Complete genome sequence of "Candidatus Phytoplasma cynodontis" GY2015, a plant pathogen associated with Bermuda grass white leaf disease in Taiwan.

The complete genome sequence of "Candidatus Phytoplasma cynodontis" strain GY2015, which consists of one 498,922-bp circular chromosome, is presented in this work. This uncultivated plant-pathogenic bacterium is associated with Bermuda grass white leaf disease in Taoyuan, Taiwan.

Phage therapy in a lung transplant recipient with cystic fibrosis infected with multidrug-resistant Burkholderia multivorans.

BACKGROUND: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died. METHODS: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis. RESULTS: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate. CONCLUSIONS: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections.

Rice OsHsp16.9A interacts with OsHsp101 to confer thermotolerance.

Class I small heat shock proteins (CI sHSPs), OsHsp16.9A and OsHsp18.0, share 74% identity in amino acid sequences and accumulate in response to heat shock treatments. Individual rice transformants overexpressing OsHsp16.9A and OsHsp18.0 exhibit distinct thermoprotection/thermotolerance modes. Under high temperature stress, OsHsp16.9A-overexpressing lines showed higher seed germination rate, seedling survival, and pollen germination than wild-type controls, while OsHsp18.0 overexpression provided higher thermoprotection/thermotolerance for seedling survival. To elucidate the functional roles of OsHsp16.9A, mass spectrometry was used to identify OsHsp16.9A-interacting proteins. OsHsp101 was consistently identified in the OsHsp16.9A protein complex in several mass spectrometry analyses of seed proteins from OsHsp16.9A-overexpressing lines. Both OsHsp16.9A and OsHsp101 proteins accumulated during similar developmental stages of rice seeds and formed a heat-stable complex under high temperature treatments in in vitro assays. Co-localization of OsHsp16.9A and OsHsp101 was observed via ratiometric bimolecular fluorescence complementation analyses. Amino acid mutation studies revealed that OsHsp16.9A glutamate residue 74 and amino acid residues 23-36 were essential for OsHsp16.9A-OsHsp101 interaction. Moreover, overexpressing OsHsp16.9A in OsHsp101 knockdown mutants did not increase the seed germination rate under heat stress, which further confirmed the functional roles of OsHsp16.9A-OsHsp101 interaction in conferring thermotolerance to rice plants.

Evolution of extended-spectrum ß-lactamase-producing ST131 Escherichia coli at a single hospital over 15 years.

Escherichia coli belonging to sequence type ST131 constitute a globally distributed pandemic lineage that causes multidrug-resistant extra-intestinal infections. ST131 E. coli frequently produce extended-spectrum ß-lactamases (ESBLs), which confer resistance to many ß-lactam antibiotics and make infections difficult to treat. We sequenced the genomes of 154 ESBL-producing E. coli clinical isolates belonging to the ST131 lineage from patients at the University of Pittsburgh Medical Center (UPMC) between 2004 and 2018. Isolates belonged to the well described ST131 clades A (8%), B (3%), C1 (33%), and C2 (54%). An additional four isolates belonged to another distinct subclade within clade C and encoded genomic characteristics that have not been previously described. Time-dated phylogenetic analysis estimated that the most recent common ancestor (MRCA) for all clade C isolates from UPMC emerged around 1989, consistent with previous studies. We identified multiple genes potentially under selection in clade C, including the cell wall assembly gene ftsI, the LPS biosynthesis gene arnC, and the yersiniabactin uptake receptor fyuA. Diverse ESBL genes belonging to the blaCTX-M, blaSHV, and blaTEM families were identified; these genes were found at varying numbers of loci and in variable numbers of copies across isolates. Analysis of ESBL flanking regions revealed diverse mobile elements that varied by ESBL type. Overall, our findings show that ST131 subclades C1 and C2 dominated and were stably maintained among patients in the same hospital and uncover possible signals of ongoing adaptation within the clade C ST131 lineage.

