The Enhancer-Binding Protein MifR, an Essential Regulator of α-Ketoglutarate Transport, Is Required for Full Virulence of Pseudomonas aeruginosa PAO1 in a Mouse Model of Pneumonia.

The opportunistic human pathogen Pseudomonas aeruginosa PAO1 has an extensive metabolism, enabling it to utilize a wide range of structurally diverse compounds to meet its nutritional and energy needs. Interestingly, the utilization of some of the more unusual compounds often associated with a eukaryotic-host environment is regulated via enhancer-binding proteins (EBPs) in P. aeruginosa. Whether the utilization of such compounds and the EBPs involved contribute to the pathogenesis of P. aeruginosa remains to be fully understood. To narrow this gap, we investigated the roles of the EBPs EatR (regulator of ethanolamine catabolism), DdaR (regulator of methylarginine catabolism), and MifR (regulator of α-ketoglutarate or α-KG transport) in the virulence of P. aeruginosa PAO1 in a pneumonia-induced septic mouse model. Deletion of genes encoding EatR and DdaR had no significant effect on the mortality of P. aeruginosa PAO1-infected mice compared to wide-type (WT) PAO1-infected mice. In contrast, infected mice with ΔmifR mutant exhibited a significant reduction (~50%) in the mortality rate compared with WT PAO1 (P < 0.05). Infected mice with ΔmifR PAO1 had lower lung injury scores, fewer inflammatory cells, decreased proinflammatory cytokines, and decreased apoptosis and cell death compared to mice infected with WT PAO1 (P < 0.05). Furthermore, molecular analysis revealed decreased NLRP3 inflammasome activation in infected mice with ΔmifR PAO1 compared to WT PAO1 (P < 0.05). These results suggested that the utilization of α-KG was a contributing factor in P. aeruginosa-mediated pneumonia and sepsis and that MifR-associated regulation may be a potential therapeutic target for P. aeruginosa infectious disease.

Optimizing a Fed-Batch High-Density Fermentation Process for Medium Chain-Length Poly(3-Hydroxyalkanoates) in Escherichia coli.

Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by ß-oxidation deficient recombinant Escherichia coli LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L-1). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L-1, 0.42 g L-1 h-1), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L-1). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.

Utilization of L-glutamate as a preferred or sole nutrient in Pseudomonas aeruginosa PAO1 depends on genes encoding for the enhancer-binding protein AauR, the sigma factor RpoN and the transporter complex AatJQMP.

BACKGROUND: Glutamate and aspartate are preferred nutrients for a variety of microorganisms. In the case for many Pseudomonas spp., utilization of these amino acids is believed to be dependent on a transporter complex comprised of a periplasmic-solute binding protein (AatJ), two permease domains (AatQM) and an ATP-binding component (AatP). Notably, expression of this transporter complex is hypothesized to be regulated at the transcriptional level by the enhancer-binding protein AauR and the alternative sigma factor RpoN. The purpose of the current study was to determine the biological significance of the putative aatJ-aatQMP operon and its regulatory aauR and rpoN genes in the utilization of L-glutamate, L-glutamine, L-aspartate and L-asparagine in Pseudomonas aeruginosa PAO1. RESULTS: Deletion of the aatJ-aatQMP, aauR or rpoN genes did not affect the growth of P. aeruginosa PAO1 on L-glutamate, L-glutamine, L-aspartate and L-asparagine equally. Instead, only growth on L-glutamate as the sole carbon source was abolished with the deletion of any one of these genes. Interestingly, growth of the aauR mutant on L-glutamate was readily restored via plasmid-based expression of the aatQMP genes, suggesting that it is the function of AatQMP (and not AatJ) that is limiting in the absence of the aauR gene. Subsequent analysis of beta-galactosidase reporters revealed that both aatJ and aatQ were induced in response to L-glutamate, L-glutamine, L-aspartate or L-asparagine in a manner dependent on the aauR and rpoN genes. In addition, both aatJ and aatQ were expressed at reduced levels in the absence of the inducing-amino acids and the regulatory aauR and rpoN genes. The expression of the aatJ-aatQMP genes is, therefore, multifaceted. Lastly, the expression levels of aatJ were significantly higher (> 5 fold) than that of aatQ under all tested conditions. CONCLUSIONS: The primary function of AauR in P. aeruginosa PAO1 is to activate expression of the aatJ-aatQMP genes in response to exogenous acidic amino acids and their amide derivatives. Importantly, it is the AauR-RpoN mediated induction of the aatQMP genes that is the pivotal factor enabling P. aeruginosa PAO1 to effectively utilize or consume L-glutamate as a sole or preferred nutrient.

