Comparative Genomic Analysis of Bacterial Data in BV-BRC: An Example Exploring Antimicrobial Resistance.

As genomic and related data continue to expand, research biologists are often hampered by the computational hurdles required to analyze their data. The National Institute of Allergy and Infectious Diseases (NIAID) established the Bioinformatics Resource Centers (BRC) to assist researchers with their analysis of genome sequence and other omics-related data. Recently, the PAThosystems Resource Integration Center (PATRIC), the Influenza Research Database (IRD), and the Virus Pathogen Database and Analysis Resource (ViPR) BRCs merged to form the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) at https://www.bv-brc.org/ . The combined BV-BRC leverages the functionality of the original resources for bacterial and viral research communities with a unified data model, enhanced web-based visualization and analysis tools, and bioinformatics services. Here we demonstrate how antimicrobial resistance data can be analyzed in the new resource.

An automated system for interrogating the evolution of microbial endosymbiosis.

Inter-kingdom endosymbiotic interactions between bacteria and eukaryotic cells are critical to human health and disease. However, the molecular mechanisms that drive the emergence of endosymbiosis remain obscure. Here, we describe the development of a microfluidic system, named SEER (S̲ystem for the E̲volution of E̲ndosymbiotic R̲elationships), that automates the evolutionary selection of bacteria with enhanced intracellular survival and persistence within host cells, hallmarks of endosymbiosis. Using this system, we show that a laboratory strain of Escherichia coli that initially possessed limited abilities to survive within host cells, when subjected to SEER selection, rapidly evolved to display a 55-fold enhancement in intracellular survival. Notably, molecular dissection of the evolved strains revealed that a single-point mutation in a flexible loop of CpxR, a gene regulator that controls bacterial stress responses, substantially contributed to this intracellular survival. Taken together, these results establish SEER as the first microfluidic system for investigating the evolution of endosymbiosis, show the importance of CpxR in endosymbiosis, and set the stage for evolving bespoke inter-kingdom endosymbiotic systems with novel or emergent properties.

Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR.

The National Institute of Allergy and Infectious Diseases (NIAID) established the Bioinformatics Resource Center (BRC) program to assist researchers with analyzing the growing body of genome sequence and other omics-related data. In this report, we describe the merger of the PAThosystems Resource Integration Center (PATRIC), the Influenza Research Database (IRD) and the Virus Pathogen Database and Analysis Resource (ViPR) BRCs to form the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) https://www.bv-brc.org/. The combined BV-BRC leverages the functionality of the bacterial and viral resources to provide a unified data model, enhanced web-based visualization and analysis tools, bioinformatics services, and a powerful suite of command line tools that benefit the bacterial and viral research communities.

Phenotype-Based Threat Assessment.

Bacterial pathogen identification, which is critical for human health, has historically relied on culturing organisms from clinical specimens. More recently, the application of machine learning (ML) to whole-genome sequences (WGSs) has facilitated pathogen identification. However, relying solely on genetic information to identify emerging or new pathogens is fundamentally constrained, especially if novel virulence factors exist. In addition, even WGSs with ML pipelines are unable to discern phenotypes associated with cryptic genetic loci linked to virulence. Here, we set out to determine if ML using phenotypic hallmarks of pathogenesis could assess potential pathogenic threat without using any sequence-based analysis. This approach successfully classified potential pathogenetic threat associated with previously machine-observed and unobserved bacteria with 99% and 85% accuracy, respectively. This work establishes a phenotype-based pipeline for potential pathogenic threat assessment, which we term PathEngine, and offers strategies for the identification of bacterial pathogens.

The role of uspE in virulence and biofilm formation by Histophilus somni.

UspE is a global regulator in Escherichia coli. To study the function of Histophilus somni uspE, strain 2336::TnuspE was identified from a bank of mutants generated with EZ::Tn5™ Tnp Transposome™ that were biofilm deficient. The 2336::TnuspE mutant was highly attenuated in mice, the electrophoretic profile of its lipooligosaccharide (LOS) indicated the LOS was truncated, and the mutant was significantly more serum-sensitive compared to the wildtype strain. In addition to forming a deficient biofilm, exopolysaccharide (EPS) production was also compromised, but the electrophoretic profile of outer membrane proteins was not altered. RNA sequence analysis revealed that the transcription levels of some stress response chaperones, transport proteins, and a large number of ribosomal protein genes in 2336::TnuspE were significantly differentially regulated compared to strain 2336. Therefore, uspE may differentially function in direct and indirect expression of H. somni genes, but its attenuation may be linked to poor biofilm formation and rapid clearance of the bacteria resulting from a compromised LOS structure. Our results support that uspE is a global stress regulatory gene in H. somni.

The Role of luxS in Histophilus somni Virulence and Biofilm Formation.

