The Entner–Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress

Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner–Doudoroff (ED) pathway due to the absence of 6‐phosphofructokinase. In order to activate the Embden–Meyerhof–Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild‐type strain as well as to an eda mutant, i.e. lacking 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase. PfkA^{E. coli} failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkA^{E. coli} was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP⁺ ratio. Pseudomonas putida cells carrying PfkA^{E. coli} became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkA^{E. coli} could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative‐related insults.     

Metabolic potential of the organic‐solvent tolerant Pseudomonas putida DOT‐T1E deduced from its annotated genome

Pseudomonas putida DOT‐T1E is an organic solvent tolerant strain capable of degrading aromatic hydrocarbons. Here we report the DOT‐T1E genomic sequence (6 394 153 bp) and its metabolic atlas based on the classification of enzyme activities. The genome encodes for at least 1751 enzymatic reactions that account for the known pattern of C, N, P and S utilization by this strain. Based on the potential of this strain to thrive in the presence of organic solvents and the subclasses of enzymes encoded in the genome, its metabolic map can be drawn and a number of potential biotransformation reactions can be deduced. This information may prove useful for adapting desired reactions to create value‐added products. This bioengineering potential may be realized via direct transformation of substrates, or may require genetic engineering to block an existing pathway, or to re‐organize operons and genes, as well as possibly requiring the recruitment of enzymes from other sources to achieve the desired transformation.     

ParI,an orphan ParA family protein from Pseudomonas putida KT2440‐specific genomic island,interferes with the partition system of IncP‐7 plasmids

Pseudomonas putida KT2440 is an ideal soil bacterium for expanding the range of degradable compounds via the recruitment of various catabolic plasmids. In the course of our investigation of the host range of IncP‐7 catabolic plasmids pCAR1, pDK1 and pWW53, we found that the IncP‐7 miniplasmids composed of replication and partition loci were exceptionally unstable in KT2440, which is the authentic host of the archetypal IncP‐9 plasmid pWW0. This study identified ParI, a homologue of ParA family of plasmid partitioning proteins encoded on the KT2440‐specific cryptic genomic island, as a negative host factor for the maintenance of IncP‐7 plasmids. The miniplasmids were destabilized by ectopic expression of ParI, and the loss rate correlated with the copy number of ParB binding sites in the centromeric parS region. Mutations in the conserved ATPase domains of ParI abolished destabilization of miniplasmids. Furthermore, ParI destabilized miniplasmid derivatives carrying the partition‐deficient parA mutations but failed to impact the stability of miniplasmid derivatives with parB mutations in the putative arginine finger. Altogether, these results indicate that ParI interferes with the IncP‐7 plasmid partition system. This study extends canonical partition‐mediated incompatibility of plasmids beyond heterogeneous mobile genetic elements, namely incompatibility between plasmid and genomic island.     

Understanding the unfavorable result after otoplasty: an integrated approach to correction

The correction of an unfavorable outcome after otoplasty requires a thorough understanding of the anatomy of prominent ear and recognition of the spectrum of secondary deformities and their origin. The goal of this article is to describe the causes of postotoplasty deformity, including both undercorrection and overcorrection. The latter presents the more complicated reconstructive problem, as both skin shortage and permanent cartilage disruption need to be addressed. The authors propose an algorithm for revision otoplasty based on clinical findings and patient concerns. Finally, a case with overcorrection secondary to both skin deficiency and cartilage disruption is illustrated showing the sequential steps needed for optimal correction.     

