BackgroundMetagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant.ResultsIn preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v).ConclusionsThe combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.

SummaryBiosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and ratelimiting step in fatty acid biosynthesis is catalyzed by a multi-subunit form of the acetyl-CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl-CoA to produce malonyl-CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the P II family. Here we show that the GlnB paralogues of the PII proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC-BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC-BCCP-GlnB interaction and ACC inhibition were relieved by 2-oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2-oxoglutarate-sensitive dissociable regulatory subunit of ACC in Bacteria.

The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energyexpensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the P II signal transduction proteins act as key players. IntroductionBiological nitrogen fixation, the reduction of atmospheric N 2 to NH 3 by nitrogen-fixing bacteria, is a key step in the nitrogen cycle. This process is catalysed by nitrogenase, the most common form of which is the molybdenum nitrogenase, composed of dinitrogenase (MoFe protein or NifDK), an a 2 b 2 tetramer encoded by the nifD and nifK genes, respectively, and dinitrogenase reductase (Fe protein or NifH), a c 2 homodimer encoded by the nifH gene. The NifH protein is responsible for ATP-hydrolysisdriven electron transport to the NifDK protein, which contains the site for the reduction of dinitrogen to ammonium (Seefeldt et al., 2009). The reduction of N 2 to two molecules of ammonium is an energy-expensive process requiring the hydrolysis of 16 ATPs.To avoid energy wastage, diazotrophs have evolved both transcriptional and post-translational mechanisms to shut-down nitrogen fixation when ammonium is available in the environment. Post-translational control of nitrogenase activity has been found in a range of diazotrophs and affords a rapid and reversible mechanism by which the organism can respond to transient changes in the environment. Here we review current knowledge of the different mechanisms of post-translational control of nitrogenase. We focus on the two best-described systems: ADP-ribosylation of NifH, which occurs in proteobacteria, and the interaction of NifI regulatory proteins with NifDK in archaea, which potentially also operates in some anaerobic diazotrophic bacteria. Regulation of nitrogenase activity by reversible ADP-ribosylation Historical perspectiveThe process of metabolic inactivation of nitrogenase in response to ammonium was first described in Azotobacter vinelandii (Burris & Wilson, 1946); this phenomenon was later identified in other prokaryotes and named nitrogenase 'switch-off' (Zumft & Castillo, 1978). Different mechanisms are used to regulate nitrogenase post-translationally depending on the organism. The best-studied mechanism operates through reversible ADP-ribosylation of NifH (reviewed by Nordlund, 2000). This system responds to not only the presence of ammonium but also a decrease in the availability of cellular energy, in response to either darkness in phototrophs such as ...

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SummaryThe PII family comprises a group of widely distributed signal transduction proteins. The archetypal function of PII is to regulate nitrogen metabolism in bacteria. As PII can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by PII may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a PII protein affinity column was used to identify PII targets in Azospirillum brasilense. One of the PII partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl-CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a PII target in Arabidopsis thaliana we hypothesized that the PII-BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the PII-BCCP interaction is conserved in Escherichia coli. The BCCP-PII interaction required MgATP and was dissociated by increasing 2-oxoglutarate. The interaction was modestly affected by the post-translational uridylylation status of PII; however, it was completely dependent on the post-translational biotinylation of BCCP.