Examining the taxonomic distribution of tetracycline resistance in a wastewater plant.

Microbial communities serve as reservoirs of antibiotic resistance genes (ARGs) and facilitate the dissemination of these genes to bacteria that infect humans. Relatively little is known about the taxonomic distribution of bacteria harboring ARGs in these reservoirs and the avenues of transmission due to the technical hurdles associated with characterizing the contents of complex microbial populations and the assignment of genes to particular genomes. Focusing on the array of tetracycline resistance (Tcr) genes in the primary and secondary phases of wastewater treatment, 17 of the 22 assayed Tcr genes were detected in at least one sample. We then applied emulsion, paired isolation, and concatenation PCR (epicPCR) to link tetracycline resistance genes to specific bacterial hosts. Whereas Tcr genes tend to vary in their distributions among bacterial taxa according to their modes of action, there were numerous instances in which a particular Tcr gene was associated with a host that was distantly related to all other bacteria bearing the same gene, including several hosts not previously identified. Tcr genes are far less host-restricted than previously assumed, indicating that complex microbial communities serve as settings where ARGs are spread among divergent bacterial phyla.

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

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 Escherichia coli, 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 E. coli 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.

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.

Fine-tuning citrate synthase flux potentiates and refines metabolic innovation in the Lenski evolution experiment.

Evolutionary innovations that enable organisms to colonize new ecological niches are rare compared to gradual evolutionary changes in existing traits. We discovered that key mutations in the gltA gene, which encodes citrate synthase (CS), occurred both before and after Escherichia coli gained the ability to grow aerobically on citrate (Cit(+) phenotype) during the Lenski long-term evolution experiment. The first gltA mutation, which increases CS activity by disrupting NADH-inhibition of this enzyme, is beneficial for growth on the acetate and contributed to preserving the rudimentary Cit(+) trait from extinction when it first evolved. However, after Cit(+) was refined by further mutations, this potentiating gltA mutation became deleterious to fitness. A second wave of beneficial gltA mutations then evolved that reduced CS activity to below the ancestral level. Thus, dynamic reorganization of central metabolism made colonizing this new nutrient niche contingent on both co-opting and overcoming a history of prior adaptation.

Draft Genome Sequence of the Bacterium Pseudomonas putida CBB5, Which Can Utilize Caffeine as a Sole Carbon and Nitrogen Source.

Pseudomonas putida CBB5 was isolated from soil by enriching for growth on caffeine (1,3,7-trimethylxanthine). The draft genome of this strain is 6.9 Mb, with 5,941 predicted coding sequences. It includes the previously studied Alx gene cluster encoding alkylxanthine N-demethylase enzymes and other genes that enable the degradation of purine alkaloids.

An alternate pathway of arsenate resistance in E. coli 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 E. coli mutant cells lacking the gene for arsenate reductase (E. coli ΔarsC). We found that overexpression of glutathione S-transferase B (GstB) complemented the ΔarsC allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type E. coli 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.

Recursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in Escherichia coli.

Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability to use abundant citrate (Cit(+)) in the growth medium after ∼31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit(+) variants achieve a much higher population density in this environment. All Cit(+) individuals share a mutation that activates aerobic expression of the citT citrate transporter, but this mutation confers only an extremely weak Cit(+) phenotype on its own. To determine which of the other >70 mutations in early Cit(+) clones were needed to take full advantage of citrate, we developed a recursive genomewide recombination and sequencing method (REGRES) and performed genetic backcrosses to purge mutations not required for Cit(+) from an evolved strain. We discovered a mutation that increased expression of the dctA C4-dicarboxylate transporter greatly enhanced the Cit(+) phenotype after it evolved. Surprisingly, strains containing just the citT and dctA mutations fully use citrate, indicating that earlier mutations thought to have potentiated the initial evolution of Cit(+) are not required for expression of the refined version of this trait. Instead, this metabolic innovation may be contingent on a genetic background, and possibly ecological context, that enabled citT mutants to persist among competitors long enough to obtain dctA or equivalent mutations that conferred an overwhelming advantage. More generally, refinement of an emergent trait from a rudimentary form may be crucial to its evolutionary success.

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%.

Caffeine junkie: an unprecedented glutathione S-transferase-dependent oxygenase required for caffeine degradation by Pseudomonas putida CBB5.

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 N1- and N3-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N7-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.

Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida 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 Pseudomonas putida CBB5 to function in Escherichia coli. In the process, we discovered that adding a glutathione S-transferase from Janthinobacterium sp. Marseille was necessary to achieve N 7 -demethylation activity. E. coli cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in de novo 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 N-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs.

Multicopy suppression underpins metabolic evolvability.

Our understanding of the origins of new metabolic functions is based upon anecdotal genetic and biochemical evidence. Some auxotrophies can be suppressed by overexpressing substrate-ambiguous enzymes (i.e., those that catalyze the same chemical transformation on different substrates). Other enzymes exhibit weak but detectable catalytic promiscuity in vitro (i.e., they catalyze different transformations on similar substrates). Cells adapt to novel environments through the evolution of these secondary activities, but neither their chemical natures nor their frequencies of occurrence have been characterized en bloc. Here, we systematically identified multifunctional genes within the Escherichia coli genome. We screened 104 single-gene knockout strains and discovered that many (20%) of these auxotrophs were rescued by the overexpression of at least one noncognate E. coli gene. The deleted gene and its suppressor were generally unrelated, suggesting that promiscuity is a product of contingency. This genome-wide survey demonstrates that multifunctional genes are common and illustrates the mechanistic diversity by which their products enhance metabolic robustness and evolvability.