The loss of function of PhaC1 is a survival mechanism that counteracts the stress caused by the overproduction of poly‐3‐hydroxyalkanoates in Pseudomonas putidaΔfadBA

The poly‐3‐hydroxylkanoate (PHA)‐overproducing mutant Pseudomonas putida U ΔfadBA (PpΔfadBA) lacks the genes encoding the main β‐oxidation pathway (FadBA). This strain accumulates enormous amounts of bioplastics when cultured in chemically defined media containing PHA precursors (different n‐alkanoic or n‐aryl‐alkanoic acids) and an additional carbon source. In medium containing glucose or 4‐hydroxy‐phenylacetate, the mutant does not accumulate PHAs and grows just as the wild type (P. putida U). However, when the carbon source is octanoate, growth is severely impaired, suggesting that in PpΔfadBA, the metabolic imbalance resulting from a lower rate of β‐oxidation, together with the accumulation of bioplastics, causes severe physiological stress. Here, we show that PpΔfadBA efficiently counteracts this latter effect via a survival mechanism involving the introduction of spontaneous mutations that block PHA accumulation. Surprisingly, genetic analyses of the whole pha cluster revealed that these mutations occurred only in the gene encoding one of the polymerases (phaC1) and that the loss of PhaC1 function was enough to prevent PHA synthesis. The influence of these mutations on the structure of PhaC1 and the existence of a protein–protein (PhaC1–PhaC2) interaction that explains the functionality of the polymerization system are discussed herein.     

Regulation of plants metabolism in response to salt stress: an omics approach

In recent era, some man-made and natural activities are responsible for causing salt stress that affects each component of environment. Among several components, plant is one of the essential components, and with the consequence of excess salts accumulation in the environment their metabolic activities may get affected. Plants exhibit hyper-osmotic stress and ion disturbance in the presence of excess salt accumulation. To combat this stress situation, some metabolites/or stress-responsive gene(s) and proteins are synthesized by the plants to mitigate the salt toxicity. Therefore, to reduce the impact of salt stress on yield and other physiological activities of plants it is essential to know the whole pathway through which plant’s metabolism get affected in the presence of salt. The present review is also dealing with the same objective and special focus is given to the omics tools, such as genomics, proteomics and metabolomics to discuss the different ways of tolerance mechanism in the plant system against salt toxicity.     

Tissue proteomics of the low‐molecular weight proteome using an integrated cLC‐ESI‐QTOFMS approach

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500–5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC‐MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues.     

Understanding bone cell biology requires an integrated approach: Reliable opportunities to study osteoclast biology in vivo

The relative simplicity of all in vitro methods to study bone cell biology will at best result in oversimplification of the development and functional capacity of the skeleton in vivo. We have shown this to be true for selected aspects of bone cell biology, but numerous other examples are available. One alternative is to undertake skeletal research in vivo. It is important that those in bone research be willing to move increasingly in this direction not only to understand the true complexitities of skeletal versatility, but also to avoid repetition and perpetuation of erroneous or irrelevant conclusions which waste resources. Toward this end we have described two situations, osteopetrosis and tooth eruption, in which reproducible abrogations or local activations of bone resorption can be examined in vivo. The application of emerging molecular and morphological techniques that permit the subcellular dissection of metabolic pathways and their precise cellular localization, such as a combination of the variety of in situ hybridzation technologies with PCR, antisense probes, and antibody blockase, will allow the investigator greater control of variables in vivo. We expect that these technologies, largely worked out in vitro, combined with highly selected, appropriate models, as we have oulined here for osteoclast biology worked out in vitro, combined with highly selected, appropriate models, as we have ourlined here for osteoclast biology, will make research in vivo less intimidating and increase the frequency with which the real biology is studied directly.     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.     

Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1‐butanol tolerance using a metagenomic DNA library

Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real‐time and minimal sample preparation. In this research, Raman spectroscopy is used to analyze E. coli with engineered solvent tolerance, which is a multi‐genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries are serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1‐butanol. DNA libraries are created using degenerate oligonucleotide primed PCR (DOP‐PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP‐PCR enabled high efficiency library cloning (with no DNA shearing or end‐polishing) and the inclusion un‐culturable organisms. Nine strains with improved tolerance are analyzed by Raman spectroscopy and vastly different solvent‐tolerant phenotypes are characterized. Common among these are improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real‐time and is adaptable to high‐throughput screening, making it relevant to metabolic engineering.     

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.