Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor.

High levels of the intracellular signalling molecule cyclic diguanylate (c-di-GMP) supress motility and activate exopolysaccharide (EPS) production in a variety of bacterial species. In many bacteria part of the effect of c-di-GMP is on gene expression, but the mechanism involved is not known for any species. We have identified the protein FleQ as a c-di-GMP-responsive transcriptional regulator in Pseudomonas aeruginosa. FleQ is known to activate expression of flagella biosynthesis genes. Here we show that it also represses transcription of genes including the pel operon involved in EPS biosynthesis, and that this repression is relieved by c-di-GMP. Our in vivo data indicate that FleQ represses pel transcription and that pel transcription is not repressed when intracellular c-di-GMP levels are high. FleN, a known antiactivator of FleQ also participates in control of pel expression. In in vitro experiments we found that FleQ binds to pel promoter DNA and that this binding is inhibited by c-di-GMP. FleQ binds radiolabelled c-di-GMP in vitro. FleQ does not have amino acid motifs that resemble previously defined c-di-GMP binding domains. Our results show that FleQ is a new type of c-di-GMP binding protein that controls the transcriptional regulation of EPS biosynthesis genes in P. aeruginosa.

Fatty aldehydes in cyanobacteria are a metabolically flexible precursor for a diversity of biofuel products.

We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis.

A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels.

Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.

Positive and negative transcriptional regulators of glutathione-dependent formaldehyde metabolism.

A glutathione (GSH)-dependent pathway is used for formaldehyde metabolism by a wide variety of prokaryotes and eukaryotes. In this pathway, S-hydroxymethylglutathione, produced by the reaction of formaldehyde with the thiolate moiety of glutathione, is the substrate for a GSH-dependent formaldehyde dehydrogenase (GSH-FDH). While expression of GSH-FDH often increases in the presence of metabolic or exogenous sources of formaldehyde, little is known about the factors that regulate this response. Here, we identify two signal transduction pathways that regulate expression of adhI, the gene encoding GSH-FDH, in Rhodobacter sphaeroides. The loss of the histidine kinase response regulator pair RfdRS or the histidine kinase RfdS increases adhI transcription in the absence of metabolic sources of formaldehyde. Cells lacking RfdRS further increase adhI expression in the presence of metabolic sources of formaldehyde (methanol), suggesting that this negative regulator of GSH-FDH expression does not respond to this compound. In contrast, mutants lacking the histidine kinase response regulator pair AfdRS or the histidine kinase AfdS cannot induce adhI expression in the presence of either formaldehyde or metabolic sources of this compound. AfdR stimulates activity of the adhI promoter in vitro, indicating that this protein is a direct activator of GSH-FDH expression. Activation by AfdR is detectable only after incubation of the protein with acetyl phosphate, suggesting that phosphorylation is necessary for transcription activation. Activation of adhI transcription by acetyl-phosphate-treated AfdR in vitro is inhibited by a truncated RfdR protein, suggesting that this protein is a direct repressor of GSH-FDH expression. Together, the data indicate that AfdRS and RfdRS positively and negatively regulate adhI transcription in response to different signals.

Link between the membrane-bound pyridine nucleotide transhydrogenase and glutathione-dependent processes in Rhodobacter sphaeroides.

The presence of a glutathione-dependent pathway for formaldehyde oxidation in the facultative phototroph Rhodobacter sphaeroides has allowed the identification of gene products that contribute to formaldehyde metabolism. Mutants lacking the glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) are sensitive to metabolic sources of formaldehyde, like methanol. This growth phenotype is correlated with a defect in formaldehyde oxidation. Additional methanol-sensitive mutants were isolated that contained Tn5 insertions in pntA, which encodes the alpha subunit of the membrane-bound pyridine nucleotide transhydrogenase. Mutants lacking transhydrogenase activity have phenotypic and physiological characteristics that are different from those that lack GSH-FDH activity. For example, cells lacking transhydrogenase activity can utilize methanol as a sole carbon source in the absence of oxygen and do not display a formaldehyde oxidation defect, as determined by whole-cell (13)C-nuclear magnetic resonance. Since transhydrogenase can be a major source of NADPH, loss of this enzyme could result in a requirement for another source for this compound. Evidence supporting this hypothesis includes increased specific activities of other NADPH-producing enzymes and the finding that glucose utilization by the Entner-Doudoroff pathway restores aerobic methanol resistance to cells lacking transhydrogenase activity. Mutants lacking transhydrogenase activity also have higher levels of glutathione disulfide under aerobic conditions, so it is consistent that this strain has increased sensitivity to oxidative stress agents like diamide, which are known to alter the oxidation reduction state of the glutathione pool. A model will be presented to explain the role of transhydrogenase under aerobic conditions when cells need glutathione both for GSH-FDH activity and to repair oxidatively damaged proteins.

Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.