IMG/PR: a database of plasmids from genomes and metagenomes with rich annotations and metadata.

Plasmids are mobile genetic elements found in many clades of Archaea and Bacteria. They drive horizontal gene transfer, impacting ecological and evolutionary processes within microbial communities, and hold substantial importance in human health and biotechnology. To support plasmid research and provide scientists with data of an unprecedented diversity of plasmid sequences, we introduce the IMG/PR database, a new resource encompassing 699 973 plasmid sequences derived from genomes, metagenomes and metatranscriptomes. IMG/PR is the first database to provide data of plasmid that were systematically identified from diverse microbiome samples. IMG/PR plasmids are associated with rich metadata that includes geographical and ecosystem information, host taxonomy, similarity to other plasmids, functional annotation, presence of genes involved in conjugation and antibiotic resistance. The database offers diverse methods for exploring its extensive plasmid collection, enabling users to navigate plasmids through metadata-centric queries, plasmid comparisons and BLAST searches. The web interface for IMG/PR is accessible at https://img.jgi.doe.gov/pr. Plasmid metadata and sequences can be downloaded from https://genome.jgi.doe.gov/portal/IMG_PR.

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background.

Synonymous mutations are changes to DNA sequence, which occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation elongation factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels as well as global polysome abundance on RNA transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.IMPORTANCEThis study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein elongation factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

Luteibacter mycovicinus sp. nov., a yellow-pigmented gammaproteobacterium found as an endohyphal symbiont of endophytic Ascomycota.

We isolated and described a yellow-pigmented strain of bacteria (strain 9143T), originally characterized as an endohyphal inhabitant of an endophytic fungus in the Ascomycota. Although the full-length sequence of its 16S rRNA gene displays 99 % similarity to Luteibacter pinisoli, genomic hybridization demonstrated <30 % genomic similarity between 9143T and its closest named relatives, further supported by average nucleotide identity results. This and related endohyphal strains form a well-supported clade separate from L. pinisoli and other validly named species including the most closely related Luteibacter rhizovicinus. The name Luteibacter mycovicinus sp. nov. is proposed, with type strain 9143T (isolate DBL433), for which a genome has been sequenced and is publicly available from the American Type Culture Collection (ATCC TSD-257T) and from the Leibniz Institute DSMZ (DSM 112764T). The type strain reliably forms yellow colonies across diverse media and growth conditions (lysogeny broth agar, King's Medium B, potato dextrose agar, trypticase soy agar and Reasoner's 2A (R2A) agar). It forms colonies readily at 27 °C on agar with a pH of 6-8, and on salt (NaCl) concentrations up to 2 %. It lacks the ability to utilize sulphate as a sulphur source and thus only forms colonies on minimal media if supplemented with alternative sulphur sources. It is catalase-positive and oxidase-negative. Although it exhibits a single polar flagellum, motility was only clearly visible on R2A agar. Its host range and close relatives, which share the endohyphal lifestyle, are discussed.

Pantailocins: phage-derived bacteriocins from Pantoea ananatis and Pantoea stewartii subsp. indologenes.

IMPORTANCE: Phage-derived bacteriocins (tailocins) are ribosomally synthesized structures produced by bacteria in order to provide advantages against competing strains under natural conditions. Tailocins are highly specific in their target range and have proven to be effective for the prevention and/or treatment of bacterial diseases under clinical and agricultural settings. We describe the discovery and characterization of a new tailocin locus encoded within genomes of Pantoea ananatis and Pantoea stewartii subsp. indologenes, which may enable the development of tailocins as preventative treatments against phytopathogenic infection by these species.

Interspecies killing activity of Pseudomonas syringae tailocins.

Tailocins are ribosomally synthesized bacteriocins, encoded by bacterial genomes, but originally derived from bacteriophage tails. As with both bacteriocins and phage, tailocins are largely thought to be species-specific with killing activity often assumed to be directed against closely related strains. Previous investigations into interactions between tailocin host range and sensitivity across phylogenetically diverse isolates of the phytopathogen Pseudomonas syringae have demonstrated that many strains possess intraspecific tailocin activity and that this activity is highly precise and specific against subsets of strains. However, here we demonstrate that at least one strain of P. syringae, USA011R, defies both expectations and current overarching dogma because tailocins from this strain possess broad killing activity against other agriculturally significant phytopathogens such as Erwinia amylovora and Xanthomonas perforans as well as against the clinical human pathogen Salmonella enterica serovar Choleraesuis. Moreover, we show that the full spectrum of this interspecific killing activity is not conserved across closely related strains with data suggesting that even if tailocins can target different species, they do so with different efficiencies. Our results reported herein highlight the potential for and phenotypic divergence of interspecific killing activity of P. syringae tailocins and establish a platform for further investigations into the evolution of tailocin host range and strain specificity.

