Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections.

Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to in vivo antibiotic exposure, and we found that treatment consistently reduced intrastrain genomic diversity. In addition, analysis of gene-level allele frequency changes suggested that some genes without conventional resistance functions may be important for bacterial fitness during in vivo antibiotic exposure. GenCap-Seq's ability to scalably enrich targeted bacterial DNA from complex samples will enable studies on the effects of intrastrain and intraspecies diversity in human infectious disease. IMPORTANCE Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods. We developed a novel approach, GenCap-Seq, to enrich microbial DNA from complex human samples like sputum and feces for genome-wide measurements of bacterial allelic diversity. The approach is inexpensive, scalable to encompass entire targeted genomes, and works in the presence of abundant untargeted nucleic acids and inhibiting substances. We used GenCap-Seq to investigate in vivo responses of diversified bacterial strains to antibiotic treatment. This method will enable new ideas about the effects of intrastrain diversity on human infections to be tested.

Putative transcription antiterminator RfaH contributes to Erwinia amylovora virulence.

The gram-negative bacterium Erwinia amylovora causes fire blight disease of apple and pear trees. The exopolysaccharide amylovoran and lipopolysaccharides are essential E. amylovora virulence factors. Production of amylovoran and lipopolysaccharide is specified in part by genes that are members of long operons. Here, we show that full virulence of E. amylovora in apple fruitlets and tree shoots depends on the predicted transcription antiterminator RfaH. RfaH reduces pausing in the production of long transcripts having an operon polarity suppressor regulatory element within their promoter region. In E. amylovora, only the amylovoran operon and a lipopolysaccharide operon have such regulatory elements within their promoter regions and in the correct orientation. These operons showed dramatically increased polarity in the ΔrfaH mutant compared to the wild type as determined by RNA sequencing. Amylovoran and lipopolysaccharide production in vitro was reduced in rfaH mutants compared to the wild type, which probably contributes to the rfaH mutant virulence phenotype. Furthermore, type VI secretion cluster 1, which contributes to E. amylovora virulence, showed reduced expression in ΔrfaH compared to the wild type, although without an increase in polarity. The data suggest that E. amylovora RfaH directly, specifically, and exclusively suppresses operon polarity in the amylovoran operon and a lipopolysaccharide operon.

Virulence Genetics of an Erwinia amylovora Putative Polysaccharide Transporter Family Member.

The Gram-negative enterobacterium Erwinia amylovora causes fire blight disease in apple and pear trees. Lipopolysaccharides and the exopolysaccharide amylovoran are essential E. amylovora virulence factors. We found that mutations in rfbX disrupted amylovoran production and virulence in apple fruits and tree shoots and that the deletion of yibD suppressed the rfbX mutant phenotype. The level of expression of yibD was about 10-fold higher in the ΔrfbX mutant than the wild type. A forward genetic suppressor screen in the ΔrfbX mutant uncovered multiple mutations in yibD and supported the conclusion that the virulence defect of rfbX mutants is due to reduced amylovoran production. The yibD and rfbX genes are expressed as a two-gene operon, yibD rfbX The rfbX gene encodes a previously uncharacterized putative polysaccharide subunit transporter, while yibD encodes a predicted glycosyltransferase. Mutation of rfbX did not have a detectable effect on lipopolysaccharide patterns; however, the overexpression of yibD in both the wild-type and ΔyibD ΔrfbX genetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, the overexpression of yibD in the ΔyibD ΔrfbX mutant inhibited bacterial growth in amylovoran-inducing medium. This growth inhibition phenotype was used in a forward genetic suppressor screen and reverse-genetics tests to identify several genes involved in lipopolysaccharide production, which, when mutated, restored the ability of the ΔyibD ΔrfbX mutant overexpressing yibD to grow in amylovoran-inducing medium. Remarkably, all the lipopolysaccharide gene mutants tested were defective in lipopolysaccharide and amylovoran production. These results reveal a genetic connection between amylovoran and lipopolysaccharide production in E. amylovoraIMPORTANCE This study discovered previously unknown genetic connections between exopolysaccharide and lipopolysaccharide production in the fire blight pathogen Erwinia amylovora This represents a step forward in our understanding of the biology underlying the production of these two macromolecules. Fire blight is an economically important disease that impacts the production of apples and pears worldwide. Few fire blight control measures are available, and growers rely heavily on antibiotic applications at bloom time. Both exopolysaccharide and lipopolysaccharide are E. amylovora virulence factors. Our results indicate that the overexpression of the yibD gene in E. amylovora disrupts both lipopolysaccharide production and exopolysaccharide production. This effect could potentially be used as the basis for the development of an antivirulence treatment for the prevention of fire blight disease.

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples.

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

The Apple Fruitlet Model System for Fire Blight Disease.

Fire blight is a perennial disease affecting apple and pear production worldwide. Development of resistant cultivars and disease control measures are crucial aspects of managing fire blight. Furthermore, the study of the causal agent, the Gram-negative bacterium Erwinia amylovora, has led to important insights into molecular plant-microbe interactions. However, fire blight does not have a suitable model host, since its host range is limited to plants with complex genetics and relatively limited resources for genetic analysis. Here, we present a rationale for using apple fruitlets as a potential fire blight model host system, and describe a protocol for quantitative fruit inoculation, bacterial growth measurement, and symptom assessment. The use of apple fruitlets is applicable to the molecular genetic analysis of E. amylovora, including high-throughput genetic screens for E. amylovora virulence-defective mutants, and is potentially useful to study host resistance and responses to E. amylovora as well.

Extragenic Suppression of Elongation Factor P Gene Mutant Phenotypes in Erwinia amylovora.

Elongation factor P (EF-P) facilitates the translation of certain peptide motifs, including those with multiple proline residues. EF-P must be posttranslationally modified for full functionality; in enterobacteria, this is accomplished by two enzymes, namely, EpmA and EpmB, which catalyze the ß-lysylation of EF-P at a conserved lysine position. Mutations to efp or its modifying enzymes produce pleiotropic phenotypes, including decreases in virulence, swimming motility, and extracellular polysaccharide production, as well as proteomic perturbations. Here, we generated targeted deletion mutants of the efp, epmA, and epmB genes in the Gram-negative bacterium Erwinia amylovora, which causes fire blight, an economically important disease of apples and pears. As expected, the Δefp, ΔepmA, and ΔepmB mutants were all defective in virulence on apples, and all three mutants were complemented in trans with plasmids bearing wild-type copies of the corresponding genes. By analyzing spontaneous suppressor mutants, we found that mutations in the hrpA3 gene partially or completely suppressed the colony size, extracellular polysaccharide production, and virulence phenotypes in apple fruits and apple tree shoots but not the swimming motility phenotypes of the Δefp, ΔepmA, and ΔepmB mutants. The deletion of hrpA3 alone did not produce any alterations in any characteristics measured, indicating that the HrpA3 protein is not essential for any of the processes examined. The hrpA3 gene encodes a putative DEAH-box ATP-dependent RNA helicase. These results suggest that the loss of the HrpA3 protein at least partially compensates for the lack of the EF-P protein or ß-lysylated EF-P.IMPORTANCE Fire blight disease has relatively few management options, with antibiotic application at bloom time being chief among them. As modification to elongation factor P (EF-P) is vital to virulence in several species, both EF-P and its modifying enzymes make attractive targets for novel antibiotics. However, it will be useful to understand how bacteria might overcome the hindrance of EF-P function so that we may be better prepared to anticipate bacterial adaptation to such antibiotics. The present study indicates that the mutation of hrpA3 could provide a partial offset for the loss of EF-P activity. In addition, little is known about EF-P functional interactions or the HrpA3 predicted RNA helicase, and our genetic approach allowed us to discern a novel gene associated with EF-P function.

An Erwinia amylovora yjeK mutant exhibits reduced virulence, increased chemical sensitivity and numerous environmentally dependent proteomic alterations.

The Gram-negative bacterium Erwinia amylovora causes fire blight, an economically important disease of apples and pears. Elongation factor P (EF-P) is a highly conserved protein that stimulates the formation of the first peptide bond of certain proteins and facilitates the translation of certain proteins, including those with polyproline motifs. YjeK and YjeA are two enzymes involved in the essential post-translational ß-lysylation of EF-P at a conserved lysine residue, K34. EF-P, YjeA and YjeK have been shown to be essential for the full virulence of Escherichia coli, Salmonella species and Agrobacterium tumefaciens, with efp, yjeA and yjeK mutants having highly similar phenotypes. Here, we identified an E. amylovora yjeK::Tn5 transposon mutant with decreased virulence in apple fruit and trees. The yjeK::Tn5 mutant also showed pleiotropic phenotypes, including reduced growth in rich medium, lower extracellular polysaccharide production, reduced swimming motility and increased chemical sensitivity compared with the wild-type, whilst maintaining wild-type level growth in minimal medium. All yjeK::Tn5 mutant phenotypes were complemented in trans with a plasmid bearing a wild-type copy of yjeK. Comprehensive, quantitative proteomics analyses revealed numerous, environmentally dependent changes in the prevalence of a wide range of proteins, in higher abundance and lower abundance, in yjeK::Tn5 compared with the wild-type, and many of these alterations could be linked to yjeK::Tn5 mutant phenotypes. The environmental dependence of the yjeK::Tn5 mutant proteomic alterations suggests that YjeK could be required for aspects of the environmentally dependent regulation of protein translation. YjeK activity may be critical to overcoming stress, including the challenging host environment faced by invading pathogenic bacteria.