Genomic Diversity of Hospital-Acquired Infections Revealed through Prospective Whole-Genome Sequencing-Based Surveillance.

Healthcare-associated infections (HAIs) cause mortality, morbidity, and waste of health care resources. HAIs are also an important driver of antimicrobial resistance, which is increasing around the world. Beginning in November 2016, we instituted an initiative to detect outbreaks of HAIs using prospective whole-genome sequencing-based surveillance of bacterial pathogens collected from hospitalized patients. Here, we describe the diversity of bacteria sampled from hospitalized patients at a single center, as revealed through systematic analysis of bacterial isolate genomes. We sequenced the genomes of 3,004 bacterial isolates from hospitalized patients collected over a 25-month period. We identified bacteria belonging to 97 distinct species, which were distributed among 14 groups of related species. Within these groups, isolates could be distinguished from one another by both average nucleotide identity (ANI) and principal-component analysis of accessory genes (PCA-A). Core genome genetic distances and rates of evolution varied among species, which has practical implications for defining shared ancestry during outbreaks and for our broader understanding of the origins of bacterial strains and species. Finally, antimicrobial resistance genes and putative mobile genetic elements were frequently observed, and our systematic analysis revealed patterns of occurrence across the different species sampled from our hospital. Overall, this study shows how understanding the population structure of diverse pathogens circulating in a single health care setting can improve the discriminatory power of genomic epidemiology studies and can help define the processes leading to strain and species differentiation. IMPORTANCE Hospitalized patients are at increased risk of becoming infected with antibiotic-resistant organisms. We used whole-genome sequencing to survey and compare over 3,000 clinical bacterial isolates collected from hospitalized patients at a large medical center over a 2-year period. We identified nearly 100 different bacterial species, which we divided into 14 different groups of related species. When we examined how genetic relatedness differed between species, we found that different species were likely evolving at different rates within our hospital. This is significant because the identification of bacterial outbreaks in the hospital currently relies on genetic similarity cutoffs, which are often applied uniformly across organisms. Finally, we found that antibiotic resistance genes and mobile genetic elements were abundant and were shared among the bacterial isolates we sampled. Overall, this study provides an in-depth view of the genomic diversity and evolutionary processes of bacteria sampled from hospitalized patients, as well as genetic similarity estimates that can inform hospital outbreak detection and prevention efforts.

Comparative Genome Analysis of 'Candidatus Phytoplasma luffae' Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution.

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of 'Candidatus Phytoplasma luffae' strain NCHU2019 that is associated with witches' broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has a remarkably repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for subfunctionalization or neofunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

Modular evolution of secretion systems and virulence plasmids in a bacterial species complex.

BACKGROUND: Many named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. RESULTS: We utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. CONCLUSIONS: We demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

Whole-Genome Sequencing Surveillance and Machine Learning of the Electronic Health Record for Enhanced Healthcare Outbreak Detection.

BACKGROUND: Most hospitals use traditional infection prevention (IP) methods for outbreak detection. We developed the Enhanced Detection System for Healthcare-Associated Transmission (EDS-HAT), which combines whole-genome sequencing (WGS) surveillance and machine learning (ML) of the electronic health record (EHR) to identify undetected outbreaks and the responsible transmission routes, respectively. METHODS: We performed WGS surveillance of healthcare-associated bacterial pathogens from November 2016 to November 2018. EHR ML was used to identify the transmission routes for WGS-detected outbreaks, which were investigated by an IP expert. Potential infections prevented were estimated and compared with traditional IP practice during the same period. RESULTS: Of 3165 isolates, there were 2752 unique patient isolates in 99 clusters involving 297 (10.8%) patient isolates identified by WGS; clusters ranged from 2-14 patients. At least 1 transmission route was detected for 65.7% of clusters. During the same time, traditional IP investigation prompted WGS for 15 suspected outbreaks involving 133 patients, for which transmission events were identified for 5 (3.8%). If EDS-HAT had been running in real time, 25-63 transmissions could have been prevented. EDS-HAT was found to be cost-saving and more effective than traditional IP practice, with overall savings of $192 408-$692 532. CONCLUSIONS: EDS-HAT detected multiple outbreaks not identified using traditional IP methods, correctly identified the transmission routes for most outbreaks, and would save the hospital substantial costs. Traditional IP practice misidentified outbreaks for which transmission did not occur. WGS surveillance combined with EHR ML has the potential to save costs and enhance patient safety.

