David A. Baltrus

Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates

Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species.

Extending assembly of short DNA sequences to handle error

UNLABELLED Inexpensive de novo genome sequencing, particularly in organisms with small genomes, is now possible using several new sequencing technologies. Some of these technologies such as that from Illuminas Solexa Sequencing, produce high genomic coverage by generating a very large number of small reads ( approximately 30 bp). While prior work shows that partial assembly can be performed by k-mer extension in error-free reads, this algorithm is unsuccessful with the sequencing error rates found in practice. We present VCAKE (Verified Consensus Assembly by K-mer Extension), a modification of simple k-mer extension that overcomes error by using high depth coverage. Though it is a simple modification of a previous approach, we show significant improvements in assembly results on simulated and experimental datasets that include error. AVAILABILITY http://152.2.15.114/~labweb/VCAKE

De novo assembly using low-coverage short read sequence data from the rice pathogen Pseudomonas syringae pv. oryzae

We developed a novel approach for de novo genome assembly using only sequence data from high-throughput short read sequencing technologies. By combining data generated from 454 Life Sciences (Roche) and Illumina (formerly known as Solexa sequencing) sequencing platforms, we reliably assembled genomes into large scaffolds at a fraction of the traditional cost and without use of a reference sequence. We applied this method to two isolates of the phytopathogenic bacteria Pseudomonas syringae. Sequencing and reassembly of the well-studied tomato and Arabidopsis pathogen, Pto(DC3000), facilitated development and testing of our method. Sequencing of a distantly related rice pathogen, Por(1_)(6), demonstrated our methods efficacy for de novo assembly of novel genomes. Our assembly of Por(1_6) yielded an N50 scaffold size of 531,821 bp with >75% of the predicted genome covered by scaffolds over 100,000 bp. One of the critical phenotypic differences between strains of P. syringae is the range of plant hosts they infect. This is largely determined by their complement of type III effector proteins. The genome of Por(1_6) is the first sequenced for a P. syringae isolate that is a pathogen of monocots, and, as might be predicted, its complement of type III effectors differs substantially from the previously sequenced isolates of this species. The genome of Por(1_6) helps to define an expansion of the P. syringae pan-genome, a corresponding contraction of the core genome, and a further diversification of the type III effector complement for this important plant pathogen species.

The Complete Genome Sequence of Helicobacter pylori Strain G27

Helicobacter pylori is a gram-negative pathogen that colonizes the stomachs of over half the worlds population and causes a spectrum of gastric diseases including gastritis, ulcers, and gastric carcinoma. The H. pylori species exhibits unusually high levels of genetic variation between strains. Here we announce the complete genome sequence of H. pylori strain G27, which has been used extensively in H. pylori research.

Exploring the costs of horizontal gene transfer

Horizontal gene transfer (HGT) is one of the most important evolutionary forces within microbial populations. Although evidence for beneficial fitness effects of HGT is overwhelming, recently acquired regions often function inefficiently within new genomic backgrounds so that each transfer event has the potential to disrupt existing regulatory and physiological networks. Identifying and exploring costs is essential for guiding general discussions about the interplay between selection and HGT, as well as generating hypotheses to explain how HGT affects evolutionary potential through, for example, changing adaptive trajectories. Focusing on costs of HGT as foundations for future studies will enhance exploration at the interface between acquired regions and recipient genomes, including the process of amelioration, and enable experimental evaluation of the role of HGT in structuring genetic diversity across populations.

Genome duplication, subfunction partitioning, and lineage divergence: Sox9 in stickleback and zebrafish

Teleosts are the most species‐rich group of vertebrates, and a genome duplication (tetraploidization) event in ray‐fin fish appears to have preceded this remarkable explosion of biodiversity. What is the relationship of the ray‐fin genome duplication to the teleost radiation? Genome duplication may have facilitated lineage divergence by partitioning different ancestral gene subfunctions among co‐orthologs of tetrapod genes in different teleost lineages. To test this hypothesis, we investigated gene expression patterns for Sox9 gene duplicates in stickleback and zebrafish, teleosts whose lineages diverged early in Euteleost evolution. Most expression domains appear to have been partitioned between Sox9a and Sox9b before the divergence of stickleback and zebrafish lineages, but some ancestral expression domains were distributed differentially in each lineage. We conclude that some gene subfunctions, as represented by lineage‐specific expression domains, may have assorted differently in separate lineages and that these may have contributed to lineage diversification during teleost evolution. Developmental Dynamics, 2003.

