Abdelali Daddaoua

Regulation of Glucose Metabolism in Pseudomonas THE PHOSPHORYLATIVE BRANCH AND ENTNER-DOUDOROFF ENZYMES ARE REGULATED BY A REPRESSOR CONTAINING A SUGAR ISOMERASE DOMAIN

In Pseudomonas putida, genes for the glucose phosphorylative pathway and the Entner-Doudoroff pathway are organized in two operons; one made up of the zwf, pgl, and eda genes and another consisting of the edd, glk, gltR2, and gltS genes. Divergently with respect to the edd gene is the gap-1 gene. Expression from Pzwf, Pedd, and Pgap is modulated by HexR in response to the availability of glucose in the medium. To study the regulatory process in greater detail we purified HexR and showed that it is a monomer in solution. Electrophoretic mobility shift assays and isothermal titration calorimetry assays were done showing that HexR recognizes the Pedd, Pzwf, and Pgap-1 promoters with affinity in the nanomolar range. DNA footprinting assays identified the binding site between +30 and +1 at Pzwf, between +16 and +41 at Pedd, and between −6 and +18 at Pgap-1. Based on DNA sequence alignment of the target sites and isothermal titration calorimetry data, two monomers of HexR bind to a pseudopalindrome with a consensus sequence of 5′-TTGTN7–8ACAA-3′. Binding of the Entner-Doudoroff pathway intermediate 2-keto-3-deoxy-6-phosphogluconate to HexR released the repressor from its target operators, whereas other chemicals such as glucose, glucose 6-phosphate, and 6-phosphogluconate did not induce complex dissociation. The phosphorylated effector is likely to be recognized by a sugar isomerase domain located at the C-terminal end of HexR, whereas the helix-turn-helix DNA binding domain of HexR exhibits high similarity to proteins of the RpiR family of regulators.

Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17.

Bovine glycomacropeptide (BGMP) is an inexpensive, non‐toxic milk peptide with anti‐inflammatory effects in rat experimental colitis but its mechanism of action is unclear. It is also unknown whether BGMP can ameliorate inflammation in proximal regions of the intestine. Our aim was therefore two‐fold: first, to determine the anti‐inflammatory activity of BGMP in the ileum; second, to characterise its mechanism of action.

Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-κB signal transduction pathways

Background and purpose:  Bovine glycomacropeptide (BGMP) is a natural milk peptide that is produced naturally in the gastrointestinal tract during digestion. Glycomacropepide has intestinal anti‐inflammatory activity, but the mechanism of action is unknown. Here we have characterized the effects of BGMP on monocytes.

The bisphosphonate alendronate improves the damage associated with trinitrobenzenesulfonic acid-induced colitis in rats

The nitrogen‐containing bisphosphonates are drugs used successfully in the treatment of osteoporosis. They act inhibiting farnesyl diphosphate synthase. This mechanism may also produce anti‐inflammatory effects. The therapeutic activity of alendronate was tested in vivo using a model of inflammatory bowel disease.

Differential transcriptional response to antibiotics by Pseudomonas putida DOT-T1E

Multi-drug resistant bacteria are a major threat to humanity, especially because the current battery of known antibiotics is not sufficient to combat infections produced by these microbes. Therefore, the study of how current antibiotics act and how bacteria defend themselves against antibiotics is of critical importance. Pseudomonas putida DOT-T1E exhibits an impressive array of RND efflux pumps, which confer this microorganism high resistance to organic solvents and antibiotics that would kill most other microorganisms. We have chosen DOT-T1E as a model microbe to study the microbial responses to a wide battery of antibiotics (chloramphenicol, rifampicin, tetracycline, ciprofloxacin, ampicillin, kanamycin, spectinomycin and gentamicin). Ribonucleic acid sequencing (RNA)-seq analyses revealed that each antibiotic provokes a unique transcriptional response profile in DOT-T1E. While many of the genes identified were related to known antibiotic targets, others were unrelated or encoded hypothetical proteins. These results indicate that our knowledge of antibiotic resistance mechanisms is still partial. We also identified 138 new small RNAs (sRNAs) in DOT-T1E, dramatically adding to the 16 that have been previously described. Importantly, our results reveal that a correlation exists between the expression of messenger RNA and sRNA, indicating that some of these sRNAs are likely involved in fine tuning the expression of antibiotic resistance genes. Taken together, these findings open new frontiers in the fight against multi-drug resistant bacteria and point to the potential use of sRNAs as novel antimicrobial targets.

The nutritional supplement Active Hexose Correlated Compound (AHCC) has direct immunomodulatory actions on intestinal epithelial cells and macrophages involving TLR/MyD88 and NF-κB/MAPK activation.

