Youjun Feng

Escherichia coli unsaturated fatty acid synthesis: Complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB

Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA.

Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters

Two transcriptional regulators, the FadR activator and the FabR repressor, control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was blocked in the absence of exogenous unsaturated fatty acids in a ΔfadR strain and found that the rates of transcription of fabA and fabB were unaffected by the lack of unsaturated thioesters. To examine the discrepancy between our in vivo results and the prior in vitro results we obtained active, natively folded forms of the E. coli and Vibrio cholerae FabRs by use of an in vitro transcription–translation system. We report that FabR bound the intact promoter regions of both fabA and fabB in the absence of unsaturated acyl thioesters, but bound the two promoters differently. Native FabR bound the fabA promoter region provided that the canonical FabR binding site is extended by inclusion of flanking sequences that overlap the neighbouring FadR binding site. In contrast, although binding to the fabB operator also required a flanking sequence, a non‐specific sequence could suffice. However, unsaturated thioesters did allow FabR binding to the minimal FabR operator sites of both promoters which otherwise were not bound. Thus unsaturated thioester ligands were not essential for FabR/target DNA interaction, but acted to enhance binding. The gel mobility shift data plus in vivo expression data indicate that despite the remarkably similar arrangements of promoter elements, FadR predominately regulates fabA expression whereas FabR is the dominant regulator of fabB expression. We also report that E. coli fabR expression is not autoregulated. Complementation, qRT‐PCR and fatty acid composition analyses demonstrated that V. cholerae FabR was a functional repressor of unsaturated fatty acid synthesis. However, in contrast to E. coli, gel mobility shift assays indicated that neither E. coli nor V. cholerae FabRs bound the V. cholerae fabB promoter, although both proteins efficiently bound the V. cholerae fabA promoter. This asymmetry was shown to be due to the lack of a FabR binding site within the V. cholerae fabB promoter region.

The Burkholderia cenocepacia BDSF quorum sensing fatty acid is synthesized by a bifunctional crotonase homologue having both dehydratase and thioesterase activities

Signal molecules of the diffusible signal factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram‐negative bacteria. DSF signals are reported to be active not only on their cognate bacteria, but also on unrelated bacteria and the pathogenic yeast, Candida albicans. DSFs are monounsaturated fatty acids of medium chain length containing an unusual cis‐2 double bond. Although genetic analyses had identified genes involved in DSF synthesis, the pathway of DSF synthesis was unknown. The DSF of the important human pathogen Burkholderia cenocepacia (called BDSF) is cis‐2‐dodecenoic acid. We report that BDSF is synthesized from a fatty acid synthetic intermediate, the acyl carrier protein (ACP) thioester of 3‐hydroxydodecanoic acid. This intermediate is intercepted by protein Bcam0581 and converted to cis‐2‐dodecenoyl‐ACP. Bcam0581 is annotated as a homologue of crotonase, the first enzyme of the fatty acid degradation pathway. We demonstrated Bcam0581to be a bifunctional protein that not only catalysed dehydration of 3‐hydroxydodecanoyl‐ACP to cis‐2‐dodecenoyl‐ACP, but also cleaved the thioester bond to give the free acid. Both activities required the same set of active‐site residues. Although dehydratase and thioesterase activities are known activities of the crotonase superfamily, Bcam0581 is the first protein shown to have both activities.

Overlapping Repressor Binding Sites Result in Additive Regulation of Escherichia coli FadH by FadR and ArcA

Escherichia coli fadH encodes a 2,4-dienoyl reductase that plays an auxiliary role in beta-oxidation of certain unsaturated fatty acids. In the 2 decades since its discovery, FadH biochemistry has been studied extensively. However, the genetic regulation of FadH has been explored only partially. Here we report mapping of the fadH promoter and document its complex regulation by three independent regulators, the fatty acid degradation FadR repressor, the oxygen-responsive ArcA-ArcB two-component system, and the cyclic AMP receptor protein-cyclic AMP (CRP-cAMP) complex. Electrophoretic mobility shift assays demonstrated that FadR binds to the fadH promoter region and that this binding can be specifically reversed by long-chain acyl-coenzyme A (CoA) thioesters. In vivo data combining transcriptional lacZ fusion and real-time quantitative PCR (qPCR) analyses indicated that fadH is strongly repressed by FadR, in agreement with induction of fadH by long-chain fatty acids. Inactivation of arcA increased fadH transcription by >3-fold under anaerobic conditions. Moreover, fadH expression was increased 8- to 10-fold under anaerobic conditions upon deletion of both the fadR and the arcA gene, indicating that anaerobic expression is additively repressed by FadR and ArcA-ArcB. Unlike fadM, a newly reported member of the E. coli fad regulon that encodes another auxiliary beta-oxidation enzyme, fadH was activated by the CRP-cAMP complex in a manner similar to those of the prototypical fad genes. In the absence of the CRP-cAMP complex, repression of fadH expression by both FadR and ArcA-ArcB was very weak, suggesting a possible interplay with other DNA binding proteins.