S-Ribosylhomocysteinase (LuxS) is required for the synthesis of the autoinducer-2 (AI-2) quorum-sensing signaling molecule in many Gram-negative bacteria. The bovine (and ovine) opportunistic pathogen Histophilus somni contains luxS and forms a biofilm containing an exopolysaccharide (EPS) in the matrix. Since biofilm formation is regulated by quorum sensing in many bacteria, the roles of luxS in H. somni virulence and biofilm formation were investigated. Although culture supernatants from H. somni were ineffective at inducing bioluminescence in the Vibrio harveyi reporter strain BB170, H. somniluxS complemented the biosynthesis of AI-2 in the luxS-deficient Escherichia coli strain DH5α. H. somni strain 2336 luxS was inactivated by transposon mutagenesis. RNA expression profiles revealed that many genes were significantly differentially expressed in the luxS mutant compared to that in the wild-type, whether the bacteria were grown planktonically or in a biofilm. Furthermore, the luxS mutant had a truncated and asialylated lipooligosaccharide (LOS) and was substantially more serum sensitive than the wild-type. Not surprisingly, the luxS mutant was attenuated in a mouse model for H. somni virulence, and some of the altered phenotypes were partially restored after the mutation was complemented with a functional luxS However, no major differences were observed between the wild-type and the luxS mutant in regard to outer membrane protein profiles, biofilm formation, EPS production, or intracellular survival. These results indicate that luxS plays a role in H. somni virulence in the context of LOS biosynthesis but not biofilm formation or other phenotypic properties examined.

Polymicrobial Biofilm Interaction Between Histophilus somni and Pasteurella multocida.

Histophilus somni and Pasteurella multocida are two of multiple agents responsible for bovine respiratory disease (BRD) in cattle. Following respiratory infection of calves with H. somni, P. multocida may also be isolated from the lower respiratory tract. Because H. somni may form a biofilm during BRD, we sought to determine if P. multocida can co-exist with H. somni in a polymicrobial biofilm in vitro and in vivo. Interactions between the two species in the biofilm were characterized and quantified by fluorescence in situ hybridization (FISH). The biofilm matrix of each species was examined using fluorescently tagged lectins (FTL) specific for the exopolysaccharide (EPS) using confocal laser scanning microscopy. Bacterial interactions were determined by auto-aggregation and biofilm morphology. Pasteurella multocida and H. somni were evenly distributed in the in vitro biofilm, and both species contributed to the polymicrobial biofilm matrix. The average biomass and biofilm thickness, and the total carbohydrate and protein content of the biofilm, were greatest when both species were present. Polymicrobial bacterial suspensions auto-aggregated faster than single species suspensions, suggesting physical interactions between the two species. Almost 300 P. multocida genes were significantly differentially regulated when the bacteria were in a polymicrobial biofilm compared to a mono-species biofilm, as determined by RNA-sequencing. As expected, host genes associated with inflammation and immune response were significantly upregulated at the infection site following H. somni challenge. Encapsulated P. multocida isolates not capable of forming a substantial biofilm enhanced an in vitro polymicrobial biofilm with H. somni, indicating they contributed to the polymicrobial biofilm matrix. Indirect evidence indicated that encapsulated P. multocida also contributed to a polymicrobial biofilm in vivo. Only the EPS of H. somni could be detected by FTL staining of bovine tissues following challenge with H. somni. However, both species were isolated and an immune response to the biofilm matrix of both species was greater than the response to planktonic cells, suggesting encapsulated P. multocida may take advantage of the H. somni biofilm to persist in the host during chronic BRD. These results may have important implications for the management and prevention of BRD.

Evaluating the impact of international airline suspensions on the early global spread of COVID-19.

Global airline networks play a key role in the global importation of emerging infectious diseases. Detailed information on air traffic between international airports has been demonstrated to be useful in retrospectively validating and prospectively predicting case emergence in other countries. In this paper, we use a well-established metric known as effective distance on the global air traffic data from IATA to quantify risk of emergence for different countries as a consequence of direct importation from China, and compare it against arrival times for the first 24 countries. Using this model trained on official first reports from WHO, we estimate time of arrival (ToA) for all other countries. We then incorporate data on airline suspensions to recompute the effective distance and assess the effect of such cancellations in delaying the estimated arrival time for all other countries. Finally we use the infectious disease vulnerability indices to explain some of the estimated reporting delays.

Phylogenomic analysis of Haemophilus parasuis and proposed reclassification to Glaesserella parasuis, gen. nov., comb. nov.

