A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa.

The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA (Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.

TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa.

Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB.

Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa.

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNAMet(CAU) and tRNATrp(CCA) are substrates for Cm formation, tRNAGln(UUG), tRNAPro(UGG), tRNAPro(CGG) and tRNAHis(GUG) for Um, and tRNAPro(GGG) for Am. tRNASer(UGA), previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNAPro(GGG) and Um in tRNAGln(UUG) by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.

Gene expression and physiological role of Pseudomonas aeruginosa methionine sulfoxide reductases during oxidative stress.

Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H2O2. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive.

Pseudomonas aeruginosa GidA modulates the expression of catalases at the posttranscriptional level and plays a role in virulence.

Pseudomonas aeruginosa gidA, which encodes a putative tRNA-modifying enzyme, is associated with a variety of virulence phenotypes. Here, we demonstrated that P. aeruginosa gidA is responsible for the modifications of uridine in tRNAs in vivo. Loss of gidA was found to have no impact on the mRNA levels of katA and katB, but it decreased KatA and KatB protein levels, resulting in decreased total catalase activity and a hydrogen peroxide-sensitive phenotype. Furthermore, gidA was found to affect flagella-mediated motility and biofilm formation; and it was required for the full virulence of P. aeruginosa in both Caenorhabditis elegans and macrophage models. Together, these observations reveal the posttranscriptional impact of gidA on the oxidative stress response, highlight the complexity of catalase gene expression regulation, and further support the involvement of gidA in the virulence of P. aeruginosa.

Analyses of the regulatory mechanism and physiological roles of Pseudomonas aeruginosa OhrR, a transcription regulator and a sensor of organic hydroperoxides.

ohrR encodes an organic hydroperoxide sensor and a transcriptional repressor that regulates organic hydroperoxide-inducible expression of a thiol peroxidase gene, ohr, and itself. OhrR binds directly to the operators and represses transcription of these genes. Exposure to an organic hydroperoxide leads to oxidation of OhrR and to subsequent structural changes that result in the loss of the repressor's ability to bind to the operators that allow expression of the target genes. Differential induction of ohrR and ohr by tert-butyl hydroperoxide suggests that factors such as the repressor's dissociation constants for different operators and the chemical nature of the inducer contribute to OhrR-dependent organic hydroperoxide-inducible gene expression. ohrR and ohr mutants show increased and decreased resistance to organic hydroproxides, respectively, compared to a parental strain. Moreover, the ohrR mutant had a reduced-virulence phenotype in the Pseudomonas aeruginosa-Caenorhabditis elegans pathogenicity model.

The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes.

The OxyR-regulated phnW gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect Pseudomonas aeruginosa from tert-butyl hydroperoxide.

The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.

Regulation of Organic Hydroperoxide Stress Response by Two OhrR Homologs in Pseudomonas aeruginosa.

Pseudomonas aeruginosa ohrR and ospR are gene homologs encoding oxidant sensing transcription regulators. OspR is known to regulate gpx, encoding a glutathione peroxidase, while OhrR regulates the expression of ohr that encodes an organic peroxide specific peroxiredoxin. Here, we show that ospR mediated gpx expression, like ohrR and ohr, specifically responds to organic hydroperoxides as compared to hydrogen peroxide and superoxide anion. Furthermore, the regulation of these two systems is interconnected. OspR is able to functionally complement an ohrR mutant, i.e. it regulates ohr in an oxidant dependent manner. In an ohrR mutant, in which ohr is derepressed, the induction of gpx expression by organic hydroperoxide is reduced. Likewise, in an ospR mutant, where gpx expression is constitutively high, oxidant dependent induction of ohr expression is reduced. Moreover, in vitro binding assays show that OspR binds the ohr promoter, while OhrR binds the gpx promoter, albeit with lower affinity. The binding of OhrR to the gpx promoter may not be physiologically relevant; however, OspR is shown to mediate oxidant-inducible expression at both promoters. Interestingly, the mechanism of OspR-mediated, oxidant-dependent induction at the two promoters appears to be distinct. OspR required two conserved cysteines (C24 and C134) for oxidant-dependent induction of the gpx promoter, while only C24 is essential at the ohr promoter. Overall, this study illustrates possible connection between two regulatory switches in response to oxidative stress.

The omlA gene is involved in multidrug resistance and its expression is inhibited by coumarins in Xanthomonas campestris pv. phaseoli.

A gene encoding the outer membrane lipoprotein, OmlA, from the bacterial phytopathogen Xanthomonas campestris pv. phaseoli was isolated and characterized. An omlA insertion mutant showed an increased susceptibility to novobiocin and coumermycin, antibiotics with gyrase inhibitor activity. The omlA mutant accumulated novobiocin. Additionally, the omlA mutant was more sensitive than the wild type to chloramphenicol, a protein synthesis inhibitor; SDS, a detergent; and menadione, a superoxide generator. The susceptibility of the mutant to unrelated chemicals indicated a general role for OmlA in maintaining membrane integrity. Transcription of omlA was downregulated in the presence of both gyrase inhibitors, suggesting that DNA supercoiling might regulate the synthesis of OmlA. The omlA gene was divergently transcribed from the gene encoding the ferric uptake regulator Fur. Although the promoters of omlA and fur overlapped, Fur did not play any regulatory role in the expression of omlA due to the fact that inactivation of Fur did not affect the expression of omlA either in the presence or absence of iron.

Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli.

katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA-ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like sigma70 -35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.

Comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress.

The molecular mechanisms by which plants acclimate to oxidative stress are poorly understood. To identify the processes involved in acclimation, we performed a comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress. Combining mRNA differential display and cDNA array analysis, we estimated that at least 95 genes alter their expression in tobacco leaves acclimated to oxidative stress, of which 83% are induced and 17% repressed. Sequence analysis of 53 sequence tags revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular protection and detoxification, energy and carbohydrate metabolism, de novo protein synthesis, and signal transduction showed altered expression. Expression of most of the genes was enhanced, except for genes associated with photosynthesis and light-regulated processes that were repressed. During acclimation, two distinct groups of coregulated genes ("early-" and "late-response" gene regulons) were observed, indicating the presence of at least two different gene induction pathways. These two gene regulons also showed differential expression patterns on an oxidative stress challenge. Expression of "late-response" genes was augmented in the acclimated leaf tissues, whereas expression of "early-response" genes was not. Together, our data suggest that acclimation to oxidative stress is a highly complex process associated with broad gene expression adjustments. Moreover, our data indicate that in addition to defense gene induction, sensitization of plants for potentiated gene expression might be an important factor in oxidative stress acclimation.

Bacterial Ohr and OsmC paralogues define two protein families with distinct functions and patterns of expression.

Xanthomonas campestris Ohr (a protein involved in organic peroxide protection) and Escherichia coli OsmC (an osmotically inducible protein of unknown function) are related proteins. Database searches and phylogenetic analyses reveal that Ohr and OsmC homologues cluster into two related subfamilies of proteins widely distributed in both Gram-negative and Gram-positive bacteria. To determine if these two subfamilies are functionally distinct, ohr and osmC in Pseudomonas aeruginosa (a bacterium with one representative from each subfamily) were analysed. Only ohr mutants are hypersensitive to organic peroxide, and this phenotype can be restored by complementation with ohr but not osmC. In addition, expression of ohr was highly induced only by organic peroxides, and not by other oxidants or stresses. In contrast, osmC was induced by ethanol and osmotic stress. A similar pattern of regulation was observed for Ohr and OsmC homologues in the Gram-positive bacterium Deinococcus radiodurans, though uninduced expression was much higher and induction lower in this species. These data clearly support the conclusion that Ohr and OsmC define two functionally distinct subfamilies with distinct patterns of regulation.

The repressor for an organic peroxide-inducible operon is uniquely regulated at multiple levels.

ohrR encodes a novel organic peroxide-inducible transcription repressor, and we have demonstrated that ohrR is regulated at the transcriptional and the post-transcriptional levels. Primer extension results show that ohrR transcription initiates at the A residue of the ATG translation initiation codon for the ohrR coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1) has high homology to the consensus sequence for Xanthomonas promoters, which is reflected in the high in vivo promoter activity of P1. Deletion of a 139 bp fragment containing the P1 promoter showed that the sequences upstream of -35 regions were required for neither the promoter activity nor OhrR autoregulation. In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands. The protected regions include the -35 and -10 regions of P1. We suggest that OhrR represses gene expression by blocking RNA polymerase binding to the promoter. There are two steps in the post-transcriptional regulation of ohrR, namely differential stability and inefficient translation of the mRNA. The bicistronic ohrR-ohr mRNA was highly labile and underwent rapid processing in vivo to give only stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore, the ohrR mRNA was inefficiently translated. We propose that, in uninduced cells, the concentration of OhrR is maintained at low levels by the autoregulation mechanism at the transcriptional levels and by the ohrR mRNA instability coupled with inefficient translation at the post-transcriptional level. Upon exposure to an organic peroxide, the compound probably interacts with OhrR and prevents it from repressing the P1 promoter, thus allowing high-level expression of the ohrR-ohr operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic per-oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.