MifS, a DctB family histidine kinase, is a specific regulator of α-ketoglutarate response in Pseudomonas aeruginosa PAO1.

The C5-dicarboxylate α-ketoglutarate (α-KG) is a preferred nutrient source for the opportunistic pathogen Pseudomonas aeruginosa. However, very little is known about how P. aeruginosa detects and responds to α-KG in the environment. Our laboratory has previously shown that the MifS/MifR two-component signal transduction system regulates α-KG assimilation in P. aeruginosa PAO1. In an effort to better understand how this bacterium detects α-KG, we characterized the MifS sensor histidine kinase. In this study we show that although MifS is a homologue of the C4-dicarboxylate sensor DctB, it specifically responds to the C5-dicarboxylate α-KG. MifS activity increased >10-fold in the presence of α-KG, while the related C5-dicarboxylate glutarate caused only a 2-fold increase in activity. All other dicarboxylates tested did not show any significant effect on MifS activity. Homology modelling of the MifS sensor domain revealed a substrate binding pocket for α-KG. Using protein modelling and mutational analysis, we identified nine residues that are important for α-KG response, including one residue that determines the substrate specificity of MifS. Further, we found that MifS has a novel cytoplasmic linker domain that is required for α-KG response and is probably involved in signal transduction from the sensor domain to the cytoplasmic transmitter domain. Until this study, DctB family histidine kinases were known to only respond to C4-dicarboxylates. Our work shows that MifS is a novel member of the DctB family histidine kinase that specifically responds to α-KG.

SfnR2 Regulates Dimethyl Sulfide-Related Utilization in Pseudomonas aeruginosa PAO1.

Dimethyl sulfide (DMS) is a volatile sulfur compound produced mainly from the degradation of dimethylsulfoniopropionate (DMSP) in marine environments. DMS undergoes oxidation to form dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO2), and methanesulfonate (MSA), all of which occur in terrestrial environments and are accessible for consumption by various microorganisms. The purpose of the present study was to determine how the enhancer-binding proteins SfnR1 and SfnR2 contribute to the utilization of DMS and its derivatives in Pseudomonas aeruginosa PAO1. First, results from cell growth experiments showed that deletion of either sfnR2 or sfnG, a gene encoding a DMSO2-monooxygenase, significantly inhibits the ability of P. aeruginosa PAO1 to use DMSP, DMS, DMSO, and DMSO2 as sulfur sources. Deletion of the sfnR1 or msuEDC genes, which encode a MSA desulfurization pathway, did not abolish the growth of P. aeruginosa PAO1 on any sulfur compound tested. Second, data collected from ß-galactosidase assays revealed that the msuEDC-sfnR1 operon and the sfnG gene are induced in response to sulfur limitation or nonpreferred sulfur sources, such as DMSP, DMS, and DMSO, etc. Importantly, SfnR2 (and not SfnR1) is essential for this induction. Expression of sfnR2 is induced under sulfur limitation but independently of SfnR1 or SfnR2. Finally, the results of this study suggest that the main function of SfnR2 is to direct the initial activation of the msuEDC-sfnR1 operon in response to sulfur limitation or nonpreferred sulfur sources. Once expressed, SfnR1 contributes to the expression of msuEDC-sfnR1, sfnG, and other target genes involved in DMS-related metabolism in P. aeruginosa PAO1.IMPORTANCE Dimethyl sulfide (DMS) is an important environmental source of sulfur, carbon, and/or energy for microorganisms. For various bacteria, including Pseudomonas, Xanthomonas, and Azotobacter, DMS utilization is thought to be controlled by the transcriptional regulator SfnR. Adding more complexity, some bacteria, such as Acinetobacter baumannii, Enterobacter cloacae, and Pseudomonas aeruginosa, possess two, nonidentical SfnR proteins. In this study, we demonstrate that SfnR2 and not SfnR1 is the principal regulator of DMS metabolism in P. aeruginosa PAO1. Results suggest that SfnR1 has a supportive but nonessential role in the positive regulation of genes required for DMS utilization. This study not only enhances our understanding of SfnR regulation but, importantly, also provides a framework for addressing gene regulation through dual SfnR proteins in other bacteria.

Targeting the alternative sigma factor RpoN to combat virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative sigma factor 54 (σ54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro, we explored its effects in a Caenorhabditis elegans-P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

DdaR (PA1196) Regulates Expression of Dimethylarginine Dimethylaminohydrolase for the Metabolism of Methylarginines in Pseudomonas aeruginosa PAO1.

Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa, the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize NG ,NG -dimethyl-l-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including NG -monomethyl-l-arginine (l-NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for dimethylarginine dimethylaminohydrolase regulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment.IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains.

Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus.

Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

Enhancing poly(3-hydroxyalkanoate) production in Escherichia coli by the removal of the regulatory gene arcA.

Recombinant Escherichia coli is a desirable platform for the production of many biological compounds including poly(3-hydroxyalkanoates), a class of naturally occurring biodegradable polyesters with promising biomedical and material applications. Although the controlled production of desirable polymers is possible with the utilization of fatty acid feedstocks, a central challenge to this biosynthetic route is the improvement of the relatively low polymer yield, a necessary factor of decreasing the production costs. In this study we sought to address this challenge by deleting arcA and ompR, two global regulators with the capacity to inhibit the uptake and activation of exogenous fatty acids. We found that polymer yields in a ΔarcA mutant increased significantly with respect to the parental strain. In the parental strain, PHV yields were very low but improved 64-fold in the ΔarcA mutant (1.92-124 mg L-1) The ΔarcA mutant also allowed for modest increases in some medium chain length polymer yields, while weight average molecular weights improved by approximately 1.5-fold to 12-fold depending on the fatty acid substrate utilized. These results were supported by an analysis of differential gene expression, which showed that the key genes (fadD, fadL, and fadE) encoding fatty acid degradation enzymes were all upregulated by 2-, 10-, and 31-fold in an ΔarcA mutant, respectively. Additionally, the short chain length fatty acid uptake genes atoA, atoE and atoD were upregulated by 103-, 119-, and 303-fold respectively, though these values are somewhat inflated due to low expression in the parental strain. Overall, this study demonstrates that arcA is an important target to improve PHA production from fatty acids.

Total Biosynthesis of Legionaminic Acid, a Bacterial Sialic Acid Analogue.

Legionaminic acid, Leg5,7Ac2 , a nonulosonic acid like 5-acetamido neuraminic acid (Neu5Ac, sialic acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac2 has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a de novo Leg5,7Ac2 biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over-expression of this de novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac2 (≈120 mg L(-1) of culture broth) to be produced. Pure Leg5,7Ac2 could be isolated and converted into CMP-activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac2 enabling study of its role in prokaryotic and eukaryotic glycobiology.

A rapid and efficient electroporation method for transformation of Halomonas sp. O-1.

Halomonas sp. O-1 is a halophilic bacterium with a high potential for industrial application due to its natural ability to produce polyhydroxyalkanoates (PHAs) using seawater-based media. However, a major barrier preventing industrial scale implementation of this organism is a lack of molecular methodologies capable of readily transforming members of the Halomonas genus. Currently, the only reliable method used for introducing DNA into Halomonas spp. is bacterial conjugation, a somewhat tedious and time-consuming technique compared to electroporation-based methodologies. Here we describe a rapid and reproducible method for the electroporation of Halomonas sp. O-1 with plasmid DNA. Electrocompetent cells were generated by growing Halomonas sp. O-1 in a yeast extract-tryptone medium with a final salinity of 3.5%, pH of 7.5, followed by several washes using 300mM sucrose. Results show that plasmids containing chloramphenicol (Cm(R)) and gentamicin (Gm(R)) resistance cassettes are suitable antibiotic selection markers for transformation and yields of 10(4) transformants per µg of DNA were obtained. This method is simple to perform and the materials used are readily available in most research labs. Additionally, this plasmid-based transformation procedure has the potential to be adapted for a number of applications including the creation of recombinant stains and the generation of deletion mutants of Halomonas spp.

Erratum for Sarwar et al., GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1.

[This corrects the article DOI: 10.1128/mSphere.00020-16.].

GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1.

