Amino Acid Racemization in Pseudomonas putida KT2440

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k_cat/K_m values with l- and d-lysine were 3 orders of magnitude greater than the k_cat/K_m values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k_cat/K_m values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.     

Acetone extraction of mcl-PHA from Pseudomonas putida KT2440

A methodology was developed for the extraction of medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) from Pseudomonas putida. It was determined that if dry P. putida biomass containing mcl-PHA was washed in 20 volumes of methanol for 5 min followed by Soxhlet extraction in 10 volumes of acetone for 5 h, almost all of the PHA could be recovered with no detectable loss of molecular weight. Biomass containing higher amounts of PHA required less methanol during the pretreatment step but more acetone in the solvent extraction step than biomass containing less PHA. Further purification could be achieved by redissolving the PHA in acetone and reprecipitating in cold methanol. UV spectroscopy at 241 and 275 nm could be used as an indication of product purity.     

Engineering Pseudomonas putida KT2440 for the production of isobutanol

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback‐resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y_Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical Y_Iso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non‐growing conditions.     

Global features of the Pseudomonas putida KT2440 genome sequence

The compositional bias of the G+C, di- and tetranucleotide contents in the 6 181 862 bp Pseudomonas putida KT2440 genome was analysed in sliding windows of 4000 bp in steps of 1000 bp. The genome has a low GC skew (mean 0.066) between the leading and lagging strand. The values of GC contents (mean 61.6%) and of dinucleotide relative abundance exhibit skewed Gaussian distributions. The variance of tetranucleotide frequencies, which increases linearly with increasing GC content, shows two overlapping Gaussian distributions of genome sections with low (minor fraction) or high variance (major fraction). Eighty per cent of the chromosome shares similar GC contents and oligonucleotide bias, but 105 islands of 4000 bp or more show atypical GC contents and/or oligonucleotide signature. Almost all islands provide added value to the metabolic proficiency of P. putida as a saprophytic omnivore. Major features are the uptake and degradation of organic chemicals, ion transport and the synthesis and secretion of secondary metabolites. Other islands endow P. putida with determinants of resistance and defenceor with constituents and appendages of the cell wall. A total of 29 islands carry the signature of mobile elements such as phage, transposons, insertion sequence (IS) elements and group II introns, indicating recent acquisition by horizontal gene transfer. The largest gene carries the most unusual sequence that encodes a multirepeat threonine-rich surface adhesion protein. Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.     

Tolerance of plastic-encapsulated Pseudomonas putida KT2440 to chemical stress

Pseudomonas putida dried in the presence of hydroxyectoine or trehalose can withstand exposure to organic solvents and therefore can be encapsulated inside plastics such as polystyrene. Here we show that P. putida in a plastic-encapsulated dried tablet exhibits remarkable tolerance to chemical stress, comparable to that of spores of Bacillus subtilis.     

Biodegradation of alkyl branched aromatic alkanoic naphthenic acids by Pseudomonas putida KT2440

The majority of the world’s crude oil reserves consist of highly biodegraded heavy and super heavy crude oils and oil sands that have not yet been fully exploited. These vast resources contain complex mixtures of carboxylic acids known as naphthenic acids (NAs). NAs cause major environmental and economic problems, as they are recalcitrant, corrosive and toxic. Although aromatic acids make up a small proportion of most NA mixtures, they have demonstrable toxicities to some organisms (e.g. some bacteria and algae) and ideally need to be removed or reduced by remediation. The present study analysed the ability of Pseudomonas putida KT2440 to degrade highly recalcitrant aromatic acids, as exemplified by the alkyl phenylalkanoic acid (4′-t-butylphenyl)-4-butanoic acid (t-BPBA) and the more degradable (4′-n-butylphenyl)-4-butanoic acid (n-BPBA). n-BPBA was completely metabolized after 14 days, with the production of a persistent metabolite identified as (4′-n-butylphenyl)ethanoic acid (BPEA) which resulted from removal of two carbon atoms from the carboxyl side chain (beta-oxidation) as observed previously with a mixed consortium. However, when n-BPBA concentration was increased two-fold, degradation decreased by 56% with a concomitant six-fold decrease in cell numbers, suggesting that at greater concentrations, n-BPBA may be toxic to P. putida KT2440. In contrast, P. putida KT2440 was unable to degrade the highly recalcitrant t-BPBA even after 49 days. These findings have implications for NA bioremediation in the environment.     

Cell density-dependent gene contributes to efficient seed colonization by Pseudomonas putida KT2440

We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440. The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function. Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene. Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant. Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase. It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth. Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates. This is the first evidence suggesting the existence of a quorum-sensing system in P. putida KT2440. The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed.     

Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440

We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440. The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function. Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene. Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant. Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase. It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth. Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates. This is the first evidence suggesting the existence of a quorum-sensing system in P. putida KT2440. The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed.     

Mercury bioremediation by engineered Pseudomonas putida KT2440 with adaptationally optimized biosecurity circuit

Hazardous materials, such as heavy metals, are the major sources of health risk. Using genetically modified organisms (GMOs) to dispose heavy metals has the advantages of strong environmental compatibility and high efficiency. However, the biosecurity of GMOs used in the environment is a major concern. In this study, a self-controlled genetic circuit was designed and carefully fine-tuned for programmable expression in Pseudomonas putida KT2440, which is a widely used strain for environmental bioremediation. The cell behaviours were controlled by automatically sensing the variation of Hg²⁺ concentration without any inducer requirement or manual interventions. More than 98% Hg²⁺ was adsorbed by the engineered strain with a high cell recovery rate of 96% from waterbody. The remaining cells were killed by the suicide module after the mission was accomplished. The escape frequency of the engineered P. putida strain was lower than 10⁻⁹, which meets the recommendation of US NIH guideline for GMOs release (<10⁻⁸). The same performance was achieved in a model experiment by using natural lake water with addition of Hg²⁺. The microbial diversity analysis further confirmed that the remediation process made little impact on the indigenous ecosystem. Thus, this study provides a practical method for environmental remediation by using GMOs.     

Response of Pseudomonas putida KT2440 to increased NADH and ATP demand

Adenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism of Pseudomonas putida KT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only when P. putida was challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, for Escherichia coli or Saccharomyces cerevisiae. The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds.