Preferential utilization of aromatic compounds over glucose by Pseudomonas putida CSV86

Pseudomonas putida CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.     

Preferential Utilization of Aromatic Compounds over Glucose by Pseudomonas putida CSV86

Pseudomonas putida CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.     

Sequential utilization of substrates by Pseudomonas putida CSV86: signatures of intermediate metabolites and online measurements

Pseudomonas putida CSV86 preferentially utilizes aromatics over glucose and co-metabolizes them with organic acids. On aromatics plus glucose, CSV86 utilized aromatics first with concomitant appearance of transient metabolites such as salicylate, benzaldehyde and benzoate. Citrate was the main extracellular metabolite observed during glucose uptake. The strain showed simultaneous utilization of organic acids and aromatic compounds. Based on the metabolite analysis and growth profiles, we hypothesize that the repression of glucose utilization could be due to organic acid intermediates generated from aromatic compound metabolism. The online measurements indicate the instantaneous metabolic state of the culture. For example, the CO₂ evolution and agitation speed show peak values during the two growth phases in the diauxic growth while dissolved oxygen values show decrease at the corresponding durations. These measurements correlated well with the offline measurements but provided a better time resolution of the process.     

Inducible uptake and metabolism of glucose by the phosphorylative pathway in Pseudomonas putida CSV86

Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and benzoate as the sole source of carbon and energy. Compared with glucose, cells grew faster on aromatic compounds as well as on organic acids. The organism failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. Whole-cell oxygen uptake, enzyme activity and metabolic studies suggest that in strain CSV86 glucose utilization is exclusively by the intracellular phosphorylative pathway, while in Stenotrophomonas maltophilia CSV89 and P. putida KT2442 glucose is metabolized by both direct oxidative and indirect phosphorylative pathways. Cells grown on glucose showed five- to sixfold higher activity of glucose-6-phosphate dehydrogenase compared with cells grown on aromatic compounds or organic acids as the carbon source. Study of [14C]glucose uptake by whole cells indicates that the glucose is taken up by active transport. Metabolic and transport studies clearly demonstrate that glucose metabolism is suppressed when strain CSV86 is grown on aromatic compounds or organic acids.     

The relative ligand binding preference of the murine ileal lipid binding protein

The ileal lipid binding protein (ILBP), a member of the intracellular lipid binding protein family, is a 14-kDa protein that has bile and fatty acids as possible physiological ligands. The ligand binding specificity of this protein is not well characterized. Therefore, we studied the lipid binding activity of purified recombinant murine ILBP (mILBP) in vitro. These studies demonstrated by direct analysis the interaction of mILBP with naturally occurring bile and fatty acids. The rank order of binding preference for fatty acids, or unconjugated and conjugated bile acids, was assessed. Among fatty acids, mILBP preferred species that had longer chain length and increased saturation, similar to other members of the intracellular lipid binding protein family. Among the bile acids, mILBP showed the greatest preference for conjugated species that contained a doubly hydroxylated steroid moiety. The results demonstrate that mILBP exhibits a preference for certain species of bile and fatty acids.     

Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean

A 43-kDa soybean protein is a receptor-like protein kinase that is capable of interaction with a 4-kDa hormone-like peptide (leginsulin). The 43-kDa protein consists of alpha and beta subunits; the beta subunit has protein kinase activity that is stimulated by the binding of the 4-kDa peptide. The protein kinase activity is believed to be an early step in a signal transduction cascade, triggered by the peptide. Animal insulin also interacts with the 43-kDa protein and stimulates the protein kinase activity, suggesting that the 4-kDa peptide and insulin bind to the 43-kDa protein with similar mechanisms. To determine the mechanism of interaction between the 4-kDa peptide and 43-kDa protein, we investigated the binding region of the 4-kDa peptide on the 43-kDa protein using surface plasmon resonance (SPR) spectroscopy. We found that the N- (amino acids 1-43) and C-terminal (amino acids 228-251) regions of the alpha subunit of the 43-kDa protein are involved in the binding. The interactions of both insulin and the 4-kDa peptide with the 43-kDa protein were compared using SPR spectroscopy, revealing that insulin binds to the C-terminal regions of the alpha subunit of the 43-kDa protein. These results suggest that the C-terminal region is especially important for the biological function. The N-terminal region is thought to play an important role in stabilizing the complex of the 43-kDa protein and the 4-kDa peptide.     

Characterization of an OprL null mutant of Pseudomonas putida.

A Pseudomonas putida oprL null mutant was generated with reverse genetics by using an in vitro-truncated oprL::xylE construct and in vivo allelic exchange. The nature of the mutation introduced in P. putida was confirmed by Southern blotting. Western blots (immunoblots) of peptidoglycan-associated proteins revealed that the OprL protein was not made in the mutant strain, whereas it was detectable as a 19-kDa band in protein preparations of the wild-type strain. The P. putida oprL, mutant exhibited altered cell morphology as revealed by electron microscopy and was more sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA than the wild-type strain. The oprL gene was conserved in a wide variety of the Pseudomonas strains belonging to rRNA group I, which suggests that this gene is important for the maintenance of the cell envelope and cell morphology in this group of microorganisms.     

Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein.