Active Hexose Correlated Compound (AHCC) is an immunostimulatory nutritional supplement. AHCC effects and mechanism of action on intestinal epithelial cells or monocytes are poorly described. AHCC was added to the culture medium of intestinal epithelial cells (IEC18 and HT29 cells) and monocytes (THP-1 cells) and assessed the secretion of proinflammatory cytokines by ELISA. Inhibitors of NFκB and MAPKs were used to study signal transduction pathways while TLR4 and MyD88 were silenced in IEC18 cells using shRNA. It was found that AHCC induced GROα and MCP1 secretion in IEC18 and IL-8 in HT29 cells. These effects depended on NFκB activation, and partly on MAPKs activation and on the presence of MyD88 and TLR4. In THP-1 cells AHCC evoked IL-8, IL-1β and TNF-α secretion. The induction of IL-8 depended on JNK and NFκB activation. Therefore, AHCC exerts immunostimulatory effects on intestinal epithelial cells and monocytes involving TLR4/MyD88 and NFκB/MAPK signal transduction pathways.

First- and second-generation biochemicals from sugars: biosynthesis of itaconic acid

Competitive prices for sugars are needed to expand the range of biochemicals that can be industrially synthesized profitably. The most commonly accepted path from these starting materials to products is the Task 42 IEA BioEnergy biorefinery classification system, which is schematically depicted in Fig. 1. The upper part of the figure shows a wide range of potential raw material sources, while the lower part shows progressive series of chemicals that can be produced from C6 or C5 sugar core backbones through chemical catalysis or microbial fermentation. Subsequent modification of these compounds through chemical catalysis gives rise to yet another group of chemicals. Figure 1 High‐level representation of pathways via the sugar platform. The aim of IEA (International Energy Agency) Bioenergy Task 42 is to initiate and actively promote information exchange on all features of biorefinery. Sugars, which serve as the core structure upon which these chemicals are built, are typically generated using starch that originates from different grains. Of the total corn produced in the world, less than 5% is used for ethanol production. Given global controversy regarding the use of food for fuel production, many biofuel companies have begun to explore the production of ethanol from lignocellulosic materials, such as straw and other agricultural waste materials. Ethanol generated from starch is known as 1G ethanol, while ethanol produced from lignocellulose is known as 2G ethanol. We have adopted the terms 1G and 2G for use with biochemicals that can be made from starch and lignocellulose, respectively. The use of lignocellulosic materials to produce biochemicals is challenging because these starting materials require intensive pretreatment (physical, chemical or biological) followed by enzymatic hydrolysis. The hydrolysis process is mediated by a set of enzymes, generically known as cellulases, which work synergistically to produce sugars. While glucose is almost the only product that results from starch hydrolyses, lignocellulose yields glucose in addition to a range of other sugars, such as xylose, arabinose, rhamnose and galactose. Regardless of whether the sugars are derived from starch or lignocellulose, fermentation of the sugars can produce downstream products than include alcohols, organic acids, alkenes, lipids and a wide range of other chemicals. This conversion can be accomplished using bacteria, fungi or yeast (genetically modified or not) using a variety of process conditions (e.g. low/high pH, aerobic/anaerobic, mesophilic/thermophilic and various nutritional regimes). The biotransformation industry modifies these and other variables to develop new processes through iterative parameter optimization with one aim: to attain the highest possible biochemical yields at rates that enable maximal recovery after downstream processing. The number of biochemicals currently produced through this manner at a commercial scale is still low. Examples of these include: ethanol, lactic acid, succinic acid, butanol, acetone, sorbitol and itaconic acid. In this issue of Microbial Biotechnology, a new pathway for production of itaconic acid is described by Geiser et al. (2015). The annual production of itaconic acid, an unsaturated dicarboxylic acid, is around 50 000 tons/year. This biochemical is used as building block for the biosynthesis of pharmaceuticals, certain resins and adhesives (Steiger et al., 2013). It should be noted that poly (itaconic acid) can be used to develop superabsorbents, anti‐scaling agents in water treatment, and can comprise components of detergents and dispersants (Klement and Buchs, 2013). Because of its industrial potential, itaconic acid was selected by US Department of Energy as one of the top 12 chemical candidates (that can be generated from biomass) to serve as a building block for the production of value added chemicals (Werpy and Petersen, 2004). The existing itaconic biosynthesis pathway was first studied in Aspergillus terreus. Early studies established that itaconic acid was produced from cis‐aconitate, a tricarboxylic acid cycle intermediate, via the action of a decarboxylase known as CadA (Okabe et al., 2009; Klement and Buchs, 2013) (Fig. 2). The microbial‐based production of itaconic acid has been reported to be as high as 45–80 g l−1 of media (Kanamasa et al., 2008; Steiger et al., 2013). In addition to Aspergillus, previous studies have shown that other fungi, such as Ustilago maydis, produce itaconic acid. Figure 2 Pathways for itaconic acid biosynthesis. cis‐Aconitate is an output chemical from the tricarboxylic acid (TCA) cycle. Blue lines represent the classic pathway described in A spergillus species (Huang et al., 2014). Red lines represent ... The new pathway revealed by Geiser et al. (2015) has been identified in U. maydis and it involves the isomerisation of cis‐ to trans‐aconitate, via a cytosolic aconitase isomerase (Adi1), followed by a decarboxylation step mediated by a novel decarboxylase (Tad1), which exhibits significant sequence similarities to lactonizing enzymes. A quick BLASTp search reveals that the isomerase described in this study and the new decarboxylase are present in a limited number of fungi with the best hit with sequences from Pseudozyma hubeiensis. The production rates reported for U. maydis are lower than those from A. terreus; however, gene regulation studies and optimization of production conditions in U. maydis are needed to reveal the biotechnological potential of the new pathway. The current limitation of the biological production of itaconic acid on an industrial scale seems to be production costs (Klement and Buchs, 2013). This limitation can be overcome through improving microbial strains, optimization of processes and the sourcing of cheaper raw materials. The newly identified pathway also provides new options for optimizing production processes, and moves us one step closer to overcoming the current affordability challenges.

