Caffeine Junkie: an Unprecedented Glutathione S-Transferase- Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5 Downloaded from

c Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N 1 -and N 3 -demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N 7 -demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners. C affeine (1,3,7-trimethylxanthine) and other N-methylated xanthines are well known for applications in food and as pharmaceuticals that improve lung function for asthmatics and chronic obstructive pulmonary disease (COPD) sufferers. More recently, these compounds have been investigated for use as natural insecticides and in treatments for cancer, septic shock, and functional neutrophil disorders (1-3). Enzymatic methods for producing and degrading these N-methylated xanthines could have broader applications for health through both biosynthesis and environmental remediation of waste and by-products. Therefore, bacteria that have evolved to live on caffeine as the sole carbon and nitrogen source are of interest, as are their metabolic pathways toward N-methylated xanthines. Pseudomonas putida CBB5 degrades caffeine, theophylline (1,3-dimethylxanthine), and related methylxanthines via sequential N-demethylation to xanthine (4-6). The ordered N-demethylation of caffeine to xanthine occurs in three steps catalyzed by enzymes belonging to the Rieske oxygenase (RO) family (5, 6), which are encoded by the Alx operon. Initially, two Rieske, nonheme Fe(II) monooxygenases, NdmA and NdmB, remove the N 1 -and N 3 -methyl groups, respectively, from caffeine to form 7-methylxanthine. Both enzymes require an unusually large 65-kDa redox-dense RO reductase, NdmD, which transfers electrons from NADH to NdmA and NdmB for oxygen activation. The final step in the caffeine degradation pathway is N 7 -demethylation of 7-methylxanthine to xanthine. This N 7 -demethylation activity was inseparable from NdmD after four chromatographic steps (6). A highly enriched protein fraction containing this activity was comprised of NdmD and two additional major protein bands, as visualized by SDS-PAGE. These two additional peptides are encoded by two genes in the Alx operon, labeled orf7 and orf8, which flank ndmD on the CBB5 genome Here, we report that ndmE encodes a new type of GST that is absolutely required for N 7 -demethylation of 7-methylxanthine, the final step of caffeine degradation in P. putida CBB5. The N 7 -demethylase RO is unusual in itself because the iron-sulfur and non-heme iron domains that compose the normally functional oxygenase are encoded by two separate genes. The non-heme iron is contained in NdmC, while the iron-sulfur domain is fused to NdmD. NdmE is proposed to facilitate the formation of the NdmCDE complex, which catalyzes the N 7 -demethylation. This is the first report of a new class of GST-dependent ROs. Additional identification of similar uncharacterized gene clusters within genome databases suggests that there is a more generalized role for GSTs in oxygenation and/or biodegradation

Local genic base composition impacts protein production and cellular fitness

The maintenance of a G + C content that is higher than the mutational input to a genome provides support for the view that selection serves to increase G + C contents in bacteria. Recent experimental evidence from Escherichia coli demonstrated that selection for increasing G + C content operates at the level of translation, but the precise mechanism by which this occurs is unknown. To determine the substrate of selection, we asked whether selection on G + C content acts across all sites within a gene or is confined to particular genic regions or nucleotide positions. We systematically altered the G + C contents of the GFP gene and assayed its effects on the fitness of strains harboring each variant. Fitness differences were attributable to the base compositional variation in the terminal portion of the gene, suggesting a connection to the folding of a specific protein feature. Variants containing sequence features that are thought to result in rapid translation, such as low G + C content and high levels of codon adaptation, displayed highly reduced growth rates. Taken together, our results show that purifying selection acting against A and T mutations most likely results from their tendency to increase the rate of translation, which can perturb the dynamics of protein folding

Innovation in an <i>E</i>. <i>coli</i> evolution experiment is contingent on maintaining adaptive potential until competition subsides

