Microbial communities are typically large, diverse, and complex, and identifying and understanding the processes driving their structure has implications ranging from ecosystem stability to human health and well-being. Phylogenetic data gives us a new insight into these processes, providing a more informative perspective on functional and trait diversity than taxonomic richness alone. But the sheer scale of high resolution phylogenetic data also presents a new challenge to ecological theory. We bring a sampling theory perspective to microbial communities, considering a local community of co-occuring organisms as a sample from a larger regional pool, and apply our framework to make analytical predictions for local phylogenetic diversity arising from a given metacommunity and community assembly process. We characterize community assembly in terms of quantitative descriptions of clustered, random and overdispersed sampling, which have been associated with hypotheses of environmental filtering and competition. Using our approach, we analyze large microbial communities from the human microbiome, uncovering significant variation in diversity across habitats relative to the null hypothesis of random sampling.

Author Summary

Microbial diversity analyses have revolutionized our knowledge of the microscopic world, from terrestrial and marine to human and urban environments. This growing field rests on the evolutionary relatedness of organisms, and at its frontier is the inference of ecological processes from phylogenetic diversity. However, the rapidly reducing cost of sequencing means that computational analysis of phylogenetic data is becoming increasingly intractable. We develop a new analytical method to address this issue, providing a computationally-efficient way to compare local phylogenetic diversity to a sample from a regional pool of organisms, under a given ecological process. Our approach has both pragmatic and far-reaching applications. Until now investigators have lacked even an analytical method to compare the diversity of unequally-sized communities without throwing data away, while on a deeper level our theory provides a new framework for connecting phylogenetic data to a wide range of ecological processes. As an application of our approach, we use our methods to distinguish between random, clustered and overdispersed sampling for human microbiome habitats. Finally, we identify a new, phylogenetic analogue of the widely used taxonomic measure of diversity, the Species Abundance Distribution, and we find that it has consistent behavior across microbiome habitats.

Background

The sulfanilamide family comprises a clinically important group of antimicrobial compounds which also display bioactivity in plants. While there is evidence that sulfanilamides inhibit folate biosynthesis in both bacteria and plants, the complete network of plant responses to these compounds remains to be characterized. As such, we initiated two forward genetic screens in Arabidopsis in order to identify mutants that exhibit altered sensitivity to sulfanilamide compounds. These screens were based on the growth phenotype of seedlings germinated in the presence of the compound sulfamethoxazole (Smex).

Results

We identified a mutant with reduced sensitivity to Smex, and subsequent mapping indicated that a gene encoding 5-oxoprolinase was responsible for this phenotype. A mutation causing enhanced sensitivity to Smex was mapped to a gene lacking any functional annotation.

Conclusions

The genes identified through our forward genetic screens represent novel mediators of Arabidopsis responses to sulfanilamides and suggest that these responses extend beyond the perturbation of folate biosynthesis.

Despite extensive study, little is known about the origins of the mutualistic bacterial endosymbionts that inhabit approximately 10% of the world's insects. In this study, we characterized a novel opportunistic human pathogen, designated “strain HS,” and found that it is a close relative of the insect endosymbiont Sodalis glossinidius. Our results indicate that ancestral relatives of strain HS have served as progenitors for the independent descent of Sodalis-allied endosymbionts found in several insect hosts. Comparative analyses indicate that the gene inventories of the insect endosymbionts were independently derived from a common ancestral template through a combination of irreversible degenerative changes. Our results provide compelling support for the notion that mutualists evolve from pathogenic progenitors. They also elucidate the role of degenerative evolutionary processes in shaping the gene inventories of symbiotic bacteria at a very early stage in these mutualistic associations.

Author Summary

Many insects harbor symbiotic bacteria that perform diverse functions within their hosts. However, the origins of these associations have been difficult to define. In this study we isolate a novel bacterium from a human infection and show that this bacterium is a close relative of the Sodalis-allied clade of insect symbionts. Comparative genomic analyses reveal that this organism maintains many genes that have been inactivated and lost independently in derived insect symbionts as a result of rapid genome degeneration. Our work also shows that recently derived Sodalis-allied symbionts maintain a significant population of “cryptic” pseudogenes that are assumed to have no beneficial function in the symbiosis but have not yet accumulated mutations that disrupt their translation. Taken together, our results show that genome degeneration proceeds rapidly following the onset of symbiosis. They also highlight the potential for diverse insect taxa to acquire closely related insect symbionts as a consequence of vectoring bacterial pathogens to plants and animals.