The Gram-negative bacterium Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs, and causes significant economic losses to the swine industry. This bacterium has been classified as a member of the family Pasteurellaceae in the genus Haemophilus, but phylogenetic relatedness has not been adequately examined to support this genus classification. Phenotypically, all 38 strains of H. parasuis tested were positive for catalase activity, oxidase activity, V-factor requirement, and acid formation from maltose and d-galactose without gas. All strains were negative for X-factor requirement, formation of indole from tryptophan, urease, l-arabinose, and α-glucosidase activity. To determine whether H. parasuis belongs to one of the current Pasteurellaceae genera 40 H. parasuis genomes, plus those of representative Pasteurellaceae, were subjected to phylogenetic analysis of concatenated, multi-protein alignments. Sequence variation at 16S rRNA and rpoB loci allowed the 15 reference serovars of H. parasuis to be integrated into the whole-genome tree. The phylogenetic analysis showed H. parasuis to be a distinct and tight clade whose sister taxon is the genus Bibersteinia. Within H. parasuis two clades were identified with individual serovars distributed between the two. As a result, H. parasuis was confirmed as a member of the family Pasteurellaceae, but was distinct from other genera in this family. Therefore, we propose the name Glaesserella parasuis, gen. nov., comb. nov. for bacterial strains currently classified as H. parasuis. The reference strain of this species is ATCC 19417 (1374)T, NCTC 4557T, DSM 21448T, CCUG 3712T.

The PATRIC Bioinformatics Resource Center: expanding data and analysis capabilities.

The PathoSystems Resource Integration Center (PATRIC) is the bacterial Bioinformatics Resource Center funded by the National Institute of Allergy and Infectious Diseases (https://www.patricbrc.org). PATRIC supports bioinformatic analyses of all bacteria with a special emphasis on pathogens, offering a rich comparative analysis environment that provides users with access to over 250 000 uniformly annotated and publicly available genomes with curated metadata. PATRIC offers web-based visualization and comparative analysis tools, a private workspace in which users can analyze their own data in the context of the public collections, services that streamline complex bioinformatic workflows and command-line tools for bulk data analysis. Over the past several years, as genomic and other omics-related experiments have become more cost-effective and widespread, we have observed considerable growth in the usage of and demand for easy-to-use, publicly available bioinformatic tools and services. Here we report the recent updates to the PATRIC resource, including new web-based comparative analysis tools, eight new services and the release of a command-line interface to access, query and analyze data.

Identification of new protein-protein and protein-DNA interactions linked with wood formation in Populus trichocarpa.

Cellular processes, such as signal transduction and cell wall deposition, are organized by macromolecule interactions. Experimentally determined protein-protein interactions (PPIs) and protein-DNA interactions (PDIs) relevant to woody plant development are sparse. To begin to develop a Populus trichocarpa Torr. & A. Gray wood interactome, we applied the yeast-two-hybrid (Y2H) assay in different ways to enable the discovery of novel PPIs and connected networks. We first cloned open reading frames (ORFs) for 361 genes markedly upregulated in secondary xylem compared with secondary phloem and performed a binary Y2H screen with these proteins. By screening a xylem cDNA library for interactors of a subset of these proteins and then recapitulating the process by using a subset of the interactors as baits, we ultimately identified 165 PPIs involving 162 different ORFs. Thirty-eight transcription factors (TFs) included in our collection of P. trichocarpa wood ORFs were used in a Y1H screen for binding to promoter regions of three genes involved in lignin biosynthesis resulting in 40 PDIs involving 20 different TFs. The network incorporating both the PPIs and PDIs included 14 connected subnetworks, with the largest having 132 members. Protein-protein interactions and PDIs validated previous reports and also identified new candidate wood formation proteins and modules through their interactions with proteins and promoters known to be involved in secondary cell wall synthesis. Selected examples are discussed including a PPI between Mps one binder (MOB1) and a mitogen-activated protein kinase kinase kinase kinase (M4K) that was further characterized by assays confirming the PPI as well as its effect on subcellular localization. Mapping of published transcriptomic data showing developmentally detailed expression patterns across a secondary stem onto the network supported that the PPIs and PDIs are relevant to wood formation, and also illustrated that wood-associated interactions involve gene products that are not upregulated in secondary xylem.

Probiotics and virulent human rotavirus modulate the transplanted human gut microbiota in gnotobiotic pigs.

We generated a neonatal pig model with human infant gut microbiota (HGM) to study the effect of a probiotic on the composition of the transplanted microbiota following rotavirus vaccination and challenge. All the HGM-transplanted pigs received two doses of an oral attenuated rotavirus vaccine. The gut microbiota of vaccinated pigs were investigated for effects of Lactobacillus rhamnosus GG (LGG) supplement and homotypic virulent human rotavirus (HRV) challenge. High-throughput sequencing of V4 region of 16S rRNA genes demonstrated that HGM-transplanted pigs carried microbiota similar to that of the C-section delivered baby. Firmicutes and Proteobacteria represented over 98% of total bacteria in the human donor and the recipient pigs. HRV challenge caused a phylum-level shift from Firmicutes to Proteobacteria. LGG supplement prevented the changes in microbial communities caused by HRV challenge. In particular, members of Enterococcus in LGG-supplemented pigs were kept at the baseline level, while they were enriched in HRV challenged pigs. Taken together, our results suggested that HGM pigs are valuable for testing the microbiota's response to probiotic interventions for treating infantile HRV infection.

