Single-base resolution quantitative genome methylation analysis in the model bacterium Helicobacter pylori by enzymatic methyl sequencing (EM-Seq) reveals influence of strain, growth phase, and methyl homeostasis.

BACKGROUND: Bacterial epigenetics is a rapidly expanding research field. DNA methylation by diverse bacterial methyltransferases (MTases) contributes to genomic integrity and replication, and many recent studies extended MTase function also to global transcript regulation and phenotypic variation. Helicobacter pylori is currently one of those bacterial species which possess the highest number and the most variably expressed set of DNA MTases. Next-generation sequencing technologies can directly detect DNA base methylation. However, they still have limitations in their quantitative and qualitative performance, in particular for cytosine methylation. RESULTS: As a complementing approach, we used enzymatic methyl sequencing (EM-Seq), a technology recently established that has not yet been fully evaluated for bacteria. Thereby, we assessed quantitatively, at single-base resolution, whole genome cytosine methylation for all methylated cytosine motifs in two different H. pylori strains and isogenic MTase mutants. EM-Seq reliably detected both m5C and m4C methylation. We demonstrated that three different active cytosine MTases in H. pylori provide considerably different levels of average genome-wide single-base methylation, in contrast to isogenic mutants which completely lost specific motif methylation. We found that strain identity and changed environmental conditions, such as growth phase and interference with methyl donor homeostasis, significantly influenced quantitative global and local genome-wide methylation in H. pylori at specific motifs. We also identified significantly hyper- or hypo-methylated cytosines, partially linked to overlapping MTase target motifs. Notably, we revealed differentially methylated cytosines in genome-wide coding regions under conditions of methionine depletion, which can be linked to transcript regulation. CONCLUSIONS: This study offers new knowledge on H. pylori global and local genome-wide methylation and establishes EM-Seq for quantitative single-site resolution analyses of bacterial cytosine methylation.

Methylome evolution suggests lineage-dependent selection in the gastric pathogen Helicobacter pylori.

The bacterial pathogen Helicobacter pylori, the leading cause of gastric cancer, is genetically highly diverse and harbours a large and variable portfolio of restriction-modification systems. Our understanding of the evolution and function of DNA methylation in bacteria is limited. Here, we performed a comprehensive analysis of the methylome diversity in H. pylori, using a dataset of 541 genomes that included all known phylogeographic populations. The frequency of 96 methyltransferases and the abundance of their cognate recognition sequences were strongly influenced by phylogeographic structure and were inter-correlated, positively or negatively, for 20% of type II methyltransferases. Low density motifs were more likely to be affected by natural selection, as reflected by higher genomic instability and compositional bias. Importantly, direct correlation implied that methylation patterns can be actively enriched by positive selection and suggests that specific sites have important functions in methylation-dependent phenotypes. Finally, we identified lineage-specific selective pressures modulating the contraction and expansion of the motif ACGT, revealing that the genetic load of methylation could be dependent on local ecological factors. Taken together, natural selection may shape both the abundance and distribution of methyltransferases and their specific recognition sequences, likely permitting a fine-tuning of genome-encoded functions not achievable by genetic variation alone.

Genome and population dynamics during chronic infection with Helicobacter pylori.

Helicobacter pylori is responsible for one of the most prevalent bacterial infections worldwide. Chronic infection typically leads to chronic active gastritis. Clinical sequelae, including peptic ulcers, mucosa-associated lymphoid tissue lymphoma or, most importantly, gastric adenocarcinoma develop in 10-15% of cases. H. pylori is characterized by extensive inter-strain diversity which is the result of a high mutation rate, recombination, and a large repertoire of restriction-modification systems. This diversity is thought to be a major contributor to H. pylori's persistence and exceptional aptitude to adapt to the gastric environment and evade the immune system. This review covers efforts in the last decade to characterize and understand the multiple layers of H. pylori's diversity in different biological contexts.

Intraprocedural gastric juice analysis as compared to rapid urease test for real-time detection of Helicobacter pylori.