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.

Adaptation is likely to be an important determinant of the success of many pathogens, for example when colonizing a new host species, when challenged by antibiotic treatment, or in governing the establishment and progress of long-term chronic infection. Yet, the genomic basis of adaptation is poorly understood in general, and for pathogens in particular. We investigated the genetics of adaptation to cystic fibrosis-like culture conditions in the presence and absence of fluoroquinolone antibiotics using the opportunistic pathogen Pseudomonas aeruginosa. Whole-genome sequencing of experimentally evolved isolates revealed parallel evolution at a handful of known antibiotic resistance genes. While the level of antibiotic resistance was largely determined by these known resistance genes, the costs of resistance were instead attributable to a number of mutations that were specific to individual experimental isolates. Notably, stereotypical quinolone resistance mutations in DNA gyrase often co-occurred with other mutations that, together, conferred high levels of resistance but no consistent cost of resistance. This result may explain why these mutations are so prevalent in clinical quinolone-resistant isolates. In addition, genes involved in cyclic-di-GMP signalling were repeatedly mutated in populations evolved in viscous culture media, suggesting a shared mechanism of adaptation to this CF–like growth environment. Experimental evolutionary approaches to understanding pathogen adaptation should provide an important complement to studies of the evolution of clinical isolates.

Author Summary

Pathogens face a hostile and often novel environment when infecting a new host, and adaptation to this environment can be critical to a pathogen's survival. The genetic basis of pathogen adaptation is in turn important for treatment, since the consistency with which therapies succeed may depend on the extent to which a pathogen adapts via the same routes in different patients. In this study, we investigate adaptation of the bacterium Pseudomonas aeruginosa to laboratory conditions that resemble the lungs of cystic fibrosis patients and to quinolone antibiotics. We find that a handful of genes and genetic pathways are repeatedly involved in adaptation to each condition. Nonetheless, other, less common mutations can play important roles in determining fitness, complicating strategies aimed at reducing the prevalence of antibiotic resistance.

Microbes have evolved many strategies to adapt to changes in environmental conditions and population structures, including cooperation and competition. One apparently competitive mechanism is contact dependent growth inhibition (CDI). Identified in Escherichia coli, CDI is mediated by Two–Partner Secretion (TPS) pathway proteins, CdiA and CdiB. Upon cell contact, the toxic C-terminus of the TpsA family member CdiA, called the CdiA-CT, inhibits the growth of CDI− bacteria. CDI+ bacteria are protected from autoinhibition by an immunity protein, CdiI. Bioinformatic analyses indicate that CDI systems are widespread amongst α, β, and γ proteobacteria and that the CdiA-CTs and CdiI proteins are highly variable. CdiI proteins protect against CDI in an allele-specific manner. Here we identify predicted CDI system-encoding loci in species of Burkholderia, Ralstonia and Cupriavidus, named bcpAIOB, that are distinguished from previously-described CDI systems by gene order and the presence of a small ORF, bcpO, located 5′ to the gene encoding the TpsB family member. A requirement for bcpO in function of BcpA (the TpsA family member) was demonstrated, indicating that bcpAIOB define a novel class of TPS system. Using fluorescence microscopy and flow cytometry, we show that these genes are expressed in a probabilistic manner during culture of Burkholderia thailandensis in liquid medium. The bcpAIOB genes and extracellular DNA were required for autoaggregation and adherence to an abiotic surface, suggesting that CDI is required for biofilm formation, an activity not previously attributed to CDI. By contrast to what has been observed in E. coli, the B. thailandensis bcpAIOB genes only mediated interbacterial competition on a solid surface. Competition occurred in a defined spatiotemporal manner and was abrogated by allele-specific immunity. Our data indicate that the bcpAIOB genes encode distinct classes of CDI and TPS systems that appear to function in sociomicrobiological community development.

