Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which—a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273—was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells

Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291T), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291T. Pseudomonas monteilii (DSM14164) clustered well with P. putida strains

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Normalised hybridisation signals of NADH dehydrogenase genes.

<p>Solid lines: <i>P</i>. <i>putida</i> S12; dotted lines: <i>P</i>. <i>putida</i> S12Δ<i>trgI</i>. ● NADH dehydrogenase (probe PS1205605_at), ♦ NADH dehydrogenase (probe PS1200059_at), ▲ NADH-quinine oxidoreductases chain A, × NADH-quinone oxidoreductases chain B, + NADH-quinone oxidoreductases chain C, ○ NADH-quinone oxidoreductases chain E, ∆ NADH-quinone oxidoreductases chain I, □ NADH-quinone oxidoreductases chain L.</p

Overrepresentation of COG groups per time-period in <i>P</i>. <i>putida</i> S12 and <i>P</i>. <i>putida</i> S12∆<i>trgI</i>.

<p>Overrepresentation of COG groups per time-period in <i>P</i>. <i>putida</i> S12 and <i>P</i>. <i>putida</i> S12∆<i>trgI</i>.</p

Summary of the transcriptomics results.

<p>Number of genes differentially expressed with an absolute log2(expression level at t = 0 / expression level at t = t) ≥0.5 in wild-type S12 (dashed line) and mutant S12Δ<i>trgI</i> (solid line) at the indicated time points are shown. The dotted line shows the number of genes that is present in both comparisons. T = 0: no toluene present.</p

Normalised hybridisation signals of NADH dehydrogenase genes.

<p>Solid lines: <i>P</i>. <i>putida</i> S12; dotted lines: <i>P</i>. <i>putida</i> S12Δ<i>trgI</i>. ● NADH dehydrogenase (probe PS1205605_at), ♦ NADH dehydrogenase (probe PS1200059_at), ▲ NADH-quinine oxidoreductases chain A, × NADH-quinone oxidoreductases chain B, + NADH-quinone oxidoreductases chain C, ○ NADH-quinone oxidoreductases chain E, ∆ NADH-quinone oxidoreductases chain I, □ NADH-quinone oxidoreductases chain L.</p

Overrepresentation of COG groups per time-period in <i>P</i>. <i>putida</i> S12 and <i>P</i>. <i>putida</i> S12∆<i>trgI</i>.

<p>Overrepresentation of COG groups per time-period in <i>P</i>. <i>putida</i> S12 and <i>P</i>. <i>putida</i> S12∆<i>trgI</i>.</p

Normalised hybridisation signals of genes involved in glucose import and metabolism.

<p>(Upper panels) ♦ <i>gtsA</i>, ▲<i>gtsB</i>, ● <i>gtsC</i>, × <i>gtsD</i>, + <i>gcd</i>, <math><mrow><mi>▲</mi></mrow></math><i>oprB-2</i>, <math><mrow><mi>●</mi></mrow></math><i>oprB-1</i>; (Lower panels) + <i>ptxS</i>, ○ <i>kguK</i>, <math><mrow><mi>■</mi></mrow></math><i>kguT</i>, × <i>gad</i> cytochrome c subunit, <math><mrow><mi>●</mi></mrow></math><i>gad</i> alpha chain, <math><mrow><mi>▲</mi></mrow></math><i>gad</i> gamma chain, + <i>gnuK</i>, ● <i>gntP</i>, × <i>gnuR</i>, ♦ <i>kguD</i>, ■ σ54-dependent transcriptional regulator.</p