There is a wealth of information on the genetic regulation and biochemical properties of bacterial C 4 -dicarboxylate transport systems. In sharp contrast, there are far fewer studies describing the transport and assimilation of C 5 -dicarboxylates among bacteria. In an effort to better our understanding on this subject, we identified the structural and regulatory genes necessary for the utilization of ␣-ketoglutarate (␣-KG) in Pseudomonas aeruginosa PAO1. The PA5530 gene, encoding a putative dicarboxylate transporter, was found to be essential for the growth of P. aeruginosa PAO1 on both ␣-KG and glutarate (another C 5 -dicarboxylate). Metabolite analysis confirmed that the PA5530 gene was necessary for the uptake of extracellular ␣-KG. Like other substrate-inducible transporter genes, expression of the PA5530 gene was induced by extracellular C 5 -dicarboxylates. It was later found that the expression of the PA5530 gene was driven solely by a ؊24/؊12 promoter recognized by the alternative sigma factor RpoN. Surprisingly, the enhancer binding protein MifR, which is known to have an essential role in biofilm development, was required for the expression of the PA5530 gene. The MifR protein is homologous to other transcriptional regulators involved in dicarboxylate assimilation, suggesting that MifR might interact with RpoN to activate the expression of the PA5530 gene in response to extracellular C 5 -dicarboxylates, especially ␣-KG. The results of this study provide a framework for exploring the assimilation of ␣-KG in other pseudomonads.

bMany pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in Pseudomonas aeruginosa PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in P. aeruginosa PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria.

BackgroundHeterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products.MethodologyWe have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways.ConclusionsWe have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products.

p-Nitrophenol (pNP) is a widely used compound for analytical determinations of several esterases (EC. 3.1.1.X), including lipases (E.C. 3.1.1.3). Most enzymatic measurements employ pNP derivatives such as esters, which are broken down by enzymatic hydrolysis, releasing pNP that is quantified by its absorbance at 410 nm. Although this type of methods was developed a few decades ago, the spectrophotometric analysis of pNP requires analytical measurements of pH and temperature to achieve reliable determinations. The aim of this paper is to offer a graphical update of how pH and temperature affect the p-nitrophenol absorbance at different wavelengths in lipase emulsified media, due to its relevance for the quantitative determination of lipase activity using spectrophotometric methods. To highlight the importance of each variable involved in this analysis, we dissolved pNP in emulsified media (for lipase activity quantification) at several pH values from 4.00 to 11.00, and measured its absorbance in a range of 270 nm – 500 nm and at several temperatures from 25°C to 50°C. The absorption patterns of pNP under the established conditions were graphed in 3D plots. The constructed 3D plots showed that, regardless of the temperature, below pH 6.00, pNP predominantly absorbs at 317 nm, due to the greater abundance of its protonated form, which is completely predominant at pH 3.50 and below. On the other hand, at pH 10.0 and above, the major absorption occurs at about 401 nm, confirming that the equilibrium is completely shifted to the pNP anionic form. These results also indicate that close to neutral pH value pNP, it displays a temperature dependence effect, increasing absorbance to 410 nm at higher temperatures. Due to many analytical determinations of enzymatic activities, the release of pNP is carried around pH 7.00. It is necessary to consider the determinant role of both pH and temperature over these measurements, how these variables must be strictly controlled, and how the calibration curves and blanks should take the reaction media pH and temperature into account.

Biorefineries are a model for greener production processes, based on the concept of bioeconomy. Instead of targeting first-generation biofuels—that compete with food supply—the focus relies on lignocellulosic material, considering many aspects, such as sustainable fuel production, as well as valorization of waste, as an alternative to the traditional petrochemical approach of goods production. Especially, in tropical countries agricultural activities lead to tremendous amounts of biomass, resulting in waste that has to be dealt with. In the case of Costa Rica, the five major crops cultivated for export are coffee, oil palm, pineapple, sugarcane, and banana. Traditional ways of waste treatment cannot cope with the increasing amount of biomass produced and therefore, bear various challenges often related to increased pollution. This review aims to bring up the recent state of waste treatment but even more, stress potential opportunities of adding value to not used residues; thus, improve sustainability in the agro industrial sector. Part I of the review already highlighted the potential of producing promising bioactive chemical compounds by novel biorefinery concepts from agricultural waste originating from coffee and oil palm cultivation. This second part focuses on the lignocellulose-rich biowaste from pineapple, sugarcane, and banana, showing biorefinery concepts, where fuel and energy production, as well as establishment of novel products and new applications, play an important role.