Author Summary

Contact dependent growth inhibition (CDI) is a phenomenon discovered in Escherichia coli in which CDI+ bacteria inhibit the growth of CDI− bacteria upon cell-to-cell contact. CDI is mediated by large toxic “exoproteins” present on the bacterial cell surface. An ‘immunity’ protein protects CDI+ cells from killing themselves. While predicted CDI systems are widespread throughout bacterial genera, the role of these systems in nature has remained elusive. Here we identify a distinct class of CDI system in Burkholderia species. The genes encoding these systems are expressed in a stochastic manner such that only a few cells in the population produce the proteins at any given time when grown in broth. We also show that these systems are required for aggregation on an abiotic surface, suggesting an important role for CDI in biofilm formation. Finally, we show that CDI mediates competition under specific conditions in a precise spatiotemporal pattern when bacteria are grown on a solid surface. Our data suggest that in nature, CDI systems may be used by bacteria to establish complex sociomicrobial communities.

Background

Hazelnut (Corylus avellana) decline disease in Greece and Italy is caused by the convergent evolution of two distantly related lineages of Pseudomonas syringae pv. avellanae (Pav). We sequenced the genomes of three Pav isolates to determine if their convergent virulence phenotype had a common genetic basis due to either genetic exchange between lineages or parallel evolution.

Results

We found little evidence for horizontal transfer (recombination) of genes between Pav lineages, but two large genomic islands (GIs) have been recently acquired by one of the lineages. Evolutionary analyses of the genes encoding type III secreted effectors (T3SEs) that are translocated into host cells and are important for both suppressing and eliciting defense responses show that the two Pav lineages have dramatically different T3SE profiles, with only two shared putatively functional T3SEs. One Pav lineage has undergone unprecedented secretome remodeling, including the acquisition of eleven new T3SEs and the loss or pseudogenization of 15, including five of the six core T3SE families that are present in the other Pav lineage. Molecular dating indicates that divergence within both of the Pav lineages predates their observation in the field. This suggest that both Pav lineages have been cryptically infecting hazelnut trees or wild relatives for many years, and that the emergence of hazelnut decline in the 1970s may have been due to changes in agricultural practice.

Conclusions

These data show that divergent lineages of P. syringae can converge on identical disease etiology on the same host plant using different virulence mechanisms and that dramatic shifts in the arsenal of T3SEs can accompany disease emergence.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45–52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Author Summary

We sequenced the genomes of seven strains of the Pseudomonas fluorescens group that colonize plant surfaces and function as biological control agents, protecting plants from disease. In this study, we demonstrated the genomic diversity of the group by comparing these strains to each other and to three other strains that were sequenced previously. Only about half of the genes in each strain are present in all of the other strains, and each strain has hundreds of unique genes that are not present in the other genomes. We mapped the genes that contribute to biological control in each genome and found that most of the biological control genes are in the variable regions of the genome, which are not shared by all of the other strains. This finding is consistent with our knowledge of the distinctive biology of each strain. Finally, we looked for new genes that are likely to confer antimicrobial traits needed to suppress plant pathogens, but have not been identified previously. In each genome, we discovered many of these new genes, which provide avenues for future discovery of new traits with the potential to manage plant diseases in agriculture or natural ecosystems.

Miniature inverted terminal repeat elements (MITEs) are nonautonomous mobile elements that have a significant impact on bacterial evolution. Here we characterize E622, a 611-bp virulence-associated MITE from Pseudomonas syringae, which contains no coding region but has almost perfect 168-bp inverted repeats. Using an antibiotic coupling assay, we show that E622 is transposable and can mobilize an antibiotic resistance gene contained between its borders. Its predicted parent element, designated TnE622, has a typical transposon structure with a three-gene operon, consisting of resolvase, integrase, and exeA-like genes, which is bounded by the same terminal inverted repeats as E622. A broader genome level survey of the E622/TnE622 inverted repeats identified homologs in Pseudomonas, Salmonella, Shewanella, Erwinia, Pantoea, and the cyanobacteria Nostoc and Cyanothece, many of which appear to encompass known virulence genes, including genes encoding toxins, enzymes, and type III secreted effectors. Its association with niche-specific genetic determinants, along with its persistence and evolutionary diversification, indicates that this mobile element family has played a prominent role in the evolution of many agriculturally and clinically relevant pathogenic bacteria.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.

Author Summary

Human bodies are complex ecological systems in which various microbial organisms and viruses interact with each other and with the human host. The Human Microbiome Project (HMP) has resulted in >700 datasets of shotgun metagenomic sequences, from which we can learn about the compositions and functions of human-associated microbial communities. CRISPR/Cas systems are a widespread class of adaptive immune systems in bacteria and archaea, providing acquired immunity against foreign nucleic acids: CRISPR/Cas defense pathways involve integration of viral- or plasmid-derived DNA segments into CRISPR arrays (forming spacers between repeated structural sequences), and expression of short crRNAs from these single repeat-spacer units, to generate interference to future invading foreign genomes. Powered by an effective computational approach (the targeted assembly approach for CRISPR), our analysis of CRISPR arrays in the HMP datasets provides the very first global view of bacterial immunity systems in human-associated microbial communities. The great diversity of CRISPR spacers we observed among different body sites, in different individuals, and in single individuals over time, indicates the impact of subtle niche differences on the evolution of CRISPR defenses and indicates the key role of bacteriophage (and plasmids) in shaping human microbial communities.

Intercontinental spread of emerging plant diseases is one of the most serious threats to world agriculture. One emerging disease is bacterial canker of kiwi fruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (PSA). The disease first occurred in China and Japan in the 1980s and in Korea and Italy in the 1990s. A more severe form of the disease broke out in Italy in 2008 and in additional countries in 2010 and 2011 threatening the viability of the global kiwi fruit industry. To start investigating the source and routes of international transmission of PSA, genomes of strains from China (the country of origin of the genus Actinidia), Japan, Korea, Italy and Portugal have been sequenced. Strains from China, Italy, and Portugal have been found to belong to the same clonal lineage with only 6 single nucleotide polymorphisms (SNPs) in 3,453,192 bp and one genomic island distinguishing the Chinese strains from the European strains. Not more than two SNPs distinguish each of the Italian and Portuguese strains from each other. The Japanese and Korean strains belong to a separate genetic lineage as previously reported. Analysis of additional European isolates and of New Zealand isolates exploiting genome-derived markers showed that these strains belong to the same lineage as the Italian and Chinese strains. Interestingly, the analyzed New Zealand strains are identical to European strains at the tested SNP loci but test positive for the genomic island present in the sequenced Chinese strains and negative for the genomic island present in the European strains. Results are interpreted in regard to the possible direction of movement of the pathogen between countries and suggest a possible Chinese origin of the European and New Zealand outbreaks.

Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses.

Author Summary

Oomycete plant pathogens are among the most devastating agricultural pests and employ arsenals of effector proteins to manipulate their plant hosts. Some of these effectors, however, are recognized in the plant and trigger an immune response. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana and this interaction has been developed as a model system for oomycete pathogenesis. Here, we employ computational predictions to identify a novel effector ATR39-1, which is highly conserved among different Hpa isolates. A two amino acid-insertion in the alternative allele ATR39-2 correlated with evasion of recognition. We identified the corresponding resistance gene RPP39 and found that the signaling gene NDR1 is required to establish full resistance. Recognition of ATR39-1 by RPP39 in the plant did not inhibit growth of the oomycete, suggesting that complex mechanisms exist to prevent effector recognition. Knowledge of such novel resistance interactions provides the backbone of our understanding of plant resistance mechanisms and will aid in the further dissection of plant immunity.

Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8−1.2×10−5 per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994–95) strains and have lost the CRISPR–associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs.

Author Summary

Documenting the evolutionary changes occurring in pathogens when they switch hosts is important for understanding mechanisms of adaptation and rates of evolution. We took advantage of a novel host–pathogen system involving a bacterial pathogen (Mycoplasma gallisepticum, or MG) and a songbird host, the House Finch, to study genome-wide changes during a host-shift. Around 1994, biologists noticed that House Finches were contracting conjunctivitis and MG from poultry was discovered to be the cause. The resulting epizootic was one of the best documented for a wildlife species, partly as a result of thousands of citizen science observers. We sequenced the genomes of 12 House Finch MG strains sampled throughout the epizootic, from 1994–2007, as well as four additional putatively ancestral poultry MG strains. Using this serial sample, we estimate a remarkably high rate of substitution, consistent with past implications that mycoplasmas are among the fastest evolving bacteria. We also find that an array of likely phage-derived sequences known as CRISPRs has degraded and ceased to recruit new repeats in the House Finch MG strains, as compared to the poultry strains in which it is diverse and rapidly evolving. This suggests that phage dynamics might be important in the dynamics of MG infection.