Glycine serves as a major source of single carbon units for biochemical reactions within bacterial cells. Utilization of glycine is tightly regulated and revolves around a key group of proteins known as the glycine cleavage system (GCS). Our lab previously identified the transcriptional regulator GcsR (PA2449) as being required for catabolism of glycine in the opportunistic pathogen Pseudomonas aeruginosa PAO1. In an effort to clarify and have an overall better understanding of the role of GcsR in glycine metabolism, a combination of transcriptome sequencing and electrophoretic mobility shift assays was used to identify target genes of this transcriptional regulator. It was found that GcsR binds to an 18-bp consensus sequence (TGTAACG-N4-CGTTCCG) upstream of the gcs2 operon, consisting of the gcvH2, gcvP2, glyA2, sdaA, and gcvT2 genes. The proteins encoded by these genes, namely, the GCS (GcvH2-GcvP2-GcvT2), serine hydroxymethyltransferase (GlyA2), and serine dehydratase (SdaA), form a metabolic pathway for the conversion of glycine into pyruvate, which can enter the central metabolism. GcsR activates transcription of the gcs2 operon in response to glycine. Interestingly, GcsR belongs to a family of transcriptional regulators known as TyrR-like enhancer-binding proteins (EBPs). Until this study, TyrR-like EBPs were only known to function in regulating aromatic amino acid metabolism. GcsR is the founding member of a new class of TyrR-like EBPs that function in the regulation of glycine metabolism. Indeed, homologs of GcsR and its target genes are present in almost all sequenced genomes of the Pseudomonadales order, suggesting that this genetic regulatory mechanism is a common theme for pseudomonads. IMPORTANCE Glycine is required for various cellular functions, including cell wall synthesis, protein synthesis, and the biosynthesis of several important metabolites. Regulating levels of glycine metabolism allows P. aeruginosa to maintain the metabolic flux of glycine through several pathways, including the metabolism of glycine to produce other amino acids, entry into the trichloroacetic acid cycle, and the production of virulence factors such as hydrogen cyanide. In this study, we characterized GcsR, a transcriptional regulator that activates the expression of genes involved in P. aeruginosa PAO1 glycine metabolism. Our work reveals that GcsR is the founding member of a novel class of TyrR-like EBPs that likely regulate glycine metabolism in Pseudomonadales.

Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN.

UNLABELLED: Although genes encoding enzymes and proteins related to ethanolamine catabolism are widely distributed in the genomes of Pseudomonas spp., ethanolamine catabolism has received little attention among this metabolically versatile group of bacteria. In an attempt to shed light on this subject, this study focused on defining the key regulatory factors that govern the expression of the central ethanolamine catabolic pathway in Pseudomonas aeruginosa PAO1. This pathway is encoded by the PA4022-eat-eutBC operon and consists of a transport protein (Eat), an ethanolamine-ammonia lyase (EutBC), and an acetaldehyde dehydrogenase (PA4022). EutBC is an essential enzyme in ethanolamine catabolism because it hydrolyzes this amino alcohol into ammonia and acetaldehyde. The acetaldehyde intermediate is then converted into acetate in a reaction catalyzed by acetaldehyde dehydrogenase. Using a combination of growth analyses and ß-galactosidase fusions, the enhancer-binding protein PA4021 and the sigma factor RpoN were shown to be positive regulators of the PA4022-eat-eutBC operon in P. aeruginosa PAO1. PA4021 and RpoN were required for growth on ethanolamine, and both of these regulatory proteins were essential for induction of the PA4022-eat-eutBC operon. Unexpectedly, the results indicate that acetaldehyde (and not ethanolamine) serves as the inducer molecule that is sensed by PA4021 and leads to the transcriptional activation of the PA4022-eat-eutBC operon. Due to its regulatory role in ethanolamine catabolism, PA4021 was given the name EatR. Both EatR and its target genes are conserved in several other Pseudomonas spp., suggesting that these bacteria share a mechanism for regulating ethanolamine catabolism. IMPORTANCE: The results of this study provide a basis for understanding ethanolamine catabolism and its regulation in Pseudomonas aeruginosa PAO1. Interestingly, expression of the ethanolamine-catabolic genes in this bacterium was found to be under the control of a positive-feedback regulatory loop in a manner dependent on the transcriptional regulator PA4021, the sigma factor RpoN, and the metabolite acetaldehyde. Previously characterized regulators of ethanolamine catabolism are known to sense and respond directly to ethanolamine. In contrast, PA4021 (EatR) appears to monitor the intracellular levels of free acetaldehyde and responds through transcriptional activation of the ethanolamine-catabolic genes. This regulatory mechanism is unique and represents an alternative strategy used by bacteria to govern the acquisition of ethanolamine from their surroundings.

Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1.

A variety of soil-dwelling bacteria produce polyhydroxybutyrate (PHB), which serves as a source of energy and carbon under nutrient deprivation. Bacteria belonging to the genus Pseudomonas do not generally produce PHB but are capable of using the PHB degradation product (R)-3-hydroxybutyrate [(R)-3-HB] as a growth substrate. Essential to this utilization is the NAD+-dependent dehydrogenase BdhA that converts (R)-3-HB into acetoacetate, a molecule that readily enters central metabolism. Apart from the numerous studies that had focused on the biochemical characterization of BdhA, there was nothing known about the assimilation of (R)-3-HB in Pseudomonas, including the genetic regulation of bdhA expression. This study aimed to define the regulatory factors that govern or dictate the expression of the bdhA gene and (R)-3-HB assimilation in Pseudomonas aeruginosa PAO1. Importantly, expression of the bdhA gene was found to be specifically induced by (R)-3-HB in a manner dependent on the alternative sigma factor RpoN and the enhancer-binding protein PA2005.This mode of regulation was essential for the utilization of (R)-3-HB as a sole source of energy and carbon. However, non-induced levels of bdhA expression were sufficient for P. aeruginosa PAO1 to grow on ( ± )-1,3-butanediol, which is catabolized through an (R)-3-HB intermediate. Because this is, we believe, the first report of an enhancer-binding protein that responds to (R)-3-HB, PA2005 was named HbcR for (R)-3-hydroxybutyrate catabolism regulator.

Deletion of the pflA gene in Escherichia coli LS5218 and its effects on the production of polyhydroxyalkanoates using beechwood xylan as a feedstock.

Engineering of microorganisms to directly utilize plant biomass as a feedstock for the biosynthesis of value-added products such as bioplastics is the aim of consolidated bioprocessing. In previous research we successfully engineered E. coli LS5218 to produce polyhydroxyalkanoates (PHAs) from xylan. In this study we report further genetic modifications to Escherichia coli LS5218 in order to increase the lactic acid (LA) fraction in poly(lactic acid-co-3-hydroxyalkanoate) P(LA-co-HA) copolymers. Deletion of the pflA gene resulted in increased content of LA repeating units in the copolymers by over 3-fold compared with the wild type; however, this increase was offset by reduced yields in cell mass. Additionally, when acetate was used as a feedstock LA monomer incorporation reached 18.5 (mol%), which suggests that acetate can be used as a feedstock for the production of P(LA-co-HA) copolymers by E. coli.

Genetic analysis of the assimilation of C5-dicarboxylic acids in Pseudomonas aeruginosa PAO1.

There is a wealth of information on the genetic regulation and biochemical properties of bacterial C4-dicarboxylate transport systems. In sharp contrast, there are far fewer studies describing the transport and assimilation of C5-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 α-ketoglutarate (α-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 α-KG and glutarate (another C5-dicarboxylate). Metabolite analysis confirmed that the PA5530 gene was necessary for the uptake of extracellular α-KG. Like other substrate-inducible transporter genes, expression of the PA5530 gene was induced by extracellular C5-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 C5-dicarboxylates, especially α-KG. The results of this study provide a framework for exploring the assimilation of α-KG in other pseudomonads.

Gene PA2449 is essential for glycine metabolism and pyocyanin biosynthesis in Pseudomonas aeruginosa PAO1.

Many 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.

Sialic acid and N-acyl sialic acid analog production by fermentation of metabolically and genetically engineered Escherichia coli.

Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity of glycoproteins. Sialic acid analogs are antiviral therapeutics as well as crucial tools in bacterial pathogenesis research, immunobiology and development of cancer diagnostic imaging. The scarce supply of sialic acid hinders production of these materials. We have developed an efficient, rapid and cost effective fermentation route to access sialic acid. Our approach uses low cost feedstock, produces an industrially relevant amount of sialic acid and is scalable to manufacturing levels. We have also shown that precursor directed biosynthesis can be used to produce a N-acyl sialic acid analog. This work demonstrates the feasibility of engineering manufacturing-friendly bacteria to produce complex, unavailable small molecules.