<div><p>Key innovations are disruptive evolutionary events that enable a species to escape constraints and rapidly diversify. After 15 years of the Lenski long-term evolution experiment with <i>Escherichia coli</i>, cells in one of the twelve populations evolved the ability to utilize citrate, an abundant but previously untapped carbon source in the environment. Descendants of these cells became dominant in the population and subsequently diversified as a consequence of invading this vacant niche. Mutations responsible for the appearance of rudimentary citrate utilization and for refining this ability have been characterized. However, the complete nature of the genetic and/or ecological events that set the stage for this key innovation is unknown. In particular, it is unclear why it took so long for citrate utilization to evolve and why it still has evolved in only one of the twelve <i>E</i>. <i>coli</i> populations after 30 years of the Lenski experiment. In this study, we recapitulated the initial mutation needed to evolve citrate utilization in strains isolated from throughout the first 31,500 generations of the history of this population. We found that there was already a slight fitness benefit for this mutation in the original ancestor of the evolution experiment and in other early isolates. However, evolution of citrate utilization was blocked at this point due to competition with other mutations that improved fitness in the original niche. Subsequently, an anti-potentiated genetic background evolved in which it was deleterious to evolve rudimentary citrate utilization. Only later, after further mutations accumulated that restored the benefit of this first-step mutation and the overall rate of adaptation in the population slowed, was citrate utilization likely to evolve. Thus, intense competition and the types of mutations that it favors can lead to short-sighted evolutionary trajectories that hide a stepping stone needed to access a key innovation from many future generations.</p></div

Evolution of rudimentary citrate utilization by activating <i>citT</i> expression is slightly beneficial in the genetic background in which it evolved and in the LTEE ancestor.

<p><b>(a)</b> The <i>rnk</i>-<i>citG</i> duplication that evolved in the LTEE creates a genomic configuration in which a novel mRNA encoding the CitT transporter is expressed from the <i>rnk</i> promoter (P<i>_rnk</i>) (right). This mutation alone is sufficient for weak citrate utilization (Cit⁺ phenotype). It is the ‘actualizing mutation’ in the evolution of this key innovation. Strain ZDB564 is the earliest Cit⁺ isolate from the LTEE. In order to measure the effect that this mutation had on competitive fitness when it evolved, a spontaneous Cit⁻ revertant of ZDB564 in which the duplication collapsed back to the ancestral state was isolated (left). <b>(b)</b> Competitive fitness of Cit⁺ versus Cit⁻ strain variants. The ZDB564 versus ZDB706 competitions measure the fitness effect of the <i>rnk</i>-<i>citG</i> duplication when it evolved. The ZDB706 and REL606 competitions test the effect of adding one copy of the evolved P_<i>rnk</i>-<i>citT</i> module into a strain (+) versus adding an empty version of the same cassette (Ø), as pictured in <b>c</b>. An additional ZDB706 competition (in population) was conducted with the two strains together mixed at a 1:99 ratio with the evolved LTEE population from at 31,000 generations to determine if the mutation had a different effect on fitness when rare in the population. Starred strains (*) have a change to the Ara⁺ marker state to allow competition with the corresponding Ara⁻ strain as illustrated in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s001" target="_blank">S1</a></b> and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s002" target="_blank">S2</a> Figs</b>. The marker change had no effect on competitive fitness in each case (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s003" target="_blank">S3 Fig</a>)</b>. Error bars are 95% confidence intervals. (<b>c</b>) Schematic of the gene cassettes used in the P_<i>rnk</i>-<i>citT</i> knock-in assay showing how they were integrated into the <i>E</i>. <i>coli</i> chromosome in a way that replaces the native <i>lac</i> locus. <b>(d)</b> <i>citT</i> mRNA expression levels measured relative to the REL606 LTEE ancestor in the evolved Cit⁺ isolate from the LTEE (ZDB564) and strains with the P_<i>rnk</i>-<i>citT</i> and corresponding empty control cassettes integrated into their chromosomes. Error bars are 95% confidence intervals.</p

Fitness consequences of evolving Cit⁺ in different evolved genetic backgrounds.

