The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle.     

A screen for recessive speciation genes expressed in the gametes of F1 hybrid yeast

Diploid hybrids of Saccharomyces cerevisiae and its closest relative, Saccharomyces paradoxus, are viable, but the sexual gametes they produce are not. One of several possible causes of this gamete inviability is incompatibility between genes from different species—such incompatible genes are usually called “speciation genes.” In diploid F1 hybrids, which contain a complete haploid genome from each species, the presence of compatible alleles can mask the effects of (recessive) incompatible speciation genes. But in the haploid gametes produced by F1 hybrids, recessive speciation genes may be exposed, killing the gametes and thus preventing F1 hybrids from reproducing sexually. Here I present the results of an experiment to detect incompatibilities that kill hybrid gametes. I transferred nine of the 16 S. paradoxus chromosomes individually into S. cerevisiae gametes and tested the ability of each to replace its S. cerevisiae homeolog. All nine chromosomes were compatible, producing nine viable haploid strains, each with 15 S. cerevisiae chromosomes and one S. paradoxus chromosome. Thus, none of these chromosomes contain speciation genes that were capable of killing the hybrid gametes that received them. This is a surprising result that suggests that such speciation genes do not play a major role in yeast speciation.     

Design and diversity in bacterial chemotaxis: a comparative study in Escherichia coli and Bacillus subtilis

Comparable processes in different species often involve homologous genes. One question is whether the network structure, in particular the feedback control structure, is also conserved. The bacterial chemotaxis pathways in E. coli and B. subtilis both regulate the same task, namely, excitation and adaptation to environmental signals. Both pathways employ many orthologous genes. Yet how these orthologs contribute to network function in each organism is different. To investigate this problem, we propose what is to our knowledge the first computational model for B. subtilis chemotaxis and compare it to previously published models for chemotaxis in E. coli. The models reveal that the core control strategy for signal processing is the same in both organisms, though in B. subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness. Furthermore, the network structures are different despite the similarity of the proteins in each organism. These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property.     

The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.     

Major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species

Sequencing and comparative genome analysis of four strains of Campylobacter including C. lari RM2100, C. upsaliensis RM3195, and C. coli RM2228 has revealed major structural differences that are associated with the insertion of phage- and plasmid-like genomic islands, as well as major variations in the lipooligosaccharide complex. Poly G tracts are longer, are greater in number, and show greater variability in C. upsaliensis than in the other species. Many genes involved in host colonization, including racR/S, cadF, cdt, ciaB, and flagellin genes, are conserved across the species, but variations that appear to be species specific are evident for a lipooligosaccharide locus, a capsular (extracellular) polysaccharide locus, and a novel Campylobacter putative licABCD virulence locus. The strains also vary in their metabolic profiles, as well as their resistance profiles to a range of antibiotics. It is evident that the newly identified hypothetical and conserved hypothetical proteins, as well as uncharacterized two-component regulatory systems and membrane proteins, may hold additional significant information on the major differences in virulence among the species, as well as the specificity of the strains for particular hosts.     

Lessons from “Lower” Organisms: What Worms, Flies, and Zebrafish Can Teach Us about Human Energy Metabolism

A pandemic of metabolic diseases (atherosclerosis, diabetes mellitus, and obesity), unleashed by multiple social and economic factors beyond the control of most individuals, threatens to diminish human life span for the first time in the modern era. Given the redundancy and inherent complexity of processes regulating the uptake, transport, catabolism, and synthesis of nutrients, magic bullets to target these diseases will be hard to find. Recent studies using the worm Caenorhabditis elegans, the fly Drosophila melanogaster, and the zebrafish Danio rerio indicate that these “lower” metazoans possess unique attributes that should help in identifying, investigating, and even validating new pharmaceutical targets for these diseases. We summarize findings in these organisms that shed light on highly conserved pathways of energy homeostasis.

     

A quantitative study of the division cycle of Caulobacter crescentus stalked cells

Progression of a cell through the division cycle is tightly controlled at different steps to ensure the integrity of genome replication and partitioning to daughter cells. From published experimental evidence, we propose a molecular mechanism for control of the cell division cycle in Caulobacter crescentus. The mechanism, which is based on the synthesis and degradation of three “master regulator” proteins (CtrA, GcrA, and DnaA), is converted into a quantitative model, in order to study the temporal dynamics of these and other cell cycle proteins. The model accounts for important details of the physiology, biochemistry, and genetics of cell cycle control in stalked C. crescentus cell. It reproduces protein time courses in wild-type cells, mimics correctly the phenotypes of many mutant strains, and predicts the phenotypes of currently uncharacterized mutants. Since many of the proteins involved in regulating the cell cycle of C. crescentus are conserved among many genera of α-proteobacteria, the proposed mechanism may be applicable to other species of importance in agriculture and medicine.     