BACKGROUND: Endofaster is an innovative technology that can be combined with upper gastrointestinal endoscopy (UGE) to perform gastric juice analysis and real-time detection of Helicobacter pylori (H. pylori). AIM: To assess the diagnostic performance of this technology and its impact on the management of H. pylori in the real-life clinical setting. METHODS: Patients undergoing routine UGE were prospectively recruited. Biopsies were taken to assess gastric histology according to the updated Sydney system and for rapid urease test (RUT). Gastric juice sampling and analysis was performed using the Endofaster, and the diagnosis of H. pylori was based on real-time ammonium measurements. Histological detection of H. pylori served as the diagnostic gold standard for comparing Endofaster-based H. pylori diagnosis with RUT-based H. pylori detection. RESULTS: A total of 198 patients were prospectively enrolled in an H. pylori diagnostic study by Endofaster-based gastric juice analysis (EGJA) during the UGE. Biopsies for RUT and histological assessment were performed on 161 patients (82 men and 79 women, mean age 54.8 ± 19.2 years). H. pylori infection was detected by histology in 47 (29.2%) patients. Overall, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (NPV) for H. pylori diagnosis by EGJA were 91.5%, 93.0%, 92.6%, 84.3%, and 96.4%, respectively. In patients on treatment with proton pump inhibitors, diagnostic sensitivity was reduced by 27.3%, while specificity and NPV were unaffected. EGJA and RUT were comparable in diagnostic performance and highly concordant in H. pylori detection (κ-value = 0.85). CONCLUSION: Endofaster allows for rapid and highly accurate detection of H. pylori during gastroscopy. This may guide taking additional biopsies for antibiotic susceptibility testing during the same procedure and then selecting an individually tailored eradication regimen.

Plant-Pathogenic Ralstonia Phylotypes Evolved Divergent Respiratory Strategies and Behaviors To Thrive in Xylem.

Bacterial pathogens in the Ralstonia solanacearum species complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments such as biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to the virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance the growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem. IMPORTANCE Plant-pathogenic Ralstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels. Ralstonia cells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. The virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant-pathogenic Ralstonia can use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenic Ralstonia species. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct microniches in the same habitat.

The Helicobacter pylori UvrC Nuclease Is Essential for Chromosomal Microimports after Natural Transformation.

Helicobacter pylori is a Gram-negative bacterial carcinogenic pathogen that infects the stomachs of half of the human population. It is a natural mutator due to a deficient DNA mismatch repair pathway and is naturally competent for transformation. As a result, it is one of the most genetically diverse human bacterial pathogens. The length of chromosomal imports in H. pylori follows an unusual bimodal distribution consisting of macroimports with a mean length of 1,645 bp and microimports with a mean length of 28 bp. The mechanisms responsible for this import pattern were unknown. Here, we used a high-throughput whole-genome transformation assay to elucidate the role of nucleotide excision repair pathway (NER) components on import length distribution. The data show that the integration of microimports depended on the activity of the UvrC endonuclease, while none of the other components of the NER pathway was required. Using H. pylori site-directed mutants, we showed that the widely conserved UvrC nuclease active sites, while essential for protection from UV light, one of the canonical NER functions, are not required for generation of microimports. A quantitative analysis of recombination patterns based on over 1,000 imports from over 200 sequenced recombinant genomes showed that microimports occur frequently within clusters of multiple imports, strongly suggesting they derive from a single strand invasion event. We propose a hypothetical model of homologous recombination in H. pylori, involving a novel function of UvrC, that reconciles the available experimental data about recombination patterns in H. pylori. IMPORTANCE Helicobacter pylori is one of the most common and genetically diverse human bacterial pathogens. It is responsible for chronic gastritis and represents the main risk factor for gastric cancer. In H. pylori, DNA fragments can be imported by recombination during natural transformation. The length of those fragments determines how many potentially beneficial or deleterious alleles are acquired and thus influences adaptation to the gastric niche. Here, we used a transformation assay to examine imported fragments across the chromosome. We show that UvrC, an endonuclease involved in DNA repair, is responsible for the specific integration of short DNA fragments. This suggests that short and long fragments are imported through distinct recombination pathways. We also show that short fragments are frequently clustered with longer fragments, suggesting that both pathways may be mechanistically linked. These findings provide a novel basis to explain how H. pylori can fine-tune the genetic diversity acquired by transformation.

Evolved to vary: genome and epigenome variation in the human pathogen Helicobacter pylori.

Helicobacter pylori is a Gram-negative, spiral shaped bacterium that selectively and chronically infects the gastric mucosa of humans. The clinical course of this infection can range from lifelong asymptomatic infection to severe disease, including peptic ulcers or gastric cancer. The high mutation rate and natural competence typical of this species are responsible for massive inter-strain genetic variation exceeding that observed in all other bacterial human pathogens. The adaptive value of such a plastic genome is thought to derive from a rapid exploration of the fitness landscape resulting in fast adaptation to the changing conditions of the gastric environment. Nevertheless, diversity is also lost through recurrent bottlenecks and H. pylori's lifestyle is thus a perpetual race to maintain an appropriate pool of standing genetic variation able to withstand selection events. Another aspect of H. pylori's diversity is a large and variable repertoire of restriction-modification systems. While not yet completely understood, methylome evolution could generate enough transcriptomic variation to provide another intricate layer of adaptive potential. This review provides an up to date synopsis of this rapidly emerging area of H. pylori research that has been enabled by the ever-increasing throughput of Omics technologies and a multitude of other technological advances.

