The phosphotransferase system formed by PtsP, PtsO, and PtsN proteins controls production of polyhydroxyalkanoates in Pseudomonas putida

Abstract

The genome of Pseudomonas putida KT2440 encodes five proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system. Two of these (FruA and FruB) form a dedicated system for fructose intake, while enzyme I(Ntr) (EI(Ntr); encoded by ptsP), NPr (ptsO), and EII(Ntr) (ptsN) act in concert to control the intracellular accumulation of polyhydroxyalkanoates, a typical product of carbon overflow.

FIG. 1.

Distribution of EI, HPr, and EII domains in PTS-related proteins of P. putida. PTS modules encoded by the chromosome of P. putida KT2440 were surveyed using the Pfam motif search (http://www.sanger.ac.uk/Software/Pfam/search.shtml), the hmmsearch program, and the hmmpfam tool (http://hmmer.wustl.edu/) on all the known ORFs of the genome (23). The identities of the corresponding proteins are indicated to the left.

FIG. 2.

Chromosomal context of the PTS-related genes of P. putida and their mutant variants. All mutants were derivatives of P. putida MAD2 (12). Nonpolar ptsN::Km, ptsO::Km, ptsP::Km, and fruB::xylE insertion and deletion variants of this strain, as well as a double ptsN::xylE ptsO::Km derivative, have been described previously (8, 9). (A) fruB and fruA form part of a cluster for uptake and metabolism of fructose spanning coordinates 908214 to 915027 of the P. putida chromosome. The fruB mutant used in this work consists of a replacement of codons 158 to 648 of the ORF by a xylE cassette. fruR is the repressor of the transport system, while fruK encodes 1-phosphofructokinase. (B) The organization and coordinates of the two PTS genes found downstream of rpoN (ptsN and ptsO) are shown, along with sites of insertion/deletion of their sequence in the corresponding mutants. rpoX is a gene of unknown function. Note that individual ptsN and ptsO strains bear insertions of kanamycin resistance genes, while the ptsN ptsO double mutant consists of a kanamycin insertion in ptsO and an xylE cassette in ptsN. (C) The ptsP gene stands alone in the P. putida chromosome, flanked downstream by a divergent gene encoding a protein with orphan function (PP5144) and upstream by a hypothetical dinucleoside polyphosphate hydrolase (PP5146) with an equally unknown role. The kanamycin insertion mutant strain lacks codons 237 to 488 as indicated.

FIG. 3.

Growth of PTS mutants of P. putida on mineral medium with diverse C sources. Growth tests were done in 96-microwell plates from overnight cultures of each strain in LB medium washed twice 10 mM MgSO₄, adjusted to an optical density at 600 nm (OD₆₀₀) of 3.0. and diluted 100-fold in 200 μl of M9 medium (22) supplemented with 10 mM succinate, glucose, fructose, or glycerol and an excess (10 mM) of NH₄⁺. The plates were incubated at 30°C for 60 to 100 h, with 2 min of heavy orbital shaking every 15 min. The growth rates shown are the mean values from ≥4 replicates. Note the lack of growth of the fruB strain on fructose and the long lag of the ptsN strain in glycerol.

FIG. 4.

Accumulation of PHAs in P. putida strains lacking PTS proteins. Cells were grown overnight in LB medium (22), regrown in the same medium for 6 hours to an A₆₀₀ of ∼0.6, washed twice in 10 mM MgSO₄, and resuspended in the same volume of mineral M63 (22) supplemented with 15 mM of sodium octanoate as the sole C source, and then cultured aerobically for 22 h at 30°C. For determination of PHA contents, cells from duplicate cultures were collected, lyophilized, and analyzed (again in duplicate for each) by a method described in detail previously (18). A CP-Sil 5CB column (ChromPack) was employed for the identification of the methanolyzed PHA monomers by gas chromatography. (A) Quantification of PHAs in PTS mutants of P. putida. The levels of the polymer are expressed as a percentage of dry cell weight. (B) Accumulation of PHAs by ptsN-less P. putida cells complemented with wild-type ptsN and its H68A and H68D variants. The ptsN::Km strain was separately transformed with plasmids pRK767 (void vector) (11), pRK154 (ptsN⁺), pRK154HA (ptsN H68A⁺), and pRK154HD (ptsN H68D⁺), the two last encoding PtsN versions that mimic, respectively, the nonphosphorylated and the phosphorylated proteins. The PHA content of each of the resulting strains following growth in mineral-octanoate medium is shown. Error bars indicate standard deviations.