Prevalence of an Insect-Associated Genomic Region in Environmentally Acquired Burkholderiaceae Symbionts.

Microbial symbionts are critical for the development and survival of many eukaryotes. Recent research suggests that the genes enabling these relationships can be localized in horizontally transferred regions of microbial genomes termed "symbiotic islands." Recently, a putative symbiotic island was found that may facilitate symbioses between true bugs and numerous Burkholderia species, based on analysis of five Burkholderia symbionts. We expanded on this work by exploring the putative island's prevalence, origin, and association with colonization across the bacterial family Burkholderiaceae. We performed a broad comparative analysis of 229 Burkholderiaceae genomes, including 8 new genomes of insect- or soil-associated Burkholderia sequenced for this study. We detected the region in 23% of the genomes; these were located solely within two Burkholderia clades. Our analyses suggested that the contiguous region arose at the common ancestor of plant- and insect-associated Burkholderia clades, but the genes themselves are ancestral. Although the region was initially discovered on plasmids and we did detect two likely instances of horizontal transfer within Burkholderia, we found that the region is almost always localized to a chromosome and does not possess any of the mobility elements that typify genomic islands. Finally, to attempt to deduce the region's function, we combined our data with information on several strains' abilities to colonize the insect's symbiotic organ. Although the region was associated with improved colonization of the host, this relationship was confounded with, and likely driven by, Burkholderia clade membership. These findings advance our understanding of the genomic underpinnings of a widespread insect-microbe symbiosis. IMPORTANCE Many plants and animals form intricate associations with bacteria. These pairings can be mediated by genomic islands, contiguous regions containing numerous genes with cohesive functionality. Pathogen-associated islands are well described, but recent evidence suggests that mutualistic islands, which benefit both host and symbiont, may also be common. Recently, a putative symbiosis island was found in Burkholderia symbionts of insects. We determined that this genomic region is located in only two clades of Burkholderia (the plant- and insect-associated species) and that although it has undergone horizontal transfer, it is most likely a symbiosis-associated region rather than a true island. This region is associated with improved host colonization, although this is may be due to specific Burkholderia clades' abilities to colonize rather than presence of the region. By studying the genomic basis of the insect-Burkholderia symbiosis, we can better understand how mutualisms evolve in animals.

Prophylactic Application of Tailocins Prevents Infection by Pseudomonas syringae.

Tailocins are phage-derived bacteriocins that demonstrate great potential as agricultural antimicrobials given their high killing efficiency and their precise strain-specific targeting ability. Our group has categorized and characterized tailocins produced by and tailocin sensitivities of the phytopathogen Pseudomonas syringae, and here we extend these experiments to test whether prophylactic tailocin application can prevent infection of Nicotiana benthamiana by P. syringae pv. syringae B728a. Specifically, we demonstrate that multiple strains can produce tailocins that prevent infection by strain B728a and engineer a deletion mutant to prove that tailocin targeting is responsible for this protective effect. Lastly, we provide evidence that heritable resistance mutations do not explain the minority of cases in which tailocins fail to prevent infection. Our results extend previous reports of prophylactic use of tailocins against phytopathogens, and establish a model system with which to test and optimize tailocin application for prophylactic treatment to prevent phytopathogen infection.

Pseudomonas Can Survive Tailocin Killing via Persistence-Like and Heterogenous Resistance Mechanisms.

Phage tail-like bacteriocins (tailocins) are bacterially produced protein toxins that mediate competitive interactions between cocolonizing bacteria. Both theoretical and experimental research has shown there are intransitive interactions between bacteriocin-producing, bacteriocin-sensitive, and bacteriocin-resistant populations, whereby producers outcompete sensitive cells, sensitive cells outcompete resistant cells, and resistant cells outcompete producers. These so-called rock-paper-scissors dynamics explain how all three populations occupy the same environment, without one driving the others extinct. Using Pseudomonas syringae as a model, we demonstrate that otherwise sensitive cells survive bacteriocin exposure through a physiological mechanism. This mechanism allows cells to survive bacteriocin killing without acquiring resistance. We show that a significant fraction of the target cells that survive a lethal dose of tailocin did not exhibit any detectable increase in survival during a subsequent exposure. Tailocin persister cells were more prevalent in stationary- rather than log-phase cultures. Of the fraction of cells that gained detectable resistance, there was a range from complete (insensitive) to incomplete (partially sensitive) resistance. By using genomic sequencing and genetic engineering, we showed that a mutation in a hypothetical gene containing 8 to 10 transmembrane domains causes tailocin high persistence and that genes of various glycosyltransferases cause incomplete and complete tailocin resistance. Importantly, of the several classes of mutations, only those causing complete tailocin resistance compromised host fitness. This result indicates that bacteria likely utilize persistence to survive bacteriocin-mediated killing without suffering the costs associated with resistance. This research provides important insight into how bacteria can escape the trap of fitness trade-offs associated with gaining de novo tailocin resistance.IMPORTANCE Bacteriocins are bacterially produced protein toxins that are proposed as antibiotic alternatives. However, a deeper understanding of the responses of target bacteria to bacteriocin exposure is lacking. Here, we show that target cells of Pseudomonas syringae survive lethal bacteriocin exposure through both physiological persistence and genetic resistance mechanisms. Cells that are not growing rapidly rely primarily on persistence, whereas those growing rapidly are more likely to survive via resistance. We identified various mutations in lipopolysaccharide biogenesis-related regions involved in tailocin persistence and resistance. By assessing host fitness of various classes of mutants, we showed that persistence and subtle resistance are mechanisms P. syringae uses to survive competition and preserve host fitness. These results have important implications for developing bacteriocins as alternative therapeutic agents.

Variable suites of non-effector genes are co-regulated in the type III secretion virulence regulon across the Pseudomonas syringae phylogeny.

Pseudomonas syringae is a phylogenetically diverse species of Gram-negative bacterial plant pathogens responsible for crop diseases around the world. The HrpL sigma factor drives expression of the major P. syringae virulence regulon. HrpL controls expression of the genes encoding the structural and functional components of the type III secretion system (T3SS) and the type three secreted effector proteins (T3E) that are collectively essential for virulence. HrpL also regulates expression of an under-explored suite of non-type III effector genes (non-T3E), including toxin production systems and operons not previously associated with virulence. We implemented and refined genome-wide transcriptional analysis methods using cDNA-derived high-throughput sequencing (RNA-seq) data to characterize the HrpL regulon from six isolates of P. syringae spanning the diversity of the species. Our transcriptomes, mapped onto both complete and draft genomes, significantly extend earlier studies. We confirmed HrpL-regulation for a majority of previously defined T3E genes in these six strains. We identified two new T3E families from P. syringae pv. oryzae 1_6, a strain within the relatively underexplored phylogenetic Multi-Locus Sequence Typing (MLST) group IV. The HrpL regulons varied among strains in gene number and content across both their T3E and non-T3E gene suites. Strains within MLST group II consistently express the lowest number of HrpL-regulated genes. We identified events leading to recruitment into, and loss from, the HrpL regulon. These included gene gain and loss, and loss of HrpL regulation caused by group-specific cis element mutations in otherwise conserved genes. Novel non-T3E HrpL-regulated genes include an operon that we show is required for full virulence of P. syringae pv. phaseolicola 1448A on French bean. We highlight the power of integrating genomic, transcriptomic, and phylogenetic information to drive concise functional experimentation and to derive better insight into the evolution of virulence across an evolutionarily diverse pathogen species.

Isolation of Endohyphal Bacteria from Foliar Ascomycota and In Vitro Establishment of Their Symbiotic Associations.

Endohyphal bacteria (EHB) can influence fungal phenotypes and shape the outcomes of plant-fungal interactions. Previous work has suggested that EHB form facultative associations with many foliar fungi in the Ascomycota. These bacteria can be isolated in culture, and fungi can be cured of EHB using antibiotics. Here, we present methods for successfully introducing EHB into axenic mycelia of strains representing two classes of Ascomycota. We first establish in vitro conditions favoring reintroduction of two strains of EHB (Luteibacter sp.) into axenic cultures of their original fungal hosts, focusing on fungi isolated from healthy plant tissue as endophytes: Microdiplodia sp. (Dothideomycetes) and Pestalotiopsis sp. (Sordariomycetes). We then demonstrate that these EHB can be introduced into a novel fungal host under the same conditions, successfully transferring EHB between fungi representing different classes. Finally, we manipulate conditions to optimize reintroduction in a focal EHB-fungal association. We show that EHB infections were initiated and maintained more often under low-nutrient culture conditions and when EHB and fungal hyphae were washed with MgCl2 prior to reassociation. Our study provides new methods for experimental assessment of the effects of EHB on fungal phenotypes and shows how the identity of the fungal host and growth conditions can define the establishment of these widespread and important symbioses.

Pseudomonas syringae CC1557: a highly virulent strain with an unusually small type III effector repertoire that includes a novel effector.

Both type III effector proteins and nonribosomal peptide toxins play important roles for Pseudomonas syringae pathogenicity in host plants, but whether and how these pathways interact to promote infection remains unclear. Genomic evidence from one clade of P. syringae suggests a tradeoff between the total number of type III effector proteins and presence of syringomycin, syringopeptin, and syringolin A toxins. Here, we report the complete genome sequence from P. syringae CC1557, which contains the lowest number of known type III effectors to date and has also acquired genes similar to sequences encoding syringomycin pathways from other strains. We demonstrate that this strain is pathogenic on Nicotiana benthamiana and that both the type III secretion system and a new type III effector, hopBJ1, contribute to pathogenicity. We further demonstrate that activity of HopBJ1 is dependent on residues structurally similar to the catalytic site of Escherichia coli CNF1 toxin. Taken together, our results provide additional support for a negative correlation between type III effector repertoires and the potential to produce syringomycin-like toxins while also highlighting how genomic synteny and bioinformatics can be used to identify and characterize novel virulence proteins.

The Pseudomonas viridiflava phylogroups in the P. syringae species complex are characterized by genetic variability and phenotypic plasticity of pathogenicity-related traits.

As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.

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

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

Bigger is not always better: transmission and fitness burden of ∼1MB Pseudomonas syringae megaplasmid pMPPla107.

BACKGROUND: Horizontal gene transfer (HGT) is a widespread process that enables the acquisition of genes and metabolic pathways in single evolutionary steps. Previous reports have described fitness costs of HGT, but have largely focused on the acquisition of relatively small plasmids. We have previously shown that a Pseudomonas syringae pv. lachrymans strain recently acquired a cryptic megaplasmid, pMPPla107. This extrachromosomal element contributes hundreds of new genes to P. syringae and increases total genomic content by approximately 18%. However, this early work did not directly explore transmissibility, stability, or fitness costs associated with acquisition of pMPPla107. RESULTS: Here, we show that pMPPla107 is self-transmissible across a variety of diverse pseudomonad strains, on both solid agar and within shaking liquid cultures, with conjugation dependent on a type IV secretion system. To the best of our knowledge, this is the largest self-transmissible megaplasmid known outside of Sinorhizobium. This megaplasmid can be lost from all novel hosts although the rate of loss depends on medium type and genomic background. However, in contrast, pMPPla107 is faithfully maintained within the original parent strain (Pla107) even under direct negative selection during laboratory assays. These results suggest that Pla107 specific stabilizing mutations have occurred either on this strain's chromosome or within the megaplasmid. Lastly, we demonstrate that acquisition of pMPPla107 by strains other than Pla107 imparts severe (20%) fitness costs under competitive conditions in vitro. CONCLUSIONS: We show that pMPPla107 is capable of transmitting and maintaining itself across multiple Pseudomonas species, rendering it one of the largest conjugative elements discovered to date. The relative stability of pMPPla107, coupled with extensive fitness costs, makes it a tractable model system for investigating evolutionary and genetic mechanisms of megaplasmid maintenance and a unique testing ground to explore evolutionary dynamics after HGT of large secondary elements.

Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 Pseudomonas syringae isolates.

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

Draft genome sequences for Pantoea ananatis ATCC 35400 and Pantoea stewartii subspecies indologenes ICMP 10132.

Here, we describe draft genome sequences for two bacterial isolates from the genus Pantoea. Pantoea ananatis ATCC 35400 was originally isolated from honeydew melon and was obtained from the American Type Culture Collection. Pantoea stewartii subspecies indologenes ICMP 10132 was originally isolated from sugarcane and classified as Pantoea ananatis, but average nucleotide identity and discriminatory PCR support species reclassification.

A beneficial synonymous substitution in EF-Tu is contingent on genetic background.

Synonymous mutations are changes to DNA sequence that occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation Elongation Factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels, as well as the polysome abundance on global transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.

Draft genome sequence of plant growth-promoting rhizobium Mesorhizobium amorphae, isolated from zinc-lead mine tailings.

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