Efflux pump‐deficient mutants as a platform to search for microbes that produce antibiotics

Pseudomonas putida DOT‐T1E‐18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild‐type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT‐T1E‐18 on solid Luria–Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT‐T1E‐18 growth was an isolate named 250J that, through multi‐locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram‐positive bacteria, Gram‐negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram‐positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin.

Identification of new residues involved in intramolecular signal transmission in a prokaryotic transcriptional repressor

TtgV is a member of the IclR family of transcriptional regulators. This regulator controls its own expression and that of the ttgGHI operon, which encodes an RND efflux pump. TtgV has two domains: a GAF-like domain harboring the effector-binding pocket and a helix-turn-helix (HTH) DNA-binding domain, which are linked by a long extended helix. When TtgV is bound to DNA, a kink at residue 86 in the extended helix gives rise to 2 helices. TtgV contacts DNA mainly through a canonical recognition helix, but its three-dimensional structure bound to DNA revealed that two residues, R19 and S35, outside the HTH motif, directly contact DNA. Effector binding to TtgV releases it from DNA; when this occurs, the kink at Q86 is lost and residues R19 and S35 are displaced due to the reorganization of the turn involving residues G44 and P46. Mutants of TtgV were generated at positions 19, 35, 44, 46, and 86 by site-directed mutagenesis to further analyze their role. Mutant proteins were purified to homogeneity, and differential scanning calorimetry (DSC) studies revealed that all mutants, except the Q86N mutant, unfold in a single event, suggesting conservation of the three-dimensional organization. All mutant variants bound effectors with an affinity similar to that of the parental protein. R19A, S35A, G44A, Q86N, and Q86E mutants did not bind DNA. The Q86A mutant was able to bind to DNA but was only partially released from its target operator in response to effectors. These results are discussed in the context of intramolecular signal transmission from the effector binding pocket to the DNA binding domain.

Regulation of carbohydrate degradation pathways in Pseudomonas involves a versatile set of transcriptional regulators

Bacteria of the genus Pseudomonas are widespread in nature. In the last decades, members of this genus, especially Pseudomonas aeruginosa and Pseudomonas putida, have acquired great interest because of their interactions with higher organisms. Pseudomonas aeruginosa is an opportunistic pathogen that colonizes the lung of cystic fibrosis patients, while P. putida is a soil bacterium able to establish a positive interaction with the plant rhizosphere. Members of Pseudomonas genus have a robust metabolism for amino acids and organic acids as well as aromatic compounds; however, these microbes metabolize a very limited number of sugars. Interestingly, they have three‐pronged metabolic system to generate 6‐phosphogluconate from glucose suggesting an adaptation to efficiently consume this sugar. This review focuses on the description of the regulatory network of glucose utilization in Pseudomonas, highlighting the differences between P. putida and P. aeruginosa. Most interestingly, It is highlighted a functional link between glucose assimilation and exotoxin A production in P. aeruginosa. The physiological relevance of this connection remains unclear, and it needs to be established whether a similar relationship is also found in other bacteria.