A New Member of the Escherichia coli fad Regulon: Transcriptional Regulation of fadM (ybaW)

Recently, Nie and coworkers (L. Nie, Y. Ren, A. Janakiraman, S. Smith, and H. Schulz, Biochemistry 47:9618-9626, 2008) reported a new Escherichia coli thioesterase encoded by the ybaW gene that cleaves the thioester bonds of inhibitory acyl-coenzyme A (CoA) by-products generated during beta-oxidation of certain unsaturated fatty acids. These authors suggested that ybaW expression might be regulated by FadR, the repressor of the fad (fatty acid degradation) regulon. We report mapping of the ybaW promoter and show that ybaW transcription responded to FadR in vivo. Moreover, purified FadR bound to a DNA sequence similar to the canonical FadR binding site located upstream of the ybaW coding sequence and was released from the promoter upon the addition of long-chain acyl-CoA thioesters. We therefore propose the designation fadM in place of ybaW. Although FadR regulation of fadM expression had the pattern typical of fad regulon genes, its modulation by the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) global regulator was the opposite of that normally observed. CRP-cAMP generally acts as an activator of fad gene expression, consistent with the low status of fatty acids as carbon sources. However, glucose growth stimulated fadM expression relative to acetate growth, as did inactivation of CRP-cAMP, indicating that the complex acts as a negative regulator of this gene. The stimulation of fadM expression seen upon deletion of the gene encoding adenylate cyclase (Deltacya) was reversed by supplementation of the growth medium with cAMP. Nie and coworkers also reported that growth on a conjugated linoleic acid isomer yields much higher levels of FadM thioesterase activity than does growth on oleic acid. In contrast, we found that the conjugated linoleic acid isomer was only a weak inducer of fadM expression. Although the gene is not essential for growth, the high basal level of fadM expression under diverse growth conditions suggests that the encoded thioesterase has functions in addition to beta-oxidation.

Crosstalk of Escherichia coli FadR with Global Regulators in Expression of Fatty Acid Transport Genes

Escherichia coli FadR plays two regulatory roles in fatty acid metabolism. FadR represses the fatty acid degradation (fad) system and activates the unsaturated fatty acid synthetic pathway. Cross-talk between E. coli FadR and the ArcA-ArcB oxygen-responsive two-component system was observed that resulted in diverse regulation of certain fad regulon β-oxidation genes. We have extended such analyses to the fadL and fadD genes, the protein products of which are required for long chain fatty acid transport and have also studied the role of a third global regulator, the CRP-cAMP complex. The promoters of both the fadL and fadD genes contain two experimentally validated FadR-binding sites plus binding sites for ArcA and CRP-cAMP. Despite the presence of dual binding sites FadR only modestly regulates expression of these genes, indicating that the number of binding sites does not determine regulatory strength. We report complementary in vitro and in vivo studies indicating that the CRP-cAMP complex directly activates expression of fadL and fadD as well as the β-oxidation gene, fadH. The physiological relevance of the fadL and fadD transcription data was validated by direct assays of long chain fatty acid transport.

Aflatoxin B1 Negatively Regulates Wnt/β-Catenin Signaling Pathway through Activating miR-33a

MicroRNAs are known to play an important role in modulating gene expression in various diseases including cancers and cardiovascular disorders, but only a few of them are associated with the pathology of aflatoxin B1 (AFB1), a potent mycotoxin. Here, we discovered a novel regulatory network between AFB1, miR-33a and β-catenin in human carcinoma cells. The level of miR-33a was up-regulated in hepatocellular carcinoma (HCC) cells treated with AFB1, while in the same cells causing the decrease in β-catenin expression when treated at their IC50 values. miR-33a, specifically miR-33a-5p, was demonstrated to down-regulate the expression of β-catenin, affect the β-catenin pathway, and inhibit cell growth. Also, by employing a luciferase assay, we found that miR-33a down-regulated β-catenin by directly binding to the 3’-UTR of β-catenin. These results suggested that AFB1 might down-regulate β-catenin by up-regulating miR-33a. This understanding opens new lines of thought in the potential role of miR-33a in the clinical therapy of cancer.

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

A Francisella virulence factor catalyses an essential reaction of biotin synthesis

We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side‐chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin‐free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl‐ACP methyl ester carboxyl‐esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl‐ACP to pimeloyl‐ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the α/β‐hydrolase family. Structure‐guided mapping combined with site‐directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.

Profligate biotin synthesis in α‐proteobacteria – a developing or degenerating regulatory system?

Biotin (vitamin H) is a key enzyme cofactor required in all three domains of life. Although this cofactor was discovered over 70 years ago and has long been recognized as an essential nutrient for animals, our knowledge of the strategies bacteria use to sense biotin demand is very limited. The paradigm mechanism is that of Escherichia coli in which BirA protein, the prototypical bi‐functional biotin protein ligase, both covalently attaches biotin to the acceptor proteins of central metabolism and represses transcription of the biotin biosynthetic pathway in response to biotin demand. However, in other bacteria the biotin protein ligase lacks a DNA‐binding domain which raises the question of how these bacteria regulate the synthesis of biotin, an energetically expensive molecule. A bioinformatic study by Rodionov and Gelfand identified a protein termed BioR in α‐proteobacteria and predicted that BioR would have the biotin operon regulatory role that in most other bacteria is fulfilled by the BirA DNA‐binding domain. We have now tested this prediction in the plant pathogen Agrobacterium tumefaciens. As predicted the A. tumefaciens biotin protein ligase is a fully functional ligase that has no role in regulation of biotin synthesis whereas BioR represses transcription of the biotin synthesis genes. Moreover, as determined by electrophoretic mobility shift assays, BioR binds the predicted operator site, which is located downstream of the mapped transcription start site. qPCR measurements indicated that deletion of BioR resulted in a c. 15‐fold increase of bio operon transcription in the presence of high biotin levels. Effective repression of a plasmid‐borne bioB‐lacZ reporter was seen only upon the overproduction of BioR. In contrast to E. coli and Bacillus subtilis where biotin synthesis is tightly controlled, A. tumefaciens synthesizes much more biotin than needed for modification of the biotin‐requiring enzymes. Protein‐bound biotin constitutes only about 0.5% of the total biotin, most of which is found in the culture medium. To the best of our knowledge, A. tumefaciens represents the first example of profligate biotin synthesis by a wild type bacterium.