Characterization and RNA-seq analysis of underperformer, an activation-tagged potato mutant.

The potato cv. Bintje and a Bintje activation-tagged mutant, underperformer (up) were compared. Mutant up plants grown in vitro were dwarf, with abundant axillary shoot growth, greater tuber yield, altered tuber traits and early senescence compared to wild type. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant remained, but the up plants exhibited a lower tuber yield and fewer axillary shoots compared to wild type. Southern blot analyses indicated a single T-DNA insertion in the mutant, located on chromosome 10. Initial PCR-based gene expression studies indicated transcriptional activation/repression of several genes in the mutant flanking the insertion. The gene immediately flanking the right border of the T-DNA insertion, which encoded an uncharacterized Broad complex, Tramtrac, Bric-a-brac; also known as Pox virus and Zinc finger (BTB/POZ) domain-containing protein (StBTB/POZ1) containing an Armadillo repeat region, was up-regulated in the mutant. Global gene expression comparisons between Bintje and up using RNA-seq on leaves from 60 day-old plants revealed a dataset of over 1,600 differentially expressed genes. Gene expression analyses suggested a variety of biological processes and pathways were modified in the mutant, including carbohydrate and lipid metabolism, cell division and cell cycle activity, biotic and abiotic stress responses, and proteolysis.

Comparative phylogenomics and evolution of the Brucellae reveal a path to virulence.

Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as "stealth pathogens" that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing "atypical" strains and a highly conserved "classical" core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.

Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States.

UNLABELLED: Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5'-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV. IMPORTANCE: The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.

An active ac/ds transposon system for activation tagging in tomato cultivar m82 using clonal propagation.

Tomato (Solanum lycopersicum) is a model organism for Solanaceae in both molecular and agronomic research. This project utilized Agrobacterium tumefaciens transformation and the transposon-tagging construct Activator (Ac)/Dissociator (Ds)-ATag-Bar_gosGFP to produce activation-tagged and knockout mutants in the processing tomato cultivar M82. The construct carried hygromycin resistance (hyg), green fluorescent protein (GFP), and the transposase (TPase) of maize (Zea mays) Activator major transcript X054214.1 on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds-ATag element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 11,000 selfed and cv M82 backcrossed progeny was produced from the functional T0 line. This population was screened using glufosinate herbicide, hygromycin leaf painting, and multiplex polymerase chain reaction (PCR). Insertion sites of transposed Ds-ATag elements were identified through thermal asymmetric interlaced PCR, and resulting product sequences were aligned to the recently published tomato genome. A population of 509 independent, Ds-only transposant lines spanning all 12 tomato chromosomes has been developed. Insertion site analysis demonstrated that more than 80% of these lines harbored Ds insertions conducive to activation tagging. The capacity of the Ds-ATag element to alter transcription was verified by quantitative real-time reverse transcription-PCR in two mutant lines. The transposon-tagged lines have been immortalized in seed stocks and can be accessed through an online database, providing a unique resource for tomato breeding and analysis of gene function in the background of a commercial tomato cultivar.

Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Comparison of genome diversity of Brucella spp. field isolates using Universal Bio-signature Detection Array and whole genome sequencing reveals limitations of current diagnostic methods.

The detection and identification of bio-threat agents and the study of host-pathogen interactions require a high-resolution detection platform capable of discerning closely related species. Diverse analysis methods are used to identify pathogens, specifically Brucella species or biovars. In this study, we compared four diagnostic approaches including serology-based biochemical test, PCR assay, microarray analysis using a Universal Bio-signature Detection Array (UBDA) and whole genome "deep" sequencing for Brucella organisms including a number of field isolates. We found that although there was frequent agreement among the different tests, some tests gave compound/contradictory results that were a consequence of species diversity due to mixed infections or minor contaminants as measured by UBDA and validated from whole genome sequence. By comparing these analysis techniques, we demonstrate that standard diagnostics used in the field are limited in their ability to identify genomic DNA contaminants in field isolates while UBDA and sequencing analysis are highly sensitive in tracing genomic differences among the isolates.

Transposon tagging in diploid strawberry.

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T1 progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T0 launch pads, putative transposants in the T1 generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T1 plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T1 plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T0 generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T2 transposon-tagged plants. The mutant collection has been catalogued in an on-line database.

Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1(T) and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1(T) and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1(T) and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1(T) maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.