Background. Recurrent bacterial infections play a key role in the pathogenesis of bronchiectasis, but conventional microbiologic methods may fail to identify pathogens in many cases. We characterized and compared the pulmonary bacterial communities of cystic fibrosis (CF) and non-CF bronchiectasis patients using a culture-independent molecular approach. Methods. Bacterial 16S rRNA gene libraries were constructed from lung tissue of 10 non-CF bronchiectasis and 21 CF patients, followed by DNA sequencing of isolates from each library. Community characteristics were analyzed and compared between the two groups. Results. A wide range of bacterial diversity was detected in both groups, with between 1 and 21 bacterial taxa found in each patient. Pseudomonas was the most common genus in both groups, comprising 49% of sequences detected and dominating numerically in 13 patients. Although Pseudomonas appeared to be dominant more often in CF patients than in non-CF patients, analysis of entire bacterial communities did not identify significant differences between these two groups. Conclusions. Our data indicate significant diversity in the pulmonary bacterial community of both CF and non-CF bronchiectasis patients and suggest that this community is similar in surgically resected lungs of CF and non-CF bronchiectasis patients.

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

Author Summary

Many bacterial pathogens disrupt key components of host physiology by injecting virulence proteins (or “effectors”) via a needle-like structure, called the type III secretion system, directly into eukaryotic cells. The YopJ / HopZ superfamily of type III secreted effector proteins is found in pathogens of both animals and plants providing an excellent opportunity to address how a family of type III secreted effectors can promote pathogenesis in hosts from two kingdoms. YopJ from the animal pathogen Yersinia pestis is an acetyltransferase that targets signaling components of innate immunity and prevents their activation. Here we show that HopZ1a, from the phytopathogen Pseudomonas syringae is an acetyltransferase that binds plant tubulin. Like YopJ, the eukaryotic cofactor phytic acid activates the acetyltransferase activity of HopZ1a. In addition, we demonstrate that activated HopZ1a can acetylate tubulin, a major constituent of the eukaryotic cytoskeleton. In plants, activated HopZ1a causes a dramatic destruction of microtubule networks, inhibits protein secretion, and ultimately suppresses cell wall-mediated defense. Our study emphasizes the functional diversification of this important type III effector family in plant and animal hosts using a conserved acetyltransferase activity.

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network.

Author Summary

Differences in gene regulation underlie many important biological processes and are thought to be important for the adaption of organisms to novel environments. Here we focus on the regulation of a group of well-studied genes, the lac operon, that control the utilization of lactose sugar, and we examine how their regulation changes during the adaptation of populations of Escherichia coli bacteria to environments that differ only in the presence of lactose. We find that lac operon regulation is altered in almost all populations that evolve in the presence of lactose and identify two classes of mutations that explain a large part of this change and that confer significant fitness benefits. Interestingly, our study indicates that other mutations, lying outside of the commonly recognized control region, cause new regulation of the lac operon. Together these findings reinforce the importance of changes in gene regulation during evolution and suggest that the biological basis of these changes can be complex and involve novel interactions between genes.

Although bacteria with multipartite genomes are prevalent, our knowledge of the mechanisms maintaining their genome is very limited, and much remains to be learned about the structural and functional interrelationships of multiple chromosomes. Owing to its bi-chromosomal genome architecture and its importance in public health, Vibrio cholerae, the causative agent of cholera, has become a preferred model to study bacteria with multipartite genomes. However, most in vivo studies in V. cholerae have been hampered by its genome architecture, as it is difficult to give phenotypes to a specific chromosome. This difficulty was surmounted using a unique and powerful strategy based on massive rearrangement of prokaryotic genomes. We developed a site-specific recombination-based engineering tool, which allows targeted, oriented, and reciprocal DNA exchanges. Using this genetic tool, we obtained a panel of V. cholerae mutants with various genome configurations: one with a single chromosome, one with two chromosomes of equal size, and one with both chromosomes controlled by identical origins. We used these synthetic strains to address several biological questions—the specific case of the essentiality of Dam methylation in V. cholerae and the general question concerning bacteria carrying circular chromosomes—by looking at the effect of chromosome size on topological issues. In this article, we show that Dam, RctB, and ParA2/ParB2 are strictly essential for chrII origin maintenance, and we formally demonstrate that the formation of chromosome dimers increases exponentially with chromosome size.

