized resistance to polymyxin B. Resistance to this peptide was unchanged in the OprH-null mutant H855 and a PhoP-null mutant H851. In contrast, PhoQ-null mutant H854 demonstrated constitutive CP28 resistance. Northern blot analysis revealed constitutive expression of phoP in this strain, implicating PhoP-PhoQ in the resistance of P. aeruginosa to cationic peptides. Furthermore, all three null-mutant strains demonstrated increased resistance to the aminoglycoside antibiotics streptomycin, kanamycin and amikacin. Two additional mutant strains, H895 and H896, were constructed that carried unmarked deletions in oprH and were found to exhibit aminoglycoside susceptibility equivalent to that of the wildtype. This result provided definitive evidence that OprH is not involved in P. aeruginosa aminoglycoside resistance and that the changes in resistance in strain H855 and a previously reported oprH mutant were due to polar effects on phoP-phoQ rather than loss of OprH expression. A role for PhoP-PhoQ in resistance to aminoglycosides is envisaged that is distinct from that in resistance to cationic peptides and polymyxin B.

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Section: A Single Mg 2m -Regulated Promoter Situated Upstream Of Oprhmentioning

confidence: 85%

Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides

2000

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“…pMC10 was then transformed into E. coli XL-1 Blue, reisolated and sequenced. pMC10 was introduced into P. aeruginosa strains by triparental conjugation (Rothmel et al, 1991). b-Galactosidase assays were carried out according to Miller (1972) and all experiments were repeated at least three times.…”

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confidence: 99%

Regulation of expression of the cyanide-insensitive terminal oxidase in Pseudomonas aeruginosa

2003

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The regulation of the cyanide-insensitive oxidase (CIO) in Pseudomonas aeruginosa, a bacterium that can synthesize HCN, is reported. The expression of a cioA-lacZ transcriptional fusion, CioA protein levels and CIO activity were low in exponential phase but induced about fivefold upon entry into stationary phase. Varying the O 2 transfer coefficient from 11?5 h 21 to 87?4 h 21 had no effect on CIO expression and no correlation was observed between CIO induction and the dissolved O 2 levels in the growth medium. However, a mutant deleted for the O 2 -sensitive transcriptional regulator ANR derepressed CIO expression in an O 2 -sensitive manner, with the highest induction occurring under low-O 2 conditions. Therefore, CIO expression can respond to a signal generated by low O 2 levels, but this response is normally kept in check by ANR repression. ANR may play an important role in preventing overexpression of the CIO in relation to other terminal oxidases. A component present in spent culture medium was able to induce CIO expression. However, experiments with purified N-butanoyl-L-homoserine lactone or N-(3-oxododecanoyl)homoserine lactone ruled out a role for these quorum-sensing molecules in the control of CIO expression. Cyanide was a potent inducer of the CIO at physiologically relevant concentrations and experiments using spent culture medium from a DhcnB mutant, which is unable to synthesize cyanide, showed that cyanide was the inducing factor present in P. aeruginosa spent culture medium. However, the finding that in a DhcnB mutant cioA-lacZ expression was induced normally upon entry into stationary phase indicated that cyanide was not the endogenous inducer of the terminal oxidase. The authors suggest that the failure of O 2 to have an effect on CIO expression in the wild-type can be explained either by the requirement for an additional, stationary-phase-specific inducing signal or by the loss of an exponential-phase-specific repressing signal. INTRODUCTIONPseudomonas aeruginosa is an opportunistic pathogen that causes a variety of nosocomial infections, including pneumonia, urinary tract infections, surgical wound infections, and bloodstream infections (for a review see Deretic, 2000). It causes life-threatening illness in patients with cystic fibrosis. Initially, P. aeruginosa colonizes the airways with other pathogens such as Haemophilus influenzae and Staphylococcus aureus. However, in most of these patients chronic lung disease develops in which the bacterial population consists almost exclusively of P. aeruginosa in the form of biofilms (Govan & Deretic, 1996). P. aeruginosa is a facultative anaerobe that preferentially obtains its energy via aerobic respiration, but it is well adapted to conditions of limited O 2 supply (Palleroni, 1984;Davies et al., 1989). It is capable of anaerobic growth with nitrate as a terminal electron acceptor and in the absence of nitrate it is able to ferment arginine, generating ATP by substrate-level phosphorylation (Palleroni, 1984;Davies et al., 1989;Van der Wauven et ...

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“…Codon usage for the partial PI6 gene sequence resembles that for mvaA and mvaB (Beach, 1987) as well as for other Pseudomonas genes (Rothmel et al, 1991). The GC content for the partial PI6 gene sequence is approximately 57% GC, a value typical for pseudomonads.…”

Section: Discussionmentioning

confidence: 92%

Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon

Rosenthal

Rodwell

1998

Protein Science

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The mvaAB operon of Pseudomonas mevalonii encodes HMG-CoA reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the rnvaAB cis-regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA-binding activity, comigrated with PI5 and P16 during DNA-affinity chromatography, size-exclusion chromatography, and sucrose density gradient centrifugation. PI5 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31-kDa polypeptide complex that contained N-terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size-exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N-termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.

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[23] Genetic systems in Pseudomonas

Cited by 46 publications

References 73 publications

Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides

Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides

Regulation of expression of the cyanide-insensitive terminal oxidase in Pseudomonas aeruginosa

Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon

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