Genetic differentiation of Xylella fastidiosa following the introduction into Taiwan.

The economically important plant pathogen Xylella fastidiosa has been reported in multiple regions of the globe during the last two decades, threatening a growing list of plants. Particularly, X. fastidiosa subspecies fastidiosa causes Pierce's disease (PD) of grapevines, which is a problem in the USA, Spain, and Taiwan. In this work, we studied PD-causing subsp. fastidiosa populations and compared the genome sequences of 33 isolates found in Central Taiwan with 171 isolates from the USA and two from Spain. Phylogenetic relationships, haplotype networks, and genetic diversity analyses confirmed that subsp. fastidiosa was recently introduced into Taiwan from the Southeast USA (i.e. the PD-I lineage). Recent core-genome recombination events were detected among introduced subsp. fastidiosa isolates in Taiwan and contributed to the development of genetic diversity. The genetic diversity observed includes contributions through recombination from unknown donors, suggesting that higher genetic diversity exists in the region. Nevertheless, no recombination event was detected between X. fastidiosa subsp. fastidiosa and the endemic sister species Xylella taiwanensis, which is the causative agent of pear leaf scorch disease. In summary, this study improved our understanding of the genetic diversity of an important plant pathogenic bacterium after its invasion to a new region.

Parasitic modulation of host development by ubiquitin-independent protein degradation.

Certain obligate parasites induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels. However, the mechanisms underlying these changes remain largely unknown. Here we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes. These effectors simultaneously prolong the host lifespan and induce witches' broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that relies on hijacking the plant ubiquitin receptor RPN10 independent of substrate ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.

Complete Genome Sequence of Xylella taiwanensis and Comparative Analysis of Virulence Gene Content With Xylella fastidiosa.

The bacterial genus Xylella contains plant pathogens that are major threats to agriculture in America and Europe. Although extensive research was conducted to characterize different subspecies of Xylella fastidiosa (Xf), comparative analysis at above-species levels was lacking due to the unavailability of appropriate data sets. Recently, a bacterium that causes pear leaf scorch (PLS) in Taiwan was described as the second Xylella species (i.e., Xylella taiwanensis; Xt). In this work, we report the complete genome sequence of Xt type strain PLS229T. The genome-scale phylogeny provided strong support that Xf subspecies pauca (Xfp) is the basal lineage of this species and Xylella was derived from the paraphyletic genus Xanthomonas. Quantification of genomic divergence indicated that different Xf subspecies share ∼87-95% of their chromosomal segments, while the two Xylella species share only ∼66-70%. Analysis of overall gene content suggested that Xt is most similar to Xf subspecies sandyi (Xfs). Based on the existing knowledge of Xf virulence genes, the homolog distribution among 28 Xylella representatives was examined. Among the 11 functional categories, those involved in secretion and metabolism are the most conserved ones with no copy number variation. In contrast, several genes related to adhesins, hydrolytic enzymes, and toxin-antitoxin systems are highly variable in their copy numbers. Those virulence genes with high levels of conservation or variation may be promising candidates for future studies. In summary, the new genome sequence and analysis reported in this work contributed to the study of several important pathogens in the family Xanthomonadaceae.

Complete Genome Sequence of "Candidatus Phytoplasma asteris" RP166, a Plant Pathogen Associated with Rapeseed Phyllody Disease in Poland.

The complete genome sequence of "Candidatus Phytoplasma asteris" RP166, which consists of one 829,546-bp circular chromosome, is presented in this work. This bacterium is associated with rapeseed phyllody disease in Poland and belongs to the 16SrI-B (i.e., aster yellows) group.

Species Boundaries and Molecular Markers for the Classification of 16SrI Phytoplasmas Inferred by Genome Analysis.

Phytoplasmas are plant-pathogenic bacteria that impact agriculture worldwide. The commonly adopted classification system for phytoplasmas is based on the restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes. With the increased availability of phytoplasma genome sequences, the classification system can now be refined. This work examined 11 strains in the 16SrI group within the genus 'Candidatus Phytoplasma' and investigated the possible species boundaries. We confirmed that the RFLP classification method is problematic due to intragenomic variation of the 16S rRNA genes and uneven weighing of different nucleotide positions. Importantly, our results based on the molecular phylogeny, differentiations in chromosomal segments and gene content, and divergence in homologous sequences, all supported that these strains may be classified into multiple operational taxonomic units (OTUs) equivalent to species. Strains assigned to the same OTU share >97% genome-wide average nucleotide identity (ANI) and >78% of their protein-coding genes. In comparison, strains assigned to different OTUs share < 94% ANI and < 75% of their genes. Reduction in homologous recombination between OTUs is one possible explanation for the discontinuity in genome similarities, and these findings supported the proposal that 95% ANI could serve as a cutoff for distinguishing species in bacteria. Additionally, critical examination of these results and the raw sequencing reads led to the identification of one genome that was presumably mis-assembled by combining two sequencing libraries built from phytoplasmas belonging to different OTUs. This finding provided a cautionary tale for working on uncultivated bacteria. Based on the new understanding of phytoplasma divergence and the current genome availability, we developed five molecular markers that could be used for multilocus sequence analysis (MLSA). By selecting markers that are short yet highly informative, and are distributed evenly across the chromosome, these markers provided a cost-effective system that is robust against recombination. Finally, examination of the effector gene distribution further confirmed the rapid gains and losses of these genes, as well as the involvement of potential mobile units (PMUs) in their molecular evolution. Future improvements on the taxon sampling of phytoplasma genomes will allow further expansions of similar analysis, and thus contribute to phytoplasma taxonomy and diagnostics.

Chromosomal and plasmid-mediated fluoroquinolone resistance in human Salmonella enterica infection in Ghana.

BACKGROUND: Salmonella infection poses significant public health threat globally, especially in resource-limited countries. Emergence and spread of antibiotic resistant strains to fluoroquinolones have led to treatment failures and increased mortality in Salmonella infection. However, there is dearth of information regarding mechanisms of resistance to fluoroquinolones in Ghana. This study therefore sought to identify chromosomal mutations and plasmid-mediated resistance as possible mechanisms of fluoroquinolone resistance from clinical isolates in Ghana. METHODS: This was a retrospective study of archived isolates biobanked at Kumasi Centre for Collaborative Research in Tropical Medicine, Ghana. Isolates were obtained from blood, stool and oropharynx samples at two hospitals, between May, 2016 and January, 2018. Salmonella identification was done using standard microbiological protocols and antibiotic susceptibility testing performed by Kirby-Bauer disc diffusion method. Isolates with intermediate susceptibility and/or resistance to nalidixic acid and/or ciprofloxacin were selected and examined for chromosomal mutations by Sanger sequencing and plasmid-mediated resistance by PCR. RESULTS: Of 133 biobanked isolates cultured, 68 (51.1%) and 16 (12%) were identified as Salmonella Typhi and non-typhoidal Salmonella (NTS), respectively. Sequence analysis of gyrA gene revealed the presence of 5 different nonsynonymous mutations, with the most frequent mutation (Ile203Ser) occurring in 12 out of 13 isolates tested. Gyrase B (gyrB) gene had 1 nonsynonymous mutation in 3 out of 13 isolates, substituting phenylalanine with leucine at codon 601 (Phe601Leu). No mutation was observed in parC and parE genes. Two NTS isolates were found to harbour qnrS plasmid-mediated resistant gene of molecular size 550 bp with high ciprofloxacin MIC of 0.5 µg/ml. CONCLUSION: This study reports for the first time in Ghana plasmid-mediated fluoroquinolone resistant gene qnrS in Salmonella clinical isolates. Nonsynonymous mutations of gyrA and gyrB genes likely to confer Salmonella reduced susceptibility to ciprofloxacin were also reported.