Natural Transformation Increases the Rate of Adaptation in the Human Pathogen Helicobacter pylori

Abstract Gene exchange between individuals can lead to profound evolutionary effects at both the genomic and population levels. These effects have sparked widespread interest in examining the specific adaptive benefits of recombination. Although this work has primarily focused on the benefits of sex in eukaryotes, it is assumed that similar benefits of genetic exchange apply across eukaryotes and prokaryotes. Here we report a direct test of this assumption using the naturally transformable human gastric pathogen Helicobacter pylori as a model organism. We show that genetic exchange accelerates adaptation to a novel laboratory environment within bacterial populations and that a general adaptive advantage exists for naturally transformable strains when transfer occurs among conspecific backgrounds. This finding demonstrates that there are generalized benefits to adaptation in both eukaryotes and prokaryotes even though the underlying processes are mechanistically different.

The Molecular Basis of Host Specialization in Bean Pathovars of Pseudomonas syringae

Biotrophic phytopathogens are typically limited to their adapted host range. In recent decades, investigations have teased apart the general molecular basis of intraspecific variation for innate immunity of plants, typically involving receptor proteins that enable perception of pathogen-associated molecular patterns or avirulence elicitors from the pathogen as triggers for defense induction. However, general consensus concerning evolutionary and molecular factors that alter host range across closely related phytopathogen isolates has been more elusive. Here, through genome comparisons and genetic manipulations, we investigate the underlying mechanisms that structure host range across closely related strains of Pseudomonas syringae isolated from different legume hosts. Although type III secretion-independent virulence factors are conserved across these three strains, we find that the presence of two genes encoding type III effectors (hopC1 and hopM1) and the absence of another (avrB2) potentially contribute to host range differences between pathovars glycinea and phaseolicola. These findings reinforce the idea that a complex genetic basis underlies host range evolution in plant pathogens. This complexity is present even in host-microbe interactions featuring relatively little divergence among both hosts and their adapted pathogens.

The Facultative Symbiont Rickettsia Protects an Invasive Whitefly against Entomopathogenic Pseudomonas syringae Strains.

ABSTRACT Facultative endosymbionts can benefit insect hosts in a variety of ways, including context-dependent roles, such as providing defense against pathogens. The role of some symbionts in defense may be overlooked, however, when pathogen infection is transient, sporadic, or asymptomatic. The facultative endosymbiont Rickettsia increases the fitness of the sweet potato whitefly (Bemisia tabaci) in some populations through mechanisms that are not yet understood. In this study, we investigated the role of Rickettsia in mediating the interaction between the sweet potato whitefly and Pseudomonas syringae, a common environmental bacterium, some strains of which are pathogenic to aphids. Our results show that P. syringae multiplies within whiteflies, leading to host death, and that whiteflies infected with Rickettsia show a decreased rate of death due to P. syringae. Experiments using plants coated with P. syringae confirmed that whiteflies can acquire the bacteria at a low rate while feeding, leading to increased mortality, particularly when the whiteflies are not infected with Rickettsia. These results suggest that P. syringae may affect whitefly populations in nature and that Rickettsia can ameliorate this effect. This study highlights the possible importance of interactions among opportunistic environmental pathogens and endosymbionts of insects.

Draft Genome Sequence of Plant Growth-Promoting Rhizobium Mesorhizobium amorphae, Isolated from Zinc-Lead Mine Tailings

Here, we describe the draft genome sequence of Mesorhizobium amorphae strain CCNWGS0123, isolated from nodules of Robinia pseudoacacia growing on zinc-lead mine tailings. A large number of metal(loid) resistance genes, as well as genes reported to promote plant growth, were identified, presenting a great future potential for aiding phytoremediation in metal(loid)-contaminated soil.