Author Summary

Vibrio cholerae, the causative agent of cholera in humans, has two circular chromosomes of uneven size, each with distinct maintenance requirements. This is in contrast to classical, Escherichia coli–centric bacterial models of a single chromosome. In this study, we took advantage of V. cholerae's atypical genome structure to address important biological issues related to the maintenance of multipartite genomes. We further used V. cholerae to determine how genome architecture and genetic organization affects the odds of topological difficulties arising during replication. Our approach consisted of performing massive genome rearrangements to create various synthetic mutants of V. cholerae with nearly identical genetic backgrounds. We created mutants of V. cholerae with a single chromosome, with two chromosomes of equal size, or with identical origins of replication. To do so, we developed a genetic engineering tool based on the multiplexing of two site-specific recombination systems to allow efficient and directional manipulations of any DNA segment. In this study, we show that Dam, RctB, and ParA2/ParB2 are only essential for chrII origin maintenance, and we demonstrate that the odds of forming chromosome dimers exponentially increases with chromosome size.

Background

Identification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system.

Results

Here we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data make it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence.

Conclusions

We demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.

Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multi-host parasites and thus for the emergence of new pathogens. Here we characterize the DMFE for a collection of twenty single-nucleotide substitution mutants of Tobacco etch potyvirus (TEV) across a set of eight host environments. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas the other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail. Within the Solanaceae hosts, the distribution contained an excess of deleterious mutations, whereas for the non-Solanaceae the fraction of beneficial mutations was significantly larger. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host.

Author Summary

Mutations are the raw material on which natural selection operates to optimize the fitness of populations. The occurrence of selection and its strength depend on the effect that mutations may have on the survival and reproduction of individuals: mutations can be lethal, deleterious, neutral, or beneficial. Thus, determining how many mutations belong to each of these categories is of importance for predicting the evolutionary fate of a population. For emerging infectious diseases, this distribution determines the likelihood that a pathogen crosses the species barrier and successfully infects a new host. We characterized such distributions across a panel of alternative hosts for a plant virus and found that fitness effects of individual mutations varied across hosts in an unpredictable way and that many mutations considered deleterious in the natural host may turn out to be beneficial in other hosts.

Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an “enhanced-quality draft” genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2–5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.

To identify enzymes that could be developed to reduce the recalcitrance of softwood resources, the transcriptomes of the softwood-degrading white-rot fungus Phanerochaete carnosa were evaluated after growth on lodgepole pine, white spruce, balsam fir, and sugar maple and compared to the transcriptome of P. carnosa after growth on liquid nutrient medium. One hundred fifty-two million paired-end reads were obtained, and 63% of these reads were mapped to 10,257 gene models from P. carnosa. Five-hundred thirty-three of these genes had transcripts that were at least four times more abundant during growth on at least one wood medium than on nutrient medium. The 30 transcripts that were on average over 100 times more abundant during growth on wood than on nutrient medium included 6 manganese peroxidases, 5 cellulases, 2 hemicellulases, a lignin peroxidase, glyoxal oxidase, and a P450 monooxygenase. Notably, among the genes encoding putative cellulases, one encoding a glycosyl hydrolase family 61 protein had the highest relative transcript abundance during growth on wood. Overall, transcripts predicted to encode lignin-degrading activities were more abundant than those predicted to encode carbohydrate-active enzymes. Transcripts predicted to encode three MnPs represented the most highly abundant transcripts in wood-grown cultivations compared to nutrient medium cultivations. Gene set enrichment analyses did not distinguish transcriptomes resulting from softwood and hardwood cultivations, suggesting that similar sets of enzyme activities are elicited by P. carnosa grown on different wood substrates, albeit to different expression levels.