Genomic Characterization of the Periwinkle Leaf Yellowing (PLY) Phytoplasmas in Taiwan.

The periwinkle leaf yellowing (PLY) disease was first reported in Taiwan in 2005. This disease was caused by an uncultivated bacterium in the genus "Candidatus phytoplasma." In subsequent years, this bacterium was linked to other plant diseases and caused losses in agriculture. For genomic investigation of this bacterium and its relatives, we conducted whole genome sequencing of a PLY phytoplasma from an infected periwinkle collected in Taoyuan. The de novo genome assembly produced eight contigs with a total length of 824,596 bp. The annotation contains 775 protein-coding genes, 63 pseudogenes, 32 tRNA genes, and two sets of rRNA operons. To characterize the genomic diversity across populations, a second strain that infects green onions in Yilan was collected for re-sequencing analysis. Comparison between these two strains identified 337 sequence polymorphisms and 10 structural variations. The metabolic pathway analysis indicated that the PLY phytoplasma genome contains two regions with highly conserved gene composition for carbohydrate metabolism. Intriguingly, each region contains several pseudogenes and the remaining functional genes in these two regions complement each other, suggesting a case of duplication followed by differential gene losses. Comparative analysis with other available phytoplasma genomes indicated that this PLY phytoplasma belongs to the 16SrI-B subgroup in the genus, with "Candidatus Phytoplasma asteris" that causes the onion yellowing (OY) disease in Japan as the closest known relative. For characterized effectors that these bacteria use to manipulate their plant hosts, the PLY phytoplasma has homologs for SAP11, SAP54/PHYL1, and TENGU. For genome structure comparison, we found that potential mobile unit (PMU) insertions may be the main factor that drives genome rearrangements in these bacteria. A total of 10 PMU-like regions were found in the PLY phytoplasma genome. Two of these PMUs were found to harbor one SAP11 homolog each, with one more similar to the 16SrI-B type and the other more similar to the 16SrI-A type, suggesting possible horizontal transfer. Taken together, this work provided a first look into population genomics of the PLY phytoplasmas in Taiwan, as well as identified several evolutionary processes that contributed to the genetic diversification of these plant-pathogenic bacteria.

Differentiations in Gene Content and Expression Response to Virulence Induction Between Two Agrobacterium Strains.

Agrobacterium tumefaciens is important in biotechnology due to its ability to transform eukaryotic cells. Although the molecular mechanisms have been studied extensively, previous studies were focused on the model strain C58. Consequently, nearly all of the commonly used strains for biotechnology application were derived from C58 and share similar host ranges. To overcome this limitation, better understanding of the natural genetic variation could provide valuable insights. In this study, we conducted comparative analysis between C58 and 1D1609. These two strains belong to different genomospecies within the species complex and have distinct infectivity profiles. Genome comparisons revealed that each strain has >1,000 unique genes in addition to the 4,115 shared genes. Furthermore, the divergence in gene content and sequences vary among replicons. The circular chromosome is much more conserved compared to the linear chromosome. To identify the genes that may contribute to their differentiation in virulence, we compared the transcriptomes to screen for genes differentially expressed in response to the inducer acetosyringone. Based on the RNA-Seq results with three biological replicates, ∼100 differentially expressed genes were identified in each strain. Intriguingly, homologous genes with the same expression pattern account for <50% of these differentially expressed genes. This finding indicated that phenotypic variation may be partially explained by divergence in expression regulation. In summary, this study characterized the genomic and transcriptomic differences between two representative Agrobacterium strains. Moreover, the short list of differentially expressed genes are promising candidates for future characterization, which could improve our understanding of the genetic mechanisms for phenotypic divergence.