Soybean 4-kDa peptide, a hormone-like peptide, is a ligand for the 43-kDa protein in legumes that functions as a protein kinase and controls cell proliferation and differentiation. As this peptide stimulates protein kinase activity, the interaction between the 4-kDa peptide (leginsulin) and the 43-kDa protein is considered important for signal transduction. However, the mechanism of interaction between the 4-kDa peptide and the 43-kDa protein is not clearly understood. We therefore investigated the binding mechanism between the 4-kDa peptide and the 43-kDa protein, by using gel-filtration chromatography and dot-blot immunoanalysis, and found that the 4-kDa peptide bound to the dimer form of the 43-kDa protein. Surface plasmon resonance analysis was then used to explore the interaction between the 4-kDa peptide and the 43-kDa protein. To identify the residues of the 4-kDa peptide involved in the interaction with the 43-kDa protein, alanine-scanning mutagenesis of the 4-kDa peptide was performed. The 4-kDa peptide-expression system in Escherichia coli, which has the ability to install disulfide bonds into the target protein in the cytoplasm, was employed to produce the 4-kDa peptide and its variants. Using mass spectrometry, the expressed peptides were confirmed as the oxidized forms of the native peptide. Surface plasmon resonance analysis showed that the C-terminal hydrophobic area of the 4-kDa peptide plays an important role in binding to the 43-kDa protein.     

Kinetics of medium-chain-length polyhydroxyalkanoate production by a novel isolate of Pseudomonas putida LS46

Six bacteria that synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were isolated from sewage sludge and hog barn wash and identified as strains of Pseudomonas and Comamonas by 16S rDNA gene sequencing. One isolate, Pseudomonas putida LS46, showed good PHA production (22% of cell dry mass) in glucose medium, and it was selected for further studies. While it is closely related to other P.?putida strains (F1, KT2440, BIRD-1, GB-1, S16, and W619), P.?putida LS46 was genetically distinct from these other strains on the basis of nucleotide sequence analysis of the cpn60 gene hypervariable region. PHA production was detected as early as 12?h in both nitrogen-limited and nitrogen-excess conditions. The increase in PHA production after 48?h was higher in nitrogen-limited cultures than in nitrogen-excess cultures. Pseudomonas?putida LS46 produced mcl-PHAs when cultured with glucose, glycerol, or C(6)-C(14) saturated fatty acids as carbon sources, and mcl-PHAs accounted for 56% of the cell dry mass when cells were batch cultured in medium containing 20?mmol/L octanoate. Although 3-hydroxydecanoate was the major mcl-PHA monomer (58.1-68.8?mol%) in P.?putida LS46 cultured in glucose medium, 3-hydroxyoctanoate was the major monomer produced in octanoate medium (88?mol%).     

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.     

Repression of Pseudomonas putida phenanthrene-degrading activity by plant root extracts and exudates

The phenanthrene-degrading activity (PDA) of Pseudomonas putida ATCC 17484 was repressed after incubation with plant root extracts of oat (Avena sativa), osage orange (Maclura pomifera), hybrid willow (Salix alba x matsudana), kou (Cordia subcordata) and milo (Thespesia populnea) and plant root exudates of oat (Avena sativa) and hybrid poplar (Populus deltoides x nigra DN34). Total organic carbon content of root extracts ranged from 103 to 395 mg l(-1). Characterization of root extracts identified acetate (not detectable to 8.0 mg l(-1)), amino acids (1.7-17.3 mg l(-1)) and glucose (1.6-14.0 mg l(-1)), indicating a complex mixture of substrates. Repression was also observed after exposure to potential root-derived substrates, including organic acids, glucose (carbohydrate) and glutamate (amino acid). Carbon source regulation (e.g. catabolite repression) was apparently responsible for the observed repression of P. putida PDA by root extracts. However, we showed that P. putida grows on root extracts and exudates as sole carbon and energy sources. Enhanced growth on root products may compensate for partial repression, because larger microbial populations are conducive to faster degradation rates. This would explain the commonly reported increase in phenanthrene removal in the rhizosphere.     

Purification of glucose-inducible outer membrane protein OprB of Pseudomonas putida and reconstitution of glucose-specific pores.

A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida. OprB was surface expressed in whole cells, had a high beta-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation. Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (KS = 110 mM) and other carbohydrates. However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.     

Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme.

The nucleotide sequences of the genes encoding the enzyme aspartate transcarbamoylase (ATCase) from Pseudomonas putida have been determined. Our results confirm that the P. putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately from overlapping genes. The P. putida ATCase does not possess dissociable regulatory and catalytic functions but instead apparently contains the regulatory nucleotide binding site within a unique N-terminal extension of the pyrB-encoded subunit. The first gene, pyrB, is 1,005 bp long and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the enzyme. The second gene is 1,275 bp long and encodes a 424-residue polypeptide which bears significant homology to dihydroorotase (DHOase) from other organisms. Despite the homology of the overlapping gene to known DHOases, this 44.2-kDa polypeptide is not considered to be the functional product of the pyrC gene in P. putida, as DHOase activity is distinct from the ATCase complex. Moreover, the 44.2-kDa polypeptide lacks specific histidyl residues thought to be critical for DHOase enzymatic function. The pyrC-like gene (henceforth designated pyrC') does not complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs. The proposed function for the vestigial DHOase is to maintain ATCase activity by conserving the dodecameric assembly of the native enzyme. This unique assembly of six active pyrB polypeptides coupled with six inactive pyrC' polypeptides has not been seen previously for ATCase but is reminiscent of the fused trifunctional CAD enzyme of eukaryotes.     

Methyl ketone production by Pseudomonas putida is enhanced by plant-derived amino acids

Plants are an attractive sourceof renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components, such as aromatics and amino acids, may improve the efficiency of biomass conversion. Pseudomonas putida is a promising host for its ability to metabolize a wide variety of organic compounds. P. putida was engineered to produce methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics. Unexpectedly, P. putida methyl ketone production using Arabidopsis thaliana hydrolysates was enhanced 2–5-fold compared with sugar controls derived from engineered plants that overproduce lignin-related aromatics. This enhancement was more pronounced (~seven-fold increase) with hydrolysates from nonengineered switchgrass. Proteomic analysis of the methyl ketone-producing P. putida suggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a nine-fold increase in methyl ketone titer (1.1 g/L).