Genetic Analysis of the Assimilation of C ₅ -Dicarboxylic Acids in Pseudomonas aeruginosa PAO1

Abstract

There is a wealth of information on the genetic regulation and biochemical properties of bacterial C 4 -dicarboxylate transport systems. In sharp contrast, there are far fewer studies describing the transport and assimilation of C 5 -dicarboxylates among bacteria. In an effort to better our understanding on this subject, we identified the structural and regulatory genes necessary for the utilization of ␣-ketoglutarate (␣-KG) in Pseudomonas aeruginosa PAO1. The PA5530 gene, encoding a putative dicarboxylate transporter, was found to be essential for the growth of P. aeruginosa PAO1 on both ␣-KG and glutarate (another C 5 -dicarboxylate). Metabolite analysis confirmed that the PA5530 gene was necessary for the uptake of extracellular ␣-KG. Like other substrate-inducible transporter genes, expression of the PA5530 gene was induced by extracellular C 5 -dicarboxylates. It was later found that the expression of the PA5530 gene was driven solely by a ؊24/؊12 promoter recognized by the alternative sigma factor RpoN. Surprisingly, the enhancer binding protein MifR, which is known to have an essential role in biofilm development, was required for the expression of the PA5530 gene. The MifR protein is homologous to other transcriptional regulators involved in dicarboxylate assimilation, suggesting that MifR might interact with RpoN to activate the expression of the PA5530 gene in response to extracellular C 5 -dicarboxylates, especially ␣-KG. The results of this study provide a framework for exploring the assimilation of ␣-KG in other pseudomonads.

Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN

“…At an OD 600 of 0.3, 0.5 ml of culture was treated with 1.0 ml of RNAprotect bacterial reagent (Qiagen), and RNA was then purified from the treated cells using the RNeasy kit (Qiagen) (25). Prior to cDNA synthesis, the purified RNA was checked for genomic DNA contamination by PCR designed to amplify the rplU gene (24,30,31). Following this quality check, reverse transcriptase reactions were conducted using 500 ng of purified RNA and the iScript cDNA synthesis kit (Bio-Rad).…”

“…The P. aeruginosa and Escherichia coli strains used in this study are given in Table S1 in the supplemental material. The ⌬PA4021, ⌬PA4022, ⌬eat, and ⌬eutB deletion mutants of P. aeruginosa PAO1 were constructed using established methods that have been described (23)(24)(25). Bacteria were grown in Lennox broth (LB) or minimal medium (22 mM KH 2 PO 4 , 42 mM Na 2 HPO 4 , 8.6 mM NaCl, 1.0 mM MgSO 4 , 5.0 M FeSO 4 , and 2 mg liter Ϫ1 cyanocobalamin [pH 7.0]).…”

“…The PA4021 gene was subcloned into two different plasmids. In the first cloning event, PA4021 was subcloned into the XbaI/SacI sites of ⌬Plac-pBBR1MCS-5 (the pBBR1MCS-5 plasmid lacking the Plac promoter) (24) to give pBRL667. For the second cloning event, the PA4021 gene was subcloned into the XbaI sites of pTrc99a with a forward orientation relative to the trc promoter to yield pBRL602.…”

“…PCR was used to amplify the 5=-regulatory regions adjacent to the open reading frames (ORFs) for exaC, PA4022, and eat. The amplified 5=-regulatory regions (850 bp for exaC, 986 bp for PA4022, 2,716 bp for eat, and 928 bp for truncated eat) were then fused to the ␤-galactosidase (lacZ) ORF of E. coli through PCR (24,27). The PA4022::lacZ, eat:: lacZ, and truncated eat::lacZ fusions were subcloned into the XbaI site of ⌬Plac-pBBR1MCS-5 (24) to yield the plasmids pBRL601, pBRL678, and pBRL597, respectively.…”

Strain Promoted Click Chemistry of 8-Azidopurine and 5-Azidopyrimidine Nucleosides and Nucleotides with Cyclooctynes and Applications to Living Cell Imaging

“…296 Campbell and Stokes noted that P. aeruginosa grown in acetate rich media displayed a significant lag when transferred into α-KG medium suggesting the possibility of an inducible α-KG transporter. 297 We found no P. aeruginosa growth, when the mifR, mifS, and mifSR deletion mutants were incubated with α-KG, indicating that either the α-KG was unable to cross the bacterial cell membrane because there is no transport protein or once the α-KG was inside the cell it was not metabolized and therefore could not be used as a carbon source.…”