Plant-Pathogenic Agrobacterium tumefaciens Strains Have Diverse Type VI Effector-Immunity Pairs and Vary in In-Planta Competitiveness.

The type VI secretion system (T6SS) is used by gram-negative bacteria to translocate effectors that can antagonize other bacterial cells. Models predict the variation in collections of effector and cognate immunity genes determine competitiveness and can affect the dynamics of populations and communities of bacteria. However, the outcomes of competition cannot be entirely explained by compatibility of effector-immunity (EI) pairs. Here, we characterized the diversity of T6SS loci of plant-pathogenic Agrobacterium tumefaciens and showed that factors other than EI pairs can impact interbacterial competition. All examined strains encode T6SS active in secretion and antagonism against Escherichia coli. The spectra of EI pairs as well as compositions of gene neighborhoods are diverse. Almost 30 in-planta competitions were tested between different genotypes of A. tumefaciens. Fifteen competitions between members of different species-level groups resulted in T6SS-dependent suppression in in-planta growth of prey genotypes. In contrast, ten competitions between members within species-level groups resulted in no significant effect on the growth of prey genotypes. One strain was an exceptional case and, despite encoding a functional T6SS and toxic effector protein, could not compromise the growth of the four tested prey genotypes. The data suggest T6SS-associated EI pairs can influence the competitiveness of strains of A. tumefaciens, but genetic features have a significant role on the efficacy of interbacterial antagonism.

Redundancy and Specificity of Type VI Secretion vgrG Loci in Antibacterial Activity of Agrobacterium tumefaciens 1D1609 Strain.

Type VI secretion system (T6SS) is a contractile nanoweapon employed by many Proteobacteria to deliver effectors to kill or inhibit their competitors. One T6SS gene, vgrG, encodes a spike protein for effector translocation and is often present as multiple copies in bacterial genomes. Our phylogenomic analyses sampled 48 genomes across diverse Proteobacteria lineages and found ∼70% of them encode multiple VgrGs, yet only four genomes have nearly identical paralogs. Among these four, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Compared to A. tumefaciens model strain C58 which harbors two vgrG genes, 1D1609 encodes four vgrG genes (i.e., vgrGa-d) with each adjacent to different putative effector genes. Thus, 1D1609 was selected to investigate the functional redundancy and specificity of multiple vgrG genes and their associated effectors. Secretion assay of single and multiple vgrG deletion mutants demonstrated that these four vgrGs are functionally redundant in mediating T6SS secretion. By analyzing various vgrG mutants, we found that all except for the divergent vgrGb could contribute to 1D1609's antibacterial activity. Further characterizations of putative effector-immunity gene pairs revealed that vgrGa-associated gene 2 (v2a) encodes an AHH family nuclease and serves as the major antibacterial toxin. Interestingly, C58's VgrG2 shares 99% amino acid sequence identity with 1D1609's VgrGa, VgrGc and VgrGd. This high sequence similarity allows 1D1609 to use an exogenous VgrG delivered from C58 to kill another competing bacterium. Taken together, Agrobacterium can use highly similar VgrGs, either produced endogenously or injected from its close relatives, for T6SS-mediated interbacterial competition.

The genome of 'Candidatus Phytoplasma solani' strain SA-1 is highly dynamic and prone to adopting foreign sequences.

Bacteria of the genus 'Candidatus Phytoplasma' are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as 'Ca. P. solani', have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of 'Ca. P. solani' strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The 'Ca. P. solani' SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.

Draft Genome Sequence of Vibrio vulnificus 86573B, a Bacterium Isolated from Diseased Tilapia in Taiwan.

Vibrio vulnificus 86573B is a biotype 1 strain isolated from a moribund tilapia collected in Kaohsiung, Taiwan, during an outbreak early in 1997. Here, we report the draft genome sequence of this bacterium to facilitate the investigation of its biology and future comparative genomic analysis.