In F. graminearum, the transcriptional regulator Tri6 is encoded within the trichothecene gene cluster and regulates genes involved in the biosynthesis of the secondary metabolite deoxynivalenol (DON). The Tri6 protein with its Cys2His2 zinc-finger may also conform to the class of global transcription regulators. This class of global transcriptional regulators mediate various environmental cues and generally responds to the demands of cellular metabolism. To address this issue directly, we sought to find gene targets of Tri6 in F. graminearum grown in optimal nutrient conditions. Chromatin immunoprecipitation followed by Illumina sequencing (ChIP-Seq) revealed that in addition to identifying six genes within the trichothecene gene cluster, Tri1, Tri3, Tri6, Tri7, Tri12 and Tri14, the ChIP-Seq also identified 192 additional targets potentially regulated by Tri6. Functional classification revealed that, among the annotated genes, ∼40% are associated with cellular metabolism and transport and the rest of the target genes fall into the category of signal transduction and gene expression regulation. ChIP-Seq data also revealed Tri6 has the highest affinity toward its own promoter, suggesting that this gene could be subject to self-regulation. Electro mobility shift assays (EMSA) performed on the promoter of Tri6 with purified Tri6 protein identified a minimum binding motif of GTGA repeats as a consensus sequence. Finally, expression profiling of F. graminearum grown under nitrogen-limiting conditions revealed that 49 out of 198 target genes are differentially regulated by Tri6. The identification of potential new targets together with deciphering novel binding sites for Tri6, casts new light into the role of this transcriptional regulator in the overall growth and development of F. graminearum.

Author Summary

Our knowledge of mechanisms involved in the activation and biosynthesis of DON comes largely from in vitro culture studies. Cumulated knowledge suggests that the physiological status of the fungus and the availability of nutrients are the main determining factors for DON production. Integration of various environmental cues to coordinate expression of secondary metabolic genes is thought to be mediated by a combination of global and pathway-specific transcription factors. While the global transcriptional factors respond to broad range of environmental cues such as the availability of carbon and nitrogen, the pathway-specific transcriptional factors regulate genes within a gene cluster. In F. graminearum, the transcriptional regulator Tri6 is encoded within the trichothecene gene cluster and regulates genes involved in the synthesis and transport of DON. In this report, we utilized ChIP-Seq to demonstrate that Tri6 can potentially bind to promoters and regulate genes not involved in the synthesis of DON and furthermore, many of these non-trichothecene genes are involved in various aspects of cellular metabolism, including transport and energy. Expression profiling revealed that many of the target genes are differentially regulated by Tri6, thus validating our hypothesis that Tri6 is a global regulator involved in cellular metabolism.

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

Author Summary

Our knowledge of the recent evolution of bacterial human pathogens has increased dramatically over the last five years. By comparison, relatively little is known about recent evolution of bacterial plant pathogens. Here, we analyze a large collection of isolates of the economically important plant pathogen Pseudomonas syringae pv. tomato with markers derived from the comparison of five genomes of this pathogen. We find that this pathogen likely evolved on a relatively recent time scale and continues to adapt to tomato by minimizing its recognition by the tomato immune system. We find that an allele of the flagellin subunit fliC that appeared in the pathogen population for the first time in the 1980s, and which is the most common allele of this gene in North America and Europe today, triggers a weaker tomato immune response than the fliC allele found in the 1960s and 1970s. These results not only impact our understanding of pathogen – plant interactions and pathogen evolution but also have important ramifications for disease prevention. Given the speed with which new pathogen strains spread and replace existing strains, limiting the movement of specific strains between geographic regions is critically important, even for pathogens known to have worldwide distribution.

Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ∼40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ∼1000 donor polymorphisms in 3–6 contiguous runs (8.1±4.5 kb in length) that collectively comprised ∼1–3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.

Author Summary

The ability of bacteria to acquire genetic information from their relatives—called natural competence—poses a major health risk, since recombination between pathogenic bacterial lineages can help bacteria develop resistance to antibiotics and adapt to host defenses. In this study we transformed competent cells of the human pathogen Haemophilus influenzae with genomic DNA from a divergent clinical isolate and used deep sequencing to identify the recombination events in four transformed chromosomes. The results show that transformation of single competent cells is more extensive than expected, and suggests that transformation can be used as a tool to map traits that vary between clinical isolates.