In Vivo Genome and Methylome Adaptation of cag-Negative Helicobacter pylori during Experimental Human Infection.

Multiple studies have demonstrated rapid bacterial genome evolution during chronic infection with Helicobacter pylori In contrast, little was known about genetic changes during the first stages of infection, when selective pressure is likely to be highest. Using single-molecule, real-time (SMRT) and Illumina sequencing technologies, we analyzed genome and methylome evolution during the first 10 weeks of infection by comparing the cag pathogenicity island (cagPAI)-negative H. pylori challenge strain BCS 100 with pairs of H. pylori reisolates from gastric antrum and corpus biopsy specimens of 10 human volunteers who had been infected with this strain as part of a vaccine trial. Most genetic changes detected in the reisolates affected genes with a surface-related role or a predicted function in peptide uptake. Apart from phenotypic changes of the bacterial envelope, a duplication of the catalase gene was observed in one reisolate, which resulted in higher catalase activity and improved survival under oxidative stress conditions. The methylomes also varied in some of the reisolates, mostly by activity switching of phase-variable methyltransferase (MTase) genes. The observed in vivo mutation spectrum was remarkable for a very high proportion of nonsynonymous mutations. Although the data showed substantial within-strain genome diversity in the challenge strain, most antrum and corpus reisolates from the same volunteers were highly similar to each other, indicating that the challenge infection represents a major selective bottleneck shaping the transmitted population. Our findings suggest rapid in vivo selection of H. pylori during early-phase infection providing adaptation to different individuals by common mechanisms of genetic and epigenetic alterations.IMPORTANCE Exceptional genetic diversity and variability are hallmarks of Helicobacter pylori, but the biological role of this plasticity remains incompletely understood. Here, we had the rare opportunity to investigate the molecular evolution during the first weeks of H. pylori infection by comparing the genomes and epigenomes of H. pylori strain BCS 100 used to challenge human volunteers in a vaccine trial with those of bacteria reisolated from the volunteers 10 weeks after the challenge. The data provide molecular insights into the process of establishment of this highly versatile pathogen in 10 different human individual hosts, showing, for example, selection for changes in host-interaction molecules as well as changes in epigenetic methylation patterns. The data provide important clues to the early adaptation of H. pylori to new host niches after transmission, which we believe is vital to understand its success as a chronic pathogen and develop more efficient treatments and vaccines.

Within-host evolution of Helicobacter pylori shaped by niche-specific adaptation, intragastric migrations and selective sweeps.

The human pathogen Helicobacter pylori displays extensive genetic diversity. While H. pylori is known to evolve during infection, population dynamics inside the gastric environment have not been extensively investigated. Here we obtained gastric biopsies from multiple stomach regions of 16 H. pylori-infected adults, and analyze the genomes of 10 H. pylori isolates from each biopsy. Phylogenetic analyses suggest location-specific evolution and bacterial migration between gastric regions. Migration is significantly more frequent between the corpus and the fundus than with the antrum, suggesting that physiological differences between antral and oxyntic mucosa contribute to spatial partitioning of H. pylori populations. Associations between H. pylori gene polymorphisms and stomach niches suggest that chemotaxis, regulatory functions and outer membrane proteins contribute to specific adaptation to the antral and oxyntic mucosa. Moreover, we show that antibiotics can induce severe population bottlenecks and likely play a role in shaping the population structure of H. pylori.

The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori.

Helicobacter pylori encodes a large number of restriction-modification (R-M) systems despite its small genome. R-M systems have been described as 'primitive immune systems' in bacteria, but the role of methylation in bacterial gene regulation and other processes is increasingly accepted. Every H. pylori strain harbours a unique set of R-M systems resulting in a highly diverse methylome. We identified a highly conserved GCGC-specific m5C MTase (JHP1050) that was predicted to be active in all of 459 H. pylori genome sequences analyzed. Transcriptome analysis of two H. pylori strains and their respective MTase mutants showed that inactivation of the MTase led to changes in the expression of 225 genes in strain J99, and 29 genes in strain BCM-300. Ten genes were differentially expressed in both mutated strains. Combining bioinformatic analysis and site-directed mutagenesis, we demonstrated that motifs overlapping the promoter influence the expression of genes directly, while methylation of other motifs might cause secondary effects. Thus, m5C methylation modifies the transcription of multiple genes, affecting important phenotypic traits that include adherence to host cells, natural competence for DNA uptake, bacterial cell shape, and susceptibility to copper.

Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco.

UNLABELLED: Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. IMPORTANCE: Plant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants, these crop hosts use salicylic acid (SA) both indirectly as a signal to activate defenses and directly as an antimicrobial chemical. We found that SA inhibits growth of R. solanacearum and induces a general stress response that includes repression of multiple bacterial wilt virulence factors. The ability to degrade SA reduces the pathogen's sensitivity to SA toxicity and increases its virulence on tobacco.

Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species.

BACKGROUND: The increased availability of genome sequences has advanced the development of genomic distance methods to describe bacterial diversity. Results of these fast-evolving methods are highly correlated with those of the historically standard DNA-DNA hybridization technique. However, these genomic-based methods can be done more rapidly and less expensively and are less prone to technical and human error. They are thus a technically accessible replacement for species delineation. Here, we use several genomic comparison methods, supported by our own proteomic analyses and metabolic characterization as well as previously published DNA-DNA hybridization analyses, to differentiate members of the Ralstonia solanacearum species complex into three species. This pathogen group consists of diverse and widespread strains that cause bacterial wilt disease on many different plants. RESULTS: We used three different methods to compare the complete genomes of 29 strains from the R. solanacearum species complex. In parallel we profiled the proteomes of 73 strains using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Proteomic profiles together with genomic sequence comparisons consistently and comprehensively described the diversity of the R. solanacearum species complex. In addition, genome-driven functional phenotypic assays excitingly supported an old hypothesis (Hayward et al. (J Appl Bacteriol 69:269-80, 1990)), that closely related members of the R. solanacearum could be identified through a simple assay of anaerobic nitrate metabolism. This assay allowed us to clearly and easily differentiate phylotype II and IV strains from phylotype I and III strains. Further, genomic dissection of the pathway distinguished between proposed subspecies within the current phylotype IV. The assay revealed large scale differences in energy production within the R. solanacearum species complex, indicating coarse evolutionary distance and further supporting a repartitioning of this group into separate species. CONCLUSIONS: Together, the results of these studies support the proposed division of the R. solanacearum species complex into three species, consistent with recent literature, and demonstrate the utility of proteomic and genomic approaches to delineate bacterial species.

In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum.

Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity.

BACKGROUND: Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains. RESULTS: These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences. CONCLUSIONS: This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation.

A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

Hydroxycinnamic Acid Degradation, a Broadly Conserved Trait, Protects Ralstonia solanacearum from Chemical Plant Defenses and Contributes to Root Colonization and Virulence.

Plants produce hydroxycinnamic acid (HCA) defense compounds to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase (Δfcs) mutant that cannot degrade HCA was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCA are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCA, namely, caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCA contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity.

Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups.

Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected.

Multiplex PCR for detection of seven virulence factors and K1/K2 capsular serotypes of Klebsiella pneumoniae.

A single multiplex PCR assay targeting seven virulence factors and the wzi gene specific for the K1 and K2 capsular serotypes of Klebsiella pneumoniae was developed and tested on 65 clinical isolates, which included 45 isolates responsible for community-acquired severe human infections. The assay is useful for the surveillance of emerging highly virulent strains.

Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2.

Hypervirulent Klebsiella pneumoniae isolates of capsular serotype K2 (hvKP-K2) that cause community-acquired invasive infections represent several unrelated clones, which all belong to phylogenetic group KpI. These clones can be recognized using multilocus sequence typing and genomic analyses, but no rapid method currently exists to differentiate them. In this work, a multiplex PCR assay was developed to identify three hvKP-K2 groups: (i) sequence type (ST)86; (ii) ST380 and ST679 (i.e. clonal group 380); and (iii) ST65 and ST375. A specific genetic marker, Kp50233, allowing K. pneumoniae sensu stricto (corresponding to phylogroup KpI) to be distinguished from closely related species, was included in the assay. This PCR assay will be useful in better defining the epidemiology and clinical features of emerging virulent K. pneumoniae clones.

Ralstonia solanacearum requires PopS, an ancient AvrE-family effector, for virulence and To overcome salicylic acid-mediated defenses during tomato pathogenesis.

UNLABELLED: During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum's natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. IMPORTANCE: The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogen Ralstonia solanacearum. To our knowledge, this is the first study of R. solanacearum effector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundant R. solanacearum effectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts. R. solanacearum is a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens.