David S. Guttman

Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months

Background: The gut microbiota is essential to human health throughout life, yet the acquisition and development of this microbial community during infancy remains poorly understood. Meanwhile, there is increasing concern over rising rates of cesarean delivery and insufficient exclusive breastfeeding of infants in developed countries. In this article, we characterize the gut microbiota of healthy Canadian infants and describe the influence of cesarean delivery and formula feeding. Methods: We included a subset of 24 term infants from the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort. Mode of delivery was obtained from medical records, and mothers were asked to report on infant diet and medication use. Fecal samples were collected at 4 months of age, and we characterized the microbiota composition using high-throughput DNA sequencing. Results: We observed high variability in the profiles of fecal microbiota among the infants. The profiles were generally dominated by Actinobacteria (mainly the genus Bifidobacterium) and Firmicutes (with diverse representation from numerous genera). Compared with breastfed infants, formula-fed infants had increased richness of species, with overrepresentation of Clostridium difficile. Escherichia–Shigella and Bacteroides species were underrepresented in infants born by cesarean delivery. Infants born by elective cesarean delivery had particularly low bacterial richness and diversity. Interpretation: These findings advance our understanding of the gut microbiota in healthy infants. They also provide new evidence for the effects of delivery mode and infant diet as determinants of this essential microbial community in early life.

Evolution of the Core Genome of Pseudomonas syringae, a Highly Clonal, Endemic Plant Pathogen

ABSTRACT Pseudomonas syringae is a common foliar bacterium responsible for many important plant diseases. We studied the population structure and dynamics of the core genome of P. syringae via multilocus sequencing typing (MLST) of 60 strains, representing 21 pathovars and 2 nonpathogens, isolated from a variety of plant hosts. Seven housekeeping genes, dispersed around the P. syringae genome, were sequenced to obtain 400 to 500 nucleotides per gene. Forty unique sequence types were identified, with most strains falling into one of four major clades. Phylogenetic and maximum-likelihood analyses revealed a remarkable degree of congruence among the seven genes, indicating a common evolutionary history for the seven loci. MLST and population genetic analyses also found a very low level of recombination. Overall, mutation was found to be approximately four times more likely than recombination to change any single nucleotide. A skyline plot was used to study the demographic history of P. syringae. The species was found to have maintained a constant population size over time. Strains were also found to remain genetically homogeneous over many years, and when isolated from sites as widespread as the United States and Japan. An analysis of molecular variance found that host association explains only a small proportion of the total genetic variation in the sample. These analyses reveal that with respect to the core genome, P. syringae is a highly clonal and stable species that is endemic within plant populations, yet the genetic variation seen in these genes only weakly predicts host association.

The Gain-of-Function Arabidopsis acd6 Mutant Reveals Novel Regulation and Function of the Salicylic Acid Signaling Pathway in Controlling Cell Death, Defenses, and Cell Growth

We isolated a dominant gain-of-function Arabidopsis mutant, accelerated cell death 6 (acd6), with elevated defenses, patches of dead and enlarged cells, reduced stature, and increased resistance to Pseudomonas syringae. The acd6-conferred phenotypes are suppressed by removing a key signaling molecule, salicylic acid (SA), by using the nahG transgene, which encodes SA hydroxylase. This suppression includes phenotypes that are not induced by application of SA to wild-type plants, indicating that SA acts with a second signal to cause many acd6-conferred phenotypes. acd6–nahG plants show hyperactivation of all acd6-conferred phenotypes after treatment with a synthetic inducer of the SA pathway, benzo(1,2,3)thiadiazole-7-carbothioic acid (BTH), suggesting that SA acts with and also modulates the levels and/or activity of the second defense signal. acd6 acts partially through a NONEXPRESSOR OF PR 1 (NPR1) gene–independent pathway that activates defenses and confers resistance to P. syringae. Surprisingly, BTH-treated acd6–nahG plants develop many tumor-like abnormal growths, indicating a possible role for SA in modulating cell growth.

ANNUAL REVIEW OF PHYTOPATHOLOGY, VOL 49

The phytopathogenic bacterium Pseudomonas syringae causes serious diseases in a wide range of important crop plants, with recent severe outbreaks on the New Zealand kiwifruit crop and among British horse chestnut trees. Next-generation genome sequencing of over 25 new strains has greatly broadened our understanding of how this species adapts to a diverse range of plant hosts. Not unexpectedly, the genomes were found to be highly dynamic, and extensive polymorphism was found in the distribution of type III secreted effectors (T3SEs) and other virulence-associated genes, even among strains within the same pathovar. An underexplored area brought to light by these data is the specific metabolic adaptations required for growth on woody hosts. These studies provide a tremendous wealth of candidates for more refined functional characterization, which is greatly enhancing our ability to disentangle the web of host-pathogen interactions that determine disease outcomes.

Next-generation mapping of Arabidopsis genes

Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F₂ population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F₂ population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F₂ lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.

Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae

ABSTRACT Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.

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.

Gut Microbial Metabolism Drives Transformation of Msh2-Deficient Colon Epithelial Cells

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating β-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:

Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system

Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells. To extend previous studies of Avr effectors that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy. With this system, we confirmed earlier findings showing that P. syringae pv. maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2. Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2+ plants. Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P. syringae grown in rps2- plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2. Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpml effector and the C-terminal effector region of AvrRpt2 was delivered to plant cells. Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design. This work establishes a readily manipulatable system to study type III effectors in a biologically realistic context.

An X-linked gene with a degenerate Y-linked homologue in a dioecious plant.

Most flowering plants are hermaphroditic, having flowers with both male and female parts. Less than 4% of plant species are dioecious (with individuals of separate sexes), and many of these species have chromosome-mediated sex determination. The taxonomic distribution of separate sexes and chromosomal sex-determination systems in the flowering plants indicates that plant sex chromosomes have evolved recently through replicated, independent events, contrasting with the ancient origins of mammalian and insect sex chromosomes. Plant sex chromosomes, therefore, offer opportunities to study the most interesting early stages of the evolution of sex chromosomes. Here we show that a gene encoding a male-specific protein is linked to the X chromosome in the dioecious plant Silene latifolia, and that it has a degenerate homologue in the non-pairing region of the Y chromosome. The Y-linked locus has degenerated as a result of nucleotide deletion and the accumulation of repetitive sequences. We have identified both the first X-linked gene and the first pair of homologous sex-linked loci to be found in plants. The homology between the active X-linked locus and the degenerate Y-linked locus supports a common ancestry for these two loci.