Adaptive gene expression divergence inferred from population genomics

Detailed studies of individual genes have shown that gene expression divergence often results from adaptive evolution of regulatory sequence. Genome-wide analyses, however, have yet to unite patterns of gene expression with polymorphism and divergence to infer population genetic mechanisms underlying expression evolution. Here, we combined genomic expression data—analyzed in a phylogenetic context—with whole genome light-shotgun sequence data from six Drosophila simulans lines and reference sequences from D. melanogaster and D. yakuba. These data allowed us to use molecular population genetics to test for neutral versus adaptive gene expression divergence on a genomic scale. We identified recent and recurrent adaptive evolution along the D. simulans lineage by contrasting sequence polymorphism within D. simulans to divergence from D. melanogaster and D. yakuba. Genes that evolved higher levels of expression in D. simulans have experienced adaptive evolution of the associated 3′ flanking and amino acid sequence. Concomitantly, these genes are also decelerating in their rates of protein evolution, which is in agreement with the finding that highly expressed genes evolve slowly. Interestingly, adaptive evolution in 5′ cis-regulatory regions did not correspond strongly with expression evolution. Our results provide a genomic view of the intimate link between selection acting on a phenotype and associated genic evolution.     

Population genomics: whole-genome analysis of polymorphism and divergence in Drosophila simulans

The population genetic perspective is that the processes shaping genomic variation can be revealed only through simultaneous investigation of sequence polymorphism and divergence within and between closely related species. Here we present a population genetic analysis of Drosophila simulans based on whole-genome shotgun sequencing of multiple inbred lines and comparison of the resulting data to genome assemblies of the closely related species, D. melanogaster and D. yakuba. We discovered previously unknown, large-scale fluctuations of polymorphism and divergence along chromosome arms, and significantly less polymorphism and faster divergence on the X chromosome. We generated a comprehensive list of functional elements in the D. simulans genome influenced by adaptive evolution. Finally, we characterized genomic patterns of base composition for coding and noncoding sequence. These results suggest several new hypotheses regarding the genetic and biological mechanisms controlling polymorphism and divergence across the Drosophila genome, and provide a rich resource for the investigation of adaptive evolution and functional variation in D. simulans.     

The evolution of spinnable cotton fiber entailed prolonged development and a novel metabolism

A central question in evolutionary biology concerns the developmental processes by which new phenotypes arise. An exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium (“cotton fiber”). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinnable fibers of its closest relative, G. herbaceum, which facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ~22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were upregulated early in G. longicalyx fiber development. Several candidate genes upregulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers, but expression was not detected by quantitative real time-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinnable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program.     

Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.     

Odorant-binding proteins OBP57d and OBP57e affect taste perception and host-plant preference in Drosophila sechellia

Despite its morphological similarity to the other species in the Drosophila melanogaster species complex, D. sechellia has evolved distinct physiological and behavioral adaptations to its host plant Morinda citrifolia, commonly known as Tahitian Noni. The odor of the ripe fruit of M. citrifolia originates from hexanoic and octanoic acid. D. sechellia is attracted to these two fatty acids, whereas the other species in the complex are repelled. Here, using interspecies hybrids between D. melanogaster deficiency mutants and D. sechellia, we showed that the Odorant-binding protein 57e (Obp57e) gene is involved in the behavioral difference between the species. D. melanogaster knock-out flies for Obp57e and Obp57d showed altered behavioral responses to hexanoic acid and octanoic acid. Furthermore, the introduction of Obp57d and Obp57e from D. simulans and D. sechellia shifted the oviposition site preference of D. melanogaster Obp57d/e^KO flies to that of the original species, confirming the contribution of these genes to D. sechellia's specialization to M. citrifolia. Our finding of the genes involved in host-plant determination may lead to further understanding of mechanisms underlying taste perception, evolution of plant–herbivore interactions, and speciation.     

Dependency map of proteins in the small ribosomal subunit

The assembly of the ribosome has recently become an interesting target for antibiotics in several bacteria. In this work, we extended an analytical procedure to determine native state fluctuations and contact breaking to investigate the protein stability dependence in the 30S small ribosomal subunit of Thermus thermophilus. We determined the causal influence of the presence and absence of proteins in the 30S complex on the binding free energies of other proteins. The predicted dependencies are in overall agreement with the experimentally determined assembly map for another organism, Escherichia coli. We found that the causal influences result from two distinct mechanisms: one is pure internal energy change, the other originates from the entropy change. We discuss the implications on how to target the ribosomal assembly most effectively by suggesting six proteins as targets for mutations or other hindering of their binding. Our results show that by blocking one out of this set of proteins, the association of other proteins is eventually reduced, thus reducing the translation efficiency even more. We could additionally determine the binding dependency of THX—a peptide not present in the ribosome of E. coli—and suggest its assembly path.     

Type III effector diversification via both pathoadaptation and horizontal transfer in response to a coevolutionary arms race

The concept of the coevolutionary arms race holds a central position in our understanding of pathogen–host interactions. Here we identify the molecular mechanisms and follow the stepwise progression of an arms race in a natural system. We show how the evolution and function of the HopZ family of type III secreted effector proteins carried by the plant pathogen Pseudomonas syringae are influenced by a coevolutionary arms race between pathogen and host. We surveyed 96 isolates of P. syringae and identified three homologs (HopZ1, HopZ2, and HopZ3) distributed among ~45% of the strains. All alleles were sequenced and their expression was confirmed. Evolutionary analyses determined that the diverse HopZ1 homologs are ancestral to P. syringae, and have diverged via pathoadaptive mutational changes into three functional and two degenerate forms, while HopZ2 and HopZ3 have been brought into P. syringae via horizontal transfer from other ecologically similar bacteria. A PAML selection analysis revealed that the C terminus of HopZ1 is under strong positive selection. Despite the extensive genetic variation observed in this family, all three homologs have cysteine–protease activity, although their substrate specificity may vary. The introduction of the ancestral hopZ1 allele into strains harboring alternate alleles results in a resistance protein-mediated defense response in their respective hosts, which is not observed with the endogenous allele. These data indicate that the P. syringae HopZ family has undergone allelic diversification via both pathoadaptive mutational changes and horizontal transfer in response to selection imposed by the host defense system. This genetic diversity permits the pathogen to avoid host defenses while still maintaining a virulence-associated protease, thereby allowing it to thrive on its current host, while simultaneously impacting its host range.     

Complete Mitochondrial Genome and Phylogeny of Pleistocene MammothMammuthus primigenius

Phylogenetic relationships between the extinct woolly mammoth(Mammuthus primigenius), and the Asian(Elephas maximus) and African savanna(Loxodonta africana) elephants remain unresolved. Here, we report the sequence of the complete mitochondrial genome (16,842 base pairs) of a woolly mammoth extracted from permafrost-preserved remains from the Pleistocene epoch—the oldest mitochondrial genome sequence determined to date. We demonstrate that well-preserved mitochondrial genome fragments, as long as ~1,600–1700 base pairs, can be retrieved from pre-Holocene remains of an extinct species. Phylogenetic reconstruction of the Elephantinae clade suggests thatM. primigenius andE. maximus are sister species that diverged soon after their common ancestor split from theL. africana lineage. Low nucleotide diversity found between independently determined mitochondrial genomic sequences of woolly mammoths separated geographically and in time suggests that north-eastern Siberia was occupied by a relatively homogeneous population ofM. primigenius throughout the late Pleistocene.     

Helicobacter pylori evolution: lineage- specific adaptations in homologs of eukaryotic Sel1-like genes

Geographic partitioning is postulated to foster divergence of Helicobacter pylori populations as an adaptive response to local differences in predominant host physiology. H. pylori's ability to establish persistent infection despite host inflammatory responses likely involves active management of host defenses using bacterial proteins that may themselves be targets for adaptive evolution. Sequenced H. pylori genomes encode a family of eight or nine secreted proteins containing repeat motifs that are characteristic of the eukaryotic Sel1 regulatory protein, whereas the related Campylobacter and Wolinella genomes each contain only one or two such “Sel1-like repeat” (SLR) genes (“slr genes”). Signatures of positive selection (ratio of nonsynonymous to synonymous mutations, d_N/d_S = ω > 1) were evident in the evolutionary history of H. pylori slr gene family expansion. Sequence analysis of six of these slr genes (hp0160, hp0211, hp0235, hp0519, hp0628, and hp1117) from representative East Asian, European, and African H. pylori strains revealed that all but hp0628 had undergone positive selection, with different amino acids often selected in different regions. Most striking was a divergence of Japanese and Korean alleles of hp0519, with Japanese alleles having undergone particularly strong positive selection (ω_J > 25), whereas alleles of other genes from these populations were intermingled. Homology-based structural modeling localized most residues under positive selection to SLR protein surfaces. Rapid evolution of certain slr genes in specific H. pylori lineages suggests a model of adaptive change driven by selection for fine-tuning of host responses, and facilitated by geographic isolation. Characterization of such local adaptations should help elucidate how H. pylori manages persistent infection, and potentially lead to interventions tailored to diverse human populations.     

Asymmetrical reinforcement and Wolbachia infection in Drosophila

Reinforcement refers to the evolution of increased mating discrimination against heterospecific individuals in zones of geographic overlap and can be considered a final stage in the speciation process. One the factors that may affect reinforcement is the degree to which hybrid matings result in the permanent loss of genes from a species' gene pool. Matings between females of Drosophila subquinaria and males of D. recens result in high levels of offspring mortality, due to interspecific cytoplasmic incompatibility caused by Wolbachia infection of D. recens. Such hybrid inviability is not manifested in matings between D. recens females and D. subquinaria males. Here we ask whether the asymmetrical hybrid inviability is associated with a corresponding asymmetry in the level of reinforcement. The geographic ranges of D. recens and D. subquinaria were found to overlap across a broad belt of boreal forest in central Canada. Females of D. subquinaria from the zone of sympatry exhibit much stronger levels of discrimination against males of D. recens than do females from allopatric populations. In contrast, such reproductive character displacement is not evident in D. recens, consistent with the expected effects of unidirectional cytoplasmic incompatibility. Furthermore, there is substantial behavioral isolation within D. subquinaria, because females from populations sympatric with D. recens discriminate against allopatric conspecific males, whereas females from populations allopatric with D. recens show no discrimination against any conspecific males. Patterns of general genetic differentiation among populations are not consistent with patterns of behavioral discrimination, which suggests that the behavioral isolation within D. subquinaria results from selection against mating with Wolbachia-infected D. recens. Interspecific cytoplasmic incompatibility may contribute not only to post-mating isolation, an effect already widely recognized, but also to reinforcement, particularly in the uninfected species. The resulting reproductive character displacement not only increases behavioral isolation from the Wolbachia-infected species, but may also lead to behavioral isolation between populations of the uninfected species. Given the widespread occurrence of Wolbachia among insects, it thus appears that there are multiple ways by which these endosymbionts may directly and indirectly contribute to reproductive isolation and speciation.     

Genetic Mechanisms of Rare Matings of the Yeast SACCHAROMYCES CEREVISIAE Heterozygous for Mating Type

Yeast heterozygous for mating type lacks the ability to conjugate as judged by the mass-mating technique and accordingly is designated "non-mater". However, the non-mater shows rare mating ability with a frequency of less than 10^-6. In the present study, the RD auxotroph mating method was mainly employed with the intention of examining the rare mating ability of various non-maters, using lactate ethanol minimal medium as a selective medium for hybridization. Crosses of aα x a, aα x a, aaα x a, aαα x a, etc. resulted in the production of respective hybrids with a relatively high frequency of about 10^-6 to 10^-7, whereas crosses of aaα x a, aαα x α, aaαα x a, aaαα x α, etc. resulted in hybrids with an extremely low frequency of about less than 10^-8. Genetic analyses revealed that the rare matings were mostly caused by the presence of cells derived from the non-maters in which mating type had converted to a homozygous genotype. Mitotic recombination was shown to be a likely explanation for most of the conversion, judging from associated exchange of an outside marker, thr4. By successive employment of the RD auxotroph mating method, it was possible to produce a series of polyploid yeasts, triploids to octoploids. The DNA content and the cell volume were observed to increase parallel to the elevated ploidy states.     

Widespread discordance of gene trees with species tree in Drosophila: evidence for incomplete lineage sorting

The phylogenetic relationship of the now fully sequenced species Drosophila erecta and D. yakuba with respect to the D. melanogaster species complex has been a subject of controversy. All three possible groupings of the species have been reported in the past, though recent multi-gene studies suggest that D. erecta and D. yakuba are sister species. Using the whole genomes of each of these species as well as the four other fully sequenced species in the subgenus Sophophora, we set out to investigate the placement of D. erecta and D. yakuba in the D. melanogaster species group and to understand the cause of the past incongruence. Though we find that the phylogeny grouping D. erecta and D. yakuba together is the best supported, we also find widespread incongruence in nucleotide and amino acid substitutions, insertions and deletions, and gene trees. The time inferred to span the two key speciation events is short enough that under the coalescent model, the incongruence could be the result of incomplete lineage sorting. Consistent with the lineage-sorting hypothesis, substitutions supporting the same tree were spatially clustered. Support for the different trees was found to be linked to recombination such that adjacent genes support the same tree most often in regions of low recombination and substitutions supporting the same tree are most enriched roughly on the same scale as linkage disequilibrium, also consistent with lineage sorting. The incongruence was found to be statistically significant and robust to model and species choice. No systematic biases were found. We conclude that phylogenetic incongruence in the D. melanogaster species complex is the result, at least in part, of incomplete lineage sorting. Incomplete lineage sorting will likely cause phylogenetic incongruence in many comparative genomics datasets. Methods to infer the correct species tree, the history of every base in the genome, and comparative methods that control for and/or utilize this information will be valuable advancements for the field of comparative genomics.     

Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli

Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.