<p><b>(a)</b> Results of the P_<i>rnk</i><i>-citT</i> knock-in assay on 23 pre-Cit⁺ evolved strains. The clones are ordered by the generation from which they were isolated. Error bars are 95% confidence intervals. Strain construction details and how the results of competition assays were combined into these fitness estimates are described in the <b>Methods</b> and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s001" target="_blank">S1</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s004" target="_blank">S4</a> Figs</b>. <b>(b)</b> Increased lag phase upon addition of the P_<i>rnk</i>-<i>citT</i> module in anti-potentiated strains. Growth curves for the ancestor, REL606, and two anti-potentiated strains, ZDB483 and ZDB14, are shown. Error bars are standard deviations of four replicate cultures.</p

Potential for evolving Cit⁺ mapped onto phylogeny.

<p>Phylogeny of isolates from the LTEE population including 20 new clones sequenced for this study to provide better resolution of the timing of mutations on the lineage leading to Cit⁺ (names in italics). In order to identify changes in the degree of potentiation due to mutations, we mapped the results of the P_<i>rnk</i><i>-citT</i> knock-in assay onto this phylogenetic tree. Colored symbols reflect the Cit⁺ to Cit⁻ relative fitness measured for those strains. The ancestor and 61 evolved isolates were used to construct this phylogenetic tree (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s006" target="_blank">S1 Table</a></b>). Two clones isolated at 50,000 generations are not shown. Two strains that evolved citrate utilization in replay experiments under the LTEE conditions in a previous study [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.ref007" target="_blank">7</a>] are marked with plus signs (++), and three strains that had evolved alleles added or removed during strain construction as described in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s007" target="_blank">S2 Table</a></b> are starred (*).</p

An Alternate Pathway of Arsenate Resistance in <i>E. coli</i> Mediated by the Glutathione S-Transferase GstB

Microbial arsenate resistance is known to be conferred by specialized oxidoreductase enzymes termed arsenate reductases. We carried out a genetic selection on media supplemented with sodium arsenate for multicopy genes that can confer growth to <i>E. coli</i> mutant cells lacking the gene for arsenate reductase (<i>E. coli</i> Δ<i>arsC</i>). We found that overexpression of glutathione S-transferase B (GstB) complemented the Δ<i>arsC</i> allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type <i>E. coli</i> arsenate reductase, arsenate resistance via GstB was not dependent on reducing equivalents provided by glutaredoxins or a catalytic cysteine residue. Instead, two arginine residues, which presumably coordinate the arsenate substrate within the electrophilic binding site of GstB, were found to be critical for transferase activity. We provide biochemical evidence that GstB acts to directly reduce arsenate to arsenite with reduced glutathione (GSH) as the electron donor. Our results reveal a pathway for the detoxification of arsenate in bacteria that hinges on a previously undescribed function of a bacterial glutathione S-transferase

Generalized bacterial genome editing using mobile group II introns and Cre‐ lox

Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns (‘targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%

Decaffeination and Measurement of Caffeine Content by Addicted <i>Escherichia coli</i> with a Refactored <i>N</i>‑Demethylation Operon from <i>Pseudomonas putida</i> CBB5

The widespread use of caffeine (1,3,7-trimethylxanthine) and other methylxanthines in beverages and pharmaceuticals has led to significant environmental pollution. We have developed a portable caffeine degradation operon by refactoring the alkylxanthine degradation (Alx) gene cluster from <i>Pseudomonas putida</i> CBB5 to function in <i>Escherichia coli</i>. In the process, we discovered that adding a glutathione <i>S</i>-transferase from <i>Janthinobacterium</i> sp. Marseille was necessary to achieve <i>N</i>₇-demethylation activity. <i>E. coli</i> cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in <i>de novo</i> guanine biosynthesis that contain the refactored operon are ″addicted″ to caffeine: their growth density is limited by the availability of caffeine or other xanthines. We show that the addicted strain can be used as a biosensor to measure the caffeine content of common beverages. The synthetic <i>N</i>-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs